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Article

Phylogenetic Analysis of Wild Pomegranate (Punica granatum L.) Based on Its Complete Chloroplast Genome from Tibet, China

by
Lide Chen
1,†,
Yuan Ren
1,†,
Jun Zhao
2,
Yuting Wang
2,
Xueqing Liu
3,
Xueqing Zhao
1 and
Zhaohe Yuan
1,*
1
Co-Innovation Center for Sustainable Forestry in Southern China, College of Forestry, Nanjing Forestry University, Nanjing 210037, China
2
Institute of Forestry Science of Tibet Autonomous Region, Lhasa 850000, China
3
Yantai Academy of Agricultural Science, Yantai 265500, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2023, 13(1), 126; https://doi.org/10.3390/agronomy13010126
Submission received: 26 October 2022 / Revised: 15 December 2022 / Accepted: 28 December 2022 / Published: 30 December 2022
(This article belongs to the Section Horticultural and Floricultural Crops)
Browse Figures
Versions Notes

Abstract

:
Pomegranates (Punica granatum L.) are one of the most fashionable fruits and can be cultivated in both tropical and subtropical regions of the world. It is a shiny and attractive pome due to its cultivation. It belongs to the Lythraceae family. In this study, we analyzed the chloroplast genome of wild pomegranate based on whole genome shotgun sequences. In wild pomegranate, we found 158,645 bp in size, 132 genes containing 84 protein genes, 37 tRNA genes, 8 rRNA genes, and 36.92% of GC content, one infA and two duplicated ycf15 pseudogenes. Moreover, 21 chloroplast genes contained intros that are detected in a large single copy (LSC), small single copy (SSC), and two inverted repeats (IRA and IRB) regions, 17 of which were involved in single introns, while four genes (ycf3, rps12, clpP and rsp12) located in LSC, IRA, and IRB region. In total, 26,272 codons are found in protein-coding genes (PCGs); relative synonymous codon usage (RSCU) analysis revealed that the most abundant amino acid is leucine containing 2773 codons (10.55%), less abundant is methionine amino acid containing 1 codon (0.0032) in the PCGs. Furthermore, a total of 233 cpSSRs were identified in the wild pomegranate cp genome, and their distribution was analyzed in three regions, namely IR, LSC, and SSC. However, 155 cpSSR were found in the LSC (66.5%), followed by 40 cpSSR in the SSC (17.2%) and 38 cpSSR in the IR (16.3%) regions. Phylogenetic validation revealed that wild pomegranate is close to the pemphis acidula species. We believe that the cp genome allocates significant information promising for breeding research of wild pomegranate to Lythraceae.

1. Introduction

Pomegranates (Punica granatum L.) are an essentially important pome of the tropical as well as subtropical areas of the entire cosmos. It originated from central Asia and since ancient times it has tremendously blessed many human cultures [1]. Pomegranate belongs to the family Lythraceae, in scientific research [2]. Due to its high adaptability and resistance, pomegranate is widely cultivated in more than 30 countries of the world, including Iran, the United States, Spain, and China [2,3,4]. Preliminary reports have indicated that pomegranate originated from the regions of Iran and Turkey, and subsequently spread throughout different parts eastward, including China, India, and other areas [2,3,4]; it then slowly moved west towards the Mediterranean countries [5]. Wild pomegranate grows throughout various parts of the world, such as the Near East, Transcaucasia, Dagestan, and Asian regions, due to genetic resources [6]. In recent times, breeding has played a significant role in the field of molecular and genetic research, however, the interspecific hybridization between wild and cultivated species has become one of the major concerns in breeding research. At molecular and evolutionary approach, both the wild species and cultivated species possess a great relationship due to their wild relativeness, and will be an essential gene bank for pomegranate [7,8,9]. At the genetic and molecular level, these wild and cultivated pomegranates have also played a significant role in the identification of DNA-based molecular markers [8]. In the long process of natural selection, artificial domestication, and breeding, pomegranate germplasm had accumulated abundant genetic variations, showing a high diversity in the color, hardness, flavor, and size of fruits. Recently, many studies based on DNA molecular markers have been carried out to determine local cultivars polymorphism [10,11,12]. However, research on wild pomegranate resources based on structural, functional, and divergence analysis are still scarce, therefore, it is necessary to conduct further investigation into the various cultivars of the family Lythraceae.
Pomegranate is an ancient fruit tree with nutritional value and health function [13,14,15]. Various anthocyanins, flavonoids, aromatic compounds, phenolic structures, and various number of ingredients can be found in different tissues of pomegranates, such as seeds [16], flowers [17], and leaves [18]. The tree possesses the blessing fruit along with a high content of anthocyanins pigments, such as violet, blue, red, and other different colors based on flavonoids, which can be found in various parts of the plant [19,20]. Additionally, it had been recommended that these fruits are fantastically valuable to humankind because it contains a high level of anthocyanin pigments and flavonoid contents that are knowns to potentially possess antioxidants compounds, which is beneficiation against numerous health diseases such as cardiac disorders, cancer, leukemia, and infections [21,22].
Recently, omic, genomic, and proteomic studies have attracted a lot of attention in the field of biological, horticultural, and scientific research. At the floral level, the chloroplast genomes have shown a lot of structural, divergence, and functional analysis of pomegranate plants, because chloroplast genomes are photosynthetic organelles belongs to plant cells, and it is hypothesized that these were originated from free-living cyanobacteria by process of endosymbiosis [23]. In plants, chloroplast not only play significant roles in photosynthetic process, but also plant growth and development [24]. Furthermore, chloroplast plays an important role in the physiological and biological functions of the plants [24]. On the other hand, chloroplast genome has played various roles in the floral defense mechanism involving the collaboration of defense compounds [25]. With the collaborative development of high-throughput sequencing technology and bioinformatics, reports on the cp genome of fruit trees have been increasing rapidly [26,27,28,29]. It is of great value to use the chloroplast genome to reveal the origin of a species and evolutionary relationships among different species [30]. Currently, more and more genomics studies on pomegranate cultivars have been carried out for different analyses. The genome of the pomegranate cultivar ‘Taishanhong’ has been determined, and the chloroplast genome of several pomegranate cultivars has been successfully published [2], which lays a foundation for the study of the genetic evolution of pomegranate. However, there are fewer studies on the genetic relationship among wild pomegranates [31,32], especially in China. Therefore, information regarding the plastid genome of wild pomegranates is needed to provide a reference for the origin and evolution of pomegranates and the phylogenetic development of the family Lythraceae. Apart from this, various online web tools, especially bioinformatics, have played and developed to recover the chloroplast genome from genomic DNA at a molecular level, for example, NOVOPlasty [33], chloroExtractor [34], and GetOrganelle [35].
The current study is the first study that provides the functional, molecular, and genetic structure and characteristics of the complete chloroplast genome of wild pomegranate. In addition, the genome sequence was compared with closely related species from the Lythraceae family. We also study: the chloroplast genome; structural features; physiological measurements; genes related to photosynthesis and self-duplication; genes containing intronic position; relative synonymous codon usage (RSCU) analysis; microsatellite analysis (forward, palindromic, reverse and complement); genomic divergence; and the phylogenetic analysis of wild pomegranate. We believe that our results obtained here will offer authentic information for understanding the evolutionary relationship of wild pomegranate, as well as the phylogenetic history of the order Myrtales.

