Comparative transcriptome analysis and marker development of two closely related Primrose species (Primula poissonii and Primula wilsonii)
© Zhang et al.; licensee BioMed Central Ltd. 2013
Received: 13 December 2012
Accepted: 11 May 2013
Published: 14 May 2013
Primula 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.
We 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.
We 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.
Adaptive radiation, ‘the rise of a diversity of ecological roles and attendant adaptations in different species within a lineage’ is one of the most important processes bridging the gap between ecology and evolution . Usually, the genetic divergence between species within adaptive radiations is very small, and only a handful of genes with large effects are responsible for differences in ecologically significant traits and reproductive isolation between species. 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. The same is true for speciation studies, which rely heavily on there being sufficient intraspecific genetic variation. Moreover, we still have little understanding of how divergent natural selection may have acted on the genomes of such species within the short evolutionary time span since their common ancestor .
Transcriptome analysis is not only an effective way to study gene expression in specific tissues at specific time, and it also provides unprecedented opportunities to address comparative genomic-level questions for non-model organisms. RNA-sequencing (RNA-seq) is an efficient new technology for large scale transcriptome investigations. With the rapid development of next-generation sequencing (NGS), RNA-sequencing becomes more efficient and less expensive, and is increasingly being used to study the evolutionary origins and ecology of non-model plants [3, 4]. For instance, a large number of microsatellite markers or single-copy nuclear genes in yam (Dioscorea alata) , buckwheat (Fagopyrum)  and big sagebrush (Artemisia tridentata)  have been identified by making use of RNA-sequencing. Since RNA-sequencing is still somewhat expensive at present, few RNA-seq studies to date have included for more than one species at the same time [6, 7]. However, comparative RNA-sequencing studies between closely related species can in principle not only provide additional genomic resources such as genus-specific SSR primers or single copy nuclear gene primers, but also give information about the processes of speciation or adaptive evolution, e.g. divergence time estimations, or detection of adaptive loci.
Primula with around 430 species, is one of the three great garden genera , and southwestern China, in which ca. 187 species of the genus are distributed, is its diversity centre [9, 10]. In this region Primula shows a typical patterns of adaptive evolution and explosive speciation; however, research has been hampered by the fact that few Primula genomic resources are available. Up to now, only a few SSR primers from the three Primula species P. vulgaris, P. obconica, and P. sieboldii have been developed [11–13], and only one large EST collection, consisting of 5,651 ESTs generated from Primula sieboldii were available . Paucity of genetic data such as genome sequences, transcriptome sequences and associated molecular markers has made Primula breeding or evolutionary analysis a challenging task.
Primula section Proliferae Pax, which contains ca. 25–30 species and is centred on southwestern China, is regarded as a taxonomically well-known group circumscribed by possession of numerous whorls of flowers . Within this section, Primula wilsonii and P. poissonii are two closely related species with very similar morphological characters, and the two diagnostic characters used to distinguish them are the corolla structure and the aromatic fragrance of fresh leaves; for P. wilsonii, the fresh leaves are fragrant and corolla limbs are slightly opened, whereas, P. poissonii has no obvious fragrance and widely opened corolla limbs . These closely related species represent a useful resource for addressing two questions: how did Primula species in southwestern China radiate within a short period of time, and what was the driving force underlying the process of rapid adaptive evolution? As the first step towards answering these questions, in this study, we obtained transcriptomes for Primula poissonii and P. wilsonii using the Illumina platform, and carried out a comprehensive analysis of them. Our aims were to 1) characterize the transcriptomes of P. poissonii and P. wilsonii, and increase the genetic resources available for Primula breeding or evolutionary analysis; 2) determine the evolutionary dynamics of the two species, including obtaining a divergence time estimation, signatures of adaptive evolution between the two species; and 3) discover genus-specific SSR markers and single-copy nuclear gene markers from both species.
