Development Of SSR And SNP Markers And Genome-wide Association Study Of Important Traits In Gossypium Hirsutum

Cotton(Gossypium SPP.)is the most important fiber crop in the world and is also an important edible oil crop.Two of the four cultivated species,G.hirsutum L.(AD)1 and G.barbadense L.(AD)2,account for 90%and 5-8%of world cotton production,respectively.G.arboreum(A2)and G.raimondii(D5)are regarded as the progenitors that led to the formation of the tetraploid cotton species 1-2 million years ago(MYA).Although much research has been conducted to characterize microsatellites and develop markers,due to the lack of complete genome sequences,the distribution of microsatellites remains ambiguous and the use of microsatellite markers in genomic studies and marker-assisted selection is limited.Natural variants of crops are generated from wild progenitor plants under both natural and human selection.Diverse cotton varieties that are able to adapt to various environmental conditions are valuable resources for cotton improvements to meet the increasing demands of the cotton fiber.China is the world’s important producer and consumer of textiles,cotton industry plays an irreplaceable role in China’s national economy.In addition to economic value,Upland cotton is also a model system for studying polyploidy,plant cell elongation and cellulosic synthesis.The overall genetic diversity within the cultivated species and genetic changes during their improvement are poorly understood.With the completion of cotton reference genome sequences,the advent of high-throughput genotyping technology now enables rapid and accurate resequencing of a large number of cotton genomes to detect the genetic basis of phenotypic variations in cotton.Comprehensive maps of cotton genome variations facilitate genome-wide association studies of complex traits and functional investigations of evolutionary changes in cotton.Understanding the genetic basis of phenotypic variation and the domestication processes in cotton can help us efficiently utilize these diverse genetic resources for cotton improvement.We developed SSR markers in the genomic sequences of Upland cotton,G.arboreum,and G.raimondii.The distribution frequency of SSRs between different genomes was compared and the whole genome primers were designed.Genome-wide re-sequencing of 318 cotton varieties identifies SNPs for genetic diversity analysis,including the nucleotide diversity index(π),genetic differentiation index(Fst)and selective sweep analysis,reveals the genetic diversity in cotton,as well as genome evolution from the landrace to modem cultivar.Finally,genome-wide association study of important agronomic traits such as cotton yield,fiber quality and disease resistance was conducted to explore the elite allelic variation.To identify microsatellites for cotton research,we mined 100,290,83,160,and 56,937 microsatellites with frequencies of 41.2,49.1,and 74.8 microsatellites per Mb in the recently sequenced Gossypium species:G.hirsutum,G.arboreum,and G.raimondii,respectively.The distributions of microsatellites in their genomes were non-random and were positively or negatively correlated with genes and transposable elements,respectively.Of the 77,996 developed microsatellite markers,65,498 were physically anchored to the 26 chromosomes of G.hirsutum with an average marker density of 34 markers per Mb.We confrmed 67,880(87%)universal and 7,705(9.9%)new genic microsatellite markers.The polymorphism was estimated in above three species by in silico PCR and validated with 505 markers in G.hirsutum.We further predicted 8,825 polymorphic microsatellite markers within G.hirsutttm acc.TM-1 and G.barbadense cv.Hai7124.In our study,genome-wide mining and characterization of microsatellites,and marker development were very useful for the saturation of the allotetraploid genetic linkage map,genome evolution studies and comparative genome mapping.Within the framework of this research,we report a comprehensive genomic assessment of the modem improved Upland cotton based on the genome-wide resequencing of 318 landrace and modem improved cultivars or lines.We aligned the sequence reads to the reference genome sequence of TM-1 and identified a total of 8,621,073 single nucleotide polymorphisms(SNPs)with an average density of 4.41 SNPs per kb.By analysing the allele frequency of each SNP site in the 318 accessions,we used 2,167,186 common SNPs with minor allele frequency(MAF)>0.05 for the subsequent analysis.A total of 190,949 common SNPs were found in genes,66,138 of which were found in the coding sequences,including 769 large-effect SNPs(stop codons gain/loss and exon-intron splice sites)in 745 genes.We examined the genetic diversity across 26 chromosomes.The fixation index values(Fst)were 0.04 between the landraces and modern improved cultivars,which is lower than between the G.hirsutum races and the cultivars(Fst=0.10).The nucleotide diversity levels of the landrace(2.59×10-4)were higher than in the case of the modern improved cultivar(1.79×10-4).We detected 25 improvement-selective sweeps through the comparison of genetic diversity between the landraces and the modem improved cultivars.In total,we identified 119 associated loci:71 for yield-related traits,45 for fibre qualities and three for the resistance to Verticillium wilt(VW).More associated loci for lint yield were detected compared to fiber quality,suggesting lint yield had stronger signatures of selection than the other traits.Two ethylene related pathway genes were found to associate with the lint yield increasing in improved cultivars.The population frequency of each elite allele in historically released cultivar groups was also evaluated.These results provide genomic bases for improving the cotton cultivars and for the further evolution analysis of polyploid crops.