Abstract
BackgroundPrimulina 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.MethodsFor 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/.ResultsThree 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.ConclusionsThese 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.
Highlights
Primulina Hance is an emerging model for studying evolutionary divergence, adaptation and speciation of the karst flora
For restrictionsite associated DNA sequences (RAD-Seq) of the three populations, DNA was first treated with restriction enzyme EcoR I (Takara, China), and several standard steps were performed, from the addition of sequencing adapters, interruption of enzyme digestion products, to break into smaller random pieces, and repairing the end based on existing protocols [18]
The results of population genetic analysis of the cp concatenated sequences from consistent CpContigs are similar to that from consistent poTs (Fig. 6c and d). These results indicated that around half of Primulina cp genome could be directly assembled from RAD-Seq data through our Sub-assembly of chloroplast from PE RAD-Seq (SACRing) pipeline
Summary
Primulina Hance is an emerging model for studying evolutionary divergence, adaptation and speciation of the karst flora. By comparing chloroplast genomes of 13 angiosperm lineages, Shaw et al, [5, 6] identified a set of 34 non-coding regions that ranked highest in their potentially informative characters (PIC), an index which is counted by the sum of nucleotide substitutions, indel and inversions between each of two ingroup species and between an ingroup species and an outgroup species. This set of most variable non-coding regions is widely used in plant evolutionary biology and systematics studies. These findings imply that lineage-specific screening is needed for the identification of the most highly variable markers in different clades
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