2. Materials & Methods

2.1. Plant Materials

For the current study, the young and fresh leaves of the wild pomegranate were collected from the Nujiang Valley (97°42′ E, 29°85′ N), which is in the Tibet Autonomous Region of China. The fresh leaves were dried with allochroic silica gel and stored at the Co-Innovation Center for Sustainable Forestry in Southern China, College of Forestry, Nanjing Forestry University, Nanjing. The total genomic DNA was extracted followed by a Cetyltrimethylammonium bromide (CTAB) method (Doyle 1986). After the extraction of DNA, the extracted DNA was stored at −20 °C for further analysis.

2.2. Genome Sequencing and Annotation

DNA libraries were sequenced using the Illumina Hiseq X Ten platform (Nanjing, China) for at least 150 bp of reads. The fastp (version 0.20.0, https://github.com/OpenGene/fastp, accessed on 6 May 2021) was utilized to filter the original data. Finally, 5.2 GB of clean data was obtained. The chloroplast genome was assembled by SPAdes v3.10.1 (http://cab.spbu.ru/software/spades/, accessed on 10 May 2021) [36] with a kmer setting at 55, 87, and 121, respectively. Quality control of the assembled data was conducted with the reference cp genome of Punica granatum (NC_035240. 1) downloaded from National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/nuccore/, accessed on 10 April 2021). For the present study, two methods were adopted to annotate the chloroplast genome to improve the accuracy of the annotation. The prodigal v2.6.3 (https://www.github.com/hyattpd/Prodigal, accessed on 12 May 2021) [37] was applied to annotate CDS. The HMMER v3.3 (http://www.hmmer.org/, accessed on 12 May 2021) [38] was used to predict rRNA, while online software ARAGORN v1.2.38 (http://130.235.244.92/ARAGORN/, accessed on 15 May 2021) [39] was utilized to predict tRNA. On the other hand, blast v2.6 (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 15 May 2021) was used to align the assembled genome with the related species genome published on NCBI. The final annotation sequence was submitted to Gene Bank (accession number MT600023) after manual correction.

2.3. Condon Usage Bias Analysis

The entire chloroplast genome of wild pomegranate was chosen to analyze the codon usage pattern. For relative synonymous codon usage (RSCU) analysis, the RSCU values were calculated by CodonW v1.4.4 according to [40]. The GC content was calculated using TBtools v0.6696 [41]. The vmatch v2.3.0 (http://www.vmatch.de/, accessed on 28 May 2021) [42] was used to identify repeat sequences (forward, palindromic, reverse and complement) with minimum length and hamming distance set as 20, 3. The MISA-web v1.0, Microsatellite identification toll (Leibniz-Institute, Saarbrucken, Germany, http://pgrc.ipk-gatersleben.de/misa/misa. html, accessed on 29 May 2021) [43] was applied to identify simple sequence repeats (cpSSRs) in the chloroplast genome with minimal repeat numbers of 1–8. In addition, the Perl script version 5.26 (Wall, L., Los Angeles, CA, USA, perl.org, accessed on 29 May 2021) was used to recognize the repeated sequences.

2.4. Genome Comparison and Sequence Divergence

For homology, modeling via the complete chloroplast genome of wild pomegranate species were compared with other species by using the default parameters of Mauve online software (Darling, A, Wisconsin-Madison, Madison, WI, USA, http://darlinglab.org/mauve, accessed on 14 June 2021) [44,45]. IRscope bio tool was used to visualize the boundaries of four different regions in the cp genomes of 8 related species including (Punica granatum, Heimia myrtifolia, Lagerstroemia indica, Lagerstroemia intermedia, Lagerstroemia speciosa, Lagerstroemia subcostata, Sonneratia alba and Trapa maximowiczii).

