Development, Evaluation And Application Of New Nonredundancy EST-SSR Markers From Gossypium Based On Public Database

In recent years exponential growth of public database especially nucleic acid data and bioinformatics technology revolution has made genomics and proteomics develop rapidly. How to use these data reasonably, efficiently and apply to genomics research is an urgent problem. However, redundancy has become a very important problem on the process of molecular markers development, and less relevant reports. Until now there is no right software to analyse the redundancy of a pair of primers meanwhile, causing the repeat research and development, a waste of time and cost. For system integration cotton EST(Expressed Sequence Tag) resources, the paper developed nonredundancy functional markers and carried out related applied research. It will accumulate technology materials for the abundant information from genome sequencing and transcriptome sequencing.A software Clustal X was used to analyse the redundancy of393753ESTs of Gossypium available in public database. By mining349815non-redundant ESTs, a total of11372SSR(Simple Sequence Repeat) loci derived from10507ESTs using a software SSRmine developed by ourselves were observed. The frequency of ESTs containing SSRs was3%, with an average of one SSR in every21kb of EST sequence. One thousand of new nonredundancy EST-SSR primers were developed based on the mentioned above EST sequences removed the redundancy which have not been developed so far in Gossypium, And we used a software SSRD developed by ourselves to obtain non-similarity primers, designated CICR (China Institute of Cotton Research)XXX through six steps, including SSR primer sequences download, pretreatment, Blastn, extraction of primer numbers of similarity score more than81%, extraction of redundant primers pairs and making redundant primers in a line, to remove homologous sequences from themselves and similar primers released in CMD(Cotton Marker Database) from different cotton species. Among them,100primers were evaluated in polymorphism information content (PIC), transferability using twelve cotton species including seven representative diploids species and five tetraploid species and preliminary mapping based on a F2population of G. hirsutum L x G. hirsutum. The results showed that a total of56from the100pairs of SSR primers could be amplified the stable and clear polymorphic bands in the12accessions mentioned above, moreover,35out of56pairs of primers were polymorphic, with the primer polymorphism ratio of35%. PIC of these primers ranged from0.097to0.888, with the average of0.482. Totally, the transferability among twelve cotton species was100%for a pair of EST-SSR primers from Gossypium barbadense L.,81%for25primers from G. arboreum and80.1%for74primers from G. hirsutum, respectively. It showed the new non-redundant EST-SSR markers efficacy is good and the method is feasible.One thousand of pairs of new primers were carried out the application study such as the genetic group mapping with a BC1population of G. hirsutum x G. barbadense, assessment of genetic diversity with67wild cotton materials and the function annotation and KEGG metabolic pathways analysis of the corresponding ESTs. Including:1. A total of380from1000pairs of SSR primers were used to amplify67accessions from wild cotton, which could produce stable and clear polymorphic bands. Six hundred and sixty DNA fragments were obtained among all materials with the average of1.73. The polymorphism information content (PIC) of these primers was from0.026-0.824, with the average of0.384, effective number of alleles (Ne) varied from1.024to5.698with the average of2.64and the Shannon-Weaver diversity index (H) with with the average of4.38. The UPGMA cluster analysis showed that when the genetic similarity coefficient was0.8, it classified materials into eight categories. The clustering result was approximately in accord with Wendel (2010) results. Meanwhile, it demonstrated that the clustering result of the same cotton species sources from different places was related with chromosome group, there was no significant correlation with geographical origin.2. A genetic proup had been constructed in the lab of Professor Yuan Youlu in Cotton Research Institute, Chinese Academy of Agricultural Sciences with a total genetic distance of over4000cM, based on the genetic linkage analysis with135BCi population of ZMS36x Hail and SSR primers screened(unpublished data). One hundred and thirty-two CICR markers were mapped the genetic proup, involving136loci (A subgenome63, D subgenome73, respectively), covering all26chromosomes and there were the most markers on Chromosome19. And there were forty-two segregation distortion loci related to CICR markers in the map.3. The function annotation of the corresponding ESTs of1000CICR markers was carried out and on level2, these EST were classified into three types including components, molecular function and biological process. Among them976EST belongded to cell components,597belonged to molecular function and1126belonged to biological processes. Two hundred and thirty-nine (account for23.9%) ESTs among them were associated with metabolic pathways. Carbohydrates and energy metabolism was most, amino acid metabolism was second.Through the preliminary evaluation and the application research, it showed that the new nonredundancy EST-SSR markers efficacy was good. The redundancy identification and evaluation methods were feasible and can be carried out related genomics application research.