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基因组学与植物改良 FROM GENOMICS TO PLANT IMPROVEMENT


FROM GENOMICS TO PLANT IMPROVEMENT

基因组学与植物改良

Proceedings of the 3rd International Conference of Plant Molecular Breeding 第三届植物分子育种国际会议论文摘要

Beijing,September 5

-9, 2010
北京,2010 年 9 月 5-9 日

Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

ICPMB2010 Organization
Honorary Presidents
Dr. JM Ribaut, Generation Challenge Program, CGIAR Dr. Huqu Zhai, Chinese Academy of Agricultural Sciences Dr. Qifa Zhang, Huazhong Agricultural University Dr. Jiayang Li, Chinese Academy of Sciences

President
Dr. Zhikang Li, Chinese Academy of Agricultural Sciences & International Rice Research Institute

Co-Presidents
Dr. Jianmin Wan, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences Dr. Aimin Zhang, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences

International Organizing Committee Chair : Zhikang Li, Chinese Academy of Agricultural Sciences & International Rice Research Institute Co-Chair : JM Ribaut, Generation Challenge Program, CGIAR Members:
Aimin Zhang, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences Andrew H. Paterson, University of Georgia, USA Christian Jung, Plant Breeding Institute, Christian-Albrechts-University of Kiel David Mackill, International Rice Research Institute, Philippines Jiayang Li, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences Jinguo Hu, USDA-ARS, USA John Z Yu, USDA-ARS, Crop Germplasm Research, Texas A&M University, USA Lijun Luo, SAGC, Shanghai Academy of Agricultural Sciences Mark J. van Haaren, Keygene N.V. Masahiro Yano, National Institute of Agrobiological Sciences, Japan Graham McLaren, The Generation Challenge Program, CGIAR Henry T. Nguyen, University of Missouri, USA Noel Ellis, John Innes Centre, UK Peter Langridge, Australia National Center for Plant Functional Genomics, Adelaide, Australia Qifa Zhang, Huazhong Agricultural University Roberto Tuberosa, University of Bologna, Italy Swapan Datta, Indian Council of Agricultural Research, India Yunbi Xu, CIMMYT, Mexico Xingwang Deng, Peking University, China; Yale University, USA 2

Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Zhonghu He, Chinese Academy of Agricultural Sciences & CIMMYT Michael Thomson, International Rice Research Institute, Philippines

Local Organizing Committee Chair : Jianmin Wan, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences Co-Chairs :
Aimin Zhang, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences Shuming Wang, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences

Members :
Daowen Wang, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences Yuxian Zhu, Peking University, China Zhen Zhu, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences

Program Committee Chair : Zhikang Li, Chinese Academy of Agricultural Sciences & International Rice Research Institute Co-Chair: Members:
Aimin Zhang, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences David Mackill, International Rice Research Institute, Ithaca, New York, USA JM Ribaut, Generation Challenge Program, CGIAR Masahiro Yano, National Institute of Agrobiological Sciences, Japan Mark J. van Haaren, Keygene N.V. Noel Ellis, John Innes Centre, UK Peter Langridge, Australia Center for Plant Functional Genomics Qifa Zhang, Huazhong Agricultural University, China Roberto Tuberosa, University of Bologna, Italy Swapan Datta, Indian Institute of Agricultural Research, India Xingwang Deng, Peking University, China; Yale University, USA Yongbiao Xue, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences Jinguo Hu, USDA-ARS, USA

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Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Contents
LECTURES
Plenary Session I……………………………………………………………….……………..10-13
Molecular breeding in developing countries: not a dream anymore. Ribaut JM Progress of rice functional genomics research and the implications in crop genetic improvement. Zhang QF Towards molecular design of super rice. Li JY Progress and challenges in molecular breeding for drought tolerance in crop plants. Nguyen HT

Plenary Session II…………………………………………………………….……………..14-18
Three genetic systems controlling rice growth and productivity–a reevaluation of the green revolution. Li ZK Genomics-assisted germplasm enhancement and its integration to breeding in rice. Yano M Molecular basis of heterosis in crop plants: From nonadditive gene expression to gene regulatory network. Sun QX Transgenic trait development and deployment circa 2010. Bedbrook J Transgenic crop research in India-current status and perspectives. Datta S

Plenary Session III……………………………………………………………….…………..19-23
Fostering molecular breeding in developing countries: The GCP approach. Delannay X The sorghum genome, the diversification of cereals, and the productivity of tropical grasses. Paterson AH Polyploidy and epigenetics: direct application and impact on crop improvement. Chen ZJ Identification of key regulators for flowering time control and their application in breeding of biennial crop species. Jung C Whole genome strategies for molecular plant breeding. Xu YB

Plenary Session IV…………………………………………………………….……………..24-28
Global epigenetic and transcriptional trends among two rice subspecies and their reciprocal hybrid. Deng XW Breeding seeds of innovation. Hervé PM
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Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Breeding by design and innovations in molecular plant breeding. Sorensen A Meeting the challenge of higher nutritional value in seeds: a novel way of increasing methionine content in seeds of the model plant of tobacco. Amir R Association mapping for enhancing maize genetic improvement. Yan JB

Concurrent session 1: Molecular breeding for abiotic stress tolerances……………….…29-35 Mapping QTLs for root morphology in relation to nutrient uptake in wheat. Tong YP
The research progress of drought tolerance and molecular breeding in maize. Wang GY Towards molecular breeding for salt tolerance through modification of root System architecture. Li X Mapping and validating QTLs for plant height developmental behaviours in bread wheat. Jing RL Discovery of genes for drought resistance improvement of rice by systematic genetic and functional genomic approaches. Xiong LZ Heat stress transcriptome analysis and functional characterization of responsive genes in wheat. Ni ZF

Concurrent session 2: Gene discovery and function……………………………………….36-42
Identification and application of the rice broad-spectrum blast resistance gene Pigm. He ZH Mutant resources for functional studies of genes related to fertility in rice. Wu CY Gene discovery from common wild rice (Oryza rufipogon Griff). Sun CQ Discovery of brown planthopper resistance gene in rice. He GC Molecular basis of cytoplasmic male sterility in rice. Liu YG Toward map-based cloning of a good eating-quality QTL derived from an elite Japanese rice cultivar Koshihikari. Hori K Map-based cloning of QTL genes for flowering time/maturity in soybean. Xia ZJ

Concurrent session 3: Molecular breeding for biotic stresses…………………….……….43-49
From QTLs for fungal disease resistance to marker-assisted selection in durum wheat. Maccaferri M Genomic approaches to plant defense research and crop improvement for insect resistance. Huang YH Improvement of maize resistance to head smut and stalk rot. Xu ML Enhancing broad spectrum resistance to rice diseases. Wang SP Molecular mapping of adult-plant resistance genes to stripe rust and powdery mildew and validation of allelic
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Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

specific markers for Lr34/Yr18/Pm38 in Chinese wheat cultivars. Xia XC Infection character and rice resistance screening of Southern rice black-streaked dwarf virus, a new Fiji virus threating rice production in Asia. Zhou GH

Concurrent session 4: New transgenic technologies, products and markets……………..50-58
New transgenic technologies. Broglie R Simultaneously changing several quality traits of Brassica napus by one transgenic event. Liu CL In situ Pistil Delivery: A High Throughput Method of Brassica Genetic Transformation. Guo XL Wheat genetic transformation in China, current status, challenges and future perspectives. Xia LQ A new effective selection marker for crop transformation. Xia M Enhancing the lysine in wheat grain by genetic transformation of a lysine rich protein gene Cflr. Ma HX Transgenic strategies for improving drought tolerance traits in chickpea. Bhatnagar-Mathur P Identification of stress-inducible and tissue-specific promoters in rice. Zhou JL

Concurrent session 5: Molecular breeding for cotton, brassica and bio-energy crops…..59-65
Progress toward genome sequencing of upland cotton, Gossypium hirsutum. Yu SX Maternal effects and genetic improvement of seed oil content in Brassica napus. Wang HZ Towards establishing a molecular breeding platform in cotton: Progress and challenges. Kumpatla SP Molecular breeding of apomixis hickory. Huang JQ Mining of novel genes for cotton fiber improvement. Yu JZ Rational design and molecular breeding of sorghum, a dedicated bioenergy crop. Huang YH Molecular breeding for cottonseed quality improvement. Zhu SJ Molecular focus in commercial plant breeding. Rossouw JD

Concurrent session 6: Maize molecular breeding……………………………………….....66-72
QTL fine mapping of leaf angle and leaf orientation value in maize. Chen YH Application of molecular techniques in maize haploid breeding. Chang MT Identification of gene marker sets for screening maize lines for resistance to aflatoxin contamination. Luo M Maize disease resistance gene discovery and utilization through association and linkage mapping. Mahuku G
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Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Forward to molecular breeding from genetics in high-oil maize. Li JS Genome-wide association study identifies known as well as novel loci for maize kernel tocopherol content and composition. Li Q

Concurrent session 7: Applied plant genomics: from genomics to field…………………..73-77
Molecular breeding in chickpea- still a dream or the reality now! Varshney RK Single-base resolution DNA methylomes of rice and new regulatory roles of DNA methylation in plant gene expression. Li X Insertion site-based polymorphism markers open new perspectives for genome saturation and marker-assisted selection in wheat. Paux E Integrating technologies for genetic improvement of quantitative traits in sorghum. Mace E Irradiation mutant mapping of wild beet translocation lines carrying resistance genes against the beet cyst nematode. Capistrano G

Concurrent session 8: Rice molecular breeding…………………………………..………..78-85
Development of 384-plex SNP marker sets for diversity analysis, mapping, and marker-assisted selection in rice. Thomson MJ Epigenetic and genetic control of drought tolerance in rice – a merging story of Larmarkism and Mendelism. Li ZK Clustered QTLs for source leaf size and yield traits in rice (Oryza sativa L). Yu SB Molecular breeding approaches for sustainable disease resistance in rice: Current and future strategies. Vera Cruz CM MAS pyramiding of disease and pest resistant genes into drought tolerant hybrid rice. Mei HW Development of single nucleotide polymorphisms (SNPs) detection platforms for genetic analyses and molecular breeding of rice. Chen HD Identification of a new blast resistant gene from Dacca6, a useful donor to improve the wide spectrum resistance of Jin23 against rice blast fungi (Magnaporthe grisea) in Southeast China. Shi BH

Concurrent session 9: Wheat molecular breeding…………………………………….……86-93
Towards systematic genetic and functional analyses of the complex gliadin gene family in common wheat. Wang DW Development and application of molecular markers for improving processing quality in common wheat.
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Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

He ZH New insights into the organization, recombination, expression and functional mechanism of low molecular weight glutenin subunit genes at the complex glu-3 loci in bread wheat. Ling HQ QTL mapping and marker assisted selection for some quality traits in bread wheat. Gupta P

Application of MAS for resistance to Fusarium head blight in a wheat breeding program Fedak G
Genomic distribution of quantitative trait loci (QTL) for yield and yield-related traits in common wheat (Triticum aestivum). Zhang LY Gene function and modulation of DREB (dehydration-responsive element binding protein) genes from soybean. Chen M

Concurrent session 10: Molecular breeding platform and new technologies…………...94-98
The integrated breeding platform: vision and practice. McLaren G Optimization of NGS-based SNP discovery approaches for facilitating molecular breeding in orphan crop species. Varshney R ISMAB: A data visualization and decision support tool for crop improvement. Shah T Bringing genomic data to breeding: what we expect from the IBP to help future breeding. Liang CZ Development and optimization of the 50K infinium chip for maize diversity analysis. Ganal M

Concurrent session 11: Germplasm and genetic diversity…………………………..…..99-106
Core collection-based genomic stocks in wheat. Jia JZ High-throughput SNP genotyping of a subset of lettuce landraces for genetic diversity assessment. Hu JG The genetic diversity, structure and classification of rice germplasm in China Li ZC Genetic Diversity Studies on Cool Season Legumes. Zong XX Molecular diversity reveals narrow genetic base of local Ghanaian accessions. Quain MD The strategy and potential utilization of temperate germplasm for tropical germplasm improvement—a case study in maize (Zea mays. L). Wen WW

Concurrent session 12: Molecular breeding in legumes and trees crops…………….....107-110
Concentration of genetic diversity for gene discovery and broadening genetic base of modern cultivar in soybean. Qiu LJ
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Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Development and application of genomic resources for molecular breeding in groundnut (Arachis hypogaea L). Pandey M The genomics path from pre-breeding to marker-assisted selection in wheat and barley. Tuberosa R Genomics tools to aid cassava breeding for drought tolerance Rabinowicz P Genetic networks controlling zygomorphic development in legumes Luo D

POSTERS……………………………………………………………………………..….111-231

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Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Plenary Session I

Molecular breeding in developing countries: not a dream anymore
Ribaut JM Generation Challenge Programme (GCP), c/o CIMMYT, Int APDO Postal 6-641, 06600 Mexico, DF, Mexico. Email: J.RIBAUT@cgiar.org

Molecular breeding (MB) is definitely an efficient approach, when the necessary minimum human and operational resources are already in place.This is because MB increases genetic gain per crop cycle, stacksfavourable alleles at target loci and reduces the number of selection cycles. In the last decade, the private sector has benefitted immensely from MB, which demonstrates its efficacy. In contrast, MB adoption is still limited in the public sector, and hardly used in developing countries. Major bottlenecks in these countries include shortage of well-trained personnel, inadequate high-throughput capacity, poor phenotyping infrastructure, lack of information systems or adapted analysis tools, or simply resource-limited breeding programmes.The emerging virtual platforms aided by the information and communication technology revolution will help to overcome some of these limitations, by providing breeders with better access to genomic resources, advanced laboratory services, and robust analytical and data management tools. It is unrealistic to project that large-scale MB breeding activities will be conducted in the near-term in developing countries. However, the exponential development of genomic resources,the ever-decreasing cost of marker technologiesand the emergence of platforms for accessing MB tools and support services, plus the increasing public–private partnerships and needs-driven demand for improved varieties to counter the global food crisis, are all grounds to predict that MB will have a significant impact on crop breeding in developing countries.

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Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Plenary Session I

Progress of rice functional genomics research and the implications in crop genetic improvement
Zhang QF National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research, Huazhong Agricultural University, Wuhan 430070, China Email: qifazh@mail.hzau.edu.cn

There has been a large global effort in rice functional genomics research aiming at characterization of the full complement of the rice genes. The Chinese program on rice functional genomic research is composed of the following components: (1) development of technological platforms, (2) functional genomics of agriculturally important traits, (3) molecular cloning of important genes and, (4) gene discovery by resequencing natural diversity of the rice species. The traits targeted for functional genomic studies include yield, grain quality, stress tolerance, disease and insect resistances, and nutrient use efficiency. Major progress has been made in a number of fronts. Totally 270,000 independent transformants have been generated for the T-DNA insertion mutant library and are now being screened for mutations of important traits. Over 50000 flanking sequences have been isolated, and their analyses identified a number of interesting features of nonrandom distributions of the T-DNA insertions in the rice genome. A large number of mutants have now been targeted for gene isolation. For genome-wide expression profiling, data have been generated from a large number of tissues covering the whole life cycle of the rice plants grown under various conditions. Map-based cloning has been applied to isolate genes of agronomic importance, including dozens of genes for yield, grain quality, fertility restoration, resistances to biotic and abiotic stresses. Hundreds of accessions of rice germplasm have been resequenced using new sequencing technologies. The implications of these developments in crop genetic improvement will be discussed in the presentation.

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Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Plenary Session I

Towards Molecular Design of Super Rice
Li JY, Wang YH Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China Email: jyli@genetics.ac.cn

Rice (Oryza sativa. L) is one of the most important staple crop, feeding more than half of the world’s population. To achieve super rice varieties, we focus on the improvement of the grain yield, grain quality, disease and insect resistance. Rice plant architecture, a collection of the important agronomic traits that determine grain production, is mainly affected by factors including tillering (tiller number and tiller angle), plant height, and panicle morphology. To elucidate molecular mechanisms that control rice plant architecture, we have identified several key genes that contribute greatly to the plant architecture of rice. Among them, the MONOCULM1 (MOC1) gene was characterized as an essential regulator involved in tiller bud initiation and outgrowth; the DWARF27 (D27) gene acts as a new component involved in the biosynthesis of strigolactones and controls rice tiller number by regulating the outgrowth of tiller buds; the LA1 gene plays an important role in determining tiller angle by negatively regulating polar auxin transport (PAT); the SHORT PANICLE1 (SP1) gene encodes a transporter that regulates the panicle size. The quantitative trait locus (QTL) gene, Ideal Plant Architecture 1 (IPA1), profoundly affects rice plant architecture and substantially enhances rice grain yield. Our studies demonstrate that the application of these genes will facilitate to breed new elite varieties by modifying tiller number, tiller angle, plant height, panicle morphology and lodging resistance. To improve the rice grain quality, we carried out a systematic examination of genetic determinations of rice grain ECQ through a comprehensive association analysis, the results of which were then further have been confirmed by gene transformation. A series of molecular markers have been developed for MAS. Our research findings provided a much clearer picture of how starch synthesis system regulates grain quality. Also, we engage in cloning insect resistance genes and developing molecular markers that are linked to quantitative trait loci for rice insect resistance. Our studies will provide a molecular basis for developing super rice varieties in the future.

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Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Plenary Session I

Progress and challenges in molecular breeding for drought tolerance in crop plants
Nguyen HT, Valliyodan B, Manavalan L Division of Plant Sciences and National Center for Soybean Biotechnology, University of Missouri, Columbia, MO 65211 Email: nguyenhenry@missouri.edu

Production of sufficient food for the growing world population during the verge of global climate changes will be one of the major challenges for the future. This demands the requirement of directed adaptation of crop species on an unprecedented magnitude. The global grain demand is expected to be double by 2050. Much effort is being made by agricultural researchers worldwide to reduce water use by crops to address the challenge which especially affect farmers in drought-prone environments across the developing world. Understanding the concept and components of drought resistance is a key factor for improving drought tolerance of crops. Research to date has shown that improvements in crop drought resistance are from the increasing dehydration avoidance, specifically increasing water availability for plant functions through changes such as earlier development, smaller leaves, and deeper roots. In addition, plasticity response of root growth under water deficit conditions, and dehydration tolerance traits such as; osmotic adjustment, cell membrane stability, and mobilization of stem carbohydrate reserves in crops also play specific roles in drought resistance mechanisms. Molecular breeding approaches through identification of quantitative trait loci (QTL) and marker-assisted selection offers an opportunity for significant improvements in the drought tolerance of crops; however the successful application of marker assisted selection to crop breeding is still in the preliminary stage. Past studies aimed at osmo-protection did not result in field performance for drought tolerance in crops. Recent work on engineering candidate genes including transcription factors and cold shock responsive proteins to enhance drought tolerance showed promising results in field conditions. Transgenic maize plants with a transcription factor show tolerance to drought based on the responses of a number of stress-related parameters, including; stomatal conductance, leaf temperature, reduced wilting, and maintenance of photosynthesis. Another example is engineering farnesylation machinery for plant drought tolerance and yield protection-through stomatal closure, and these transgenic plants showed promising field performance. Enhanced drought tolerance has also been observed in transgenic plants expressing a cold shock protein under field conditions. Research advances in the area of integrated functional genomics will certainly be helpful to improve the molecular breeding and plant transformation approaches to achieve a significant progress in the generation of crop plants with enhanced drought resistance.

