Development of a universal and simplified ddRAD library preparation approach for SNP discovery and genotyping in angiosperm plants

作者：Guoqian Yang, Yunmei Chen, Jinpeng Wang, Cen Guo, Lei Zhao, Xiaoyan Wang, Ying Guo, Li Li, Li D, Zhenhua Guo · 发表于：Plant Methods · 年份：2016 · DOI：10.1186/s13007-016-0139-1 · 被引用次数：103 · 研究领域：Genomics and Phylogenetic Studies、Genetic diversity and population structure、Environmental DNA in Biodiversity Studies

BACKGROUND: The double digest restriction-site associated DNA sequencing technology (ddRAD-seq) is a reduced representation sequencing technology by sampling genome-wide enzyme loci developed on the basis of next-generation sequencing. ddRAD-seq has been widely applied to SNP marker development and genotyping on animals, especially on marine animals as the original ddRAD protocol is mainly built and trained based on animal data. However, wide application of ddRAD-seq technology in plant species has not been achieved so far. Here, we aim to develop an optimized ddRAD library preparation protocol be accessible to most angiosperm plant species without much startup pre-experiment and costs. RESULTS: We first tested several combinations of enzymes by in silico analysis of 23 plant species covering 17 families of angiosperm and 1 family of bryophyta and found AvaII + MspI enzyme pair produced consistently higher number of fragments in a broad range of plant species. Then we removed two purifying and one quantifying steps of the original protocol, replaced expensive consumables and apparatuses by conventional experimental apparatuses. Besides, we shortened P1 adapter from 37 to 25 bp and designed a new barcode-adapter system containing 20 pairs of barcodes of varying length. This is an optimized ddRAD strategy for angiosperm plants that is economical, time-saving and requires little technical expertise or investment in laboratory equipment. We refer to this simplified protocol as Mi...