International Conference on Climate Change and Food Security Discovering Drought-tolerant Gene Resources for Crop Improvement Ruilian Jing email@example.com 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
China: PrecipitationAbout 50% of land area is arid and semi-arid in China, where6 667 000 ha of rainfed wheat are grown with low and variable yield.Developing drought-tolerant cultivars is an efficient way to stabilizewheat production and ensure food security in China and the world.
Total drought area 10.5 Mha 8.8 Mha Average year: 1.7 Mha drought areaProvinces suffered from severe drought stress in the early spring 2009
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 populationCrop drought-tolerance improvementis a challenging task for breeders.Discover and use drought-tolerantgene resources in the crop breedingcan contribute to improvement forwater-limited environments.
? Germplasm Resources? ? Gene Resources ? ? ?How can we discoverbeneficial 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?
Drought tolerance at seedling stageDrought tolerant genotypes survived in the soil moisture of ~17% relative water content
Drought tolerance in the field 2009 Henan Shanxi Henan
Sensing, signalling andcell-level responses to drought stress ABA-mediated responses Non-ABA-mediated responses Other mechanisms (Chaves, et al., 2003)
Fructan functionsFructans represented 85% of the water soluble carbohydrate(WSC) --- main carbon source for grain yield in cereal cropsFructans 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
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-FFTlevan 6-kestotriose SUCROSE 1-kestotriose inulinβ(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)
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.
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/0Among 30 hexaploid cultivars, 14 polymorphism sites in 6-SFT-A1 genenucleotide sequences were identified, which included 13 SNPs and 1 InDel.
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, 20113000 bp2000 bp1200 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
Phylogenetic tree representing the haplotyperelationship of 6-SFT-A1 HapⅠ Hap Ⅱ Hap ⅢThree haplotypes were identified using the 34 wheat germplasm. Hap I wasmainly detected among wheat accessions showing mid-drought resistanceand drought susceptiple. Hap III was found in the most of high droughtresistant and resistant wheat germplasm.
6-SFT-A1 is associated with seedling biomassunder drought stress condition in a historical population with 154 accessions CK T Well-watered (CK) Drought stress (T)
Agronomic traits associated with 6-SFT-A1 in a historical population with 154 accessionsEnvironment 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
Single nucleotide polymorphism in 6-SFT-A2No. Site Location Type Change Hap I Hap II Hap III1 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
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-A2Hap Ⅲ - + on chromosome 4A (Hanxuan 10×Lumai 14)
Phylogenetic tree representing thehaplotype relationship of 6-SFT-A2 Hap Ⅱ HapⅠ Hap Ⅲ
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) withHap III of 6-SFT-A2 is significant higher than that of Hap I underdifferent water regimes in five years.
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±5.5Hap III of 6-SFT-A2 is associated with higher thousand grainweight in the historical population consisted of 154 accessions.
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
Phylogenetic tree representing thehaplotype relationship of 6-SFT-D Hap Ⅰ Hap Ⅱ Hap Ⅲ
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
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 favourablehyplotypes for increasing grain weight, their combinationis optimum for improving grain weight in wheat.
Relationship between TGW and water soluble carbohydrate in stemEarly grain filling stage Middle grain filling stage CK Cut spike 0.3% KI (200 mL/m2) KI: potassium iodide
Analysis of thousand grain weight (TGW) Reduction (CK – KI) Env. Treatment Range (g) Mean±SD Max (g) Min (g) Mean±SDWell-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.
QTLs QTLs for stem WSC in DH population for WSC Additive Epistatic Total58 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 36.80QTLs for TGW Second section 17 40.97 10 8.60 49.5720 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 population22 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 11.01WMC41; 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 16.11Xgwm67, Xgwm213, Xgwm499, WMC380; 6A: CWM487; 7A: A3446-280, A2454-280) 7 5 22.69 5 6.48 29.17
6-SFT-A2 mapping 4A 4A 4A H10 L14 TGW TGW epistatic QTL, stage 5Linkage map of 6-SFT-A2 on 4A Su et al., 2009 Yang et al., 2007 (Hanxuan 10×Lumai 14) Plant Science Genetics
SummaryA number of gene/QTLs involved in thedrought tolerance.Favourable alleles of target genes hide inthe germplasm resources.Recombining favourable alleles of targetgenes could improve crop plants.Molecular marker assistant selection is anefficient approach for drought toleranceimprovement in crop plants.
Acknowledgements Collaborators Yuchen DONG Jizeng JIA Xueyong ZHANG Xiuying KONG Chenyang HAOFinancial SupportNational High Tech ProgramNational Key Program for Basic Research