Vicia L. is a genus in the Fabaceae family containing approximately 180–210 species. These species are widely distributed across temperate regions of the northern hemisphere and extend to temperate regions of South America and tropical Africa (Hanelt and Mettin, 1989). Vicia species are used as green manure, cover, forage, and honey crops, making it an economically important genus and a valuable genetic resource (Montemurro et al., 2013). Despite their high economic value, very few genetic and genomic data are available for species of Vicia, other than Vicia villosa (hairy vetch) and Vicia faba (broad bean). Vicia bungei, native to South Korea and China, is phenotypically and ecologically similar to Vicia americana (Endo et al., 2000). Although several chloroplast genomes of different Vicia species have been obtained through next-generation sequencing (NGS) (Cooper et al., 2017; Li et al., 2018; Xin and Yang, 2020), to date, no genetic or genomic studies have been conducted on V. bungei, despite the potentially valuable genetic resources present in wild varieties of this species. In addition, more effective molecular markers are required to support the phylogenetic and population genetic studies underlying the identification, conservation, utilization, and breeding of Vicia species.

The chloroplast genome has long been a focus of research into plant molecular evolution and systematics because of its small size, high copy number, and conservation among species. It has been extensively characterized at the molecular level (Kim et al., 2005). Recent technical advances in NGS technologies mean that the number of completely sequenced chloroplast genomes has increased rapidly, and such sequences play a progressively important role in the identification of molecular markers and in molecular phylogenetic analyses (Kim et al., 2016). Chloroplast genetic markers are potentially more effective indicators of population subdivision and differentiation than are nuclear markers (Schaal et al., 1998; Petit et al., 2005). Chloroplast simple sequence repeats (cpSSRs), generally defined as microsatellites with tandem repeats of 1–6 bp, are valuable resources for assessing genetic and genome diversity, as well as in phylogenetic and systematic evolutionary analyses; cpSSRs have several advantageous characteristics, including haploidy, non-recombination, uniparental inheritance, and low nucleotide substitution rate (Powell et al., 1995). The chloroplast genome can provide unique insight into evolutionary processes as it retains ancient genetic patterns (Provan et al., 2001). Moreover, as the genetic information in angiosperm chloroplasts is inherited maternally, chloroplast markers serve as useful indicators of maternal ancestry (Raveendar et al., 2015).

A new complete chloroplast genome sequence for V. bungei has been generated and compared with sequences of related genera in the Fabaceae family. This comparison enabled the development of cpSSR markers for future population genetics studies, explaining the structure. Our findings help explaining the structure of the complete V. bungei chloroplast genome and reveal evolutionary relationships within the genus Vicia.

Most chloroplast genomes have a circular structure that contains a large single-copy region, an inverted repeat A region, a small single-copy region, and an inverted repeat B region (Raveendar et al., 2015; Zhu et al., 2016). The chloroplast genomes of some legumes, including V. sepium, have lost one of the two inverted repeats, and these species form the IRLC (Li et al., 2018). The chloroplast genome of V. bungei indicates that this economically important crop is also a member of the IRLC, suggesting that it may be part of the same evolutionary clade as V. sepium and V. sativa, which may share a similar evolutionary history.

A chloroplast gene is rarely lost arbitrarily. Instead, the gene is either transferred to the nuclear genome, or its function is replaced by a nuclear gene (Wang et al., 2018). Recent sequencing and analyses of some IRLC plastomes (Kim et al., 2005; Li et al., 2018; Moghaddam et al., 2022) have revealed important evolutionary patterns in this clade, including loss of the genes rpl22 and rps16, the deletion of one intron of clpP (Jansen et al., 2008; Li et al., 2020), multiple sequence inversions (Schwarz et al., 2015; Williams et al., 2015), and gene transfers to the nucleus (Moghaddam and Kazempour-Osaloo, 2020; Wu et al., 2021). Vicia bungei plastome lacked one intron of clpP, which was consistent with the finding of Jansen et al. (2008) and confirmed the parallel loss of this clpP intron in V. bungei and in members of the papilionoid IRLC. Recently, Williams et al. (2015) demonstrated that the rates of synonymous and non-synonymous mutations are accelerated in the clpP sequence of Acacia, suggesting there may be a functional nuclear-encoded copy of this gene in at least some mimosoid legumes. The mechanism by which rpl22, rps16, and one intron of clpP gene have been lost from V. bungei requires further in-depth research.

