Rapid development and characterization of fifteen novel microsatellite loci in vulnerable tree aquilaria malaccensis using next generation ion torrent PGM™ sequencing

Abstract

Aquilaria malaccensis is one of the 15 tree species of the genus Aquilaria widely distributed in the Indo-Malaysian region. Its resin (Agar or Gaharu) is widely known for its use in the production of highly valued incense and traditional medicine throughout Asia. The species has been listed as ‘Vulnerable’ in the IUCN red list of threatened species due to its over exploitation. In the present study, for the first time, next generation sequencing platform (Ion Torrent PGM™) was used to obtain a large number of sequences containing microsatellites for A. malaccensis. A total of 527,425 high quality reads were identified, which contained 4138 SSRs. In these SSRs, the number of dinucleotide repeats were the most frequent one (74.77%). Primers were developed for 93 loci; of these 82 loci were successfully amplified and 47 were polymorphic across a panel of 42 genotypes. Fifteen loci with at least three or more alleles were chosen for detailed characterization. The observed heterozygosity ranged from 0.25 to 0.90, whereas the expected heterozygosity ranged from 0.24 to 0.89, with 5.9 alleles per locus on average. Simple matching dissimilarity coefficients and cluster analysis revealed significant diversity among the genotypes. The study suggested that PGM™ sequencing is a rapid technique for developing microsatellite markers. The microsatellite markers developed in the present study would be useful in comprehensive genetic diversity studies aimed towards the conservation and germplasm management of Aquilaria.