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Article

Characterization of the Chloroplast Genome Structure of Gueldenstaedtia verna (Papilionoideae) and Comparative Analyses among IRLC Species

by
Ogyeong Son
1 and
Kyoung Su Choi
2,*
1
Educational Research Division, Exhibition Research Department, Daegu National Science Museum, Yugaeup, Daegu 43023, Republic of Korea
2
Plant Research Team, Animal and Plant Research Department, Nakdonggang National Institute of Biological Resources, Sangju 37242, Republic of Korea
*
Author to whom correspondence should be addressed.
Forests 2022, 13(11), 1942; https://doi.org/10.3390/f13111942
Submission received: 7 October 2022 / Revised: 13 November 2022 / Accepted: 15 November 2022 / Published: 17 November 2022
(This article belongs to the Section Genetics and Molecular Biology)
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Abstract

:
The genus Gueldenstaedtia belongs to Papilionaceae’s inverted repeat-lacking clade (IRLC) and includes four species distributed throughout Asia. We sequenced the chloroplast genome of G. verna and compared it with those of the IRLC clade. The genome was 122,569 bp long, containing 77 protein-coding genes, 30 tRNAs, and 4 rRNAs. Comparative analyses showed that G. verna lost one inverted repeat region, the rps16 gene, an intron of rpoC1, and two introns of clpP. Additionally, G. verna had four inversions (~50 kb inversion, trnK–psbK; ~28 kb inversion, accD–rpl23; ~10 kb inversion, rps15–trnL; ~6 kb inversion, trnL–trnI) and one reposition (ycf1). Its G + C content was higher than that of other IRLC species. The total length and number of repeats of G. verna were not significantly different from those of the other IRLC species. Phylogenetic analyses showed that G. verna was closely related to Tibetia. A comparison of substitution rates showed that ycf2 and rps7 were higher than one, suggesting that these were positive selection genes, while others were related to purified selection. This study reports the structure of the chloroplast genome of a different type, i.e., with four inversions and one reposition, and would be helpful for future research on the evolution of the genome structure of the IRLC.

1. Introduction

The chloroplast (cp) genomes of angiosperms have been used for phylogenetic analysis [1,2], nucleotide substitution analysis [3,4], cp genome evolution analysis [5,6], and DNA molecular marker analysis [7,8] over the past decades. Previous studies have demonstrated that the cp genome structure of angiosperms comprises large single-copy regions, small single-copy regions, and two inverted repeat (IR) regions [9,10]. Gene content and order are highly conserved, including 79 protein-coding genes, 29 tRNA genes, and 4 rRNA genes [10]. However, some angiosperms show differences in gene content, order, and structure. For example, members of Fabaceae [11], Geraniaceae [3,12], Companulaceae [13,14,15], and Orobanchaceae [16,17,18] showed rearrangement of gene order, inversion, loss in IR regions, expansion of IR regions, loss of genes, or pseudogenes.
Fabaceae is one of the largest families of angiosperms containing essential species for agricultural activities. Fabaceae is classified into six subfamilies: Caesalpinioideae, Cercidoideae, Detarioideae, Dialioideae, Duparquetioideae, and Faboideae (Papilionoideae) [19]. Among the subfamilies, Faboideae, one of its monophyletic clades, known as the inverted repeat lacking clade (IRLC), has lost one copy of the IR region (25 kb) in the cp genome [20]. The IRLC includes 52 genera and over 4000 species divided into seven tribes, and the cp genomes of IRLC species show a loss or pseudogenization of genes (rps16, rpl22, infA, accD, and ycf4), loss of introns (clpP, atpF, and rpoC1), inversions, and gene transfer to the nucleus [20,21,22,23,24]. Recently, Choi et al. [25] suggested the IR re-emergence in one IRLC species, Medicago minima.
Gueldenstaedtia is a genus of papilionoid legumes established by Fischer and named after Gueldenstaedt [26]. Sanderson and Wojciechowski’s [27] molecular analysis included G. himalaica under the Astragalus genus due to its close relation with Chesneya dshungarica, although it was supported by low bootstrap values (30%). Later, Zhu [28] suggested dividing the genus Gueldenstaedtia into two subgenera, Gueldenstaedtia and Tibetia, once the two groups were distinct in seeds and other morphological traits, pollen characteristics, and chromosome data [28,29]. Only four species of the genus Gueldenstaedtia (G. monophylla, G. thihangensis, G. henryi, and G. verna) are distributed throughout Asia [29]. Recently, molecular phylogenetic analyses using a nuclear internal transcribed spacer (ITS) and plastids matK, trnL-F, and psbA-trnH showed that Gueldenstaedtia and Tibetia (GUT clade) are closely related, being supported by the highest bootstrap value (100%). Both analyses also placed Chesneya as a sister clade to GUT [30]. In previous studies, some of the cp genomes in IRLC species independently showed genomic rearrangements, such as intron loss and gain, pseudogenization, and inversions [23,31]. There are reports of cp genome analyses of Tibetia species (T. himalaya, NC_053369 and T. liangshanensis, NC_036109), but the cp genome of Gueldenstaedtia has never been analyzed.
In this study, we report the novel and complete cp genome of G. verna in Fabaceae. We aimed to (1) compare the cp genomes within Fabaceae considering inversion, gene, and intron loss; (2) suggest a new phylogenetic position for the genus Gueldenstaedtia; and (3) determine the nucleotide substitution rates of G. verna.

