Abstract
BackgroundPrimula species are important early spring garden plants with a centre of diversity and speciation in the East Himalaya-Hengduan Mountains in Western China. Studies on population genetics, speciation and phylogeny of Primula have been impeded by a lack of genomic resources. In the present study, we sequenced the transcriptomes of two closely related primrose species, Primula poissonii and Primula wilsonii, using short reads on the Illumina Genome Analyzer platform.ResultsWe obtained 55,284 and 55,011 contigs with N50 values of 938 and 1,085 for P. poissonii and P. wilsonii, respectively, and 6,654 pairs of putative orthologs were identified between the two species. Estimations of non-synonymous/synonymous substitution rate ratios for these orthologs indicated that 877 of the pairs may be under positive selection (Ka/Ks > 0.5), and functional enrichment analysis revealed that significant proportions of the orthologs were in the categories DNA repair, stress resistance, which may provide some hints as to how the two closely related Primula species adapted differentially to extreme environments, such as habitats characterized by aridity, high altitude and high levels of ionizing radiation. It was possible for the first time to estimate the divergence time between the radiated species pair, P. poissonii and P. wilsonii; this was found to be approximately 0.90 ± 0.57 Mya, which falls between the Donau and Gunz glaciation in the Middle Pleistocene. Primers based on 54 pairs of orthologous SSR-containing sequences between the two Primula species were designed and verified. About half of these pairs successfully amplified for both species. Of the 959 single copy nuclear genes shared by four model plants (known as APVO genes), 111 single copy nuclear genes were verified as being present in both Primula species and exon-anchored and intron-spanned primers were designed for use.ConclusionWe characterized the transcriptomes for the two Primula species, and produced an unprecedented amount of genomic resources for these important garden plants. Evolutionary analysis of these two Primula species not only revealed a more precise divergence time, but also provided some novel insights into how differential adaptations occurred in extreme habitats. Furthermore, we developed two sets of genetic markers, single copy nuclear genes and nuclear microsatellites (EST-SSR). Both these sets of markers will facilitate studies on the genetic improvement, population genetics and phylogenetics of this rapidly adapting taxon.
Highlights
Primula species are important early spring garden plants with a centre of diversity and speciation in the East Himalaya-Hengduan Mountains in Western China
Contig with lengths between 200 and 500 bp were overrepresented, making up about 56% of the total number of contigs for P. poissonii, and 53% for P. wilsonii, the most abundant size class was 500–1000 bp, constituting about 24% and 24% of the total, respectively (Figure 1)
Orthologous contigs, substitution rates, and transcriptome divergence between two Primula species We identified 28,482 pairs of putative orthologous contigs between P. poissonii and P. wilsonii using the reciprocal best hit method with BLASTN algorithm
Summary
Speciation and phylogeny of Primula have been impeded by a lack of genomic resources. Due to the lack of availability of molecular markers for rapidly evolving taxa, especially from nuclear genome, most plant molecular systematic studies on adaptive radiation have hitherto failed to provide resolved phylogenies. Transcriptome analysis is an effective way to study gene expression in specific tissues at specific time, and it provides unprecedented opportunities to address comparative genomic-level questions for nonmodel organisms. Comparative RNA-sequencing studies between closely related species can in principle provide additional genomic resources such as genusspecific SSR primers or single copy nuclear gene primers, and give information about the processes of speciation or adaptive evolution, e.g. divergence time estimations, or detection of adaptive loci
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