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Article

The Genome and Transcriptome Analysis of the Vigna mungo Chloroplast

by
Wanapinun Nawae
1,
Chutintorn Yundaeng
1,
Chaiwat Naktang
1,
Wasitthee Kongkachana
1,
Thippawan Yoocha
1,
Chutima Sonthirod
1,
Nattapol Narong
1,
Prakit Somta
2,
Kularb Laosatit
2,
Sithichoke Tangphatsornruang
1 and
Wirulda Pootakham
1,*
1
National Omics Center (NOC), National Science and Technology Development Agency, 111 Thailand Science Park, Khlong Nueng, Khlong Luang, Pathum Thani 12120, Thailand
2
Department of Agronomy, Faculty of Agriculture at Kamphaeng Saen, Kasetsart University, Nakhon Pathom 73140, Thailand
*
Author to whom correspondence should be addressed.
Plants 2020, 9(9), 1247; https://doi.org/10.3390/plants9091247
Submission received: 27 August 2020 / Revised: 11 September 2020 / Accepted: 17 September 2020 / Published: 21 September 2020
Browse Figures
Versions Notes

Abstract

:
Vigna mungo is cultivated in approximately 5 million hectares worldwide. The chloroplast genome of this species has not been previously reported. In this study, we sequenced the genome and transcriptome of the V. mungo chloroplast. We identified many positively selected genes in the photosynthetic pathway (e.g., rbcL, ndhF, and atpF) and RNA polymerase genes (e.g., rpoC2) from the comparison of the chloroplast genome of V. mungo, temperate legume species, and tropical legume species. Our transcriptome data from PacBio isoform sequencing showed that the 51-kb DNA inversion could affect the transcriptional regulation of accD polycistronic. Using Illumina deep RNA sequencing, we found RNA editing of clpP in the leaf, shoot, flower, fruit, and root tissues of V. mungo. We also found three G-to-A RNA editing events that change guanine to adenine in the transcripts transcribed from the adenine-rich regions of the ycf4 gene. The edited guanine bases were found particularly in the chloroplast genome of the Vigna species. These G-to-A RNA editing events were likely to provide a mechanism for correcting DNA base mutations. The V. mungo chloroplast genome sequence and the analysis results obtained in this study can apply to phylogenetic studies and chloroplast genome engineering.

Graphical Abstract

1. Introduction

Vigna mungo (L.) Hepper or black gram is a diploid plant with 2n = 2x = 22 chromosomes. It belongs to the family Leguminosae, subfamily Papilionoideae, clade Millettioid [1], and is a tropical legume crop species that is cultivated in Asia, Africa, and America [2]. Black gram is an economically important Vigna species, which provides high-protein food [3].
Chloroplast is an essential organelle that harbors about 120–130 genes in its own genome [4]. The genera Phaseolus [5], Glycine [6], Vigna [7], and Cajanus [8] are examples of the tribe Phaseoleae species that have their complete chloroplast genome sequence reported. Structural variations including inverted repeat region (IR) expansion or contraction, genome rearrangement, loss of gene or intron, and pseudogenes among these legumes are described [9]. The chloroplast genome of Vigna radiata has been sequenced [7,10], where Lin et al., 2015 additionally used RNA-seq data to identify RNA editing events [10]. Many recent chloroplast genome studies focus their analysis on identifying positively selected genes, RNA editing events, and polycistronic transcription units [11,12,13]. The identification of the positively selected genes is important for evolutionary studies because these genes had a fixation of advantageous point mutations (positively selected sites) as an adaptation to the selective force (positive selection) from different ecological conditions [14,15]. The non-synonymous (Ka) and the synonymous (Ks) nucleotide substitution rates and the Ka/Ks ratio are commonly used to detect positive selection or adaptive evolution events [13]. For example, the adaptive evolution of chloroplast genes as identified by Ka/Ks ratios was reported to be responsible for the adaptation of rice species to diverse ecological habitats related to sunlight preferences [13]. Detailed sequences of genes and intergenic regions from the chloroplast genome are often used to study the phylogenetic relationship of plant species [16,17]. Recently, the phylogenetic tree generated from the sequence of 80 plastid genes from 2,514 plant species was used to estimate the origin and divergence time of angiosperms [18]. Moreover, chloroplast genomes are the targets for genetic engineering in the agricultural, pharmaceutical, and medical applications [19,20,21]. For example, the engineering of chloroplast genomes could increase the tolerance of plants to high temperature [22] and salt stress [23] and to confer insecticidal activity to the transgenic plants [24]. Pharmaceutically, the chloroplast genome engineering enabled the low-cost production of, for example, polio vaccine [25], interferon-α2b [26], and proinsulin [27]. Therefore, more information on the sequence, structure, and transcription of the chloroplast genomes enable us to better understand plant evolution and to effectively use this organelle in a broader biotechnological application.
In this study, we sequenced the chloroplast genome of V. mungo. We identified positively selected genes in the V. mungo chloroplast genome by performing comparative analysis with chloroplast genomes from the related legume species that preferred different climates to grow. We additionally used PacBio isoform sequencing (Iso-seq) reads to show polycistronic transcription units and used Illumina short reads to identify RNA edited sites in different V. mungo tissues.