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Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Plenary Session II

Three Genetic systems Controlling Rice Growth and productivity – A Reevaluation of the Green Revolution
Zhang F1, Xu JL1, Gao YM1, Yu SB2, Fu BY1, Ali J2 and Li ZK1,2 , * 1 Institute of Crop Sciences/National Key Facility for Crop Gene Resources and Genetic Improvement, Chinese Academy of Agricultural Sciences, Beijing 100081, China; 2 IRRI, DAPO Box 7777, Metro Manila, The Philippines. *Email: zhkli@yahoo.com.cn

The well-known Green Revolution (GR) since 1960s has more than doubled the productivity of rice, which results from loss of function alleles at the GAox-2 locus encoding gibberellin 20-oxidase. Over 95% of the current worldwide rice breeding programs are carried out in the mutant sd1 genetic backgrounds without functional gibberellin acids (GA). To better understand the effects of sd1 on rice yield and related traits, the phenotypic data of the IR64/Azucena DH population across 11 diverse environments were reanalyzed using a new molecular-quantitative genetics model. Three genetic systems controlling rice growth and productivity in rice were revealed, resulting in the discovery of 157 functional genetic units (FGUs) affecting 9 traits related to rice growth, development and productivity. The first one was the GA-mediated pathways controlled by SD1 and its 43 downstream FGUs for increased plant height (PH), increased biomass, reduced spikelet fertility (SF), delayed heading (HD), reduced harvest index (HI), reduced panicle number (PN), increased grain weight (GW) and reduced yield. Their effects gain yield (GY) and spikelet number per panicle (SN) varied depending on the environments. Of these downstream FGUs, 3 PH QTLs (QPh2b, QPh3b and QPh4a) had effects highly correlated with the mean PH values of the SD1 subpopulation, suggesting their positive responses to the overall soil fertility levels of the test environments. Together, the GA-mediated pathways explained 38.6%, ranging from 16.0% for SF to 54.8% for PH. The second system was the GA-repressed pathways that were expressed only in the mutant (sd1) background, which comprised of 39 FGUs for PH, SF, biomass, HD, SN, PN, HI, GW, and yield. The effect directions of most these pathways could not be determined based on available QTL information. The GA-repressed pathways collectively explained 32.3% of the total genotypic variation of the 9 traits in the DH population, ranging from 14.7% for PN to 59.3% for SN. The third one was the GA independent pathways controlled by 75 FGUs that affected all measured traits. Together, the GA-independent pathways explained 29.2% of the total genotypic variation of the 9 traits in the DH population, ranging from 6.0% for PH to 55.8% for PN. Because the overall effects of the GR are reflected by the differences between the GA-mediated and GA-repressed pathways, detailed Comparison between them indicated that the former had larger effects on PH, HD, PN, HI and GY, whereas the latter influenced more SN and SF. Based on these results, the advantages and potential consequences of the GR gene, sd1, were discussed in the context of the global rice improvement and its challenges. Alternative breeding strategies for developing “Green Super Rice” cultivars that have high yield potential with less input are proposed based on our discoveries.

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Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Plenary Session II

Genomics-assisted germplasm enhancement and its integration to breeding in rice
Yano M*, Hori K, Uga Y, Fukuoka S, Ebana K, Yonemaru J and Yamamoto T QTL Genomics Research Center, National Institute of Agrobiological Sciences, Tsukuba, Japan. *E-mail: myano@nias.affrc.go.jp

Progresses on recent genomics in rice have provided a new tools and opportunities to enhance activity in crop improvement. Elucidation of the association between nucleotide and phenotypic changes is inevitable to this end and has been a big challenge in molecular genetics and breeding of rice. Toward this goal, we have been involved in the genetic dissection of natural phenotypic variations in rice and have identified several genes involved in complex traits, including heading date, shattering habit, pre-harvest sprouting, root morphology, disease resistance, seed size and eating quality. To enhance the power of genetic dissection of complex phenotypes, we are developing several mapping populations, such as recombinant inbred lines and chromosome segment substitution lines, which will allow us to extract the useful alleles from natural variants. Recently, QTL for durable resistance to rice blast has been cloned from Japanese upland rice. This finding has opened new opportunity to introduction of the unique blast resistance gene without a linkage drag of low eating quality. We have also detected a major QTL for deeper rooting on chromosome 9. This finding has open new opportunity to enhance drought avoidance in rice. To facilitate allele mining using novel plant materials, we have also embarked on the genome-wide discovery of single nucleotide polymorphisms (SNPs). In particular, to overcome a shortage of SNPs among temperate japonica cultivars, we have attempted whole-genome sequencing of several Japanese cultivars using next-generation sequencing approaches. This SNP discovery has led to the development of an array-based SNP genotyping system in Japanese rice cultivars. Large-scale genotyping of these SNPs has made it possible to visualize pedigree haplotypes of particular chromosome segments in the Japanese landraces and modern cultivars. These efforts in genomics have opened up new opportunities to accelerate not only the genetic dissection of complex traits, but also integration of genomics to breeding in rice.

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Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Plenary Session II

Molecular basis of heterosis in crop plants: From nonadditive gene expression to gene regulatory network
Sun QX, Ni ZF, Yao YY, Peng HR, Du JK China Agricultural University, Beijing 100183, China. *Email: qxsun@cau.edu.cn Whole genome expression analysis in hybrid and its parental inbreds provides a platform to identify nonadditively expressed genes in hybrids, which have given some insights into the understanding of mechanisms of heterosis. In this study, two wheat (Triticum aesticum L.) hybrid F1 derived from same female parent but displaying contrasting heterosis in primary root are used for expression analysis by using wheat genome array. The expression polymorphism analysis between the parental inbreds indicates that up to 4% genes display expression difference, but more than 3 times more present-absent genes between the two parental inbreds are detected in highly heterotic Hybrid A than in nonheterotic Hybrid B. Differential expression (DE) analysis in hybrids and their parental inbreds identify 1019 (4.94%) and 698 (3.23%) DE genes in Hybrid A and B, respectively. It is interesting to note that heterotic Hybrid A tends to have more DE genes of dominance and partial dominance expression modes than nonheterotic Hybrid B which, however, tends to have more DE genes of negative partial dominance expression mode. By adopting the “Wooden Barrel Principle”, we propose that accumulation of dominance and partial dominance expression in wheat hybrid could be a major determinant of root heterosis. We also find that a substantial number of stress-related genes as well as retrotransposon-like and transposon-like genes are also included in the DE genes. We propose that as compared to the interspecific hybridization which can be a source of genomic shock as described by Barbara McClintock, hybrids derived from less distantly-related two inbreds can be a source of “mild genomic shock” or “intrinsic stress” in the hybrid genome, which, in turn, could cause expression changes of genes, especially stress-related genes and retrotransposon. Heterosis in internode elongation and plant height are commonly observed in hybrid plants, and higher GAs contents were found to be correlated with the heterosis in plant height. By using the uppermost internode tissues of wheat, we examined expression patterns of genes participating in both GA biosynthesis and GA response pathways between a hybrid and its parental inbreds. Our results indicated that among the 18 genes analyzed, genes encoding enzymes that promote synthesis of bioactive GAs, and genes that act as positive components in the GA response pathways were up-regulated in hybrid, whereas genes encoding enzymes that deactivate bioactive GAs, and genes that act as negative components of GA response pathways were down-regulated in hybrid. Moreover, the putative wheat GA receptor gene TaGID1, and two GA responsive genes participating in internode elongation, GIP and XET, were also up-regulated in hybrid. A model for GA and heterosis in wheat plant height was proposed. This model is also validated by using 16 wheat hybrids with different level of heterosis in plant height. Our results provided molecular evidences not only for the higher GA levels and more active GA biosynthesis in hybrid, but also for the heterosis in plant height of wheat and possibly other cereal crops. Moreover, overexpression of 6 differentially expressed genes suggested that up-regulated genes in hybrids could enhance the trait performance but the down-regulated genes in hybrids can have negative effects on the trait performance.
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Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Plenary Session II

Transgenic Trait Development and Deployment Circa 2010
Bedbrook J Vice President, DuPont Agricultural Biotechnology

Transgenic traits providing weed and insect pest control solutions, first introduced in the mid 1990’s have been rapidly adopted globally in corn, soybean and cotton. Next generation transgenic traits providing new functionalities, including; grain quality attributes, abiotic stress tolerance, disease resistance and seed production systems are close to commercialization. In this paper I describe DuPont’s approaches to genetic based gene discovery, event selection, trait development and commercial deployment for these next generation traits.

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Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Plenary Session II

Transgenic Crop Research in India-Current status and perspectives
Datta SK Crop Science Division, ICAR, Krishi Bhavan, New Delhi-110114, India Email: swpndatta@yahoo.com

Functional genomics provides powerful tool for the identification of desirable genes and their introduction into crops for the trait improvement. The ability to introduce beneficial genes under the control of specific promoters through transgenic approaches is the path towards targeted crop improvement. Development and commercialization of transgenic crops expressing a wide range of agronomic traits during mid-nineties has virtually revolutionized the face of global agriculture. Safety of transgenic crops, especially GM food crops is a major concern. To address all the issues related to biosafety, environmental safety, risk assessment, biodiversity and socio-economic impact the GM crops, Government of India has entrusted the task to the Ministry of Science and Technology to develop one window regulatory mechanism to approve and release the GM in the field through NBRA (National Biotechnology Regulatory Authority). The environmental release of transgenic cotton with insect-pest resistance in 2002 is a landmark in Indian agriculture. It has placed India at the forefront of global cotton production and trade. At the global level, cultivation of transgenic crops in the past twelve years has conferred significant social, economic and environmental benefits to mankind. Such a sea change in the production of major food crops is the need of the hour. Bt cotton, which confers resistance to important insect pests of cotton, was first adopted in India as hybrids in 2002. The number of events, as well as the number of Bt cotton hybrids and companies marketing approved hybrids increased from one event and 20 hybrids in 2005 by more than three-fold in 2009 to six events and 282 hybrids. India currently produces >30 million bales of cotton per year and occupies # 2 position in terms of global cotton production and now #1 in Bt cotton areas. Other Crops such as Bt rice, Bt brinjal, transgenic tomato, Sorghum, Brassica, Groundnut etc are at the different stages of development.

18

Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Plenary Session III

Fostering Molecular Breeding in Developing Countries -the GCP approach
Xavier Delannay Generation Challenge Program, CGIAR Email:x.delannay@cgiar.org

An important focus of the Generation Challenge Programme (GCP) since its inception has been to promote an increased use of molecular marker technologies in developing country breeding programmes. This started with the implementation in applied breeding programmes of marker-assisted selection for new important traits that had been mapped with funding assistance from the GCP. More recently, the GCP has focused on the implementation of new integrated breeding programmes in developing country crops through the use of molecular breeding technologies such as marker-assisted recurrent selection (MARS). The use of MARS should help accelerate the improvement of crops growing under suboptimal conditions of Africa and Asia, which is also a focus of the GCP. This development will be greatly facilitated by the Integrated Breeding Platform that is concurrently being developed by the GCP. Examples will be shown of practical applications of molecular breeding being used or being put in place in developing countries for crops such as rice, cassava, sorghum, cowpea and chickpea.

19

Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Plenary Session III

The sorghum genome, the diversification of cereals, and the productivity of tropical grasses
Paterson AH Plant Genome Mapping Laboratory, University of Georgia 111 Riverbend Road, Rm 228, Athens, GA 30602 Email: paterson@plantbio.uga.edu

Sorghum, an African grass related to sugarcane and maize, is grown for food, feed, fiber, and fuel, is representative of tropical grasses that are among the most efficient biomass accumulators thanks to ‘C4’ photosynthesis. An initial analysis of the sorghum genome placed ~98% of genes in their chromosomal context using whole genome shotgun sequence validated by genetic, physical, and synteny information. Genetic recombination is largely confined to about one-third of the sorghum genome with gene order and density similar to those of rice. Retrotransposon accumulation in recombinationally-recalcitrant heterochromatin explains the ~75% larger genome size of sorghum than rice. While gene and repetitive DNA distributions have been preserved since paleopolyploidization ~70 million years ago, most duplicated gene sets lost one member before sorghum/rice divergence. Concerted evolution makes one duplicated chromosomal segment appear only a few million years old. About 24% of genes are grass-specific and 7% are sorghum-specific. Recent gene and miRNA duplications may contribute to sorghum’s drought tolerance. The sorghum sequence offers new means to improve sorghum itself and new or existing biofuel crops, and to try to control weedy and invasive plants.

20

Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Plenary Session III

Polyploidy and epigenetics: direct application and impact on crop improvement
Chen ZJ
Institute for Cellular and Molecular Biology, The University of Texas at Austin, Texas 78712, USA. Email: zjchen@mail.utexas.edu

Polyploidy, or whole-genome duplication (WGD), is common in some animals and many plants, including important crops such as wheat, cotton, canola, sugar cane, and switchgrass. The common occurrence of polyploidy suggests an evolutionary advantage of having multiple sets of genetic material for adaptive evolution and crop domestication. However, increased gene and genome dosages in autopolyploids (duplications within species) and allopolyploids (combination of two or more divergent genomes among species) often cause genome instabilities, chromosome imbalances, regulatory incompatibilities, and reproductive failures. Therefore, new allopolyploids must establish a compatible relationship between alien cytoplasm and nuclei and between two divergent genomes, leading to rapid changes in genome structure, gene expression, and developmental traits such as fertility, inbreeding, apomixis, flowering time, and hybrid vigor. Although the underlying mechanisms for these changes are poorly understood, some themes are emerging. There is compelling evidence for epigenetic changes during early stages of polyploid formation. Using Arabidopsis allopolyploids and hybrids as model systems, we found that changes in cis- and trans-acting effects, chromatin modifications, RNA-mediated pathways, and regulatory networks modulate differential expression of homoeologous genes and phenotypic variation such as flowering time. We have shown that nonadditive gene expression, small RNAs, and epigenetic regulation of circadian-mediated metabolic pathways, play central roles in growth vigor in hybrids and allopolyploids. Understanding epigenetic mechanisms for polyploidy and hybrid vigor will facilitate the use and exploitation of the increased biomass and yield in hybrids and allopolyploids for food, feed, and fuels.

21

Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Plenary Session III

Identification of key regulators for flowering time control and their application in breeding of biennial crop species
Jung C*, Wafa SAE, Büttner B, Schulze-Buxloh G, Müller A

Plant Breeding Institute, Christian-Albrechts-University of Kiel
*Email: c.jung@plantbreeding.uni-kiel.de

Floral transition is a major developmental switch that is tightly controlled by regulatory pathways that integrate endogenous and environmental cues to ensure flowering under favourable conditions. Sugar beet (Beta vulgaris) is a biennial crop which bolts and flowers after a period of cold temperatures over winter, however annual types without vernalization requirement exist. We have identified >30 flowering time regulators from the beet genome by different approaches. The existence of an FLC-like gene in beet suggests similar regulatory pathways as in Arabidopsis. In a complementary approach additional components of the floral transition gene network in sugar beet are being identified by homology to genes from model species and genome-wide transcript profiling. We found a number of ESTs with homology to Arabidopsis genes. Using RACE and BAC cloning we identified full length cDNA and genomic sequences. We functionally characterized these sequences by expression analysis and transformation into Arabidopsis. We found evidence for the existence of autonomous and vernalization pathways in beet similar to Arabidopsis, however substantial differences between both species exist. Annuality is controlled by the bolting locus B. We have identified by map based cloning sequences from the B locus with homology to floral transition genes from other species that suggest that they mediate bolting time control in response to environmental cues. Another QTL for early bolting was mapped with molecular markers demonstrating for the first time that at least two loci cause early bolting in beets. A beet TILLING platform now also enables the identification of mutants and functional characterization of candidate genes. New beet prototypes with altered vernalization requirement have been produced either by EMS mutagenesis or transformation. These mutants in combination with transgenic beets with altered bolting behaviors are needed for the breeding of winter beets which are sown before winter. Apart from winter hardiness these beets must be completely bolting resistant to prevent bolting after winter. Different approaches to establish fully bolting resistant beet prototypes are presented.

22

Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Plenary Session III

Whole Genome Strategies for Molecular Plant Breeding
Xu YB1*, Lu YL2 and Gao SB2 Institute of Crop Science and CIMMYT, Chinese Academy of Agricultural Sciences, National Key Facilities for Crop Genetic Resources and Improvement, 12 Zhongguancun South St., Beijing 100081, China. 2Maize Research Institute, Sichuan Agricultural University, Ya’an, Sichuan 625014, China. *Email: y.xu@cgiar.org
1

Molecular breeding for complex traits in plants needs to understand and manipulate many factors influencing plant growth and development including genotypes, environments and their interaction. Molecular breeding procedures can be facilitated and revolutionized through whole genome strategies, which are featured by utilizing full genome sequence and genome-wide molecular markers to address all genomic and environmental factors through a representative or complete set of genetics and breeding germplasm. The strategies should be developed for understanding specific genomic region, genes, haplotypes, linkage disequilibrium block or alleles and their contribution to specific phenotypes and breeding products. Genotyping-by-sequencing and genomewide selection are two important components of the strategies. These strategies need to be integrated with precision phenotyping and powerful population management systems. Examples of such integrated systems include joint linkage-linkage-disequilibrium mapping for marker development and gene discovery, breeding-to-genetics approaches by using existing genetic and breeding materials, and simultaneous genomewide improvement for multiple traits. As components of whole genome strategies, molecular breeding platforms and methodologies should be backed up with strong supporting systems such as breeding informatics and decision support tools. Some basic strategies will be discussed using maize as an example, including (1) seed DNA-based genotyping for simplifying marker-assisted selection, reducing breeding cost and increasing scale and efficiency, (2) selective genotyping and phenotyping for capturing most important contributing factors with optimized breeding design, (3) flexible genotyping systems refined for different selection methods including marker assisted selection, marker assisted recurrent selection and genomic selection, and (4) sequence-based strategies for marker development, allele mining, gene discovery and molecular breeding.

