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Article

Characteristics of the Complete Chloroplast Genome of Pourthiaea (Rosaceae) and Its Comparative Analysis

by
Zhengying Cao
1,2,
Wenzhi Zhao
1,2,
Yaxuan Xin
1,2,
Weixiang Shen
1,2,
Fei Wang
1,2,
Qishao Li
1,2,
Yuxiang Tu
1,2,
Haorong Zhang
1,2,
Zhanghong Dong
1,2 and
Peiyao Xin
1,2,*
1
Southwest Research Center for Engineering Technology of Landscape Architecture National Forestry and Grassland Administration, Southwest Forestry University, Kunming 650224, China
2
Key Laboratory of National Forestry and Grassland Administration on Biodiversity Conservation in Southwest China, Southwest Forestry University, Kunming 650224, China
*
Author to whom correspondence should be addressed.
Horticulturae 2022, 8(12), 1144; https://doi.org/10.3390/horticulturae8121144
Submission received: 7 November 2022 / Revised: 26 November 2022 / Accepted: 1 December 2022 / Published: 5 December 2022
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Abstract

:
Pourthiaea is of great ornamental value because it produces white flowers in spring and summer, red fruit in autumn, and their fruit does not fall in winter. In order to explore the genetic structure and evolutionary characteristics of the chloroplast genome of Pourthiaea, comparative genomics analysis and phylogenetic analysis were conducted using ten published chloroplast genomes of Pourthiaea from the NCBI database. The results showed that the chloroplast genomes of the ten species of Pourthiaea showed typical circular tetrad structures, and the genome sizes were all within the range of 160,159–160,401 bp, in which the large single copy was 88,047–88,359 bp, the small single copy was 19,234–19,338 bp, and the lengths of a pair of inverted repeats were 26,341–26,401 bp. The GC contents ranged from 36.5% to 36.6%. A total of 1017 SSR loci were identified from the chloroplast genomes of the ten species of Pourthiaea, including six types of nucleotide repeats. The gene types and gene distribution of the IR boundary regions of the chloroplast genomes of different species of Pourthiaea were highly conservative, with little variation. Through the sequence alignment of chloroplast genomes, it was found that the chloroplast genomes of the ten species of Pourthiaea were generally highly conservative. The variation mainly occurred in the spacer regions of adjacent genes. Through nucleic acid diversity analysis, three hypervariable regions were screened at Pi > 0.006, namely trnQ(UUC)-psbk-psbl, accD-psal, and ndhF-rpl32-trnL (UAG). Phylogenetic analysis showed that the ten species of the genus Pourthiaea were clustered in the same branch and formed sister groups with the genus Stranvaesia, and that the support rate for the monophyly of the genus Pourthiaea was high. This study can serve as a reference for the breeding, genetic evolution, and phylogeny of Pourthiaea.

