Leaf Transcriptome Analysis and Development of EST-SSR Markers in Arrowhead (Sagittaria trifolia L. Var. Sinensis)

Abstract

As a native in Southeast Asia, Sagittaria trifolia L. var. sinensis (arrowhead) is an aquatic perennial herb of Alismataceae family. For the aim of discovering genes and development molecular markers, high-throughput transcriptome sequencing is applied to generate enormous transcript sequences from arrowhead leaf generate. Through trimmed and assembled, 51, 836 contigs were obtained with mean length of 680 bp. Sequence similarity analyses against four public databases (NR, GO, KEGG, KOG) found 23, 595 contigs that could be annotated with gene descriptions, conserved protein domains, or gene ontology terms. In this study, 3, 861 simple sequence repeat (SSR) markers of arrowhead were identified in 3, 174 contigs of transcriptome data,and 2, 476 primer pairs were designed for marker development. Of these, 100 primers were randomly selected, synthesized and used for validation of the amplification and assessment of polymorphisms in 79 arrowhead accessions. 78 loci reliably amplified a clear single band of expexted size, and 68 loci were scoreable and polymorphic with 2 to 11 alleles per locus. The polymorphism information content valued ranged from 0.01 to 0.84. The SSR markers developed from arrowhead, could be successfully applied to Sagittaria pygmaea, with a transferability rate of 91.14% (72/79). The phylogenetic tree was also constructed to analyse the genetic diversity in arrowhead accessions. The large number of SSR markers developed in this study will be useful in the researches of population genetics, genetic diversity, and germplasm characterization in arrowhead.