Complete chloroplast genome sequences of the ornamental plant Prunus Cistena and comparative and phylogenetic analyses with its closely related species

doi:10.21203/rs.3.rs-2970286/v1

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Complete chloroplast genome sequences of the ornamental plant Prunus Cistena and comparative and phylogenetic analyses with its closely related species

https://doi.org/10.21203/rs.3.rs-2970286/v1

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Background Prunus Cistena

is an excellent color leaf configuration tree for urban landscaping in the world, which has purplish red leaves, light pink flowers, plant shape and high ornamental value. Genomic resources for P. Cistena are scarce, and a clear phylogenetic and evolutionary history for this species has yet to be elucidated. Here, we sequenced and analyzed the complete chloroplast (cp) genome of P. Cistena and compared it with related species of Prunus genus based on chloroplast genome.

Results

The complete chloroplast genome of P. Cistena was 157,935 bp in length, which had a typical tetrad structure. The overall GC content was 36.72%, while IR regions displayed a higher GC content than LSC and SSC regions. It encoded 130 genes, including 85 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. The ycf3 and clpP genes had two introns; the longest intron was trnK-UUU gene in the LSC region. There were 253 SSRs, and the mononucleotide SSRs were the most abundant. The chloroplast sequences and gene arrangements of P. Cistena were conserved, which overall structure and gene order were similar to other Prunus species. The atpE, ccsA, petA, rps8 and matK gene had significant positive selection in Prunus species. P. Cistena had a close evolutionary relationship with the P. jamasakura. The coding region and IR region were more conserved than noncoding region, and the cpDNA sequences highly conserved throughout the genus Prunus.

Conclusions

The current genomic datasets provide valuable information for further species identification, evolution, and phylogenetic research of genus Prunus.

Forestry

Prunus Cistena

Chloroplast genome

Genetic diversity

Phylogenetic analysis

Prunus Cistena belongs to the genus Prunus in the Rosaceae family, which is popular ornamental tree in China. The genus Prunus consists of more than 250 species, most of which present many high ornamental and economic values for their flowers and leafs, which are loved by people all over the world [1–3]. They have great potential for development and application, the research on this economically important group is becoming more and more extensive [4–5]. P. Cistena is a hybrid of P. cerasifera and P. jamasakura, which has purplish red or dark purplish red leaves and light pink flowers. It also has plant shape and high ornamental value, which is an excellent color leaf configuration tree for urban landscaping.

Chloroplasts are unique organelles of green plants and play a crucial role in photosynthesis, amino acid synthesis, and carbon sequestration [5–7]. The chloroplast genomes of higher plants are characterized by a quadripartite structure typically consisting of two inverted repeats (IRs) with a length of 20–28 kb, separated by a large single-copy (LSC) region with a length of 80–90 kb and a small single-copy (SSC) region with a length of 16–27 kb, exhibiting highly conserved gene content and order [8–10]. It has the characteristics of haploid inheritance, relatively small genome, slow mutation rate, and sufficient polymorphism in plants, which provides an ideal model for genomic evolution and molecular markers for resolving phylogenetic relationships [11–13].

In recent years, with the rapid development of high-throughput sequencing technologies, the assembly of the chloroplast genome of conventional species has become convenient and inexpensive. More and more studies have shown that variation in chloroplast genomes provides effective information that can be used to resolve phylogenetic relationships at different levels for species identification [14–17]. The chloroplast genomes of many species of the Prunus genus have been sequenced, including P. pseudocerasus[2], P. campanulata [3] and P. phaeosticta [5], etc. The molecular structures and phylogenetic relationships of 20 Prunus subgenus Cerasus species were comparative analyzed based on complete chloroplast genomes [4]. However, the chloroplast genomes of P. Cistena had not been reported, leading to limitations in the mining of its genetic information and comprehensive analysis of the interspecific relationships of P. Cistena and other species in Prunus genus.

Therefore, the characteristics of the complete chloroplast genome sequences of P. Cistena were reported for the first time in this study. To understand the relationships, the phylogenetic tree of P. Cistena and other species within the Rosaceae family was analyzed. A comparative genomic analysis among six species of Prunus genus was performed to investigate the phylogenetic relationship. The findings of this paper provide a foundation for future genomic research on phylogenetic relationships and evolution within the genus Prunus.