2. Results and Discussion

2.1. General Features of Vigna Mungo Chloroplast Genome

We obtained 4.4 Gb from 15 million Illumina paired-end (PE) reads for chloroplast genome assembly. The raw reads were deposited to the National Center for Biotechnology Information (NCBI) database under the BioProject accession number of PRJNA623719. The assembled chloroplast genome of V. mungo was 151,294 base pairs (bp) long (Figure 1A). We have deposited the V. mungo chloroplast genome to the GenBank Nucleotide Database with the accession number MT418597. The average depth coverage across the genome was 61.98× (Figure S1). The genome has a circular quadripartite structure with one large single copy (LSC; 80,984 bp), one small single copy (SSC; 17,448 bp), and two inverted repeat regions (IRa and IRb; 26,431 × 2 bp). The IRa and IRb were repeated sequences appearing in the inverted direction in the circular structure of the chloroplast genome. The overall GC content of the chloroplast genome is 35.24%. The GC content of the LSC, SSC, and each of IR regions were 32.59%, 28.54%, and 41.52%, respectively, which were consistent with that of the chloroplast genome of V. radiata (mungbean) [7]. The V. mungo chloroplast genome has 108 genes including 75 protein-coding genes, 29 tRNA genes, and four rRNA genes (Table 1). The LSC region contained 57 protein-coding genes and 21 tRNA genes, while the SSC region had 12 protein-coding genes and one tRNA gene. In each of the IR regions, there are six protein-coding genes, four rRNA genes, and seven tRNA genes. The gene density was slightly higher in the LSC region (963 genes/Mb) than in the SSC (745 genes/Mb) and the IR regions (643 genes/Mb). Nine protein-coding genes and five tRNA genes had one intron. ycf3 and clpP were two genes that had two introns. We found ψrpl33 andψrps16 pseudogenes in the LSC region and ψycf1 pseudogene, which spanned the IRb/SSC (JSB) boundary. The ψrpl33 was shown be specific to Phaseolinae chloroplast genomes, while ψrps16 was found to be absent in the Ceratonia siliqua and Glycine max chloroplast genomes [28].

2.2. Comparative Chloroplast Genome Analysis

We compared the V. mungo chloroplast genome sequenced in this study with 11 other Leguminosae chloroplast genomes that were downloaded from the GenBank database. Five of these species were V. radiata, Vigna angularis, Vigna unguiculata, and Phaseolus vulgaris, and G. max was from the tribe Phaseoleae. The first four species are member of the subtribe Phaseolinae, while G. max is from the subtribe Glycininae. The other four species were Glycyrrhiza glabra, Cicer arietinum, Pisum sativum, and Medicago sativa from the IR-lacking clade. Two other species were Arachis hypogaea from the tribe Dalbergieae and Ceratonia siliqua from the subfamily Caesalpinioideae.
We generated phylogenetic tree for these species using 69 orthologous genes (Table S1). As a result, the species from the tribe Phaseoleae formed a sister clade to the species from the IR-lacking clade (Figure 2). These two clades shared commons ancestors with Arachis and Ceratonia, respectively. V. mungo formed a monophyletic group with V. radiata at the deepest branch of the Phaseolinae clade, indicating a close evolutionary relationship between these two species. We also calculated the phylogenetic tree of these species with 57 other species to confirm the placement of these Fabales species in relative to other diverse species. This calculation used 38 orthologous protein sequences from the chloroplast genomes. In resulted phylogenetic tree (Figure S2), the Fabales species formed a sister clade to the Rosales, Fagales, and Cucurbitales clades, which was consistent with the tree that was calculated from 80 genes from 2881 chloroplast genomes [18].
As a structural analysis, the IR expansion/contraction of the V. mungo chloroplast genome were examined. During the evolution of land plant, the IR expansion caused the movement of the entire or a portion of genes from the SC regions into the IR regions and vice versa for the IR contraction [29]. In this study, we found that V. mungo and other Phaseolinae species had two copies of rps19 inside their IR regions (Figure 3). In contrast, G. max, A. hypogaea, and C. siliqua had one copy of rps19, although they had two IR regions (Table S1). rps19 of these three species spanned the LSC/IRb boundaries (Figure 3). In addition, the length of the IR regions in the V. mungo chloroplast genome was 857, 394, and 607 bp longer than the IR regions from the G. max, A. hypogaea, and C. siliqua chloroplast genomes, respectively. Altogether, our results indicated that the IR regions were expanded in the chloroplast genome of V. mungo.
We used Mauve aligner [30] to align the chloroplast genome of the species in the Fabales clade to investigate genome rearrangement. The alignment calculated the locally collinear blocks (LCBs), which presented the high similarity conserved regions among the compared genomes. For Mauve alignment, we removed the IRa sequence from the analyzed chloroplast genomes that had two IR regions. This modification allowed the program to calculate the LCBs for the repetitive sequence of the IR regions. We obtained 18 LCBs from the alignment using the C. siliqua chloroplast genome as a reference sequence (Figure 4). We identified the rearrangement of the V. mungo chloroplast genome based on the relative position of these LCBs. We found an inversion of the DNA segment covering from LCB2 to LCB5 in the chloroplast genome of V. mungo and all other Faboideae species, except for P. sativum (Figure 4). This inversion coincided with the 51-kb inversion that was reported to occur early in the diversification of papilionoid legumes [31]. Compared to the A. hypogaea chloroplast genome, the G. max had the inversion of the DNA segment covering from LCB11 to LCB18, which covered the SSC region and the IR regions. In the chloroplast genome of V. mungo and other Phaseolinae species, this DNA segment was reinverted to the same arrangement as the chloroplast genome of A. hypogaea (Figure 4). Additional DNA segment that changed it location with this reinversion was LCB10, which was a part of LSC regions. The inversion of this LCB10-to-LCB18 DNA segment correspond to the 78-kb inversion reported in other Phaseolinae species [5,7]. These results supported that the 51-kb inversion and the 78-kb inversion were characteristics of the chloroplast genomes from the subtribe Phaseolinae.