2. Results

2.1. Characterization of the Chloroplast Genome of Gieldenstaedtia verna

A total of 32,505,084 reads were obtained after whole-genome sequencing (Figure S1). The size of the cp genome of G. verna (Genbank accession number: OP525440) was 122,569 bp, and it showed an IR loss (Figure 1). The GC content was 36.0%, and the total genes included 77 protein-coding genes (PCGs), 30 transfer RNA (tRNA), and 4 ribosomal RNA (rRNA). Among these genes, Rps16 has been lost in G. verna, thirteen genes (atpF, ndhA, ndhB, petB, petD, rpl2, rpl16, rps12, trnG-UCC, trnL-UAA, trnV-UAC, trnI-GAU, and trnA-UGC) contained a single intron, and one gene (ycf3) contained two introns.

2.2. Comparison of cp Genomes within Fabaceae

The total length of IRLC species ranged from 121,020 bp (Lathyrus sativus) to 131,179 bp (Wisteria brachybotrys), and the GC content ranged from 33.8% (Medicago hybrida) to 36.0% (G. verna) (Table 1).
A few genes in the cp genomes of IRLC species were lost. The rps16 gene was lost in all IRLC species. Genes rps18, rpl23, atpE, and ycf4 were lost independently (Table 2). Only the rps16 gene was lost in G. verna. The intron content in IRLC cp genomes was more variable than the gene content. The intron of atpF was lost in L. frutescens, and the intron of rpl16 was lost in T. aureum. The intron of rpoC1 was lost in G. verna. L. japonicus had two introns for clpP, whereas IRLC species lost one or two introns for clpP. Two Tibetia species (T. himalacia and T. liangsharensis), G. verna, and Glycyrrhiza lepidota did not have introns for clpP (Figure 2, Table 2). Five tRNA genes containing one intron were identified. However, the intron of trnG-UCC lost in four species (M. hybrid, T. aureum, L. culinaris, and V. sativa).
Among sequences longer than 30 bp, the repetitive sequence analysis detected sequences with 35 (Lessertia frutescens) to 236 (Tibetia himalaica) repeats. The length of the repeats varied between 30 bp and 517 bp (V. sativa), mostly forward repeats, except in L. frutescens and Lotus japonicas (not IRLC species) (Table 1). The abundance of repetitive sequences in seven species, including L. japonicus (not an IRLC species), was below 3%, whereas another seven species had around 3% or slightly more. T. himalaica had the highest percentage of repetitive sequences (9.5%).

2.3. Phylogenetic Analysis

We conducted a maximum likelihood (ML) phylogenetic analysis based on 67 protein-coding genes from 19 species, including an outgroup (L. japonicus), with 67,345 bp alignment (Figure 3, Table 1). The IRLC formed a monophyletic group, subdivided into two clades: (1) Glycyrrhiza and Wisteria species and (2) Tribes Galegeae, Carganeae, Cicereae, Trifolieae, and Fabeae species. G. verna and genus Tibetia formed a single sister clade to the tribe Galegeae (L. frutescens and Astragalus mongholicus var. nakainus). The IRLC monophyletic group, both subclades, and the Caraganeae sister clades were well-supported by bootstrap value (100%).