1. Introduction

Pourthiaea refers to deciduous trees or shrubs of Maleae in Rosaceae. The native area of this genus includes the Eastern Himalayas to East Asia and IndoChina, mainly distributed across East Asia, South Asia, and Southeast Asia [1]. The Pourthiaea genus was established by Decaisne in 1874 and is noticeably different from other genera because of its deciduous leaves, pedicels and peduncles with nodular protrusions, and fruit flesh with stone cells [2]. The leaves of this genus are papery, and the flowers are mostly in umbels, corymbos, or compound corymbos [3]. White flowers bloom in spring and summer, while the fruit appears red in autumn and does not fall in winter [2]. Therefore, Pourthiaea has high ornamental value and is usually cultivated as an ornamental plant.
Rosaceae is the most widely distributed family of angiosperms, with 91 genera and approximately 4828 species [4]. It includes three subfamilies: Dryadoideae, Rosoideae, and Amygdaloideae [5,6]. In the Amygdaloideae, the intergeneric relationship of Maleae has attracted much attention [7,8,9]. The genus Pourthiaea, which was investigated in this study, is one of the most controversial genera of Maleae. The relationship between Pourthiaea and its related genera has always been complex, especially with regard to Photinia. In 1820, Lindley established Photinia [10]. Since then, three evergreen species and two deciduous species originally belonging to Crataegus have been moved into the genus [11,12]. The species under Photinia include two types: evergreen and deciduous. Until 1874, Decaisne believed that the deciduous species of Photinia were obviously different from those of other genera in that they had tuberous protrusions on the pedicels and peduncles, and stone cells in the flesh, and thus established Pourthiaea [13]. In the past, some botanists incorporated the genus Pourthiaea into Photinia based on morphological data [14,15,16,17], while some botanists believe that it should be an independent genus [18,19,20]. With the development of molecular biology, molecular systematics based on molecular biology has provided a new theoretical basis for taxonomy. In 2011, Guo et al. [21] first used two chloroplast gene fragments (trnL-trnF and psbA-trnH) and one nuclear gene fragment (nrITS) to analyze the phylogenies of Photinia, Pourthiaea, Stranvaesia, and other related genera. The results showed that Pourthiaea formed a branch line with high support and did not share a branch with Photinia, which provides solid evidence for the independence of Pourthiaea. Since then, many scholars have used different nuclear gene fragments, chloroplast gene fragments, whole chloroplast genomes (cpDNA), or ribosomal DNA (nrDNA) to study the phylogenetic relationship between Pourthiaea and its relatives [6,8,22,23], and the results have shown that Pourthiaea and Photinia are located in different branches. Not only the relationship between genera but also the division of species within the genus Pourthiaea is controversial, and the phenomenon of synonyms is serious. Liu et al. [2] revised the classification of the P. villosa complex, in which only one species, P. villosa, was identified, but it included 56 synonyms. Then, they classified the P. blinii complex, P. salicifolia complex, P. sorbifolia complex, and P. arguta complex [24] and finally provided a taxonomic list of Pourthiaea [25] for further taxonomic and evolutionary research.
Chloroplasts widely exist in eukaryotes and are an important site for photosynthesis and other metabolic processes. As semiautonomous organelles, they have a complete genome [26]. The chloroplast genomes of most higher plants each have a double-stranded circular tetrad structure, including a large single copy (LSC), a small single copy (SSC), and two inverted repeat sequences (IRs) [27]. The genome size is approximately 120–160 kb, and differences are mainly caused by the two inverted repeat sequences [28]. The structure and sequence of chloroplast genomes is of great value in revealing the origins and evolution of, and genetic relationship between, species [29]. Firstly, chloroplast genomes are highly conserved and contain rich genetic information, and their structures are simpler than those of nuclear genomes, so it is easier to obtain their full-length sequences. Secondly, the nucleotide substitution rate is moderate (approximately one third the rate of nuclear genes and three times that of the mitochondria) [30]. Moreover, the significantly different molecular evolution rates of the coding and noncoding regions can be applied to the study of different taxonomic categories [31]. At present, in the public database National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/, accessed on 15 June 2022), there are chloroplast genome data for the genus Pourthiaea that can be queried. However, some recent studies have attempted to solve the phylogenetic relationship between Pourthiaea and its relatives, while no studies have been conducted on the chloroplast genome characteristics of the genus.
In this study, the chloroplast genome sequences of ten species of Pourthiaea that have been published in GenBank thus far were downloaded, and their characteristics were analyzed comparatively to improve our understanding of the chloroplast genome structure of Pourthiaea. The purposes of this study were to compare the genomic structural variation of Pourthiaea; to study the contraction and expansion of IRs in the chloroplast genome of Pourthiaea and screen hypervariable sites, repeated sequences, and SSRs; and to reveal the relationships among species of Pourthiaea and between Pourthiaea and related genera through phylogenetic analysis based on plant chloroplast genomes. Our research results can provide resources for the subsequent interspecific identification, marker development and utilization, genetic breeding, and phylogenetic study of Pourthiaea.

2. Materials and Methods

2.1. Sequence Data Acquisition

The chloroplast genome sequences and annotation information of P. villosa, P. amphidoxa, P. pilosicalyx, P. blinii, P. zhejiangensis, P. tomentosa, P. hirsuta var. lobulata, P. arguta var. salicifolia, P. arguta, and P. sorbifolia, as well as the chloroplast genome sequences of 13 species of Photinia, 3 species of Stranvaesia, and 2 outgroups (Table A1), were downloaded from the NCBI database for bioinformatics analysis.

2.2. Analysis of the Basic Characteristics of Chloroplast Genomes

According to the chloroplast genome sequences of Pourthiaea plants published by the NCBI and their annotations, the chloroplast genome information of the 10 species of Pourthiaea was statistically analyzed in Geneious software, including the lengths and GC contents of the whole-genome sequences, the four main divisions (LSC, Ira, SSC, and IRb), and gene annotation results. Moreover, the physical map of the chloroplast genomes of Pourthiaea plants was drawn in the DRAW Organelle Genome Maps (OGDRAW)(https://chlorobox.mpimp-golm.mpg.de/OGDraw.html, accessed on 28 June 2022) [32] online drawing tool using the GenBank format file, and the annotated genes of the chloroplast genome were statistically analyzed.

2.3. Detection of Repetitive Sequences and SSRs

The simple sequence repeats (SSRs) of chloroplast genomic sequences of the 10 species of Pourthiaea were detected using MISA (https://webblast.ipk-gatersleben.de/misa/, accessed on 2 July 2022) [33], and the parameters were set with reference to a study by Wu et al. [34]: the minimum repeat number of mononucleotides, dinucleotides, trinucleotides, tetranucleotides, pentanucleotides, and hexanucleotides were set to 10, 5, 4, 3, 3, and 3, respectively. The palindromic repeats, complement repeats, forward repeats, and reverse repeats in the sequences were calculated using an online program called REPUTER (https://bibserv.cebitec.uni-bielefeld.de/REPUTER/manual.html, accessed on 3 July 2022) [35], and the specific parameters were determined according to the default setting [36]: the maximum computed repeats was 50 and the minimum repeat size was 8. Moreover, the Tandem Repeat Finder (http://tandem.bu.edu/trf/trf.html, accessed on 3 July 2022) [37] was used to detect tandem repeats, using the default parameter settings.