Characteristics of P. Cistena cp Genomes

The complete cp genome of P. Cistena showed a typical circular double-stranded DNA with quadripartite structure, and length was 157,935 bp. It was consisted of one LSC region (85,947 bp), one SSC (region 19,116 bp), and a pair of IR regions (26,436 bp) (Fig. 1). The overall GC content was about 36.72%, while IR regions displayed a higher GC content (42.53%) than the GC content of LSC (34.59%) and SSC (30.22%). The GC content distribution pattern was similar to that of other plants [18–20].

The cp genome encoded a total of 130 genes, including 85 protein-coding genes (PCGs), 37 tRNA genes, and 8 rRNA genes (Table 1). Nine of 85 protein coding genes were for large subunits of ribosome, 12 were for small subunits of ribosome, 4 were for RNA polymerase, 20 were for photosystem and 6 were for ATP synthase. There were 20 duplicated genes in IRs, including seven protein-coding genes, nine tRNA genes and four rRNA genes (Table 1). Among these intron-containing genes, 21 out of 23 included a single intron, and 2 genes (ycf3, clpP) had two introns (Table S1). In comparison with other introns, the longest intron was within the trnK-UUU gene in the LSC region, reaching 2547 bp. The intron of the trnL-UAA gene was the shortest, only 514 bp. The rps12 gene was identified as trans-spliced with a single 5′-end at the LSC region while repeated 3′-end exons was located in the IRs (Fig. 1 and Table S1).

Table 1

Annotated genes in the *Prunus Cistena* CP genomes
Category	Gene group	Gene name
Photosynthesis	Subunits of photosystem I	psaA, psaB, psaC, psaI, psaJ
	Subunits of photosystem II	psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, psbM, psbN, psbT, psbZ
	Subunits of NADH dehydrogenase	ndhA^, ndhB^(2), ndhC, ndhD, ndhE, ndhF, ndhG, ndhH, ndhI, ndhJ, ndhK
	Subunits of cytochrome b/f complex	petA, petB^, petD^, petG, petL, petN
	Subunits of ATP synthase	atpA, atpB, atpE, atpF^*, atpH, atpI
	Large subunit of rubisco	rbcL
Self-replication	Proteins of large ribosomal subunit	rpl14, rpl16^, rpl2^(2), rpl20, rpl22, rpl23(2), rpl32, rpl33, rpl36
	Proteins of small ribosomal subunit	rps11, rps12^*(2), rps14, rps15, rps16^, rps18, rps19, rps2, rps3, rps4, rps7(2), rps8
	Subunits of RNA polymerase	rpoA, rpoB, rpoC1^*, rpoC2
	Ribosomal RNAs	rrn16(2), rrn23(2), rrn4.5(2), rrn5(2)
	Transfer RNAs	trnA-UGC^(2), trnC-GCA, trnD-GUC, trnE-UUC, trnF-GAA, trnG-GCC, trnG-GCC^, 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(2), trnS-UGA, trnT-GGU, trnT-UGU, trnV-GAC(2), trnV-UAC^, trnW-CCA, trnY-GUA, trnfM-CAU
Other genes	Maturase	matK
	Protease	clpP^**
	Envelope membrane protein	cemA
	Acetyl-CoA carboxylase	accD
	c-type cytochrome synthesis gene	ccsA
Genes of unknown function	Conserved hypothetical chloroplast ORF	ycf1(2), ycf2(2), ycf3^**, ycf4
: contains one intron; *: contains two introns; (2): gene with a copy number greater than 1, the number of copies in parentheses.

The relative occurrence of the synonymous codons in the coding sequences of P. Cistena cpDNA was calculated using 26,526 codons. It suggested that the four most frequently used codons were AUU-I (1117), AAA-K (1064), GAA-E (1029) and AAU-N (998), accounting for 4.21%, 4.01%, 3.88% and 3.76% among all codons, respectively (Table S2 and Fig. 2). Similar to the previous results for other study, most of the codons ending with A or T have RSCU values greater than 1, while, most of those ending with C or G have RSCU values of less than 1[21–23].

Analysis of repeat sequence and simple sequence repeats (SSRs)

In the cpDNA of P. Cistena, a total of 49 long repeats were recognized, including 19 forward, 23 palindrome, 6 reverse, and 1 complement repeats (Fig.S1). The length of the repeat sequence ranged mainly from 30 to 26436 bp. Furthermore, a total of 27, 23 and 7 long repeats were detected in the LSC, IRs and SSC regions, respectively.

