| Cotton is the most important source of natural fiber in the world. Gossypium barbadense and Gossypium hirsutum are two cultivated tetraploid species. G. barbadense provides vital fiber material for top-grade and high value new textile, and plays crucial economic role in optimizing cotton industrial utilization. Meanwhile G. barbadense is the major gene donor for high quality fiber. However, compared with G. hirsutum which is more adaptable and productive, G. barbadense has longer growth period, smaller boll and lower yield. The genomics study and bioinformatics analysis of fiber development will effectively unearth the gene of fiber quality and illustrate the mechanism of fiber development. Further, new elite fiber quality accessions will be enhanced by molecular breeding by design. In this paper, new and nonredundant molecular markers were developed and exploited based on EST sequence of fiber development and bioinformatics analysis among paralogous loci from different cotton species were carried out based on EST sequences. The main results were as following:1. Larger numbers of SSRs exist in the transcriptome, they have high polymorphism and been widely used in the development and exploitation of molecular marker. Based on Gossypium barbadense fiber ESTs and homology analysis against G. hirsutum ESTs,638SSRs and13,275InDels information were mined. Then380non-redundant EST-SSRs of G. barbadense and2,160EST-InDels molecular markers between G. barbadense and G. hirsutum were exploited.90pairs EST-InDels marker primer pairs selected randomly were synthesized for interspecific or intersubgenome polymorphism confirmation by PCR electrophoresis detection. The results indicated that64primer pairs had certain polymorphism between the two species or between subgroup within species;20pairs primer did not show polymorphism, and6pairs had no amplification product.9polymorphism loci were selected to confirm the efficiency and accuracy of InDel.2. The InDel difference of EST sequence between two species on the molecular level has correlation with its function. Gene function annotation, Gene Ontology analysis and KEGG metabolic pathway analysis were carried out with those13,275ESTs containing InDel. Bioinformatics analysis indicated that fiber development genes related to sucrose metabolism and secondary wall synthesis process such as sucrose synthase gene, cellulose synthase gene and glycosyl transferase gene had InDels difference between G. barbadense and G. hirsutum. These differences on the transcriptional level may be related to the fiber quality difference between the two species. These results laid the foundation for discovering the key genes of fiber development and elucidating the genes network function mechanism.3. The tetraploid cotton species in Gossypium were formed about1-2million years ago by naturally happened genomic hybridization of two diploid A and D genome and then polyploidization. Different species in Gossypium have a common descent. EST homology analysis of different cotton species also showed the Higher-level sequence conservation between different cotton species and between the A-and D-subgenomes in tetraploid cotton. The Clustering analysis based on four species ESTs sequence in Gossypium, including two tetraploid cotton species, G. barbadense and G. hirsutum, and two diploid cotton species, G. arboreum and G. raimondii, indicated that50.77%sequence of D genome ESTs from G. raimondii can cluster with one contig of tetraploid, while only17.15%ESTs of A genome from G. arboreum cluster with tetraploid. These results implied that there were a close genetic relationship between G. raimondii and tetraploid Dt genome, whereas remote affinity existed between G. arboreum and tetraploid At genome in transcriptional level.4. High reliability SNP/InDels site information was detected by SNPs/InDels mining of contig from four genomes of teraploid and diploid. Further haplotype analysis were carried out and identified A/At, D/Dt homologous gene. The results showed that except A/At, D/Dt variation had high conservatism, most of the contig had a lot of new intragenomic variation sites within At or Dt genome. These demonstrated that allotetraploid At and Dt genome evolve separately during the gene evolution and more adaptive evolutional structural variation has emerged. As a result, SNP markers distinguishing1340homologous genes between G. barbadense and G. hirsutum were further developed.5. Because of polyploidy genome of Gossypium, there were a large number of homologous sequences which caused redundancy of different source SSRs molecular markers. According to Gossypium SSRs markers published by CMD, in addition to NAU series markers newly developed by our laboratory, total15,180SSR primers pairs were used for redundancy analysis. As a result,1,530redundancy groups were obtained and there were4,034primers pairs. Based on the redundancy primer groups we obtained,1,530universe SSRs molecular markers can be developed. Integrated with the rest non-redundant SSRs markers, non-redundant SSRs12,676primer paris were obtained.6. Based on released G. raimondii genomic sequencing data of JGI, all the developed SSRs were physically mapped. The results indicated that9,697SSRs marker primers were successfully anchored to the G. raimondii scaffolds with scattered in different chromosomes. Among them,8,809pairs anchored within one chromosome,26pairs existed different positions on the same chromosome, while the rest862pairs scattered in different chromosomes. According to amplification products size,9,124SSRs molecular markers were mapped with high accuracy whose product size were smaller than500bp,369SSRs were mapped with moderate accuracy whose product size were between500and1000bp, and204were mapped with low reliability whose product size were larger than2000bp. The physical mapping results of molecular markers will provide integrated genomic resources for further molecular breeding studies such as map-based cloning, QTLfine mapping, and so on.7. Based on released G. raimondii genomic sequencing data of JGI,3,414marker loci from high density tetraploid cultivated cotton species genetic map constructed by our laboratory were further integrated with G. raimondii whole genome.2,696marker loci (81.10%) were successfully anchored to13chromosomes of G. raimondii. Among them, most were specifically anchored (2,236,82.94%). The results demonstrated that D/Dt genome had high colinearity, while At2/At3, At4/At5had certain chromosomal translocation. Integrated data laid foundation for tetraploid whole genome sequencing, fine mapping and map-based cloning of elite genes and QTL, and comparative genomics in Gossypium. |
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