2.3. Positively Selected Genes

In this study, we calculated the non-synonymous (Ka) to synonymous (Ks) substitution ratio (Ka/Ks) for each of the 60 protein-coding genes shared by the analyzed legumes. The Ka/Ks ratio showed the strength and mode of natural selection on the protein-coding genes [32]. The Ka/Ks > 1 indicated that the corresponding genes experienced positive selection while the genes experiencing neutral or purifying (negative) selection were indicated by Ka/Ks = 1 or Ka/Ks < 1, respectively [32,33]. The average Ka/Ks ratio of the 61 protein-coding genes of the V. mungo chloroplast genome was 1.77. We found V. mungo chloroplast genome had 19 positively selected genes (Ka/Ks > 1 [34,35]) including eight photosynthetic genes (42%), four ribosome genes (21%), three RNA polymerase genes (16%), and four other genes (21%) (Figure 1A and Table 2). We found that rbcL and ycf2 had the highest number of positively selected sites (11 sites), followed by ndhF (7 sites), atpF and rps2 (5 sites), ndhH (4 sites), matK, psbT and rpoC1 (3 sites), and other genes (<2 sites) (Table 2).
Amino acid changes due to the selection pressure can drive evolution within a specific taxonomic lineage [36]. Positive selection is a process, in which advantageous mutations increase fitness of plants to the ecological habitats [37]. Leguminosae consists of 19,500 species with a worldwide distribution [38], which can be grouped, based on growing seasons, into cool-season food legumes and warm-season food legumes [39]. For the legumes analyzed in this study, P. sativum (dry pea) and C. arietinum (chickpea) are members of the cool-season food legumes, while V. radiata (mungbean), V. mungo (black gram), and P. vulgaris (common bean) grow well under hot and humid conditions [39]. We used legume species that prefer different climatic conditions for calculating the Ka/Ks ratio by hypothesizing that these species experienced different ecologically selective pressures. Many positively selected genes, e.g., rbcL, matK, ndhF, atpF, and rpoC2, in the V. mungo chloroplast genome were found to be involved in ecological adaptations in other plant species. For example, an adaptive evolution in the rbcL gene is linked with photosynthetic performance under temperature, drought and carbon dioxide concentration variations [40]. Rubisco had a small carboxylase turnover rate and low CO2 affinity, which limits the rate of carbon assimilation and net photosynthesis [41]. The improvable activities of Rubisco might explain the rbcL positive selection that was widespread in many land plants [41]. The positive selection of matK was also identified in many plants suggesting that this gene experienced different ecological selective pressures [19,42]. In the genus Citrus, positive selection of matK and ndhF was considered to play roles in the adaptation of Australian species to hot and dry climate [19]. In addition, positive selection of ndhF was believed to be able to delay drought-induced senescence in Haberlea rhodopensis [43]. The gene sequence comparisons between deciduous Quercus species and evergreen Quercus species revealed that the positive selection of atpF and rpoC2 played a role in the adaptation of the deciduous Quercus species to the winter or the dry season [44]. The positive selections identified in this study were likely related to the different climatic preferences among the analyzed legume species.
In addition to climatic conditions, some legumes prefer different light spectrums as shown by a study indicating that the red light significantly suppressed pod growth in soybean, but promoted the growth in cowpea [45]. A study with 22 chloroplast genomes from the genus Oryza that were grouped into shade-tolerant and sun-loving rice species showed a correlation between the positive selection of photosynthetic genes, e.g., rbcL, ndh, and psb, and the adaptation of rice species to different sunlight levels [13]. The positive selection of these genes was also observed in our Ka/Ks analysis, which included soybean and cowpea (Table 2). Light spectrums might be other possible selection pressure exerted to the chloroplast gene of the analyzed legumes.

