Data from: Conserved genetic regions across angiosperms as tools to develop single copy nuclear markers in gymnosperms: an example using cycads

Several individuals of the Caribbean Zamia clade and other cycad genera were used to identify single copy nuclear genes for phylogeographic and phylogenetic studies in Cycadales. Two strategies were employed to select target loci: 1) a tblastX search of Arabidopsis conserved ortholog sequence (COS) set and, 2) a tblastX search of Arabidopsis-Populus-Vitis-Oryza Shared Single Copy genes (APVO SSC) against the EST Zamia databases in Genbank. From the first strategy, 30 loci were selected, and from the second, 16 loci. In both cases the matching Genbank accessions of Zamia were used as a query for retrieving highly similar sequences from Cycas, Picea, Pinus species or Ginko biloba. After retrieving and aligning all the sequences in each locus, intron predictions were completed to assist in primer design. PCR was carried out in three rounds to detect paralogous loci. A total of 29 loci were successfully amplified as a single band of which 20 were likely single copy loci. These loci showed different diversity and divergence levels. A preliminary screening allowed us to select 8 promising loci (40S, ATG2, BG, GroES, GTP, LiSH, PEX4 and TR) for the Z. pumila complex and 4 loci (COS26, GroES, GTP and HTS) for all other cycad genera.

本研究选取加勒比泽米演化支的多个个体及其他苏铁类属物种，旨在筛选适用于苏铁目（Cycadales）谱系地理学与系统发育研究的单拷贝核基因。研究采用两种策略筛选目标基因座：1）对拟南芥保守直系同源序列（COS）集进行tblastX搜索；2）针对GenBank数据库中的泽米属表达序列标签（EST）库，以拟南芥-杨树-葡萄-水稻共享单拷贝基因（APVO SSC）为参考进行tblastX搜索。第一种策略筛选得到30个基因座，第二种策略筛选得到16个基因座。两种策略均以匹配到的泽米属GenBank登录号序列作为查询序列，从苏铁属（Cycas）、云杉属（Picea）、松属（Pinus）物种及银杏（Ginkgo biloba）中检索高度相似的序列。检索并比对每个基因座的所有序列后，完成内含子预测以辅助引物设计。通过三轮聚合酶链式反应（PCR）检测旁同源基因座。最终共有29个基因座成功扩增为单一条带，其中20个被初步判定为单拷贝基因座。这些基因座展现出不同的多样性与分化水平。经初步筛选，为矮泽米复合群（Z. pumila complex）筛选出8个优质基因座（40S、ATG2、BG、GroES、GTP、LiSH、PEX4及TR），为其余所有苏铁类属筛选出4个优质基因座（COS26、GroES、GTP及HTS）。