2.4. Inversion in cp of G. verna

A comparison between G. verna and two Tibetia species (T. liangsharensis and T. himalaica) detected four inversions and one reposition in G. verna (Figure 4). A large inversion of approximately 50 kb was located between the genes trnK and psbK (Figure 4A, Figure S2). Three inversions of approximately 28 kb, 10 kb, and 6 kb were located between accD and rpl23 (Figure 4B), rps15 and trnL (Figure 4E), and trnL and trnI (Figure 4C), respectively. Additionally, G. verna showed a reposition of the ycf1 gene (Figure 4D).

2.5. Substitution Analysis

We analyzed the substitution rates of 71 protein-coding genes from 18 IRLC species using Lotus japonicus as a reference (Figure 5 and Table S1). The median value of synonymous substitutions (dS) was higher than that of the non-synonymous substitutions (dN). The dS median ranged from 0.31 (G. lepidota) to 0.06 (L. sativus), and the dN ranged from 0.19 (G. lepidota) to 0.26 (L. culinaris). Among the analyzed genes, the highest dN rates were from clpP (0.55) and ycf1 (0.45) in G. verna. The psbI gene had a higher dS rate in L. frutescens, P. sativum, and L. sativus than that in other species. Most of the genes’ dN/dS values were less than 1. The exceptions were ycf1 in W. brachybotrys, W. sinensis, T. haimalaica, and T. liangsharensis; rps18 in L. culianris; and rps7 and ycf2 in G. verna.

3. Discussion

The complete cp genome size, structure, and gene content are highly conserved in angiosperms [10]. However, rare genome characteristics, such as gene loss, inversion, and IR loss, have been reported in Fabaceae [11,21,22,31]. Gueldenstaetia is a genus from the Caraganaceae family, which, together with Fabaceae, belongs to the IRLC [32]. In this study, we showed the novel and complete cp genome of G. verna and compared it with previously reported cp genomes from related species.
Previous studies have shown that six genes (accD, infA, rpl22, rps16, rps18, and ycf1) were absent in Trifolium subterraneum [11], the genes rps16 and rpl22 were lost in Astragalus membranaceus [33], and the rpl2 gene was absent in Trifolium resupinatum [34]. Magee et al. [22] reported that four legume species (Glycine max, Trifolium subterraneum, Cicer arietinum, and Medicago truncatula) lost the ycf4 gene. The introns of rps12 and clpP were also not found in the IRLC [22,35,36]. This study found that the rps16 gene and two introns (clpP and rpoC1) were absent in G. verna (Figure 2, Table 2). Previous studies have shown that the rps16 gene and intron 1 of clpP were absent from the IRLC species [37,38,39], except Glycyrrhiza glabra [37]. However, in G. verna and the genus Tibetia, the clpP gene lost two introns. Intron II of clpP has been lost independently in land plants [40], including G. verna, two Tibetia species, and Glycyrrhiza glabra. The loss of the rpoC1 intron has been reported in some taxa, such as one species of Medicago, four species of Passiflora, and other species of Scaevola, Goodenia, and Cactaceae [40,41]. This study showed that the genus Tibetia has an intron of rpoC1, which has been lost in G. verna (Figure 2).
The IRLC exhibits many rearrangements, such as two inversions in Astragalus [31], Trifolium, and Vicia [23] (Figure S2). Our results showed that Glycyrrhiza, Wisteria, Astragalus, Lessertia, and Tibetia had similar cp genome structures (Figure S2), whereas some variations, such as inversion and reposition, were detected in G. verna (using Tibetia as the reference, Figure 4). Hiratsuka et al. [42] and Walker et al. [43] suggested that the cp genome structure is correlated with tRNA through intermolecular recombination between tRNA sequences, while Fullerton et al. [44] reported that the G + C content affects inversion. We detected four inversions and one repositioning in G. verna, although its total G + C content was higher than that of other IRLC species. Our results do not support the G + C content hypotheses, and future studies are needed to describe the cp genome structure variation better. Repetitive sequences in G. verna are not longer or more numerous than those in other species of the IRLC (Table 1). T. himalaica had the highest number of repetitive sequences; however, the cp genome structure of T. himalaica is similar to closely related species (Figure S2). Previous studies [23,45,46] have reported that repetitive sequences are located in duplications of tRNA and flanking inversion regions in cp genomes. However, no such association was found among the repetitive sequences in G. verna.
Previous molecular phylogenetic studies [47] using nrDNA ITS and cpDNA matK, trnL-F, and psbA-trnH markers grouped Gueldenstaedtia and Tibetia into one clade. Our results revealed that Guldenstaedtia and Tibetia were in the same clade and well-supported (100% bootstrap value).
The dS of cpDNA is lower than that of nrDNA and higher than that of mtDNA [48]. The substitution rates of genes in the single-copy (SC) region are higher than those in the IR region [49]. Recently, many scholars [37,50,51,52] have attempted to solve the questions associated with genome evolution, such as structure, inversion, and rearrangement, using substitution rates. For example, Schwarz et al. [50] suggested that the dN and dS substitution rates are correlated with plastome size and rearrangements. We observed many inversions and repositionings in G. verna. The dN of ycf1 and accD were higher than in other species (Figure 5A). However, ycf1 and accD did not exhibit positive selection (dN/dS < 1). Two genes (rps7 and ycf2) were positively selected with dN/dS > 1 (Figure 5). Our study showed that the substitution rates of G. verna did not support the previously reported ones [50]. In addition, localized hypermutation regions, such as accD, clpP, and ycf1, have been reported to accelerate substitution rates [23,24], whereas the substitution rates of the three genes in G. verna were not positively correlated (dN/dS < 1). This implies that the rate accelerations of cp genes in G. verna are different from those in other species, and a more comprehensive sampling of this taxon is needed to determine the evolution of cp genes in Gueldenstaedtia.

