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

Chao Feng, Meizhen Xu, Chen Feng, Eric J. B. von Wettberg, Ming Kang

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. 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/ . 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 Primulin a. The second method was also successfully applied in population genetic studies of 21 individuals from three natural populations of Primulina. 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.