Key Laboratory of Chinese Medicine Resources Conservation, State Administration of Traditional Chinese Medicine of China, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, 100193, China.
BMC Plant Biol. 2022 Aug 25;22(1):412. doi: 10.1186/s12870-022-03682-x.
Cistanche is an important genus of Orobanchaceae, with critical medicinal, economic, and desertification control values. However, the phylogenetic relationships of Cistanche genus remained obscure. To date, no effective molecular markers have been reported to discriminate effectively the Cistanche closely related species reported here. In this study, we obtained and characterized the plastomes of four Cistanche species from China, to clarify the phylogenetic relationship within the genus, and to develop molecular markers for species discrimination. RESULTS: Four Cistanche species (Cistanche deserticola, Cistanche salsa, Cistanche tubulosa and Cistanche sinensis), were deep-sequenced with Illumina. Their plastomes were assembled using SPAdes and annotated using CPGAVAS2. The plastic genomes were analyzed in detail, finding that all showed the conserved quadripartite structure (LSC-IR-SSC-IR) and with full sizes ranging from 75 to 111 Kbp. We observed a significant contraction of small single copy region (SSC, ranging from 0.4-29 Kbp) and expansion of inverted repeat region (IR, ranging from 6-30 Kbp), with C. deserticola and C. salsa showing the smallest SSCs with only one gene (rpl32). Compared with other Orobanchaceae species, Cistanche species showed extremely high rates of gene loss and pseudogenization, as reported for other parasitic Orobanchaceae species. Furthermore, analysis of sequence divergence on protein-coding genes showed the three genes (rpl22, clpP and ycf2) had undergone positive selection in the Cistanche species under study. In addition, by comparison of all available Cistanche plastomes we found 25 highly divergent intergenic spacer (IGS) regions that were used to predict two DNA barcode markers (Cis-mk01 and Cis-mk02 based on IGS region trnR-ACG-trnN-GUU) and eleven specific DNA barcode markers using Ecoprimer software. Experimental validation showed 100% species discrimination success rate with both type of markers.
Our findings have shown that Cistanche species are an ideal model to investigate the structure variation, gene loss and pseudogenization during the process of plastome evolution in parasitic species, providing new insights into the evolutionary relationships among the Cistanche species. In addition, the developed DNA barcodes markers allow the proper species identification, ensuring the effective and safe use of Cistanche species as medicinal products.
肉苁蓉是列当科的一个重要属，具有重要的药用、经济和防治荒漠化价值。然而，肉苁蓉属的系统发育关系仍然不清楚。迄今为止，还没有有效的分子标记能够有效地鉴别这里报道的肉苁蓉近缘种。本研究对来自中国的 4 种肉苁蓉进行了质体基因组的测序和特征分析，以阐明属内的系统发育关系，并开发用于物种鉴别分子标记。
用 Illumina 对 4 种肉苁蓉（肉苁蓉、盐生肉苁蓉、管花肉苁蓉和肉苁蓉）进行了深度测序。使用 SPAdes 组装它们的质体基因组，并使用 CPGAVAS2 进行注释。详细分析了这些质体基因组，发现它们都具有保守的四分体结构（LSC-IR-SSC-IR），全基因组大小在 75-111 Kbp 之间。我们观察到小单拷贝区（SSC，0.4-29 Kbp）显著收缩和反向重复区（IR，6-30 Kbp）扩张，其中 C. deserticola 和 C. salsa 具有最小的 SSC，只有一个基因（rpl32）。与其他列当科物种相比，肉苁蓉物种表现出极高的基因丢失和假基因化率，这与其他寄生列当科物种的情况相似。此外，对蛋白质编码基因序列分歧的分析表明，在所研究的肉苁蓉物种中，三个基因（rpl22、clpP 和 ycf2）经历了正选择。此外，通过比较所有可用的肉苁蓉质体，我们发现 25 个高度分化的基因间 spacer（IGS）区域，可用于预测两种 DNA 条形码标记物（基于 IGS 区域 trnR-ACG-trnN-GUU 的 Cis-mk01 和 Cis-mk02）和使用 Ecoprimer 软件的 11 种特定 DNA 条形码标记物。实验验证表明，两种标记物的物种鉴别成功率均为 100%。
我们的研究结果表明，肉苁蓉属是研究寄生物种质体基因组进化过程中结构变异、基因丢失和假基因化的理想模型，为肉苁蓉属物种的进化关系提供了新的见解。此外，开发的 DNA 条形码标记物可用于准确鉴定物种，确保肉苁蓉属作为药用植物的有效和安全使用。