2.4. Analysis of the IR/SC Boundary Region

The chloroplast genomic sequences of the 10 species of Pourthiaea from GenBank were uploaded to IRs cope (https://irscope.shinyapps.io/irapp/, accessed on 10 July 2022) [38] in gb format for visual mapping of the IR/SC boundary region genes of the chloroplast genomes, and the correctness of the IRScope mapping was checked against sequence annotations with Geneious. The contraction and expansion of the IR region were analyzed by comparing the gene types and gene positions of the boundary regions of the 10 species.

2.5. Analysis of Genomic Differences

The program mVISTA (http://genome.lbl.gov/vista/mvista/submit.shtml, accessed on 18 July 2022) [39] was used to compare the chloroplast genome sequences of Pourthiaea. The chloroplast genome of P. villosa was taken as a reference for comparison with the remaining 9 species, and the genomes were aligned with the annotations through visual mapping in mVISTA to identify the differences among the 10 chloroplast genomes.

2.6. Analysis of Nucleotide Polymorphisms

The chloroplast genomic sequences of the 10 species of Pourthiaea were aligned using MAFTF v7 [40]. Next, the nucleotide polymorphisms (Pis) among genomes were calculated using DnaSP v6 software [41], and the parameters were set to a window length of 600 and a step size of 200 to screen out the loci with higher Pi values as hypervariable regions of the chloroplast genome of Pourthiaea. The specific loci were determined according to the annotation results of the chloroplast genome.

2.7. Phylogenetic Analysis

Phylogenetic tree reconstruction was carried out using the chloroplast gene sequences of 26 species (including 10 species of Pourthiaea, 13 species of Photinia, and 3 species of Stranvaesia) downloaded from the NCBI, with two species (Eriobotrya henryi and Rhaphiolepis lanceolata) from the genus Eriobotrya and the genus Rhaphiolepis functioning as the outgroup. The phylogenetic trees were constructed using the maximum likelihood (ML) and Bayesian inference (BI) methods. After aligning these sequences using MAFFT v7 [40], the alignment results were calibrated in BioEdit software [42]. An ML phylogenetic tree was built in IQ-TREE v1.6.7 software [43] using the best model TVM+F+R2, and 1000 repetitions were run to ensure the stability of the evolutionary tree [44]. A BI phylogenetic tree was built using MrBayes v3.2.6 [45]. JModelTest v2.1.10 [46] was used to select the most appropriate alternative DNA model, and the most appropriate model “TVM+I+G” (freqA = 0.3187, freqC = 0.1813, freqG = 0.1738, freqT = 0.3262, R (a) [AC] = 0.9547, R (b) [AG] = 0.9110, R (c) [AT] = 1.0136, R (d) [CG] = 0.2569, R (e) [CT] = 0.9110, R (f) [GT] = 1.000, p-inv = 0.7650, gamma shape = 0.0300) was used to construct the phylogenetic tree. All phylogenetic analysis results were visualized and adjusted using FigTree (version 1.4.3).

3. Results and Analysis

3.1. Basic Characteristics of Chloroplast Genomes

The basic characteristics of the chloroplast genomes were analyzed, including chloroplast genome size, gene number, GC content, and other information (Figure 1, Table 1). The results showed that the chloroplasts of the ten species of Pourthiaea all had typical circular DNA molecular characteristics. The genomes all had total lengths ranging from 160,159 to 160,401 bp and a conservative tetrad structure, including a large single copy region (88,047–88,359 bp), a small single copy region (19,234–19,338 bp), and a pair of reverse repeat regions (26,341–26,401 bp). The GC contents of the whole chloroplast genomes of the ten species of Pourthiaea ranged from 36.5% to 36.6%. The GC contents in the LSC region ranged from 34.1 to 36.6%, the GC contents in the SSC region ranged from 30.2% to 30.4%, and the GC contents in the IR region varied from 42.6% to 42.7%. The GC contents in the IR region were higher than those in the SSC region and LSC region.
A total of 130–131 genes were annotated in the chloroplast genomes of the ten species of Pourthiaea, including 83–84 protein coding genes. The number of tRNA genes was 37–38, the number of rRNA genes was stable at eight, and there were also two pseudogenes, namely, ψycf1 and ψrps19 (Table 2). These genes can be divided into several different categories according to different functions. Among them, 21 genes were located in the IR region. They contained two copies, including nine protein coding genes (psaI, ndhB, rps123, rps7, rpl2, rps19, rps12, ycf1, and ycf2), four rRNA genes (rrn4.5, rrn5, rrn16, and rrn23), and eight tRNA genes (trnA-UGC, trnG-GCC, trnI-CAU, trnI-GAU, trnL-CAA, trnR-ACG, trnN-GUU, and trnV-GAC). Gene transcription regulation is considered to be affected by introns and exons. Introns can accumulate more mutations and play an important role in gene expression regulation. In the chloroplast genome of Pourthiaea, 18 genes (12 protein coding genes and six tRNA genes) contained at least one intron, and three genes (clpP, ycf3, and rps12) among them harbored two introns. In addition, rps12 had a trans-splicing structure, with the 5′ end located in the LSC region and the 3′ end containing an intron located in the IR region.

