Next generation sequencing-based molecular marker development: A case study in Betula alnoides

Abstract

Betula alnoides is a fast-growing valuable indigenous tree species with multiple uses in the tropical and warm subtropical regions in South-East Asia and southern China. It has been proved to be tetraploid in most parts of its distribution in China. In the present study, next generation sequencing (NGS) technology was applied to develop numerous SSR markers for B. alnoides, and 64,376 contig sequences of 106,452 clean reads containing 164,357 candidate SSR loci were obtained. Among the derived SSR repeats, mono-nucleotide was the main type (77.05%), followed by di- (10.18%), tetra- (6.12%), tri- (3.56%), penta- (2.14%) and hexa-nucleotide (0.95%). The short nucleotide sequence repeats accounted for 90.79%. Among the 291 repeat motifs, AG/CT (46.33%) and AT/AT (44.15%) were the most common di-nucleotide repeats, while AAT/ATT (48.98%) was the most common tri-nucleotide repeats. A total of 2549 primer sets were designed from the identified putative SSR regions of which 900 were randomly selected for evaluation of amplification successfulness and detection of polymorphism if amplified successfully. Three hundred and ten polymorphic markers were obtained through testing with 24 individuals from B. alnoides natural forest in Jingxi County, Guangxi, China. The number of alleles (NA) of each marker ranged from 2 to 19 with a mean of 5.14. The observed (HO) and expected (HE) heterozygosities varied from 0.04 to 1.00 and 0.04 to 0.92 with their means being 0.64 and 0.57, respectively. Shannon-Wiener diversity index (I) ranged from 0.10 to 2.68 with a mean of 1.12. Cross-species transferability was further examined for 96 pairs of SSR primers randomly selected, and it was found that 48.96–84.38% of the primer pairs could successfully amplify each of six related Betula species. The obtained SSR markers can be used to study population genetics and molecular marker assisted breeding, particularly genome-wide association study of these species in the future.