1. International Conference on Climate Change and Food Security
Discovering Drought-tolerant Gene
Resources for Crop Improvement
Ruilian Jing
jingrl@caas.net.cn
The National Key Facility for Crop Gene Resources and
Genetic Improvement / Institute of Crop Science
Chinese Academy of Agricultural Sciences (CAAS)
Beijing • November 7–8, 2011

2. 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.

3. Total drought area
10.5 Mha
8.8 Mha
Average year: 1.7 Mha drought area
Provinces suffered from severe drought
stress in the early spring 2009

4. 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.

5. ? Germplasm Resources
?
?
Gene Resources
?
?
?
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?

6. Drought tolerance at seedling stage
Drought tolerant genotypes survived in the soil
moisture of ~17% relative water content

7. Drought tolerance
in the field
2009
Henan
Shanxi Henan

8. Sensing, signalling and
cell-level responses to
drought stress
ABA-mediated responses
Non-ABA-mediated responses
Other mechanisms
(Chaves, et al., 2003)

9. Fructan functions
Fructans represented 85% of the water soluble carbohydrate
(WSC) --- main carbon source for grain yield in cereal crops
Fructans involved in tolerance to abiotic stresses
High water solubility: osmotic adjustment
A source of hexose sugars: allow continued leaf expansion during
periods of drought
Direct protective effect to membrane stabilization
Bolouri-Moghaddam, et al., FEBS J., 2010, 277, 2022-2037

10. 6-SFT (Sucrose: fructan 6-fructosyltransferase)
gene function in the process of fructan synthesis
6-SFT 1-FFT
levan neoseries 6G-kestotriose inulin neoseries
β(2-1) β(2-1)
6G-FFT
6-SFT 6-SFT 1-SST 1-FFT
levan 6-kestotriose SUCROSE 1-kestotriose inulin
β(2-1) β(2-1)
6-SFT
6-SFT 1-FFT
mixed-type levan bifurcose mixed-type levan
β(2-1) and β(2-6) 6-SFT β(2-1) and β(2-6)
FEH
1-FFT
levan
β(2-6)
Model for 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)

11. Three copies for 6-SFT were detected in wheat
6-SFT-A1
6-SFT-A2
6-SFT-D1
6-SFT-A1
6-SFT-A2
6-SFT-D1
6-SFT-A2 specific primer
6-SFT-A1
6-SFT-A2
6-SFT-D1
6-SFT-A1 specific primer
6-SFT-D1 specific primer
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.

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

13. 6-SFT-A1 mapping
1781 bp G/A
4A
3269 bp
MluⅠdigest
M G A G G G G G G G G Y N
Wu et al.
2010, 2011
3000 bp
2000 bp
1200 bp
Polymorphism and mapping of 6-SFT-A1 in RILs (Yanzhan 1×Neixiang 188)
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 were
located in 6-SFT-A1 region (Wu et al., 2010, JXB; 2011, PLoS ONE).
Yue et al., Scientia Agricultura Sinica. 2011, 44:2216-2224

14. Phylogenetic tree representing the haplotype
relationship of 6-SFT-A1
HapⅠ
Hap Ⅱ
Hap Ⅲ
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.

15. 6-SFT-A1 is associated with seedling biomass
under drought stress condition in a historical
population with 154 accessions
CK T
Well-watered (CK) Drought stress (T)

16. Agronomic traits associated with 6-SFT-A1 in
a historical population with 154 accessions
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 5.60
Well-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

17. Single nucleotide polymorphism in 6-SFT-A2
No. Site Location Type Change Hap I Hap II Hap III
1 600 Intron 2 SNP G/A G G A
2 730 Intron 2 SNP T/C T C T
3 807 Intron 2 SNP T/A C A C
4 858 Intron 2 SNP C/A C C A
5 1207 Exon 3 SNP G/A G A A
6 1237 Exon 3 SNP A/T A C T
7 1591 Exon 3 SNP C/T C C T
8 1870 Exon 3 SNP G/A G G A
9 2053 Intron 3 Indel T/0 T 0 T
10 2056 Intron 3 Indel 0/C 0 C 0
11 2546 Exon 4 SNP C/T C C T
12 2918 Exon 4 SNP G/C G G C
13 2951 Exon 4 SNP G/A G A G

18. Molecular marker design for 6-SFT-A2
4A
1870bp G/A 2951bp G/A
2660b
Mbo II Digest p Msg I Digest
G G G A G G G G G G G A
+ - + -
Hap Ⅰ + +
Hap Ⅱ + - Linkage map of 6-SFT-A2
Hap Ⅲ - + on chromosome 4A
(Hanxuan 10×Lumai 14)

19. Phylogenetic tree representing the
haplotype relationship of 6-SFT-A2
Hap Ⅱ
HapⅠ
Hap Ⅲ

20. Thousand grain weights of DHLs with
two 6-SFT-A2 haplotypes
50
**
*
**
**
**
45
**
40 * *
35
30
TGW(g)
25
20
15
10
5
0
2001
2001 2005
2005 2006H
2006DS 2006S
2006WW 2009H
2009DS 2009S
2009WW 2010H
2010DS 2010S
2010WW
Hap I (Hanxuan 10) Hap III (Lumai 14)
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.