3.2. Repetitive Sequences and SSR Analysis

The simple repetitive sequences in the chloroplast genomes of the ten species of Pourthiaea were identified using MISA software. A total of 1017 SSR loci were detected, between 95 (P. arguta var. salicifolia) and 107 (P. blinii) loci for each species, including six types of nucleotide repeats, namely mononucleotide, dinucleotide, trinucleotide, tetranucleotide, pentanucleotide, and hexanucleotide repeats, the numbers of which were 762, 181, 3, 60, 10, and 1, respectively (Figure 2A), accounting for 74.93%, 17.80%, 0.29%, 5.90%, 0.98%, and 0.10% of the total SSRs, respectively (Figure 2B). Among them, mononucleotide repeats were the most abundant, followed by dinucleotide repeats, and hexanucleotide repeats were the least abundant, which only existed in P. villosa. From the perspective of distribution regions, SSRs were not evenly distributed in various regions in the whole genome. Specifically, SSRs identified in the LSC region were the most abundant (75–83), followed by the SSC region (10–17), while those in the IR region were the least abundant (6–8) (Figure 2C). In the SSRs, there were four types of mononucleotide repeats (A, C, G, and T), three types of dinucleotide repeats (AT, TA, and TC), two types of trinucleotide repeats (AAT and TAA), four types of tetranucleotide repeats (AATA, ATTT, TAAA, and TTTA), four types of pentanucleotide repeats (TCCAA, TGATT, CCTTG, and GGCAA), and one type of hexanucleotide repeat (TAAATA); there were ten motifs in total, among which the dominant motif was A/T (731), followed by AT/AT (171), and AAATAT/ATATTT (1) was the least common, accounting for 71.88%, 16.81%, and 0.10% of the total SSRs, respectively (Figure 2E). Furthermore, 943 tandem repeats, 153 palindromic repeats (CRISPRs), and 347 dispersed repeats (including 236 forward repeats, 104 inverted repeats, and seven complement repeats) were identified in the chloroplast genomes of the ten species of Pourthiaea. (Figure 2D).

3.3. Analysis of the IR/SC Boundary Region

The analysis of the contraction and expansion of the SC/IR boundary in the chloroplast genomes of Pourthiaea (Figure 3) showed that the gene types and gene distribution of the LSC/IRb (JLB), IRb/SSC (JSB), SSC/IRa (JSA), and IRa/LSC (JLA) connection boundaries of the chloroplast genomes of the ten species of Pourthiaea varied little. The genes rps19, ycf1, ndhF, and trnH were distributed at the LSC/IR and SSC/IR boundaries. The LSC/IRb and IRa/LSC boundaries of the ten species of Pourthiaea were all related to the rps19 gene. The LSC/IRb boundary was located in the gene coding region of rps19, and the gene length was 279 bp, of which 159 bp were located in the LSC region and 120 bp expanded to the IRb region. The IRa/LSC boundary was located in the noncoding region between the rps19 gene and the trnH gene, of which the trnH gene was 6–150 bp away from the boundary. The ndhF gene of P. arguta was the closest to the boundary, while that of P. hirsuta var. lobulata was farthest from the boundary. The IRb/SSC and SSC/IRa boundaries of these ten species of Pourthiaea were all related to the ycf1 gene. The IRb/SSC boundary was located in the overlapping region of the ycf1 gene and the ndhF gene, of which the ycf1 gene had 7 bp crossing the boundary, located in the SSC region, and the ndhF gene had 14 bp crossing the boundary, located in the IRb region. The SSC/IRa boundaries of all ten chloroplast genomes were located in the coding region of the ycf1 gene, where the length of the ycf1 gene was 5640 bp, in which 1076 bp expanded into the IRa region. In general, in the ten species of Pourthiaea, except for the trnH gene located at the IRa/LSC boundary, the genes distributed at the other three boundaries were exactly the same. The above analysis results showed that the SC/IR boundary regions in the chloroplast genomes of the ten species of Pourthiaea varied little, and the IR region was highly conserved.

3.4. Analysis of Genomic Differences

With the chloroplast genome of P. villosa as a reference, the chloroplast genomes of nine species of Pourthiaea, including P. amphidoxa, were aligned and analyzed in full sequences based on the mVISTA software (Figure 4). The results showed that the chloroplast genomes of the ten species of Pourthiaea were relatively conserved overall: the IR region was more conserved than the LSC and SSC regions, and the coding region was more conserved than the noncoding region. Variation mainly occurred in the spacer regions of adjacent genes, such as rpl2-trnH (GUG), trnR (UCU)-atpA, trnT (GGU)-psbD, and trnT (UGU)-trnL (UAA).

3.5. Analysis of Nucleotide Polymorphisms

The nucleotide polymorphisms (Pi values) of the ten chloroplast genome sequences of Pourthiaea after MAFFT alignment were calculated in DnaSP v6 software. The results showed that the Pi values in the chloroplast genomes of the ten species of Pourthiaea ranged from 0 to 0.00667, with an average of 0.000959. When Pi > 0.006, three hypervariable regions were detected. Among them, two were detected in the LSC region: trnQ (UUC)-psbk-psbl (Pi = 0.00604) and accD-psal (Pi = 0.00633); and one was detected in the SSC region: ndhF-rpl32-trnL (UAG) (Pi = 0.00667) (Figure 5). In general, the Pi values of the ten chloroplast genomes were small, indicating that nucleotides varied little among the ten species of Pourthiaea.