Results and discussion
De novoassembly and functional annotation of contigs
Summary of assembly and annotation results for P. poissonii and P. wilsonii using Trinity
Total number of reads
55,056,996 × 2
55,468,564 × 2
Total number of contigs
Mean length of contigs
Median length of contigs
N50 value of contigs
Length range of contigs
200 ~ 16,932
200 ~ 12,384
Contigs with BLASTX hit
Contigs with annotation
Orthologous contigs, substitution rates, and transcriptome divergence between two Primulaspecies
We identified 28,482 pairs of putative orthologous contigs between P. poissonii and P. wilsonii using the reciprocal best hit method with BLASTN algorithm. After incorporating the Vitis peptide sequences , 7,006 pairs of putative orthologs were obtained using the RBM triangulation method . This reduction in ortholog numbers was caused mainly by the exclusion of the relatively young orthologs specific to Primula, which were discarded as being low similarity to Vitis. After excluding alignments with unexpected stop codons, lengths less than 150 bp or Ks values above 0.1, 6,654 pairs of orthologs were retained for subsequent analysis.
Using the Vitis proteins as reference, the coding regions of 6,654 pairs of orthologs from P. poissonii and P. wilsonii were extracted, in some cases, 5′-UTR (1,315 pairs of orthologs) or 3′-UTR (2,051 pairs of orthologs) were also determined. The average genetic divergence of coding regions between the two Primula species is 0.011 ± 0.007 according to the K2P model. The genetic divergence between the two species is 0.019 ± 0.017 for 5′-UTR and 0.018 ± 0.013 for 3′-UTR regions. The accelerated substitution rate observed in the 5′UTR and 3′ UTR relative to the coding region, is indicative of relaxed functional constraint on the evolution of the UTR than on the coding region at the genome level, which is consistent with the evidence from other model species-pairs .
Among the 6,654 pairs of orthologs between P. poissonii and P. wilsonii, 165 pairs were identical, 1,327 pairs had only either synonymous or nonsynonymous substitutions, and 5,162 pairs had both types of substitutions, for which the Ka/Ks ratio were calculated. The mean values of Ka, Ks, and the Ka/Ks ratio of all orthologous pairs were 0.007 ± 0.005, 0.027 ± 0.017 and 0.322 ± 0.324, respectively. Of the 5,162 pairs of orthologs, 233 pairs with a Ka/Ks value > 1 were found. Taking a more appropriate threshold of 0.5 for the Ka/Ks ratio as an indicator of positive selection , 644 pairs with a Ka/Ks value between 0.5 and 1 were also found.
The peak synonymous rates (Ks) for orthologous transcript pairs can be used to estimate the times of divergence between species. To obtain a rough estimate of the divergence time (T) between P. poissonii and P. wilsonii, we followed the simple formula: T = K/2r , where r is the mean rate of synonymous substitution, and is considered to be 1.5E-8 substitutions/synonymous site/year for all dicots ; K is genetic divergence expressed in terms of mean number of synonymous substitutions between orthologs. The age of the speciation event between P. poissonii and P. wilsonii is approximately 0.90 ± 0.57 Mya, which falls between the Donau and Gunz glaciation in the Middle Pleistocene. Bearing in mind disputes about the substitution rate , this divergence time is only an appropriate estimate based on the coding region of orthologous genes, nonetheless, it is useful because there is as yet no adequate fossil dating the divergence of the two Primula species.