23

Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Plenary Session IV

Global Epigenetic and Transcriptional Trends among Two Rice Subspecies and Their Reciprocal Hybrids
He GM, Zhu XP, Elling AA, Chen LB, Chen RS and Deng XW* Peking-Yale Joint Center of Plant Molecular Genetics and Agrobiotechnology, College of Life Sciences, Peking University, Beijing 100871, China. Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA. Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing 100101, China *Email: xingwang.deng@yale.edu

The behavior of transcriptomes and epigenomes in hybrids of heterotic parents is of fundamental interest. Here we report highly integrated maps of the epigenome, mRNA and small RNA transcriptomes of two rice subspecies and their reciprocal hybrids. We found that gene activity was correlated with DNA methylation and both active and repressive histone modifications in transcribed regions. Differential epigenetic modifications correlated with changes in transcript levels among hybrids and parental lines. Distinct patterns in gene expression and epigenetic modifications in reciprocal hybrids were observed. Through analyses of single nucleotide polymorphisms from our sequence data, we observed a high correlation of allelic bias of epigenetic modifications or gene expression in reciprocal hybrids with their differences in the parental lines. The abundance of distinct small RNA size classes differed between the parents and more small RNAs were down-regulated than up-regulated in the reciprocal hybrids. Together, our data reveal a comprehensive overview of transcriptional and epigenetic trends in heterotic rice crosses, and provides a very useful resource for the rice community.

24

Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Plenary Session IV

Breeding Seeds of Innovation
Hervé PM Bayer Cropscience, Bioscience NV Belgium

At Bayer Cropscience, we help farmers worldwide meet the ever-increasing demand for affordable and high quality food, feed, fiber and energy crops. We aim at providing sustainable crop solutions from seed to harvest, with outstanding seeds and modern crop protection products. Major technology platforms based on the complementary of modern breeding methods and plant biotechnology are used to develop new seeds and innovative traits solutions. An update of our Seeds & Traits Pipeline and key examples of successful molecular breeding solutions for our core crops will be presented.

25

Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Plenary Session IV

Breeding by design and innovation in molecular plant breeding
S?rensen AP, van Schriek M, Hofstede R, Guerra J, Prins M and Buntjer JB Keygene N.V., Agro Business Park 90, P.O. Box 216, 6700 AE Wageningen, the Netherlands

Since the concept of Breeding by Design (BBD) was launched by KeyGene in 2003, DNA technologies have developed with a dramatic acceleration; especially high-throughput sequencing technologies are revolutionizing the DNA research arena. The possibilities for genetic research to elucidate the molecular mechanism of phenotypic expression have increased significantly. As a consequence, implementing BBD or BBD like approaches for trait and variety improvement programs is ongoing. We will discuss here a selection of current genomic tools and applications. Whole genome sequence scaffolds and whole genome BAC based physical maps of commercial crop species are being developed, following the examples of model plant organisms. The discovery of total germplasm variation at the genotypic level and at the gene haplotype level is practically feasible for many crop species. Phenotypic evaluation of germplasm variability is performed with high precision digital imaging systems and supported by statistical tools for evaluation of reproducibility, heritability and interrelatedness of phenotypic scores. The current challenge for plant geneticists clearly lies in the ability to integrate and aggregate the different and large data sources, in order to make firm and robust associations between the phenotypic variability and the genotypic variability, after which these can immediately be exploited by modern plant breeders. Furthermore novel technologies for generation of mutant alleles of interesting plant genes are in development and will increase the genetic variability of germplasm available for variety improvement programs. We will present some of the approaches taken by Keygene to assist plant breeders in these novel opportunities.

26

Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Plenary Session IV

Meeting the challenge of higher nutritional value in seeds: a novel way of increasing methionine content in seeds of the model plant of tobacco
Godo I, Matityahu I, Hacham Y, Amir R Laboratory of Plant Science, Migal Galilee Technology Center, P.O. Box 831, Kiryat Shmona 12100, Israel

The sulfur-containing amino acid, methionine, is an essential amino acid whose level limits the nutritional value of crop plants. Yet, aside from its nutritional importance, methionine is also a fundamental metabolite in plant cells because it indirectly regulates a variety of cellular processes as the precursor of S-adenosyl methionine (SAM). This study describes the first modification of methionine biosynthesis in seeds using the model plant, tobacco (Nicotiana tabacum). Overexpression of the unregulated form of cystathionine gamma synthase (AtD-CGS), the first unique enzyme of methionine biosynthesis pathway from Arabidopsis in tobacco plants, led to an over 10-fold increase in methionine content. However, in these transgenic plants, the methionine level inside their seeds increased only by 15% compared to wild-type seeds. Similar results were obtained when AtD-CGS was seed-specific expressed in tobacco plants. This suggests that the CGS expression level does not limit methionine synthesis in tobacco seeds. To further study the factors regulating methionine synthesis in seeds, the receptacle of developing pods were fed with homoserine, the substrate of CGS. Seeds from these pods demonstrated three-fold higher levels of methionine, suggesting that homoserine content limits methionine synthesis. To further test this assumption, we next crossed between plants seed specific expressing AtD-CGS with those seed-specific expressing the feedback-insensitive bacterial aspartate kinase (bAK), which evidence suggests their seeds have a higher homoserine content. Seeds obtained from the progenies of this cross showed a three-fold higher level of methionine compared to wild-type seeds. In addition, the level of threonine, an important essential amino acid that limits the nutritional quality of cereals, accumulated significantly in these seeds. Our next goal was to reveal if the developing transgenic seeds are tolerant to metabolic perturbations that occur with changes in methionine and threonine levels. To this end, we performed metabolic profiling to wild-type and transgenic seeds expressing AtD-CGS, bAK and AtD-CGS/bAK using GC-MS. A principal component analysis of about 150 metabolites from each transgenic line shows that these lines differ significantly from one another. Of these metabolites, only 12 compounds significantly changed and contributed to this diversity. These include the main amino acids, glutamine and asparagine, and several sugars, trehalose, galactose, glycerol and melbiose. A further study should be performed to reveal the relationships between these metabolites and methionine metabolism. In general, this study demonstrates a novel way of increasing methionine content in seeds, which consequently contributes to enhancing their nutritional value.

27

Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Plenary Session IV

Association mapping for enhancing maize genetic improvement
Yan JB 1,2 *, Li JS 2 1 International Maize and Wheat Improvement Center, (CIMMYT), Apartado Postal 6-640, 06600 Mexico, DF, Mexico China 2 National Maize Improvement Center of China, CAU, Beijing 100193, China E-mail: j.yan@cgiar.org

Association mapping through linkage disequilibrium (LD) analysis is a powerful tool for the dissection of complex agronomic traits and for the identification of alleles that can contribute to the enhancement of a target trait. With the developments of high throughput genotyping techniques and advanced statistical approaches as well as the assembling and characterization of multiple association mapping panels, maize has become the model crop for association analysis. In this talk, we summarize the progress in maize association mapping and the impacts of genetic diversity, rate of LD decay, population size and population structure. We also report the use of candidate genes and gene-based markers in maize association mapping studies which has generated particularly promising results. In addition, we examine recent developments in genome-wide genotyping techniques which promise to improve the power of association mapping and significantly refine our understanding of the genetic architecture of complex quantitative traits. Already these seem to be suggesting that the structure of agronomic traits in maize has more in common with important traits in humans and animals than it does with similar traits in Arabidopsis and rice. The new challenges and opportunities associated with genome-wide analysis studies will be discussed.

28

Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Concurrent session 1: Molecular breeding for abiotic stress tolerances

Mapping QTLs for root morphology in relation to nutrient uptake in wheat
He X, Li JJ, Ren YZ, Zhao XQ, Li B, Li ZS and Tong YP* State Key Laboratory for Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Sciences, Chinese Academy of Sciences, Beijing 100101. *Email: yptong@genetics.ac.cn

Nitrogen (N) and phosphorus (P) fertilizers are required to maximize crop yields in many agricultural systems. However, the recovery rates of fertilizer N and P were low. To increase N and P recovery rates, systematic approaches are required, including optimizing management practices and breeding crops with improved N and P use efficiency. Previous studies have shown that vigorous early root growth is a major factor influencing N and P uptake in wheat. However, roots, the ‘unseen half’ of wheat plants, are difficult to be selected directly by wheat breeders. Therefore, identifying QTLs/genes regulating root traits can help wheat breeders to develop wheat varieties with ideal root system for efficient use of nutrients through MAS approach. A RIL population of derived from two Chinese wheat varieties Xiaoyan 54 and Jing 411 was used to map QTLs for root traits in relation to N and P uptake. A hydroponic culture and a soil column experiment were carried to phenotype the RIL population at seedling stage. For the hydroponic culture, the maximal root length (MRL), root dry weight (RDW), shoot dry weight (SDW), N (NUP) and P (PUP) uptake of this RIL population were investigated under sufficient nutrient supply, low N and low P conditions. Phenotype variation explained by individual QTL varied from 4.6% to 32.7%. For the soil column experiment, root distribution in the soil profiles, SDW, NUP and PUP were investigated under sufficient nutrient condition. Phenotype variation explained by individual QTL varied from 5.2% to 22.5%. To develop MAS for breeding wheat root traits, we analyzed the effects of pyramiding multi-QTLs on RDW, as well as SDW, NUP and PUP investigated in these tow experiments. The results showed that pyramiding the three QTLs linked with Xgwm157-2D, Xgwm533.2-3B and Xbarc90-4B, respectively, significantly increased RDW, SDW, NUP and PUP under different N and P supply levels in the hydroponic culture. The RILs harboring the positive alleles at these three loci had, averagely, 33%-69% higher SDW, RDW, NUP and PUP than those with the negative alleles under different N and P conditions. In the soil column experiment, pyramiding the three QTLs linked with Xgwm157-2D, Xgwm533.2-3B and Xbarc70.1-4A, respectively, significantly increased SDW, RDW, NUP and PUP. The RILs harboring all the three positive alleles had, averagely, 30% higher SDW, 25% higher RDW in the 0-30 cm soil layer, 48% higher RDW in the 30-60 cm soil layer, 43% higher RDW in the 60-90 cm soil layer, 31% higher total RDW, 31% higher NUP and 30% higher PUP than those with the negative alleles.

29

Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Concurrent session 1: Molecular breeding for abiotic stress tolerances

The research progress of drought tolerance and molecular breeding in maize
Zheng J, Fu JJ, Liu YJ, Jian M, Wang GY


Institute of Crop Sciences and National Center for Plant Gene Research, Chinese Academy of Agricultural Sciences, Beijing 100081, China.


Email: gywang@caas.net.cn

Drought stress greatly affects maize growth and its yield potential. In order to understand the molecular basis in response to drought stress, and further to improve the drought tolerance in maize, transcriptome analysis, QTL mapping and transgenic approaches were performed in our lab. Genome-wide gene expression profiling was analyzed between the drought-tolerant line Han21 and drought-sensitive line Ye478. Our data identified a common set of ~2,600 regulated genes under drought stress between the two lines, and showed that the drought tolerant line has fewer genes with altered expression. The potential components of the abscisic acid signaling pathway were significantly identified from the common set of genes. A total of 827 genes with significantly differential expression between the two lines under drought stress were identified. A F2 population of Han21×Ye478 was used to construct the genetic linkage map and QTL mapping. Drought tolerant NILs (near-isogenic lines) were also screened out from the backcross population of Han21×Ye478 under severe drought stress conditions. Additionally, the transgenic maize that overexpressed HDG11, which encodes a homeodomain-START transcription factor, had increased the drought tolerance with improved maize root system and reduced stomatal density.

30

Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Concurrent session 1: Molecular breeding for abiotic stress tolerances

Towards molecular breeding for salt tolerance through modification of root System architecture
Zhao YK, Wang T, Wang ZJ and Li X* Plant Cell & Chromosome Engineering, Center of Agricultural Resources Research, Institute of Genetics and Developmental Biology, 286 Huaizhong Road, Shijiazhuang, Hebei, P.R. China. *Email: xli@genetics.ac.cn

Salinity is a major constraint to crop growth and production. Root system architecture has been considered as one of most important traits of crops in response to various abiotic stresses. Research on root traits is a major breeding objective in genetic improvement in nutrient use efficiency and drought tolerance. Some quantitative trait loci (QTLs) and genes conferring superior root system architecture have been identified. However, the role of developmental plasticity of root system architecture under salt stress is largely unknown, and the genes and QTLs mediating this trait remains to be identified. To investigate the response of plant root system to salt stress, we have conducted a systematic study using Arabidopsis plants. We found that the root system architecture is highly sensitive to salt stress. The SOS (Salt Overly Sensitive) genes are essential for root plastic development in response to salt stress. Loss of function in the SOS genes are hypersensitive to salt, particularly the mutant plants exhibited developmental failure in lateral root initiation and emergence. In contrast, the transgenic plants overexpressing the SOS genes showed enhanced tolerance to salt stress and developed more root mass. Further, we have identified the STS1 (Sensitive To Salt1) gene as an upstream regulator in the root traits mediated by the SOS signaling pathway in response to salt stress. STS1 gene encodes a WD40 repeat protein and is induced by salt stress. Interestingly, STS1 interacts with ABI2, a key regulator of ABA signaling pathway, suggesting that ABA may play an important role in the root trait. The SOS and STS1 genes are found to be conserved in Arabidopsis and winter wheat. The functions of the genes and the SOS and ABA signaling in developmental plasticity of root system architecture in various winter wheat with different salt tolerance are under investigation. These results will further our understanding of the genetics of salt tolerance in crops and to provide novel insights into improvement of their performance under salt stress conditions.

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Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Concurrent session 1: Molecular breeding for abiotic stress tolerances

Mapping and validating QTLs for plant height developmental behaviours in bread wheat
Wu XS, Wang ZH, Zhang JN, Wei TM, Shi W, Zhang B, Jing RL* The National Key Facility for Crop Gene Resources and Genetic Improvement; Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081, China. * Email: jingrl@caas.net.cn

Plant height (PH), a crucial trait related to yield potential in crop plants, is known to be typically quantitatively inherited. However, its full expression can be inhibited by a limited water supply. As a trait easily measured, plant height is also a suitable model trait for exploring drought tolerance from jointing stage to flowering time in wheat (Triticum aestivum L.). In this study, we mapped and validated QTLs for plant height developmental behaviours in wheat by a doubled haploid (DH) population, a recombinant inbred line (RIL) population, a collection of accessions and backcross lines. The genetic basis of the developmental behaviour of PH was assessed in a 150-line doubled haploid population (Hanxuan 10 × Lumai 14) grown in 10 environments (year × site × water regime combinations) by unconditional and conditional quantitative trait locus (QTL) analyses in a mixed linear model. QTLs with additive and epistatic effects that expressed selectively during ontogeny were identified. Total of seven genomic regions covering PH QTL clusters on different chromosomes identified from the DH population were used as the candidate QTLs and extensively analyzed in a RIL population derived from the same cross as the DH. Five additive QTLs and eight pairs of epistatic QTLs significantly affecting plant height development were detected by unconditional QTL mapping method. Six additive QTLs and four pairs of epistatic QTLs were validated using conditional mapping approach. Among them, three additive QTLs and three pairs of epistatic QTLs were common QTLs detected by both methods. Three QTLs were expressed under both drought and well-water conditions. Total of 270 historical winter wheat accessions planted in northern China were genotyped using 60 PH candidate markers on six chromosomes. A list of association was identified in the regions of gene Rht, indicating a consistency of association analysis with linkage mapping. A total of 68 backcross lines of BC3F3-4 were used to validate the QTLs detected in the genetic populations and natural collection. The results showed that some lines pyramiding multi-allele with effect of increasing or decreasing plant height exhibited superiority over the opposite lines. This case, mapping and validating QTLs for plant height developmental behaviours in wheat indicates the possibility of molecular breeding for plant complex quantitative traits.

32

Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Concurrent session 1: Molecular breeding for abiotic stress tolerances

Discovery of genes for drought resistance improvement of rice by systematic genetic and functional genomic approaches
Xiong LZ National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China E-mail: lizhongx@mail.hzau.edu.cn

Drought resistance is a very complex trait with distinct molecular and physiological mechanisms in different plant species. Irrigated rice has been domesticated in full irrigation ecosystem and it is extreme sensitive to drought. With a long-term goal of improving drought resistance in irrigated rice, we have adopted a strategy by integrating the approaches including germplasm exploitation, genetic and functional genomics approaches to identify loci/genes effective for drought resistance improvement of rice. In this paper, we described the approaches and the major progresses made to discover genes for drought resistance improvement. On the basis of genetic dissection of drought resistance of rice, more than 30 QTLs have been targeted for construction of near isogenic lines and marker-assisted molecular breeding. Several drought resistance-associated genes were identified through drought screening of T-DNA insertion mutants of rice. Hundreds of genes differentially involved in drought responses and adaptation were identified through comparative expression profiling analysis. More than 200 drought-responsive candidate genes were transformed into rice for drought resistance testing, and a few genes (such as SNAC1, OsSKIPa, and OsLEA3-1) showed significant effect in improving drought resistance of transgenic rice. Finally, problems and perspectives of drought resistance improvement in rice will be discussed.