3.6. Phylogenetic Analysis

With E. henryi Nakai and R. lanceolata Hu as the outgroups, the phylogenetic relationship between Pourthiaea and its related genera (Photinia and Stranvaesia) was reconstructed based on the complete chloroplast genomes using ML and BI phylogenetic analysis methods (Figure 6 and Figure 7). The results showed that the topological structures of the phylogenetic BI tree and ML tree were basically the same. The bootstrap support (BS) and posterior probability (PP) values of most branches were high, but the branch support values of the BI tree were higher than those of the ML tree. The 26 species of plants were divided into three branches. Thirteen species of Photinia, three species of Stranvaesia and ten species of Pourthiaea were clustered into one branch, while Photinia and Stranvaesia formed sister groups, and there was no nesting phenomenon among the three genera. The ten species of the genus Pourthiaea were divided into two small branches, one branch of which consisted of P. amphidoxa and P. pilosicalyx, which were sisters to each other. The genetic relationship was well supported (ML-BS = 100%, BI-PP = 1.00). The other eight species, including P. villosa, were clustered into one branch, in which P. hirsuta var. lobulata showed slightly different locations in the ML and BI trees. In the BI tree, P. hirsuta var. lobulata was separated (BI-PP = 1.00), so it was far from the other seven species. In the ML tree, P. villosa and P. arguta var. salicifolia were separated out, and though the genetic relationship between P. hirsuta var. lobulata and the other five species was closer, the support of their genetic relationship was not high (ML-BS = 48%). Similarly, the sister relationship of P. Zhejiangensis and P. tomentosa was not highly supported in the ML tree (ML-BS = 44%), but it was well supported in the BI tree (BI-PP = 1.00).