Functions under positive selection and implications for adaptive evolution between two Primulaspecies
Gene Ontology terms significantly over-represented in the test dataset versus the reference set
P-value (Fisher’s exact test)
Frequency in test set
Frequency in reference set
thiolester hydrolase activity
response to topologically incorrect protein
regulation of ARF protein signal transduction
glycine metabolic process
response to hydrogen peroxide
ribonucleoprotein complex biogenesis
cellular component biogenesis at cellular level
response to oxidative stress
response to DNA damage stimulus
cellular component biogenesis
RNA methyltransferase activity
ubiquitin thiolesterase activity
NADPH: quinone reductase activity
thiolester hydrolase activity
large ribosomal subunit
Partial list of candidate orthologs under positive selection between P. wilsonii and P. poissonii
Arabidopsis thaliana gene accession
KU70, atp-dependent dna helicase 2 subunit ku70
KU80, atp-dependent dna helicase 2 subunit ku80-like
SMC5, structural maintenance of chromosomes protein
ETG1, mini-chromosome maintenance complex-binding
ROR1, replicon protein a2
GPX7, glutathione peroxidase
ARP3, actin-related protein 3
LEW1, nogo-b receptor-like
SBP1, s-ribonuclease binding protein 1
FER2, ferritin subunit precursor
AP3, mads-domain transcription factor
MYB12, transcription factor myb12
Some orthologs related to abiotic stress were also found to be positively selected. For example, pw23141 is homologous to GPX7, which regulates cellular photooxidative tolerance and immune response ; pw12283 is homologus to ARP3, related to light-induced stomatal opening ; pw37795 is homologous to LEW1, the product of which catalyzes the biosynthesis of dolichol  and confers acclimation to drought stress, which may partially explain why the two Primula species were able to inhabit habitats with different level of aridity; pw10616 is homologous to SBP1 and pw09970 is homologous to FER2, which are involved in the cadmium stress  and iron deficiency , respectively; these results shed further light on how the two Primula species differentially adapted to extreme environments. In addition, two positively selected genes are worth notice, one gene pw13953, is homologous to AP3, a key component in the ABC mode of flower development , and may provide a clue about the origin of the differences in corolla structure between P. poissonii and P. wilsonii; the other gene pw42171, is homologous to MYB12, which functions as a R2R3-MYB transcription factor in phenylpropanoid biosynthesis , also may give some hints on the leaf fragrance differentiation between P. poissonii and P. wilsonii.
Overall, in this study, we detected a dozens of gene under positive selection between the Primula species pairs, and these findings will not only shed light on how differentiations between two Primula species occurs, but also open the door to increased understanding of how plants living in plateau environments adapt to different characteristics of high altitude, such as strong radiation, aridity and so on.
Identification of microsatellites and single copy genes
Summary of microsatellite loci in Primula poissonii and P. wilsonii
Contigs containing SSRs
SSRs (repetitive units >16 bp)
One noteworthy fact about the SSR primer development based on the Illumina platform is the lower proportion of contigs suitable for primer design compared with Sanger sequencing, in our study, 367 out of the 421 pairs of contigs with SSRs were excluded from primer design because they had insufficient flanking regions caused by NGS assembly algorithms or sequencing . As an alternative, the 454/Roche sequencing platform, which delivers longer reads, has promise as a way of reducing bias.
Using the APVO gene sets  to carry out TBLASTN queries against our Primula dataset orthologs, 612 of the APVO genes were found to give hits against orthologous contigs between P. poissonii and P. wilsonii over at least 600 bp; these are most likely to be single copy genes in the two Primula species. When we set a threshold identity of 75% with Arabidopsis thaliana and specified facultative intron sizes not less than 300 bp in Arabidopsis thaliana, we were successful in obtaining primers for 111 of the 612 APVO genes (Additional file 1: Table S3); we randomly selected four primer pairs to test, and of these, three of the pairs amplified successfully and when the products were sequenced directly, they yielded the expected gene model, and only one primer pair failed due to the presence of extremely long intron variants, which made the products unsuitable for Sanger sequencing.. The availability from this study of dozens of single copy nuclear genes with heterogeneous rates of variation, will undoubtedly facilitate phylogeny resolution for the radiative Primula species, and open a doorway to understanding the dynamics of speciation using a population genetics approach.
We developed sets of two types of molecular markers, and these two widely-used marker types, each of which has its own advantages, were applied for characterizing population structure, parentage analysis, genotyping, gene flow inferences and phylogenetic construction. The large number of novel single nuclear gene will greatly increase the resolution of phylogenetic reconstruction for this adaptive taxon. Moreover, these markers with their diverse evolutionary rates will provide unprecedented opportunities to answer the following important questions: What demographic histories underlie the phylogeographic patterns of Primula species? Which evolutionary forces drive the explosive radiation of Primula species in the extreme habitats?
