Program Of SSR MINING1.0 And Identification And Characterization Of EST-SSRs Of Soybean (Glycine Max)

EST-SSR is a type of molecular marker developed from the databank of sequence of expression sequence tags(EST)and/or cDNAs.As a new kind of molecular marker, EST-SSR markers have more advantages than the traditional genomic-derived SSRs because they are part of expressed genes. Therefore, EST-SSRs might be involved in gene functioning directly or indirectly.The ability of SSR markers will be greatly enhanced. In this study, an efficient arithmetic had been put forward by the analysis of the features of SSR marker itself and permutation and combination theory and corresponding software with Visual Basic 6.0 were developed. Then, primers based on the mining results from the nonredundant EST database in PlantGDB of soybean were designed. The distribution of SSR, design primers, filtered valid EST-SSR markers were analyzed with 8 soybean cultivars and part of these EST-SSRs were mapped on the scaffold and linkage group of recent published soybean genome. Using BLAST, partial EST sequences were related to specific biological functions.1. After summarizing and anglicizing the problems of existed SSR markers developing software, a program SSR MINING 1.0 were accomplished to with Visual Basic 6.0. Then new SSR markers were searched from 22161 soybean EST sequences published between 2004. 08. 02 and 2005. 06. 12 and 2070 SSR markers were detected, and with the help of DNAMAN, 30 pairs of primer had been designed for 30 SSR markers among them, then synthesized and verified by the experiments, 14 polymorphic SSR markers were got. SSR MINING 1.0 is an efficient, flexible, interface friendly, and easy to use SSR markers developing software.2. 200516 nonredundant soybean ESTs were found from the database PlantGDB, till April 5th, 2006. 12,833 SSRs were discovered by the module TROLL that could insert in staden package. There was one SSR in every 7.25Kb EST in 93.1Mb soybean EST database. The frequencies of di-, tri-, tetra-, penta- SSR motifs and compoud SSR were 25.2%, 46.5%, 6.9%, 16.1%, and 5.3% of total number of discovered SSRs respectly.3. According to the selected EST-SSR sequences, primers were successfully designed from 9638 discovered EST-SSRs. 124 pairs primers of these EST-SSRs were selected and synthesised. With genomic DNA of 8 soybean cultivars which represent a wide-range of diversity as template, PCR products of 81 pairs were successfully amplified while 44 of them showed polymorphism, and could be used as new EST-SSR markers of soybean and related species.4. The ESTs sequences corresponding to EST-SSR markers were analyzed by the programs BLAST of NCBI and Soybase to get the useful information of possible function and expression pattern etc. After sequence similarity assignment, 29 ESTs have homologous sequences or proteins, and the locations were determined of 14 EST-SSR in genes.5. 44 EST-SSRs were mapped on the scaffold and linkage groups based on the recent published soybean genome sequence by local BLAST program. Among them, 31 had been located on scaffolds, and only 29 could be located on related linkage groups.