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Development of Agricultural Science              Discovering Favourable Gene Resources                       for Crop Impr...
OutlineWhy ?Where ?How ?  Phenotyping  Genotyping  Utilization
Feeding the 9 billion people expected to inhabit ourplanet by 2050 will be an unprecedented challenge                     ...
“Take one world already beingexhausted by 6 billion people. Findthe ingredients to feed another 2billion people. Add deman...
Area-Total yield-Yield per unit of cereal crops         in China during 1952-2008                                         ...
Abiotic & biotic stresses on crop plants Drought, Waterlogging/Submergence Heat, Cold Mineral deficiency/Mineral toxici...
To keep pace with food consumerdemand, muti-favourable genesshould be pyramided in crop cultivars.To discover favourable g...
 The “green revolution  gene” is an allele of gene  which control plant height. 0nly a base pair difference  between wil...
Nature allele variation is widely                            present in plant germplasm.                            Differ...
Hybrid rice successful utilization inChina due to a cytoplasmic male sterility      gene discovered in wild rice      Wild...
Soybean is originated from China. Wild     soybean is high diversity.
Wild rice                       野生稻聚群      Salsa(Suaeda heteroptera) 碱蓬(又名盐蒿)
Hexaploid Agropyron                      六倍体冰草                       (Xinjiang)Psathyrostachys 陕西华山新麦草    (Shaanxi)
Investigation andcollection of plant    germplasm
National Long-term GenebankFounded in 1986   -18℃, RH≤50%±7%                  712 Species; 397,000 accessions
National Medium-term Genebank                          0-40C
Distribution of 32 National Germplasm Nursries                                                                            ...
Geographic distribution of crop germplasm in China                              WheatRice                          Soybean
National Germplasm Nursry: Grape, Jujube           10 species, 636 accessions
National Germplasm Nursry: perennial grasses            147 species, 432 accessions  国家内蒙多年生牧草圃:147个种、432份
CIMMYT
Germplasm Resources??                            Gene Resources    ?        ?    ?        ?How can we discoverbeneficial g...
Rice genome sequencing and    functional genomics植物功能基因组学研究进展迅速  完成了水稻基因组测序为分离和发掘新基因奠定了基础                             2002
Forward- vs. reverse-genetics approaches                    QTL mapping,            Association mapping, Positional       ...
Phenotyping--- base for discovering genes   (Case: drought tolerance)
An unexpectedly abruptdecline in the supply ofwater for China’s farmersposes a rising threat toworld food security.       ...
China: PrecipitationAbout 50% of land area is arid and semi-arid in China, where6 667 000 ha of rainfed wheat are grown wi...
Total drought area                                                           10.5 Mha                                     ...
Qingtu Lake in Min Qin, from lake to desert in 40 years                            Reeds and remaining shells
Abandoned village
The Aral Seain CentralAsia, once the4th largestsaline water,has shrunkby 75% insurface areasince 1960s
The Chad Lake in central africa, once the 6th largest lake in the world, 90% reduction in size from 1972 to 2006
The Lake Faguibine in Mali, change from 1974 to 2006
Drought seriously limits crop production in                many areas of the world, especially in China.                Mo...
Water shortage in agriculture                ‘Blue Revolution –                more crop for every                drop’   ...
1. Understanding the molecularmechanisms of water stress responses Difference in dehydration tolerance and droughttoleranc...
 How can plant maintain turgidity with  declining soil water availability? The  molecular details about how the metabolic...
2. Breeding cultivars to cope with         specific objectivesDrought breeding should be localized withspecific objectives...
2. Breeding cultivars to cope with       specific objectivesMolecular breeding is more efficient butthe available magic dr...
Whole-plant responses to drought stressLeft: long-term or acclimation responses; right: short-term responses              ...
Sensing, signalling andcell-level responses to     drought stress ABA-mediated responses Non-ABA-mediated responses Oth...
Dissecting yield into bite size “physiological markers”                       Traits                                      ...
Early generation selection methodologies Visual selection ++     Leaf porometry Canopy temperature    Spectral reflectance
Factors affecting Canopy Temperature          Depression (CTD) in plants                                                  ...
5500Grain yield (kg/ha)                      5000                      4500                      4000                     ...
Selection for canopy temperature: to enrich    favourable alleles before yield testing
Use of CTD in early generation selection- F4 bulks under drought stress (R. Trethowan)- Following visual selection, CTD sc...
Complementing breeder selection withcanopy temperature(Van Ginkel et al., 2008)                    14                     ...
Sampling soil core  To sample roots  To measure soil moisture profiles                       CIMMYT
Models to quantify yield under abiotic stress  Drought yield =    Water Uptake x WUE x HI (partitioning)    (Passioura, 19...
Generic model of stress adaptation under drought & heatPhoto-protection                 Water use efficiency (WUE) &      ...
Physiological breeding: strategic crossing for drought             YLD = WU x WUE x HI (Passioura, 1979)   Photo-Protectio...
2001
PlantphenotypingmethodologyDrought phenotypingin crops: from theoryto practicewww.generationcp.org/drought_phenotyping2011
Genotyping--- to discover gene resources   (Case: drought tolerance)
OutlineLinkage mappingAssociation mappingFunctional marker mappingPerspective of MAS
Evaluation of drought                               Drought tolerant genotypestolerance at seedling stage   survived in th...
Drought toleranceevaluation in the field         2009                              Henan                     Shanxi   Henan
Linkage mapping               Association mappingHanxuan 10 × Lumai 14                                   Historical winter...
Parents Hanxuan 10 Drought tolerant cultivar grown under rainfed condition in semi-arid region Lumai 14 High yield potenti...
Water Regime Treatments for PhenotypingTreatment: Rainfed/Drought stressControl: Well wateredTraits for QTL MappingAgronom...
QTLs for accumulation and remobilization of    stem water-soluble carbohydrates
Yang et al., Genetics. 2007, 176: 571-584
Integrated mapping of QTLs controlling      drought tolerance in wheat        S le : 7 re p e a ta tio n                  ...
QTL validation in different populations   DH (Hanxuan 10×Lumai 14)   RIL (Opata85×W7984)
E1: 2001 Fenyang, Shanxi                                                          E2: 2005 Haidian, Beijing               ...
QTL mappingMixed linear model was used to divide geneticeffects into additive main effects (a), epistatic maineffects (aa)...
QTLs for plant height during ontogeny in DHLs                                 Additive QTLs                Epistatic QTL p...
Uneven distributions of PH QTLs on chromosomes            Unconditional plant height            Conditional plant height  ...
Common QTLs for plant height development between          unconditional and conditional analysis in DHLsStage      QTLs   ...
0.5                                                                        Component contribution                         ...
