The complete chloroplast genome of Glycyrrhiza uralensis Fisch. isolated in Korea (Fabaceae)

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Korean J. Pl. Taxon. 2021;51(4):353-362

Publication date (electronic) : 2021 December 31

doi : https://doi.org/10.11110/kjpt.2021.51.4.353

Mi-Hee KIM ^{1 , **} , Suhyeon PARK ^{2 , **} , Junho LEE ³ , Jinwook BAEK ¹ , Jongsun PARK ^{2 ,}, Gun Woong LEE ^{1 ,}

¹Jeonju AgroBio-Materials Institute, Jeonju 54810, Korea

²InfoBoss Inc. and InfoBoss Research Center, Seoul 06278, Korea

³FromBio Co. Ltd., Suwon 16681, Korea

^*Author for correspondence: starflr@infoboss.co.kr (J. Park), gwlee@jami.re.kr (G. W. Lee)

**These authors contributed equally to this work.

Received 2021 October 27; Revised 2021 December 26; Accepted 2021 December 27.

Abstract

Materials and Methods

Identification of intraspecific variations

Single nucleotide polymorphisms (SNPs) and insertions and deletions (INDELs) were identified from the multiple sequence alignment of the six G. uralensis chloroplast genomes conducted by MAFFT 7.450 (Katoh and Standley, 2013) with ‘Find variations/SNPs’ implemented in Geneious Prime 2020.2.4 (Biomatters Ltd.). In addition, multiple sequence alignments from eleven Glycyrrhiza species were used in the same way (Table 1). Moreover, pair-wise alignments of the selected chloroplast genomes (Table 1) were also utilized in the same way. This method has been used in the previous studies investigating intraspecific variations on organelle genomes (Choi et al., 2021; Park et al., 2021c, 2021d). INDEL region was defined as the continuous INDELs.

Table 1.

List of number of intraspecific variations identified from chloroplast genomes of the twelve Glycyrrhiza species including G.uralenesis.

Results and Discussion

The chloroplast genome of G. uralensis (GenBank accession: MZ066516) isolated in Korea is 127,689 bp long, which is shorter than those of typical plant chloroplast genomes. This is caused by lack of one of inverted repeat regions in the typical plant chloroplast genomes (Fig. 1), which is a common phenomenon in genus Glycyrrhiza (Kang et al., 2018). However, Abrus chloroplast (GenBank accession: MT328396), neighbor genus of Glycyrrhiza (Hayashi et al., 1998), presents a typical structure of chloroplast genome, indicating that this atypical structure is specific to Glycyrrhiza. It contains 110 genes consisting of 76 protein-coding genes (PCGs), four ribosomal RNAs (rRNAs), and 30 transfer RNAs (tRNAs) (Fig. 1).

Fig. 1.

Circular map of chloroplast genome of Glycyrrhiza uralensis isolated in Korea. Gene shown outside are transcribed clockwise, and inside the circle are transcribed counter clockwise. Genes are color-coded to distinguish different functional groups. The dark grey and the light grey plot in the inner circle correspond to the GC content and AT content, respectively. Inside chloroplast genome, the three arcs of which colors are grey, light green, and light blue indicate large single copy (LSC), small single copy (SSC), and inverted repeat (IR) regions.

A total of 150 SNPs and 10 INDEL regions covering 75 bp were identified from the six G. uralensis chloroplast genomes. Eighteen out of 76 PCGs, including matK, atpB, ndhK, psaB, psbC, rpoB, rpoC1, rpoC2, atpF, atpA, rpl20, psbB, rps3, ycf2, ycf1, ndhL, ccsA, and ndhF, cover at least one SNPs. The number of PCGs containing at least one SNP is slightly larger than that of Chenopodium album L. (Park et al., 2021b); however, the ratio of non-synonymous to synonymous SNPs is 15:19, which is typical in anigosperms. The genes containing non-synonymous SNPs, matK, atpB, ndhK, rpoC1, rps3, ycf1, and ccsA, can be a candidate to develop Glycyrrhiza-specific molecular marker to evaluate their generic background.

To evaluate the numbers of SNPs and INDELs identified from G. uralensis chloroplast genomes, we investigated intraspecific variations of eleven Glycyrrhiza species (Table 1) using the available complete chloroplast genomes. The numbers of SNPs ranged from 11 to 1,163 and those of INDEL regions were from 8 to 281 (15 to 2,339 bp in length) (Fig. 2). Many of numbers of intraspecific variations of the other plant species showed similar or smaller than those of G. uralensis except Goodyera schlechtendaliana Rchb. F. (Fig. 2, Table 2). It indicates that the number of SNPs of G. uralensis is in the middle, but INDEL coverage is relatively small. However, genetic diversity of chloroplast genomes should be investigated further based on well-designed sampling strategy considering with the number of samples and their locations together.

Fig. 2.