4. Discussion

The chloroplast genome is a set of DNA sequences carrying genetic information which plays an important role in studying phylogeny, genetic diversity, speciation mechanisms, and so on [47,48]. In this study, through comparative analysis of the chloroplast genomes of ten species of Pourthiaea, it was found that the chloroplast genomes of Pourthiaea were all of a traditional tetrad structure with a total length of 160,401–160,159 bp, and 130–131 genes were annotated. Their genome lengths and gene contents are consistent with the characteristics of chloroplast genomes in angiosperms [49]. GC content plays an important role in genome recognition [50]. The GC contents of the chloroplast genomes of the ten species of Pourthiaea changed little, ranging from 36.5% to 36.6%, with differences not exceeding 0.1%. GC content was differed across different regions of the chloroplast genomes, among which the IR region had the highest GC content, and the SSC region showed the lowest value. Some studies show that this may be because there are four kinds of rRNA genes with high GC contents distributed in the IR region, while the GC content of the NADH deoxidase gene distributed in the SSC region is very low [51]. GC content affects the stability of sequences. The higher the GC content of a genome, the greater the DNA density, and the more conservative and inflexible the sequence [52]. The higher GC content of the IR region makes it more conserved than the LSC region and the SSC region, which is of great significance for protecting the base contents of chloroplast genomes and stabilizing the genome structures.
Chloroplast genome SSRs are characterized by unilineal inheritance, strong conservatism and simple structure, and they have the advantages of nuclear genome SSR codominance, high polymorphism, and wide distribution, so they are mostly used in population genetics, species evolution, gene flow, and other applications [53,54]. Through SSR analysis of the chloroplast genomes of the ten species of Pourthiaea, six SSRs were detected, from mononucleotide to hexanucleotide repeats, among which mononucleotide repeats were the most abundant and hexanucleotide repeats were the least abundant, which is consistent with the results of Shen et al. [55] on thirteen species of Rosa in the same family. From the chloroplast genomes of the ten species of Pourthiaea, 95–107 SSRs were detected in total, with the fewest in P. arguta var. salicifolia. Hexanucleotide repeats only existed in P. villosa, while trinucleotide repeats were found only in P. amphidoxa and P. pilosicalyx. In the SSRs from the chloroplast genomes of the ten species of Pourthiaea, the dominant repeat motif was A/T, with 731 in total, while there were only 31 G/C, and the poly-A/T type was much larger than the G/C type, which is consistent with the A/T enrichment in the complete chloroplast genome of angiosperms [56]. In this study, 1017 SSRs were identified from the chloroplast genomes of the ten species of Pourthiaea, including six types of nucleotide repeats. The repeat motifs were diverse in type, indicating that there was rich SSR polymorphism information in the chloroplast genomes of the species of Pourthiaea, which could provide a reference for the identification and genetic diversity analysis of species of Pourthiaea [57].
There are four boundaries (JLA, JLB, JSA, and JSB) in chloroplast genomes, which are located in the middle of two IRs and two single copy regions [58], and the contraction and expansion of the IR region can be analyzed by comparing the gene distribution of the four boundary regions. The base substitution rate of the IR region gene is only a quarter of the rate of the SC region, which is of great significance for maintaining the stability of the structures of chloroplast genomes [59]. The contraction and expansion of the IR region of chloroplast genomes is a common phenomenon, which can be observed in plants with close or distant genetic relationships [60,61]. By analyzing the IR boundary of the chloroplast genomes of the ten species of Pourthiaea, it was found that the rps19, ndhF, and ycf1 genes crossed the boundary and expanded to the IR region, which is consistent with the research results of Li et al. [62] on the IR/SC boundary of ten species of Pourthiaea and five related species. The contraction and expansion of the IR region may cause the production of pseudogenes, gene duplication, and the deletion of single-copy genes [63,64,65]. In this study, pseudogenes ycf1 and rps19 were observed at the IRb/SSC and IRa/LSC boundaries, and these two pseudogenes were also found in Crataegus plants of the same family [36]. It has been pointed out that contraction and expansion of the IR region and changes in gene spacer length will lead to changes in chloroplast genome length [66]. By comparing the IR boundary in the chloroplast genomes of the ten species of Pourthiaea, it was found that the genes distributed at the three boundaries (LSC/IRb, Irb/SSC, and SSC/Ira) were completely the same across the ten species of Pourthiaea. The difference at the boundary of IRa/LSC was the distance between the trnH gene and the boundary ranged from 6 to 150 bp. Therefore, it was speculated that the variation in the chloroplast genome of Pourthiaea was mainly caused by the variation in gene spacers. Multiple sequence alignment analysis and nucleotide polymorphism analysis also showed that the variation in gene spacers was much greater than that in protein coding regions. The contraction and expansion of the IR boundary may be helpful in the study of evolutionary models. Compared with more diversified species, species with close relatives will show high similarity at the four connection boundaries of chloroplast genomes [67]. In this study, the four boundaries of the chloroplast genomes of the ten species in the genus Pourthiaea were highly conservative. In general, the analysis of the contraction and expansion of the IR region broadens our understanding of the structure and evolution of the chloroplast genome of Pourthiaea.
Multiple sequence alignment analysis showed that the sequence variation of the chloroplast genomes of the ten species of Pourthiaea was small. The sequence variation in the IR region was smaller than that in the SC region, and the variation in the coding region was smaller than that in the noncoding region, which is consistent with the results of chloroplast genomes of other angiosperms reported [68,69]. Through nucleic acid diversity analysis, it was found that the genome Pi values ranged from 0 to 0.00,667, which was relatively low overall. At Pi > 0.006, three variation sites were screened out, including two in the LSC region and one in the SSC region. There were also two relatively variable sites with Pi values of 0.00333 and 0.005 in the IR region, which is below 0.006, and high nucleic acid variability in the IR region has also been found in studies of 7 Dracaena species and 16 other representatives of Asparagales. [70]. It is different from the highly conserved IR region of most plant chloroplast genomes, which may be caused by the differential evolution rate of chloroplast genomes in different regions and species [71]. The hypervariable regions screened here can be used as specific DNA barcode candidate fragments for species identification [72].
Chloroplast genomes play an important role in phylogenetic analysis and have been widely used in the phylogenetic study of angiosperms. Based on the whole chloroplast genome sequences, the phylogenetic analysis of Pourthiaea and its two related genera was carried out using the ML and BI methods. The topological structures of the two were basically the same. However, in the ML tree, the support rates of some internal nodes of the Pourthiaea branch were low, while the support rates of all branches in the BI tree were high. Therefore, the BI method was selected as the main method used to construct the phylogenetic tree. Pourthiaea is closely related to Photinia, and its phylogenetic location is controversial. The phylogenetic results of this study showed that Photinia and Pourthiaea were clustered in different branches, and the clustering between genera was clear, without the nesting phenomenon of species between genera and the monophyly of Pourthiaea was highly supported (ML-BS = 100%, BI-PP = 1.00). These results were consistent with the research results of Zhang [6] and Liu [23], supporting the independence of the genus Pourthiaea. Wang [22] and Liu [23] transferred S. amphidoxa and S. tomentosa, which originally belonged to the genus Stranvaesia and the genus Pourthiaea, but the two were distantly related and located in two different branchlets in the phylogenetic tree based on the complete chloroplast genomes. Specifically, P. amphidoxa and P. pilosicalyx were sisters, while P. tomentosa and P. zhejiangensis were sisters. However, it is different from the results of Liu [23], who used nrDNA to reconstruct the phylogenetic relationships within the genus. In the phylogenetic tree based on nrDNA sequences, P. amphidoxa, P. pilosicalyx, and P. tomentosa were closely related and clustered into a single branch, in which P. pilosicalyx and P. tomentosa were sisters. The phylogenetic relationships based on cpDNA and nrDNA are conflicting. In many studies, the phylogenetic relationships based on nuclear genes and cytoplasmic genes are inconsistent, which may be caused by convergent evolution, incomplete lineage sorting, or hybridization/introgression [73]. Hybridization/introgression may cause chloroplast capture events and coevolution of nuclear genes, thus distorting the phylogenetic relationships of related species [74]. Liu et al. [75] found strongly supportive but inconsistent nuclear and chloroplast topological structures in the phylogenetic analysis of Maleae and speculated that the cause might be hybridization and/or chloroplast capture events. Furthermore, incomplete lineage sorting and sampling error may also be important reasons for inconsistent phylogenetic relationships. Therefore, a clear definition of species may require further use of genomic data at the population level and more sampling.