In addition, 253 simple sequence repeats (SSRs) were detected in the cpDNA, among which 161, 14, 65, 11, 1 and 1 were mono-, di-, tri-, tetra-, penta- and hexonucleotide repeats, respectively (Fig. 3A). The mononucleotide SSRs were the most abundant and accounted for 63.63% of SSRs, trinucleotides SSRs followed, accounting for 25.69%. There were 170 SSRs in LSC region, 45 in SSC region and 38 in IR region (Fig. 3B). There were 35 SSRs in exons, 32 in intron and 103 in intergenic region in the LSC region. In the SSC region, 26 SSRs were located in exons, 4 in introns, and 15 in intergenic region. In the IR region, a total of 19, 4 and 15 SSRs were located in the exons, introns, and intergenic regions, respectively. The high variation in numbers of SSRs might provide abundant information for molecular marker studies and plant breeding.

Adaptive evaluation analysis

The Ka/Ks ratio was used to evaluate the degree of selection constraint on each gene and estimate the selective pressure of protein-coding genes. Ka/Ks > 1 indicates the gene is under positive selection, Ka/Ks = 1 indicates the gene is under neutral selection and Ka/Ks < 1 indicates purification selection [24].

In the present study, KaKs Calculator was used to calculate the non-synonymous (Ka) to synonymous (Ks) ratio (Ka/Ks) of 78 effectively shared protein-coding genes in P. Cistena and five other Prunus species (Table S3). The results showed that the Ka/Ks values between P. Cistena and Prunus species ranged from 0 (ndhC) to 1.84989 (matK). The Ka/Ks value of atpE, ccsA, petA, rps8 gene between P. Cistena and P. padus was > 1, which had positive selection effects. The matK gene was positively selected within P. Cistena, P. salicina, P.japonica and P. simonii. The Ka/Ks values of other genes were < 1, which had strong purification selection pressure in Prunus genera (Fig. 4).

Phylogenetic analysis

The cpDNAs of P. Cistena and another 29 species within the Rosaceae family were selected to explore the genetic relationship between P. Cistena and its relatives, Punica granatum was used as outgroup. Multiple alignments of all of the 31 cpDNAs were computed using MAFFT, and then a maximum likelihood (ML) tree was determined using RAxML implementing the GTRGAMMA model. All relationships were strongly supported by high bootstrap values ranging from 93 to 100 (Fig. 5).

The phylogenetic tree with most branches having high levels of support showed three divergent clades. The first clade was formed by Prinsepia, Crataegus, Pyrus, Photinia, Malus and Prunus species. The phylogenetic analyses had established that three Rosa species and two Rubus species fell into the second clade. The outgroup Punica granatum alone was located at the third clade (Fig. 5). Prunus species were clustered into a group in which P. Cistena was closely related to P. jamasakura.

Comparative chloroplast genomic analysis

To advance our understanding of the cpDNAs of the genus Prunus, further investigation was conducted to make critical comparisons of the IR/SSC and IR/LSC border positions in the six selected Prunus species, to access the degree of IR expansion or contraction among them. The difference at the boundaries was observed in the six cpDNAs, of which the LSC, IRa/b, and SSC regions have an average length of 86,351 bp, 26,367 bp and 19,035 bp, respectively (Fig. 6A).

The rpl22 of P. cistena, P. salicina, P. jamasakura, P. japonica and P. simonii were located completely at the junction of the LSC region (Fig. 6A). The LSC/IRb border was inside the rps19 gene in the six cpDNAs. The 217 bp fragment of the rps19 was located within the IRb region in P. cistena and P. jamasakura, whereas the remaining 62 bp section of the gene can be found within the LSC region. There were 240bp fragment of the rps19 within the LSC region in P. padus, while the remaining 39 bp section within the IRb region. The IRb/SSC boundary was located inside the ycf1 and ndhF gene. The fragment length of ycf1 ranged from 1036bp to 1051bp in IRb region, while the length of remaining section was 3 bp ~ 94bp in the six cpDNAs. The 1 bp ~ 10 bp fragment of the ndhF gene was located within the IRb region, whereas the remaining 2219 bp ~ 2250bp section can be found within the SSC region. The ycf1 gene was located across the SSC/IRa junction, which length in the SSC region was 4561 bp ~ 4614 bp. The trnN-GUU and rpl2 was completely found in the IRa region, but rpl2 was also in the IRb region in P. padus. The trnH-GUG was completely found in the LSC region.