Rht1       (Cadalen, 1998;                                                  QTLs        Epi.       Huang, 2006;       Sour...
Rht8 (Korzun, 1998)                          Rht5                          (Ellis,                         2005)          ...
(Cadalen et al. 1998)                             (Ellis, 2005)PH QTL clusters on other chromosomes
(McCartney ,2005;                                                 Cadalen, 1998;                                          ...
Association analysis of candidate PH QTLs270 historical winter wheat accessions60 candidate SSR markers in six chromosome ...
Associations of plant height developmental     behavior and the candidate markersTotal of 46 marker-trait associations wer...
2D              PIC            2D   0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0     23.2                                      ...
4D            PIC            4D   0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0      28                                         ...
Phenotypic effects of marker alleles                                                           Xcfd23-4D                  ...
Allele effect on plant height of associated                 locus in drought environment                                  ...
Validation of Xwmc349 allelic effect        in ILs with Jinmai 47 background                                              ...
Summary Plant height is a typically quantitative trait  controlled by additive effects and epistatic  effects. A list of...
Flow chart of suppression subtractive        hybridization (SSH)   Wheat seedling           Data analysis(-0.5MPa PEG-6000...
Assessment of contigs from the cDNA libraries      responding to water stress by suppression           subtractive hybridi...
Classification of genes that respond to water stress              1h, 6h, 12h, 24h and 48h                                ...
Putative key classifications of differential expressed genes from 648 Uni-genes  Protein                  Protein kinase  ...
Case 1: TaPP2Ac (protein phosphatase 2Ac)                             TaPP2Ac                             identified from ...
PP2A structureStructural subunit A; Variable subunit B (B’, B’’, B’’’);Catalytic subunit C
PP2Ac Function CTR 1, a negative regulator of the ethylene response pathway inArabidopsis, encodes a member of the Raf fam...
Expression of TaPP2Ac-1
Overexpression of transgenic TaPP2Ac-1 tobacco under water stress condition     Before water stress           Water stress...
Transgenic TaPP2Ac-1 tobacco plants enhance    drought tolerance under water deficit          Time of drought stress (d)  ...
DT of transgenic TaPP2Ac-1 Arabidopsis                               Salt tolerance                                 WT: wi...
Chromosome location of TaPP2Ac-1 by the wild relative species and nulli-tetrasomics lines of Chinese Spring   (A) TaPP2Ac-...
4D                                                                          M: DNA marker                                 ...
Case 2: TaABC1L mapping in RILs   Genetic mapping of TaABC1L gene based     on CAPS marker and AS-PCR marker              ...
Case 3: TaSnRK2.7 Cloning, location and functionalanalysis of a gene involved in abiotic-stressed responses      Minimal A...
Southern blotting                                         One copy of TaSnRK2.7 might exist in each of                    ...
TaSnRK2.7 was expressed strongly in seedling                              roots, weakly in booting spindles, and marginall...
Subcellular localization                            TaSnRK2.7-GFP was present in the                            cell membr...
Drought tolerant                                         Drought sensitivePhylogenetic tree representing TaSnRK2.7 haploty...
Case 4: Ta6-SFT Cloning, location and functional analysis of a gene involved in fructan synthesis                         ...
Specific primer design based on thepolymorphism in the sequencing of gene 6-SFT                        10            20   ...
Single nucleotide mutation in 6-SFT-A1   No.    Site   Location    Type       Change      Amino acid change    1     116  ...
6-SFT-A1 mapping                              1781 bp G/A                                   3269 bp                       ...
Phylogenetic tree representing the haplotyperelationship of 6-SFT-A1                                                      ...
The high correlation between seedling biomass under   drought stress and the molecular marker wasidentified, which was des...
Agronomic traits associated with 6-SFT-A1 in   a historical population with 154 accessionsEnvironment        Trait        ...
Single nucleotide polymorphism in 6-SFT-A2No.   Site   Location   Type    Change   Hapl I   Hapl II   Hapl III1     600   ...
Molecular marker design for 6-SFT-A2                                                              4A              1870bp G...
Phylogenetic tree representing thehaplotype relationship of 6-SFT-A2                                     HaplⅡ            ...
Thousand grain weights of DHLs with            two 6-SFT-A2 haplotypes           50                                       ...
TGW of three haplotypes of 6-SFT-A2 in           a historic population   Year    Haplotype      TGW (g)      P-Value    R2...
Single nucleotide polymorphism in 6-SFT-D                C          A                         G            C              ...
Phylogenetic tree representing thehaplotype relationship of 6-SFT-D                               HaplⅠ                   ...
HaplⅠ of 6-SFT-D is a favourable haplotype    for TGW in a historical population                  50                  45  ...
2008H                                                    2008S     50                                                     ...
TGW in genotypes with different haplotype    combinations of 6-SFT-A2 and 6-SFT-D      Haplotype*             2008D       ...
Relationship between TGW and       water soluble carbohydrate in stem                               CK                    ...
Analysis of thousand grain weight (TGW)                                                              Reduction (CK – KI)  ...
WSC QTL for stem WSC in DH population      QTLs58 additive, 34 pairs Additive QTL; contribution rate 36.80%               ...
Lower section, WSC       additive QTL, stage 5       Lower section, WSC       epistatic QTL, stage 3                      ...
6-SFT-A2 mapping                                   4A                 4A               4A  H10 L14                        ...
SummaryA number of QTLs and QTL clusters for drought tolerance have been identified by linkage mapping.A few of function...
In the Future To integrate the QTLs and functional markers  mapped in multi-population To identify beneficial alleles in...
Acknowledgements                                  Collabrators                                  Yuchen DONG               ...
Thank you!
“There’s no single gene that’s going to be thepanacea to our drought problem. We’re tryingto cherry-pick the various mecha...
果聚糖的作用     Water soluble carbohydrate (WSC) in wheat stem is mainly composed offructans, sucrose, glucose and fructose, wi...
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Discovering favourable gene resources for crop improvement

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Discovering favourable gene resources for crop improvement
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  1. 1. Development of Agricultural Science Discovering Favourable 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) Specific Topic for Student Abroad • Oct. 20, 2011
  2. 2. OutlineWhy ?Where ?How ?  Phenotyping  Genotyping  Utilization
  3. 3. Feeding the 9 billion people expected to inhabit ourplanet by 2050 will be an unprecedented challenge Special issue 2010 Special issue 2007 Special issue 2003 Special issue 2008
  4. 4. “Take one world already beingexhausted by 6 billion people. Findthe ingredients to feed another 2billion people. Add demand for morefood, more animal feed and morefuel. Use only the same amount ofwater the planet has had sincecreation. And don’t forget torestore the environment thatsustains us. Stir very carefully.”Margaret Catley-Carlson2008-2009 Chair of World EconomicForum Global Agenda Council onWater Security World Economic Forum, Davos January 2009
  5. 5. Area-Total yield-Yield per unit of cereal crops in China during 1952-2008 Area (10 M ha.) Total yield (100 M ton) Unit yield (ton·ha-1) 1950’ 1960’-1990’ 1998- Zhensheng Li
  6. 6. Abiotic & biotic stresses on crop plants Drought, Waterlogging/Submergence Heat, Cold Mineral deficiency/Mineral toxicity Salinity …… Diseases and Insect pests http://www.plantstress.com
  7. 7. To keep pace with food consumerdemand, muti-favourable genesshould be pyramided in crop cultivars.To discover favourable generesources for improving crop plants.
