Next-generation polyploid phylogenetics: rapid resolution of hybrid polyploid complexes using PacBio single-molecule sequencing

Difficulties in generating nuclear data for polyploids have impeded phylogenetic study of these groups. We describe a high-throughput protocol and an associated bioinformatics pipeline (PURC: “Pipeline for Untangling Reticulate Complexes”) that is able to generate these data quickly and conveniently, and demonstrate its efficacy on accessions from the fern family Cystopteridaceae. We conclude with a demonstration of the downstream utility of these data by inferring a multilabeled species tree for a subset of our accessions. We amplified four ~1kb-long nuclear loci and sequenced them in a parallel-tagged amplicon sequencing approach using the PacBio platform. PURC infers the final sequences from the raw reads via an iterative approach that corrects PCR and sequencing errors and removes PCR-mediated recombinant sequences (chimeras). We generated data for all gene copies (homeologs, paralogs, and segregating alleles) present in each of three sets of 50 mostly-polyploid accessions, for fo...

多倍体的核数据生成难题长期阻碍了该类群的系统发育研究。本研究开发了一套高通量实验流程及配套生物信息学分析流程PURC（全称：Pipeline for Untangling Reticulate Complexes，即“解析网状进化复合体流程”），可快速便捷地生成所需核数据，并通过冷蕨科（Cystopteridaceae）类群的种质材料验证了该流程的有效性。最后，我们通过对部分种质材料构建多标记物种树，展示了该数据集的下游应用价值。我们针对4个长度约1千碱基对的核基因座进行扩增，并采用PacBio平台的并行标签扩增子测序策略完成测序。PURC流程通过迭代策略从原始测序读段中推导最终序列，可校正PCR及测序错误，并去除PCR介导的重组序列（嵌合体）。我们为三组各50份以多倍体为主的种质材料中存在的所有基因拷贝（包括异源同源基因、旁系同源基因及分离等位基因）生成了测序数据，用于后续……