Transcriptome Sequencing Under Drought Stress, EST-SSR Development And High Density Genetic Linkage Map Construction In Gossypium Darwinii

Cotton is an important economic crop and has great significance in our national economy.Cultivated allotetraploid species Gossypium hirsutum account for90%of the world cotton production.But because of the genetic basis of the cultivated species is too narrow, resulting in cotton in terms ofyield, quality or stress resistance breeding stagnant. So it is very necessary to adopt a variety ofapproaches, in particular explore good genes from wild species of cotton to enrich the genetic diversityof germplasm resources of cotton in China. G. darwinii belongs to tetraploid wild cotton, which hasmany good characteristics like good fibre fineness, drought resistance and verticillium wilt resistance.But there is not any basic and applied research reported which focus on such excellent characteristicsof this cotton species. This paper conducted the transcriptome sequencing under drought stress,EST-SSR function markers development and high density interspecific genetic map construction of G.darwinii. The results are as follows:1. Solexa sequencing the transcriptome of G. darwinii seedlings under drought stressIn this study, we employed RNA-seq to analyse the transcriptomes from leaves of G. darwiniiseedlings with and without drought stress. A total of86.7million valid reads with average lengths of95.79bp were generated from the two samples and58960transcripts with length more than500bpwere assembled. Based on sequence similarity search with known proteins these transcripts wereannotated with COG, KEGG and GO functional categories. According to gene expression abundanceRPKM value we conducted differential expression analysis, obtained19,165differentially expressedgenes (32.5%) between two samples, including7,392up regulated genes and11,773downregulated genes. Through the analysis of the high abundance of gene expression, we found somemajor gene may be involved in mediating Cotton Drought stress resistence biological pathway.Successful implementation of G. darwinii drought stress transcriptome sequencing, on the one handwe can obtain a large amount of the cotton seed EST sequence to further enrich the EST sequenceinformation in NCBI database, on the other hand the data will facilitate genes discovery, large scaledrought stress response genes expression analysis and EST-SSR function markers development.2. Development and applicaton of G. darwinii EST-SSR markersA total of51,049non-redundant ESTs were obtained after removed redundant from the lengthmore than300bp EST sets of G. darwinii. From these non-redundant ESTs we detected1,388SSRloci, and first time developed780EST-SSR primers from G. darwinii species. Then the characteristicsof EST-SSR loci, polymorphism and transferability among different species of EST-SSR primers wereanalyzed in detail. UPGMA Cluster analysis using new primers revealed that A111is an independentnew species and has a close genetic relationship with G genome. Using135pairs of polymorphismprimers between G. hirsutum CRI12-4and G. darwinii to screen F2population including188individuals,141genotyping loci data were obtained, of which139loci were integrated into the genetic map. The map of EST-SSR Numbers increase from1,180to1,319, increased by11.8%, andimplements the further saturation of our interspecific genetic map.3. Construction a high density genetic linkage map in interspecific population of G. hirsutum×G. darwiniiIn this study, an interspecific high density linkage map of allotetraploid cotton was constuctedin F2population (G. hirsutum×G. darwinii). This map was based entirely on genome-wide simplesequence repeat (SSR) markers. A total of2,763markers were maped in26linkage groups(chromosomes) covering a genetic length of4,176.7cM, with the average interval distance betweenloci was1.5cM. The number of markers varied from49to169per chromosome, with the average106.3markers in each chromosome. The length of the chromosomes ranged from84.7to238.5cM,with average160.6cM each chromosome. At genome length was2,160.7cM, with the averagedistance of1.6cM; Dt genome length was2,016cM, with the average distance of1.4cM. Therewere601segregation distortion SSR loci in this map and the numbers of segregation distortion locilocated in At chromosomes was obviously less than the numbers in Dt chromosomes. To ourknowledge, this map is one of the three most dense linkage maps in cotton, and also is the firstinterspecific high density genetic map between upland cotton and wild cotton completely based onSSR markers. This map not only plays an important role on G. darwinii genome research, but alsolays groundwork for further genetic analysis of important stress resistance trait, map-based cloning,marker-assisted selection in wild cotton, and genome organization architecture in cotton as well asfor comparative genomics between cotton and other species.