  8. 8.  The “green revolution gene” is an allele of gene which control plant height. 0nly a base pair difference between wild type and mutant type. Finding and utilization of this one base mutation resulted in a “Green Revolution” Nature, 1999, 400: 256-261
  9. 9. Nature allele variation is widely present in plant germplasm. Difference of fruit weight of tomato between wild type and cultivar type is a few base pair change in the promoter region of gene fw2.2.Science, 2000, 289: 85-88
  10. 10. Hybrid rice successful utilization inChina due to a cytoplasmic male sterility gene discovered in wild rice Wild rice Hybrid rice
  11. 11. Soybean is originated from China. Wild soybean is high diversity.
  12. 12. Wild rice 野生稻聚群 Salsa(Suaeda heteroptera) 碱蓬(又名盐蒿)
  13. 13. Hexaploid Agropyron 六倍体冰草 (Xinjiang)Psathyrostachys 陕西华山新麦草 (Shaanxi)
  14. 14. Investigation andcollection of plant germplasm
  15. 15. National Long-term GenebankFounded in 1986 -18℃, RH≤50%±7% 712 Species; 397,000 accessions
  16. 16. National Medium-term Genebank 0-40C
  17. 17. Distribution of 32 National Germplasm Nursries 29 黑龙江 吉林 25 19 14 蒙 辽宁 24 新 疆 9 北京 23 内 26 Ⅰ 22 甘 Ⅲ 32 山 天津 河 宁 17 北 肃 陕 西 山东 Ⅱ 夏 12 青 海 28 16 西 10 江苏 河 南 13  安 7 西 藏 徽 上海 湖北 5 4 四 川 重庆 18 6 11 浙 江 江 8 湖 南 西 福 21 贵 州 建 台 15 云 南 广 东 湾 27 广 西 20 1  3 图例: 2  资源库 30南海诸岛  资源圃 海南  31
  18. 18. Geographic distribution of crop germplasm in China WheatRice Soybean
  19. 19. National Germplasm Nursry: Grape, Jujube 10 species, 636 accessions
  20. 20. National Germplasm Nursry: perennial grasses 147 species, 432 accessions 国家内蒙多年生牧草圃:147个种、432份
  21. 21. CIMMYT
  22. 22. 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?
  23. 23. Rice genome sequencing and functional genomics植物功能基因组学研究进展迅速 完成了水稻基因组测序为分离和发掘新基因奠定了基础 2002
  24. 24. Forward- vs. reverse-genetics approaches QTL mapping, Association mapping, Positional cloning, Mutagenesis, etc. Forward genetics Candidate Reverse genetics sequencePhenotype Genotype Genetic engineering, RNAi, TILLING, Insertional mutagenesis, etc.
  25. 25. Phenotyping--- base for discovering genes (Case: drought tolerance)
  26. 26. An unexpectedly abruptdecline in the supply ofwater for China’s farmersposes a rising threat toworld food security. WORLD•WATCH July/August 1998
  27. 27. 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.
  28. 28. Total drought area 10.5 Mha 8.8 Mha Average year: 1.7 Mha drought areaProvinces suffered from drought stress in the early spring 2009
  29. 29. Qingtu Lake in Min Qin, from lake to desert in 40 years Reeds and remaining shells
  30. 30. Abandoned village
  31. 31. The Aral Seain CentralAsia, once the4th largestsaline water,has shrunkby 75% insurface areasince 1960s
  32. 32. The Chad Lake in central africa, once the 6th largest lake in the world, 90% reduction in size from 1972 to 2006
  33. 33. The Lake Faguibine in Mali, change from 1974 to 2006
  34. 34. 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.
  35. 35. Water shortage in agriculture ‘Blue Revolution – more crop for every drop’ Norman E. Borlaug Nobel Peace Prize Laureate 1970
  36. 36. 1. Understanding the molecularmechanisms of water stress responses Difference in dehydration tolerance and droughttolerance• The former is the capability to maintain functions and minimize damages under dehydration . In reality, crop plants cannot survive for long under prolonged dehydration. What we see are a short-term stress responses.• The later is the ability to grow and yield under less soil moisture. This should be the trait of crops in drought-prone areas.
  37. 37.  How can plant maintain turgidity with declining soil water availability? The molecular details about how the metabolic genes are regulated in responses? How can plant maintain their membrane integrity under oxidative stress which is a secondary stress derived from water stress? How are the other physiological functions maintained or regulated as an integrative response to water stress?
  38. 38. 2. Breeding cultivars to cope with specific objectivesDrought breeding should be localized withspecific objectives to specific areas, suchas less irrigation, rainfed in semi-arid.Conventional breeding is time consuming and laborcostly since it is a natural selection under droughtcondition. However, large scale gene recombinationcan be easily achieved.
  39. 39. 2. Breeding cultivars to cope with specific objectivesMolecular breeding is more efficient butthe available magic drought-resistantgenes are very limited. Genes for root traits should be tapped. Drought tolerance is fundamentally related to the capability to maintain water balance, much less to the ability to tolerate dehydration.