2.4. Polycistronic Transcription Units

The transcriptional regulation of chloroplast genes has characteristics found in both prokaryotes and eukaryotes [46]. Plastid genes are co-transcribed into polycistronic transcription units, which resemble operons in bacteria [46,47]. Primary polycistronic transcripts then undergo post-transcriptional modifications (for example, RNA editing), which is a characteristic of eukaryotes [46,48]. In this study, we investigated both polycistronic transcription units and RNA editing events in the V. mungo chloroplast genome.
This study used PacBio Isoform Sequencing (Iso-Seq) to identify polycistronic transcription units of the V. mungo chloroplast genome. We retrieved 10 monocistronic units, seven dicistronic units, and 11 polycistronic units (Figure 1B). We found three overlapping pairs of polycistronic units (accD-psaI, petL-psaJ, and psbB-psbN). The degradation of a long polycistronic unit into smaller oligocistronic units could result from intercistronic processing activities [49]. For example, in Arabidopsis thaliana, the binding of HCF152 to the intergenic region between psbH and petB was involved in the digestion of the psbB-psbT-psbH-petB-petD polycistronic units by exonucleases to psbB-psbT-psbH tricistronic units and petB-petD dicistronic units [50]. In this study, we found coexistence of psbB-psbT-psbN-psbH-petB and psbN-psbH-petB-petD polycistronic units, suggesting that there may be binding sites in the intergenic region between psbT and psbN for a protein with a similar function to HCF152 in the V. mungo chloroplast genome.
The Iso-Seq data also provided evidence that the accD polycistronic transcripts in V. mungo were transcribed from the rps16 promoter, locating further upstream of accD, due to a 51-kb inversion between accD/rps16 and rbcL/trnK-UUU. We observed that the promoter sequence of accD in V. mungo is truncated and lacks the GAA-box compared to that of C. siliqua, a legume without the 51-kb inversion. These results suggested that the 51-kb inversion affect transcriptional regulation of the accD polycistronic transcripts.
The iso-seq reads also allow for the detection of alternative splicing events. There are two genes (ycf3 and clpP) with two introns in the V. mungo chloroplast genome. We have mapped our iso-seq reads to the genome. However, the results showed no evidence of an alternative splicing event in either of these genes.

2.5. RNA Editing

In this study, we identified 34 RNA editing events in 18 plastid genes from leaf, shoot, flower, fruit, and root tissues of V. mungo using RNA-seq data (Figure 1A and Table 3). Ninety-one percent of these RNA editing events were C-to-U editing, and the remaining were G-to-A editing. RNA editing occurred at different efficiency levels in different tissues [51,52]. A majority of editing events in V. mungo were found in leaves, which was consistent with a study in A. thaliana [52]. The number of RNAseq reads from leaves that were uniquely mapped to the chloroplast genome (44,607 reads) was higher than the number of reads from other tissues (16,631 reads from shoots, 17,501 reads from flowers, 4356 reads from fruits, and 8334 reads from roots). The results suggested different expression levels in different tissues and might explain the highest number of the edited sites in leaves compared to other tissues of this study. We also found that different tissues had different RNA edited events. It was only the C-to-U editing of clpP (clpP-556) that was commonly found in all five tissues (Table 3). Our Ka/Ks analysis also showed that clpP was one of the positively selected gene of the V. mungo chloroplast genome (Table 2). clpP has been reported to express in chloroplasts and non-photosynthetic plastids and has been shown to be involved in chloroplast development and cell survival [53]. The disruption of clb19, which was involved in clpP editing, impaired chloroplast development, caused yellow phenotype, and increased early seedling lethality rate [54]. clb19 and the corresponding clpP-559 editing in the A. thaliana chloroplast genome (equivalent to the clpP-556 editing in the V. mungo chloroplast genome) are absent from core asterids and Poaceae but are retained in most of rosids [55]. Vicia faba and C. arietinum are two legumes that lost CLB19 and clpP-559 editing [55]. Our results, together with the results from these studies, suggested that the sequence and the RNA editing of clpP can be used as a good marker for studying phylogenetics.
RNA-seq data allow for the detection of edited sites with low editing efficiency [56,57]. We found three G-to-A editing events in ycf4 from leaves (ycf4-20, ycf4-309, and ycf4-316), although they had low editing efficiency. G-to-A editing has also been reported in ndhF, rpoC2, and ycf1 genes of the A. thaliana [52] and Betula platyphylla [58]. In the V. mungo chloroplast genome, we found the edited G bases in two A-rich regions (Figure S3), where the sequences were translated to long lysine chains. The ycf4-20 and ycf4-316 editing resulted in an amino acid change from arginine and glutamic acid to lysine, respectively (Table 3). The ycf4-309 editing event was a synonymous editing, which did not change lysine amino acid in the protein sequence. The conversion of G to A in these A-rich regions might facilitate three-dimensional folding or interaction of the protein products translated from the edited transcripts of ycf4. All analyzed Vigna species have these three edited G bases in ycf4, but the P. vulgaris chloroplast genome has only one G base homologous to ycf4-20 (Figure S3). The edited G bases were likely specific to the chloroplast genome of Vigna species and might have resulted from mutation events. The observed G-to-A editing events in the V. mungo ycf4 transcripts support the idea that RNA editing is a mechanism to correct mutations in the genomic coding sequences, which accumulated during evolution process [57,59].