2.5. Phylogenetic Analysis

To visualize the phylogenetic position of wild pomegranate species, phylogenetic visualization was performed by using the multiple sequence alignment, the published chloroplast genome of the 60 species was downloaded from the National Center of Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov, accessed on 28 May 2021) using an in a house python script and the linkage tree was constructed by using MAFFT v7.427 [46]. RAxML v8.2.10 [47] (https://cme.h-its.org/exelixis/software.html, accessed on 14 June 2021) was used to construct the maximum likelihood evolutionary tree (GTRGAMMA model, rapid Bootstrap analysis, Bootstrap = 1000) replicates.

3. Results

3.1. Structural Organization and Gene Content of Wild Pomegranate cp Genome

As shown in Figure 1 and Table 1, the complete chloroplast genome of the wild pomegranate genotypes demonstrates the structural organization, and the “cp genome” of wild pomegranate was sequenced through the Illumina paired-end subsequently de novo assembly in SPAdes by using the GetOrganelle pipeline via presently determined in Bandage. Moreover, the size of the genome was 158,645 base pairs (bp). The structure of wild pomegranate “cp genome” demonstrates that it is composed of four regions, namely (a) large single copy (LSC), (b) small single copy (SSC), and two inverted repeat regions (c) (IRA) via (IRB). The length of these four regions between wild and cultivated pomegranates was also differentially expressed. In addition, the length of wild pomegranates in different regions containing, genome, LSC, SSC, and IR size (bp) (158,645, 89,028, 18,686, and 25,465), while the corresponding lengths of cultivated pomegranates of different regions containing, genome, LSC, SSC and IR size (bp) (158,638, 89,021, 18,684, and 25,467), respectively. Based on the difference in gene sequences length also demonstrates variations in the number of annotated genes via other components, for example, in wild pomegranate, several genes (132), proteins genes (84), tRNA genes (37), rRNA genes (8), GC content (36.92), and GenBank accession (MT600023). While in cultivated pomegranate, several genes (113), proteins genes (79), tRNA genes (30), rRNA genes (8), GC content (36.92), and GenBank accession (MK603513) in family Lythraceae (Table 1). Taken together, the base composition statistically analysis of the “cp genome” is mentioned in Table S1. Such as adenine, thymine, cytosine, and guanine (31.09%, 31.99%, 18.78%, and 18.14%). The total GC content of the “cp genome” was 36.92%, while LSC, SSC, and IR regions were 34.89%, 30.63%, and 42.78%, respectively (Table S1).
Additionally, the functional categorization of wild pomegranate chloroplast genome based on gene functions, mostly photosynthesis and self-replication components was the most reliable. The most relevant genes were categorized into photosynthesis (44) and self-replication (59) genes. Among them, based on the specific function of photosynthesis related genes were divided into photosystem I, photosystem II, NADH dehydrogenase, cytochrome b/f complex, ATP synthase, and rubisco (8, 12, 11, 6, 6 and 1) genes. Furthermore, the genes based on the specific function of self-replication were divided into proteins of large ribosomal subunit, proteins of small ribosomal subunit, subunits of RNA polymerase, ribosomal RNA, and transfer RNA (9, 12, 4, 4, and 30) genes (Table 2). While, other genes were categorized into maturase, protease, envelope membrane protein, Acetyl-CoA carboxylase, and c-type cytochrome synthesis (1, 1, 1, 1 and 1) genes. Interestingly, a total of three pseudo genes (one infA and two ycf15) were detected in the cp genome of the wild pomegranate. It is predicted that infA and the ycf15 genes may be generated because of the interruption of internal stop codons. On the other hand, there were some other genes with unknown functions (Table 2). In our research, a total of 21 chloroplast genes contained intros that are detected in LSC, SSC, IRA, and IRB regions, 17 of which were involved in single introns, while four genes (ycf3, rps12, clpP, and rsp12) located in LSC, IRA, and IRB region (Table 3). Furthermore, the smallest intron in the entire chloroplast genome of wild pomegranate was considered to be as trnL-UAA (517 bp) whereas the longest intron for trnK-UUU (2889 bp).

3.2. Codon Usage (RSCU) Analysis

The relative synonymous codon usage (RSCU) of the wild chloroplast genome was analyzed with Perl script for comparable calculations. The relative numbers of amino acids, codons, number count frequency, and RSCU values are shown in Table 4 and Figure 2, the chloroplast genome of wild pomegranate, 26,272 codons were found in protein-coding genes (PCGs) based on the codon usage bias. Among them, codon AUG-encoded with methionine amino acid was the higher RSCU value (1.9968), while, the lower RSCU value was found in the GUG-encoded methionine (0.0032). The most abundant amino acid was leucine containing 2773 codons (10.55%) in the PCGs, whereas less abundant was cysteine amino acid containing 299 codons (1.13%) in the PCGs. The UAA, UAG, and UGA were termination codons that could not encode any amino acids. Furthermore, the magnitude of the RSCU value had a significant relationship with the codons themselves. It can be found that almost all RSCU values ending with A/U ending codons are greater than 1 (RSCU > 1), while the RSCU values ending with C/G ending codons are lesser than 1(RSCU < 1). The UGG codon only encoded Trp with no codon usage bias (RSCU = 1).

