Comparative Transcriptional Profiling Provides Insights into the Evolution and Development of the Zygomorphic Flower of <em>Vicia sativa</em> (Papilionoideae)

<div><p>Background</p><p><i> Vicia sativa</i> (the common vetch) possesses a predominant zygomorphic flower and belongs to the subfamily Papilionoideae, which is related to <i>Arabidopsis thaliana</i> in the eurosid II clade of the core eudicots. Each vetch flower consists of 21 concentrically arranged organs: the outermost five sepals, then five petals and ten stamens, and a single carpel in the center.</p> <p>Methodology/Principal Findings</p><p> We explored the floral transcriptome to examine a genome-scale genetic model of the zygomorphic flower of vetch. mRNA was obtained from an equal mixture of six floral organs, leaves and roots. <i>De novo</i> assembly of the vetch transcriptome using Illumina paired-end technology produced 71,553 unigenes with an average length of 511 bp. We then compared the expression changes in the 71,553 unigenes in the eight independent organs through RNA-Seq Quantification analysis. We predominantly analyzed gene expression patterns specific to each floral organ and combinations of floral organs that corresponded to the traditional ABC model domains. Comparative analyses were performed in the floral transcriptomes of vetch and <i>Arabidopsis</i>, and genomes of vetch and <i>Medicago truncatula</i>.</p> <p>Conclusions/Significance</p><p> Our comparative analysis of vetch and <i>Arabidopsis</i> showed that the vetch flowers conform to a strict ABC model. We analyzed the evolution and expression of the TCP gene family in vetch at a whole-genome level, and several unigenes specific to three different vetch petals, which might offer some clues toward elucidating the molecular mechanisms underlying floral zygomorphy. Our results provide the first insights into the genome-scale molecular regulatory network that controls the evolution and development of the zygomorphic flower in Papilionoideae.</p> </div

Expression patterns of homologous transcripts between vetch and <i>Arabidopsis</i> petals.

<p>The unigenes enriched in the three types of petals and their homologs in <i>Arabidopsis</i> (E-value<1.0E-05) are arranged in the same row. Most of the <i>Arabidopsis</i> homologs display equivalent expression among the floral organs. The bar represents the scale of the expression levels of the transcripts. The scale of the log2 ratios ranges from −2.5 (black) to 2.5 (yellow). The abbreviations for the different tissues are the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057338#pone-0057338-g001" target="_blank">Figure 1</a>. The <i>Arabidopsis</i> data were obtained from Schmid et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057338#pone.0057338-Schmid1" target="_blank">[3]</a>.</p

Expression patterns of the homologous transcripts identified between vetch and <i>Arabidopsis</i> organs.

<p>(<b>a</b>) Expression patterns of the unigenes (5,891) enriched in vetch and their homologs (arranged in the same row) in <i>Arabidopsis</i> (E-value<1.0E-05). (<b>b</b>) Expression patterns of the genes (3,367) enriched in <i>Arabidopsis</i> and their homologs in vetch (E-value<1.0E-05). The bar represents the scale of the expression levels of the transcripts. The scale of the log2 ratios ranges from −2.5 (black) to 2.5 (yellow). The abbreviations for the different tissues are the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057338#pone-0057338-g001" target="_blank">Figure 1</a>. The <i>Arabidopsis</i> data were obtained from Schmid et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057338#pone.0057338-Schmid1" target="_blank">[3]</a>.</p

Eight organs isolated from vetch.

<p>(<b>a</b>–<b>i</b>) Sepal, dorsal petal, lateral petal, ventral petal, stamen, carpel, leaf, root and transverse section (2 µm) of a vetch flower at late pre-anthesis, which is identical to stage 12 in <i>Arabidopsis </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057338#pone.0057338-Sanders1" target="_blank">[30]</a>. C, carpel; DP, dorsal petal; LP, lateral petal; SE, sepal; ST, stamen; VP, ventral petal. Scale bars = 1.0 mm in (<b>a</b>–<b>g</b>). 5.0 mm in (<b>h</b>) and 500 µm in (<b>i</b>).</p

Neighbor-joining consensus tree and expression patterns of TCP genes.

