Studies Of EST-SSR Derived From Gossypium Barbadense And Construction Of High Density Genetic Linkage Map By Interspecific Population For QTL Mapping

Cotton(Gossypium L.) is one of the most important economic crops that provide natural textile fiber and edible oil throughout the world.In the past few years, improvement in the quality of cotton fiber has become more important because of profound innovations in spinning technology.While cotton yield traits are negatively related with fiber quality traits which make it difficult to improve yield and quality at the same time by traditional breeding ways.With the rapid development of genomic technologies,molecular breeding has become an important method for the genetic improvement of cotton fiber.New sets of molecular markers should be developed to facilitate the development of a high-resolution integrated genetic map for structural and functional studies of cotton fiber genes and marker assisted selection(MAS) of genes that could enhance cotton fiber quality.1.Studies of new EST-SSRs derived from Gossypium barbadenseThe plant EST(expressed sequence tags) program is one of the most important parts which compose the plant genome program.The expansion of EST.data base is a foundation of development of EST markers.EST-SSRs can be used in studies of plant genetics and evolution;they also have higher levels of transferability to related species than genomic SSR markers.Furthermore,the functions of EST-SSRs are usually presumable.However,existing cotton EST-SSR markers are mostly derived from G. arboreum and G.hirsutum,while G.barbadense derived EST-SSRs are scarce.It is well-known that G.barbadens is one of the most valuable cotton species and it is an ideal candidate for providing new genetic material for improving fiber quality in G.hirsutum.In this paper,one hundred and nineteen EST-SSRs were developed based on 98 unique ESTs from a cDNA library constructed in our laboratory using developing fibers from G.barbadense cv.Pima3-79.The characteristics of these EST-SSRs were defined, and their transferability,applications in genetic diversity analysis and linkage mapping were evaluated. Among the SSRs,trinucleotide AAG appeared at a high frequency of 11.76%. Thirty-six accessions(consisting of 13 diploids of the A genome,11 dipioids of the D genome and 12 allotetraploids of the AD genome) were employed to test new EST-SSRs. Seventy-six EST-SSRs were successfully amplified,and yielded 313 polymorphic fragments,with an average of 4.11 fragments per primer pair.Cross-species transferability of the 76 EST-SSRs in diploid Gossypium species ranged from 78.95%(in G.thurberi) to 94.74%(in G.arboreum and G.herbaceum),whereas in tetraploid species from 93.42%(in G.mustelinum) to 100%(in G.darwinii).Polymorphism in diploid species G.arboreum was the same as in tetraploid species G.hirsutum,both were 11.84%. The PIC ranged from 0.17 to 0.95 with an average of 0.53.Based on Jaccard's genetic similarity coefficient,these 36 accessions were clustered into three groups.Twenty-one EST-SSRs exhibited polymorphisms in BC₁ population[(Emian22×Pima3-79)×Emian22],24 polymorphic loci were generated,while 22 of the 24 polymorphic loci were integrated with our interspecific BC₁ backbone genetic linkage map,and anchored in 12 chromosomes.This study effectively proved that EST-SSRs from G.barbadense are valuable for genetic diversity analysis and genetic mapping.2.Construction of dense genetic linkage map by interspecific population for QTL mappingIn the past decade,several successive cotton molecular maps have been constructed using diverse DNA molecular markers and mapping populations.A detailed genetic linkage map covering a large region of the cotton genome based on some informative, portable and practical markers will provide a platform for evolutionary comparisons with other species,comparative mapping,QTL mapping and MAS,and structural and functional studies of the cotton genome.Simple sequence repeats(SSRs),alternatively called microsatellites,are regarded as useful and efficient markers for map construction of cotton,which are abundant,locus-specific,often codominant and multiallelic.In this study,an interspecific linkage map of allotetraploid cotton was developed in a BC₁ population[(Emian22×Pima3-79)×Emian22].This map was based entirely on genome-wide simple sequence repeat(SSR) markers.Totally 6310 SSR markers (including 13 sets of markers) were employed to screen polymorphisms between 'Emian22' and 'Pima3-79',approximately 16%(1026/6310) of all SSR markers revealed polymorphic bands,then yielded 1040 polymorphic loci in BC₁ population.The molecular map was generated using MAPMAKER version 3.0b program(LOD scores≥8.0,recombination frequency≤0.40),with 44 linkage groups assigned to 26 chromosomes,and 917 loci spanning 5452.2 cM of the genome.The average distance between loci was 5.9 cM,covering the A-subgenome and D-subgenome uniformly. Significant segregation distortion Was observed for 107 loci(11.7%),of which 29(27.1%) loci skewed toward the 'Emian22' parent,while 78(72.9%) loci skewed toward the heterozygote.The frequency of distorted loci of genomic SSRs(11.7%) was coincident with EST-SSRs(11.5%).In total,11 SDRs were detected in seven linkage groups,2 SDRs(35 loci) appeared in the A-subgenome,and 9 SDRs(72 loci) appeared in the D-subgenome.Many of the distorted loci concentrated in Chr02,Chr16 and Chr18.This map was characterized and analyzed in detail,including the distributions of genomic SSRs,expressed sequence tag(EST)-SSRs,and distorted markers.Furthermore, the relationship between motif characteristics(sizes,types,lengths) and the level of polymorphism in EST-SSRs was also surveyed.The results showed that polymorphism rates of tetranucleotide motifs(11.33%) was higher than dinucleotide motifs(7.90%) and trinucleotide motifs(6.72%),the percentages of polymorphic hexanucleotide and pentanucleotide were both only 4.00%;ACAT,AC,and ACT motif types had the highest level of polymorphism(17.24%,16.67%and 12.99%,respectively),while ACG and AGG were the lowest(both 3.33%);loci with lengths of 27 bp,33 bp,and 24 bp were more likely to be polymorphic.These results will provide information to assist in designing future EST-SSRs.The method of composite interval mapping was applied to search for QTL(LOD≥3.0) by the phenotypes of BC₁ plants and corresponding BC₁F₂ families.On the basis of this analysis yielded 11 QTL:2 QTL controlling fiber length,1 QTL controlling fiber uniformity,1 QTL controlling micronaire,3 QTL controlling fiber elongation,4 QTL controlling fiber strength.Among them,6 were mapped on At chromosome and 5 were mapped on Dt chromosome,explained 9.80%～16.82%of phenotypic variance.In three of the QTL,Pima3-79 alleles had positive additive genetic effect,the additive genetic effect values ranged from 0.33 to 1.10.