Chloroplast Genome Comparison and Phylogenetic Analysis of the Commercial Variety Actinidia chinensis ‘Hongyang’

Actinidia chinensis ‘Hongyang’, also known as red yangtao (red heart kiwifruit), is a vine fruit tree native to China possessing significant nutritional and economic value. However, information on its genetic diversity and phylogeny is still very limited. The first chloroplast (cp) genome of A. chinensis ‘Hongyang’ cultivated in China was sequenced using de novo technology in this study. A. chinensis ‘Hongyang’ possesses a cp genome that spans 156,267 base pairs (bp), exhibiting an overall GC content of 37.20%. There were 132 genes that were annotated, with 85 of them being protein-coding genes, 39 transfer RNA (tRNA) genes, and 8 ribosomal RNA (rRNA) genes. A total of 49 microsatellite sequences (SSRs) were detected, mainly single nucleotide repeats, mostly consisting of A or T base repeats. Compared with 14 other species, the cp genomes of A. chinensis ‘Hongyang’ were biased towards the use of codons containing A/U, and the non-protein coding regions in the A. chinensis ‘Hongyang’ cpDNA showed greater variation than the coding regions. The nucleotide polymorphism analysis (Pi) yielded nine highly variable region hotspots, most in the large single copy (LSC) region. The cp genome boundary analysis revealed a conservative order of gene arrangement in the inverted repeats (IRs) region of the cp genomes of 15 Actinidia plants, with small expansions and contractions of the boundaries. Furthermore, phylogenetic tree indicated that A. chinensis ‘Hongyang’ was the closest relative to A. indochinensis. This research provides a useful basis for future genetic and evolutionary studies of A. chinensis ‘Hongyang’, and enriches the biological information of Actinidia species.


Introduction
Actinidia chinensis 'Hongyang' (A.chinensis Planch.var.chinensis) is the initial commercially grown cultivar of red-fleshed kiwifruit [1].Its popularity among consumers stems from its attractive red inner pericarp and exceptional fruit qualities [2].The kiwifruit fruit has rich nutritional value, and its soluble solid content reaches 19.6%.It also has high levels of vitamin C. Therefore, the kiwifruit is generally referred to as the 'king of fruit ' [3].Moreover, the fruits have free radical scavengers, anti-aging, cancer prevention, and other physiological effects favored by the majority of consumers [4].Although research on kiwifruit chloroplast (cp) genome analysis is rapidly increasing [5][6][7][8][9], little in-depth analysis exists concerning the genetic characteristics and evolution of the commercial variety A. chinensis 'Hongyang'.
Chloroplasts, found in plants, serve as the central hubs for photosynthesis as well as the production of fatty acids, amino acids, and starch.These vital organelles also facilitate the transfer and manifestation of genetic information [10].Cp genomes are usually between 120 and 170 kb in size and encompass 120-130 genes [11], and cpDNA forms a unique shape resembling a twisted loop, showcasing a distinctive four-part structure.This structure comprises a spacious section known as the large single copy region (LSC), a compact single copy region (SSC), and a pair of inverted repeat (IRs) sequences [12][13][14].In angiosperms, the cpDNA contains a highly conserved set of protein-coding genes (PCGs), transfer RNA (tRNA) genes, and ribosomal RNA (rRNA) genes [15].Nevertheless, angiosperm cpDNA has experienced numerous changes due to its ability to adapt to ever-changing surroundings.These alterations encompass not only variations in size and structure but also the contraction and expansion of inverted repeats [16].Cp genomes are mostly inherited maternally in angiosperms, and molecular phylogenies based on the coding or non-coding areas of cpDNA sequences have proven powerful at the genus and species levels [17].
Due to its relatively stable genome structure and slow rate of nucleotide substitution, cpDNA has gained significant popularity in the fields of plant systematics, phylogeny, and evolution [18].
Advanced sequencing technology has enabled the sequencing and functional characterization of the cpDNAs of numerous species [19].However, although the cpDNA sequences of some Actinidia species have been sequenced, limited knowledge exists regarding the cpDNA information of the cultivated A. chinensis 'Hongyang' in China.Furthermore, there has been a lack of comprehensive analysis in a single study, particularly concerning the genetic variability and evolutionary correlation with other affiliated species.To delve into the genetic characteristics and evolutionary background of the cultivated A. chinensis 'Hongyang' and enrich the molecular biological information of the Actinidia genus, we performed a comprehensive comparative analysis of A. chinensis 'Hongyang' cpDNA with other closely associated Actinidia species.
The primary aim of this research was to analyze the genomic features of A. chinensis 'Hongyang' cpDNA, compare it with cpDNA from 14 other Actinidia species, and analyze the frequency of codon utilization patterns, in addition to the presence of repeats and small sequence repeats.This would allow us to identify the unique characteristics of the A. chinensis 'Hongyang' cpDNA.By comparing the complete cpDNA sequences of A. chinensis 'Hongyang' with those of 28 other Actinidia species, we were able to determine their phylogenetic relationships.We have identified potential SSR markers in Actinidia plants that could be utilized to investigate genetic variation for DNA barcoding purposes.These findings lay the groundwork for future genomic studies on the genetic variation and evolutionary patterns of A. chinensis 'Hongyang' and its closely related taxa.