3. Materials and Methods

3.1. DNA and RNA Extraction

Black gram samples were obtained from Kasetsart University (Nakhon Pathom, Thailand). Frozen tissues (CN80 accession) were homogenized, and DNA was extracted using QIAGEN Genomic-tip 100/G based on the manufacturer’s protocol (Qiagen, Hilden, Germany). We assessed DNA integrity with the Pippin Pulse Electrophoresis System (Sage Science, Beverly, MA, USA). Total RNA was isolated from leaf, root, stem, flower, and three-week-old pod using the CTAB buffer (2% CTAB, 1.4 M 91 NaCl, 2% PVP, 20 mM EDTA pH 8.0, 100 mM Tris-HCl pH 8.0, 0.4% SDS). RNA was extracted from the aqueous phase three times using 25:24:1 phenol:chloroform:isoamylalcohol. Poly(A) enrichment with Dynabeads mRNA Purification Kit (ThermoFisher Scientific, Waltham, MA, USA) was used to get mRNAs.

3.2. Preparation of DNA and RNA Libraries and Sequencing

For DNA sequencing, we prepared sequencing library from a total of 1.25 ng of high molecular weight DNA using the Chromium Genome Library Kit & Gel Bead Kit v2, the Chromium Genome Chip Kit v2, and the Chromium i7 Multiplex Kit following the manufacturer’s instructions (10X Genomics, Pleasanton, CA, USA). We used a single lane of Illumina HiSeq X Ten (2 × 150 bp paired-end reads) to sequenced the library.
For RNA sequencing, RNA integrity was assessed with a Fragment Analyzer System (Agilent, Santa Clara, CA, USA). To obtain short-read RNA sequences, six RNA libraries (one for each tissue type) were prepared according to the protocol reported in Pootakham et al., 2018. Briefly, 200 ng of poly(A) mRNA was used to construct a library using the Ion Total RNA Sequencing Kit (ThermoFisher Scientific, Waltham, MA, USA). The libraries were sequenced on the Ion S5 XL using the Ion 540 TM chip (ThermoFisher Scientific, Waltham, MA, USA). For RNA isoform sequencing, two PacBio Iso-seq libraries were prepared following protocols described in [60] using the SMARTer PCR cDNA Synthesis Kit (Clontech, Mountain View, CA, USA) and size-selected using the BluePippin Size Selection System (Sage Science, Beverly, MA, USA) into 1–2 kb, 2–3 kb, and 3–6 kb bins. The Iso-seq sequencing was performed on the PacBio RSII sequencing system (Pacific Biosciences, Menlo Park, USA, outsourced to NovogenAIT, Singapore) using P6-C4 polymerase and chemistry and 360 min movie times according to the manufacturer’s protocol.

3.3. Genome Assembly and Annotation

The raw reads were trimmed and filtered for high quality reads by fastp program with default parameters [61]. We used the GetOrganelle pipeline [62] to de novo assemble the genome sequence. In the pipeline, we set word size ratio to 0.4 for extracting chloroplast reads from total DNA reads and used a combined k-mer of 95,105,125 together with k-mer gradient for de novo assembly with SPAdes [63]. We ran the “evaluate_assembly_using_mapping.py” script from the GetOrganelle software package to evaluate depth of coverage across the assembled chloroplast genome. We used CPGAVAS2 [64], GeSeq [65], and Geneious [66] for chloroplast genome annotation. The annotated chloroplast genome was visualized with OGDRAW [67].

3.4. Comparative Analysis of V. mungo Chloroplast Genome

We used Mauve aligner software [30] with default parameters to align the V. mungo chloroplast genome with chloroplast genome of V. radiata (NC_013843.1), V. angularis (NC_021091.1), V. unguiculata (NC_018051.1), P. vulgaris (NC_009259.1), G. max (NC_007942.1), G. glabra (NC_024038.1), C. arietinum (NC_011163.1), P. sativum (NC_014057.1), M. sativa (NC_042841.1), A. hypogaea (NC_037358.1), and C. siliqua (NC_026678.1). The sequence of the C. siliqua chloroplast genome was used as a reference for visualizing the LCB relative locations. To analyze IR expansion/contraction, IRscope [68] was used to compare size of the LSC region, the SSC region, and IR region among analyzed species.

3.5. Positive Selection

The codon-based alignment of the protein-coding gene sequences were conducted with the integrated MUSCLE alignment program of MEGA-CC software [69,70]. The positive selected genes and sites were identified with CODEML program of PAML 4 package [71] via the EasyCodeML interface [72]. The likelihood ratio tests (LRT) with a p-value cutoff of 0.05 was used to compared between the fitness of the alignment data to the M8 model (with the Ka/Ks > 1 parameter) and the M7 model (without the Ka/Ks > 1 parameter). Thereby, the identified positively selected genes had a high probability to experience positive selection than neutral selection.