  40. 40. Whole-plant responses to drought stressLeft: long-term or acclimation responses; right: short-term responses (Chaves, et al., 2003)
  41. 41. Sensing, signalling andcell-level responses to drought stress ABA-mediated responses Non-ABA-mediated responses Other mechanisms (Chaves, et al., 2003)
  42. 42. Dissecting yield into bite size “physiological markers” Traits G-C G-C T-A T-A G-C Y G-C T-A G-C T-A G-C T-A T-A G-C I • Ground cover G-C T-A G-C T-A G-C T-A E • Plant height G-C T-A G-C T-A G-C T-A T-A G-C • Root depth G-C T-A L • Transpiration efficiency T-A G-C T-A G-C T-A G-C G-C T-A D • Stem carbohydrates G-C T-A G-C T-A G-C T-A T-A G-C • Spike photosynthesis G-C T-A G-C T-A G-C T-A • …… T-A G-C P G-C T-A G-C T-A G-C T-A G-C T-A O T-A G-C T-A G-C T-A G-C G-C T-A T T-A G-C T-A G-C T-A G-C G-C G-C T-A E • Interception of radiation T-A G-C T-A T-A G-C T-A G-C T-A G-C G-C N • Canopy cooling T-A G-C T-A G-C G-C T-A T-A T • Membrane thermostability T-A G-C T-A G-C T-A G-C G-C T-A G-C • Photoprotection G-C T-A T-A G-C T-A I • …… G-C T-A G-C T-A G-C T-A T-A G-C A G-C T-A G-C T-A G-C T-A T-A L G-C T-A Environment G-C Genes T-A G-C T-A M Reynolds, 2010
  43. 43. Early generation selection methodologies Visual selection ++ Leaf porometry Canopy temperature Spectral reflectance
  44. 44. Factors affecting Canopy Temperature Depression (CTD) in plants Radiation Biological Environmental Clouds Partitioning o (T C) H2O CTD Evaporation Metabolism WindVascularTransport H2O (soil water availability) (M Reynolds, 2001)
  45. 45. 5500Grain yield (kg/ha) 5000 4500 4000 3500 3000 2500 2000 1500 5.0 6.0 7.0 8.0 9.0 Canopy temperature depression (oC) The relationship of grain yield to CTD, mean of 2sowings dates, Tlaltizapán, 1992-93, 23 genotypes. (Amani, Fischer and Reynolds, 1996)
  46. 46. Selection for canopy temperature: to enrich favourable alleles before yield testing
  47. 47. Use of CTD in early generation selection- F4 bulks under drought stress (R. Trethowan)- Following visual selection, CTD scores used to influence gene frequency 29 28.5 C a n o p y te m p p o s t 28 27.5 flo w e rin g 27 26.5 26 25.5 25 24.5 20 21 22 23 24 25 26 Canopy temperature vegetative
  48. 48. Complementing breeder selection withcanopy temperature(Van Ginkel et al., 2008) 14 BREEDER 12 BREEDER+CTDIndividual number 10 8 6 4 2 0 6.3 6.8 7.3 7.8 8.3 Yield (t/ha)
  49. 49. Sampling soil core To sample roots To measure soil moisture profiles CIMMYT
  50. 50. Models to quantify yield under abiotic stress Drought yield = Water Uptake x WUE x HI (partitioning) (Passioura, 1977) Irrigated yield = Light Interception x RUE x HI (partitioning) WUE: water use efficiency RUE: radiation use efficiency HI: harvest index
  51. 51. Generic model of stress adaptation under drought & heatPhoto-protection Water use efficiency (WUE) & Radiation use efficiency (RUE) Pigments for dissipation of excess light energy, e.g. •Transpiration efficiency (drought only) carotenoids measured • CID using spectral reflectance •Heat tolerant metabolism (growth rate) (RARSc) • Stay green (CHL) • CO2 fixation rate (COND)Early growth (pre-grainfill) Access to water by roots (indicated by cooler canopies)• Ground cover: measured with spectral indices (NDVI & WI) • Under drought estimates water use• Growth rate (BMA) • Under hot, irrigated conditions:• Stem carbohydrates (CHO) estimates CO2 fixation and thus radiation use efficiency
  52. 52. Physiological breeding: strategic crossing for drought YLD = WU x WUE x HI (Passioura, 1979) Photo-Protection Transpiration Efficiency Leaf morphology WUE of leaf photosynthesis • wax/pubescence • posture/rolling low 12C/13C discrimination Pigments Spike/awn photosynthesis • chl a:b • carotenoids Antioxidants Partitioning (HI) Water Uptake Partitioning to stem Rapid ground cover carbohydrates • protects soil moisture Access to water by roots Harvest index • Ψ leaf • Rht alleles • cool canopy • (osmotic adjustment)(Reynolds & Tuberosa, 2008. COPB)
  53. 53. 2001
  54. 54. PlantphenotypingmethodologyDrought phenotypingin crops: from theoryto practicewww.generationcp.org/drought_phenotyping2011
  55. 55. Genotyping--- to discover gene resources (Case: drought tolerance)
  56. 56. OutlineLinkage mappingAssociation mappingFunctional marker mappingPerspective of MAS
  57. 57. Evaluation of drought Drought tolerant genotypestolerance at seedling stage survived in the soil moisture of ~17% relative water content
  58. 58. Drought toleranceevaluation in the field 2009 Henan Shanxi Henan
  59. 59. Linkage mapping Association mappingHanxuan 10 × Lumai 14 Historical winter DHLs wheat collection RILs DT QTLs DT QTLs Introgression lines (BC3F3-4) Donor 1 Jinmai 47 × Donor 2 . . . Elite alleles
  60. 60. Parents Hanxuan 10 Drought tolerant cultivar grown under rainfed condition in semi-arid region Lumai 14 High yield potential cultivar grown under irrigated condition Hanxuan 10 DH Lines Lumai 14 (Hanxuan 10 × Lumai 14)
  61. 61. Water Regime Treatments for PhenotypingTreatment: Rainfed/Drought stressControl: Well wateredTraits for QTL MappingAgronomic traits (coleoptile length, early vigor, heading date,flowering date, plant height, spike number per plant, kernel perspike, spike length, seed setting, thousand-grain weight, plantmorphology and grain yield)Physiological traits (stay-green, chlorophyll fluorescence, leafwater status, canopy temperature, accumulation and remobilizationof stem water-soluble carbohydrates)Anatomical structure (number and area of vascular bundles)