5. Conclusions

In this study, the characteristics of the chloroplast genomes of the ten species of Pourthiaea were compared, and the phylogenetic relationship between Pourthiaea and two related genera was inferred based on chloroplast genomes. The results showed that the chloroplast genomes of these ten species of Pourthiaea were highly conserved, and there were close similarities among different species in the genus. Meanwhile, three hypervariable regions (trnQ(UUC)-psbk-psbl, accD-psal, and ndhF-rpl32-trnL(UAG)) and 1017 SSR loci were screened out, which, as potential resources for the development of DNA barcodes and effective molecular markers, can be used for further study of the demarcation, phylogeny, population genetics, and evolution of Pourthiaea, as well as the molecular breeding and protection of Pourthiaea and related genera. Phylogenetic analysis based on complete chloroplast genomes showed that the ten species of Pourthiaea were monophyletic, with clear boundaries separating them from Photinia and Stranvaesia. In summary, this study deepened the understanding of the chloroplast genome structure of Pourthiaea and simultaneously provided some basis for further determining the origin and genetic relationships of Pourthiaea.

Author Contributions

Sequence data acquisition and annotation proofreading, Z.C., W.Z. and F.W.; data analysis, Z.C., Y.X., W.S., Q.L., Y.T., H.Z. and Z.D.; thesis writing, Z.C.; project fund support provider, P.X. All authors have read and agreed to the published version of the manuscript.

Funding

Yunnan Science and Technology Talents and Platform Program (No. 202205AF150022).

Data Availability Statement

The sequence data used in this paper are all from GenBank.

Conflicts of Interest

The authors declare no conflict of interest.

Appendix A

Table
Table A1. GenBank accession number of 28 species.
Table A1. GenBank accession number of 28 species.
GenusLatin NameAccession Number
PourthiaeaP. villosaMN061989
P. amphidoxaMN061992
P. pilosicalyxMN216024
P. bliniiMN061990
P. zhejiangensisMN061988
P. tomentosaMN061995
P. hirsuta var. lobulataMN061986
P. arguta var. salicifoliaMN061987
P. argutaMN061991
P. sorbifoliaMN061994
StranvaesiaS. nussiaMK920284
S. bodinieriMK920276
S. oblanceolataMK920280
PhotiniaPh. serratifoliaMN577865
Ph. lochengensisMN577888
Ph. lanuginosaMN577890
Ph. × fraseriMZ128520
Ph. crassifoliaMZ984217
Ph. prunifoliaMK920279
Ph. glabraMZ984218
Ph. integrifoliaMN577879
Ph. taishunensisMK920278
Ph. beckiiMN577889
Ph. glomerataOM772659
Ph. prionophyllaMN577891
Ph. davidsoniaeMT230547
EriobotryaE. henryiNC_045345
RhaphiolepisR. lanceolataMN577867