4. Materials and Methods

4.1. Sampling, DNA Extraction, and Sequencing

Fresh G. verna leaves were collected from Bolli-ri, Hwawon-eup, Dalseong-gun, Daego, Korea. The specimens were deposited at the Daegu National Science Museum. Total genomic DNA was extracted using a DNeasy Plant Mini Kit (Qiagen Inc., Valencia, CA, USA). Genomic DNA was sequenced using the Illumina HiSeq X platform (San Diego, CA, USA). We obtained 32,505,084 total reads from the 150 bp paired-end sequences with a quality value ≥Q30, accounting for 89.1%.

4.2. Genome Assembly, Genome Annotation, and Comparison of Genome Structure

The de novo assembly of the chloroplast genome was performed using GetOrganelle v.1.7.6.1 [53]. For coverage calculations (Figure S1), the reads were aligned using Bowtie2 [54]. The read coverage of G. verna is shown in Figure S1. Geseq (https://chlorobox.mpimp-golm.mpg.de/OGDraw.html, accessed on 10 November 2022) [55] was used to annotate the cp genome of G. verna, and tRNA gene sequences were annotated using tRNAscan-SE 2.0 [56]. Protein-coding genes and tRNAs were double-checked by identifying open reading frames and comparing with reference genomes (Table 1) in Geneious Prime [57]. Genome mapping was performed using OrganellarGenomeDRAW (OGDRAW) (Version 1.3.1) [58], and the chloroplast genome of G. verna was deposited in GenBank.
We compared the cp genome of G. verna with published data (Table 1) of other IRLC Fabaceae species. Alignments of 16 species, including the outgroup, were found to detect genome rearrangements, such as inversion and repositioning, using Mauve v.1.1.3 on Geneious [57].

4.3. Repeat Analysis

The simple sequence repeats (SSRs) of G. verna were identified using the REPuter program [59]. Additionally, SSRs of 16 species (including the outgroup Lotus japonicus) were also detected (Table 1). Forward, palindromic, reverse, and complement sequences were identified with a Hamming distance of 3, minimum repeat size of 30 bp, and sequence identity ≥ 90%.

4.4. Phylogenetic Analysis

The chloroplast genome sequences of 19 taxa, including one outgroup (Lotus japonicus, following Xiong et al. [32] and Xia et al. [60]), were included in the phylogenetic analyses. The 67 protein-coding genes shared across taxa were extracted from each chloroplast genome and concatenated. The sequences were aligned using MAFTT [61]. The ML analysis was conducted using RAxML (version 8) [62]; the GTR + GAMMA + I model was performed using a rapid bootstrap of 1000 replications.

4.5. Substitution Rate Estimation

The dN and dS rates were estimated for each of the 67 cp genes using CODEML in PAML v4.8 [63]. The phylogenetic tree generated in the previous section was used as a constraint tree for all the rate comparisons. In PAML, codon frequencies were determined using the F3 × 4 model, and gapped regions were excluded with the “cleandata = 1” parameter option. The transition/transversion ratio and dN/dS values were estimated using initial values of 2.0 and 0.4, respectively.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/f13111942/s1, Figure S1: Coverage of G. verna. Whole genome resequencing reads were mapped to the assembled G. verna.; Figure S2: Gene rearrangement analyses among IRLC species by Mauve alignment; Table S1: Nonsynonymous (dN) and synonymous (dS) substitutions among IRLC species.