  62. 62. QTLs for accumulation and remobilization of stem water-soluble carbohydrates
  63. 63. Yang et al., Genetics. 2007, 176: 571-584
  64. 64. Integrated mapping of QTLs controlling drought tolerance in wheat S le : 7 re p e a ta tio n F v /F o - W W (1 2 .0 9 % ) (1 7 .4 3 % ~ 2 2 .4 4 % ) R A L V B -D S (1 3 .1 6 % ) H e i (8 .4 9 % ~ 3 1 .0 4 % ) K w e (3 3 .3 9 % ) S le (9 .1 5 % ~ 1 8 .7 3 % ) K g n (1 5 .5 5 % ~ 2 9 .0 6 % ) K w e i (1 4 .0 6 % ) T s p ( 1 0 .0 7 % ~ 1 2 .2 5 ) P y i ( 8 .5 7 % ) H e i ( 9 .3 9 % ~ 2 1 .1 3 % ) P y i (1 0 .6 2 % ~ 1 9 .2 3 % ) S le ( 1 3 .6 2 % ) S s p ( 8 .8 6 % ) N U P - H N 2 (1 4 .0 % ) N U P - L N 1 (6 .0 % ) R P A T V B ( 1 3 .2 3 % ) R D W - H ( 1 1 .0 % ) S le ( 7 .8 % ~ 2 1 .9 7 % ) S p i ( 6 .5 9 ~ 1 0 .3 7 % ) C h lC (1 1 .6 8 % ) K g n ( 2 2 .6 2 % ) N S V B ( 2 1 .3 8 % ) S s p (6 .4 3 % ~ 1 4 .3 8 % ) N T V B ( 2 0 .3 6 % ) S p i (9 .3 7 % ) T s p (8 .0 9 % ~ 3 4 .9 3 % ) S s p (1 0 .8 9 % ~ 3 0 .9 7 % ) N L V B -D S H e i (9 .3 2 % ~ 2 1 .7 9 ) (1 6 .0 5 % ) F m -D S ( 2 6 .5 8 % ) T s p ( 1 5 .7 1 % ~ 2 4 .5 3 % ) F v -D S (2 2 .9 9 % ) H e i ( 2 4 .5 3 % ~ 4 3 .4 5 % ) R F W (1 0 .3 7 % ) N U P -H (4 .3 % )
  65. 65. QTL validation in different populations DH (Hanxuan 10×Lumai 14) RIL (Opata85×W7984)
  66. 66. E1: 2001 Fenyang, Shanxi E2: 2005 Haidian, Beijing E3: 2005 Changping, Beijing DS E4: 2006 Haidian, Beijing E5: 2006 Changping, Beijing Plant height E6: 2001 Fenyang, Shanxi phenotyping E7: 2005 Haidian, Beijing WW E8: 2005 Changping, Beijing E9: 2006 Haidian, Beijing E10: 2006 Changping, Beijing Condition PH ---net increase effect in the given period S1|S0 S2|S1 S3|S2 S4|S3 S5|S4S0 S1 S2 S3 S4 S5 Unconditional PH--- accumulated effect
  67. 67. QTL mappingMixed linear model was used to divide geneticeffects into additive main effects (a), epistatic maineffects (aa) and their environment interactioneffects (QE, including ae and aae). Cao et al, 2001
  68. 68. QTLs for plant height during ontogeny in DHLs Additive QTLs Epistatic QTL pairs Traits Stages a b c d Number A AE Number AA AAEUnconditional S1 12 12 6 7 7 2plant height S2 11 11 7 18 18 4 S3 12 12 7 19 19 3 S4 10 10 4 22 22 1 S5 10 10 5 20 20 2 Total 55 55 29 86 86 12Conditional S1|S0 12 12 6 7 7 2plant height S2|S1 3 3 3 4 4 3 S3|S2 4 4 3 4 2 3 S4|S3 1 1 1 3 1 2 S5|S4 6 5 5 5 3 3 Total 26 26 18 23 17 13 a QTL number with additive main effects; b QTL number with additive environment interaction effects; c QTL pair number with additive epistatic effects; d QTL pair number with epistatic environment interaction effects.
  69. 69. Uneven distributions of PH QTLs on chromosomes Unconditional plant height Conditional plant height Chrom. Homeologous Genome Chrom. Homeologous Genome group group 1A 2 36 1 19 1B 18 51 7 23 1D 2 22 22 1 9 11 2A 8 3 2B 4 1 2D 10 22 6 10 3A 3 1 3B 12 4 3D 3 18 5 4A 5 1 4B 2 2 4D 2 9 2 5 5A 7 3 5B 5 4 5D 1 13 7 6A 6 4 6B 6 4 6D 12 8 7A 5 6 7B 4 1 7D 4 13 2 9Total 109 53
  70. 70. Common QTLs for plant height development between unconditional and conditional analysis in DHLsStage QTLs Marker interval A AE1 AE2 AE3 AE4 AE5 AE6 AE7 AE8 AE9 AE10 *** ** *S1|S0 QPh.cgb-2D.1 WMC453.1-WMC18 1.85 -0.83 0.35 0.77 *** * * *S3|S2 QPh.cgb-2D.1 WMC453.1-WMC18 -0.50 -0.86 0.48 -0.74 0.52 0.39 -0.36 0.74 0.51 -0.54 *** ** * S1 QPh.cgb-2D.1 WMC453.1-WMC18 1.85 -0.83 0.35 0.77 *** *** *** *** S2 QPh.cgb-2D.1 WMC453.1-WMC18 3.89 -2.44 1.87 -1.06 2.13 *** *** S3 QPh.cgb-2D.1 WMC453.1-WMC18 3.02 -1.92 1.03 -0.64 *** *** S4 QPh.cgb-2D.1 WMC453.1-WMC18 4.70 -0.86 -2.96 0.75 0.83 -0.89 1.28 0.85 *** S5 QPh.cgb-2D.1 WMC453.1-WMC18 2.84 -0.86 0.49
  71. 71. 0.5 Component contribution 0.5Component contribution 0.4 0.4 0.3 0.3 0.2 0.2 0.1 0.1 0 0 S0 S1 S2 S3 S4 S1| S2| S3 | S4| S5| S1 S3 S2 S4 S5 h^2(A) h^2(AE) h^2(AA) h^2(AAE) Contributions of different genetic effects to plant height during ontogeny in DHLs
  72. 72. Rht1 (Cadalen, 1998; QTLs Epi. Huang, 2006; Sourdille, 2003; Liu, 2006; a e ae aa e aae McCartney, 2005) S1 S2 Rht12 (Ellis, 2005) Un. S3 S4 Rht9 (Schnurbusch, 2003; S5 Ellis, 2005) S1 S2 Con. S3 Rht2 (Cadalen,1998; S4 Huang, 2003, 2006; S5 McCartney, 2005; Sourdille, 2003)PH QTL clusters matched up to Rht genes in DHLs
  73. 73. Rht8 (Korzun, 1998) Rht5 (Ellis, 2005) Rht13 (Ellis 2005)PH QTL clusters near Rht genes in DHLs
  74. 74. (Cadalen et al. 1998) (Ellis, 2005)PH QTL clusters on other chromosomes
  75. 75. (McCartney ,2005; Cadalen, 1998; Sourdille, 2003; Quarrie, ,2006) (Schnurbusch, 2003)PH QTL clusters on other chromosomes Wu et al., JXB, 2010, 61: 2923-2937
  76. 76. Association analysis of candidate PH QTLs270 historical winter wheat accessions60 candidate SSR markers in six chromosome regions Rht8 Rht1 Rht12 Rht9 Rht2 Rht13 Ave. Dis. = 4.7 cM Wheat, Consensus SSR, 2004