<p>(<b>a</b>) Unrooted protein tree for the TCP protein domain data set produced using MEGA 4.0.1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057338#pone.0057338-Tamura1" target="_blank">[73]</a>. The tree summarizes the evolutionary relationships among the 24 <i>AtTCPs </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057338#pone.0057338-MartinTrillo1" target="_blank">[41]</a>, 16 <i>VsTCPs</i>, and other representative sequences. The bootstrap values are denoted above the nodes; bootstrap values of less than 50 are not shown on the phylogenetic tree. The representative sequences include <i>OsPCF1</i> (NP_001052212) and <i>OsPCF2</i> (NP_001062409) in rice; <i>ZmTB1</i> (AF377742) in maize; and <i>AmCYC</i> (Y16313) in snapdragon. (<b>b</b>) The expression patterns of the TCP genes in vetch organs. The abbreviations for the different tissues are the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057338#pone-0057338-g001" target="_blank">Figure 1</a>.</p

Global Transcriptome Sequencing Using the Illumina Platform and the Development of EST-SSR Markers in Autotetraploid Alfalfa

<div><p>Background</p><p>Alfalfa is the most widely cultivated forage legume and one of the most economically valuable crops in the world. The large size and complexity of the alfalfa genome has delayed the development of genomic resources for alfalfa research. Second-generation Illumina transcriptome sequencing is an efficient method for generating a global transcriptome sequence dataset for gene discovery and molecular marker development in alfalfa.</p> <p>Methodology/Principal Findings</p><p>More than 28 million sequencing reads (5.64 Gb of clean nucleotides) were generated by Illumina paired-end sequencing from 15 different alfalfa tissue samples. In total, 40,433 unigenes with an average length of 803 bp were obtained by <i>de</i><i>novo</i> assembly. Based on a sequence similarity search of known proteins, a total of 36,684 (90.73%) unigenes were annotated. In addition, 1,649 potential EST-SSRs were identified as potential molecular markers from unigenes with lengths exceeding 1 kb. A total of 100 pairs of PCR primers were randomly selected to validate the assembly quality and develop EST-SSR markers from genomic DNA. Of these primer pairs, 82 were able to amplify sequences in initial screening tests, and 27 primer pairs successfully amplified DNA fragments and detected significant amounts of polymorphism among 10 alfalfa accessions.</p> <p>Conclusions/Significance</p><p>The present study provided global sequence data for autotetraploid alfalfa and demonstrates the Illumina platform is a fast and effective approach to EST-SSR markers development in alfalfa. The use of these transcriptome datasets will serve as a valuable public information platform to accelerate studies of the alfalfa genome.</p> </div

Plot showing the dependence of unigene length on the number of reads assembled into each unigene.

<p>Plot showing the dependence of unigene length on the number of reads assembled into each unigene.</p

The tissues used in this study and all samples shown are from the alfalfa cultivar “Golden queen”.

<p>(A) Callus cells. (B) Germinated seeds (36 hours after seed germination). (C) Germinated seeds (48 hours after seed germination). (D) Roots from a 20-day-old seedling. (E) Cotyledons from a 7-day-old seedling. (F) Unifoliate leaves from a 20-day-old seedling. (G) Compound leaves. (H) Young stem (less lignified). (I) Middle stem (moderately lignified). (J) Old stem (more lignified). (K) Shoot apex. (L) Young inflorescence (diameter 0.4-0.5 cm). (M) Mature inflorescence (diameter 2 cm), (N) Young pod (16 days after pollination). (O) Mature pod (24 days after pollination).</p

Alfalfa unigenes mapped to Mt3.5.2 pseudomolecules with a threshold of 95% identity and 90% coverage.

<p>White lines represent alfalfa unigenes that were not mapped to the <i>M. truncatula</i> genome sequence assembly, and black lines represent alfalfa unigenes that were mapped to the <i>M. truncatula</i> genome.</p

Gene Ontology (GO) classification of assembled unigenes in <i>M. sativa</i>.

<p>A total of 14,415 unigenes with BLAST matches to known proteins were assigned to three main categories: cellular component, molecular function and biological process.</p