Sample Collection, Extraction of cpDNA, and Sequencing
The newly sprouted foliage of an A. chinensis 'Hongyang' was gathered from the kiwifruit germplasm resource nursery (29.24N, 105.87 E) of Chongqing University of Arts and Sciences.We utilized the plant total DNA extraction kit (Beijing Solaibao Technology Co., Ltd., Beijing, China) for the isolation of complete genomic DNA samples.The quality of the DNA products was tested, and the DNA was then fragmented.The short-insert libraries of 350 bp were created using the fragmented DNA, and the libraries that met the quality standards were sequenced on the BGISEQ-500 platform sequencer with PE150 bp, following the instructions provided by the manufacturer.

CpDNA Assembly and Annotation
To trim the raw reads, Fastp [20] was employed, followed by aligning the highquality reads to the reference cp genomes of A. chinensis sourced from GenBank using Bowtie2 v. 2.3.4.3 [21].We utilized NOVOPlasty v4.2.1 [22] to conduct a de novo assembly of the sequence.The annotation of the assembled cp genomes was carried out with the assistance of DOGMA [23], ARAGORN [24], GeSeq [25], and tRNAscan v2.0 [26].Manual adjustments and confirmations were made using Geneious v9.1.8[27].OGDRAW v.1.3.1 [28] was utilized to create the cp genome map.Geneious [27] software was used to extract the LSC, SSC, and IR regions from the cp genome sequences.Statistics on AT and GC content were obtained using BioEdit [29].The latest cp genome annotation has been uploaded to the GenBank (Number: MW596239).

Analysis of the Cp Genome for Repetitive Sequences
Employing the REPuter online tool [30], we investigated the occurrence of forward, reverse, palindromic, and complement repeat sequences within the cp genome of A. chinensis 'Hongyang'.The maximum number of repetitive sequences was set to 100, the minimum repetitive element size was 20 bp, the Hamming distance was 3, and the edit distance was selected as the default.The MISA v2.1 software [31] was employed to conduct a search for SSRs.The parameters for mono-, di-, tri-, tetra-, penta-, and hexa-nucleotides were established as 10, 5, 4, 3, 3, and 3, respectively.

Codon Preference Analysis
Codon W v1.4.4 [32] software is employed for codon analysis and the determination of relative synonymous codon usage (RSCU) values for comparative mapping.RSCU represents the relative likelihood of using synonymous codons to encode a specific amino acid; when RSCU > 1, it indicates that the codon is commonly utilized; if RSCU = 1, it implies that the codon lacks any bias in usage; and if RSCU < 1, it signifies that the codon is seldom used.

Sequence Variation Analysis
In order to understand the divergent regions of cp genome sequences in Actinidia plants, we utilized the online tool mVISTA [33] to conduct a comprehensive visual alignment analysis on the cp genome data of Actinidia plants, encompassing a total of 15 samples, with A. chinensis 'Hongyang' as the reference sequence.The sliding window size was 100 bp, and the minimum width of the conservative region was 100 bp.The IR region's expansion and contraction were analyzed using the online tool IRscrope [34].In order to assess the variability across various regions of the genome, the estimation of nucleotide diversity (Pi) was carried out using DnaSP v5.10 [35].By employing a window length of 400 bp and a step size of 20 bp, the data were analyzed using the sliding window technique.

Phylogenetic Relationship Analysis
Our study involved analyzing the cp genomes of 29 samples within the Actinidiaceae family using phylogenetic methods.In this analysis, Clematoclethra scandens, subsp.Hemsleyi, and Saurauia tristyla were used as outgroups.The cp genome data of these species were obtained from the GeneBank within the NCBI (Table S1).In order to overcome the difficulties in aligning the entire genome, we utilized Phylosuit v1.1.13[36] to extract PCG sequences for the phylogenetic analyses.We utilized MAFFT v7.450 [37] for sequence alignment, followed by the removal of regions exhibiting site coverage less than 95% for consistency.

Chloroplast Genomic Structure of A. chinensis 'Hongyang'
The newly sequenced A. chinensis 'Hongyang' cp genome was 156,267 bp in total length (Figure 1), with the LSC, SSC, and two IRs regions of 87,866 bp, 20,335 bp, and 24,033 bp, respectively.The total GC content of the cp genome of A. chinensis 'Hongyang' was 37.20%, and the GC contents of the LSC, SSC, and IR regions were 35.30%, 31.15%, and 42.89%, respectively (Table 1).In addition, we compared the cpDNA of A. chinensis 'Hongyang' and other 14 Actinidia species, with genome lengths ranging from 156,124-157,611 bp; the LSC region length range was 86,483-88,666 bp; the SSC region length range was 20,307-22,574 bp; and the IR region length range was 23,377-24,308 bp (Table 2).The comparison revealed that although there were differences in overall genome size, the GC content was similar across species, all around 37.20% (Table 2).
24,033 bp, respectively.The total GC content of the cp genome of A. chinensis 'H was 37.20%, and the GC contents of the LSC, SSC, and IR regions were 35.30% and 42.89%, respectively (Table 1).In addition, we compared the cpDNA of A. 'Hongyang' and other 14 Actinidia species, with genome lengths ranging from 157,611 bp; the LSC region length range was 86,483-88,666 bp; the SSC region len was 20,307-22,574 bp; and the IR region length range was 23,377-24,308 bp (Tab comparison revealed that although there were differences in overall genome siz content was similar across species, all around 37.20% (Table 2).