  77. 77. Associations of plant height developmental behavior and the candidate markersTotal of 46 marker-trait associations were detected, amongthem 13 associations were highly significant (P<0.001).Five loci were also worked in DHLs. 6.5 6.0 5.5 -Log (P)>3 -Lg(P value) 5.0 4.5 4.0 3.5 3.0 2.5 2.0 m 2 2 1 1 m 2 1 3 4 3 3 2 H H H H TC PH PH PH PH PH H PH H -P -P -P -P -P -P -D 5- 9- 9- 2- 5- 5- 09 09 09 09 43 49 49 24 24 30 49 49 43 c1 c1 c1 c1 fd c3 m m m m m fd m ar ar Xc ar ar m w w w w w Xc w Xb Xb Xb Xb Xw Xg Xg Xg Xg Xg Xg Associations -Lg (P) value at different associations
  78. 78. 2D PIC 2D 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 23.2 Xgwm261 PH PH4 PH4 2 32.1 Xgwm455 Rht8 (Korzun, 1998) 37.1 Xwmc470 40.7 Xgwm484 PH3 43.1 Xcfd43 PH2 PH3 PH4 DTC2 DTC3 46.7 Xbarc168 PH1 48.2 Xgwm102cM 63.6 Xgwm249 PH3 PH4 63.7 Xwmc18 65.2 Xcfd17 65.8 Xcfd116 66.7 Xcfd84 67.1 Xwmc144 68.8 Xcfd160 73.1 Xgwm157 81 Xbarc228 DTC4 82.8 Xwmc41 DTC 4 90.9 Xgwm539 Ht= 0.5806
  79. 79. 4D PIC 4D 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 28 Xwmc112 PH4 DTCm 32.9 Xcfd23 PHm PHm Rht2 33.1 Xgwm133 PHm DTCm 34.5 Xgwm192 PHm PH1 PH3 Cadalen,1998; Huang, 2003, 2006; McCartney, 2005; Sourdille, 2003 42.9 Xwmc331cM 54 Xwmc399 PHm PH1 PH2 PH3 PH4 Plant height under well-watered PHm PH1 PH2 PH3 PH4 Plant height under drought-stress DTCm DTC1 DTC2 DTC3 DTC4 Drought tolerance coefficient 78.8 Xcfd233 82 Xgwm194 PH1 Ht= 0.6489 Zhang et al., Planta, 2011 (DOI 10.1007/s00425-011-1434-8)
  80. 80. Phenotypic effects of marker alleles Xcfd23-4D Xgwm495-4B A217 A220 A223 A155 A159 A163 A165 A167 A179 A181 10P la n t h e ig h t a t m a tu r e ( c m ) 5 0 -5 -10 -15 -20 PH under drought-stress PH under well-watered
  81. 81. Allele effect on plant height of associated locus in drought environment Average effect Percentage of effect (%) Locus Allele Effect Typical accession Positive effect Negative effect Positive effect Negative effectXbarc168 160 + 37.5 中苏68 Zhongsu68 160:174 + 17.8 科遗29 Keyi 29 160:172 + 17.6 庆丰1号 Qingfeng 1 162:174 + 29.2 华北187 Huabei 187 +26.0 0 +34.5 0Xgwm285 220 - 17.8 西安8号 Xian 8 222 - 13.9 中麦9号 Zhongmai 9 228 - 17.6 鲁麦14 Lumai 14 238 - 3.3 鲁麦1号 Lumai 1 254 - 11.5 衡95观26 Heng 95 Guan 26 256 - 5.0 衡5229 Heng 5229 0 -11.5 0 -11.0Xgwm126 193 + 20.1 冀麦32 Jimai 32 195 + 32.8 燕大1817 Yanda 817 199 + 12.8 科遗29 Keyi 29 +21.9 0 +29.6 0Xgwm95 108 - 5.2 太原566 Taiyuan 566 118 - 24.7 陕229 Shan 229 120 - 24.1 豫麦13 Yumai 13 122 - 31.7 西安8号 Xian 8 0 - 21.4 0 - 18.3Xgwm212 99 - 3.2 矮孟牛Ⅳ型(8057) Aimengniu 103 + 9.9 丹麦1号 Danmai 1 + 9.9 - 3.2 + 10.4 - 3.3Xwmc396 151 + 14.6 晋麦16 Jinmai 16 153 + 3.2 晋麦44 Jinmai44 157 + 0.4 冀麦32 Jimai 32 510 -2.1 鲁215953 Lu 215953 + 6.0 - 2.1 + 6.7 - 2.4 Wei et al., Acta Agron Sin, 2010, 36:895-904
  82. 82. Validation of Xwmc349 allelic effect in ILs with Jinmai 47 background Average varianceAllele Effect on PH IL No. Range of PH to receptorA99 -12.4 24 -19 ~ +14 0A103 -11.1 6 -9 ~ +6 -2Xwmc349 allele: A99(-12.4), A101, A103(-11.1), A105(+6.0)
  83. 83. Summary Plant height is a typically quantitative trait controlled by additive effects and epistatic effects. A list of marker-PH association was identified in the chromosome regions of PH QTLs or Rht genes detected in DHLs. Allele effects have to be validated in multi- genetic backgrounds.
  84. 84. Flow chart of suppression subtractive hybridization (SSH) Wheat seedling Data analysis(-0.5MPa PEG-6000) Test sequencesmRNA preparation SSH 1, 6, 12, 24, 48h cDNA synthesis cDNA libraries Rsa I digestion Transformation Adaptor ligation Vector ligationFirst hybridization Second PCR amplificationSecond hybridization First PCR amplification
  85. 85. Assessment of contigs from the cDNA libraries responding to water stress by suppression subtractive hybridization (SSH) Known Unknown cDNA Valid Total Uni- functional contigs functionallibrary ESTs contigs contigs contigs Number %SSH 1h 1697 938 146 114 792 84.43SSH 6h 1824 566 265 203 301 53.18SSH 12h 1833 516 166 133 350 67.83SSH 24h 1131 786 441 202 345 43.89SSH 48h 1148 635 414 234 221 34.80Total 6733 3441 1432 886 2009 58.38Wheat seedlings were treated with -0.5MPa PEG-6000 for 1, 6, 12, 24, 48h, respectively.
  86. 86. Classification of genes that respond to water stress 1h, 6h, 12h, 24h and 48h G1: Alcohol dehydrogenase G2: Aldehyde dehydrogenase SSH 1h G3: Ca2+-binding proteins G4: Calmondulin binding proteins 1 / 19 G5: Carbohydrate metabolism-related proteins G6: Cellular structure and organization-related proteins G7: Cytochrome p450 1 1 G8: Detoxification enzymes G9: Fatty acid metabolism-related proteins G10: Ferritin SSH 48h SSH 6h G11: Membrane proteins 1/26 G12: Osmoprotectant synthesis-related proteins 0 / 19 G13: Plant defence-related proteins G14: Protease inhibitor 13 / 27 G15: Protection factors of macromolecules G16: Protein kinases 2 2 G17: Protein phosphatases G18: Protein synthesis-related proteins G19: Proteinases G20: Proteins involved in biosynthesis and metabolism of hormones G21: Proteins regulated by various hormones G22: Reproductive development-related proteins SSH 24h 1 SSH 12h G23: Respiration-related proteins 0/17 1/24 G24: RNA-binding proteins G25: Secondary metabolism-related proteins G26: Senescence-related proteins G27: Transcription factorsBlack represents the number of shared/total classification in 5 cDNA libraries;Red represents the number of classification shared by 2 bordering upon libraries;Blue represents the number of special/total classification in the library.
