Transcriptome characterisation of Pinus tabuliformis and evolution of genes in the Pinusphylogeny
© Niu et al.; licensee BioMed Central Ltd. 2013
Received: 8 November 2012
Accepted: 15 April 2013
Published: 18 April 2013
The Chinese pine (Pinus tabuliformis) is an indigenous conifer species in northern China but is relatively underdeveloped as a genomic resource; thus, limiting gene discovery and breeding. Large-scale transcriptome data were obtained using a next-generation sequencing platform to compensate for the lack of P. tabuliformis genomic information.
The increasing amount of transcriptome data on Pinus provides an excellent resource for multi-gene phylogenetic analysis and studies on how conserved genes and functions are maintained in the face of species divergence. The first P. tabuliformis transcriptome from a normalised cDNA library of multiple tissues and individuals was sequenced in a full 454 GS-FLX run, producing 911,302 sequencing reads. The high quality overlapping expressed sequence tags (ESTs) were assembled into 46,584 putative transcripts, and more than 700 SSRs and 92,000 SNPs/InDels were characterised. Comparative analysis of the transcriptome of six conifer species yielded 191 orthologues, from which we inferred a phylogenetic tree, evolutionary patterns and calculated rates of gene diversion. We also identified 938 fast evolving sequences that may be useful for identifying genes that perhaps evolved in response to positive selection and might be responsible for speciation in the Pinus lineage.
A large collection of high-quality ESTs was obtained, de novo assembled and characterised, which represents a dramatic expansion of the current transcript catalogues of P. tabuliformis and which will gradually be applied in breeding programs of P. tabuliformis. Furthermore, these data will facilitate future studies of the comparative genomics of P. tabuliformis and other related species.
Conifers are widely distributed globally as the largest and most diverse group of gymnosperms  that evolved independently from angiosperms >300 million years ago . Modern conifers are divided into eight families including 68 genera and 630 species, which form an integral part of the economy in many parts of the world . Chinese pine (Pinus tabuliformis Carr.) is a widespread indigenous conifer species and an economically and ecologically important hard pine in northern China [4, 5]. Because of its irreplaceable economic development and environmental protection status, a genetic improvement program for P. tabuliformis was initiated in the 1970s, and considerable progress has been made in many basic physiological aspects . The study of natural genetic variation in P. tabuliformis has traditionally been investigated using a common garden approach, whereas the pace of development of genomic resources has been slow, as only 288 P. tabuliformis entries are included in the NCBI database. Information regarding the genetic control of many important traits and fine-scale genetic variations is extremely limited, and more is needed given the renewed emphasis to accelerate the pace of P. tabuliformis breeding and shorten the breeding cycle.
Despite the economic and ecological importance of the genus Pinus, the progress of entire genome sequencing and associated marker development has been limited [6, 7]. Huge genomes with highly heterozygous and large amounts of repetitive DNA elements are the major obstacles towards sequencing the genomes of all Pinus spp. [8, 9]. The genome sizes of conifers are larger than those of most other plant species. The genome in all extant members of the genus Pinus is 18,000–40,000 Mbp . In contrast, several representative genera of angiosperm trees have genome sizes of 540–2,000 Mb . Therefore, researchers have focused on the transcribed part of the genome using dedicated technologies [6, 7]. Transcriptome analysis and construction of large-scale expressed sequence tag (EST) collections in pines are a promising means of providing genomic resources [2, 9, 11], as this technique produces expressed sequence portions of chromosomes at a fraction of the cost of sequencing the complete genome . It also facilitates the analysis of the transcribed part of the genome, which is not easy to predict from the entire genome . Next-generation sequencing is a viable and favourable alternative to Sanger sequencing and provides researchers with a relatively rapid and affordable option for developing genomic resources in non-model organisms [14–16]. The Roche 454 massively parallel pyrosequencing platform, GS FLX Titanium, can generate one million reads with an average read length of 400 bases at 99.5% accuracy per run [17, 18].
In addition to the discovery of new genes and investigations of gene expression, thousands of simple sequence repeats (SSRs), single nucleotide polymorphisms (SNPs) and insertions and deletions (Indels) have been detected in transcriptome data [6, 19]. It is possible to use these genome-wide and abundant markers to develop very dense genetic maps that can be applied to conduct marker-assisted selection breeding programs .
Moreover, the increasing availability of transcriptome data represents an excellent resource for comparative genomic analysis. Although there has been much work on the chloroplast DNA sequences (cpDNA) and mitochondria DNA sequences (mtDNA), based on phylogenetic analysis of Pinus[21–23], less emphasis has been placed on multi-gene phylogenetic analysis and on determination of how conserved genes and functions are maintained despite species divergence.
In the current study, we used the Roche 454 GS-FLX Titanium pyrosequencing platform to obtain a comprehensive transcriptome of P. tabuliformis from normalised cDNA libraries of adult trees (xylem, phloem, vascular cambium, needles, cones and strobili). As a result, thousands of molecular markers were characterised. Evolutionary studies based on these data and other shared transcriptome data of five pine species and one spruce species were conducted. These data provide compelling new insights into the transcriptome of P. tabuliformis and evolution of genes in the Pinus phylogeny.
