Discovering Drought-tolerant Gene Resources for Crop Improvement

Ruilian Jingjingrl@caas.net.cn

The National Key Facility for Crop Gene Resources and Genetic Improvement / Institute of Crop Science

Chinese Academy of Agricultural Sciences (CAAS)

International Conference on Climate Change and Food Security

Beijing • November 7–8, 2011

China: Precipitation

About 50% of land area is arid and semi-arid in China, where 6 667 000 ha of rainfed wheat are grown with low and variable yield. Developing drought-tolerant cultivars is an efficient way to stabilize wheat production and ensure food security in China and the world.

10.5 Mha

8.8 Mha

Provinces suffered from severe drought stress in the early spring 2009

Total drought area

Average year: 1.7 Mha drought area

Drought seriously limits crop production in many areas of the world, especially in China.More than 70% water is used in the crop production in China.

Water shortage

Big population

Crop drought-tolerance improvement is a challenging task for breeders. Discover and use drought-tolerant gene resources in the crop breeding can contribute to improvement for water-limited environments.

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How can we discover beneficial genes？

More than 7 million accessions have been collected and conserved in the germplasm banks in the world. How to find the favourable genes from the huge number of plant germplasm resources for plant breeding?

Germplasm Resources

Gene Resources

Drought tolerance at seedling stage

Drought tolerant genotypes survived in the soil moisture of ~17% relative water content

Drought tolerance in the field

2009

Henan

Shanxi Henan

Sensing, signalling and cell-level responses to

drought stress

ABA-mediated responses

Non-ABA-mediated responses

Other mechanisms

(Chaves, et al., 2003)

Fructan functions

Bolouri-Moghaddam, et al., FEBS J., 2010, 277, 2022-2037

Fructans represented 85% of the water soluble carbohydrate (WSC) --- main carbon source for grain yield in cereal crops

Fructans involved in tolerance to abiotic stressesHigh water solubility: osmotic adjustmentA source of hexose sugars: allow continued leaf expansion during periods of droughtDirect protective effect to membrane stabilization

Model for fructan synthesis

levan neoseriesβ(2-1)

6G-kestotriose inulin neoseriesβ(2-1)

levanβ(2-1)

6-kestotriose SUCROSE 1-kestotriose inulinβ(2-1)

mixed-type levanβ(2-1) and β(2-6)

bifurcose mixed-type levanβ(2-1) and β(2-6)

levanβ(2-6)

1-SST6-SFT

6-SFT

6G-FFT

6-SFT

6-SFT

6-SFT1-FFT

1-FFT

FEH

1-FFT

1-FFT6-SFT

6-SFT (Sucrose: fructan 6-fructosyltransferase) gene function in the process of fructan synthesis

The fructan class of water soluble carbohydrates has been assigned a possible role in conferring tolerance to drought. 6-SFT is capable of producing 6-kestose as well as elongating 6-kestose and 1-kestose and producing both levan and branched fructan.(Vijn et al., Plant Physiology, 1999, 120, 351-359)

Three copies for 6-SFT were detected in wheat

Two copies were located on genome A, one on genome D.Specific genome primers were designed based on the polymorphism in the sequences of gene 6-SFT.

6-SFT-A2 specific primer

6-SFT-A1 specific primer

6-SFT-D1 specific primer

6-SFT-A16-SFT-A26-SFT-D1

6-SFT-A16-SFT-A26-SFT-D1

6-SFT-A16-SFT-A26-SFT-D1

No. Site Location Type Change Amino acid change 1 116 Exon1 SNP C/T

2 333 Intron1 SNP C/G

3 541 Intron2 SNP G/C

4 563 Intron2 SNP T/A

5 1053 Intron2 SNP A/G

6 1609 Exon3 SNP A/G

7 1727 Exon3 SNP A/G Asn /Asp

8 1781 Exon3 SNP A/G Thr/Ala

9 1783 Exon3 SNP A/G

10 1831 Exon3 SNP T/C

11 2140 Intron3 SNP G/C

12 2157 Intron3 SNP G/T

13 2311 Intron3 SNP C/T

14 2358 Intron3 Indel T/0

Single nucleotide mutation in 6-SFT-A1

Among 30 hexaploid cultivars, 14 polymorphism sites in 6-SFT-A1 gene nucleotide sequences were identified, which included 13 SNPs and 1 InDel.

MluⅠdigest

M G A G G G G G G G G Y N

2000 bp3000 bp

1200 bp

4A

The CAPS marker was developed based on the SNP at 1781 bp. 6-SFT-A1 was mapped on chromosome 4A. QTLs for plant height, 1000-grain weight werelocated in 6-SFT-A1 region (Wu et al., 2010, JXB; 2011, PLoS ONE).