In this study, we characterized the transcriptomes for the two Primula species, and obtained an unprecedented amount of genomic resources for these important garden plants. Evolutionary analysis of these two species not only yielded a more precise divergence time, but also provided some novel insights into how differential adaptations occurred in extreme habitats. In addition, we developed two sets of genetic markers of popular types, single copy nuclear genes and nuclear microsatellites (EST-SSR). These marker sets will facilitate studies on the genetic improvement, population genetics and phylogenetics of this rapidly adapting taxon.
Plant material, RNA extraction and sequencing
Primula poissonii (2n = 22) is widespread in the mountain areas of northwest Yunnan and southwest Sichuan, China. It inhabits alpine meadows at an altitude of 3200–3500 m. Primula wilsonii (2n = 22) is distributed in central Yunnan and grows on open lands in evergreen broad-leaved forest at an altitude of ca. 2500 m . During July 2011, we sampled P. poissonii from Zhongdian plateau in northwest Yunnan (28°06'55.24"N, 99°47'48.49''E, alt. 3314 m), and P. wilsonii from Ailao Mountain in central Yunnan (24°32'34.47''N, 101°01'41.48''E, alt. 2450 m), respectively, and fresh leaves and whole flowers of multiple individual plants for both species were stored in RNAlater solution (Takara Biotechnology Co. Ltd., Dalian, China) to preserve the RNA state for use immediately after harvesting. After mixing an approximately equivalent weight of fresh leaves and flowers, total RNA was extracted using a modified CTAB method and precipitated with 5 M LiCl2 at -20°C overnight, and the resulting RNA pellets were suspended in about 100 μl DEPC-treated water. After assessing RNA quality by means of electrophoresis and an Eppendorf AG 2231 BioPhotometer Plus (Hamburg, Germany), quantified total RNA (concentration ≥ 100 ng/μL; rRNA ratio ≥ 1.5) were delivered to The Beijing Genome Institute (Shenzhen, China) for further treatments. The cDNA library for transcriptome sequencing was prepared using a cDNA Synthesis Kit (Illumina Inc., San Diego, CA, USA) following the manufacturer’s recommendations. The cDNA library was then sequenced using a HiSeq2000 (Illumina Inc, San Diego, CA, USA) to obtain short sequences of 90 bp from both ends of each cDNA.
Sequence cleaning, assembly, contig annotation
Raw reads were firstly subjected to cleaning by removal of adaptors, reads with too many Ns, and reads with quality scores lower than 20. The cleaned reads were assembled de novo using Trinity  with the default parameters and contigs with length less than 200 bp were discarded due to a low annotation rate . The filtered reads for P. poissonii and P. wilsonii were deposited in the NCBI Sequence Read Archive (SRA) under the accession number SRR629689 and SRR640158, respectively.
Functional annotation was implemented using the online program Blast2GO v.2.6.0 . All the assembled contigs were firstly subjected to BLASTX against the NCBI’s non-redundant protein database with an E-value threshold of 1E-6. The predicted gene name for each contig was assigned according to the best BLASTX hit. Gene Ontology  terms were retrieved from BLASTX hits at E-value threshold 1E-6. Finally, the distributions of level-2 GO terms for all contigs were plotted with the program WEGO . In addition, we download the Vitis vinfera proteome , and queried against all the assembled contigs using TBLASTN with an E-value threshold of 1E-10 .
Identification of orthologous contigs and estimation of substitution rates
The reciprocal best matches (RBM) method  is widely used for identifying orthologs, and a modified version program RBM triangulation , allows a third species to be incorporated, which can increase reliability and detect large numbers of conserved orthologs, so we used the following approach for this study. First, we used BLASTN with the RBM method to find orthologs between the two Primula species setting the E-value cutoff at 1E-10. Next, to avoid misspecification caused by the absence of a true ortholog from either Primula species, the third species Vitis was added as positive control. All the reciprocal best hit orthologs were subjected to BLASTX against the Vistis peptide sequences at a threshold E-value of 1E-10, and only those pairs of orthologs with the same reciprocal best hit with Vitis were kept.