3.3. Analysis of Types via the Number of SSRs in the Chloroplast Genome of Wild Pomegranate

In our study, using MISA software, the occurrence of single sequence repeats (SSRs) in the chloroplast genome of wild pomegranate was analyzed. As shown in Figure 3 and Table S2, there were a total number of 60 long repeat sequences were found, including 29 forward, 31 palindromic, 0 reverse, and 0 complements. The repeat length of these sequences was in the range of 30 to 25,466 in bp. The longest-repeat sequences that occurred most frequently were 30 bp (5 sites), followed by 31 bp (4 sites) and 70 bp (4.2 sites). However, among these categories according to the localization result of the first location, 81.4% of the repeats were found in the protein-coding gene (PCG) region, 13.6% of the repeats were detected in the intergenic region (IGS), and the remaining 5% were in the IGS, IGS; ndhA, and trnS-GCU genes. In addition, repeat I start, repeat II start, type of repeat sequences (F, P, R and C), size in bp, distance, E-value, gene, and regional location is described in Table S3.
In this study, a total of 233 cpSSRs were identified in the wild pomegranate chloroplast genome, and their distribution was visualized in three regions namely IR, LSC, and SSC. However, 155 cpSSR were found in the LSC (66.5%), followed by 40 cpSSR in the SSC (17.2%) and 38 cpSSR in the IR (16.3%) regions. In addition, the number of exons and intron, type of repeat sequences, time, and the number are mentioned in Table S4. Furthermore, the size of these cpSSRs ranged from 8 bp to 32 bp, and repeat motif, length (bp), start position, end position, region, and the location of the genes are explained in Table S5. Based on sequence repeats the number of nucleotide repeats is also significantly different. These sequences were divided into mononucleotides, dinucleotides, trinucleotides, tetranucleotides, and pentanucleotides repeats. Among them, the highest repeats were found in mononucleotides (165), dinucleotide (7) trinucleotide (61), tetranucleotide (8), and pentanucleotide (1) were ranked lowest, respectively (Figure 4). According to the statistical analysis, 99.14% of cpSSR (231) contains A/T bases, showing typical A/T bases bias evolutionary relationship. Based on this assumption, it can be concluded that mononucleotide repeats have contributed the most relevant genetic as well molecular variations in the chloroplast genome of wild pomegranate.

3.4. Structural and Variant Assessment of Chloroplast Genomes

In Figure 5, IR analysis shows that cp genomes of different species were divided into four junctions (a) JLB, (b) JSB, (c) JSA, and (d) JLA. In these junctions, the four component regions of the wild pomegranate cp genomes along with other species are identified as (a) LSC, (b) IRB, (c) SSC, and (d) IRA regions. In the junction of JLB, LSC region contains the largest number of 89,028 bp, and JSA, SSC region contains smaller number of 18,685 bp. Furthermore, rsp19, rps12, trnN, ndhF, ycf1, and trnH genes were present in the different junctions of the chloroplast genome of wild pomegranate along with other species. Among them, the length of ycf1 gene in the region of SSC was the longest 4547 bp and the length of rsp19 gene in the region of LSC was the smallest 189 bp. On the other hand, the chloroplast genomes of wild pomegranate and two varieties (‘Tunisia’ and ‘Nana’) showed highly conserved characteristics and features in all regions as well as borders. Based on the chloroplast genome of the wild pomegranate analysis and comparison with other cultivars IR borders have shown species relatively conservative, in contrast, the distance in bp is slightly different. Our results hypothesized that the inverted repeat expansion and mutual contraction of the cp genomes have an evolutionary and biological relationship in terms of cp genomes related to other cultivars (Figure 5).
The cp genomes of wild pomegranate and nine other species related family of Lythraceae were compared by using Mauve alignment (Figure 6). The results displayed that in two cultivars ‘Tunisia and Nana’ the genomic sequences display perfect homology conservation with no inversion or rearrangements with wild pomegranate, as shown in Figure 6, confirming that gene distribution and gene structure are highly similar to each other. While other species have shown slight differences with wild pomegranate cultivars but are very close, it may be because of the evolutionary relationship between these chloroplast genomes of the species have to face some hurdles and may need some time for the recovery process.

3.5. Phylogenetic Analysis

To reveal the phylogenetic relationship of Punica granatum and determine the relationships within Myrtales family members, the tree is constructed by using RAxML v8.2.10 and utilized to construct a maximum likelihood evolutionary tree based on the GTRGAMMA model and rapid bootstrap analysis (bootstrap = 1000) (Figure 7). For the comparative analysis of 56 species from the order, Myrtales was selected to perform the evolutionary relationship, and four species (Oenothera argillicola, Oenothera parviflora, Oenothera biennis, and Ludwigia octovalvis) from the order Onagraceae were set as outgroups. A total of 54 nodes were generated in the phylogenetic tree, and most of them had 100% bootstrap support. The result showed that wild and cultivated pomegranates formed a clade with zero branch length and illustrated the phylogenetic placement of the genus Punica in Myrtales.

