Data_Sheet_1_Transcriptome Sequencing and Development of Novel Genic SSR Markers From Pistacia vera L..xlsx

In this study, we aimed to develop novel genic simple sequence repeat (eSSR) markers and to study phylogenetic relationship among Pistacia species. Transcriptome sequencing was performed in different tissues of Siirt and Atl cultivars of pistachio (Pistacia vera). A total of 37.5-Gb data were used in the assembly. The number of total contigs and unigenes was calculated as 98,831, and the length of N50 was 1,333 bp after assembly. A total of 14,308 dinucleotide, trinucleotide, tetranucleotide, pentanucleotide, and hexanucleotide SSR motifs (4–17) were detected, and the most abundant SSR repeat types were trinucleotide (29.54%), dinucleotide (24.06%), hexanucleotide (20.67%), pentanucleotide (18.88%), and tetranucleotide (6.85%), respectively. Overall 250 primer pairs were designed randomly and tested in eight Pistacia species for amplification. Of them, 233 were generated polymerase chain reaction products in at least one of the Pistacia species. A total of 55 primer pairs that had amplifications in all tested Pistacia species were used to characterize 11 P. vera cultivars and 78 wild Pistacia genotypes belonging to nine Pistacia species (P. khinjuk, P. eurycarpa, P. atlantica, P. mutica, P. integerrima, P. chinensis, P. terebinthus, P. palaestina, and P. lentiscus). A total of 434 alleles were generated from 55 polymorphic eSSR loci with an average of 7.89 alleles per locus. The mean number of effective allele was 3.40 per locus. Polymorphism information content was 0.61, whereas observed (Ho) and expected heterozygosity (He) values were 0.39 and 0.65, respectively. UPGMA (unweighted pair-group method with arithmetic averages) and STRUCTURE analysis divided 89 Pistacia genotypes into seven populations. The closest species to P. vera was P. khinjuk. P. eurycarpa was closer P. atlantica than P. khinjuk. P. atlantica–P. mutica and P. terebinthus–P. palaestina pairs of species were not clearly separated from each other, and they were suggested as the same species. The present study demonstrated that eSSR markers can be used in the characterization and phylogenetic analysis of Pistacia species and cultivars, as well as genetic linkage mapping and QTL (quantitative trait locus) analysis.

本研究旨在开发新型基因内简单序列重复（genic simple sequence repeat, eSSR）标记，并探究黄连木属（Pistacia）物种间的系统发育关系。研究对开心果（Pistacia vera）的Siirt与Atl两个栽培品种的不同组织开展转录组测序，共使用37.5 Gb的测序数据进行组装。组装后共获得98831个重叠群（contigs）与单基因簇（unigenes），N50长度为1333 bp。共检测到14308个二核苷酸、三核苷酸、四核苷酸、五核苷酸及六核苷酸SSR基序（重复次数范围为4~17），其中丰度最高的SSR重复类型依次为三核苷酸（29.54%）、二核苷酸（24.06%）、六核苷酸（20.67%）、五核苷酸（18.88%）与四核苷酸（6.85%）。
本研究随机设计250对引物，在8个黄连木属物种中进行扩增验证，其中233对可在至少1个供试物种中扩增得到聚合酶链式反应（polymerase chain reaction, PCR）产物。选取可在所有8个供试黄连木属物种中均实现扩增的55对引物，对11个P. vera栽培品种及隶属于9个黄连木属物种的78份野生黄连木基因型进行分型分析，所涉物种包括P. khinjuk、P. eurycarpa、P. atlantica、P. mutica、P. integerrima、P. chinensis、P. terebinthus、P. palaestina及P. lentiscus。
55个多态性eSSR位点共扩增得到434个等位基因，平均每个位点含7.89个等位基因，平均有效等位基因数为3.40。多态性信息含量（polymorphism information content, PIC）为0.61，观测杂合度（observed heterozygosity, Ho）与期望杂合度（expected heterozygosity, He）分别为0.39与0.65。非加权配对算术平均法（unweighted pair-group method with arithmetic averages, UPGMA）聚类与群体结构（STRUCTURE）分析将89份黄连木基因型划分为7个类群。与P. vera亲缘关系最近的物种为P. khinjuk；相较于P. khinjuk，P. eurycarpa与P. atlantica的亲缘关系更近。P. atlantica与P. mutica、P. terebinthus与P. palaestina的物种对无法明确区分，研究提示二者应为同一物种。
本研究证实，eSSR标记可用于黄连木属物种与栽培品种的分型鉴定及系统发育分析，同时也可应用于遗传连锁图谱构建与数量性状位点（quantitative trait locus, QTL）定位分析。