Although the structure of plastid genomes is largely conserved across land plants (Wicke et al., 2011), other exceptions to this pattern, beyond these IRLC legumes, include Geraniaceae (Guisinger et al., 2011) and Campanulaceae (Haberle et al., 2008). Some Fabaceae plastomes have been highly rearranged owing to multiple rounds of translocations and/or inversions. As a result, the plastomes of the IRLC have undergone considerable diversification in both gene order and gene/intron content (Wang et al., 2018; Li et al., 2020; Xin and Yang, 2020; Moghaddam et al., 2022).

The plastome of V. bungei most closely resembled those of V. sepium and V. sativa. This finding is consistent with previous research that indicated similar evolutionary evaluation analysis of V. sepium and V. sativa (Li et al., 2020). Plastome differences between some major clades provide valuable information for resolving phylogenetic relationships based on DNA sequence analyses. The study of genomic variation and phylogeny of Fabaceae species provides an increased understanding of general chloroplast genome evolution. In our results of multiple alignments among six Vicia species cp genomes, we observed 11 major variant regions of V. bungei in cp genome (Supplementary Figure 3). However, our research focus on the development of cpSSR markers for Vicia species identification. Thus, these variant regions would be very useful for further evolutionary studies as well as cpSSR marker design.

In other flowering plant chloroplast genomes, the most common SSR motifs are mono-, di-, and trinucleotide repeats (George et al., 2015). Nevertheless, data mining, under the recursive criteria adopted here, revealed that the majority of SSRs present in the V. bungei chloroplast genome sequences contained relatively long penta- (53.9%) or hexanucleotide (22.7%) motifs. In this study, a total of 432 potential SSR were found in the chloroplast genome of V. bungei. The number of repeat motifs was richer in V. bungei chloroplast genome compared with the V. sepium chloroplast genome (Li et al., 2020). These results provide a firm foundation for further investigations of chloroplast genome evolution in Vicia L. and other IRLC legumes such as Medicago and Pisum and species.

Raveendar et al. (2015) used SSR markers to detect genetic diversity in V. sativa and 22 other Vicia species. Li et al. (2018) used chloroplast genome sequences to determine the genetic diversity in V. sepium and 21 closely related Fabaceae species. Han et al. (2021) used barcoding loci (ITS2, matK, and rbcL) as DNA markers to differentiate 19 Vicia taxa. Previous studies of phylogenetic relationships among species of the subgenus Vicia did not detect any intraspecific variation when cDNA SSR, cpSSR, or DNA barcoding markers were applied to representative plants of each species. By contrast, our UPGMA cluster analysis classified seven clusters at the interspecies level and revealed intraspecific differences within clusters. These results suggest that the 39 cpSSR markers developed in V. bungei differentiated efficiently between Vicia species genotypes and also provide estimates of their genetic diversity. These cpSSR markers will thus be useful, not only for authenticating Vicia species but also for providing the baseline data essential for advancing systematic breeding in the field and the development of conservation strategies, as well as for guiding the collection of germplasm.

Complete chloroplast genomes have helped to reveal intraspecies relationships, but also allow to measure divergence within interspecies. Growing genomic resources for Vicia spp. provide tools to extend our knowledge on this critically important forage crop species. To develop cpSSR markers that can be utilized to classify Vicia species and analyze the genetic diversity of related species, potential cpSSR motifs were mined from the chloroplast genome of V. bungei, and finally, 39 cpSSR markers were developed. The UPGMA cluster analysis detected intra-and interspecific variation between 20 accessions with 39 cpSSR markers to distinguish Vicia species. The chloroplast genome and cpSSR markers found in this study would provide useful information for genetic diversity analyses in Vicia species.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.

I-HJ and SH performed the experiments, analyzed the data, prepared figures and tables, and wrote the manuscript. DS, HR, TH, YL, DK, Y-SS, and J-WC reviewed drafts of the manuscript. All authors have read and approved the final manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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