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

22. Single nucleotide polymorphism in 6-SFT-D
C A G C
A G A T
475 841 2243 2850
Haplotype 475 bp 841 bp 2243 bp 2850 bp
Ⅰ C A G C
Ⅱ C A G T
Ⅲ A G A C
C C C C C T C T C T C T C T C C C C C C T C T C

23. Phylogenetic tree representing the
haplotype relationship of 6-SFT-D
Hap Ⅰ
Hap Ⅱ
Hap Ⅲ

24. HapⅠ of 6-SFT-D is a favourable haplotype
for TGW in a historical population
50
45
*
40
*
35 Ⅰ
TGW(g) 30
Ⅱ
25
20
15
10
5
0
2009 2010
Year Haplotype TGW (g) P-Value R2 (%)
2009 Ⅰ 40.4 ± 4.6 0.0351 2.46
Ⅱ 38.3 ± 5.7
2010 Ⅰ 34.5 ± 7.4 0.0385 1.94
Ⅱ 31.7 ± 6.7

25. 2008H 2008S
50 45
45 40
40
35
35
30
30
25 25
I+I I+II II+I II+II III+I III+II I+I I+II II+I II+II III+I III+II
2009H 2009S
50 46
44
45
42
40 40
38
35 36
34
30
32
25 30
I+I I+II II+I II+II III+I III+II I+I I+II II+I II+II III+I III+II
2010H 2010S
50 50
45 45
40 40
35 35
30 30
25 25
I+I I+II II+I II+II III+I III+II I+I I+II II+I II+II III+I III+II

26. TGW in genotypes with different haplotype
combinations of 6-SFT-A2 and 6-SFT-D
Haplotype* 2009D 2009W 2010D 2010W
I+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
* Combines of three haplotypes of 6-SFT-A2 and two haplotypes of 6-SFT-D.
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.

27. Relationship between TGW and
water soluble carbohydrate in stem
Early grain filling stage Middle grain filling stage
CK
Cut spike
0.3% KI
(200 mL/m2)
KI: potassium iodide

28. Analysis of thousand grain weight (TGW)
Reduction (CK – KI)
Env. Treatment Range (g) Mean±SD
Max (g) Min (g) Mean±SD
Well-watered CK 27.50~49.76 39.42±5.06
29.40 4.62 16.14±5.53
KI 11.13~38.46 23.28±5.23
Rain-fed CK 26.63~48.13 36.95±4.60
24.87 1.23 7.82±5.82
KI 14.78~43.58 29.13±6.16
TGWKI
Well-watered: × 100% = 59.32%
TGWCK
TGW KI
Rain-fed: TGW CK × 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.

29. QTLs QTLs for stem WSC in DH population
for WSC
Additive Epistatic Total
58 additive, 34 pairs of epistatic QTL; contribution rate 36.80%
Trait
Number R2(%) Number (lower section)
(peduncle), 49.57% (second section), 49.24% R2(%) (%)
Peduncle 21 31.93 9 4.87 36.80
QTLs for TGW
Second section 17 40.97 10 8.60 49.57
20 additive, 17 pairs 20 epistatic QTL; contribution rate
Lower section of 37.73 15 11.51 49.24
66.36%
QTLs for TGW in DH population
22 common intervals of WSC QTL and TGW QTL.
Additive Epistatic Total
(1A:Stage
WMC59; 1B: WMC156, CWM65, A1133-370, WMC269.2; 1D:
Number R2(%) Number R2(%) (%)
WMC222; 2B: WMC441; 2D: WMC453.1, Xgwm539, A4233-175,
2 4 6.99 6 4.02 11.01
WMC41; 3A: Xgwm391; 4A: A3446-205; 5A: Xgwm156, Xgwm595; 5B:
3 4 5.13 5 3.82 8.95
4 4 13.03 1 3.08 16.11
Xgwm67, Xgwm213, Xgwm499, WMC380; 6A: CWM487; 7A: A3446-
280, A2454-280) 7
5 22.69 5 6.48 29.17

30. Lower section, WSC
additive QTL, stage 5
Lower section, WSC
epistatic QTL, stage 3
TGW epistatic QTL, stage 4
Lower section, WSC
epistatic QTL, stage 5
TGW additive QTL, stage 2, 3, 4
Second section, WSC
epistatic QTL, stage 1
TGW epistatic QTL, stage 5
Lower section, WSC
epistatic QTL, stage 5
QTLs for WSC and TGW on chromosome 4A

31. 6-SFT-A2 mapping
4A 4A
4A
H10 L14
TGW
TGW epistatic
QTL, stage 5
Linkage map of 6-SFT-A2 on 4A Su et al., 2009 Yang et al., 2007
(Hanxuan 10×Lumai 14) Plant Science Genetics

32. Summary
A number of gene/QTLs involved in the
drought 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.

33. Acknowledgements
Collaborators
Yuchen DONG
Jizeng JIA
Xueyong ZHANG
Xiuying KONG
Chenyang HAO
Financial Support
National High Tech Program
National Key Program for Basic Research