3.6. RNA Editing Sites

We mapped RNA short reads from the Illumina deep RNA sequencing to the V. mungo chloroplast genome with Bowtie 2 software [73]. RNA editing sites were then identified with REDItools software [74]. We selected sites that had at least 15 RNA support reads and the frequency of the corresponding polymorphism at the DNA level (from DNA read mapping) lower than 0.01.

3.7. Polycistronic Analysis

To find genes on a polycistronic transcription unit, we aligned PacBio Iso-seq reads to the V. mungo chloroplast genome using BLASTN [75] with a 10−3 E-value cutoff. We selected the Iso-seq reads that had a 100% coverage and >95% identity alignment with the chloroplast genome sequence.

4. Conclusions

In this study, we sequenced the genome and transcriptome of the V. mungo chloroplast. The sequence and structure of the assembled chloroplast genome was consistent with the chloroplast genome of the closely related species of V. mungo. Our comparisons between the chloroplast genome of V. mungo and other legume species that grew in different habitats revealed many positively selected genes. Our RNA sequencing results showed that the 51-kb DNA inversion conserved among Papilionoideae legume species could affect the transcriptional regulation of the accD polycistronic transcription. Finally, we found RNA editing events that change guanine to adenine in the RNA molecules that were transcribed from the adenine-rich regions of the ycf4 gene in the V. mungo chloroplast genome. The chloroplast genome sequence and the knowledge gained from this study can be used for plant phylogenetic studies and for the engineering of the chloroplast genome of V. mungo and other related legume species.

Supplementary Materials

The following are available online at https://www.mdpi.com/2223-7747/9/9/1247/s1, Figure S1: Read coverage of the assembled genome, Figure S2: Phylogenetic tree of 69 plant species, Figure S3: Edited sites on ycf4, Table S1: Orthologous genes calculated from the chloroplast genome of twelve legume species with OrthoFinder program.