4. Discussion

The cp genome had been authentically analyzed to be a productive biological and agricultural tool for fast and exact crop recognition as a super-barcode [48,49]. The complete cp genome of wild pomegranate was de novo assembled using whole genome sequencing data by using GetOrganelle [35]. In the present study, we successfully assembled and annotated the complete cp genome of wild pomegranate (Figure 1). The complete cp genome of wild pomegranate was found to be 158,645 bp in size, respectively. The wild pomegranate displayed a critical quadripartite structural arrangement that involves a pair of IRs (IRA and IRB) separated by a large single copy (LSC) and a small single copy (SSC) (Figure 1). The results show that there is 132 number of genes along with the same gene order, containing 84 protein genes, 37 tRNA genes, 8 rRNA genes and GC content 36.92% (Table 1). In our study, we found one infA pseudo gene and two ycf15 duplicated pseudo genes (Table 2). Which are inconsistent with a previous study [50,51,52]. Maybe, due to the accumulation of premature termination codons, another reason is that it may be due to the incomplete duplication of genes in the IRs via SSC region that is similar to previous findings [50,51,52]. In our study, we found 21 chloroplast genes that are located in the LSC, SSC, and IRs regions, whereas four genes (ycf3, rps12, clpP, and rps12) located in LSC, IRA, and IRB regions, having four introns. In addition, the smallest intron in all the cp genome was trnL-UAA (517 bp), while the longest intron was for trnK-UUU (2889 bp) (Table 3). Our results are in agreement with previous findings [53]. Based on the relative synonymous codon usage (RSCU), the wild cp genome of pomegranate was abundant in AT-rich, it is not wondering that AT-ending codons could be pre-eminent in the protein-coding genes. These results are in agreement with previous findings [54], furthermore, the Leu amino acid coded with CUA, CUC, CUG, CUU, UUA, and UUG codons were the abundant codon with a RSCU value 10.55% (Table 4).
Overall, the investigations of numerous cp genomes displayed that repetitive sequences were important for inducing insertion via deletions (indels) and substitutions [55]. Generally, in plants and animals these repetitive sequences not only play an essential role in the rearrangement and stabilization of the cp genome sequence, but also influence the copy number differences among various species [56]. Microsatellites or simple sequence repeats (SSRs) had been identified as a preliminary foundation of DNA based molecular markers because they carry a higher polymorphism rate via plentiful changes at the plants level. However, these SSR markers are valuable for detecting the population diversity, genetic diversity, and polymorphisms at the species level, intraspecific, intraspecific and cultivar levels, for differentiation between the species [57,58]. In the current study, a total of 60 repetitive sequences were detected in the wild pomegranate cp genome, including 29 forward, 31 palindromic, 0 reverse, and 0 complements (Table S2 and Figure 3). Furthermore, a total of 233 cpSSRs were identified in the wild pomegranate chloroplast genome, and their distribution was visualized in three regions namely IR, LSC, and SSC. However, 155 cpSSR were found in the LSC (66.5%), followed by 40 cpSSR in the SSC (17.2%), and 38 cpSSR in the IR (16.3%) regions. In addition, the number of exons and intron, the type of repeat sequences, time, and the number, are mentioned in Table S4. Furthermore, the size of these cpSSRs ranged from 8 bp to 32 bp, and repeat motif, length (bp), start position, end position, region, and location of the genes are explained in Table S5. These identified cpSSRs were categorized into mononucleotide, dinucleotide, trinucleotide, tetranucleotide, pentanucleotide, and repeat sequences. Among them, the most plentiful single-sequence repeats (SSRs) were single-nucleotide repeats (Figure 4). According to D.C et al., these findings are in agreement with his published study [59].
To date, a comparative determination of cp genome sequences revealed intrinsic biological and genetic information, such that rsp19, rps12, trnN, ndhF, ycf1, and trnH genes were present in the different junctions of the cp genomes of wild pomegranate, along with other species. Among these, ycf1 had greater nucleotide diversity, as confirmed by y regions subject to a positive selection [60]. Taken together, these reports are similar findings on the cp genome of some Prunus species that might be due to the high GC content of the eight identified rRNA genes in the IR boundaries. Moreover, the contraction and expansion of these junctions, such as SSC, LSC, and the IRs region during angiosperm evolution, are mostly accountable for the declared fluctuation in cp genome length [61,62]. This ensured that the contraction and expansion of the IRs region perhaps had a leading role in the creation of pseudogenes.
However, IR analysis shows that cp genomes of different species were divided into four junctions: (a) JLB, (b) JSB, (c) JSA, and (d) JLA. In these junctions, the four component regions of the wild pomegranate cp genomes along with other species are identified as: (a) LSC, (b) IRB, (c) SSC, and (d) IRa regions. In the junction of JLB, LSC region contains the largest number of 89,028 bp, and JSA, SSC region contains a smaller number of 18,685 bp. Furthermore, rsp19, rps12, trnN, ndhF, ycf1, and trnH genes were present in the different junctions of the chloroplast genome of wild pomegranate along with other species. Among them, the length of ycf1 gene in the region of SSC was the longest 4547 bp and length of rsp19 gene in the region of LSC was the smallest 189 bp (Figure 5).
Comparative analysis displayed that the cp genomes of wild pomegranate and related species in the family of Lythraceae were compared by using Mauve alignment. The results displayed that in two cultivars ‘Tunisia and Nana’ the genomic sequences display perfect homology conservation with no inversion or rearrangements with wild pomegranate, as shown in Figure 6, indicating highly conserved regions; Luo et al. confirmed that most cp genomes in angiosperm floras are generally stable, respectively. Moreover, based on various evolutionary analyses as well as genetic backgrounds, the cp genome structure, shape, frequency, diameter, size, and numbers, can vary. Our results are inconsistent with his published study [63].
Gu et al. displayed that phylogenetic determination using genomic repetitive sequences has resolved various lineages within angiosperms plants [64]. Therefore, to access the evolutionary history of Lythraceae family species the phylogenetic tree based on the cp genome revealed that wild pomegranate MT600023.1 accessions was closely related to pemphis acidula NC_041439.1 is closely related (Figure 7). However, the phylogenetic tree containing cp genomes of 60 species have linkage with each other demonstrating that they have diverged. It has been predicted that this could be due to the biological heterozygosity in wild pomegranates. Phylogenetic tree evolutionary analysis has been conducted on many species for biological, molecular, genetic, and divergence studies such as the Japanese Apricot [59], Balsaminaceae [65], and ornamental species [63]. These results are indicating the high possibility of limiting accessions based on their supplementary information for Lythraceae, or Myrtaceae families. In the recent study, we analyzed the characterization of cp genome using complete genomic sequencing data. Our analysis displayed that cp genome sequences may not be appropriate for examining the genetic diversity of wild pomegranate genotypes. Therefore, we hypothesize that our genomic sequencing data of wild pomegranate species are appropriate resources for wild pomegranate breeding at molecular programs.
Comparative genomic analysis revealed that the wild pomegranate was highly conserved in gene quantity, distribution, structure, and other characteristics among closely related species (Figure 5 and Figure 6). Additionally, phylogenetic analysis indicated the taxonomic status/classification of wild pomegranate in Myrtles (Figure 7). The results of this research shed new light on the phylogeny, population genetics, and evolutionary history of the genus of Punica. Given that, the chloroplast genome of higher plants has a stable conserved sequence structure, their length typically in the range of 120–160 kb. The assembly results showed that the cp genome of wild pomegranate was 158,645 bp in length, contained 132 genes, and the GC content was 36.92% (Table 1). However, the length of the cp genome of the relative species in the Lythraceae family is in the range of 152,025~159,219 bp, the GC content ranged from 36.40%~37.60%, and the gene ranged from 107~130. These results were consistent with the previous analysis of Lythraceae [66]. The GC content detection results in each region found that the IR region had the highest GC content (42.78%), which may be caused by the presence of high GC rRNA sequences in this region [67,68]). Codon usage bias analysis revealed that isoleucine codons have the highest codon abundance (8.7%) among the 26,272 codons in the wild pomegranate chloroplast genome, which was consistent with Artemisia annua [69], Paeonia ostia [70], and Forsythia suspensa [71]. Besides, codons with RSCU values greater than 1 (RSCU > 1) showed a bias ending in A/U, except for AUG and UGG, which is consistent with previous studies [71].
Previous studies have shown that repeat sequences are rich in genetic information, showing self-replication, self-splicing, and recombination during genome evolution [72]. Repeat sequences were found mainly in the intergenic region (IGS) and the protein-coding region (PCGs) [73,74]. However, only 13.6% of repeat sequences were detected in the wild pomegranate chloroplast genome in the IGS region, while 81.4% were detected in the protein-coding region (Table S3). As one of the most effective markers of genomics and systematic genomics, cpSSR (cp simple repeat sequence) embodied/represented the evolutionary pressure exerted on plants in their evolutionary process [75,76,77]. Evolutionary events in angiosperm lead to the expansion and contraction of their IR region, resulting in subtle differences in IR borders and genome sizes between different species [78,79].
A comparative analysis of wild pomegranate and closely related species revealed that the length of the IR region is similar (Figure 6), which indicates that the whole genome of the cp genome in the Lythraceae is highly conserved. Previous studies have attracted controversy about the classification of pomegranates, raising questions about whether pomegranate belongs to the Punicaeceae, Lythraceae, or Myrtaceae families. In this study, a maximum likelihood tree based on the whole chloroplast genome sequence was constructed, indicating that wild pomegranate is most closely related to Pemphis acidula (Figure 7). In addition, an analysis of the complete genomic data set clarified the botanical location of the genus Punica and explained at the organelle level for most species’ relationships under order Myrtales. Although many phylogenetic studies based on cpDNA sequences have been reported successively, there are still fewer studies on wild resources. Using the cp genome approach to obtain the chloroplast genome characteristics of wild pomegranate will provide supplementary information for pomegranate origin and evolution of the pomegranate study.