To indicate the degree of the genome divergence, the mVISTA and Mauve software was used for sequence alignment and collinearity analysis among the selected six species (Fig. 6B, Fig.S2). The nucleotide sequence similarity of the six cpDNAs was extremely high, suggesting that there was no variation in the cp genome of P. cistena compared with its ancestral species. At the same time, it can be found that divergence existed in the highly conserve regions, and the coding region and IR region were more conserved than noncoding region.

The chloroplast genome structure of P. cistena and proximal species was also analyzed by CGVIEW (Fig.S3). It indicated a high degree of similarity between genomes. The nucleotide diversity (pi) values of the selected eight cpDNAs, calculated within the slide window, ranged from 0 to 0.02398, with an average of 0.00296 (Fig.S4). There were three highly variable regions, LSC. rps18, LSC. rbcL and SSC.ycf1 with pi values higher than 0.01. The results showed low pi values, which suggested the cpDNA sequences were highly conserved at the sequence level throughout the genus.

The cp genome was a valuable resource for molecular phylogenetic studies, and had been authentically analyzed to be a productive biological and agricultural tool for fast and exact crop recognition [25–27]. In the present study, we successfully sequenced and assembled the complete chloroplast genome of P. Cistena, which was in a length of 157,935 bp (Fig. 1) and had highly similar in size, overall structure, gene order, and content with other Prunus species. These findings are consistent with previous study on Prunus genus, which showed that the chloroplast sequences and gene arrangements were conserved [2, 4]. The GC contents in LSC and SSC regions were significantly lower than that in the IR region, which was caused by a relatively high GC content in rRNA and tRNA genes, occupying more area than that of protein-coding genes in IR regionsp [19, 28–30].

Introns regulate the expression of some genes and play an important role in alternative splicing [19, 31]. Here, the results indicated that two genes (ycf3 and clpP) had two introns in the P. Cistena chloroplast genomes, which was consistent with research on other plants [4, 31, 32–33]. Furthermore, ycf3 is known as a photosynthesis-related gene as reported before [31, 34]. Therefore, the attendance of the ycf3 gene might result in an extra study of Prunus chloroplast. The rps12 genes were located at the 5’ end on the LSC region and meanwhile the duplicated 3’ ends was set in the IRs regions due to the phenomenon it has been called trans-spliced gene [22, 26, 35]. The ycf1 gene expected a basic part in the chloroplast genome. There were some related studies exposed ycf1 as a paramount pseudogene in plants, leading to incomplete duplication of the gene within IRs [31, 36]. The ycf1 gene repeated twice in the chloroplast genome of P. Cistena, which was similar to other Prunus plants [22, 37]. It has been testified that the introns play a significant role in regulating the expression of genes, which might control the gene expression level in different spatiotemporal [19, 31, 38]. The attendance of intron or the loss of genes can be discovered in the chloroplast genomes, and the regulating function of intron need to be exposed for future research [19, 31].

The repeat sequences may raise the genetic diversity of the species and advance the cpDNA rearrangements [22, 39]. Generally, the cpSSRs are valuable genetic tools for population genetics and evolution studies, because of their codominant nature, high polymorphism and low substitution rate [40–41]. The results of the study demonstrated that the genes which have long repeats may be produced as a genetic marker for identifying the related species. There were 253 SSRs in chloroplast genome of P. Cistena, and detected six types of SSR. The mononucleotide SSRs were the most abundant, which was consistent with other Prunus plants [4, 33]. The SSRs identified in the chloroplast genome of Prunus can be used for developing lineage-specific cpSSR markers [12].

The pattern of Ks and Ka nucleotide substitutions is a known phenomenon for quantifying genomic evolution, and the Ka/Ks value shows selection pressures on genes [24, 42]. The Ka/Ks value of P. Cistena cp genome was determined in the work when compared to five closely related species of genus Prunus. The average Ka/ Ks ratio was less than one, representing that the genes in the P. Cistena were subjected to clear purifying selection. The Ka/Ks ratio showed low rates of evolution (Ka/Ks < 1) for most protein coding genes among the 6 Prunus chloroplast genomes tested. These findings agreed with those of other high plant chloroplast genomes [43–44]. The Ka/Ks value of atpE, ccsA, petA, rps8 and matK gene were more than one, indicating that it has undergone significant positive selection in Prunus species. Similar findings have been obtained in prior research [44, 45].