33

Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Concurrent session 1: Molecular breeding for abiotic stress tolerances

Heat stress transcriptome analysis and Functional Characterization of Responsive Genes in wheat
Qin DD1, 2, Peng HR1, 2, Ni ZF1, 2, Yao YY 1, 2, Zhou CL1, 2, Sun QX 1, 2, * 1 Department of Plant Genetics & Breeding and State Key Laboratory for Agrobiotechnology, China Agricultural University, Beijing100193, China 2 Key Laboratory of Crop Heterosis and Utilization (MOE), Key Laboratory of Crop Genomics and Genetic Improvement (MOA) and Beijing Key Laboratory of Crop Genetic Improvement, China Agricultural University, Beijing100193, China Email: qxsun@cau.edu.cn

Wheat (Triticum aestivum L.) is a major crop around the world, and heat stress during the late stage affects its yield and quality badly. So, it’s urgent to elucidate the mechanisms of wheat heat tolerance, and identify thermotolerance-related genes for future thermotolerant wheat breeding programme. In this study, using Affymetrix Genechip? Wheat Genome Array, we analyzed genome-wide gene expression profiles of the leaves between two wheat genotypes with contrasting thermotolerance under heat treatment, namely, heat susceptible ‘Chinese Spring’ and heat tolerant ‘TAM107’. A total of 6560 (~10.7%) probe sets were identified as heat responsive in our study. Except for heat shock proteins and heat shock factors, these genes also included transcription factors, components involved in hormone biosynthesis and signaling, calcium signal pathway, RNA metabolism, primary and secondary metabolisms, as well as other stresses related proteins. Further analysis showed that, 313 probe sets were differentially regulated between the two genotypes, 1314 were between heat treatments with and without pre-acclimation, while 4533 between short and prolonged heat treatments. Furthermore, two genes, TaMBF1c (Multiprotein bridging factor 1, MBF1) and TaGAST (Gibberellin stimulated transcript), which were strong induced by heat stress in both genotypes were cloned and functionally characterized. The complete ORF encoding TaMBF1c included 471bp, the deduced amino acid sequence revealed existence of MBF1 and helix-turn-helix conserved domains at the N- and C-terminus, respectively, and was highly homologous to rice ERETC and AtMBF1c. TaMBF1c contained no intron in it. The 1074bp promoter region of it contained three heat shock elements (HSEs), identifying it as a potential heat shock factor regulated gene. Northern blot analyses showed that there was no detectable expression of TaMBF1c under control condition, and the expression of it was rapidly and significantly induced by heat stress not only at seedling stage but also at flowering stage, and was only slightly induced by drought and H2O2 stresses, ABA and ACC application, however, not by rhythm, salt and MeJA treatments. In addition, ectopic over-expression of TaMBF1c in yeast imparts high temperature stress tolerance to wild type yeast cells. The most important is that thermotolerance was significantly increased in TaMBF1c overexpressed transgenic rice.
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Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Another heat-induced gene TaGAST was also gotten by in silico cloning and RT-PCR. Bioinformatic analysis showed that the sequence of TaGAST encoded a protein with 99 amino acids which had a GASA domain in the C-terminal. In addition, the promoter region of TaGAST was cloned using BD Genome Walker method, and HSE and several cis-elements involving in other abiotic stress response were found in this region.Consistently, the expression of TaGAST was at low level in seedling leaves of the two wheat genotypes mentioned above, but strongly induced by stress factors, such as PEG, high salinity, oxidation and high temperature, and also the phytohormones such as ABA, ACC and MeJA treatment.The results suggested that this gene might be involved in various abiotic stress respons.In order to investigate the role of TaGAST in plant thermotolerance, it was over-expressed in Arabidopsis by Agrobacterium-mediated transformation method. The transgenic lines overexpressing TaGAST showed no phenotypic difference compared to wild type under normal growth condition, but showed membrane-thermostabler than WT. And had significantly higher survival rate under heat stress. All the above results indicate that these two genes have potential importance in improving thermotolerance of wheat and other cereals, and the transgenic of wheat is underway.

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Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Concurrent session 2: Gene discovery and function

Identification and application of the rice broad-spectrum blast resistance gene Pigm
He ZH*, Deng YW National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for biological Sciences, CAS, Shanghai 200032, China *E-mail zhhe@sibs.ac.cn).

Rice blast is one of the most destructive diseases of rice. The identification and utilization of broad-spectrum resistance genes has been the most effective and economical approach to control the disease. A native Chinese variety, GM4, was identified with broad-spectrum and durable resistance. Genetic and mapping analysis indicated that blast resistance to nine isolates of different races in GM4 is controlled by the same dominant locus designated as Pigm, which was identified resistance gene cluster including 13 NBS-LRR members on chromosome 6 by map-based cloning strategy, allelic to two known blast resistance genes Pi2 and Pi9. Sequence comparison of the orthologous and paralogous genes between the Pigm/Pi9/Pi2 loci showed that the Pigm loci had undergone duplication result from LTR retrotransposon, unequal cross and illegitimate recombination during the evolution of the resistance gene cluster. Furthermore, our analysis showed that Pigm confers resistance to blast isolates from different cultivated regions than Pi9/Pi2/Pizt/Piz. In the Pigm locus, Pigm-1 controls leaf blast resistance, Pigm-2 confers neck blast which leads to large loss of grain yield. Genetic and transcriptional analysis suggested that broad-spectrum resistance might be attributed to the different expression patterns of diverse R genes. We have succeeded in developing elite hybrid rice lines with broad-spectrum blast resistance with molecular markers-assisted selection for Pigm, indicating good potential of the gene in rice molecular breeding. All the elite hybrid rice lines harboring the Pigm exhibited a high resistance or immunity to blast in natural blast nurseries nationwide from 2008 to 2010.

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Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Concurrent session 2: Gene discovery and function

Mutant resources for functional studies of genes related to fertility in rice
Wu CY

National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research, Huazhong
Agricultural University, Wuhan 430070, China Email: cywu@mail.hzau.edu.cn

T-DNA tagging strategy is a high throughput approach for function analysis the plant genome. We have generated more than 100 thousand independent transgenic lines using the enhancer trap construct, consisting of the GAL4/VP16-UAS elements with GUS (or GFP) as the reporter. The system has three built-in strategies for functional analysis of the rice genome. First, T-DNA insertions cause gene mutations, providing an efficient approach for gene identification and isolation. Second, expression of the reporter gene indicates the presence of an enhancer element in the neighboring genomic region, which can be used for isolation and characterization of the enhancer. Third, the lines showing spatial- or temporal-specific expression of the reporter gene can be used to drive ectopic expression of a transgene, thus useful for unveiling latent functions of unknown or known genes. Employing our rice T-DNA insertional mutant library, we identified two genes, designed PAIR3 and OsRPA1a, which play essential roles in DNA metabolism during meiosis process. Both pair3 and Osrpa1a mutants exhibit a phenotype of completely sterile compared with their wild types. Genetic analysis of those mutants revealed that the T-DNA insertion tag co-segregated with the sterility phenotype. During meiotic prophase I, the pair3 mutant fails in homologous chromosome pairing and synapsis, resulting in no formation of bivalents and subsequent random segregation of the univalents in anaphase I. PAIR3 encodes a protein that contains putative coiled-coil motifs, but does not have any close homologs in other organisms. Primary results suggest that PAIR3 plays a crucial role in homologous chromosome pairing and synapsis in meiosis. Another mutant osrpa1a exhibits abnormal chromosomal fragmentation occurred in male meiocytes after anaphase I. Further study identified that the leaves of Osrpa1a were hypersensitive to DNA mutagens. Genetic complementation and RNAi results confirmed that OsRPA1a was responsible to the mutant phenotypes in Osrpa1a. Our data suggest that OsRPA1a plays an essential role in DNA repair but may not participate in, or at least is dispensable for, DNA replication and homologous recombination in rice.

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Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Concurrent session 2: Gene discovery and function

Gene discovery from common wild rice (Oryza rufipogon Griff.)
Sun CQ State Key Laboratory of Plant Physilogy and Biochemistry, National Center for the Evaluation of Agricultural Wild Plants (Rice), China Agricultural University, Beijing 100193, P R China. Email: suncq@cau.edu.cn

Common wild rice (Oryza rufipogon Griff.), ancestor species of cultivated rice (O. sativa L.), constitute an important gene pool for rice improvement. To discover favorable genes from wild rice which have been lost or weakened in cultivated rice has become more and more important for modern breeding strategy. In recent years, we have developed two sets of introgression lines (ILs) derived from the cross between O. rufipogon from Jiangxi and Yunnan province of China, as the donor, and elite cultivars, as the recipient. Several QTLs for yield-related traits, quality traits and tolerence to abiotic stress were mapped using introgression lines. Some major QTLs were fine-mapped and cloned. Two key genes, PROG1 and SHA1, controlling rice domestication were identified. PROG1 controlling prostrate growth of wild rice on chromosome 7 encodes a single Cys2-His2 zinc-finger protein. prog1 variants identified in O. sativa disrupt the prog1 function and inactivate prog1 expression, leading to erect growth, greater grain number and higher grain yield in cultivated rice. SHA1 controlling seed shattering of wild rice on chromosome 4 encodes a member of the trihelix family of plant-specific transcription factors. The predicted amino acid sequence of SHA1 in wild rice is distinguished from that in cultivated rice by only a single amino acid substitution (K79N) caused by a single nucleotide change (g237t).

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Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Concurrent session 2: Gene discovery and function

Discovery of brown planthopper resistance gene in rice
He GC College of Life Sciences, Wuhan University, Wuhan 430072, China Email: gche@whu.edu.cn

The brown planthopper (Nilaparvata lugens Stal; BPH) is an insect that feeds on the leaf sheath of rice (Oryza sativa L.) plants, ingesting nutrients specifically from the rice phloem using its stylet mouthparts. In the last decade, the BPH has frequently caused widespread destruction of rice crops and heavy losses of yields. The most economic and efficient method for controlling the BPH is to use the host resistance as part of IPM. To date, more than 19 BPH resistance genes in rice have been reported. Resistance of Bph1, bph2 and Bph3 has been reported to be overcome by new biotypes of BPH. Wild rice germplasm is an important gene pool for rice breeding. Two major loci for BPH resistance, Bph14 and Bph15, were detected in the F2 population and RIL population of Minghui63 X B5. Bph14 was mapped on the long arm of chromosome 3 and Bph15 on the short arm of chromosome 4. These loci were also found to confer resistance to the white-backed planthopper. Analysis of recombination events in the Bph14 region delimited the gene to genomic segment of 34-kb between SM1 and G1318. Two predicted genes encoding putative resistance proteins, designated Ra and Rb respectively, were identified after sequencing this region. Transgenic experiment showed that Ra confers the resistance phenotype and is the Bph14 gene. The Bph14 gene encodes a putative 1,323 amino acid protein containing a coiled-coil, nucleotide-binding and leucine-rich repeat (CC-NB-LRR) motif. Comparison analysis showed that in the LRR domain 54 residues and two deletions of Bph14 were unique. Electronic penetration graphs (EPG) revealed that BPH insects spent more time walking, but less time ingesting phloem, on the plants carrying resistance genes Bph14 and Bph15 than they did on susceptible plants. Tests with [14C]sucrose showed that insects ingested much less phloem sap on the resistant plants than on susceptible plants. In the plants infested with the BPH, callose was found deposited on the sieve plates of the target sieve tubes, where the stylets had been inserted. Counts of the bright callose plugs revealed more callosic sieve plates in the resistant than in susceptible plants. Moreover, with prolonged BPH feeding, the callose deposition decreased quickly in susceptible plants. It was found that the genes encode for callose decomposing enzyme β-1,3-glucanase were differetially regulated in the resistant and susceptible rice plants. In the susceptible rice the β-1,3-glucanase gene Osg1 and Gns5 were enhanced, and thereby facilitated the BPH’s continued feeding from the phloem in the susceptible plants, while in the resistant plants, these genes expression unchanged. As a result, BPH feeding on the resistant rice plants were suppresed.

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Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Concurrent session 2: Gene discovery and function

Molecular basis of cytoplasmic male sterility in rice
Wang Z, Zou Y, Luo D, Liu Z, Xu H, Wu H, Guo J, Zhang Q, Ye S, Chen Y, Liu YG* Key Laboratory of Plant Functional Genomics and Biotechnology of Education Department, Guangdong Province, College of Life Sciences, South China Agricultural University, Guangzhou 510642, China. *Email: ygliu@scau.edu.cn

The successful breeding and commercial cultivation of hybrid rice is one of the most important achievements in agriculture. Hybrid rice has been developed and released in 1970s in China, which has about 20% yield advantage over improved inbred varieties. Since the late of 1980’s, hybrid rice has occupied ~55% (~15-17 million hectares) of the total rice planting area each year in China. Therefore, hybrid rice has contributed tremendously to the food security in China, and given a great impact to agriculture. The successful development of hybrid rice is mainly due to the development and utilization of cytoplasmic male sterility (CMS) systems. Three types of CMS systems, Wild-abotive (WA), Boro II (BT), and Hong-Lian (HL), have been used for the hybrid breeding. To reveal the molecular basis of the cytoplasmic male sterility systems in rice, we have identified and functionally studied the genes conferring the CMS and restoration. We found that a mitochondrial open reading frame of previously unknown function in Boro II cytoplasm, orf79, encodes a cytotoxic peptide that causes the male sterility. Furthermore, we isolated two restorer genes, Rf1a and Rf1b, at the previously reported single locus Rf1, revealing that Rf1 is a complex locus. Rf1a and Rf1b encode PPR (Pentatrico Peptide Repeat) proteins, and they target to mitochondria to cleave and degrade the orf79 mRNA, respectively, thus silence orf79 and restore the mal fertility. When both restorers are present in the hybrids, Rf1a preferentially cleave the orf79 mRNA, showing an epistatic effect over Rf1b. The study further revealed that Rf1a has a role to promote the editing of the mitochondrial atp6 mRNA, suggesting that this may be its primary function, while the action as the fertility restorer be a new function. CMS-WA is the most widely used system for hybrid rice. We identified a novel mitochondrial gene conferring CMS-WA. Transformation of rice and Arabidopsis with this gene caused male sterility. CMS-WA is restored by Rf loci, Rf3 and Rf4, via suppressing the function of this CMS gene with different mechanisms. Evolutionary analysis revealed that this CMS gene was generated through rearrangement of multiple fragments of the mitochondrial genomes and unknown sources in this locus during the evolution of wild rice species. Further, we studied the molecular mechanism of the CMS induction involving in the cytoplasmic-nuclear interaction.

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Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Concurrent session 2: Gene discovery and function

Toward map-based cloning of a good eating-quality QTL derived from an elite Japanese rice cultivar Koshihikari
Hori K1*, Takeuchi Y2, Nagasaki H1, Ando I2, Yano M1 1 National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan 2 National Institute of Crop Science, 2-1-18 Kannondai, Tsukuba, Ibaraki 305-8518, Japan *E-mail: horikiyo@affrc.go.jp

Eating quality is an important trait to consider in rice breeding, because it determines consumer preference and the rice price. The eating quality of cooked rice is a complex trait determined by multiple genes and is largely affected by environmental factors. Although several physicochemical properties of the rice grain, such as amylose and protein contents, pasting properties and gel consistency are used to evaluate eating quality, a sensory test of cooked rice is still required for the final selection procedure in rice breeding. The sensory test is time-consuming and labor-intensive because trained panels evaluate each breeding line for appearance, taste, and texture of the cooked rice by eating it. A japonica rice cultivar Koshihikari has a good eating quality including high glossiness, a high level of stickiness, good taste, and low hardness of cooked rice. We evaluated the eating quality of cooked rice using the sensory test in a set of reciprocal backcrossed inbred lines (BILs) from crosses between Nipponbare and Koshihikari in 2006 and 2007. The major quantitative trait loci (QTL) for eating quality were detected on the short arm of chromosome 3 in the two BILs. The Koshihikari allele of the QTL increased eating quality. To validate the eating quality QTL, we developed a substitution line with a Koshihikari segment on the short arm of chromosome 3 in a Nipponbare genetic background, and evaluated the eating quality of the substitution line using sensory tests in 2008 and 2009. The eating quality of the substitution line was improved as compared with Nipponbare in both seasons. In order to screen for putative candidate genes of the eating quality QTL, a large chromosome segment (11.3 Mbp) of the genome was sequenced. Sequence comparison between Nipponbare and Koshihikari revealed insertion/deletion polymorphisms and single nucleotide polymorphisms in the sequences of 13 predicted genes in the candidate region of the QTL. RT-PCR revealed that nine of the 13 genes were expressed in the endosperm during the ripening period after pollination. For fine-mapping of the eating quality QTL, we developed additional substitution lines to replace different Koshihikari segments on the short arm of chromosome 3 in the Nipponbare background. Sensory tests of these substitution lines are now underway to narrow down the candidate region for the eating quality QTL.

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Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Concurrent session 2: Gene discovery and function

Map-based cloning of QTL genes for flowering time/maturity in soybean
Xia ZJ1,2*, Watanabe S2, Liu B1, Tsubokura Y2, Harada K2 1 Northeast Institute of Geography and Agroecology, CAS, Harbin 150081, China; 2 National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602, Japan. * Email: xiazhj@neigaehrb.ac.cn

Flowering represents the transition from the vegetative to the reproductive phase in plants. Various external cues, such as photoperiod and temperature, are known to initiate plant flowering under the appropriate seasonal conditions. In soybean [Glycine max (L.) Merrill], several maturity loci, designated as E loci have been characterized by classical methods. Of these E loci (E1 to E7), the E1, E3, and E4 loci have been suggested to be related to photoperiod sensitivity under various light conditions. Successful cloning of these genes is very important to understand the flowering gene network in soybean. As well, deciphering these identities can help us to understand the natural evolution and domestication, and impose very important impact on soybean breeding and production. As the draft soybean sequence has been made publically available, it provides us great genetic resources, especially for positional cloning. As clonings of E3 (Watanabe et al. Genetics. 182, 1251-1262) and E4 (Liu et al. Genetics 180: 995–1007) have been accomplished, we are focused on isolation of E2 and E1 loci. Map-based cloning strategy using residual heterozygous lines was applied to the isolation of the gene responsible for these two loci. One candidate gene consisting of 1170 amino-acid was identified for E2 locus. Common premature stop codon at the tenth exon (521aa) was observed in Misuzudaizu allele and other NILs originated from Harosoy (e2/e2; PI548573). Furthermore, a mutant line harboring another premature stop codon (735aa) showed early flowering phenotype than original variety Bay (E2/E2; PI553043). In contrast to other plant species, null mutation of E2 gene in soybean did not show any notable deleterious effects on the phenotype and can be a good genetic resource to control maturity in wide geographic area in soybean breeding program. Most importantly, we are cloning E1/FT1 using a population derived from a cross between two Harosoy NILs. With identified recombinants, we have successfully narrowed down E1 locus from about 300 kb to 17 kb, where a single candidate gene was identified. Further functional confirmation is in progress. With these four major flowering time genes identified, the flowering time gene networks in soybean will be greatly understood.