With the Vitis peptide sequence as reference, 5′UTR or 3′UTR sequences for some Primula contigs were determined. According to the best-match Vitis peptide sequences, the coding region sequences (CDS) of all the contigs were extracted with custom Perl scripts, and subsequently aligned using the MUSCLE algorithm  implemented in MEGA5 . Alignments with unexpected stop codons, or less than 150 base pairs in length, were discarded after checking manually. For the remaining orthologs, synonymous substitution rates (Ks) and non-synonymous rates (Ka) were estimated using a maximum-likelihood method  implemented by yn00 in the PAML toolkit . For the closely related species pair, P. poissonii and P. wilsonii, orthologs with Ks > 0.1 were excluded to avoid paralogs . Divergence in the CDS sites and UTRs were calculated using the K2P model with a custom Perl script.
On the basis of the Ka/Ks value, setting a threshold at 0.5, the orthologs were sub-categorized into two dataset: a test set with Ka/Ks above 0.5, and a reference dataset with Ka/Ks value less than 0.5. The significance of the difference in GO term abundance between the two datasets was tested using the Fisher’s exact test with the GOSSIP package  implemented in BLAST2GO V.2.6.0 .
Simple sequence repeats (SSRs) identification and mining of single copy nuclear genes
The program MISA (http://pgrc.ipk-gatersleben.de/misa/)  was used to identify and localize microsatellite motifs in the two Primula species, and only those contigs with motifs containing at least five repeats were selected. The alignments of 6,631 pairs of orthologs were extracted as the input file for the MISA program. Using the detailed information on SSR loci obtained from the output of the MISA program, primers for each SSR-containing sequence with a repetitive at least 16 bp in length were designed with Program Primer Premier 5 (PREMIER Biosoft Int., Palo Alto, CA). To validate the SSRs identified in silico identified SSRs, primer pairs shared between the two Primula species were synthesized (Invitrogen Trading Shanghai Co., Ltd, Shanghai, China), and amplified with one individual of each species as templates. PCRs were performed in a 25 μl volume containing 25 ng of template genomic DNA. The PCR reactions were carried out under the following conditions: initial denaturation at 95°C for 2 min, 35 cycles at an annealing temperature ranging from 45 ~ 60°C for 50 s, and a final extension at 72°C for 10 min. The PCR products were checked on 1.5% agarose gel.
Duarte et al. (2010) identified about 959 sets of single copy nuclear genes shared by Arabidopsis, Populus, Vitis and Oryza (known as APVO genes). We extracted the protein sequences encoded by the APVO gene from the TAIR10 database and queried them against the Primula orthologous EST database using TBLASTN  with a threshold E-value of 1E-10. All the queries with hits were considered to be single copy nuclear genes in the Primula species, and the consensus contigs of best- matched orthologous pairs of the two Primula species were extracted for degenerate primer design using the SeqMan 5.0 program (DNASTAR Inc, Madison, WI, USA). The consensus sequences were queried against the Arabidopsis thaliana protein database using BLASTX with an identity threshold of above 0.75, then subjected to exon-anchoring and intron-spanning primer design according to the corresponding Arabidopsis thaliana gene models with Program Primer Premier 5 (PREMIER Biosoft Int., Palo Alto, CA, USA). To validate these primers, four of them were randomly chosen to amplify with genomic samples from one individual of each of the two Primula species, and the products were sequenced.
We thanks Dr Nolan Kane (University of British Columbia, Canada) for providing data on substitution rate estimations for Asteraceae, thanks are also given to the members of the Adaptive Evolution Lab in Sun Yet-sen University (Guangzhou, China) for helps with bioinformatics analysis. We’d really like to thank Dr. Joelle Hoggan for helping us polish the manuscript. This work is financially supported by the National Natural Science Foundation of China (Grant Nos. 31170205, 31270009).
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