Association between SSR markers and fiber related traits in an exotic germplasm derived from multiple crosses among tetraploid species in Gossypium Linghe Zeng and William R. Meredith Crop Genetics & Production Unit, USDA-ARS, Stoneville, MS

Association Mapping Association - “Polymorphic fragments are identified with significant higher frequencies in one type of phenotype than the contrasting phenotype” (Schafer and Hawkings) Definition – an approach to identify marker-phenotype associations based on linkage disequilibrium in natural populations, cultivars, breeding lines, or breeder’s populations

Association mapping vs. linkage-based QTL mapping Linkage-based Association mapping 1. Identify polymorphic Identify polymorphic markers markers in a given in cultivars, breeding lines, segregating population natural populations, or breeder’s populations 2. Require linkage info not necessary among markers before association analysis 3. Genetic distance LD, population structure 4. Two alleles multiple alleles 5. Contrasting phenotype limited phenotype

Procedures 1. Identify polymorphic markers in cultivars or natural populations 2. Estimate association: mean comparisons of marker class (1, 0) using experiment wise t tests (permutation>1000) 3. Analyze population structure 4. Analyze genetic differentiation (Ht, Fst) in population 5. Analyze association with population structure as a factor

Objectives * Screen polymorphic SSR markers in a germplasm derived from multiple crosses among tetraploid species in Gossypium * analyze population structure and their effects on associations. * Identify and confirm associations between SSR markers and fiber related traits with consideration of population structure

* Initiated by P.A. Miller in 1967 SP Germplasm * Initiated by P.A. Miller in 1967 * Crossing 12 cultivars and strains in G. hirsutum with G. barbadense, G. tomentosum, G. mustelinum, and G. darwinii. * Partial random mating in F2 and subsequent 10 generations at Raleigh, NC * Predominant selfing for 12 generations at Stoneville, MS (2,000 plants) * 260 plants were samples and 20 bolls were collected from each plant in 2004.

Field Evaluation * Experiment design: 260 lines; Randomized complete block with three locations and 1-2 reps at each location * Three environments: Env1=Field Location1, 2005; Env2=Field Location2, 2005; Env3=Field Location1, 2006. * Single row plots (4.57 m long, 1 m row space); 30 bolls collected each plot; saw gin; lint percent and boll wt were measured separately from each plot * Fiber quality was analyzed at Starlab, Knoxville, TN

Effects of genotype and environment (fiber properties) Source Micronaire Elongation Strength Length Length (50%) (2.5%) G 0.801*** 3.09*** 10.9*** 0.0015*** 0.012*** E 49.6*** 3.29** 11.7*** 0.034*** 0.30*** G x E 0.118* 0.570 1.65*** 0.00 0.0011*** Error 0.101 0.520 1.19 0.00 0.0007

An example of SSR by capillary electrophoresis (ABI3730XL DNA Analyzer) BNL285

Population Structure Analysis Software: STRUCTURE v. 2.1, AFLP-SURV 1.0 Admixture model: assume mixed ancestral backgrounds in SP lines Allele frequency model: Allele Frequencies Correlated Burning length: 100,000 Replication: 50,000 K (number of groups): 1 to 10; p(X︱Y) Genetic differentiation: Ht, Fst using AFLP-SURV 1.0 (Vekemans, 2002)

* Phenotype = structure + marker + structure*marker + residue Association Model * Phenotype = structure + marker + structure*marker + residue * ‘Within Group’ – Reduce genetic differentiation (Fst values) among groups by removing sub-structures * Significant ‘Within Group’ – confirm association (independent of population structure) * Significant S x M, non-significant ‘Within Group’ – false association due to substructure * Non-significant S x M, non-significant ‘Within Group’ – false association, attributed to ?