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Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Concurrent session 3: Molecular breeding for biotic stresses

From QTLs for disease resistance to marker-assisted selection in durum wheat
Maccaferri M1*, Sanguineti MC1, Bassi F1, Mantovani P1, Terracciano I1, Ammar K2, Massi A3, Chen F4, Bini F1, Ratti C1, Rubies-Autonell C1, Vallega V5, Simkova H6, Kolmer J7, Kema G8, Keller B9, Czembor J10, Tuberosa R1 1 Dept. of Agroenvironmental Science & Technology (DiSTA), U. of Bologna, 40127 Bologna, Italy; 2CIMMYT, 56134 Texcoco, Mexico; 3Società Produttori Sementi Bologna (PSB), 40050 Argelato (BO), Italy; 4Institute of Crop Science, CAAS, Beijing 100081, China; 5CRA Experimental Institute for Cereal Research, 00191 Rome, Italy; 6Institute of Experimental Botany, CZ-77200 Olomouc, Czech Republic; 7Cereal Disease Laboratory, USDA-ARS, St. Paul, MN 55108, USA; 8Plant Research International (PRI), Wageningen, 6700 AA, The Netherlands; 9Institute of Plant Biology U. of Zurich, 8008 Zurich, Switzerland; 10Dept. of Genetics & Plant Breeding, IHAR, 05870 Blonie, Poland *E-mail: marco.maccaferri@unibo.it Durum wheat (Triticum durum Desf.) is a main cereal crop for the Mediterranean area where production is challenged by fungal pathogens (e.g. leaf rusts, powdery mildew, Fusarium, etc.) and in some areas, also by Soil-Borne Cereal Mosaic Virus (SBCMV). The identification of effective sources of resistance, their genetic mapping and the development of molecular markers suitable for marker-assisted selection are thus major objectives of durum wheat improvement. The positional cloning of the relevant loci would also be of great applicative value. In collaboration with CIMMYT and plant pathologists, we have undertaken a program for identifying and mapping useful genetic variation through a joint linkage and association mapping approach. The development and phenotypic characterization of two RIL populations (from Meridiano × Claudio and Colosseo × Lloyd), each including ca. 180 RILs, allowed us to identify major genes/QTLs for: (i) leaf rust resistance on chr. 7BL (C×L; Maccaferri et al., 2008; TAG 117: 1225–1240; Mol. Breed. 2010, DOI 10.1007/s11032-009-9353-0.), (ii) powdery mildew resistance (M×C) on chrs. 6BL and 7BL, and (iii) SBCMV resistance (M×C) on chr. 2BS. Association mapping with a panel of 210 elite accessions has been used to validate and to further dissect these major resistance loci. A fine-mapping effort is underway for the target loci. The analysis of rice-wheat synteny provides the bases for SNP development and candidate gene identification. Based on the obtained results, marker-assisted selection is being implemented in collaboration with the seed company Produttori Sementi Bologna. First, the proprietary germplasm has been haplotyped at the major genes/QTLs in order to identify the valuable lines that already harbor the favourable resistance alleles. Molecular selection is currently being used to screen segregant F2:4 breeding populations. High-throughput, PCR-based codominat markers have been developed (BIOEXPLOIT project) for MAS activities.

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Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Concurrent session 3: Molecular breeding for biotic stresses

Genomic approaches to plant defense research and crop improvement for insect resistance
Huang YH USDA-ARS Plant Science Research Laboratory, 1301 N. Western Road, Stillwater, OK 74075 and Department of Botany, Oklahoma State University, Stillwater, Oklahoma 74078, USA Email: yinghua.huang@ars.usda.gov

Plant-insect interaction is a complex and dynamic process, leading to a variety of beneficial and deleterious outcome. Mechanisms of plant defense against attack by insect pests, including constitutive and induced defenses, have been evolving for millions of years and are therefore shared across many plant families. Understanding of the interactions between two kingdoms and the defense mechanisms operating in host plants, such as the insect genes required for attacking and the plant genes responsible for defense, can be utilized to design crops with enhanced resistance. Research in life sciences at the post-genomics age is currently undergoing a dramatic transformation, and genomic approaches are beginning to revolutionize our understanding of plant defense. These sophisticated genomic tools, including genome sequencing, gene expression profiling, functional genomics, marker-assisted selection, genetic transformation etc., are already applied to examine the complex process of plant and pest interactions. Genome sequencing projects of a few crops, including rice and sorghum, have been completed. The resulted genetic blueprints provided by DNA sequences will allow the identification of genes involved in host defense and regulatory factors that control the induction of resistance genes. Molecular mapping has permitted locating chromosomal regions harboring the resistance to insects and identifying genetic markers enabling more efficient plant breeding or genetic manipulation. Transgenic plants already have been an unimagined tools and capacities in determination of gene functions in plant-insect interactions and in pursuit of molecular breeding for insect resistance. These successful samples highlighted the recent progress in the studies of plant-insect interactions and provided valuable data on the genome structure of host plants and the diversity of defense mechanisms used by plants to protect themselves from insects. However, our understanding of gene function remains behind the pace of genome sequencing and gene discovery. Over the next ten years, further progress and new breakthroughs are anticipated through integrated biology studies and the use of newly genomics tools; then the resulting new knowledge will lead to the development of more durable insect resistance for crop plants and novel strategies for crop pest management.

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Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Concurrent session 3: Molecular breeding for biotic stresses

Improvement of maize resistance to head smut and stalk rot
Xu ML National Maize Improvement Center of China, China Agricultural University, 2 west Yuanmingyuan Road, Beijing, 100193, China

Stalk rot and head smut are among the most destructive diseases in maize production in China. Disease control by using fungicide has been proved inefficient due to soil-borne infection, while, genetic strengthening of maize against stalk rot and head smut is the most cost-effective and environmentally friendly way to curtail the spread of the diseases. Highly resistant maize inbred lines, ‘Ji1037’ against head smut and ‘1145’ against stalk rot, have been screened from maize germplasm and used to prepare mapping populations for QTL analyses. The individuals with resistance QTL were backcrossed to the susceptible parent to generate next backcross generation. This process has been repeatedly conducted to produce advanced backcross populations. In each backcross generation, recombinants were selected and then backcrossed to the susceptible parent to generate their backcross progeny. A progeny testing was introduced to deduce phenotype of parental recombinant. If the progeny with the donor region are more resistant than those without the donor region, indicating the presence of resistance QTL, or otherwise, no any resistance QTL was present in the parental recombinant. Analysis of both resistance and donor region for every recombinant allowed fine-mapping of the resistance QTL. With this approach, the major resistance QTL to stalk rot and head smut has been mapped into intervals of 170kb and 190kb, respectively. The markers in the mapped regions were used to screen two BAC libraries constructed from the inbred lines ‘1145’ and ‘Huangzao 4’ to build BAC contigs covering the resistance QTLs. The minimal tilling BACs were subjected to sequencing and gene prediction. By comparison of the predicted genes among ‘1145’, ‘Huangzao 4’ and B73 (whole genome sequence is publicly available) allowed identification of candidates for resistance QTLs. The candidate resistance genes, three for each disease, were then cloned into expression and RNAi vectors for functional validation via genetic transformation. The makers developed on or closely-linked to the resistance genes have been extensively used for marker-assisted selection to genetic improvement of maize resistance to the diseases. This has been proved very successful since highly resistant to head smut and stalk rot has been observed in ten improved inbred lines and a number of isogenic lines, respectively. We have collaborated with many institutions in China to accelerate application of this marker technology to strengthen maize resistance to both head smut and stalk rot.

45

Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Concurrent session 3: Molecular breeding for biotic stresses

Enhancing broad spectrum resistance to rice diseases
Wang SP National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research, Huazhong Agricultural University, Wuhan 430070, China Email: swang@mail.hzau.edu.cn

Broad-spectrum resistance refers to resistance against two or more types of pathogen species or the majority of races of the same pathogen species. Bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo), bacterial streak caused by X. oryzae pv. oryzicola (Xoc), and fungal blast caused by Magnaporthe grisea are devastating diseases of rice worldwide. Application of host resistance to these pathogens is the most economical and environment-friendly approach to solve this problem. Quantitative resistance conferred by quantitative trait loci (QTLs) is presumably valuable sources for broad-spectrum disease resistance. However, molecular evidence that supports this hypothesis is rare. Resistance QTLs are also only identified genetic resources from rice against Xoc. Although a large number of QTLs against these rice diseases have been identified, these sources have not been used effectively in rice improvement because of the complex genetic control of quantitative resistance and because of the genes underlying most of resistance QTLs being unknown. We have characterized several minor resistance QTLs in rice by the strategy of validation and functional analysis of the QTL. Functional characterization of the encoding proteins of these resistance QTLs indicates that these genes belong to defense-responsive genes group. These QTLs can mediate broad-spectrum resistance and can be used in breeding programs by manipulating their expression.

46

Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Concurrent session 3: Molecular breeding for biotic stresses

Molecular mapping of adult-plant resistance genes to stripe rust and powdery mildew and validation of allelic specific markers for Lr34/Yr18/Pm38 in Chinese wheat cultivars
Xia XC1,*, He ZH1, 2, Lan CX1, Liang SS1, Ni XW1, Li ZF1 1 Institute of Crop Science/National Wheat Improvement Center, Chinese Academy of Agricultural Sciences (CAAS), 12 Zhongguancun South Street 100081, Beijing, China; 2International Maize and Wheat Improvement Center (CIMMYT) China Office, c/o CAAS, 12 Zhongguancun South Street, Beijing 100081, China. *Email: xiaxianchun@caas.net.cn

Stripe rust and powdery mildew, caused by Puccinia striiformis f. sp. tritici (PST) and Blumeria graminis f. sp. tritici, respectively, are devastating wheat diseases in China. The 145 Chinese wheat cultivars and advanced lines from the autumn-sown wheat regions were investigated. Ninety-eight lines of them were tested for seedling response to 26 PST races in the greenhouse, and 135 were tested for adult-plant resistance (APR) in the field. The results indicated that genes Yr2, Yr3a, Yr4a, Yr6, Yr7, Yr9, Yr26, Yr27, and YrSD, either singly or in combinations, were postulated in 72 lines, whereas known resistance genes were not identified in the other 26 accessions. The resistance genes Yr9 and Yr26 were found in 42 and 19 accessions, respectively; and 33 cultivars and advanced lines showed APR to stripe rust at two locations in both seasons. Using the allele-specific markers (cssfr1–cssfr5) for Lr34/Yr18/Pm38, we tested 231 Chinese improved wheat cultivars and 422 landraces. The resistance-allele specific PCR fragments were detected in 14 improved wheat cultivars, with a frequency of 6.1%, whereas these fragments were amplified in 359 landraces, with a frequency of 85.1%. However, about 25% of these landraces with resistance-allele specific PCR fragments showed highly susceptible to stripe rust. DNA sequencing for them didn’t find any mutations in the coding region of the gene, and their susceptibility might be resulted from the mutation in the promoter region or the presence of an inhibitor of the gene Lr34/Yr18/Pm38. A total of 540 simple sequence repeat (SSR) markers were screened to map quantitative trait loci (QTLs) for APR to stripe rust in a doubled haploid (DH) population of 137 lines derived from the cross Pingyuan 50× Mingxian 169. The DH lines were planted in randomized complete blocks with three replicates in Gansu and Sichuan provinces during the 2005-06, 2006-07, and 2007-08 cropping seasons, providing data for four environments. Artificial inoculations were carried out in Gansu and Sichuan with the prevalent Chinese PST race CYR32. Inclusive composite interval mapping (ICIM) detected three QTLs for APR to stripe rust on chromosomes 2BS, 5AL, and 6BS, designated QYr.caas-2BS, QYr.caas-5AL, and QYr.caas-6BS, respectively, explaining from 4.5 to 19.9% of the phenotypic variance. The 406 SSR markers were used to map QTLs for APR to powdery mildew in a DH population with 181 lines derived from the cross Bainong 64×Jingshuang 16. With the method of composite
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Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

interval mapping, four QTLs for APR to powdery mildew were detected on chromosomes 1A, 4DL, 6BS and 7A, designated QPm.caas-1A, QPm.caas-4DL, QPm.caas-6BS and QPm.caas-7A, respectively, explaining from 6.3 to 22.7% of the phenotypic variance. Two QTLs QPm.caas-4D and QPm.caas-6BS were stably detected across different environments with a high genetic effect on powdery mildew resistance, accounting for 15.2 to 22.7% and 9.0 to 13.2% of phenotypic variance, respectively. A population of 200 F3 lines from the cross Lumai 21×Jingshuang 16 was tested for powdery mildew reaction in Beijing and Anyang in the 2005-06 and 2006-07 cropping seasons, providing data for 4 environments. A total of 1,375 SSR markers were screened for associations with powdery mildew reactions. Three QTLs for APR to powdery mildew were detected by ICIM. These were designated QPm.caas-2BS, QPm.caas-2BL and QPm.caas-2DL, respectively, and explained from 5.4 to 20.6% of the phenotypic variance across 4 environments. QPm.caas-2BS and QPm.caas-2DL were likely new QTLs for APR to powdery mildew, flanked by SSR markers Xbarc98 - Xbarc1147 and Xwmc18 - Xcfd233, respectively.

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Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Concurrent session 3: Molecular breeding for biotic stresses

Transmission and molecular characteristics of Southern rice black-streaked dwarf virus, a new Fijivirus threating rice production in Asia
Zhou GH*, Wang Q, Pu LL, Zhang SG Laboratory of Plant Virology, South China Agricultural University, Guangzhou, China * Email: ghzhou@scau.edu.cn

Southern Rice Black Streak Dwarf Virus (SRBSDV, genus Fijivirus, Family Reoviridae), a new virus infecting rice discovered in 2001 in Guangdong province, spread raphidly throughout southern China and northern Vietnam, and became one of the most important virus threating rice production in Southeast Asia. The virus was transmitted by the white back planthopper (WBPH, Sogatella furcifera, Hemiptera: Delphacidae), a long-distance migration rice pest, in a persistent manner. The virus replicates in the WBPH body, but can not be transmitted to offspring through egg. About 60% WBPH individual nymph at 4-5th instar stage was viruliferous in the population developed on the infected rice plant. Both nymph and adult transmit the virus while the former with higher effieciency. Acquisition and inoculation peorids were at least 10-30 minutes, and incubation peorid were 5-7 day when the virus was artificially transmitted through WBPH nymph. The complete genome sequences of this virus, containing ten linear segments of double-stranded RNA, were determined. Based on the nucleotide identity analysis, SRBSDV was most closely related to MRDV and RBSDV. Comparsing to RBSDV, S1, S2 and S10 were most conserved with identities of 78.5-79.2% while S5 and S6 were the least conserved with 70.6-71.6% identities. No reconbination was found between this virus with related viruses. Transmission and molecular characteristics revealed in this study provided a base for virus resistance screening and breeding, especially by molecular strategy.

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Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Concurrent session 4: New transgenic technologies, products and markets

New Transgenic Technologies
Richard Broglie Director, DuPont Agricultural Biotechnology

Methods to insert genes at defined, previously characterized genetic loci are highly desirable molecular tools in plant biotechnology for excision and removal of marker genes and for controlled regulation of foreign gene expression. Homologous recombination and DNA-recombinase-mediated site-specific integration are promising technologies to accomplish this. This paper will describe the successful targeting of transgenes to predefined sites in soybean and corn using the yeast FLP-FRT recombination system. Experiments will also be presented which demonstrate the feasibility of using re-designed homing endonucleases to introduce targeted mutations in crop plants.

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Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Concurrent session 4: New transgenic technologies, products and markets

Simultaneously changing several quality traits of Brassica napus by one transgenic event
Peng Q, Hu Y, Wei R, Zhang Y, Guan C, Ruan Y, Liu CL* Hunan Provincial Key Laboratory of Crop Germplasm Innovation and Utilization, Hunan Provincial Institute of Oil Crops, Hunan Agricultural University, Changsha 410128, China Development Biology Department, Life Science and Technology College, Hunan Agricultural University, Changsha 410128, China * Email: liucl100@126.com

The fatty acid composition in the seed oil was significantly modified following the introduction of transgenes. To further enhance the desirable characteristics of rapeseed oil, it would be beneficial to develop a new approach for the simultaneous silencing of two or more target genes. Our goals in the current study were to 1) increase oleic acid to more than 75%, 2) reduce polyunsaturated fatty acids (PUFA) to about 10% and erucic acid to zero, and 3) accomplish these changes in a single-transformation event. In a single transformation, two fragments amplified from the fatty acid 12-desaturase 2 (BnaFAD2) and fatty acid elongase 1 (BnaFAE1) genes of Brassica napus were linked together to form a fusion fragment. The fusion fragment was then used to assemble unique intron-spliced hairpin interfering constructs. In the transgenic plant FFRP4-4, the expression of BnaFAD2 and BnaFAE1 genes was completely inhibited. The composition of oleic acid in FFRP4-4 rose to 85%, PUFA dropped to 10% and erucic acid was undetectable. All hybrid F1 seeds obtained from the reciprocal crossing of FFRP4-4 and GX-parents (with different genetic backgrounds) contained more than 80% oleic acid, about 10% PUFA and very low, or undetectable, erucic acid. The results confirmed that the fusion fragment silencing construct can simultaneously and effectively silence the target genes on a consistent basis. The strategy provides a useful tool for detecting gene function and advancing genetic engineering techniques for the improvement of agricultural crops.

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Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Concurrent session 4: New transgenic technologies, products and markets

In situ Pistil Delivery: A High Throughput Method of Brassica Genetic Transformation
Guo XL1, Dong CH1, Kurup S2, King G2, Shi L1, Cai L3, Fu H4, Wang Z1, Wang HZ1, Liu SY1* Oil Crops Research Institute of Chinese Academy of Agriculture Sciences and Key Laboratory of Oil Crop Biology, Ministry of Agriculture, No. 2 Xudong Road 2, Wuhan, Hubei, China, 430062 2 Rothamsted Research, Harpenden, Herts, AL5 2JQ, UK. 3College of Plant Science and Technology, Huazhong Agricultural University, No.1 Shizishan Street, Wuhan, Hubei, China, 430070. 4Ningbo Institute of Technology, Zhejiang University, No. 1 Qianhu South Street, Ningbo, Zhejiang, China, 315100. *Email: liusy@oilcrops.cn,
1

Conventional Agrobacterium-mediated transformation methods rely on tissue culture and explants regeneration. Here we described a new high throughput method without tissue culture or additional equipment for producing transgenic Brassica plants. This method of in situ pistil delivery involved placement of a liquid drop containing binary vector DNA on the cut surface of pollinated Brassica stigmas. Transformants, as Brassica napus L. cv Zhong Shuang 9 (ZS9) for example, could be obtained from pistils pollinated only within 24 hours, indicating the timing of transformation was crucial to the efficiency. High transformation frequency (Basta-resistant seedlings per 100 pistils) of 4.1% was achieved when ZS9 pistils were transformed at 18 hours post-pollination. The method was validated by introducing different binary vectors into seven other B. napus, two B. rapa and three B. oleracea lines/genotypes. The transformation frequencies ranged from 0.4% to 3.1% in B. napus, 10.3% to 16.3% in B. rapa and 0.7% to 2.7% in B. oleracea tested lines. Transgenic events were confirmed by PCR, β-glucuronidase (GUS) histochemical assay and Southern blots. A typical 3:1 (Basta-resistant: Basta-sensitive) segregation ratio is indicated in transgenic ZS9 subsequent generations, with no cytoplasmic effect detected after reciprocal hybridization between transgenic lines and the wild type. It appeared likely that gene delivery occurs after double fertilization for both embryo and endosperm can express the foreign GUS gene. Moreover, there are co-segregation evidences demonstrating the potential of this in situ pistil delivery method for mutagenesis and transgenic breeding.