Author Contributions

Conceptualization, O.S. and K.S.C.; methodology, O.S.; software, O.S. and K.S.C.; validation, O.S. and K.S.C.; formal analysis, K.S.C.; investigation, O.S. and K.S.C.; resources, O.S.; data curation, O.S. and K.S.C.; writing—original draft preparation, O.S. and K.S.C.; writing—review and editing, K.S.C.; visualization, O.S. and K.S.C.; supervision, K.S.C.; project administration, O.S.; funding acquisition, O.S. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Collect and Research Native Plants on the Korean Peninsula for the Natural History exhibition of the Daegu National Science Museum (DNSM).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. The complete chloroplast genome of Gueldenstaedtia verna. The genes are transcribed clockwise on the inside and counterclockwise on the outside. The darker gray in the inner circle corresponds to the GC content.
Figure 1. The complete chloroplast genome of Gueldenstaedtia verna. The genes are transcribed clockwise on the inside and counterclockwise on the outside. The darker gray in the inner circle corresponds to the GC content.
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Forests 13 01942 g002 550
Figure 2. Introns of clpP and rpoC1 in 19 inverted repeat-lacking clade (IRLC) species. (A) Introns of clpP in IRLC species. Arrows indicate intron locations. (B) Introns of rpoC1 in IRLC species. (C) Loss of rpoC1 intron in G. verna.
Figure 2. Introns of clpP and rpoC1 in 19 inverted repeat-lacking clade (IRLC) species. (A) Introns of clpP in IRLC species. Arrows indicate intron locations. (B) Introns of rpoC1 in IRLC species. (C) Loss of rpoC1 intron in G. verna.
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Figure 3. The maximum likelihood tree constructed using 67 protein-coding genes from 19 species. The colored boxes indicate loss of the gene or intron.
Figure 3. The maximum likelihood tree constructed using 67 protein-coding genes from 19 species. The colored boxes indicate loss of the gene or intron.
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Figure 4. Comparison of the chloroplast genome structures of two Tibetia species with G. verna. The (A–C,E) boxes indicate approximately 50 kb, 28 kb, 6 kb, and 10 kb, respectively. The (D) box indicates the repositioning of the ycf1 gene.
Figure 4. Comparison of the chloroplast genome structures of two Tibetia species with G. verna. The (A–C,E) boxes indicate approximately 50 kb, 28 kb, 6 kb, and 10 kb, respectively. The (D) box indicates the repositioning of the ycf1 gene.
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Figure 5. Boxplot showing the variation in non-synonymous substitutions (dN) (A), synonymous substitutions (dS) (B), and dN/dS (C) for the IRLC species. The median values are indicated above the whiskers. The red circles (C) show the genes of IRLC species with dN/dS > 1. LJ, Lotus japonicus; GL, Glycyrrhiza lepidota; WB, Wisteria brachybotrys; WS, Wisteria sinensis; AM, Astragalus mongholicus var. nakainus; LF, Lessertia frutescens; GV, Gueldenstaetia verna; TH, Tibetia himalacia; TL, Tibetia liangsharensis; CA, Cicer arietinum; MH, Medicago hybrida; TA, Trifolium aureum; LS, Lens culinaris; VS, Vicia sativa; PS, Pisum sativum; LS, Lathyrus sativus.
Figure 5. Boxplot showing the variation in non-synonymous substitutions (dN) (A), synonymous substitutions (dS) (B), and dN/dS (C) for the IRLC species. The median values are indicated above the whiskers. The red circles (C) show the genes of IRLC species with dN/dS > 1. LJ, Lotus japonicus; GL, Glycyrrhiza lepidota; WB, Wisteria brachybotrys; WS, Wisteria sinensis; AM, Astragalus mongholicus var. nakainus; LF, Lessertia frutescens; GV, Gueldenstaetia verna; TH, Tibetia himalacia; TL, Tibetia liangsharensis; CA, Cicer arietinum; MH, Medicago hybrida; TA, Trifolium aureum; LS, Lens culinaris; VS, Vicia sativa; PS, Pisum sativum; LS, Lathyrus sativus.
