Rapid development and characterization of fifteen novel microsatellite loci in vulnerable tree aquilaria malaccensis using next generation ion torrent PGMâ„¢ 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 PGMâ„¢) 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 PGMâ„¢ 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.