5. Conclusions

To conclude, the high throughput sequencing of plant species probably contains various readings that are taken from the cp genome that furnishes a distinctive opportunity to assemble the entire cp genomes. In the current study, we study the complete cp genome of wild pomegranate, which contains 158,645 bp in size. In cp genome, we found 132 genes involving 84 protein genes, 37 tRNA genes, 8 rRNA genes, and 36.92% of GC content. There is one infA and two duplicated ycf15 pseudogenes found. In addition, 21 chloroplast genes contained intros that are detected in LSC, SSC, IRA, and IRB regions, 17 of which were involved in single introns, while four genes (ycf3, rps12, clpP, and rsp12) are located in the LSC, IRA, and IRB region. Based on the RSCU value, the most abundant amino acid was leucine containing 2773 codons (10.55%) in the PCGs, whereas the less abundant was cysteine amino acid containing 299 codons (1.13%) in the PCGs. A total of 233 cpSSRs were identified in the wild pomegranate chloroplast genome, and their distribution was analyzed in three regions namely IR, LSC, and SSC. However, 155 cpSSR were found in the LSC (66.5%), followed by 40 cpSSR in the SSC (17.2%), and 38 cpSSR in the IR (16.3%) region. The complete cp genome sequences were assembled with ‘Tunisia and Nana’ cultivars. Phylogenetic validation revealed that wild pomegranate was close to the pemphis acidula species. Furthermore, we study: the cp genome, structural, and physiological measurements; genes related to photosynthesis and self-duplication; genes containing intronic position; RSCU analysis; cpSSRs (forward, palindromic, reverse and complement); genomic divergence; and the phylogenetic analysis of wild pomegranate. Finally, yet importantly, our analysis provides positive information to understand the evolutionary correspondence among Lythraceae.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy13010126/s1, Table S1: For wild pomegranate number and percentages of base pairs containing for each region; Table S2: Repetitive sequences of forward, palindromic, reverse and complement; Table S3: Size (bps), distance, E-value, genes and regional location; Table S4: LSC, SSC, and IRs (IRA via IRB) regional location, Table S5: Repeat motif, length region, and cpSSR location.

Author Contributions

Conceptualization, L.C.; methodology, L.C.; investigation, J.Z., Y.W. and X.L.; data curation, Y.R.; writing—original draft, L.C.; writing—review and editing, L.C., Y.R., and Z.Y.; project administration, Z.Y.; supervision, X.Z.; funding acquisition, X.Z. and Z.Y. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (31901341).

Institutional Review Board Statement

Not applicable.