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Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Concurrent session 4: New transgenic technologies, products and markets

Wheat genetic transformation in China -current status and future prospects
Xia LQ 1*, He Y 1, Liang H 2, Wang DW 2, Ma YZ 1, Jones H 3 1 Institute of Crop Sciences /The National Key Facility for Crop Gene Resources and Genetic Improvement, Chinese Academy of Agricultural Sciences (CAAS), 12 Zhongguancun South Street, Beijing 100081, China. 2 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. 3 Department of Plant Science, Rothamsted Research, Harpenden, AL5 2JQ, UK. * Email: xialq@mail.caas.net.cn

Genetic transformation is fundamental to wheat functional genomics and improvement through genetic engineering. At the present time, biolistic and Agrobacterium-mediated transformation are two mainly employed ways for foreign gene transfer into wheat in China, although other DNA delivery methods were also tried with varying degree of success. Agrobacterium-mediated transformation is perceived to have several advantages over other forms of transformation (such as biolistic), including the ability to transfer large segments of DNA with minimal rearrangement, lower transgene copy number, low cost and may facilitate the removal of plant selectable marker genes by segregation, when the presence of unnecessary DNA and transgene arrangement/copy number are scrutinized as part of the regulatory processes. Cereal species, particularly wheat (Triticum aestivum L.), have lagged behind dicots in their ability to be transformed by Agrobacterium and remain to be genotype-dependent, although there has also been a big progress in recent years, but it has been confined mainly to a few responsive varieties with quite different transformation frequencies. Here we not only review the current status of wheat genetic transformation in China, but also report the successful Agrobacterium-mediated transformation of Chinese wheat varieties and durum wheat cv Stewart by using the pGreen/pSoup system with improved transformation frequency, in which the effect of the concentration of acetosyringone and auxin in the inoculation, co-cultivation and induction media on T-DNA transfer and regeneration efficiency were thoroughly investigated. Furthermore, the research and development (R&D) initiative on genetically modified (GM) plants in China provides an opportunity and necessarily accentuates the development of a safe, precise and effective wheat genetic transformation system for later commercialization at the same time.

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Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Concurrent session 4: New transgenic technologies, products and markets

A new effective selection marker for crop transformation
Liu JH, Qiao L, Kong XF, Wang LY, Zhou JL, Xia M* National Center for Molecular Crop Design, Beijing, China

Selectable marker genes are very important for the crop transformation technologies because the effective selection marker genes allow us to identify and select the plants with the integrated DNA and to monitor the transformed progeny. Paraquat (methyl viologen)-resistant gene, pqr-5, isolated from bacteria Ochrobactrum anthropi KT-q077, was successfully used as an efficient selection marker for the development of transgenic plants in Arabidopsis, rice, corn, soybean and cotton. Transgenic plants resistant to paraquat were positive upon PCR and RT-PCR by pqr-5 gene-specific primers. The plants without expression of pqr-5 gene were killed quickly by paraquat, while the transformed plants were resistant to paraquat with the recommended concentration as a weedkiller. This selection strategy is faster, clearer and more visible in contrast to some traditional selections. The existence of paraquat-resistant marker gene in transgenic crops contain useful novel traits while marker genes usually are not needed once transgenic plants have been identified. Therefore, the lack of phenotype variation, characterization of paraquat herbicide and low cost of paraquat makes this selection marker an attractive alternative for the crop transformation.

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Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Concurrent session 4: New transgenic technologies, products and markets

Enhancing the Lysine in wheat grain by genetic transformation of a Lysine rich protein gene Cflr
Ma HX*, Sun XB, Fang R, Xu L, Zhang P, Yu GH and Zhang X Institute of Biotechnology, Jiangsu Academy of Agricultural Sciences * Email: hxma@jaas.ac.cn

Wheat is the major source of protein for the nutrition of humans and livestock, but is deficient in certain essential amino acids when used as food for monogastic animals. In particular, they contain low levels of lysine (the first limiting amino acid) resulting from deficiencies of this amino acids in the prolamin storage proteins, which account for about half of the total nitrogen in the mature grains. Genetic engineering has great potential for increasing the essential amino acid content of wheat grains. Expression of additional genes for lysine-rich proteins may lead to increased accumulation of lysine in proteins. To obtain a new lysine-rich protein gene from species of Solanaceae, a 390bp fragment was amplified from pepper (Capsicum frutescens) cultivar ‘Jiangshu 7’ by using RT-PCR with its mature pollen cDNA as the template and the conservative sequences of potato and tomato lysine-rich protein genes as the primers. A full-length cDNA with completed open reading frame of 223 amino acids was cloned by using the strategy of RACE (rapid amplification of cDNA ends). This cDNA was designated as Cflr (Capsicum frutescens Lysine Rich), which contained 920 bp with an untranslated region of 84 bp at 5’ end and a polyA tail at the 3’ end. BLAST search against NCBI showed that the Cflr gene shared 50% -60% identity with the lysine-rich protein genes from potato and tomato in nucleotide and 40% -50% in amino acid. The lysine content of CFLR protein was 21·2%, which was the highest in the reported natural Lysine-rich proteins, and the Threonine content was also high with the content at 10·3%. Analysis of semi-quantitative RT-PCR indicated that Cflr gene was transcribed in mature pollen and petal largely, less in leaf and hardly in immature anther, stem and root. The Lysine rich protein gene was transformed to the immature embryo in wheat cultivar Yangmai 12 by using gold particle bombardment with the expression vector being constructed with lysine-rich protein gene Cflr from pepper, the GluB-1 promoter from rice and the Bar gene for screening. The resistant calli differentiated to seedlings and 114 transgenic plants were obtained. PCR testing and Southern blot showed that Lysine rich protein gene had been transformed to the genome of Yangmai 12, and it could be stably expressed in its next generation. Analysis of real time PCR and semi-quantitative RT-PCR indicated that the Cflr gene had been expressed in T1 transgenic wheat cultivar Yangmai 12, and expression abundance of transgenic plants were different. The content of protein and lysine in T2 plant seeds of 81 transgenic plant lines were analyzed. The test results showed that lysine content in grains of transgenic wheat was ranged from 0.32% to 0.97% with the average of 0.55% improved 71% compared to control.

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Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Concurrent session 4: New transgenic technologies, products and markets

Transgenic strategies for improving drought tolerance traits in chickpea
Bhatnagar-Mathur P, Krithika A, Rao JS, Vadez V, and Sharma KK International Crops Research Institute for Semi-Arid Tropics (ICRISAT), Patancheru, Andhra Pradesh, Patancheru, AP 502 324

Water deficit is the most prominent abiotic stress that severely limits crop yields, thereby reducing opportunities to improve livelihoods of poor farmers in the semi-arid tropics (SAT) where most of chickpea cultivation is done. In chickpea, annual losses of over 3.7 million tones have been estimated to be due to water deficit conditions alone. Sustained long-term efforts in developing chickpeas with better drought tolerance through conventional breeding have been met with only limited success mainly because of an insufficient understanding of the underlying physiological mechanisms and lack of sufficient polymorphism for drought tolerance-related traits. At ICRISAT we have used transgenic technology to speed the process of molecular introgression of stress responsive genes such as P5CSF129A and DREB1A in chickpea. Over 50 transgenic events with 35S:P5CSF129A and 18 independently transformed plants of rd29A:DREB1A were developed and analyzed at molecular and physiological levels. The putative transformants were confirmed for the presence and expression of the transgenes by using PCR, RT-PCR, semi-quantitative RT-PCR and Southern blot analysis. The evaluation of the transgenic chickpea plants was done under progressive water stress conditions to understand the physiological effect of the inserted genes at the whole plant level. The transgenic events carrying the P5CSF129A and DREB1A genes were phenotyped under both well-watered and water-limited conditions in greenhouse based on transpiration efficiency (biomass produced per kg of water), photosynthetic activity, stomatal conductance and root length. Several events had superior transpiration efficiency, photosynthetic activity, stomatal conductance and total transpiration under water limited conditions in comparison to the untransformed parent. All the selected transgenic events had a transpiration decline upon soil drying in drier soil than in the untransformed controls. Since it is critical to assess how these putative differences in the transpiration pattern under water deficit conditions relate to the component traits of yield. The focus is on phenotyping different transgenic events in conditions close to field like conditions using a trait-based approach dissecting yield into components under water stress. The current status and future plans on engineering drought tolerance in chickpea will be discussed.

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Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Concurrent session 4: New transgenic technologies, products and markets

Identification of stress-inducible and tissue-specific promoters in rice
Li ZX, Liu M, Li XJ, Xia M, Zhou JL* National Center for Molecular Crop Design, Beijing, China

For monocotyledonous crops transformation, there is a shortage of efficient promoters for stress-inducible or tissue-specific expression of transgene. Here, we report isolation and identification of several drought-inducible or tissue-specific promoters in rice. We selected candidate genes from the available microarray data sets and then did RT-PCR assay for all genes. Promoters of the genes with stress-inducible or tissue-specific expression pattern were isolated from rice genome. These promoters were linked to the GUS reporter gene, transformed into rice, and their activity was analyzed in transgenic plants at the stages of callus and young seedling (for tress-inducible promoters), flower, leaf and roots (for tissue specific promoters). So far, we have isolated three drought-inducible promoters, one callus-specific promoter and one root-specific promoter. The functional assay of these promoters is ongoing. Together, we have identified and analyzed several stress-inducible or tissue-specific promoters which would be helpful for bio-safe vector construction and crops transformation.

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Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Concurrent session 5: Molecular breeding for cotton, brassica and bio-energy crops

Progress toward genome sequencing of upland cotton Gossypium hirsutum
Yu SX Cotton Research Institute, CAAS, Beijing 100081, China

Genome sequence analysis of a plant species provides detailed resources for genomics research, including structural, functional, and evolutionary genomic studies, significantly expanding the molecular foundation for improvement of its agronomic and biological Traits. To better understand the relevance of genome structure, genome size variation, and polyploidization to cotton fitness and evolution, we screened 362 BACs,about 27.5Mb, from the libraries of Gossypium hirsutum L. based on DNA markers genetically mapped on chromosomes 12 and 26. We sequenced these clones and assembled the DNA sequences into contigs. We identified full length LTRs, analyzed their phylogenetic relationship and found two times amplification of the major types of LTR, one accrued before allopolyploidization, the other after the allopolyploidization. We investigated the colinearity conservation between these cotton BAC sequences and with other model plant genomes, revealing that a high level component of TEs caused increased recombination in Upland cotton. The phylogenetic tree of single copy orthologues genes among cotton, Arabidopsis, Poplar, grape, rice and maize shows that poplar was a nearer relative to cotton than others.

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Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Concurrent session 5: Molecular breeding for cotton, brassica and bio-energy crops

Maternal effects and genetic improvement of seed oil content in Brassica napus
Wang HZ*, Liu GH, Hua W, Wang XF, Liu J, Zhan GM Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences, Wuhan 430062, P.R. China. * Email: wanghz@oilcrops.cn

Seed oil content is an important agronomical trait in rapeseed. Our genetic crossing results have proposed that maternal genotypes contribute significantly to the seed oil content in Brassica napus. And silique wall as a maternal organ was found to play more important role than leaf. In our study, using one high and one low oil content rapeseed lines zy036 (oil content 50%) and 51070 (oil content 36%), we explained the mechanisms how the maternal organs act on the regulation of seed oil content. By three alternative methods, 1) F2/F3 individual correlation analysis, 2) altering the local photosynthesis in the silique wall and 3) transcriptome and comparative genomic analysis, we found the photosynthetic activity in silique wall was highly associated with the filial seed oil contents. Further, OC1 and WRI, two known oil synthesis related genes in seeds, were suggested to be involved in the regulation pathway of silique wall photosynthesis affecting oil synthesis. Additionally, one related functional gene (GK) was isolated from zy036 and the transgenic results in Arabidopsis implied the feasibility of enhancing the seed oil content by increasing the silique wall photosynthetic capacity in rapeseed through genetic engineering. At the same time, the carbon transport ability from silique wall to seed was proved to be highly related with seed oil content by the physiology and biochemistry studies. In this study, we also found the maternal plant heat-resistant gives a stable condition for oil synthesis in seed, and one regulated gene (hsp17) in this pathway was also isolated and proved. In sum, three pathways in the maternal organs were considered to be involved in the regulation of seed oil content. The knowledge acquired in the study would pave the way for high oil content rapeseed breeding. Now, the highest oil content winter rapeseed cultivar (Zhongshuang 11, 49%) was generated in the national regional test, and 5 new rapeseed lines with oil content above 55% were already bred by our genetic breeding group.

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Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Concurrent session 5: Molecular breeding for cotton, brassica and bio-energy crops

Towards establishing a molecular breeding platform in Cotton: Progress and Challenges
Kumpatla SP Department of Trait Genetics & Technologies, Dow AgroSciences LLC 9330 Zionsville Road, Indianapolis, IN, USA

Cotton is the world's leading fiber crop and the second most valuable oilseed crop. Despite its economic importance, the pace of genetic mapping and genomics in cotton has been slower compared to other important crop species. This is due to the lack of development of molecular and genetic resources such as large number of molecular markers, high density genetic linkage maps and comprehensive physical maps as well as the non-availability of whole genome sequence of cultivated tetraploid cotton genome. The polyploid nature, complexity of the genome and low level of intra-specific polymorphism hindered the much needed establishment of marker assisted breeding in cotton. Global efforts since late nineties, though slow, gradually resulted in the development of thousands of simple sequence repeat (SSR) markers using multiple molecular and computational approaches and generation of several linkage maps. However, the number of markers that could be used for high fidelity molecular breeding is still very low leading several researchers to initiate efforts in the discovery of single nucleotide polymorphism (SNP) markers. As predicted, SNPs are abundant in cotton genome but identification of SNPs with simple inheritance in the tetraploid like cotton poses another challenge for researchers. Several efforts are currently underway for large scale discovery of SNPs using high throughput sequencing and bioinformatics methods as well as to identify informative SNPs. The non availability of tetraploid cotton genome sequence poses a major challenge to these efforts. Progress in marker and map development and challenges in establishing a marker assisted breeding platform for routine use in cotton will be discussed.

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Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Concurrent session 5: Molecular breeding for cotton, brassica and bio-energy crops

Cytological studies of apomixis in Hickory (Carya cathayensis Sarg.)
Huang JQ*, Zhang B, Wang ZJ, Huang YJ, Xia GH Zhejiang Agriculture and Forestry University, A Nurturing Station for the State Key Laboratory of Subtropical Silviculture,Lin'an, Zhejiang Province 311300 People’s Republic of China * Email: Huangjq@zjfc.edu.cn

Hickory (Carya cathayensis), which is an important oil tree with high economic value which bears edible nut in China show the tendency of apomictic embryos develop as its polyembryony phenomemon and previous research. Up to now, it has not been reported the origin of mature embryos and its mechanism of reproduction. Hickory was identified here as a new apomictic species cytologically by paraffin section and flow cytometry detection. Developmental pattern of embryo sac formation in hickory represented typical polygonum-type and female gametophyte developed from functional megaspore which was selected from tetrad after meiosis of megasporocyte. Zygote embryos had not been found in numerous histological researches and embryos all originated from nucellar cells. Initial cells of nucellar embryo located both at the micropylar end and chalazal end of embryo sac and presented with cell enlargement and vacuolization inhomogenously. Mature nucellar embryo only developed at the nucellus beak region although bud like nucellar embryo also distributed at the chalazal end of embryo sac in some ovules absence of endosperm development which was abortion later. Ploidy of Endosperm in hickory was identical with leaves of mother tree and was diploid by flow cytometry detection. Development of endosperm in hickory was spontaneous type.

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Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Concurrent session 5: Molecular breeding for cotton, brassica and bio-energy crops

Mining of Novel Genes for Cotton Fiber Improvement
Yu JZ *, Percy RG , Kohel RJ , Abdurakhmonov IY 1 USDA-ARS Southern Plains Agricultural Research Center, Crop Germplasm Research Unit, College Station, Texas, USA; Institute of Genetics and Plant Experimental Biology, Uzbek Academy of Sciences, Tashkent, Uzbekistan. *Email john.yu@ars.usda.gov
2 1 1 1 2

Molecular breeding for cotton lags behind that for other major crops. Low levels of DNA polymorphism and genetic bottlenecks through cotton domestication complicate the efforts to improve the genetic gains in such traits as fiber quality and yield potential. While the cotton research community continues to develop essential tools including polymorphic markers and genomic sequences, we begin to take advantage of natural genetic variation that exists in largely untapped cotton germplasm collections using a set of core DNA markers. Association analysis of the first thousand unrelated cotton accessions, representing dozens of national origins and several ecotypes or landraces, with a number of fiber traits identifies unique genotypes and diagnostic markers for cotton molecular breeding programs. Effective mining and genome-wide selection for beneficial genes or genomic fragments conferring cotton fiber and other traits become possible and this approach will expand with additional molecular tools developed by the cotton research community.

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Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Concurrent session 5: Molecular breeding for cotton, brassica and bio-energy crops

Rational design and molecular breeding of sorghum, a dedicated bioenergy crop
Huang YH USDA-ARS Plant Science Research Laboratory, 1301 N. Western Road, Stillwater, OK 74075 and Department of Botany, Oklahoma State University, Stillwater, Oklahoma 74078, USA Email: yinghua.huang@ars.usda.gov Sorghum (Sorghum bicolor L.) is a C4 grass species grown worldwide because its high-efficiency in photosynthesis, excellent tolerance to drought and prosperity on marginal lands. This plant is a dedicated biofuel crop and has already become a viable feedstock for bioethanol. It offers several options in ethanol production including from its grain, sugar in sweet sorghum stalk, and vegetative biomass of the entire plant. Sorghum grain currently is the No. 2 (behind corn) source of bioethanol in the U.S. Sweet sorghum variety, similar to sugarcane, has recently emerged as a renewable feedstock for simple ethanol conversion. To date, cellulosic biomass is becoming an attractive energy feedstock as the supplies are more abundant, for which sorghum shows great potential due to its unique characteristics, such as high water-use efficiency, high biomass productivity under sustainable low-input conditions, and amenable to manipulation of its cell-wall characteristics. Sorghum has been bred for biofuel production only recently. But the recent advances in genomics research and plant biotechnology provide opportunities to facilitate the development of new sorghum cultivars and hybrids for high productivity of biomass with desirable quality of cellulose. This paper reviews the significant advances in sorghum genomics studies and the recent breakthroughs in sorghum plant biotechnology research, and demonstrates a the potential of the genomic and biotechnological tools for both enhancing biomass productivity and manipulating cell wall structure and composition, which can help improve the energy efficiency of bioenergy crops through the combination of rational design and molecular breeding approach. In this presentation, the author will highlight crop-breeding options to address biofuel needs in the future and will also discuss opportunities and challenges in developing biomass crops for the second generation of biofuels. Furthermore, the fundamental knowledge learned from the sorghum as a model bioenergy crop can be readily applied to other fuelstock candidates such as Miscanthus and switchgrass for their genetic improvement.

