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QTL mapping and cloning of plant


植物QTL的定位与克隆
任仲海

园艺科学与工程学院
College of Horticulture Science and Engineering

QTL: Quantitative Trait Locus

园艺科学与工程学院
College of Horticulture Scie

nce and Engineering

第一个被克隆的QTL(fw2.2)

Fig. 1. (A) Fruit size extremes in the genus Lycopersicon. (B) Phenotypic effect of the fw2.2 transgene in the cultivar Mogeor.

Fig. 2. High-resolution mapping of the fw2.2 QTL.

Frary et al., Science, 2000, 289: 85- 88.

园艺科学与工程学院
College of Horticulture Science and Engineering

水稻中已克隆的主效QTL

园艺科学与工程学院
College of Horticulture Science and Engineering

临时性群体(F2群体)的QTL定位

园艺科学与工程学院
College of Horticulture Science and Engineering

技术路线

园艺科学与工程学院
College of Horticulture Science and Engineering

水稻耐盐相关生理性状

Table 1 Traits analyzed

园艺科学与工程学院
College of Horticulture Science and Engineering

水稻耐盐QTL的粗定位

Lin et al.,Theor Appl Genet (2004) 108:253–260

园艺科学与工程学院
College of Horticulture Science and Engineering

构建控制K+浓度基因SKC1 的大规模遗传群体
SKC1

1

2

3

4

5

6

SKC5

SKC6

X

BC2F1

7

8

9

10

11

12

Nona Bokra Segment Koshihikari Segment

园艺科学与工程学院
College of Horticulture Science and Engineering

盐胁迫下SKC1精细定位群体的K+离子浓度的测定

140 mM NaCl 园艺科学与工程学院
College of Horticulture Science and Engineering

精细定位

园艺科学与工程学院
College of Horticulture Science and Engineering

开发新标记确定133个交换个体更精确的交换位置

园艺科学与工程学院
College of Horticulture Science and Engineering

用K159和K061-2筛选交换个体

园艺科学与工程学院
College of Horticulture Science and Engineering

交换个体和A、B对照的K+含量相比较可以确定SKC1的精确位置

园艺科学与工程学院
College of Horticulture Science and Engineering

互补实验

A和C,盐处理前, B和D,盐处理7天。 □为空质粒对照株系, ■为SKC1转基因株系。

园艺科学与工程学院
College of Horticulture Science and Engineering

SKC1近等基因系的培育及耐盐鉴定

NIL(SKC1)基因型示意图

园艺科学与工程学院
College of Horticulture Science and Engineering

NIL(SKC1)及越光地上部分Na+、K+离子浓度及其耐盐性

NIL(SKC1)及越光经125 mM NaCl处理32天。

□为越光,■为SKC1近等基因系。

园艺科学与工程学院
College of Horticulture Science and Engineering

拟南芥耐盐相关QTL, RAS1,的定位克隆
永久性群体(RIL群体)

Ren et al., (2010) PNAS 107 :5669-5674.

园艺科学与工程学院
College of Horticulture Science and Engineering

Sha 和 Ler的耐盐性差异非常显著,Sha对盐有很高的耐受性

A

B Treated with 120mM NaCl for 17 days

Ler

Sha C Hydroponic culture with salt treatment: 50 mM NaCl for 1day, 100 mM NaCl for 5days, 150 mM NaCl for1day, 200 mM NaCl -17days.