Author Contributions

Conceptualization, S.T. and W.P.; formal analysis, W.N. and C.N.; investigation, W.N., T.Y., N.N., S.T. and W.P.; resources, P.S. and K.L.; writing—original draft preparation, W.N. and C.Y.; writing—review and editing, W.N., S.T., W.P., P.S. and K.L.; visualization, W.N., W.K. and C.S.; supervision, S.T. and W.P.; funding acquisition, S.T. and W.P. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Omics Center, the National Science and Technology Development Agency (NSTDA), Thailand, grant number 1000221.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Structure and expression of the chloroplast genome of Vigna mungo. (A) The structure of the V. mungo chloroplast genome is shown. The genes inside the circle are transcribed clockwise, and genes outside are transcribed counter-clockwise. Genes from different functional groups are shown in different colors. Genes with either RNA editing or positive selection are labeled with blue or green text colors, respectively. Genes with both RNA editing and positive selection are labeled with red text colors. (B) Polycistronic transcription units (black bars) are shown relative to the position of genes on the V. mungo chloroplast genome, which is visualized in a linear form for simplicity.
Figure 1. Structure and expression of the chloroplast genome of Vigna mungo. (A) The structure of the V. mungo chloroplast genome is shown. The genes inside the circle are transcribed clockwise, and genes outside are transcribed counter-clockwise. Genes from different functional groups are shown in different colors. Genes with either RNA editing or positive selection are labeled with blue or green text colors, respectively. Genes with both RNA editing and positive selection are labeled with red text colors. (B) Polycistronic transcription units (black bars) are shown relative to the position of genes on the V. mungo chloroplast genome, which is visualized in a linear form for simplicity.
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Figure 2. Phylogenetic relationship of the analyzed legumes. The phylogenetic tree was calculated from orthologous genes from the chloroplast genome of V. mungo and other eleven Leguminosae species. Ceratonia siliqua was used as an outgroup in the phylogenetic tree calculation. Bootstrap values are shown in blue color, and branch lengths are shown in black color.
Figure 2. Phylogenetic relationship of the analyzed legumes. The phylogenetic tree was calculated from orthologous genes from the chloroplast genome of V. mungo and other eleven Leguminosae species. Ceratonia siliqua was used as an outgroup in the phylogenetic tree calculation. Bootstrap values are shown in blue color, and branch lengths are shown in black color.
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Figure 3. IR expansion/contraction. Large single copy (LSC), small single copy (SSC), and IR regions among eight chloroplast genomes that have two IR region are compared. The numbers inside the LSC (cyan), SSC (green), and IR (orange) regions show the length of the corresponding regions. The numbers outside the regions show the distances relative to the region boundaries.
Figure 3. IR expansion/contraction. Large single copy (LSC), small single copy (SSC), and IR regions among eight chloroplast genomes that have two IR region are compared. The numbers inside the LSC (cyan), SSC (green), and IR (orange) regions show the length of the corresponding regions. The numbers outside the regions show the distances relative to the region boundaries.
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Figure 4. Chloroplast genome rearrangement. Eighteen locally collinear blocks (LCBs) are shown with different colors. Each LCB is numbered according to their order in the C. siliqua chloroplast genome. The LCB numbers are shown on top of the LCBs of the C. siliqua chloroplast genome. The LCBs with conserved sequences among the compared chloroplast genomes are represented in the same color. For the chloroplast genomes that have two IR regions, only the IRb region is considered in the alignment. The IR region of each genome is marked with pink bar. The line graph inside each LCB shows the sequence similarity level. Corresponding LCBs from different species are connected by lines that have the same color as the LCB that they connect to.
Figure 4. Chloroplast genome rearrangement. Eighteen locally collinear blocks (LCBs) are shown with different colors. Each LCB is numbered according to their order in the C. siliqua chloroplast genome. The LCB numbers are shown on top of the LCBs of the C. siliqua chloroplast genome. The LCBs with conserved sequences among the compared chloroplast genomes are represented in the same color. For the chloroplast genomes that have two IR regions, only the IRb region is considered in the alignment. The IR region of each genome is marked with pink bar. The line graph inside each LCB shows the sequence similarity level. Corresponding LCBs from different species are connected by lines that have the same color as the LCB that they connect to.
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Table
Table 1. Annotated genes of the V. mungo chloroplast genome.
Table 1. Annotated genes of the V. mungo chloroplast genome.
Category of GenesGroup of GenesName of Genes
Genes for photosynthesisSubunits of photosystem IIpsbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, psbM, psbN, psbT, psbZ, ycf3 **
Subunits of photosystem IpsaA, psaB, psaC, psaI, psaJ
Subunits of NADH-dehydrogenasendhA *, ndhB *, ndhC, ndh, ndhE, ndhF, ndhG, ndhH, ndhI, ndhJ, ndhK