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Table
Table 1. Characters of 15 inverted repeat-lacking clade (IRLC) species and 1 legume species (LS, Lotus japonicus).
Table 1. Characters of 15 inverted repeat-lacking clade (IRLC) species and 1 legume species (LS, Lotus japonicus).
TaxonGenome Size (bp)GC ContentsGeneNumber of Repeat a
(F/R/C/P) b		Length of Total Repeats (bp)Repeats Percentage
(%)
Coding GenestRNArRNA
Lotus japonicus150,51936.0%7830461 (26/3/2/33)28341.8%
Glycyrrhiza lepidota127,93934.2%7730492 (59/4/4/25)43153.3%
Wisteria sinensis130,56134.4%77304109 (70/14/0/25)46223.5%
Wisteria brachybotrys131,17934.4%7730489 (50/11/2/26)36152.7%
Astragalus mongholicus var. nakaianus123,63334.1%7730467 (37/7/1/22)25972.1%
Lessertia frutescens122,70034.2%7730435 (16/3/0/16)13941.1%
Gueldenstaedtia verna122,56936.0%7730474 (51/0/0/23)37273.0%
Tibetia himalaica124,20134.5%77304236 (227/1/0/8)11,9179.5%
Tibetia liangshanensis122,37234.7%7730493 (82/1/0/10)42603.4%
Cicer arietinum125,31933.9%7630475 (45/4/1/25)35482.8%
Medicago hybrida125,20833.8%76304105 (80/5/0/20)40623.2%
Trifolium aureum126,97034.6%7730451 (34/3/0/14)28342.2%
Lens culinaris122,96734.4%75304105 (89/0/0/16)45613.7%
Vicia sativa122,46735.2%7630478 (65/0/1/12)60044.9%
Pisum sativum122,16934.8%7530461 (54/1/0/6)25642.0%
Lathyrus sativus121,02035.1%7630478 (50/0/0/28)33432.7%
a Tandem repeats ≥ 30 bp. b F, forward repeat; R, reverse repeat; C, complement repeat; P, palindromic repeat.
Table
Table 2. Gene loss and number of introns of 15 IRLC species and 1 legume species (LS, Lotus japonicus).
Table 2. Gene loss and number of introns of 15 IRLC species and 1 legume species (LS, Lotus japonicus).
LJGLWBWSAMLFGVTHTLCAMHTALCVSPSLS
Gene loss
rps16xxxxxxxxxxxxxxxx
rps18ooooooooooooxooo
rpl23oooooooooooooxxx
atpEooooxooooooooooo
ycf4ooooooooooxoxxxx
Number of introns
atpF1111101111111111
clpP2011110001111111
ndhA1111111111111111
ndhB1111111111111111
petB1111111111111111
petD1111111111111111
rpl21111111111111111
rpl161111111111101111
rps122111111111111111
rpoC11111110111111111
ycf32222222222222222
trnG-UCC1111111111000011
trnL-UAA1111111111111111
trnV-UAC1111111111111111
trnK-UUU1111111111111111
trnI-GAU1111111111111111
trnA-UGC1111111111111111
x: loss of gene; o: intact gene; LJ, Lotus japonicus; GL, Glycyrrhiza lepidota; WB, Wisteria brachybotrys; WS, Wisteria sinensis; AM, Astragalus mongholicus var. nakainus; LF, Lessertia frutescens; GV, Gueldenstaetia verna; TH, Tibetia himalaica; TL, Tibetia liangsharensis; CA, Cicer arietinum; MH, Medicago hybrida; TA, Trifolium aureum; LS, Lens culinaris; VS, Vicia sativa; PS, Pisum sativum; LS, Lathyrus sativus.
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Son, O.; Choi, K.S. Characterization of the Chloroplast Genome Structure of Gueldenstaedtia verna (Papilionoideae) and Comparative Analyses among IRLC Species. Forests 2022, 13, 1942. https://doi.org/10.3390/f13111942

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Son O, Choi KS. Characterization of the Chloroplast Genome Structure of Gueldenstaedtia verna (Papilionoideae) and Comparative Analyses among IRLC Species. Forests. 2022; 13(11):1942. https://doi.org/10.3390/f13111942

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Son, Ogyeong, and Kyoung Su Choi. 2022. "Characterization of the Chloroplast Genome Structure of Gueldenstaedtia verna (Papilionoideae) and Comparative Analyses among IRLC Species" Forests 13, no. 11: 1942. https://doi.org/10.3390/f13111942

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