Transcriptome sequencing and de novoassembly
Sequencing, assembly and data analysis
Raw results (after trimming)
Total number of reads
Total number of isotigs
Total read length (bp)
295 125 234
Total isotigs length (bp)
21 076 176
Minimum read length (bp)
Isotig N50 (bp)
Median read length (bp)
Maximum isotig length (bp)
Maximum read length (bp)
Mean read length (bp)
Number of singletons
GC content (%)
Total number of unigenes
The unigene coverage distribution revealed that most unigenes had a read-depth coverage <20-fold (Figure 1c, d). The steep decline in read-depth coverage suggests that cDNA normalisation was effective, which is typical for a normalised library . Isotig lengths were related to the number of sequences assembled into each isotig. The average unigene length exhibited a gradual increase with increasing read depth (Figure 1c, d).
Functional annotation of the transcriptome
Identification of SSRs, SNPs and Indels
Orthologue identification and functional characterisation between six conifer species
Large-scale transcriptome characterisations have been carried out for Pinus taeda, Pinus contorta, Pinus sylvestris and Pinus pinaster. The shared transcriptomes of Pinus in the PlantGDB and NCBI databases are valuable sources of information for multi-gene comparative and phylogenetic analyses .
Phylogenetic and speciation analysis
Evolutionary pattern of Pinusspp. genes
Transcriptome sequencing in Pinus spp.
Platform or approach
Reads or ESTs
GS XL R70
needles and conelets
Sanger and GS-FLX
3 968 794
Despite the fact that a large number of pine ESTs have been obtained from cDNA libraries based on traditional sequencing technology, the methods used were inefficient. Four P. taeda cDNA libraries were sequenced and yielded a total of 142,533 ESTs (Table 2); however, only one normalised cDNA library yielded 822,891 ESTs in P. tabuliformis (Table 1). Although traditional sequencing yields longer EST sequences, it has little advantage compared to new assembly technology based on the large-scale ESTs. Additionally, most previous studies used a cDNA library of one tissue (Table 2), whereas we used a normalised cDNA library comprising multiple tissues and individuals. These large-scale ESTs will provide more comprehensive pine transcriptome information and facilitate the assembly of Pinus spp. ESTs in the future.
Next-generation sequencing technology yields a large number of sequences at considerably lower costs compared to traditional sequencing methods, and, therefore, provides a valuable starting point to expedite analysis of less-studied species [18, 36, 37]. Normalised cDNA libraries were used to sample large numbers of transcripts to maximise sequence diversity. Next-generation sequencing of normalised libraries is more efficient than that of non-normalised libraries, particularly for rare transcripts . The capacity to deliver large numbers of gene-based markers from transcriptome sequencing projects is a major advantage of next-generation sequencing technology [18, 20, 36]. Because of cost and throughput, conventional markers such as restriction fragment length polymorphism and random amplified polymorphic DNA are being replaced with SSRs and SNPs . The genome-wide and abundant EST-based SSRs and SNPs/Indels markers obtained by next-generation sequencing represent an effective approach to marker discovery in many plant species, as these markers facilitate generation of dense genetic maps and have the advantage of higher cross-species transferability [6, 39–41]. However, relevant studies in Pinus spp. are limited. In this study, 724 distinct EST-SSR loci and more than 92,000 SNPs/InDels were identified. It is possible to use these markers in a broad range of applications, including genetic mapping, genotype identification, marker-assisted selection breeding, and molecular tagging of genes. Among the EST-derived SSRs, tri-nucleotide repeat units were predominant. Considering the importance of maintaining reading frames to generating a polypeptide within a partially or fully active, it is no surprise that this observation is common for tri-nucleotide expansions (or their multiples) within translated regions [6, 42, 43]. As usual, comparisons of P. tabuliformis transcriptome SNPs show an excess of transitional over transversional substitutions. Similarly, A and T were the most frequent insertion and deletion types of Indels. Part of this bias is due to the relatively high rate of mutation of methylated cytosines to thymines [44, 45].
Comparative phylogenetic analysis at the genome level dramatically improves the precision and sensitivity of evolutionary inference . However, comparative genomics in plants has been limited by the considerable phylogenetic distances between sequenced organisms . Transcriptome sequencing using massively parallel sequencing technologies provides an attractive approach to obtaining large-scale sequence data for non-model organisms necessary for comparative genomic analysis [24, 48]. Phylogenetic utility of transcriptome sequence data yields well-resolved and highly supported tree topologies for many groups of animals [49–51]; however, few such studies have been conducted with plant taxa . Phylogenetic analysis of the genus Pinus has been limited mostly to plastid genome (cpDNA and mtDNA) sequences [21–23]. The results of this study are consistent with previous data on plastid genome phylogeny [21, 22], but transcriptome analyses, producing more robust results, are presented for the first time. Given that this study was not limited to particular genes or motifs, the results presented here are more representative of Pinus evolution than previous studies.