Both molecular and morphological evidence suggests that genus Prunus had a complex evolutionary history [4]. Based on complete chloroplast genome sequences of 30 species in Rosaceae family, we determined the phylogenetic relationship of P. Cistena using phylogenetic analysis, and the topological structure was relatively consistent. The result indicated that Prunus species were clustered into a group, and P. Cistena had a close evolutionary relationship with the P. jamasakura. It was further confirmed that P. jamasakura was the parent of P. Cistena. This study provided a valuable basis for future phylogenetic analyses of species within Rosaceae.

Typically, the IR region is the most conserved region of the chloroplast genome. The modification of the cpDNA size could be driven by the important evolutionary events, expansion and shrinkage of the IR regions, which lead to fluxes in the LSC/IR junctions, pseudogenes initiation and gene duplication [4, 46]. In the cpDNAs of angiosperm, the borders between the LSC/SSC and IR regions are highly and relatively conserved [18, 47]. We compared the cpDNAs of six Prunus species in this study. The rps19 genes were located in the boundary of LSC/IRb, which length in the IRb regions were longer than that in LSC region, except for the P. padus. The junction of LSC/IRb boundary was between rpl2 and rps19 in P. padus, whereas the junctions were located between rpl22 and rps19 in the other species. The latter phenomenon had also been reported in other plants [48–49]. The ycf1 gene was located in the IRa/SSC and IRb/SSC regions. The fragment length of ycf1 was between 4.561bp to 4, 614bp in IRa region, and ranged from 1, 036bp to 1, 051bp in IRb region. These overlapping segments of ycf1 were pseudogenized and observed at the junction of JSB in all of the six selected Prunus species, which was consistent with the findings of earlier reports [22, 31].

The nucleotide sequence similarity of the six Prunus cpDNAs was extremely high. The coding region and IR region were more conserved than noncoding region. It further indicated that the cpDNA sequences were highly conserved throughout the genus Prunus [4, 50]. The rps18 gene in LSC region had the highest Pi value in the chloroplast genomes of P. Cistena, could be used for phylogenetic analysis and population genetic study of Prunus species. The results increase the current knowledge of the evolutionary patterns in the Rosaceae.

The complete chloroplast genome of P. Cistena was sequenced and assembled employing the Illumina Hiseq platform, and compared with the cp genome of other Prunus species. The chloroplast sequences and gene arrangements of P. Cistena were conserved, which size, overall structure, gene order and content were similar to other Prunus species. The atpE, ccsA, petA, rps8 and matK gene had significant positive selection in Prunus species. Phylogenetic relationships among 30 Rosaceae species strongly supported the known classification of P. Cistena. The coding region and IR region were more conserved than noncoding region, and the cpDNA sequences highly conserved throughout the genus Prunus. The hotspot genes (ycf1 and rps18) were identified that contained rich information for species identification and phylogenetic reconstruction of Prunus, as well as provide insights into the evolution of Prunus species.

DNA extraction and sequencing

The fresh leaves of P.Cistena were collected from the nursery of Shandong Institute of Pomology, Taian City, Shandong Province, China. The samples were immediately frozen in liquid nitrogen and stored at − 80℃. Total genomic DNA was extracted from fresh leaves using the plant genomic DNA Kit (DP180123) of China Tiangen Biotechnology (Beijing) Co., Ltd. After the genomic DNA of the sample was qualified, the isolated DNA was fragmented into small pieces using sonication. The fragmented DNA was subjected to fragment purification, end repair, adding A at the 3' end and ligating sequencing adapters. Based on agarose gel electrophoresis, the suitable fragments were purified and selected as templates for next-step PCR amplification to create the final DNA library. Qualified libraries were sequenced by paired-end (PE) using the Illumina NovaSeq 6000 platform of Nanjing Genepioneer Biotechnologies, and the sequencing read length was 150.

Chloroplast genome assembly and annotation

The raw reads were filtered using Fastp v0.20.0 software (https://github.com/OpenGene/fastp) to remove the adaptors, reads with an average quality value less than Q5, N number of reads greater than 5. The obtained clean data were assembled using SPAdes v3.10.1, SSPACE v2.0 and Gapfiller v2.1.1 software [51]. The CP genome of P. simonii (NCBI accession number MW406463.1) was used for quality control after assembly.

In order to improve the accuracy of the annotation, two methods were used to annotate the chloroplast genome. First, the CDS, rRNA and tRNA genes were aligned using prodigal v2.6.3 (https://www.github.com/hyattpd/Prodigal), HMMER v3.1b2 (http://www.hmmer.org/) and aragorn v1.2.38 (http://130.235.244.92/ARAGORN/), respectively. Second, the gene sequences of closely related species published on NCBI was extracted, and aligned with the assembled sequences using blast v2.6 (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The two annotation results were manually checked for differential genes, erroneous and redundant annotations were removed, and multi-exon boundaries were determined to obtain the final annotation. The chloroplast genome maps were visualized using OGDRAW software [52].