Data Availability Statement

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Diagrammatic map of the wild pomegranate chloroplast genome. The dense line in the exterior region represents inverted repeats (IR) namely (IRA via IRB). The genes that are shown in the anterior region are transcribed clockwise, and other genes that are outside are transcribed counterclockwise. Furthermore, the genes that belong to various functional groups are color-coded. And, the dark grey in the anterior region represents the GC content.
Figure 1. Diagrammatic map of the wild pomegranate chloroplast genome. The dense line in the exterior region represents inverted repeats (IR) namely (IRA via IRB). The genes that are shown in the anterior region are transcribed clockwise, and other genes that are outside are transcribed counterclockwise. Furthermore, the genes that belong to various functional groups are color-coded. And, the dark grey in the anterior region represents the GC content.
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Figure 2. Relative synonymous codon usage (RSCU) bar chart of the wild chloroplast genome. Note: the box below represents all codons encoding each amino acid, and the height of the column above represents the sum of all codons RSCU values.
Figure 2. Relative synonymous codon usage (RSCU) bar chart of the wild chloroplast genome. Note: the box below represents all codons encoding each amino acid, and the height of the column above represents the sum of all codons RSCU values.
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Figure 3. Representation of types and number of repeat sequences in the chloroplast genome of wild pomegranate. The abbreviation stands for ‘F’ forward repetition, ‘P’ palindromic repetition, ‘R’ reverse repetition, and ‘C’ for complementary repetition. Different color indicates the individual repeats.
Figure 3. Representation of types and number of repeat sequences in the chloroplast genome of wild pomegranate. The abbreviation stands for ‘F’ forward repetition, ‘P’ palindromic repetition, ‘R’ reverse repetition, and ‘C’ for complementary repetition. Different color indicates the individual repeats.
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Figure 4. The repeat evaluation and frequency distribution of several SSR types in wild pomegranate.
Figure 4. The repeat evaluation and frequency distribution of several SSR types in wild pomegranate.
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Figure 5. Illustration of the borders and analysis of LSC, SSC, and IRs regions among demonstrative wild pomegranate chloroplast genomes. The relative information of the genes is presented by boxes.
Figure 5. Illustration of the borders and analysis of LSC, SSC, and IRs regions among demonstrative wild pomegranate chloroplast genomes. The relative information of the genes is presented by boxes.
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Figure 6. Illustration of the chloroplast genomes of 10 Lythraceae family cultivars rearranged by MAUVE software. The top of the alignment represents the genome of wild pomegranate, and below that represents the other species. The locally collinear blocks (LCBs) are demonstrated by the similar color and lines interconnect blocks. Here, the vertical row demonstrates the degree of conservation among positions. Moreover, short squares represent the locations of the gene in each genome, the white portion represents the CDs, the green represents tRNA, and the red color represents the rRNA.
Figure 6. Illustration of the chloroplast genomes of 10 Lythraceae family cultivars rearranged by MAUVE software. The top of the alignment represents the genome of wild pomegranate, and below that represents the other species. The locally collinear blocks (LCBs) are demonstrated by the similar color and lines interconnect blocks. Here, the vertical row demonstrates the degree of conservation among positions. Moreover, short squares represent the locations of the gene in each genome, the white portion represents the CDs, the green represents tRNA, and the red color represents the rRNA.
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Figure 7. Phylogenetic trees of wild pomegranate species and relative genomes of the 59 species based on whole chloroplast genomes. The tree is constructed by using RAxML via the Maximum likelihood method of 1000 replications. The bootstrap interference values are displayed above the branches or near the nodes.
Figure 7. Phylogenetic trees of wild pomegranate species and relative genomes of the 59 species based on whole chloroplast genomes. The tree is constructed by using RAxML via the Maximum likelihood method of 1000 replications. The bootstrap interference values are displayed above the branches or near the nodes.
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Table
Table 1. Summary of the wild pomegranate chloroplast genomes with other nine related species.
Table 1. Summary of the wild pomegranate chloroplast genomes with other nine related species.
Parameters Wild PomegranatePunica granatum cultivar ‘Nana’Punica granatum cultivar ‘Tunisia’Heimia myrtifoliaLagerstroemia indicaLagerstroemia
