The development of EST-SSR markers in Lilium regale and their cross-amplification in related species

Abstract

Simple-sequence repeat (SSR) markers are a common choice for assaying genetic diversity and genetic mapping due to their highly polymorphic and co-dominant nature. In this paper, we developed SSR markers derived from expressed sequence tags (EST-SSRs) based on transcriptome sequences in Lilium regale. In total, 59,046 UniGene sequences (approximately 27.37 Mb) were evaluated, where 1,716 SSR sites in the 1,606 UniGene sequences were identified by data mining. Of these SSRs, the trinucleotide repeats were the most common repeat types (631, 36.8 %), followed by hexa- (23.7 %), di- (23.4 %), penta- (11.5 %), tetra- (3.3 %), hepta- (0.9 %), deca- (0.3 %), and octanucleotide repeats (0.1 %). TC/GA and CCG/CGG predominated in dimeric and trimeric repeat motifs, respectively. In total, 1,072 primer pairs were designed, of which 494 were amplified to yield the expected PCR products in L. regale. The polymorphism and transferability study was performed in six species endemic to China and ten cultivars derived from intraspecific and interspecific hybridization. Of the 494 primer pairs, 172 exhibited clear polymorphisms with stable cross-species amplifications in the 16 individuals, which contained 537 alleles for the 181 loci. The polymorphism information content values for the 172 primers ranged from 0.111 to 0.830, with an average value of 0.493. The phylogenetic dendrogram derived from the amplification profiles of the 172 polymorphic primers was congruent with the genetic background of the hybrids and the species. In addition, the putative functions of the 109 UniGenes containing the 118 polymorphic and transferable EST-SSRs were identified. Thus, the informative EST-SSR markers will be very useful in subsequent Lilium genetic improvement projects.