  87. 87. Putative key classifications of differential expressed genes from 648 Uni-genes Protein Protein kinase Ca2+-binding phosphatase 4.01% protein 2.16% 0.62% Transcription Calmondulin factor binding protein 0.46% 6.17% Detoxification enzyme 3.40% Plant defence- Others related protein 65.74% 8.33% Membrane protein 9.11% Pang et al., Acta Agronomica Sinica. 2007, 33:333-336
  88. 88. Case 1: TaPP2Ac (protein phosphatase 2Ac) TaPP2Ac identified from cDNA libraries at 6h and 12h, plays important roles in cellular growth and signalling, ubiquitously expressed in plants.
  89. 89. PP2A structureStructural subunit A; Variable subunit B (B’, B’’, B’’’);Catalytic subunit C
  90. 90. PP2Ac Function CTR 1, a negative regulator of the ethylene response pathway inArabidopsis, encodes a member of the Raf family of protein kinase. (Kieber et al., Cell, 1993, 72: 427-441)
  91. 91. Expression of TaPP2Ac-1
  92. 92. Overexpression of transgenic TaPP2Ac-1 tobacco under water stress condition Before water stress Water stress 12 d WT GFP TaPP2Ac WT GFP TaPP2Ac Water stress 18 d Water stress 24 dWT GFP TaPP2Ac WT GFP TaPP2Ac
  93. 93. Transgenic TaPP2Ac-1 tobacco plants enhance drought tolerance under water deficit Time of drought stress (d) Physiological trait RWC: relative water content; MSI: membrane stability index; WRA: water retention ability; WUE: water use efficiency
  94. 94. DT of transgenic TaPP2Ac-1 Arabidopsis Salt tolerance WT: wild type &: transgenic line P: GFP Xu et al., Annals of Botany. 2007, 99:439-450
  95. 95. Chromosome location of TaPP2Ac-1 by the wild relative species and nulli-tetrasomics lines of Chinese Spring (A) TaPP2Ac-1-1 with PCR specific primer on A genome; (B) TaPP2Ac-1-3 with PCR specific primer on D genome; (C) TaPP2Ac-1-2 with PCR specific primer on S genome; (D) TaPP2Ac-1-2 with PCR-RFLP (TaqI) on S genome. M: DNA marker; H: Hanxuan 10; L: Lumai 14; O: Opata 85; W: W7984; AB: Triticun durum DS107(AABB); A: T. urartu UR203(AA); B: Ae.speltoides 2046(SS); D: T.tauschii Y2009(DD); CS: Chinese Spring; N4AT4B, N4AT4D, N4BT4D and N4DT4B: nulli-tetrasomics lines of CS.
  96. 96. 4D M: DNA marker O: Opata85 W: W7984Schematic illustration of PCR-RFLP product of specific-sequence of D genome between two parents of RIL population EcoR V Hind III Noc I ABD ABD ABD AB AB AB D D D A A A S S S Three copies of TaPP2Ac was identified in hexaploid wheat by Southern Blotting Map of TaPP2Ac-1 on chromosome 4DL
  97. 97. Case 2: TaABC1L mapping in RILs Genetic mapping of TaABC1L gene based on CAPS marker and AS-PCR marker Wang et al., JXB, 2011, 62:1299-1311
  98. 98. Case 3: TaSnRK2.7 Cloning, location and functionalanalysis of a gene involved in abiotic-stressed responses Minimal ABA signaling pathway Structure predictiona. In the absence of ABA, the phosphatase PP2C is free to 10-33: Protein kinases ATP-bindinginhibit autophosphorylation of a family of SnRK2 kinases. region signatureb. ABA enables the PYR/PYL/RCAR family of proteins to bind to 119-131: Serine/Threonine protein kinasesand sequester PP2C. This relieves inhibition on SnRK2, which active-site signaturebecomes auto-activated and can subsequently phosphorylateand activate downstream transcription factors (ABFs) to initiatetranscription at ABA-responsive promoter elements (ABREs).(Sheard and Zheng, 2009. Nature 462, 575-576)
  99. 99. Southern blotting One copy of TaSnRK2.7 might exist in each of the three genomes of common wheat. Chromosome locationPhylogenetic tree of TaSnRK2.7 and of TaSnRK2.7-A copy SnRK2s from other plant species Phosphorus utilization efficiencyTaSnRK2.7 was clustered in subclass I, Accumulation efficiency of stembootstrap values are in percentages. water-soluble carbohydrates Zhang et al., Gene, 2011, 478:28-34
  100. 100. TaSnRK2.7 was expressed strongly in seedling roots, weakly in booting spindles, and marginally in seedling leaves and heading spikes. The expression levels of TaSnRK2.7 increased significantly under salt, PEG and cold stress conditions, but might be not activated by ABA.Expression patterns of TaSnRK2.7 in various tissues (A) and in response to various treatments (B)
  101. 101. Subcellular localization TaSnRK2.7-GFP was present in the cell membrane, cytoplasm and nucleusStress tolerance assays of TaSnRK2.7 over-expressingtransgenic Arabidopsis Zhang et al., JXB, 2011, 62:975-988
  102. 102. Drought tolerant Drought sensitivePhylogenetic tree representing TaSnRK2.7 haplotype relationship among 50 wheat accessions Zhang et al., Gene, 2011, 478:28-34
  103. 103. Case 4: Ta6-SFT Cloning, location and functional analysis of a gene involved in 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)
  104. 104. Specific primer design based on thepolymorphism in the sequencing of gene 6-SFT 10 20 30 40 50 6-SFT-A1 TACCAAACTCTCTTAGAGTTCACGAGCGGCGCTGCGATGGGGTCACACGGCAAGCCACC 6-SFT-A2 TACCAAACTCTCTTAGAGTTCACGAGGGGCGCTGCGATGGGGTCACACGGCAAGCCACC 6-SFT-D1 TACCAAACTCTCTTAGAGTTCACGAGCGGCGCTGCGATGGGGTCACACGGCAAGCCACC 550 560 570 580 590 6-SFT-A1 ACGGGATCTCTCTCT--AGGCATAATCAAAA----TTGCTTAACTCACACCAA 6-SFT-A2 ACGGGATCTCTCTCTCTAGACATAATCAAAAGGGATTGTTTAACTCACACCAA 6-SFT-D1 ACGGGATCTCTCTCT--AGACATAATCAAAA----TTGCTTAACTCGCACCAA 6-SFT-A2 specific primer 3380 3390 3400 3410 3420 3430 6-SFT-A1 TGTCACTGTGAACTACAGTATATTACTTTGTTGGGCGTAGAATCGATATAGTTTGGGTGGGTGG 6-SFT-A2 TGTCATAGTGAACT-----ATATTACTTTGTTGGGCGTAGAATCAATATAGTTTGAGTGGGTGG 6-SFT-D1 TGTCACAGTGAACTA-----TATTACTTTGTTGGGTGTAGGATCGATATAGTTTGGGTGGGTGG 6-SFT-A1 specific primer 6-SFT-D1 specific primer Three copies for 6-SFT were detected in wheat. Two copies were located on genome A, one on genome D.