Scattered plot of number of single nucleotide polymorphisms (SNPs) and insertion and deletion (INDEL) coverage (bp) identified from the twelve Glycyrrhiza species and nine species. X-axis is number of SNPs and Y-axis is INDEL coverage. Blue dot indicates the number of SNPs and INDEL coverage identified from G. uralensis, orange dots were from eleven Glycyrrhiza species, and grey dots were from those of nine plant species identified based on pair-wise alignment of complete chloroplast genomes.

Table 2.

List of number of intraspecific variations identified from chloroplast genomes of the various plant species.

Nucleotide diversity of six G. uralensis chloroplast genomes was calculated to investigated hotspot of nucleotide diversity. Average nucleotide diversity was 0.0002216 (Fig. 3A) which is higher than those of A. thaliana (0.000017) isolated from several countries (Park et al., 2020b) and C. album (0.0000625) isolated in the Korean Peninsula (Park et al., 2021b). This high intraspecific nucleotide diveristy may be caused by different genetic backgrounds of G. uralensis, in which accessions are separated in two indepdendent clades in the phylogenetic tree (Fig. 3A). Interestingly 5′ region of atpB contained three non-synonymous SNPs (Fig. 3B), which is congruent to the studies presenting that atpB is one of positive selected genes (Xie et al., 2018; Lee and Park, 2021; Zhang et al., 2021). In addition, intron region of ndhA also displayed high nucleotide diversity, caused by one big insertion region from MT120791 (Fig. 3C). This phenomenon was also observed in various chloroplast genomes, such as A. thaliana (Park et al., 2020b), Viburnum amplificatum Kern (Park et al., 2020c), and Coffea arabica L. (Park et al., 2019b). Furthermore, intergenic regions of petN-trnC, trnC-rpoB, atpI-atpH, atpA-trnG, trnQ-accD, accD-psaI, psbE-petL, and trnR-trnL presented high nucleotide diversity (Fig. 3A), which can be used for developing molecular markers to distinguish G. uralensis populations or cultivars.

Fig. 3.

Nucleotide diversity calculated from the six G. uralensis chloroplast genomes. A. X-axis indicates coordination of chloroplast genome and Y-axis indicates nucleotide diversity of each window. Average nucleotide diversity was displayed as dark blue color and labels on graph indicate nearby gene names of the peak of which nucleotide diversity is above 0.001. B. Enlarged diagram shows multiple sequence alignment in 5′ region of atpB. C. Enlarged diagram shows multiple sequence alignment in 5′ region of ndhA intron.

Fifty-seven Glycyrrhiza chloroplast genomes with one outgroup species of Abrus pulchellus subsp. cantoniensis (Hance) Verdc., were used for constructing phylogenetic trees. Both phylogenetic trees display that six G. uralensis are clustered into the two clades including one G. glabra (GenBank accession: MG736059) with high supportive values of ML and BI trees except one low bootstrap value of ML (Fig. 4). Phylogenetic position of G. uralenesis is congruent to the previous phylogenetic studies (Hayashi et al., 1998, 2000; Liu et al., 2019; Duan et al., 2020). In addition, one G. glabra chloroplast (GenBank accession: MG736059) was found in the clade of G. uralensis (Fig. 4). One of possibility of this chloroplast can be misidentification because G. glabra was in the same clade with G. inflata in the previous studies (Hayashi et al., 1998, 2000; Liu et al., 2019; Duan et al., 2020). Moreover, two G. uralensis chloroplast genomes (GenBank accessions: KU862308 and MN199032) isolated in Korea and China (Jia et al., 2019), respectively, were clustered separately (Fig. 4), suggesting that geographical origin of G. uralensis might not be related to these two clades due to its cultivation in East-Asian countries (Yamamoto et al., 2003; Hayashi et al., 2005). Interestingly, non-synonymous SNPs identified only in the two G. uralensis chloroplast genomes (GenBank accessions: KU862308 and MN199032) were found in atpB (Fig. 3B) as well as ndhK, rpoC2, and ycf1. In addition, synonymous SNPs from those chloroplast genomes were also identified in psbA, psbC, rpoB, rpoC1, rpoC2, atpA, accD, psbB, and ndhF, which may contribute to forming the two separate clades. G. uralensis chloroplast genome assembled in this study provides insights of its intraspecific features, which will be helpful for understanding the structure of cultivated and native G. uralensis populations in detail.

Fig. 4.

Maximum-likelihood (ML) and Bayesian inference (BI) phylogenetic trees based on the complete chloroplast genomes of 57 Glycyrrhiza and one outgroup taxon, Abrus pulchellus subsp. cantoniensis. Phylogenetic tree was drawn based on ML tree. The numbers above branches indicate bootstrap values of ML tree and posterior probabilities of BI tree, respectively. Locations of each chloroplast genome were displayed after species name, originated from the published papers (Sabir et al., 2014; Kang et al., 2018; Bang et al., 2019; Jia et al., 2019; Duan et al., 2020) or source section of GenBank files of unpublished chloroplast genomes. Star mark in the NC_042146 indicates that location is not available and it is from the location of institute. The clade containing MZ066516 chloroplast was enlarged to make it easy to show the topology.

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