Subunits of cytochrome b/f complexpetA, petB *, petD *, petG, petL, petN
Subunits of ATP synthaseatpA, atpB, atpE, atpF *, atpH, atpI
Subunit of rubiscorbcL
Self-replicationLarge subunit of ribosomerpl14, rpl16 *, rpl2, rpl20, rpl23, rpl32, rpl36, rpl33ψ
DNA dependent RNA polymeraserpoA, rpoB, rpoC1 *, rpoC2
Small subunit of ribosomerps11, rps12 *, rps14, rps15, rps16 *,ψ, rps18, rps19, rps2, rps3, rps4, rps7, rps8
Other genesSubunit of Acetyl-CoA-carboxylaseaccD
c-type cytochrom synthesis geneccsA
Envelop membrane proteincemA
ProteaseclpP **
MaturasematK
Ribosomal RNAs rrn23S, rrn4.5S, rrn16S, rrn5S
Transfer RNAsAlaTrnA-UGC *
ArgtrnR-ACG, trnR-UCU
AsntrnN-GUU
AsptrnD-GUC
CystrnC-GCA
GlntrnQ-UUG
GlutrnE-UUC
GlytrnG-UCC
HistrnH-GUG
IletrnI-CAU, trnI-GAU *
LeutrnL-CAA, trnL-UAA *, trnL-UAG
LystrnK-UUU *
MettrnM-CAU, trnfM-CAU
PhetrnF-GAA
ProtrnP-UGG
SertrnS-GCU, trnS-GGA, trnS-UGA
ThrtrnT-GGU, trnT-UGU
TrptrnW-CCA
TyrTrnY-GUA
ValtrnV-GAC, trnV-UAC *
UnkownConserved open reading framesycf1, ycf1 ψ, ycf2, ycf4
* gene with one intron, ** gene with two introns, ψ pseudogene.
Table
Table 2. Positively selected genes and sites in the V. mungo chloroplast genome.
Table 2. Positively selected genes and sites in the V. mungo chloroplast genome.
GeneFunctionKa/Ks of GeneLRTs (2ΔLnL) LRT p-ValueSelective SitePr(Ka/Ks > 1)Ka/Ks of Site
atpFPhotosynthesis1.940.390.0239R0.967 *1.884
62N0.996 **1.931
79T0.971 *1.89
83L0.952 *1.86
167M0.984 *1.912
ccsAOther genes2.400.640.0094Q0.982 *2.048
clpPOther genes2.870.830.0412I0.983 *2.836
matKOther genes2.820.070.0080V0.956 *2.725
493Y0.988 *3.08
494L0.989 *3.083
ndhFPhotosynthesis1.550.550.0064K0.989 *1.54
289K0.995 **1.546
507I0.993 **1.544
616L0.965 *1.512
638L0.989 *1.54
740N0.951 *1.497
741K0.951 *1.496
ndhHPhotosynthesis1.030.150.003I0.955 *1.502
176S0.994 **1.026
269I0.989 *1.021
294C0.986 *1.019
psbDPhotosynthesis2.170.250.01122G1.000 **2.168
psbEPhotosynthesis3.940.170.0559N0.999 **3.937
psbLPhotosynthesis5.270.140.001M1.000 **5.267
psbTPhotosynthesis1.710.010.0028K1.000 **1.711
33K1.000 **1.711
34V0.952 *3.873
rbcLPhotosynthesis1.640.160.0028D0.985 *1.613
86H1.000 **3.108
95S0.993 **1.624
97F0.999 **1.633
142T0.999 **3.106
228S0.958 *3.001
251M0.999 **1.634
375I0.990 *1.619
449S0.997 **3.102
470E0.999 **1.634
475I0.998 **3.105
rpl20subunit of ribosome2.920.060.0584K0.979 *3.167
rpoBRNA polymerase1.490.620.00446I0.965 *1.473
rpoC1RNA polymerase1.301.390.00562W0.991 **1.294
568P0.964 *1.472
569K0.973 *1.275
rpoC2RNA polymerase2.750.650.00734S0.990 *2.728
735K0.980 *2.706
rps2subunit of ribosome1.390.820.0067G0.998 **1.384
129F0.954 *1.332
130Q0.960 *1.339
131S0.959 *1.337
235S0.991 **1.376
rps4subunit of ribosome3.810.680.0025K0.983 *4.044
99A0.956 *3.661
rps8subunit of ribosome1.541.110.0255N0.969 *1.502
93Q0.996 **1.537
ycf2Other genes3.310.050.003G0.986 *2.481
117S0.992 **2.491
120S0.963 *2.444
429R0.958 *2.435
484Q0.962 *2.441
627V0.997 **2.498
692K0.984 *2.478
693T0.966 *2.448
716T0.960 *2.439
1040L0.976 *2.465
1528S0.982 *2.474
LRT = likelihood ratio tests; Pr(Ka/Ks > 1) = the probability of a site to have Ka/Ks > 1 with a significant level of 0.05 * or 0.01 **. Ka: non-synonymous; Ks: synonymous.
Table
Table 3. RNA edited sites in the chloroplast genome of V. mungo.
Table 3. RNA edited sites in the chloroplast genome of V. mungo.
TissueGenePosition on GeneBase ChangeAmino Acid ChangeEditing Efficiency *Codon PositionCoverage
LeafndhC323C -> TS -> L0.89227
Leafrps1480C -> TS -> L0.9220
LeafrpoB551C -> TS -> L0.53219
LeafrpoB566C -> TS -> L0.61218
LeafrpoC141C -> TS -> L0.75216
Leafrps2134C -> TT -> I0.95264
Shootrps2134C -> TT -> I0.95221
Rootrps2134C -> TT -> I0.93215
Leafrps2248C -> TS -> L0.81216
LeafatpF92C -> TP -> L0.92238
LeafpsaI79C -> TH -> Y0.78154
LeafpsbF44C -> TS -> F0.7237
LeafpsbE124C -> TP -> L0.92134
ShootpsbE124C -> TP -> L0.88121
LeafpetL5C -> TP -> L0.43220
Leafrps18221C -> TS -> L0.67226
LeafclpP2041C -> TH -> Y0.82116
ShootclpP2041C -> TH -> Y0.71123
FlowerclpP2041C -> TH -> Y0.74136
PodclpP2041C -> TH -> Y0.391100
RootclpP2041C -> TH -> Y0.66169
LeafpsbN104C -> TS -> F0.31278
FlowerpsbN104C -> TS -> F0.47254
LeafrpoA803C -> TS -> L0.32232
LeafndhD620C -> TS -> L0.28261
LeafndhD824C -> TS -> L0.35215
LeafndhD1115C -> TT -> I0.67231
LeafndhE74C -> TP -> L0.95218
LeafndhA20C -> TS -> F0.85240
LeafndhA341C -> TS -> L0.82242
ShootndhA341C -> TS -> L0.48222
Leafycf420G -> AR -> K0.14255
Leafycf4309G -> AK -> K0.24340
Leafycf4316G -> AE -> K0.24121
* Editing efficiencies were calculated as a ratio of reads with the edited site to total reads mapped to that locus.

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Nawae, W.; Yundaeng, C.; Naktang, C.; Kongkachana, W.; Yoocha, T.; Sonthirod, C.; Narong, N.; Somta, P.; Laosatit, K.; Tangphatsornruang, S.; et al. The Genome and Transcriptome Analysis of the Vigna mungo Chloroplast. Plants 2020, 9, 1247. https://doi.org/10.3390/plants9091247

AMA Style

Nawae W, Yundaeng C, Naktang C, Kongkachana W, Yoocha T, Sonthirod C, Narong N, Somta P, Laosatit K, Tangphatsornruang S, et al. The Genome and Transcriptome Analysis of the Vigna mungo Chloroplast. Plants. 2020; 9(9):1247. https://doi.org/10.3390/plants9091247

Chicago/Turabian Style

Nawae, Wanapinun, Chutintorn Yundaeng, Chaiwat Naktang, Wasitthee Kongkachana, Thippawan Yoocha, Chutima Sonthirod, Nattapol Narong, Prakit Somta, Kularb Laosatit, Sithichoke Tangphatsornruang, and et al. 2020. "The Genome and Transcriptome Analysis of the Vigna mungo Chloroplast" Plants 9, no. 9: 1247. https://doi.org/10.3390/plants9091247

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