Chloroplast Genome Composition of Actinidia
Based on an in-depth analysis of the cpDNA of A. chinensis 'Hongyang', 132 genes were identified, including 85 PCGs, 39 tRNA, and 8 rRNA genes (Tables 2 and 3).These include eight tRNA genes, namely trnL(CAA), trnH(GUG), trnI(CAU), trnV(GAC), trnI(GAU), trnA(UGC), trnR(ACG), and trnN(GUU); seven PCGs (psbA, ycf2, ycf15, ndhB, rps7, rps12, ycf1); and four rRNA genes (rrn4.5, rrn5, rrn16, rrn23), which were duplicated in the IR region.The LSC region contains 22 tRNA genes and 61 PCG genes.In contrast, the SSC region of Actinidia species contains only 1 tRNA and 10 PCGs.When comparing the total number of PCGs and tRNA genes among 15 Actinidia species, there were minimal differences observed.A. valvata has a higher number of genes (134); A. chinensis has the most PCGs (86); and A. arguta, A. latifolia, and A. valvata have the highest number of tRNA genes.Additionally, A. chinensis and A. rufa displayed the most abundant PCG content, while A. arguta, A. latifolia, and A. valvata had the highest tRNA content.On the other hand, A. zhejiangensis had the lowest tRNA content.The number of rRNA genes in the cpDNA of Actinidia plants is relatively conserved and the same as those reported for most angiosperms [41].

Category Gene Group Gene Name Number
Self-replication Proteins of large ribosomal subunits rpl14, rpl16 *, rpl2 *, rpl20, rpl22, rpl23, rpl32, rpl33, rpl36 Proteins of the small ribosomal subunit rps11, rps12 **(2), rps14, rps15, rps16 *, rps18, rps19, rps2, rps3, rps4, rps7 (2), rps8 Studies have revealed that introns play a crucial role in governing the regulation of gene expression.In plants, numerous introns possess the capacity to amplify the expression of alien genes with precision in terms of timing and spatial positioning, leading to desirable agricultural characteristics [42].Within the genes encoded by A. chinensis 'Hongyang', a total of 21 genes have introns.The ycf3 gene consists of two introns, while rps12 has two copies.The first exon of rps12 is present in the LSC region, whereas exons 2 and 3 are located in the IR region.Eight tRNAs (trnK(UUU), trnG(UCC), trnL(UAA), trnV(UAC), trnI(GAU), trnA(UGC), trnI(GAU), trnA(UGC)), and ten PCGs (rps16, atpF, rpoC1, petB, petD, rpl16, rpl2, ndhB, ndhA, ndhB) contain only one intron.The trnK(UUU) gene contained the largest intron (2489 bp), and the trnL(UAA) gene contained the smallest intron (504 bp) (Table 4).Codons are essential for the correct expression of genetic information.We analyzed and compared the codon usage preferences and RSCU of the cp genome from A. chinensis 'Hongyang' and its relatives.Based on tRNA and PCG sequences, the frequency of codon usage in the cpDNA of A. chinensis 'Hongyang' was determined and compared with those of 14 closely related plants (Figure 2).The findings indicated that all genes in the cpDNA of A. chinensis 'Hongyang' were encoded by 25,939 codons; a total of 64 codons were detected, of which UAA, UAG, and UGA were stop codons; and the utilization rate of UAA was high.The highest coding rate among amino acids was for leucine (Leu), which used 2792 codons, accounting for 10.76% of the total, and the lowest coding rate was for cysteine (Cys), which used 278 codons.Of all the codons, AAA was the largest in number, 1114 in total, and the use frequency was 4.29.The number of UGAs and UAGs was the smallest, only 18, and the frequency of usage was 0.07.There were 31 preferred codons (RSCU > 1) in the cp genome of A. chinensis 'Hongyang'.Among the 31 codons, there were twenty-nine codons ending with A/T(U), accounting for 93.55% of the total, two codons ending with G bases (UUG, AUG), and no codons ending with C bases.There existed a pronounced inclination towards the A/U base.

Codon Usage in the Chloroplast Genome of A. chinensis 'Hongyang'
Codons are essential for the correct expression of genetic information.We analyzed and compared the codon usage preferences and RSCU of the cp genome from A. chinensis 'Hongyang' and its relatives.Based on tRNA and PCG sequences, the frequency of codon usage in the cpDNA of A. chinensis 'Hongyang' was determined and compared with those of 14 closely related plants (Figure 2).The findings indicated that all genes in the cpDNA of A. chinensis 'Hongyang' were encoded by 25,939 codons; a total of 64 codons were detected, of which UAA, UAG, and UGA were stop codons; and the utilization rate of UAA was high.The highest coding rate among amino acids was for leucine (Leu), which used 2792 codons, accounting for 10.76% of the total, and the lowest coding rate was for cysteine (Cys), which used 278 codons.Of all the codons, AAA was the largest in number, 1114 in total, and the use frequency was 4.29.The number of UGAs and UAGs was the smallest, only 18, and the frequency of usage was 0.07.There were 31 preferred codons (RSCU > 1) in the cp genome of A. chinensis 'Hongyang'.Among the 31 codons, there were twentynine codons ending with A/T(U), accounting for 93.55% of the total, two codons ending with G bases (UUG, AUG), and no codons ending with C bases.There existed a pronounced inclination towards the A/U base.The cp genome codon usage preference analysis of kiwifruit species indicated that the cp genome codon usage preference was basically the same, and they all preferred UAA as the stop codon.Leu, arginine (Arg), and serine (Ser) indicated high codon bias, while tryptophan (Trp) showed no codon bias.Most synonymous codons with RSCU > 1 end with adenine (A) or thymine (U) (except UUG and AUG), showed that there is a high prevalence of codons that conclude with A or U.The cp genome codon usage preference analysis of kiwifruit species indicated that the cp genome codon usage preference was basically the same, and they all preferred UAA as the stop codon.Leu, arginine (Arg), and serine (Ser) indicated high codon bias, while tryptophan (Trp) showed no codon bias.Most synonymous codons with RSCU > 1 end with adenine (A) or thymine (U) (except UUG and AUG), showed that there is a high prevalence of codons that conclude with A or U.