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Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Concurrent session 5: Molecular breeding for cotton, brassica and bio-energy crops

Molecular breeding for cottonseed quality improvement
Zhu SJ Department of agronomy, Zhejiang University, Hangzhou 310029. Email: shjzhu@zju.edu.cn

Cottonseed, about 2/3 of the seed cotton yield, is a very important by-product in cotton production. The world seed cotton production is about 28-30 millions tons annually, which will produce about 20 millions tons of cottonseed. The cottonseed, contented about 27-45% of protein and 28-40% of oil, is a huge natural resource in plant protein and edible oil, although it has not been utilization completely due to the existing of gossypol in the seeds. So the improvement of cottonseed quality is one of the important aims in cotton breeding, but a few reports have been published up to now. An immotalized F2 (IF2) population with 188 crosses in upland cotton was constructed by random mating among recombinant inbred lines (RIL) derived from a cross of HS46 × MARCABUCAG8US-1-88. The map genomic regions associated with seed index (SI), kernel index (KI), kernel percentage (KP), ratio of kernel and hull (K/H), protein content (PC), oil protein (OC), and gossypol content (GC) was identified to facilitate the selection of these traits in cottonseed breeding. A linkage map consisting of 388 molecular markers from this population was used to identify QTL using QTLNetwork-2.0 software. Seventeen main effects and fifteen epistasis QTLs were identi?ed for these traits. Phenotypic variation explained by each individual QTL ranged from 2.42 to 23.04%. Epistatic QTL for these traits were detected and important as well. In addition, the difference between MAS and direct selection by NIR analysis was compared to evaluate effect of these QTLs in the breeding program targeting development of cotton with improved nutrient quality. The efficiency of MAS for oil content was 56.4~92.5% and one SSR market located in chromosome 15 was linkage closely with the oil content, which can be used in MAS of cotton seed oil content. Using this marker in MAS, the oil content of more than 1000 upland cotton germplasms have been selected, and 19 of them were the trait of high oil content which can be used in cottonseed improvement for oil content.

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Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Concurrent session 5: Molecular breeding for cotton, brassica and bio-energy crops

Molecular focus in commercial plant breeding
Rossouw JD Asia Corn Breeding Lead, Monsanto Singapore Co (Pty) Ltd, 151 Lorong Chuan, #06-08 New Tech Park, Singapore, 556741; Dr. R.G Cantrell, Cotton Discovery Breeding Lead, Monsanto, St. Louis, MO 63167, USA. Email: jdross@monsanto.com

Plant breeders have various breeding ‘tools’ to their advantage but the challenge is how to incorporate these successfully into a commercial plant breeding program to deliver products to their farmer customers. Commercial plant breeding programs are equipment with excellent scientist and research staff, global germplasm and new breeding technologies like molecular markers to drive genetic gains. Commercial plant breeding has evolved over the last few decades to where we are today by continuously adding new technologies. During 1970-1990 the focus were on quantitative genetics, statistics and experimental designs and having winter nurseries to have two to three breeding cycles per year to speed the breeding process. The followed computer technologies, testing mechanization, analytics, transgenic traits, dihaploids and molecular breeding became very important to drive breeding and genetic gains. Molecular breeding has yielded some great success in commercial plant breeding with successfully combining it with other aspects of plant breeding like people, germplasm and other technologies that have been around the last few decades. Monsanto has a strong history of technology innovation in cotton and corn demonstrated by the growth in global acreage planted to Monsanto germplasm and biotech traits for insect and weed control. As core crops within Monsanto; biotechnology, germplasm, and molecular breeding are being integrated to develop new commercial products to drive simultaneous genetic gain for lint yield and fiber quality for cotton and yield and disease resistance in corn. Looking forward GWS (Genome Wide Selection) and Whole Genome Sequencing will be the next step in molecular technologies. GWS also opens the door on more innovation that needs to happen across multiple disciplines to support plant breeding in the future.

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Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Concurrent session 6: Maize molecular breeding

QTL fine mapping of leaf angle and leaf orientation value in maize
Chen YH Henan Agricultural Unviersity Email: chy9890@163.com

A major limiting factor for high productivity of maize in dense planting is light penetration through the canopy. Plant architecture with a narrower leaf angle (LA) and an optimum leaf orientation value (LOV) is desirable to increase light capture for photosynthesis and production per unit area. However, the genetic control of the plant architecture traits remains poorly understood in maize. In this study, QTL for LA were mapped using a set of 229 F2:3 families derived from the cross between compact and expanded inbred lines, evaluated in three environments. The results showed that one key genome region controlling leaf angle was identi?ed: qLA1. The QTL at nearest marker umc2226 on chromosome 1.02 accounted for 20.4% of the phenotypic variance. Based on this, using the BC3F2 population of 300 individuals from one BC3F1-86 plant, we mapped qLA1 to a 4.9-cM interval between bnlg1803 and bnlg1484, which explained 35.7% of LA variation. The BC3F2:3 families from the BC3F2 population showed a very wide variation in LA, which suggested that it was qLA1 affected the variation of LA in this population. Within a qLA1 interval, single/low-copy bacterial artificial chromosome sequences were exploited to develop 19 polymorphic markers to saturate the qLA1 region. A step by step narrowing-down strategy was adopted to pursue fine mapping of the qLA1 locus. Recombinants within the qLA1 region, screened from each backcross generation, were backcrossed to ‘Shen137’ to produce the next backcross progenies. These progenies were individually genotyped and evaluated for leaf angle. Sequential fine mapping of BC3F2, BC3F3 and BC4F2 generations enabled us to progressively refine the qLA1 locus to a ~100-kb interval flanked by the markers SSR40 and SSR53. Because of qLA1 accounting for 35.7% of LA variation, therefore, the locus was capable of improving performance of plant architecture with regard to leaf angle.

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Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Concurrent session 6: Maize molecular breeding

Application of Molecular Techniques in Maize Haploid Breeding
Chang MT1*, Li JS1 and Cai Z2 1 National Maize Improvement Center of China, China Agricultural University, Beijing 100193, China; 2 Jilin Academy of Agricultural Science, China. * Email: cshzhang2000@yahoo.com.cn

Haploid breeding is a relatively direct and rapid breeding method. Using doubled haploids (DH) to select the best maize pure elite inbred is gradually replacing the conventional maize breeding methodology. Haploids are those plants which derived from unfertilized egg cells or sperm cells. Those plants are also called haploid sporophytes. The chromosome number of a haploid plant is only half of their sporophyte. The size of their cells, chloroplasts and plastids are relatively smaller, and has less DNA content. Haploid plants can be identified by their morphology, chromosome number, flow cytometry, isozyme and molecular maker analysis. The first haploid seed plant was a dwarf sea island cotton and was discovered in 1920 by O. S. Atteck. The first haploid maize plant was discovered in 1929 by Stadler and Randolph. The frequency of spontaneous occurred haploids is very low and it is about 1 in ten thousands. It is possible to use various methods to increase the frequency of haploids, including physical treatment, chemical treatment, genetic induction, microspore and megaspore culture, anther culture, wild crosses, unisexual propagation or apomixis, and pollen treatments. Practical applications of haploids include instant fixation of genetic pure elite inbred lines, tools for population improvement, trait conversion and cytoplasmic male sterility conversion, materials for basic genetic, quantitative genetic, trait analysis, gene location and gene mutation study, and useful materials for molecular breeding and basic plant research studies. Combined haploid method and molecular technique, including genomewide analysis, QTL determination, and molecular marker assisted breeding; it will be possible to speed up the success of whole breeding process. Successful applications has been applied in transgenic trait conversion and forward breeding, backward breeding, molecular marker assisted recurrent selection, genomic wide breeding prediction, and application of DH method and SNP whole genome aggressive breeding strategy.

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Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Concurrent session 6: Maize molecular breeding

Identification of gene marker sets for screening maize lines for resistance to aflatoxin contamination
Luo M 1*, Brown RL 1, Chen ZY 2, Menkir A 3, and Bhatnagar D 1 1 Southern Regional Research Center, United States Department of Agriculture–Agricultural Research Service, New Orleans, LA 70124; 2 Department of Plant Pathology and Crop Physiology, Louisiana State University Agricultural Center, Baton Rouge, LA 70803; 3 International Institute of Tropical Agriculture, Ibadan, Nigeria. * E-mail: Meng.Luo@ars.usda.gov

Aflatoxins are the secondary metabolites of Aspergillus flavus which can be highly toxigenic and carcinogenic to humans or animals consuming contaminated food or feeds. Presently, a significant number of countries have established or proposed regulations for controlling aflatoxins in food and feeds; the US Food and Drug Administration (FDA) has limits of 20 ppb, total aflatoxins, on interstate commerce of food and feed, and 0.5 ppb of aflatoxin M1 on the sale of milk. However, many countries, especially in the developing world, experience contamination of domestic-grown commodities to alarmingly greater levels. Aflatoxin contamination caused by A. flavus is a major concern in maize production prior to harvest and during storage, and also in many other crops, such as peanuts, cottonseed, tree nuts, rice etc. Severe aflatoxin contamination during maize production is often correlated to drought stress and high temperatures. Although many maize lines have been screened for resistance to A. flavus infection and aflatoxin biosynthesis, levels of resistance are not yet adequate to prevent unacceptable aflatoxin concentrations. To rapidly identify resistant genotypes and to easily follow resistance traits in breeding populations, efforts have been made to develop genetic markers associated with resistant traits. However, very few markers are available so far. To solve this bottleneck in developing genetic markers, we are developing gene markers based on correlating the expression of the selected genes with the aflatoxin-resistance phenotype. Previous research indicated that multi-genes were involved in the resistance. Our comparative proteomics work identified kernel resistance-associated proteins (RAPs) and demonstrated their role in kernel resistance. Our microarray investigation demonstrated that RAP genes were expressed during the observed period from 25 to 45 day after pollination, but were expressed at a higher level at late maturation phase. In this presentation, we show that gene marker sets could be used to assess aflatoxin resistant lines, but are dependent on the particular state of kernel development. These results came from investigating kernels of two closely-related maize genotypes, generated by an aflatoxin-resistance breeding collaboration between the International Institute of Tropical Agriculture (IITA) and the Southern Regional Research Center (SRRC) of the USDA-ARS.

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Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Concurrent session 6: Maize molecular breeding

Maize disease resistance gene discovery and utilization through association and linkage mapping
Mahuku G *, Yan J, Magorokosho C, Makumbi D, Crossa J, and Mataka A International Maize and Wheat Improvement Center (CIMMYT), Km. 45 Carr. Mexico-Veracruz. Col El Batan. Texcoco-Edo Mexico. Mexico CP 56130 * Email: g.mahuku@cgiar.org

Assessing the association between DNA variants and disease has been used widely to identify regions of the genome and candidate genes that contribute to disease resistance. Identifying the relevant genes has been difficult, in part because each causal gene only makes a small contribution to overall heritability. Genetic association studies assess correlations between genetic variants and trait differences on a population scale, and offer a powerful approach for identifying and mapping causal genes with modest effects. In CIMMYT, we are using nine environments spread in Africa and Latin America to phenotype over 300 advanced maize lines against maize streak virus (MSV), northern corn leaf blight (Excerohilum turcicum), southern corn leaf blight (Bipolaris maydis), common rust (Puccinia sorghi), gray leaf spot (Cercospora zeae-maydis), and fusarium ear rots (Fusarium verticillioides). The objectives of this study are to (i) identify disease resistance donors effective across environments and to use association mapping strategies to detect markers near to esistance genes in the maize genome and subsequent development of gene-based markers for use to aid selection in our breeding programs. Our results showed high repeatability (r = 0.7 to 0.99) within locations compared to between locations (r = 0.30 to 0.40). These results revealed that while some sources of resistance are good across localities, pathogen populations are also highly variable and might be different between locations. Therefore, potential sources of resistance should be evaluated in multiple environments to select best genes to use in a breeding program. Marker-trait association using the general and mixed linear models identified 49 SNPs that were significantly associated with disease resistance. Thirty-four of these SNP markers were unique. Haplotype analysis using significant SNPs revealed that additive gene action appears to be more important in disease resistance than epistasis. The use of this information to elucidate the genetics of disease resistance in maize, organization of disease resistance genes in the maize genome and prospects for development of gene-based markers are discussed.

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Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Concurrent session 6: Maize molecular breeding

Forward to Molecular Breeding for High-oil Maize
Yang XH 1, Hao XM 1, Ma HL 1, Gou YQ 1, Yan JB 1, 2, Song TM 1, Li JS 1* 1 National Maize Improvement Center of China, China Agricultural University, Beijing 100193, China; 2 International Maize and Wheat Improvement Center (CIMMYT), Apdo. Postal 6-641, 06600 Mexico, D.F, Mexico * Email lijiansheng@cau.edu.cn

Maize oil is a highly valued crop as animal feed, human food and biodiesel. Phenotypic variation of oil content in maize kernel could be related to oil components like fatty acids and physical characteristics of the kernel as embryo size and embryo to endosperm weight ratio. To determine the genetic basis of oil content and composition in maize grain, a recombinant inbred population derived from a cross between normal line B73 and high-oil line By804 was phenotyped using gas chromatography, and was genotyped with 228 molecular markers. A total of 42 individual QTL, associated with fatty acid compositions and oil content, were detected in 21 genomic regions. Five major QTL were identified for measured traits, one each of which explained 42.0% of phenotypic variance for palmitic acid, 15.0% for stearic acid, 27.7% for oleic acid, 48.3% for linoleic acid, and 15.7% for oil content respectively in the RIL population. Thirty-six loci were involved in 24 molecular marker pairs of epistatic interactions across all traits, which explained phenotypic variances ranging from 0.4 to 6.1%. These results demonstrated that a few major QTL with large additive effects could play an important role in attending fatty acid compositions and increasing oil content in used germplasm. A larger number of minor QTL and a certain number of epistatic QTL, both with additive effects, also contributed to fatty acid compositions and oil content. In order to further identify the causal variants for QTL controlling oil content in maize kernel, seven more oil related traits (embryo oil content, embryo oil density, embryo to endosperm weight ratio, embryo volume, embryo width, and embryo length and embryo width to length ratio) were measured in the same RIL population. A total of 58 QTLs were identified for oil content in maize kernel and its component traits in 26 genomic regions across all chromosomes. Eight main-effect QTLs were identified for kernel oil content, embryo oil content, embryo oil concentration, embryo to endosperm weight ratio, embryo weight and embryo width to length ratio, each accounting for over 10% of the phenotypic variation in 6 genomic regions. Over 90 % of QTLs identified for kernel oil content co-localized with QTLs for component traits, validating their molecular contribution to kernel oil content. As kernel oil content is dependent on many factors, identifying QTL associated with component traits of oil content provided additional information on the genetic basis of kernel oil QTL. On chromosome 1, the largest effect QTL for kernel oil content (qKO1-1) was associated with embryo width, while on chromosome 9, the QTL for kernel oil content (qKO9) was related to embryo to endosperm weight ratio. Embryo oil density and embryo
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Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

width were identified as the most important component traits controlling the second largest QTL for kernel oil content on chromosome 6 (qKO6) and a minor QTL for kernel oil content on chromosome 5 (qKO5-2), respectively. The dissection of kernel oil QTLs will facilitate future cloning and/or functional validation of kernel oil content, and help to elucidate the genetic basis of oil content in maize kernel. Based on above results, two major QTL regions, qKO1-1 in chromosome bin 1.04 and qKO6 in chromosome bin 6.04, were targeted for introgression of the favorable alleles from the high-oil line into two elite inbred lines through molecular maker assistant selection. Chang7-2 and Zheng58, which are the parents of widely extended hybrid Zhengdan 958 in China, were used for recurrent parents. The BC5F2:3 lines were evaluated for oil content and background recovery. For qKO1-1, the introgressed genomic fragment was about 20cM containing the targeted QTL, and the oil content of maize ears with high-oil homologous genotype was increased by 0.6 and 0.3% for the improved lines from chang7-2 and zheng58, respectively. For qKO6, a function marker was developed from the validated gene DGAT1-2 controlling qKO6 and used in maker-assisted selection. In BC5F2:3, the introgressed genomic fragment contained the entire gene DGAT1-2. The oil content of maize ears with high-oil homologous genotype was increased by 0.8 and 0.5% for the improved lines from chang7-2 and zheng58, respectively. These results show MAS is a promising way to improve the quantity traits using QTL with large effects in maize, especially for the cloned QTL with validated function and developed function markers.

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Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Concurrent session 6: Maize molecular breeding

Genome-wide association study identifies known as well as novel loci for maize kernel tocopherol content and composition
Li Q 1*, Xu ST 1, Yang XH 1, Gao SB 2, Zhang ZX 3, Cai Y 1, Zhang DL 1, Zhou Y 1, Li JS 1, *, Yan JB 1, 4, *
1 2 3

National Maize Improvement Center of China, China Agricultural University, 100193 Beijing, China. Maize Research Institute, Sichuan Agricultural University, 625014 Yaan, Sichuan, China. National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, 430070 Wuhan, Hubei, International Maize and Wheat Improvement Center (CIMMYT), Apdo. Postal 6-641, 06600 Mexico, D.F., Mexico

China.
4

* Email: lijiansheng@cau.edu.cn; yjianbing@gmail.com

Tocopherols are important antioxidants for both human health and plant development. Here, we try to identify genetic variants that can control maize kernel tocopherol content and composition using both genome-wide association study (GWAS) and candidate gene based association study. Three kinds of tocopherols, namely δ-, γ- and α-, were scored in a panel of 513 lines across three locations. The contents ranged from 0.55 to 8.93 ?g/g for δ-tocopherol, 3.35 to 141.07 ?g/g for γ-tocopherol, 0.38 to 60.92 ?g/g for α-tocopherol, 7.46 to 198.35 ?g/g for total tocopherol; respectively. And the ratio of α-tocopherol to γ-tocopherol varies between 0.02 and 3.49. GWAS with about 50,000 SNPs identified 20 variants that are associated with at least one of the five traits (P < 1.02 × 10-6). The strongest association was on chromosome 5 for α-tocopherol, where a candidate gene from tocopherol biosynthesis pathway was located. Re-sequencing of this gene in a subset of 155 lines identified a total of 206 polymorphisms, most of which are in significant linkage disequilibrium. An insertion/deletion polymorphism (InDel) was significantly associated with α-tocopherol and also segregated in a recombinant inbred line population where a quantitative trait locus for the same trait was identified. This InDel can increase/decrease α-tocopherol content by 11.27-25.48 ?g/g in four F2 populations with different genetic background. Other significant variants are from genes with no obvious function in the tocopherol biosynthesis pathway, including transcriptional factor, genes targeted to chloroplast or mitochondria and so on. Re-sequencing of other five candidate genes from tocopherol biosynthesis pathway didn’t identify any strong associations, indicating these genes might play limited role in tocopherol phenotypic diversity.