Understanding the factors that affect the evolutionary patterns and rates of genes is central in many research fields . For the past 30 years, it was thought that the rate of gene evolution was determined by protein function . Studies on yeast and bacteria indicate that the expression level of a protein affects the evolution rate more than its functional category, at least in unicellular species [54, 55]. In this study, we have shown that sequence polymorphisms of the 191 putatively orthologous sets of ESTs of six Pinus species are widespread using GO terms. This suggests that selection of protein function does not contribute to the variation in the rates of gene evolution. However, most of the important factors are correlated with each other. More systematic analyses of genomic data are required to further demonstrate the effect of a range of factors on the evolutionary patterns and rates of genes.
This study is the first comprehensive sequencing effort and analysis of gene function in the transcriptome of P. tabuliformis and represents the most extensive expressed sequence resource available for P. tabuliformis to date. GO and KEGG analyses were carried out, and all unigenes were classified into functional categories so as to understand their functions and regulation pathways. An enormous number of SSR and SNP/Indel loci were detected. These data can be used to develop oligonucleotide microarrays or serve as a reference transcriptome for future RNA-seq experiments in large-scale gene expression assays. These data will accelerate our understanding of genetic variation in populations and the genetic control of important traits in P. tabuliformis. Additionally, the generation of such large-scale sequence data is a potentially invaluable scientific resource for mapping, marker-assisted breeding and conservation-genetic-oriented studies in P. tabuliformis and comparative evolutionary analysis of Pinus plants.
Sample collection, cDNA library creation and 454 sequencing
Samples used for sequencing
Samples in May
Number of individuals
Samples in July
Number of individuals
Cambium (stress side)
Cambium (stress side)
Cambium (tension side)
Cambium (tension side)
Needles (juvenile + mature)
Total RNA isolation from samples of all selected plant tissues, and cDNA library construction and normalisation were performed as described previously . The pooled library was sequenced in a full 454 plate run on the GS-FLX Titanium platform (Roche, Indianapolis, IN, USA).
Assembly and annotation
All generated ESTs were pre-screened to remove adaptor-ligated regions and contaminants by Seqclean and to trim low-quality regions by LUCY2 . Because no reference P. tabuliformis genome exists, cleaned and qualified reads were assembled de novo in Newbler 2.5.3, which performs best for restoring full-length transcripts [13, 58].
The assembled isotig and singleton sequences were combined and clustered with CD-HIT (version 4.0) [59, 60]. The sequences with similarity >95% were divided into one class, and the longest sequence of each class was treated as a unigene during later processing. Descriptive annotations and GO classifications were performed as described previously .
We simultaneously instituted a search for putative unigenes against the NCBI protein database using a BLASTx and annotated each sequence with GO terms using Blast2GO.
Identification of SSRs, SNPs and InDels
Assembled isotigs with coverage of at least four reads were screened for SSRs, SNPs and InDels using Misa and ssahaSNP software, respectively . Similar criteria for screening high-quality SNPs have been used in previous studies [20, 62]. Only perfect repeats of two to six nucleotide repeats were identified. The minimum repeat-unit size for di-nucleotides was set at six and at five for tri- to hexa-nucleotide repeats.
Identification of orthologues between six conifer species
The shared transcriptome data of five conifer species in the PlantGDB and NCBI databases were downloaded. The numbers of unigenes for each species were as follows: Picea glauca (48,619), P. contorta (13,570), P. pinaster (15,648), P. sylvestris (73,609) and P. taeda (77,540). Along with 46,584 unigenes of P. tabuliformis, clustering was carried out among the transcribed sequences using UCLUST software . Aligned sequences (at least 100 bp) showing 90% identity were defined as pairs of putative orthologues among six species. The best-hit sequence of each cluster was then used in subsequent analyses. Orthologues of P. taeda and P. tabuliformis were searched using the same approach. Sequences of P. tabuliformis were annotated with GO terms using Blast2GO.
Estimation at the level of synonymous substitution and non-synonymous substitution between orthologues
Because unigenes are derived from EST sequences, have no annotated open reading frames and may contain frame shift sequencing errors, each member of a pair of sequences was searched using BLASTX against all plant protein sequences available in GenBank. The approach used was as described previously . PAML software was used to estimate the non-synonymous substitutions per non-synonymous site (Ka) and the synonymous substitutions per synonymous site (Ks) .
Because the genus Pinus has a rich history of phylogenetic analysis and the relationships among the species in the genus are well understood [21–23], the precise topology is not critical for the purposes of this study. We chose to focus our analyses on the evolutionary pattern and rate of genes. The synonymous substitution and non-synonymous substitution between the orthologues of six conifer species were analysed as described previously. Phylograms were derived using pairwise substitution rates of orthologous transcripts as a distance metric with the neighbour-joining method . Picea glauca was used as an out-group to root trees.
The raw 454 EST data obtained in this study were deposited in the NCBI Sequence Read Archive (SRA) under the accession number SRA 056887.
This work was supported by grants from The Fundamental Research Funds for the Central Universities and the Special Fund for Scientific Forestry Research in the Public Interest (No.201104022).
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