Identification of repeat sequences and cpSSR

Interspersed repetitive sequences were identified using vmatch v2.3.0 (http://www.vmatch.de/) software combined with a Perl script. Repeat sequences included forward, reverse, complement, and palindromic repeats, and the parameters were set as minimal repeat size of 30 bp and hamming distance of 3.

Simple sequence repeats of chloroplast genomes (cpSSR) were analyzed using MISA v1.0 software (MIcroSAtellite identification tool, http://pgrc.ipk-gatersleben.de/misa/misa.html). The parameters were set to: minimum number of eight repeated motifs for mononucleotide, five repeated motifs for dinucleotide, four repeated motifs for trinucleotide, and three repeated motifs for tetra-, penta- and hexanucleotide repeats.

Codon usage, selectivepressure and nucleotide diversity

Relative Synonymous Codon Usage (RSCU) is the ratio of the actual number of synonymous codons used to translate a specific amino acid to the expected number [18]. RSCU is a useful tool for understanding genetic and evolutionary processes. The CodonW v1.4.2 software was used to count RSCU and analyze codon preference in chloroplast genomes.

The mafft v7.310 (https://mafft.cbrc.jp/alignment/software/) was used to perform indel identification. Ka/Ks values (representation of selective pressure) were calculated by KaKs_Calculator v2.0(https://sourceforge.net/projects/kakscalculator2/)software. If the ratio is greater than 1, it means that there is a positive selection effect, which is less than 1, indicating that there is purification selection.

Phylogenetic analysis

To reveal the evolutionary relationship among the six prunus species of Rosaceae class, a total of 29 complete cp genomes of the family Rosaceae were collected from GenBank, with Punica granatum as the outer group. The sequences were aligned by MAFFT v7.427 (-auto mode), and the maximum likelihood (ML) phylogenetic tree was constructed by RAxML v8.2.10 (https://cme.h-its.org/exelixis/software.html) software with GTRGAMMA model and 1000 bootstrap replicates.

Genomic comparison with related species

Perl-IRscope (https://github.com/xul962464/perl-IRscope) was used to analyze and visualize the borders between LSC/IRs and SSC/IRs among the six cp genomes, including P.Cistena, P. padus, P. salicina, P.jamasakura, P.japonica and P.simonii. The homology and collinearity of chloroplast sequence were analyzed using Mauve (http://darlinglab.org/mauve) and mVISTA (http://genome.lbl.gov/vista/index.shtml) software. CGVIEW (http://stothard.afns.ualberta.ca/cgview_server/) was used to compare the complete P.Cistena cp genome structure to that of five related species. DnaSP v5.0 software with default settings was used to calculate the nucleotide diversity (Pi) value for each gene.

Author Contributions:

Lijuan Feng designed the experiments and organized the manuscript; Guopeng Zhao, Mengmeng An analyzed and interpreted the data. Chuanzeng Wang and Yanlei Yin Original edited the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Shandong Key Research and Development Projects (2021LZGC007, 2022TZXD009), Agricultural Science and Technology Innovation Engineering Discipline Team of Shandong Academy of Agricultural Sciences (CXGC2022D02, CXGC2023A12).

Data Availability Statement: The chloroplast genome sequences have been deposited in GenBank under the accession numbers: ON585706. Row data are available at SRA, under the accession number: SRR19352552.

Ethics approval and consent to participate

For the collection of samples for this study, no special licenses were needed.

The relevant Chinese laws were followed as this research was conducted.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

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Complete chloroplast genome sequences of the ornamental plant Prunus Cistena and comparative and phylogenetic analyses with its closely related species

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Abstract

Figures

Background

Results

Analysis of repeat sequence and simple sequence repeats (SSRs)

Adaptive evaluation analysis

Phylogenetic analysis

Comparative chloroplast genomic analysis

Discussion

Conclusion

Materials and Methods

DNA extraction and sequencing

Chloroplast genome assembly and annotation

Identification of repeat sequences and cpSSR

Codon usage, selectivepressure and nucleotide diversity

Phylogenetic analysis

Genomic comparison with related species

Declarations

References

Supplementary Materials

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