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Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Concurrent session 7: Applied plant genomics: from genomics to field

Molecular breeding in chickpea- still a dream or the reality now!
RK Varshney1,2,* 1 International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru-502324, India; 2 Theme- Comparative and Applied Genomics, Generation Challenge Programme (GCP), c/o CIMMYT, Int APDO Postal 6-641, 06600 Mexico DF, Mexico * Email: r.k.varshney@cgiar.org

Chickpea (Cicer arietinum L.) is the most important cool season food legume crop, cultivated in arid and semi-arid region of the world. Terminal drought and Helicoverpa are two major constraints to chickpea production. Until a few years ago, implementation of molecular breeding programme was not possible in chickpea, due to dearth of sufficient genomic resources. With an objective to enhance molecular breeding in chickpea, concerted efforts of ICRISAT in collaboration with several partners around the world, lead to development of large scale genomic resources (http://www.icrisat.org/gt-bt/ICGGC/homepage.htm). For instance, 1,655 novel SSRs have been isolated from the SSR-enriched library (311) and BAC-end sequences (1,344), a DArT array has been developed with >16,000 features and >20,000 ESTs from drought and salinity stress challenged tissues based on Sanger sequencing. Further, a set of 443,969 sequence tags were generated through FLX-454 sequencing from a pool of normalized cDNAs assembled from developmental stages and abiotic stresses challenged tissues of a reference chickpea genotype (ICC 4958). Analysis of Sanger as well as FLX-454 sequence data provided 103,215 tentative unique sequences (TUSs). Alignment of >118.2 million sequence tags generated from drought responsive (ICC 4958 and ICC 1882) and Helicoverpa challenged tissues (ICCC 37 and ICC 506) with TUSs provided >90,000 nucleotide variants (SNPs) in these genotypes. In collaboration with University of California-Davis, a pilot Illumina GoldenGate assay for 768 SNPs coming from conserved genes across legumes has been developed. By using above mentioned resources, an integrated genetic map with >1400 marker loci has been developed based on the inter-specific mapping population (C. arietinum ICC 4958 × C. reticulatum PI 489777). With an objective of identification of candidate markers associated with drought tolerance (root traits), two intra-specific mapping populations (ICC 4958 × ICC 1882 and ICC 283 × ICC 8261) have been phenotyped for root traits in two environments and genotyped with >300 SSR markers. Marker-trait analysis has revealed several QTLs including one major QTL in both mapping populations that contributed up to 36% phenotypic variation. This genomic region is being introgressed into three elite chickpea lines (JG 11, ICC 92318 and KAK 2), using marker-assisted backcrossing (MABC) approach, to develop superior cultivars with enhanced drought tolerance. Due to availability of these genomic resources, molecular breeding in chickpea comes of age and is expected to be the integral part of chickpea breeding in near future.

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Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Concurrent session 7: Applied plant genomics: from genomics to field

Single-base resolution DNA methylomes of rice and new regulatory roles of DNA methylation in plant gene expression
Li X Kunming Institute of Zoology, Chinese Acadmey of Sciences, No.32 Jiaochang Donglu Kunming 650223, Yunnan, China Email: lixin@mail.kiz.ac.cn

DNA methylation plays important functional roles in plants and animals. To examine rice genome methylation landscape and assess its functional significance, we generated the first single-base resolution genome methylation maps for Asian cultivated rice Oryza sativa ssp. japonica, indica and their wild relatives, Oryza rufipogon and Oryza nivara. The overall methylation level of rice genomes is four times higher than that of Arabidopsis. Methylation in promoters represses gene expression while gene-body methylation generally appears to promote gene expression, which is consistent with the results reported for Arabidopsis but different from the previous epigenetic studies on rice. Most interestingly, we discovered that methylation in gene transcriptional termination regions can significantly repress gene expression, and the effect is even stronger than that of promoter methylation, which suggests a new direction in the study of DNA methylation. Through integrated analysis of genomic, DNA methylomic and transcriptomic differences between cultivated and wild rice, we found that the genetic factor reflected by DNA sequence divergence may be the major determinant for methylation differences at the whole genome level, but DNA methylation difference can only account for limited gene expressional variation between cultivated and wild rice. We also identified a number of genes with significant methylation level difference between wild and cultivated rice.

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Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Concurrent session 7: Applied plant genomics: from genomics to field

Insertion Site-Based Polymorphism markers open new perspectives for genome saturation and marker-assisted selection in wheat
Cubizolles N1, Laugier C1, Mastrangelo AM2, Faure S1, Choulet F1, Roger D3, Gauthier V3, Martinant JP3, Sourdille P1, Balfourier F1, Le Paslier MC4, Chauveau A4, Cakir M5, Gandon B3, Guerreiro L6, Gielen J7, Jaubertie JP8, Jack P9, Robert O10, Korzun V11, Feuillet C1 and Paux E1* INRA UBP UMR1095, 63100 Clermont-Ferrand, France; 2Centro di Ricerca per la Cerealicoltura, 71122 Foggia, Italy; 3Limagrain Verneuil Holding, 63204 Riom, France; 4INRA-EPGV UR1279, 91057 Evry, France; 5 Murdoch University, Murdoch WA 6150, Australia; 6Arvalis-Institut du Végétal, 75116 Paris, France ; 7 Syngenta Seeds, 31790 Saint Sauveur, France; 8AgriObtentions, 78041 Guyancourt, France ; 9RAGT Seeds, Ickleton, Essex CB10 1TA, UK; 10Bioplante, 59930 La Chapelle d'Armentières, France ; 11KWS LOCHOW, 37574 Einbeck, Germany. *Email: etienne.paux@clermont.inra.fr
1

In wheat, the deployment of marker-assisted selection has long been hampered by the lack of markers compatible with high-throughput cost-effective genotyping techniques. Recently, Insertion Site-Based Polymorphism (ISBP) markers have appeared as very powerful new tools for genomics and genetic studies in hexaploid wheat. To demonstrate their possible use in wheat breeding programmes, we assessed their potential to meet the five main requirements for utilization in MAS: flexible and high-throughput detection methods, low quantity and quality of DNA required, low cost per assay, and tight link to target loci and high level of polymorphism in breeding material. Toward this aim, we developed a programme, IsbpFinder, for the automated design of ISBP markers and adapted three detection methods (melting curve analysis, SNaPshot? Multiplex System and Illumina BeadArray technology) for high throughput and flexible detection of ISBP or ISBP-derived SNP markers. We demonstrate that the high level of polymorphism of the ISBPs combined with cost-effective genotyping methods can be used to efficiently saturate genetic maps, discriminate between elite cultivars, and design tightly linked diagnostic markers for virtually all target loci in the wheat genome. All together, our results suggest that ISBP markers have the potential to lead to a breakthrough in wheat marker-assisted selection.

75

Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Concurrent session 7: Applied plant genomics: from genomics to field

Integrating technologies for genetic improvement of quantitative traits in sorghum
Mace E*, Sakrewski K, Hunt C, Shatte T, Cruickshank A, Henzella R, Jordan D Department of Employment, Economic Development and Innovation (DEEDI), Queensland, Australia; * Email: emma.mace@deedi.qld.gov.au

Overcoming abiotic stresses and improving yield in crops remains one of greatest challenges in plant breeding. Understanding the genetic factors, and their interactions with the environment, that underlie such quantitative phenotypes is complex and requires integration across technologies, including high throughput genotyping and sequencing. Sorghum, a C4 cereal, is in a unique position to make use of a range of technologies with its rich history of genome analysis, from genetic linkage and physical mapping, through to the recent completion and annotation of the whole genome sequence, providing opportunities to fast-track progress towards better understanding gene function in cereals. A set of coordinated germplasm and technology resources is described which have been developed within the sorghum breeding program (DEEDI) to enhance the genetic dissection and improvement of both qualitative and quantitative traits in sorghum. These resources link to the whole genome sequence and include a Nested Association Mapping (NAM) population associated with phenotypic data across multiple environments and a mutagenised population consisting of approximately 5000 individuals, and encompass both forward and reverse genetics approaches to gene function determination in sorghum, representing a unique resource for the sorghum community to deliver advances in efficiency in research and development programs focused on complex traits. We detail a study focusing on the determination of the impact of allelic variation of key flowering time genes, and their interaction with the environment, detailing the combined use of the NAM populations, structured mapping populations and reverse genetics resources.

76

Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Concurrent session 7: Applied plant genomics: from genomics to field

Irradiation mutant mapping of wild beet translocation lines carrying resistance genes against the beet cyst nematode
Capistrano G *, J?ger SC, Harloff H, Jung C Plant Breeding Institute, Olshausenstr. 40, 24098 Kiel, Germany *e-mail: g.capistrano@plantbreeding.uni-kiel.de

The beet cyst nematode Heterodera schachtii Schmidt (BCN) is a severe pest in sugar beet (Beta vulgaris L.) and the only sources of complete resistance are the wild species Patellifolia procumbens and the related species P. patellaris. Sugar beet translocation lines A906001 and TR363 carrying a translocation of P. procumbens chromosome 1 are fully resistant to BCNs. The nematode resistance gene Hs1pro-1 had been cloned from the translocation line A906001 using a YAC based 1st generation physical map. However TR363 does not carry this gene. Hs1pro-1 gave complete resistance in a complementation study done with sugar beet hairy roots but only partial resistance was found in whole sugar beet plants transformed using this gene. These were strong indications for a second resistance gene on this translocation. Because the whole translocation is excluded from recombination, chromosome breakage mutants were produced. Seeds were 400 Gy gamma irradiated to produce breakages within the translocation and thus narrow down to the resistance gene. The mutants were screened with translocation specific markers for the identification of lines with smaller translocations. 2,670 seeds were irradiated and, after screening of 578 M1 offspring with three molecular markers evenly spread around the translocation, two mutants were found. These mutants, named TR320 and TR659, are susceptible to the BCN and had lost most of the translocation region. A BAC based physical map was established with a minimal tiling path encompassing 18 BACs covering 1,484 kb of the translocation region which had been estimated to be 1,500 kb in size. The BAC contigs contain only two small gaps. By colinearity analysis between four wild beet translocation lines, the region housing the nematode resistance gene could be narrowed down to ca. 180 kb. A candidate gene residing within this critical region is functionally characterized by transformation into sugar beet hairy roots and A. thaliana.

77

Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Concurrent session 8: Rice molecular breeding

Development of 384-plex SNP marker sets for diversity analysis, mapping, and marker-assisted selection in rice
Thomson MJ*1, Zhao K2, Wright M2, McNally KL1, Leung H1, McCouch SR2 1 International Rice Research Institute, DAPO Box 7777, Metro Manila, Philippines; 2 Cornell University, Ithaca, NY 14853, USA *Email: m.thomson@cgiar.org

Marker-assisted selection can enable more precise and rapid breeding strategies, but limitations in current genotyping techniques has prevented markers from being integrated into many mainstream breeding programs. Multiplexed single nucleotide polymorphism (SNP) markers have the potential to increase the speed, efficiency and cost-effectiveness of marker genotyping, provided that an optimal SNP density is used for each application. To test the usefulness of multiplexed SNP genotyping in cultivated rice, we designed four GoldenGate oligo pool assay (OPA) sets using Veracode technology for the Illumina BeadXpress Reader. Validated markers from existing 1,536 Illumina SNPs and 44K Affymetrix SNP chips developed at Cornell University were used to select subsets of SNPs for maximum information and even distribution for each application. A 96-plex OPA was developed for assigning a sample into one of the five Oryza sativa population subgroups in rice: indica, tropical japonica, temperate japonica, aus and aromatic. One 384-plex OPA was designed to have evenly-spaced polymorphic markers for QTL mapping, background selection and diversity analysis for indica and aus germplasm, while two additional 384-plex OPAs were selected for use in indica/japonica crosses and for DNA fingerprinting. The cost-effectiveness of each SNP set for different mapping populations will depend on the percent polymorphism across different combinations of mapping parents. After initial testing and validation on diverse germplasm accessions and populations, two of the SNP sets were re-designed to replace poor-performing markers. Further research is required to identify relevant SNPs for additional germplasm pools such as wild Oryza species. The availability of optimized SNP sets will help increase the efficiency of DNA fingerprinting, QTL mapping and marker-assisted selection to enable more rapid variety development to meet the challenges of rice improvement in the future.

78

Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Concurrent session 8: Rice molecular breeding

Epigenetic and Genetic Control of Drought Tolerance in Rice – A merging story of Larmarkism and Mendelism
Li ZK *,1,2, Dwivedi DK2,3, Lafitte R2, Liu SH1,2, Gao YM1,2, Xu JL1,2, Zhang F1, Fu BY1,2, Zheng TQ1,2, Ali J2, Wang Y1, Cui YR1, Domingo JR2, and Venus E2 Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China; 2 International Rice Research Institute, DAPO Box 7777, Metro Manila, Philippines; 3 Department of Biotechnology, N. D. University of Agriculture and Technology, Narendra Nagar (Kumarganj) 224 229, Faizabad (U.P.), India. *Email: z.li@cgiar.org
1

Drought is the most important factor limiting rice yields in the rainfed areas of Asia and Africa. To overcome this problem, major efforts have been taken to develop drought tolerant (DT) rice varieties and understand the genetic basis of (DT) in rice. Three sets of experiments were performed. First, we developed 221 IR64 introgression lines (ILs) with significantly improved DT selected from 13 BC2F2 populations from crosses between IR64 (recipient) and 7 diverse donors. Genetic characterization using SSR markers revealed an average of over-representation of the donor homozygotes by 1.7 times and a genomewide reduction of heterozygosy by 68% in the ILs. The second set of experiments included genotypic and phenotypic characterization of 455 DT F3 pyramiding lines (PLs) selected under severe drought from 9 F2 populations derived from crosses between 15 DT IR64 ILs. Genetic analyses with SSR markers revealed a genomewide epigenetic segregation (ES) in the DT PLs characterized with complete loss of heterozygosity at most segregating loci, strong non-random associations between or among unlinked loci of ES, and the presence of cryptic (inversions and translocations) variation in virtually all genomic regions of ES. Comparisons in segregation patterns between the drought selected PLs and random F2 progeny of the same 9 populations revealed two types of epiloci, ~30% of which inherited ES from their parental ILs and the remaining 70% were drought induced. LD analyses revealed strong nonrandom associations among the favorable alleles at these epiloci, forming putative genetic networks underlying DT in rice. The PLs from each population consisted of only a few major group (multi-locus) genotypes (GGs), which showed significantly improved DT over IR64, but differed considerably in yield and some other traits under irrigated conditions. The third set of experiments included 667 2nd round PLs selected under severe drought from the F2 populations of 14 crosses between 8 1st round PLs. Surprisingly, the drought selected F2 progeny of these populations restored completely or partially to the Mendelian segregation. Based on the results, we proposed a hypothesis and putative mechanism(s) based on which how and under what conditions Mendelian and ES (Larmarkian) segregation could be converted to each other. Finally, we proposed an efficient molecular breeding strategy for genetic improvement of DT and other abiotic stress tolerances in plants by designed QTL pyramiding and taking advantage of epigenetic control of these traits.

79

Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Concurrent session 8: Rice molecular breeding

Clustered QTLs for source leaf size and yield traits in rice
Wang P1, Zhou GL1, Cui KH1, Li ZK1,2, Yu SB1* 1 National Key Laboratory of Crop Genetic Improvement, and The College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, China. 2 National Key Facility for Crop Gene Resources & Genetic Improvement, Chinese Academy of Agricultural Sciences, Beijing 100081, China *Email: yusb@mail.hzau.edu.cn

Improvement of plant type plays an important role for super-high yield breeding in rice. In the present study, a set of backcross recombinant inbred lines derived from the cross of ‘9311’ and ‘Zhenshan97’, both elite indica hybrid rice parents, were developed to identify quantitative trait loci (QTL) for flag leaf size, panicle and and yield traits. Forty-seven QTLs were detected in common for 14 traits in the two environmental trials, of which 9 genomic regions with clustered QTLs affecting plant type and yield traits. Four co-localized QTLs on chromosomes 1, 6, 7 and 8 involving QTLs for flag leaf size (flag leaf length, width and area) contained the QTLs for yield traits such as panicle weight (PW) and secondary branch number (SBN), and in all cases alleles from ‘9311’ increased source leaf size were associated with increased sink size and yield (SBN and PW). Using a subset of overlapping substitution lines for the QTL region on chromosome 1, we validated and narrowed the QTLs into a 990 kbp interval (RM3746-RM10435) with the pleiotropic effects on flag leaf size, PW, SBN and spikelet number per panicle. These QTL clusters with large effects on source leaf size and yield-related traits provide good targets for marker assisted breeding for high-yield potential in rice.

80

Proceedings of the 3rd International Conference of Plant Molecular Breeding, Sept 5-9, 2010, Beijing, China

第三届植物分子育种国际学术会议摘要 2010 年 9 月 5-9 日,中国,北京

Concurrent session 8: Rice molecular breeding

Molecular breeding approaches for sustainable disease resistance in rice: Current and future strategies
Vera Cruz CM1, Jena KK1, Choi IR1, Mauleon RP1, Kobayashi N1, Fukuta Y2, Ali J1, Li Z1,3, Satoh K3, Kikuchi S3, Leung H1 1 International Rice Research Institute, DAPO Box 7777, Metro Manila, Philippines; 2 JIRCAS, Tsukuba, Japan; 3 Chinese Academy of Agricultural Sciences, 12 Zhongguancun South Street, Beijing 100081, China; 4 NIAS, Japan

Rice diseases have always been a significant factor in rice supply as new rice varieties and hybrids are planted to millions of hectares. Minor diseases and new forms of pathogens also evolve with changes in crop production practices and climate. Under these circumstances, disease resistance is still considered the most effective and desirable disease

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plant genomics | bmc genomics | 基因组学 | 基因组学与应用生物学 | 药物基因组学 | 功能基因组学 | 比较基因组学 | 10x genomics |