Development and characterization of cDNA library based novel EST-SSR marker in radish (Raphanus sativus L.)

Abstract

To develop novel expressed sequence tags-simple sequence repeats (EST-SSR) markers in radish, a taproot cDNA library was constructed. A total of 10,052 clones from the cDNA library were sequenced and a large number of high quality ESTs was generated. Among these ESTs, a set of 179 SSRs, distributing in 176 unigene sequences, were detected, of which, 50.84% were di-nucleotide, followed by tri- (29.61%), hexa- (9.49%), penta- (6.15%) and tetra-nucleotide (3.91%) repeats, respectively. GA/TC (25.69%) was the most frequent repeat type of all repeat types. Out of 125 primer pairs designed, 110 (88%) could generate unambiguous amplification products. Totally 28 EST-SSR primer pairs were selected for genetic diversity analysis in 32 radish genotypes. It was found that two to seven alleles could be detected with a mean of 3.5 per primer pair and the polymorphism information content (PIC) values of these primers ranged from 0.000 to 0.825 with an average of 0.500. The genotype data were analyzed with NTSYS-pc software, which grouped the 32 accessions into three main clusters at the similarity index value of 0.60, which was mainly in accordance with the different biological characterizations of the accessions. In addition, the 28 EST-SSR primer pairs were further used to test the transferability on 12 accessions from three genera of Raphanus, Brassica and Arabidopsis in Brassicaceae. The results showed that 18 primer pairs (64.2%) could produce target PCR bands in the 12 accessions. The EST-SSR markers developed herein represented a valuable resource for the genetic diversity analysis, genetic mapping and marker-assisted selection in radish.