Characterization of transcriptome and development of novel EST-SSR makers based on next-generation sequencing technology in Neolitsea sericea (Lauraceae) endemic to East Asian land-bridge islands

Abstract

Neolitsea sericea (Lauraceae), endemic to East Asian land-bridge islands, is an economically important tree species because of its characteristic aroma and timber uses. However, due to the lack of efficient molecular markers, the genetic diversity and historical demography of this endemic species is not clearly understood. In this study, we performed high-throughput transcriptome sequencing of N. sericea leaves using the Illumina HiSeq™ 2000 sequencing platform, and generated large transcript sequences for functional characterization and development of gene-associated SSR markers. A total of 68,624 unigenes (mean length 733 bp) were assembled from about 54.7 million reads, and 41,130 (59.94 %) unigenes of all the assembled unigenes showed similarity to public databases. From 68,624 unigenes, 13,213 expressed sequence tag–simple sequence repeats (EST-SSRs) were identified. Di-nucleotide SSRs were the most abundant motif (36.5 %), followed by mono- (32.3 %) and tri-nucleotide (26.5 %) repeats. From the 13,213 EST-SSRs, 1191 primer pairs were designed for marker mining. After selecting 131 of these pairs at random for further validation, 13 polymorphic pairs were identified as polymorphic SSR loci. These 13 EST-SSR markers showed intermediate levels of genetic diversity (e.g., N A = 7.15; mean H E = 0.51) when surveyed across six populations from East China (2), Taiwan (1), Korea (1), and the Ryukyus (1) and Honshu (1) of Japan. Both genetic distance and structure analyses identified two genetic clusters largely congruent with recent findings revealed by variations in chloroplast DNA sequences and genomic SSRs. The EST-SSR markers developed in our research will be an information resource for future studies on ecological, evolutionary, and conservation genomics in N. sericea.