title: Optimization of the genotyping-by-sequencing strategy for population genomic analysis in conifers.
abstract: Flexibility and low cost make genotyping-by-sequencing (GBS) an ideal tool for population genomic studies of nonmodel species. However, to utilize the potential of the method fully, many parameters affecting library quality and single nucleotide polymorphism (SNP) discovery require optimization, especially for conifer genomes with a high repetitive DNA content. In this study, we explored strategies for effective GBS analysis in pine species. We constructed GBS libraries using HpaII, PstI and EcoRI-MseI digestions with different multiplexing levels and examined the effect of restriction enzymes on library complexity and the impact of sequencing depth and size selection of restriction fragments on sequence coverage bias. We tested and compared UNEAK, Stacks and GATK pipelines for the GBS data, and then developed a reference-free SNP calling strategy for haploid pine genomes. Our GBS procedure proved to be effective in SNP discovery, producing 7000-11 000 and 14 751 SNPs within and among three pine species, respectively, from a PstI library. This investigation provides guidance for the design and analysis of GBS experiments, particularly for organisms for which genomic information is lacking.
authors.affiliation: Department of Ecology and Environmental Science, Umeå University, Umeå, SE-90187, Sweden.|State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing, 100093, China.
authors.forename: Jin
authors.initials: J
authors.lastname: Pan
authors.affiliation: Department of Ecology and Environmental Science, Umeå University, Umeå, SE-90187, Sweden.
authors.forename: Baosheng
authors.initials: B
authors.lastname: Wang
authors.affiliation: Beijing Computing Centre, Beijing, 100094, China.|Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, 100101, China.
authors.forename: Zhi-Yong
authors.initials: ZY
authors.lastname: Pei
authors.affiliation: State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing, 100093, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhao
authors.affiliation: Key Laboratory of Tropical Forest Ecology, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Menglun, Yunnan, 666303, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Gao
authors.affiliation: National Engineering Laboratory for Forest Tree Breeding, Key Laboratory for Genetics and Breeding of Forest Trees and Ornamental Plants of Ministry of Education, Beijing Forestry University, Beijing, 100083, China.
authors.forename: Jian-Feng
authors.initials: JF
authors.lastname: Mao
authors.affiliation: Department of Ecology and Environmental Science, Umeå University, Umeå, SE-90187, Sweden.
authors.forename: Xiao-Ru
authors.initials: XR
authors.lastname: Wang
mesh_terms: D004262:DNA Restriction Enzymes / Q000378:metabolism; D018744:DNA, Plant / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D060005:Genotyping Techniques / Q000379:methods*; D056186:Metagenomics; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA / Q000379:methods*; D064028:Tracheophyta / Q000145:classification* / Q000235:genetics*
chemical_list: D018744:DNA, Plant; D004262:DNA Restriction Enzymes
keywords: SNP validation; conifer genome; genotyping-by-sequencing; library preparation parameters; sequencing depth