3269 bp

1781 bp G/A

Wu et al. 2010, 2011

Yue et al., Scientia Agricultura Sinica. 2011, 44:2216-2224

6-SFT-A1 mapping

Polymorphism and mapping of 6-SFT-A1 in RILs (Yanzhan 1×Neixiang 188)

Three haplotypes were identified using the 34 wheat germplasm. Hap I was mainly detected among wheat accessions showing mid-drought resistance and drought susceptiple. Hap III was found in the most of high drought resistant and resistant wheat germplasm.

Phylogenetic tree representing the haplotype relationship of 6-SFT-A1

HapⅠ

Hap Ⅲ

Hap Ⅱ

6-SFT-A1 is associated with seedling biomass under drought stress condition in a historical

population with 154 accessions

Well-watered (CK) Drought stress (T)

CK T

Environment Trait Hap I Hap III P-Value R2 (%)

Rain-fed Peduncle length 7.4±1.0 8.0±1.4 0.0045 7.63

Plant height 79.2±13.2 88.1±14.3 0.0058 5.60Well-watered Peduncle length 24.9±3.6 27.0±4.2 0.0001 11.02

Plant height 82.6±6.4 85.0±5.4 0.0337 3.93

Agronomic traits associated with 6-SFT-A1 in a historical population with 154 accessions

Single nucleotide polymorphism in 6-SFT-A2No. Site Location Type Change Hap I Hap II Hap III

1 600 Intron 2 SNP G/A G G A2 730 Intron 2 SNP T/C T C T3 807 Intron 2 SNP T/A C A C4 858 Intron 2 SNP C/A C C A5 1207 Exon 3 SNP G/A G A A6 1237 Exon 3 SNP A/T A C T7 1591 Exon 3 SNP C/T C C T8 1870 Exon 3 SNP G/A G G A9 2053 Intron 3 Indel T/0 T 0 T10 2056 Intron 3 Indel 0/C 0 C 011 2546 Exon 4 SNP C/T C C T12 2918 Exon 4 SNP G/C G G C13 2951 Exon 4 SNP G/A G A G

2660bp

1870bp G/A 2951bp G/A

G G G A G G G G G G G A

+ - + -

Hap Ⅰ + +Hap Ⅱ + -Hap Ⅲ - +

Msg I DigestMbo II Digest

Molecular marker design for 6-SFT-A2

4A

Linkage map of 6-SFT-A2 on chromosome 4A

(Hanxuan 10×Lumai 14)

HapⅠ

Hap Ⅱ

Hap Ⅲ

Phylogenetic tree representing the haplotype relationship of 6-SFT-A2

Hap I (Hanxuan 10) Hap III (Lumai 14)

0

5

10

15

20

25

30

35

40

45

50

2001 2005 2006H 2006S 2009H 2009S 2010H 2010S

TGW

(g)

*

**

*

**

** * ****

2006DS 2009DS 2010DS 2010WW2009WW2006WW20052001

Thousand grain weights of DHLs with two 6-SFT-A2 haplotypes

Thousand grain weight (TGW) of doubled haploid lines (DHLs) with Hap III of 6-SFT-A2 is significant higher than that of Hap I under different water regimes in five years.

Year Haplotype TGW (g) P-Value R2 (%)

Ⅰ 34.8±4.8 0.0397* 4.79

2009 Ⅱ 33.0±5.6

Ⅲ 35.6±4.9

Ⅰ 38.1±5.3 0.0310* 5.12

2010 Ⅱ 37.0±5.7

Ⅲ 39.7±5.5

TGW of three haplotypes of 6-SFT-A2 in a historical population

Hap III of 6-SFT-A2 is associated with higher thousand grain weight in the historical population consisted of 154 accessions.

Single nucleotide polymorphism in 6-SFT-D

Haplotype 475 bp 841 bp 2243 bp 2850 bp

Ⅰ C A G CⅡ C A G TⅢ A G A C

475 841 2243 2850

CA

AG

GA

CT

C C C C C T C T C T C T C T C C C C C C T C T C

Hap Ⅱ

Hap Ⅰ

Hap Ⅲ

Phylogenetic tree representing the haplotype relationship of 6-SFT-D

HapⅠ of 6-SFT-D is a favourable haplotype for TGW in a historical population

Year Haplotype TGW (g) P-Value R2 (%)

Ⅰ 40.4 ± 4.6 2009 Ⅱ 38.3 ± 5.7

0.0351 2.46

Ⅰ 34.5 ± 7.4 2010 Ⅱ 31.7 ± 6.7

0.0385 1.94

0

5

10

15

20

25

30

35

40

45

50

TGW(g)