Detection of Repeated Sequences and SSRs in A. chinensis 'Hongyang' cpDNA
We analyzed the cp genome of A. chinensis 'Hongyang' and identified a remarkable 99 long repeat sequences.Among them, 39 were found to be palindromic repeats, 4 were reverse repeats, and 56 were forward repeats.Interestingly, no complementary sequences were detected (Table S2).There were 29 repeats of 20-29 bp in length, 23 repeats of 30-39 bp in length, 23 repeats of 40-99 bp in length, and 24 repeats of more than 100 bp in length, and the IR region contains the longest repeated sequence of 287 bp.The majority of the recurring sequences were found within the intergenic spacer region (IGS), while the genes contained only a limited number of recurring sequences (Table S2).
We analyzed the cp genome of A. chinensis 'Hongyang' and identif 99 long repeat sequences.Among them, 39 were found to be palindrom reverse repeats, and 56 were forward repeats.Interestingly, no complem were detected (Table S2).There were 29 repeats of 20-29 bp in length, 23 bp in length, 23 repeats of 40-99 bp in length, and 24 repeats of more than and the IR region contains the longest repeated sequence of 287 bp.Th recurring sequences were found within the intergenic spacer region (IGS contained only a limited number of recurring sequences (Table S2).

Comparative Analysis of Fifteen Species Chloroplast Genomic Structure
To verify the possibility of genome differentiation, the cp genom 'Hongyang' was selected as a reference, and the cp genomes of 15 Acti visualized and compared using mVISTA (Figure 4).All cp DNA sequen quence consistency and generally showed high interspecific conservatio gion displayed a higher level of conservatism compared to the non-cod the inverted repeat region exhibited even greater conservatism than the regions.The coding sequences with a high degree of variation were main the ycf1, accD, ycf2, and rps4 regions.The non-coding regions with a high tion were distributed in the trnG(UCC)-trnR(UCU), petN-psdM, trnT(G trnV(UAC), rbcL-accD, trnF(GAA)-ndhJ, rps12-psbB, trnC(GCA)-pe trnL(UAA), petA-psbJ, and ndhA-ndhH regions.