园艺科学与工程学院
College of Horticulture Science and Engineering

用Ler/Sha 重组自交系(RIL)粗定位耐盐相关QTL
在含120 mM NaCl的MS培养基上萌发后16天,统计绿苗数和根长。

园艺科学与工程学院
College of Horticulture Science and Engineering

盐胁迫条件下,和绿苗及根长有关的QTL

园艺科学与工程学院
College of Horticulture Science and Engineering

遗传连锁图显示绿苗数和根长QTL的分布
Chr 1
0.0 nga59

Chr 2
0.0
9.3 M2_5 nga1145 M2_26 M2_38

Chr 3
0.0 13.0 13.9 nga172 CHIB nt204

0.0 1.8 3.6 7.1

Chr 4

M4_41; M4_39 fry M4_8 M4_3 C6L9_78 nga1111

0.0 2.7

Chr 5

nga225 nga158

22.2 28.6

18.2 F21M12 F3F19 25.5

19.7 32.3

31.2 38.3

m5_14 nga139

44.0 48.7 57.7 63.9 76.0 89.5

M1_10 M1_7 CIW12 SO392 T27K12 F6D8_94 GENEA F5I14

44.5 45.4 46.7

M2_11 F3P11 M2_36

38.9

M3_19 47.7 M4_35 M4_15 CIW7

59.4 72.9 76.5 77.4 79.2 87.4 M2_17 ER M2_41 nga1126 nga361 71.1

M3_23 M3_32

56.6
70.3

56.5

flc

92.1

88.0 M3_21 93.6 106.4 111.7 M4I22 M4_14 M4_9 95.6

SO262 m5_1

104.0
115.6

112.8

M2_9

109.4 119.6

F8J2 M3_18

115.9 118.0 133.6

M5_9 k9d7 nga129 JV61_62

131.0

M1_13 137.9 nga6

126.6 138.4

M4_33 M4_37 146.7

151.8 152.7

nga692 ADH; M1_2

Green seedlings%

Root length

园艺科学与工程学院 K8A10 180.3
College of Horticulture Science and Engineering

169.8

MBK5

Chr 1
0.0 nga59

22.2
F21M12 F3F19 28.6

44.0 48.7 M1_10 M1_7 CIW12 SO392 T27K12

57.7
63.9

Bay-0

76.0

89.5

F6D8_94

104.0

GENEA

Sha

115.6

F5I14

绿苗数相关的 QTL,GS1,在另一个群体, Bay-0/Sha RIL, 中得到证实

131.0

M1_13

120 mM NaCl

园艺科学与工程学院

College of Horticulture Science and Engineering

151.8 152.7

nga692 ADH; M1_2

选择合适的自交系来构建精细定位群体
Chr 1

GS1

Chr 2
nga59 0.0 9.3 M2_5 nga1145 M2_26 M2_38 0.0 13.0 13.9

Chr 3
nga172 CHIB nt204

0.0

0.0 1.8 3.6 7.1

Chr 4

M4_41; M4_39 fry M4_8 M4_3 C6L9_78 nga1111

0.0 2.7

Chr 5

nga225 nga158

22.2 28.6

18.2 F21M12 F3F19 25.5

19.7

32.3
44.5 46.7 M2_11 M2_36 38.9 M3_19 47.7 59.4 72.9 76.5 77.4 79.2 87.4 M2_17 ER M2_41 nga1126 nga361 71.1 M3_23 M3_32

31.2 38.3

m5_14 nga139

44.0 48.7
57.7 63.9 76.0 89.5

M1_10 M1_7 CIW12 SO392

M4_35
M4_15 CIW7 56.5 flc

56.6
70.3

T27K12 F6D8_94 GENEA F5I14

92.1

M3_21

88.0 93.6 106.4 111.7 M4I22 M4_14 M4_9 95.6

SO262
m5_1

104.0 115.6

112.8

M2_9

109.4 119.6

F8J2 M3_18

115.9 118.0 133.6

M5_9 k9d7 nga129 JV61_62

131.0

M1_13 137.9 M1_2

126.6 nga6
138.4

M4_33 M4_37 146.7

152.7

Background of Ler

Background of Sha

169.8

MBK5 K8A10

Genotype of the R79

园艺科学与工程学院
180.3

College of Horticulture Science and Engineering

R79耐盐性鉴定

园艺科学与工程学院
College of Horticulture Science and Engineering

技术路线

R79

?

Ler

F1 ? Ler MAS(marker assistant selection) Developing more markers Constructing genetic map F2 BC1F1 ? Ler MAS BC2F1

F3

?

Ler MAS BC3F1 MAS Near Isogenic Line (NIL)

BC2F2
Phenotype analysis Large population MAS Recombinant lines BC2F3 Phenotype analysis

Fine mapping

High resolution linkage analysis

Cloning of the gene College of Horticulture Science and Engineering

Functional analysis 园艺科学与工程学院 of the gene

开发新标记,构建精细定位群体
Chr1 Chr1
nga59
Chr 1
0.0 nga59

R79

?
F1

Ler

F10O3-18

? 1 2 3 4 …… 190 …… F2 1: 2 :1 ?
Sha 分离 纯合
Ler 纯合

Developing more markers Constructing genetic map

22.2 28.6

F21M12 F3F19

T7A14-44 F3F20-64

44.0 48.7 57.7 63.9 76.0

M1_10 M1_7 CIW12 SO392

F7G19-17 F14J9-13 F21M12 T10O24-59 T28K15-14 F3F19 F7A19-94 F9L1-113 F19K19-16 T13M22-6

F3

T27K12
F6D8_94 GENEA

89.5
104.0 115.6

Phenotype analysis

F5I14

131.0

M1_13

Fine mapping
151.8 152.7
nga692 ADH; M1_2

M1_10

园艺科学与工程学院
College of Horticulture Science and Engineering

F3代耐盐性鉴定及精细定位
A
91 92
93

103

104

105
A. Significant segregation in this fine mapping population containing 190 lines from the cross between Ler and R79. This phonotype were analyzed with F3 generation. This picture shows 6 lines of the 190 lines.

