De novo transcriptome assembly using Illumina sequencing and development of EST-SSR markers in a monoecious herb Sagittaria trifolia Linn

Abstract

Background Sagittaria trifolia Linn. is a widespread macrophyte in Asia and southeast Europe and cultivated in parts of Asia. Although a few genomic studies have been conducted for S. trifolia var. sinensis, a crop breed, there is limited genomic information on the wild species of S. trifolia. Effective microsatellite markers are also lacking. Objective To assemble transcriptome sequence and develop effective EST-SSR markers for S. trifolia. Methods Here we developed microsatellite markers based on tri-, tetra-, penta-, and hexa-nucleotide repeat sequences by comparatively screening multiple transcriptome sequences of eleven individuals from ten natural populations of S. trifolia. Results A total of 107,022 unigenes were de novo assembled, with a mean length of 730 bp and an N50 length of 1,378 bp. The main repeat types were mononucleotide, trinucleotide, and dinucleotide, accounting for 55.83%, 23.51%, and 17.56% of the total repeats, respectively. A total of 86 microsatellite loci were identified with repeats of tri-, tetra-, penta-, and hexa-nucleotide. For SSR verification, 28 polymorphic loci from 41 randomly picked markers were found to produce stable and polymorphic bands, with the number of alleles per locus ranging from 2 to 11 and a mean of 5.2. The range of polymorphic information content (PIC) of each SSR locus varied from 0.25 to 0.80, with an average of 0.58. The expected heterozygosity ranged from 0.29 to 0.82, whereas the observed heterozygosity ranged from 0.25 to 0.90. Conclusion The assembled transcriptome and annotated unigenes of S. trifolia provide a basis for future studies on gene functions, pathways, and molecular mechanisms associated with this species and other related. The newly developed EST-SSR markers could be effective in examining population genetic structure, differentiation, and parentage analyses in ecological and evolutionary studies of S. trifolia.