De Novo SNP Discovery and Genotyping of Masson Pine (Pinus massoniana Lamb.) via Genotyping-by-Sequencing

Abstract

Masson pine (Pinus massoniana Lamb.) is an important tree species in China, but its genomic research has been hindered due to a large genome size. Genotyping-by-sequencing (GBS) has been a powerful approach to revolutionize the field of genomic research by facilitating the discovery of thousands of single nucleotide polymorphisms (SNPs) and genotyping in non-model organisms, at relatively low cost. Here, we performed de novo SNP discovery and genotyping in 299 trees via the genotyping-by-sequencing (GBS) approach. The effort produced 9.33 × 109 sequence reads, 265,525 SNP-associated contigs, and 6,739,240 raw SNPs. Further filtering and validation of the SNP-associated contigs for reliable SNPs were performed using blasting against the Pinus tabuliformis reference genome, functional annotation, technical replicates, and custom parameter settings for the optimization. The 159,372 SNP-associated contigs were aligned and validated for SNP prediction, in which 60,038 contigs were searched with hits in the NCBI nr database. We further improved the SNP discovery and genotyping with multiple technical replicates and custom parameter settings filtering. It was found that the use of blasting, annotation, technical replicates, and specific parameter settings removed many unreliable SNPs and identified 20,055 more precise and reliable SNPs from the 10,712 filtered contigs. We further demonstrated the informativeness of the identified SNPs in the inference of some genetic diversity and structure. These findings should be useful to stimulate genomic research and genomics-assisted breeding of Masson pine.