Comparative Analysis of Fifteen Species Chloroplast Genomic Structure
To verify the possibility of genome differentiation, the cp genome of A. chinensis 'Hongyang' was selected as a reference, and the cp genomes of 15 Actinidia plants were visualized and compared using mVISTA (Figure 4).All cp DNA sequences had high sequence consistency and generally showed high interspecific conservation.The coding region displayed a higher level of conservatism compared to the non-coding region, while the inverted repeat region exhibited even greater conservatism than the two single-copy regions.The coding sequences with a high degree of variation were mainly distributed in the ycf1, accD, ycf2, and rps4 regions.The non-coding regions with a high degree of variation were distributed in the trnG(UCC)-trnR(UCU), petN-psdM, trnT(GGU)-psbD, ndhC-trnV(UAC), rbcL-accD, trnF(GAA)-ndhJ, rps12-psbB, trnC(GCA)-petN, trnT(UGU)-trnL(UAA), petA-psbJ, and ndhA-ndhH regions.In addition, researchers determined nucleotide diversity (Pi) values by analyzing 400 bp sections of the Actinidia cp genome to pinpoint areas of sequence divergence.The findings revealed a range of Pi values, spanning from 0 to 0.0611, across the 15 genomes studied.We identified nine regions with high variability (Pi > 0.0200): rps12-psbB, petA-psbJ, trnT(UGU)-trnL(UAA), petN-psbM, rbcL-accD, trnF(GAA)-ndhJ, trnG(UCC)-trnR(UCU), trnC(GCA)-petN, and ycf1 (Figure 5).Out of these, only ycf1 is situated within the SSC area, while the other eight can be found within the LSC area.In addition, researchers determined nucleotide diversity (Pi) values by analyzing 400 bp sections of the Actinidia cp genome to pinpoint areas of sequence divergence.The findings revealed a range of Pi values, spanning from 0 to 0.0611, across the 15 genomes studied.We identified nine regions with high variability (Pi > 0.0200): rps12-psbB, petA-psbJ, trnT(UGU)-trnL(UAA), petN-psbM, rbcL-accD, trnF(GAA)-ndhJ, trnG(UCC)-trnR(UCU), trnC(GCA)-petN, and ycf1 (Figure 5).Out of these, only ycf1 is situated within the SSC area, while the other eight can be found within the LSC area.In addition, researchers determined nucleotide diversity (Pi) values by analyzing 400 bp sections of the Actinidia cp genome to pinpoint areas of sequence divergence.The findings revealed a range of Pi values, spanning from 0 to 0.0611, across the 15 genomes studied.We identified nine regions with high variability (Pi > 0.0200): rps12-psbB, petA-psbJ, trnT(UGU)-trnL(UAA), petN-psbM, rbcL-accD, trnF(GAA)-ndhJ, trnG(UCC)-trnR(UCU), trnC(GCA)-petN, and ycf1 (Figure 5).Out of these, only ycf1 is situated within the SSC area, while the other eight can be found within the LSC area.The fluctuation of the IR in plant cp genomes is a widespread occurrence that has significantly influenced species evolution.It is believed to be the primary factor contributing to the varying sizes of plant cpDNA throughout evolution [45] to explore the extent of IR expansion or contraction among Actinidia species by comparing the IR/LSC and IR/SSC boundaries of 15 kiwifruit species (Figure 6).It was found that A. chinensis 'Hongyang' had similar boundaries to A. chinensis, A. chinensis 'Jinguo', and A. arguta in the LSC, SSC, and IR regions, but there were still minor differences.Compared to the remaining 11 species, there were large variations in the ranges of each region.All 15 kiwifruit species have ycf1, trnN, trnH, rpl23, ndhF, and psbA genes.The rpl23 genes were all located in the LSC region and migrated to a lesser extent.The ndhF genes were all located in the SSC region, with greater migration of ndhF in A. latifolia and lesser migration in the remaining species.The psbA gene is present across all taxa, extending from the IRa/SSC boundary.It has a varying length of 269-314 bp in the IRa region and 748-793 bp in the LSC region, indicating a contraction of the cpDNA IRa/SSC boundary in Actinidia species.The IRb region of A. rufa forms the pseudogene psbA with incomplete replication.The ycf1 gene spans the IR/SSC junction of A. chinensis, A. chinensis 'Jinguo', A. deliciosa, and A. rufa, with a small degree of contraction or expansion.The ycf1 gene of A. chinensis 'HFY01', A. macrosperma, A. melanandra, A. polygama, A. deliciosa, and A. latifolia was present only at the IRa/SSC boundary, and the ycf1 gene of A. kolomikta, A. eriantha, and A. zhejiangensis was present only at the IRb/SSC boundary.Some of these species have incompletely replicated ycf1 pseudogenes in the IRb region.The trnN and trnH genes in the cpDNA of Actinidia samples were fully replicated at the IR/SSC boundary and are contained in the IR area with a small degree of offset.
Genes 2023, 14, x FOR PEER REVIEW 10 of 16 The fluctuation of the IR in plant cp genomes is a widespread occurrence that has significantly influenced species evolution.It is believed to be the primary factor contributing to the varying sizes of plant cpDNA throughout evolution [45] to explore the extent of IR expansion or contraction among Actinidia species by comparing the IR/LSC and IR/SSC boundaries of 15 kiwifruit species (Figure 6).It was found that A. chinensis 'Hongyang' had similar boundaries to A. chinensis, A. chinensis 'Jinguo', and A. arguta in the LSC, SSC, and IR regions, but there were still minor differences.Compared to the remaining 11 species, there were large variations in the ranges of each region.All 15 kiwifruit species have ycf1, trnN, trnH, rpl23, ndhF, and psbA genes.The rpl23 genes were all located in the LSC region and migrated to a lesser extent.The ndhF genes were all located in the SSC region, with greater migration of ndhF in A. latifolia and lesser migration in the remaining species.The psbA gene is present across all taxa, extending from the IRa/SSC boundary.It has a varying length of 269-314 bp in the IRa region and 748-793 bp in the LSC region, indicating a contraction of the cpDNA IRa/SSC boundary in Actinidia species.The IRb region of A. rufa forms the pseudogene psbA with incomplete replication.The ycf1 gene spans the IR/SSC junction of A. chinensis, A. chinensis 'Jinguo', A. deliciosa, and A. rufa, with a small degree of contraction or expansion.The ycf1 gene of A. chinensis 'HFY01', A. macrosperma, A. melanandra, A. polygama, A. deliciosa, and A. latifolia was present only at the IRa/SSC boundary, and the ycf1 gene of A. kolomikta, A. eriantha, and A. zhejiangensis was present only at the IRb/SSC boundary.Some of these species have incompletely replicated ycf1 pseudogenes in the IRb region.The trnN and trnH genes in the cpDNA of Actinidia samples were fully replicated at the IR/SSC boundary and are contained in the IR area with a small degree of offset.

Phylogenetic Analysis of 29 Taxa of the Actinidiaceae Based on cpDNA Sequences
In order to determine the genetic relationship of A. chinensis 'Hongyang' in the Actinidiaceae, we used 27 Actinidia species and C. scandens subs.hemsleyi and S. tristyla of the Actinidiaceae as outgroups.The construction of phylogenetic trees employed both maximum likelihood and Bayesian methods.Phylogenetic reliability was tested with 1000 replicates using the bootstrap method.The results indicated that both trees possess an identical complementary structure in terms of topology, except for the terminal branches.The support rate of the branch nodes of the evolutionary tree is basically greater than 60%, and only one node has a value lower than 60%.The high-resolution ML/BI phylogenetic tree indicated that A. chinensis 'Hongyang' was closely related to A. indochinensis and A. setosa (Figure 7).

Phylogenetic Analysis of 29 Taxa of the Actinidiaceae Based on cpDNA Sequences
In order to determine the genetic relationship of A. chinensis 'Hongyang' in the Actinidiaceae, we used 27 Actinidia species and C. scandens subs.hemsleyi and S. tristyla of the Actinidiaceae as outgroups.The construction of phylogenetic trees employed both maximum likelihood and Bayesian methods.Phylogenetic reliability was tested with 1000 replicates using the bootstrap method.The results indicated that both trees possess an identical complementary structure in terms of topology, except for the terminal branches.The support rate of the branch nodes of the evolutionary tree is basically greater than 60%, and only one node has a value lower than 60%.The high-resolution ML/BI phylogenetic tree indicated that A. chinensis 'Hongyang' was closely related to A. indochinensis and A. setosa (Figure 7).