  105. 105. 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.
  106. 106. 6-SFT-A1 mapping 1781 bp G/A 3269 bp MluⅠdigest Wu et al. M G A G G G G G G G G Y N 2010, 20113000 bp2000 bp1200 bp Segregation 6-SFT-A1 of in RILs Linkage map of 6-SFT-A1 on 4A (Yanzhan 1×Neixiang 188) (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
  107. 107. Phylogenetic tree representing the haplotyperelationship of 6-SFT-A1 Hapl Ⅰ Hapl Ⅱ Hapl ⅢThree haplotypes were identified using the 34 wheat germplasm. Haplotype Iwas mainly detected among wheat accessions showing mid-drought resistanceand drought susceptiple. Haplotype III was found in the most of high-resistantand resistant wheat germplasm.
  108. 108. The high correlation between seedling biomass under drought stress and the molecular marker wasidentified, which was designed based on the specific SNP/InDel in Haplotype III of 6-SFT-A1 CK T Well-watered (CK) Drought stress (T)
  109. 109. 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
  110. 110. Single nucleotide polymorphism in 6-SFT-A2No. Site Location Type Change Hapl I Hapl II Hapl 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
  111. 111. 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 + - + -Hapl Ⅰ + +Hapl Ⅱ + - Linkage map of 6-SFT-A2Hapl Ⅲ - + on chromosome 4A (Hanxuan 10×Lumai 14)
  112. 112. Phylogenetic tree representing thehaplotype relationship of 6-SFT-A2 HaplⅡ HaplⅠ Hapl Ⅲ
  113. 113. 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 Hapl I (Hanxuan 10) Hapl III (Lumai 14)Thousand grain weight (TGW) of doubled haploid lines (DHLs) withHapl III of 6-SFT-A2 is significant higher than that of Hapl I underdifferent water regimes in five years.
  114. 114. TGW of three haplotypes of 6-SFT-A2 in a historic population Year Haplotype TGW (g) P-Value R2 (%) Ⅰ 34.8±4.8 0.0397* 4.79 2008 Ⅱ 33.0±5.6 Ⅲ 35.6±4.9 Ⅰ 38.1±5.3 0.0310* 5.12 2009 Ⅱ 37.0±5.7 Ⅲ 39.7±5.5Hapl III of 6-SFT-A2 is associated with higher thousand grainweight in the historic population consisted of 154 accessions.
  115. 115. 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
  116. 116. Phylogenetic tree representing thehaplotype relationship of 6-SFT-D HaplⅠ HaplⅡ Hapl Ⅲ
  117. 117. HaplⅠ 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
  118. 118. 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 2009S50 46 4445 4240 40 3835 36 3430 3225 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
  119. 119. TGW in genotypes with different haplotype combinations of 6-SFT-A2 and 6-SFT-D Haplotype* 2008D 2008W 2009D 2009W 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.Hapl Ⅲ of 6-SFT-A2 and HaplⅠ of 6-SFT-D are favourablehyplotypes for increasing grain weight, their combinationis optimum for improving grain weight in wheat.
  120. 120. Relationship between TGW and water soluble carbohydrate in stem CK Cut spike 0.3% KI (200 mL/m2)Early grain filling stage Middle grain filling stage
  121. 121. 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% TGWcontrol TGWKI Rain-fed: TGWcontrol × 100% = 79.13% Stem-reserved WSC significantly contributes to TGW. The contribution under the drought stress condition is higher significantly than that under well-watered condition.
  122. 122. WSC QTL for stem WSC in DH population QTLs58 additive, 34 pairs Additive QTL; contribution rate 36.80% epistatic Epistatic Total Trait(peduncle), 49.57% (secondR2(%) Number (lower section) Number section), 49.24% R2(%) (%) Peduncle 21 31.93 9 4.87 36.80TGW QTL Second section 17 40.97 10 8.60 49.57 Lower section20 additive, 17 pairs 20 epistatic37.73 QTL; contribution 11.51 66.36% 15 rate 49.24 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
  123. 123. 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 5QTL for WSC and TGW on chromosome 4A
  124. 124. 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
  125. 125. SummaryA number of QTLs and QTL clusters for drought tolerance have been identified by linkage mapping.A few of functional markers have been developed.Some useful alleles of target genes/QTLs were tested in common wheat collections.Few markers were corresponding in diversity genetic backgrounds.
  126. 126. In the Future To integrate the QTLs and functional markers mapped in multi-population To identify beneficial alleles in germplasm resources by association mapping of candidate genes/QTLs To introgress DT into elite wheat backgrounds by molecular marker assisted recurrent selection
  127. 127. Acknowledgements Collabrators Yuchen DONG Jizeng JIA Xueyong ZHANG Xiuying KONG Chenyang HAOFinancial SupportNational High Tech ProgramNational Key Program for Basic Research
  128. 128. Thank you!
  129. 129. “There’s no single gene that’s going to be thepanacea to our drought problem. We’re tryingto cherry-pick the various mechanisms andrecombine them into one elite cultivar.” --- Dr. Ryan Whitford, a scientist with theACPFG’s Drought Focus Group, 2011
  130. 130. 果聚糖的作用 Water soluble carbohydrate (WSC) in wheat stem is mainly composed offructans, sucrose, glucose and fructose, with fructans being the majorcomponent at the late stage of the WSC accumulation phase. At the stage of maximum WSC content, fructans represented 85% of theWSC in wheat stem internodes.  Fructan’s high water solubility: osmotic adjustment.  Fructan as a source of hexose sugars: allow continued leaf expansion during periods of drought.  Direct protective effects of fructan: membrane stabilization. Bolouri-Moghaddam, et al., 2010, FEBS J., 277, 2022-2037
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