B

C
Number of lines

35

30
25 20 15 10 5 0 0 0.1

H Sha Ler

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

More

Green seedlings %

B. The peak LOD value indicates that GS1 is closely linked to the maker F21M12

C. Phonotype distribution according to the maker F21M12 College of Horticulture Science and Engineering

园艺科学与工程学院

R79

?

Ler

构建大规模群体,

F1 ? Ler MAS BC1F1 ? Ler MAS BC2F1 BC2F2 Large population MAS Recombinant lines BC2F3 Phenotype analysis High resolution linkage analysis Cloning of the gene

园艺科学与工程学院
College of Horticulture Science and Engineering

GS1的高精度连锁分析

园艺科学与工程学院
College of Horticulture Science and Engineering

候选基因
F21M12
AT1G09890.1 similar to expressed protein [Arabidopsis thaliana] (TAIR:At1g09910.1); similar to PREDICTED OSJNBb0011H15.45 gene product [Oryza sativa (japonica cultivar-group)] (GB:XP_507326.1)

AT1G09900.1 AT1G09910.1 AT1G09920.1

pentatricopeptide (PPR) repeat-containing protein, contains Pfam profile PF01535: PPR repeat
expressed protein TRAF-type zinc finger-related, contains Pfam PF02176: TRAF-type zinc finger; similar to TNF receptor associated factor 4 (GI:7274404) (Mus musculus) (GI:7274404); similar to Cysteine rich motif Associated to Ring and Traf domains protein (mCART1)(GI:1041446) (Mus musculus) oligopeptide transporter OPT family protein, similar to SP|P40900 Sexual differentiation process protein isp4 {Schizosaccharomyces pombe}; contains Pfam profile PF03169: OPT oligopeptide transporter protein

AT1G09930.1

AT1G09932.1

phosphoglycerate/bisphosphoglycerate mutase-related, weak hit to Pfam profile PF00300: phosphoglycerate mutase family phosphoglycerate/bisphosphoglycerate mutase family protein, contains Pfam profile PF00300: phosphoglycerate mutase family glutamyl-tRNA reductase 2 / GluTR (HEMA2), identical to glutamyl-tRNA reductase 2, chloroplast (SP|P49294)

AT1G09935.1
AT1G09940.1 AT1G09950.1

F21M12-126
transcription factor-related, low similarity to tumor-related protein (Nicotiana glauca x Nicotiana langsdorffii) GI:688423, SP|P23923 Transcription factor HBP-1b {Triticum aestivum}

F21M12-130

园艺科学与工程学院
College of Horticulture Science and Engineering

At1g09950 序列比对

园艺科学与工程学院
College of Horticulture Science and Engineering

培育 NIL(GS1)

R79

?
F1

Ler

?

Ler

BC1F1

?

Ler MAS

BC2F1

?

Ler MAS

BC3F1

BC3F2

MAS

MAS: Marker Assistant Selection

园艺科学与工程学院
College of Horticulture Science and Engineering

GS1近等基因系种子萌发及幼苗对 NaCl 和 ABA 都不敏感 a Ler NIL(RAS1)

MS
120mM NaCl 2.5 μM ABA

园艺科学与工程学院
College of Horticulture Science and Engineering

Ler 的RAS1 等位基因是显性
Ler F1(Ler/NIL) NIL(RAS1)

MS

120mM NaCl

2.5 μM ABA

园艺科学与工程学院
College of Horticulture Science and Engineering

互补实验 :互补成功证明At1g09950就是RAS1

园艺科学与工程学院
College of Horticulture Science and Engineering

Genotype of the R63

GS3

园艺科学与工程学院
College of Horticulture Science and Engineering

Ler

×
F1-1

R79

Ler

×
F1-2

R63

Ler

F1-1

R79

Ler

F1-2

R63

Treated on MS with 100 mM NaCl for 13 days. So GS1 and GS3 are partially dominant genes.

园艺科学与工程学院

College of Horticulture Science and Engineering

The location of GS1 on arabidopsis chromosome 1 in 190 F2 plants from a cross between R79 and Ler. The maker F21M12 is co-segregating.

园艺科学与工程学院
College of Horticulture Science and Engineering

A small QTL was detected in another region on chromosome 1 in this population.
nga59

GS1

F21M12

M1_10 M1_7

SO392

T27K12

F6D8_25 GENEA F5I14

M1_13
M1_2 nga692

园艺科学与工程学院
College of Horticulture Science and Engineering

Thanks for your attention!

园艺科学与工程学院
College of Horticulture Science and Engineering


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