Discussion
The cp genome sequence contains much information that can be used to solve complex evolutionary relationships and has been widely used in plant phylogeny and the evolutionary relationship reconstruction of crop varieties [46].The genome of A. chinensis 'Hongyang' exhibits a conventional four-part structure with similar gene composition and high resemblance in PCGs, tRNA, and rRNA, aligning with the cp genomes of other Actinidiaceae species [5].Structure variations were found in the Actinidiaceae plants' cp genomes, such as the clpP gene, which was deleted, and the trnfM-CAU gene in the LSC area, which was a homologous replication [47].A number of plant lineages have shown that some genes are missing or were transferred to the plant cp genome during the long evolutionary process [48][49][50][51].However, a comparative study conducted on the cp genome sequences of A. chinensis 'Hongyang' and 14 other Actinidia plants revealed that the Actinidia cp genome exhibited a remarkable level of conservation in terms of both genome size and gene content.This finding aligns with previous research conducted at the Zingiber [52], Sinosenecio [53], and Camellia [54] genus levels.Numerous molecular mechanisms have preserved plastid conservatism, such as exclusive inheritance from one parent, the infrequent fusion of plastids, and the presence of efficient repair mechanisms [55,56].The geographical distribution of the kiwifruit is very wide in East Asia, and the genus is known for its high morphological and ecological variation [57].Therefore, the maintenance of cp genome sequence conservation may help maintain its normal function and adapt to environmental changes.
The preference for codons is shaped by the forces of natural selection, genetic mutations, and random genetic changes over time.It is a product of the species' adaptation to

Discussion
The cp genome sequence contains much information that can be used to solve complex evolutionary relationships and has been widely used in plant phylogeny and the evolutionary relationship reconstruction of crop varieties [46].The genome of A. chinensis 'Hongyang' exhibits a conventional four-part structure with similar gene composition and high resemblance in PCGs, tRNA, and rRNA, aligning with the cp genomes of other Actinidiaceae species [5].Structure variations were found in the Actinidiaceae plants' cp genomes, such as the clpP gene, which was deleted, and the trnfM-CAU gene in the LSC area, which was a homologous replication [47].A number of plant lineages have shown that some genes are missing or were transferred to the plant cp genome during the long evolutionary process [48][49][50][51].However, a comparative study conducted on the cp genome sequences of A. chinensis 'Hongyang' and 14 other Actinidia plants revealed that the Actinidia cp genome exhibited a remarkable level of conservation in terms of both genome size and gene content.This finding aligns with previous research conducted at the Zingiber [52], Sinosenecio [53], and Camellia [54] genus levels.Numerous molecular mechanisms have preserved plastid conservatism, such as exclusive inheritance from one parent, the infrequent fusion of plastids, and the presence of efficient repair mechanisms [55,56].The geographical distribution of the kiwifruit is very wide in East Asia, and the genus is known for its high morphological and ecological variation [57].Therefore, the maintenance of cp genome sequence conservation may help maintain its normal function and adapt to environmental changes.
The preference for codons is shaped by the forces of natural selection, genetic mutations, and random genetic changes over time.It is a product of the species' adaptation to their environment through the course of evolution [58].In this study, the codon preference of A. chinensis 'Hongyang' was analyzed as a representative of Actinidia, and the codon usage preferences and RSCU of A. chinensis 'Hongyang' and its close relatives' cpDNA were also compared.The cp genomes of A. chinensis 'Hongyang' and other Actinidia plants are biased toward the use of codons containing A/U.The optimal codons of the plant cp genome can effectively improve the accuracy and efficiency of amino acid translation [59], so the optimal codons of A. chinensis 'Hongyang' screened in this study can provide a reference for subsequent studies to promote the expression of foreign genes in its chloroplasts.
The polymorphism of SSRs within the cp genome exhibits significant variation among species; these genetic markers have proven to be extremely valuable in this study of population genetics and the understanding of evolutionary processes [60].The statistical results of SSRs in the A. chinensis 'Hongyang' cp genome showed that the cp genome had a high abundance of SSR types, with the highest percentage of poly T or poly A repeats, while poly C repeats were less common, which was similar to other angiosperms [61].The discovery of SSR loci in the cp genome of A. chinensis 'Hongyang' has implications for Actinidia plant identification, population-level polymorphism detection, and evolutionary analysis.
The cp genome's highly diverse regions have the potential to serve not only as markers for species identification and phylogenetic analysis but also as valuable sources of data for investigating population-level species differentiation and population structures [62,63].In previous studies, fragments such as ITS in the nuclear genome and matK, rbcL, and trnH-psbA in the cp genome have been widely used for interspecific level classification, molecular phylogeny, and DNA barcode applications in higher plants [64].The vegetative characteristics exhibit a high level of resemblance, which makes distinguishing Actinidia plants extremely difficult [65].Nevertheless, the existing classical DNA barcodes fall short of accurately distinguishing Actinidia species and unraveling their evolutionary relationships.Therefore, it is imperative to pinpoint remarkably diverse regions at the genus level, as they hold promising prospects as markers for forthcoming investigations into the identification of different varieties.In light of the findings from mVISTA and the variation in nucleotides, nine significant polymorphisms among fifteen Actinidia species were identified, including rps12-psbB, petA-psbJ, trnT(UGU)-trnL(UAA), petN-psbM, rbcL-accD, trnF(GAA)-ndhJ, trnG(UCC)-trnR(UCU), trnC(GCA)-petN, and ycf1.The immense variability of these areas could serve as valuable molecular markers for distinguishing Actinidia plants and conducting phylogenetic analysis in further studies.Among them, Actinidia have been reported to possess rpl32-trnL-UAG, ndhF-rpl32, and ycf1 that are suitable for species identification [17,66], but the validity of these fragments for the identification of Actinidia plants is still necessary to determine further validation.
The variations in cp genome size are greatly influenced by the expansion and contraction of boundaries within the IR region; these boundary changes are crucial for maintaining structural stability and promoting the evolution of the cp genome [65].In this research, we found that the cp genomes of 15 Actinidia plants, although relatively conserved in structure and size, still showed some variation in the boundary positions of IR and SC between species.The findings are in agreement with a previous report on Actinidiaceae [5].A shift in the IR/SSC boundary always results in an increase or decrease in the length of the IR area [52].Many plant species, including Erodium [67], Pilea [68], and Pelargonium [69], exhibited a significant expansion of the IR region, resulting in the incorporation of numerous genes from the SC into this region.In contrast to the cp genomes of 15 Actinidia samples, the length of the IR region remained consistent across all species we examined.However, an interesting discovery was made in A. rufa, where a pseudogene was observed at the boundary of LSC/IRs.The pseudogene of ycf1 originated at the junction of IR in Actinidia taxa, which was also observed in other angiosperms [50,52,56].Overall, the different degrees of expansion and contraction of cp genomes at the IR junction of Actinidia plants may have been important factors in the formation of polymorphisms in boundaries and genome lengths in Actinidia plants during the evolutionary process.
The controversy surrounding the interspecies relationships within the Actinidia genus is primarily attributed to the inadequate sampling of taxa and the absence of molecular markers [17].However, the cp genome offers extensive informative loci for phylogenetic studies.It greatly enhances the accuracy of phylogenetic trees for certain taxonomic groups or those with slow molecular evolution and limited molecular variation.Additionally, it effectively resolves phylogenetic relationships across various taxonomic levels in flowering plants [70].In contrast to previous studies, the molecular markers of the Actinidia species are greatly enhanced in this study [71].Our study provides a robust phylogenetic hypothesis based on cp genome data that contributes to our understanding of Actinidia interspecies relationships (Figure 7).The species-level evolutionary tree of Actinidia reveals that the four traditionally recognized sections are not monophyletic with strong support, which is consistent with the results of Tang et al. [17] based on four noncoding cpDNA analyses.However, unlike the outcomes of the analyses of Tang et al. [17], our results strongly supported the notion that A. zhejiangensis C.F.Liang is sister to A. rufa (Siebold and Zuccarini) Planchon ex.Miquel rather than being grouped with A. hemsleyana Dunn; this conflict may be related to the fact that fewer markers were used in their research.As a result, categorizations based on sections fail to accurately represent the authentic evolutionary connections present within this collective.Overall, the robust support for resolving the lineages of Actinidia has laid a strong foundation for further classification at the inquisitive level and for exploring the biogeography.