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Figure 1. Circular map of the chloroplast genome of Pourthiaea with annotated genes. Genes shown inside and outside of the circle are transcribed in clockwise and counterclockwise directions, respectively. Genes belonging to different functional groups are color-coded. The GC and AT contents are denoted by the dark gray and light gray colors in the inner circle, respectively.
Figure 1. Circular map of the chloroplast genome of Pourthiaea with annotated genes. Genes shown inside and outside of the circle are transcribed in clockwise and counterclockwise directions, respectively. Genes belonging to different functional groups are color-coded. The GC and AT contents are denoted by the dark gray and light gray colors in the inner circle, respectively.
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Figure 2. Comparison of chloroplast genome repeat sequences in species of Pourthiaea. (A) Number of different types of SSRs in the chloroplast genomes of ten species of Pourthiaea. (B) Proportion of different SSR types in the chloroplast genomes of ten species of Pourthiaea. (C) Number of SSRs in four chloroplast genome regions of ten species of Pourthiaea. (D) Number of scattered and tandem repeats in chloroplast genomes of ten species of Pourthiaea. (E) SSR type information of chloroplast genomes of ten species of Pourthiaea.
Figure 2. Comparison of chloroplast genome repeat sequences in species of Pourthiaea. (A) Number of different types of SSRs in the chloroplast genomes of ten species of Pourthiaea. (B) Proportion of different SSR types in the chloroplast genomes of ten species of Pourthiaea. (C) Number of SSRs in four chloroplast genome regions of ten species of Pourthiaea. (D) Number of scattered and tandem repeats in chloroplast genomes of ten species of Pourthiaea. (E) SSR type information of chloroplast genomes of ten species of Pourthiaea.
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Figure 3. Comparison of LSC, SSC, and IR boundary regions of ten species of Pourthiaea. The genes around the edge are displayed above or below the main line. JLB, JSB, JSA, and JLA represent the connection sites of LSC/IRb, IRb/SSC, SSC/Ira, and IRa/LSC, respectively.
Figure 3. Comparison of LSC, SSC, and IR boundary regions of ten species of Pourthiaea. The genes around the edge are displayed above or below the main line. JLB, JSB, JSA, and JLA represent the connection sites of LSC/IRb, IRb/SSC, SSC/Ira, and IRa/LSC, respectively.
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Figure 4. Global alignment of the chloroplast genomes of ten species of Pourthiaea.
Figure 4. Global alignment of the chloroplast genomes of ten species of Pourthiaea.
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Figure 5. Nucleotide polymorphism analysis of chloroplast genomes of ten species of Pourthiaea.
Figure 5. Nucleotide polymorphism analysis of chloroplast genomes of ten species of Pourthiaea.
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Figure 6. ML tree based on the chloroplast genome sequences of 28 species.
Figure 6. ML tree based on the chloroplast genome sequences of 28 species.
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Figure 7. BI tree based on the chloroplast genome sequences of 28 species.
Figure 7. BI tree based on the chloroplast genome sequences of 28 species.
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Table
Table 1. Chloroplast genome characteristics of ten species of Pourthiaea.
Table 1. Chloroplast genome characteristics of ten species of Pourthiaea.
SpeciesSize (bp)Gene NumberGC Content (%)
LSCSSCIRTotalCDStRNArRNATotalLSCSSCIRMean
P. villosa88,30719,30626,394160,4018337812836.530.342.736.5
P. amphidoxa88,35919,23426,382160,3578337812834.130.442.636.5
P. pilosicalyx88,34219,30626,341160,3308437812934.430.342.736.6
P. blinii88,17119,33826,399160,3078338812934.230.242.736.5
P. zhejiangensis88,18319,32926,394160,3008337812836.630.242.636.6
P. tomentosa88,18119,32126,394160,2908337812834.130.342.636.5
P. hirsuta var. lobulata88,12719,30826,394160,2238338812934.230.342.736.5
P. arguta var. salicifolia88,12419,28726,394160,1998337812834.330.442.736.6
P. arguta88,04719,31426,399160,1598338812934.230.342.736.5
P. sorbifolia88,23919,33026,401160,3718337812834.230.342.636.5
Table
Table 2. List of genes in the chloroplast genomes of Pourthiaea.
Table 2. List of genes in the chloroplast genomes of Pourthiaea.
CategoryGroup of GenesGenes NamesAmount
Photosynthesis genePhotosystems IpsaA, psaB, psaJ, psaI (×2)5
Photosystems IIpsbA, psbB, psbC, psbD, psbE, psbF, psbH, psbJ, psbK, psbI, psbM, psbN, psbT, psbZ14
Cytochrome b/f complex petA, petB *, petD *, petG, petL, petN6
ATP synthaseatpA, atpB, atpE, atpF *, atpH, atpI6
NADH dehydrogenasendhA *, ndhB * (×2), ndhC, ndhD, ndhE, ndhF, ndhG, ndhH, ndhI, ndhJ, ndhK12
Rubisco Large subunitrbcL1
Self-replication geneRNA polymeraserpoA, rpoB, rpoC1 *, rpoC24
Ribosomal proteins (SSU)rps2, rps3, rps4, rps7 (×2), rps8, rps11, rps12 ** (×2), rps14, rps15, rps16 *, rps18, rps19 (×2)16
Ribosomal proteins (LSU)rpl2 * (×2), rpl14, rpl16, rpl20, rpl22, rpl23(×2), rpl32, rpl33, rpl3611
Transfer RNAstrnA-UGC * (×2), trnC-GCA, trnD-GUC, trnE-UUC, trnF-GAA, trnfM-CAU, trnG-GCC * (×2), 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-GUA37
Ribosomal RNAsrrn4.5 (×2), rrn5 (×2), rrn16 (× 2), rrn23 (×2)8
Other genesMaturasematK1
Envelop membrane proteincemA1
Subunit of acetyl-CoA-carboxylaseaccD1
c-type cytochrome synthesis geneccsA1
ProteolysisclpP **1
Hypothetical chloroplast reading frames (ycf)Ycf1 (×2), ycf2 (×2), ycf3 **, ycf46
Note: * Gene contains one intron; ** gene contains two introns; (×2) indicates the number of the repeat unit is 2.
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Cao, Z.; Zhao, W.; Xin, Y.; Shen, W.; Wang, F.; Li, Q.; Tu, Y.; Zhang, H.; Dong, Z.; Xin, P. Characteristics of the Complete Chloroplast Genome of Pourthiaea (Rosaceae) and Its Comparative Analysis. Horticulturae 2022, 8, 1144. https://doi.org/10.3390/horticulturae8121144

AMA Style

Cao Z, Zhao W, Xin Y, Shen W, Wang F, Li Q, Tu Y, Zhang H, Dong Z, Xin P. Characteristics of the Complete Chloroplast Genome of Pourthiaea (Rosaceae) and Its Comparative Analysis. Horticulturae. 2022; 8(12):1144. https://doi.org/10.3390/horticulturae8121144

Chicago/Turabian Style

Cao, Zhengying, Wenzhi Zhao, Yaxuan Xin, Weixiang Shen, Fei Wang, Qishao Li, Yuxiang Tu, Haorong Zhang, Zhanghong Dong, and Peiyao Xin. 2022. "Characteristics of the Complete Chloroplast Genome of Pourthiaea (Rosaceae) and Its Comparative Analysis" Horticulturae 8, no. 12: 1144. https://doi.org/10.3390/horticulturae8121144

APA Style

Cao, Z., Zhao, W., Xin, Y., Shen, W., Wang, F., Li, Q., Tu, Y., Zhang, H., Dong, Z., & Xin, P. (2022). Characteristics of the Complete Chloroplast Genome of Pourthiaea (Rosaceae) and Its Comparative Analysis. Horticulturae, 8(12), 1144. https://doi.org/10.3390/horticulturae8121144

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