Screening for polymorphic SSR * 84 SSR primer pairs * 296 polymorphic fragments * 3.52 polymorphic fragments per primer pair * Assumption was made for convenience: Each polymorphic fragment is a locus

Results of structure analysis(1) K = 6 based on p(X︱Y) Structure No. lines probability Group1 24 >0.75 Group2 22 >0.75 Group3 16 >0.75 Group4 16 >0.75 Group5 12 >0.75 Group6 50 >0.75 ‘Mixed group’ 120 <0.75

Results of structure analysis(2) Structure lines Ht Fst p values Overall 260 0.300 0.307 <0.0001 Group6 170 0.307 0.054 <0.0001 + ‘Mixed’

Averages of fiber related traits among population structures (1) Structure lines Lint percent Boll wt Micronaire (number) (%) (g/boll) Group 1 24 31.7 5.30 4.18 Group 2 22 32.0 5.26 4.05 Group 3 16 30.9 5.59 4.48 Group 4 16 34.9 5.33 4.26 Group 5 12 35.1 5.05 3.96 Group 6 50 32.1 5.77 4.01 Mixed group 120 32.8 5.32 4.17 Significance p<0.0001 p<0.001 p<0.01

Association between SSR and traits (1) Marker Class Line Lint % Boll wt Micronaire BNL285239 1 43 30.9*** 5.66* 4.13 0 217 33.0 5.36 4.15 Interaction p=0.57 p<0.01 ‘Within group p<0.01 p<0.01 BNL4062244 1 96 33.6*** 5.40 4.24* 0 164 32.1 5.40 4.09 Interaction p<0.001 p=0.61 ‘Within group’ p=0.87 p<0.001 CIR152338 1 95 32.6 5.41 4.27*** 0 154 32.6 5.40 4.06 Interaction p=0.163 ‘Within group’ p=0.318

Number of markers with ‘confirmed’ association or ‘false’ association Significance Significance False assoc. (t tests) (‘Within grp’) (structure) Lint% 6 3 3 Boll wt 14 5 3 MIC 9 6 0 E1 8 3 3 T1 4 1 1 Length(50%) 6 2 3 Length(2.5%) 14 7 4

A list of markers with confirmed association BNL285239 – lint%(r2=0.29) BNL2495195 - elongation(r2=0.23) BNL1317191 - lint%(r2=0.15) CIR148147 - elongation(r2=0.21) JR307102 – lint%(r2=0.25) CIR293298 - elongation(r2=0.21) BNL285239 - boll wt(r2=0.15) BNL2986158 - strength(r2=0.19)* BNL542256 - boll wt(r2=0.22) BNL569144 – length(50%)(r2=0.27) BNL1317191 - boll wt(r2=0.32) BNL2495195 – length(50%)(r2=0.23)* BNL2986158 - boll wt(r2=0.19) BNL40971 – length(2.5%)(r2=0.24) CIR81222 - boll wt(r2=0.26) BNL542256 – length(2.5%)(r2=0.18) BNL160583 - micronaire(r2=0.20) BNL569144 – length(2.5%)(r2=0.26) BNL4062244 - MIC(r2=0.18) BNL2495195 – length(2.5%)(r2=0.25)* CIR17129 - micronaire(r2=0.38)* BNL2986158 – length(2.5%)(r2=0.36) CIR9985-MIC(r2=0.21) BNL3408130 - length(2.5%)(r2=0.13) CIR10590-MIC(r2=0.17) CIR196194 – length(2.5%)(r2=0.22)

Conclusion * Twenty-seven marker-trait associations confirmed with seven fiber related traits * No effect of population structure for marker-micronaire association * Moderate effect of population structure for boll wt, elongation, and length(2.5%) * Dramatic effect population structure for lint percent and length(50%)

ACKNOWLEDGEMENT Dr. Brian Scheffler and Ms. Sheron Simpson (MSA Genomics Laboratory). Dr. Jack C. McCarty (USDA-ARS, Mississippi State University Osman A. Gutiérrez (Mississippi State Universtiy) Dr. Jodi Scheffler, Dr. Jeffery Ray (USDA-ARS, Stoneville, MS Ms. Deborah Boykin (USDA-ARS, Mid South area Statistician)

Thanks