Conclusions
In this research, the cp genome information of A. chinensis 'Hongyang' was analyzed and annotated.The structural features of the cp genomes of the 'Hongyang' kiwifruit variety were found to be remarkably similar to those of 14 other kiwifruit species.Additionally, through the analysis of the cp genomes of 15 different species of Actinidia, nine regions were identified as highly variable, making them promising candidates for molecular markers in species identification.The utilization of complete cp genome sequences has effectively elucidated the phylogenetic connections between 29 distinct Actinidia species.We found that A. zhejiangensis forms a monophyletic group with A. rufa rather than being grouped with A. hemsleyana, and this conflict may be due to the fact that fewer markers were used in previous research.The findings of our research have confirmed that the sectional categorizations do not reflect the actual evolutionary relationships.Overall, this study has significantly enhanced our knowledge of genome resources while shedding new light on the phylogenetic relationships in Actinidia taxonomy.

Supplementary Materials:
The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/genes14122136/s1,Table S1: Taxonomic and accession information for samples used in this study; Table S2: SSR length and type of A. chinensis 'Hongyang'; Table S3: Repeat sequences in the cp genome of A. chinensis 'Hongyang'.

Figure 1 .
Figure 1.The cp genomes of A. chinensis 'Hongyang' are depicted in the gene map.The gen inside the circular structure are transcribed in a clockwise manner, whereas the genes sit side the circle are transcribed in an anticlockwise manner.Gray arrows represent the d gene transcription.Genes with diverse functional groups are visually distinguished by c inner loop of the map, represented by a darker gray color, indicates the GC content.The plays SSC, LSC, and IR sequences.

Figure 1 .
Figure 1.The cp genomes of A. chinensis 'Hongyang' are depicted in the gene map.The genes located inside the circular structure are transcribed in a clockwise manner, whereas the genes situated outside the circle are transcribed in an anticlockwise manner.Gray arrows represent the direction of gene transcription.Genes with diverse functional groups are visually distinguished by colors.The inner loop of the map, represented by a darker gray color, indicates the GC content.The map displays SSC, LSC, and IR sequences.

