Identification of SNP markers for inferring phylogeny in temperate bamboos (Poaceae: Bambusoideae) using RAD sequencing

Phylogenetic relationships among temperate species of bamboo are difficult to resolve, owing to both the challenge of detecting sufficiently variable markers and their polyploid history. Here, we use restriction site–associated DNA sequencing to identify candidate loci with fixed allelic differences segregating between and within two temperate species of bamboos: Arundinaria faberi and Yushania brevipaniculata. Approximately 27 million paired-end sequencing reads were generated across four samples. From pooled data, we assembled 67 685 and 70 668 de novo contigs from partial overlap among paired-end reads, with an average length of 240 and 241 bp for the two species, respectively, which were used to investigate functional classification of RAD tags in a blastx search. Analysed separately by population, we recovered 29 443 putatively orthologous RAD tags shared across the four sampled populations, containing 28 023 sequence variants, of which c. 13 000 are segregating between species, and c. 3000 segregating between populations within each species. Analyses based on these RAD tags yielded robust phylogenetic inferences, even with data set constructed from surprisingly few loci. This study illustrates the potential for reduced-representation genome data to resolve difficult phylogenetic relationships in temperate bamboos.

温带竹类物种间的系统发育关系往往难以解析，这既受限于难以检测到足够多的多态性标记，也与其多倍化演化历史密切相关。本研究采用限制性酶切位点相关DNA测序（restriction site–associated DNA sequencing, RAD-seq）技术，在两种温带竹类——法氏箬竹（Arundinaria faberi）和短锥玉山竹（Yushania brevipaniculata）——的种间及种群内，筛选出携带固定等位基因差异的候选位点。本研究对4个样本开展双端测序，共获得约2700万条读段。基于混合测序数据，通过双端读段的部分重叠区域从头组装得到67685条和70668条重叠群（contig），两种竹类的重叠群平均长度分别为240 bp和241 bp；随后以这些重叠群为参考，通过BLASTx（blastx）比对对RAD标签（RAD tag）进行功能分类分析。按种群单独分析后，我们在4个采样种群中共检索得到29443个推定直系同源的RAD标签，共包含28023个序列变异位点，其中约13000个位点在物种间存在分离，约3000个位点在各物种种群间存在分离。基于这些RAD标签开展的分析可获得稳健的系统发育推断结果，即便所用数据集仅包含数量极少的位点。本研究证实，简化基因组数据（reduced-representation genome data）具备解析温带竹类复杂系统发育关系的应用潜力。