Ⅰ

Ⅱ

**

2009 2010

2008H

25

30

35

40

45

50

I+I I+II II+I II+II III+I III+II

2008S

25

30

35

40

45

I+I I+II II+I II+II III+I III+II

2009S

30

32

34

36

38

40

42

44

46

I+I I+II II+I II+II III+I III+II

2010S

25

30

35

40

45

50

I+I I+II II+I II+II III+I III+II

2009H

25

30

35

40

45

50

I+I I+II II+I II+II III+I III+II

2010H

25

30

35

40

45

50

I+I I+II II+I II+II III+I III+II

Haplotype* 2009D 2009W 2010D 2010WI+I 38.50 37.34 38.64 40.01 I+II 36.77 35.01 34.80 37.96 II+I 37.30 34.63 37.89 39.65 II+II 35.55 35.36 38.58 38.49 III+I 39.46 37.18 39.55 40.60 III+II 40.39 36.58 39.31 38.37

Hap Ⅲ of 6-SFT-A2 and HapⅠ of 6-SFT-D are favourable hyplotypes for increasing grain weight, their combination is optimum for improving grain weight in wheat.

* Combines of three haplotypes of 6-SFT-A2 and two haplotypes of 6-SFT-D.

TGW in genotypes with different haplotype combinations of 6-SFT-A2 and 6-SFT-D

CK

Cut spike

0.3% KI(200 mL/m2)

Early grain filling stage Middle grain filling stage

Relationship between TGW and water soluble carbohydrate in stem

KI: potassium iodide

Analysis of thousand grain weight (TGW)

Env. Treatment Range (g) Mean±SDReduction (CK – KI)

Max (g) Min (g) Mean±SD

Well-watered CK 27.50~49.76 39.42±5.0629.40 4.62 16.14±5.53

KI 11.13~38.46 23.28±5.23Rain-fed CK 26.63~48.13 36.95±4.60

24.87 1.23 7.82±5.82KI 14.78~43.58 29.13±6.16

Well-watered: × 100% = 59.32%

Rain-fed: × 100% = 79.13%

Stem-reserved WSC significantly contributes to TGW. The contribution under drought stress condition is significantly higher than that under well-watered condition.

CK

KI

TGWTGW

CK

KI

TGWTGW

QTLs for stem WSC in DH population

QTLs for TGW in DH population

TraitAdditive Epistatic Total

(%)Number R2(%) Number R2(%)Peduncle 21 31.93 9 4.87 36.80

Second section 17 40.97 10 8.60 49.57Lower section 20 37.73 15 11.51 49.24

StageAdditive Epistatic Total

(%)Number R2(%) Number R2(%)2 4 6.99 6 4.02 11.013 4 5.13 5 3.82 8.954 4 13.03 1 3.08 16.115 7 22.69 5 6.48 29.17

QTLs for WSC58 additive, 34 pairs of epistatic QTL; contribution rate 36.80%(peduncle), 49.57% (second section), 49.24% (lower section)

QTLs for TGW20 additive, 17 pairs of epistatic QTL; contribution rate 66.36%

22 common intervals of WSC QTL and TGW QTL.(1A: WMC59; 1B: WMC156, CWM65, A1133-370, WMC269.2; 1D:

WMC222; 2B: WMC441; 2D: WMC453.1, Xgwm539, A4233-175,

WMC41; 3A: Xgwm391; 4A: A3446-205; 5A: Xgwm156, Xgwm595; 5B:

Xgwm67, Xgwm213, Xgwm499, WMC380; 6A: CWM487; 7A: A3446-

280, A2454-280)

QTLs for WSC and TGW on chromosome 4A

Lower section, WSCadditive QTL, stage 5

Lower section, WSCepistatic QTL, stage 3

Lower section, WSCepistatic QTL, stage 5

Second section, WSCepistatic QTL, stage 1

Lower section, WSCepistatic QTL, stage 5

TGW epistatic QTL, stage 4

TGW additive QTL, stage 2, 3, 4

TGW epistatic QTL, stage 5

Linkage map of 6-SFT-A2 on 4A (Hanxuan 10×Lumai 14)

H10 L14

Su et al., 2009Plant Science

Yang et al., 2007Genetics

6-SFT-A2 mapping

4A

4A

TGW epistatic QTL, stage 5

TGW

4A

Summary

A number of gene/QTLs involved in thedrought tolerance.Favourable alleles of target genes hide in the germplasm resources.Recombining favourable alleles of target genes could improve crop plants.Molecular marker assistant selection is an efficient approach for drought tolerance improvement in crop plants.

Acknowledgements

Yuchen DONGJizeng JIAXueyong ZHANGXiuying KONGChenyang HAO

Collaborators

Financial SupportNational High Tech ProgramNational Key Program for Basic Research