Figure 2 .
Figure 2. RSCU analysis in cp genes of Actinidia plants.(A): Preference for codon usage and the proportion of amino acids in the PCGs of the 15 Actinidia cp genomes.(B): The histogram above each amino acid on the X-axis corresponds to the order of the 15 species.

Figure 2 .
Figure 2. RSCU analysis in cp genes of Actinidia plants.(A): Preference for codon usage and the proportion of amino acids in the PCGs of the 15 Actinidia cp genomes.(B): The histogram above each amino acid on the X-axis corresponds to the order of the 15 species.

Figure 3 .
Figure 3. Type and number of SSR nucleotides in the cpDNA of A. chinensis 'Ho

Figure 3 .
Figure 3. Type and number of SSR nucleotides in the cpDNA of A. chinensis 'Hongyang'.

Figure 4 .
Figure 4.The mVISTA was utilized to analyze and compare the cp genomes of fifteen samples.The gene orientation and the positions of IRs were indicated by gray arrows and bold black lines, respectively.The Y-scale depicted the percentage of identity, ranging from 50% to 100%.To enhance clarity, different genomic regions were color-coded, with protein-coding sequences (exons) shown in blue, rRNA in cyan, and a conserved non-coding sequence (CNS) in pink.

Figure 5 .
Figure 5.Comparison of nucleotide variability (Pi) values among fifteen different species'cp genomes.The Pi values are depicted on the Y-axis, and the genes exhibiting high Pi values are plotted on the X-axis.

Figure 4 .
Figure 4.The mVISTA was utilized to analyze and compare the cp genomes of fifteen samples.The gene orientation and the positions of IRs were indicated by gray arrows and bold black lines, respectively.The Y-scale depicted the percentage of identity, ranging from 50% to 100%.To enhance clarity, different genomic regions were color-coded, with protein-coding sequences (exons) shown in blue, rRNA in cyan, and a conserved non-coding sequence (CNS) in pink.

Figure 4 .
Figure 4.The mVISTA was utilized to analyze and compare the cp genomes of fifteen samples.The gene orientation and the positions of IRs were indicated by gray arrows and bold black lines, respectively.The Y-scale depicted the percentage of identity, ranging from 50% to 100%.To enhance clarity, different genomic regions were color-coded, with protein-coding sequences (exons) shown in blue, rRNA in cyan, and a conserved non-coding sequence (CNS) in pink.

Figure 5 .
Figure 5.Comparison of nucleotide variability (Pi) values among fifteen different species'cp genomes.The Pi values are depicted on the Y-axis, and the genes exhibiting high Pi values are plotted on the X-axis.

Figure 5 .
Figure 5.Comparison of nucleotide variability (Pi) values among fifteen different species'cp genomes.The Pi values are depicted on the Y-axis, and the genes exhibiting high Pi values are plotted on the X-axis.

Figure 6 .
Figure 6.Comparison of the boundaries of LSC, SSC, and IR regions across fifteen cp genomes.Figure 6.Comparison of the boundaries of LSC, SSC, and IR regions across fifteen cp genomes.

Figure 6 .
Figure 6.Comparison of the boundaries of LSC, SSC, and IR regions across fifteen cp genomes.Figure 6.Comparison of the boundaries of LSC, SSC, and IR regions across fifteen cp genomes.

Figure 7 .
Figure 7.The molecular phylogenetic trees were reconstructed of the genus Actinidia using the PCGs data.(A): ML tree.(B): BI tree.The red pentagrams represent the newly sequenced species in this study.Bootstrap values are indicated by numbers positioned above the branches.Lei: section Leiocarpae Dunn; Ste: section Stellatae; Mac: section Maculatae; Str: section Strigosae.

Figure 7 .
Figure 7.The molecular phylogenetic trees were reconstructed of the genus Actinidia using the PCGs data.(A): ML tree.(B): BI tree.The red pentagrams represent the newly sequenced species in this study.Bootstrap values are indicated by numbers positioned above the branches.Lei: section Leiocarpae Dunn; Ste: section Stellatae; Mac: section Maculatae; Str: section Strigosae.

Author Contributions:
Conceptualization, X.L. and Y.-Q.L.; methodology, X.L. and H.-L.L.; software, H.L., Y.G., X.-Q.F. and Q.-H.L.; validation, X.L., H.-L.L. and C.S.; formal analysis, C.S. and H.L.; investigation, X.L.; resources, W.-L.Z. and C.S.; data curation, H.-L.L. and Z.-X.L.; writing-original draft preparation, X.L. and H.L.; writing-review and editing, X.L. and H.-L.L.; visualization, C.S. and H.L.; supervision, X.L., Z.-X.L. and Y.-Q.L.; Each and every author has thoroughly reviewed and given their full consent to the final version of this manuscript for publication.All authors have read and agreed to the published version of the manuscript.Funding: The funding for this study was provided by the National Natural Science Foundation of China, grant number 31670688; the Science and Technology Research Program of the Chongqing Education Commission, China, grant numbers KJQN202101315 and KJQN201801336; and the Chongqing Municipal Science and Technology Commission, China, grant number cstc2019jscx-msxm1571.Institutional Review Board Statement: Not applicable.Informed Consent Statement: Not applicable.

Table 1 .
Composition of bases in various regions of the cp genome of A. chinensis 'Hongy

Table 1 .
Composition of bases in various regions of the cp genome of A. chinensis 'Hongyang'.

Table 2 .
Cp genome size and gene number of 15 Actinidia species.

Table 4 .
The length of the exon and intron in genes of the A. chinensis 'Hongyang' cp genome.