Development And Characterization Of EST-SSRs Markers In Sugarcane

Sugarcane, the most important sugar crop in the world, is also an important energy crop. The cane sugar accounts for more than90%of the sugar production in China. Genetic diversity among sugarcane germplasm is the important basis for sugarcane variety improvement, and SSR markers technology is a powerful tool for assessing genetic diversity in sugarcane. In the present study, the SSR information was mined from sugarcane ESTs which were downloaded from the NCBI dbEST database, and55new sugarcane EST-SSRs markers were developed accordingly, among which26were applied in genetic diversity analysis of56sugarcane germplasm. The main results and conclusions are as follows.(1) The frequency and characteristics of SSR were analyzed and clarified in sugarcane ESTs. A totoal of262,113ESTs of sugarcane in the dbEST database of NCBI were downloaded and analyzed, revealing62565non-redundant Unigene sequences with total length of about50,059kb and9,482SSRs. The frequency of SSR in these unigenes was15.15%with one SSR found in every5.28kb. Tri-nucleotide repeat was the main type, accounting for45.92%of the total SSRs. The dominant types were di-nucleotide and tri-nucleotide SSRs, accounting for21.22%and8.18%, respectively. The polymorphism of SSRs was assessed by bioinformatics methods. The searching and analysis of SSRs lays foundations for the development and application of EST-SSRs markers in sugarcane.(2) EST-SSRs markers were developed in sugarcane. Firstly, based on the results of SSR analysis in sugarcane ESTs, EST-SSRs primers were designed using Primer5.0software. The SSR sequences used in primer design were mainly low-level repeat motifs with length ranged from100bp to500bp and the length of repeat motif≥20bp. Secondly, the total of55pairs of primers were selected from the168pairs of designed primers and44with a high of proportion (80.0%) of above EST-SSRs, which gave amplified fragments of foreseen size, were selected according to the amplification results detected in agarose gel electrophoresis. Finally,38EST-SSRs with clear bands were selected for the detection in capillary electrophoresis. Among them,26pairs of EST-SSRs markers were finally determined for further identification.(3)26EST-SSRs markers were used for assessing the genetic diversity of56sugarcane germplasm, which further validated the feasibility of these newly developed markers. Through EST-SSRs marker analysis,3,138genetic similarity (GS) coefficients, which ranged from0.738to 0.919, were obtained. The number of alleles ranged from8to21with an average of13alleles per locus, while polymorphism information content (PIC) values ranged from0.428to0.928with an average of0.843. According to the breeding institutes, these56sugarcane germplasm could be assorted into8different series. Among these8series, the percentage of polymorphic bands (PPB) ranged from47.73%to80.40%, the genetic diversity (h) from0.183to0.281, while the Shannon’s Information index (1) was from0.191to0.281. A total of26EST-SSRs markers, were demonstrated to have good polymorphism and also the ability to detect gene-rich sites and more specific sites, through their application in the genetic diversity analysis of56sugarcane germplasm. From above, these26newly developed EST-SSRs markers should be feasible in the evaluation of sugarcane germplasm.