intermedia		Lagerstroemia
speciose		Lagerstroemia
subcostata		Sonneratia albaTrapa maximowicizz
Genome size (bp)158,645158,638158,639159,219152,025152,330152,476152,049153,061155,577
LSC size (bp)89,02889,02189,02288,57184,04683,98784,05183,89078,22688,528
SSC size(bp)18,68618,68418,68418,82116,91416,87316,97916,90918,03218,272
IR size (bp)25,46525,46725,46725,91425,62325,73625,72325,62523,90224,389
Number of genes132113113112113130129129107110
Protein genes84797978798585842977
tRNA genes37303030303736372429
rRNA genes8888888888
GC content (%)36.9236.9236.9237.5936.9537.637.637.637.2936.4
GenBank accessionMT600023MK603513MK603512MG921615NC_030484NC_034662NC_031414NC_034952NC_039975NC_037023
Table
Table 2. Categorization and list of genes within wild pomegranate chloroplast genome.
Table 2. Categorization and list of genes within wild pomegranate chloroplast genome.
CategoryGene GroupGene Name
PhotosynthesisSubunits of photosystem IpsaA, psaB, psaC, psaI, psaJ
Subunits of photosystem IIpsbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, psbM, psbN, psbT, psbZ
Subunits of NADH dehydrogenasendhA *, ndhB * (2), ndhC, ndhD, 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
Large subunit of rubiscorbcL
Self-replicationProteins of large ribosomal subunitrpl14, rpl16 *, rpl2 (2), rpl20, rpl22, rpl23 (2), rpl32, rpl33, rpl36
Proteins of small ribosomal subunitrps11, rps12 ** (2), rps14, rps15, rps16 *, rps18, rps19, rps2, rps3, rps4, rps7 (2), rps8
Subunits of RNA polymeraserpoA, rpoB, rpoC1 *, rpoC2
Ribosomal RNAsrrn16 (2), rrn23 (2), rrn4.5 (2), rrn5 (2)
Transfer RNAstrnA-UGC * (2), trnC-GCA, trnD-GUC, trnE-UUC, trnF-GAA, trnG-GCC, trnG-UCC *, trnH-GUG, trnI-CAU (2), trnI-GAU * (2), trnK-UUU *, trnL-CAA (2), trnL-UAA *, trnL-UAG, trnM-CAU, trnN-GUU (2), trnP-UGG, trnQ-UUG, trnR-ACG (2), trnR-UCU, trnS-GCU, trnS-GGA, trnS-UGA, trnT-GGU, trnT-UGU, trnV-GAC (2), trnV-UAC *, trnW-CCA, trnY-GUA, trnfM-CAU
Other genesMaturasematK
ProteaseclpP **
Envelope membrane proteincemA
Acetyl-CoA carboxylaseaccD
c-type cytochrome synthesis geneccsA
Translation initiation factor# infA
Genes of unknown functionConserved hypothetical chloroplast ORF# ycf15 (2), ycf1, ycf2 (2), ycf3 **, ycf4
Note: Gene *: Contains one intron; Gene **: Contains two introns; # Gene: Pseudogene; Gene (2): Gene whose copy number is more than 1.
Table
Table 3. Characteristics of the genes that contain intron and exon in the chloroplast (cp) genome of wild pomegranate.
Table 3. Characteristics of the genes that contain intron and exon in the chloroplast (cp) genome of wild pomegranate.
GeneLocationExon IIntron IExon IIIntron IIExon II
trnK-UUULSC37248935
rps16LSC40855227
trnG-UCCLSC2373848
atpFLSC145759410
rpoC1LSC4327491608
ycf3LSC124727230767153
trnL-UAALSC3551750
trnV-UACLSC3860335
rps12IRa114-23254826
clpPLSC71607292811228
petBLSC6779642
petDLSC8766475
rpl16LSC9994399
ndhBIRb777683756
rps12IRb232-26548114
trnI-GAUIRb3794935
trnA-UGCIRb3880335
ndhASSC5531049539
trnA-UGCIRa3880335
trnI-GAUIRa3794935
ndhBIRa777683756
Table
Table 4. Relative synonymous codon usage (RSCU) in the chloroplast genome of wild pomegranate.
Table 4. Relative synonymous codon usage (RSCU) in the chloroplast genome of wild pomegranate.
Amino AcidCodonCountRSCUAmino AcidCodonCountRSCU
TerUAA501.7856MetGUG10.0032
TerUAG190.6786AsnAAC2770.4362
TerUGA150.5358AsnAAU9931.5638
AlaGCA3911.1348ProCCA3131.1648
AlaGCC2220.6444ProCCC1990.7404
AlaGCG1440.418ProCCG1260.4688
AlaGCU6211.8028ProCCU4371.626
CysUGC730.4882GlnCAA7171.547
CysUGU2261.5118GlnCAG2100.453
AspGAC2220.403ArgAGA4791.818
AspGAU8801.597ArgAGG1790.6792
GluGAA10411.4946ArgCGA3781.4346
GluGAG3520.5054ArgCGC990.3756
PheUUC5210.7108ArgCGG1100.4176
PheUUU9451.2892ArgCGU3361.275
GlyGGA7311.6364SerAGC1220.3594
GlyGGC1860.4164SerAGU4131.2156
GlyGGG2960.6624SerUCA4131.2156
GlyGGU5741.2848SerUCC3150.9276
HisCAC1450.4632SerUCG1810.5328
HisCAU4811.5368SerUCU5941.749
IleAUA7030.9267ThrACA4101.2396
IleAUC4630.6102ThrACC2510.7588
IleAUU11101.4631ThrACG1490.4504
LysAAA10651.501ThrACU5131.5512
LysAAG3540.499ValGUA5211.4676
LeuCUA3870.8376ValGUC1860.524
LeuCUC1870.4044ValGUG1930.5436
LeuCUG1640.3546ValGUU5201.4648
LeuCUU5901.2768TrpUGG4591
LeuUUA8921.9302TyrUAC1920.3966
LeuUUG5531.1964TyrUAU7761.6034
MetAUG6071.9968
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Chen, L.; Ren, Y.; Zhao, J.; Wang, Y.; Liu, X.; Zhao, X.; Yuan, Z. Phylogenetic Analysis of Wild Pomegranate (Punica granatum L.) Based on Its Complete Chloroplast Genome from Tibet, China. Agronomy 2023, 13, 126. https://doi.org/10.3390/agronomy13010126

AMA Style

Chen L, Ren Y, Zhao J, Wang Y, Liu X, Zhao X, Yuan Z. Phylogenetic Analysis of Wild Pomegranate (Punica granatum L.) Based on Its Complete Chloroplast Genome from Tibet, China. Agronomy. 2023; 13(1):126. https://doi.org/10.3390/agronomy13010126

Chicago/Turabian Style

Chen, Lide, Yuan Ren, Jun Zhao, Yuting Wang, Xueqing Liu, Xueqing Zhao, and Zhaohe Yuan. 2023. "Phylogenetic Analysis of Wild Pomegranate (Punica granatum L.) Based on Its Complete Chloroplast Genome from Tibet, China" Agronomy 13, no. 1: 126. https://doi.org/10.3390/agronomy13010126

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