报春苣苔属是典型的喀斯特岩溶洞穴植物，具有非常丰富的物种多样性和特有性，是研究喀斯特植物适应性进化和物种形成的理想模式。

中国科学院华南植物园植物科学研究中心博士冯超等在研究员康明的指导下，以报春苣苔属植物为材料，率先搭建该属三个完整的叶绿体基因组并进行结构注释，提出两种高变异区段挖掘的新策略。策略一命名为Con_Sea：基于多物种叶绿体基因组的联配结果，找出真正的保守区和变异区（Con_Sea），并开发新的叶绿体标记。结合生信分析及实验验证，研究发现基于Con_Sea区域开发的叶绿体新标记的多态性程度高于传统的基于非编码区开发的标记。策略二简称SACRing：直接从PE RAD-Seq数据中分离并组装出简化叶绿体基因组，并基于各样本叶绿体基因组的共有区段，进行群体遗传或系统发育研究。研究人员采用三个自然群体的21个个体的RAD真实数据，进一步证实了该方法的可行性。基于该流程组装的简化叶绿体基因组Contig片段可覆盖完整基因组的一半，变异位点可有效区分三个自然群体的不同个体，并满足群体遗传研究要求。为方便无生信或编程背景的研究人员使用，这两种策略均已整合成perl及bash脚本（https:/github.com/scbgfengchao/）。该研究为从非模式植物中大规模获取叶绿体基因组信息提供了新思路，研究开发的分子标记将进一步促进报春苣苔属乃至苦苣苔科的系统发育研究。

研究工作得到国家自然科学基金-广东联合基金重点项目和国家自然科学基金青年基金等的资助。相关研究结果在线发表在BMC Evolutionary Biology上。（来源：中国科学院华南植物园）

The complete chloroplast genome of Primulina and two novel strategies for development of high polymorphic loci for population genetic and phylogenetic studies

Abstract

Background  Primulina Hance is an emerging model for studying evolutionary divergence, adaptation and speciation of the karst flora. However, phylogenetic relationships within the genus have not been resolved due to low variation detected in the cpDNA regions. Chloroplast genomes can provide important information for phylogenetic and population genetic studies. Recent advances in next-generation sequencing (NGS) techniques greatly facilitate sequencing whole chloroplast genomes for multiple individuals. Consequently, novel strategies for development of highly polymorphic loci for population genetic and phylogenetic studies based on NGS data are needed.

Methods  For development of high polymorphic loci for population genetic and phylogenetic studies, two novel strategies are proposed here. The first protocol develops lineage-specific highly variable markers from the true high variation regions (Con_Seas) across whole cp genomes, instead of traditional noncoding regions. The pipeline has been integrated into a single perl script, and named "Con_Sea_Identification_and_PIC_Calculation". The second method assembles chloroplast fragments (poTs) and sub-super-marker (CpContigs) through our "SACRing" pipeline. This approach can fundamentally alter the strategies used in phylogenetic and population genetic studies based on cp markers, facilitating a transition from traditional Sanger sequencing to RAD-Seq. Both of these scripts are available at https://github.com/scbgfengchao/.

Results  Three complete Primulina chloroplast genomes were assembled from genome survey data, and then two novel strategies were developed to yield highly polymorphic markers. For experimental evaluation of the first protocol, a set of Primulina species were used for PCR amplification. The results showed that these newly developed markers are more variable than traditional ones, and seem to be a better choice for phylogenetic and population studies in Primulina. The second method was also successfully applied in population genetic studies of 21 individuals from three natural populations of Primulina.

Conclusions  These two novel strategies may provide a pathway for similar research in other non-model species. The newly developed high polymorphic loci in this study will promote further the phylogenetic and population genetic studies in Primulina and other genera of the family Gesneriaceae.

原文链接：https://bmcevolbiol.biomedcentral.com/articles/10.1186/s12862-017-1067-z