Chloroplast Genome Profiling and Phylogenetic Insights of the “Qixiadaxiangshui” Pear ( Pyrus bretschneideri Rehd.1)

: The “Qixiadaxiangshui” pear ( Pyrus bretschneideri Rehd.1) is a highly valued cultivar known for its crisp texture, abundant juice, and rich aroma. In this study, we reported the first complete chloroplast genome sequence of the “Qixiadaxiangshui” pear, which is 159,885 bp in length with a GC content of 36.58%. The genome exhibits a typical circular quadripartite structure, comprising a large single-copy region (LSC), a small single-copy region (SSC), and a pair of inverted repeat regions (IRs). A total of 131 genes were identified, including 84 protein-coding genes, 8 rRNA genes, and 37 tRNA genes. We also identified 209 simple sequence repeats (SSRs) and several mutation hotspots, such as ndhC-trnM-CAU and trnR-UCU-atpA , which can be applied in molecular identification and phylogenetic studies of Pyrus . Comparative genomic analysis showed high conservation among ten pear cultivars. Phylogenetic analysis indicated that the “Qixiadaxiangshui” pear is closely related to germplasm Dangshansuli, Wonwhang, and Yali, suggesting a recent common ancestor. These findings provided valuable insights into the genetic diversity and evolutionary dynamics of the Pyrus species and contribute to the conservation and breeding of pear germplasm resources.


Introduction
The chloroplast, a crucial organelle in plant cells, is responsible for converting light energy into chemical energy, making it a focal point in botanical studies [1].The chloroplast genome, with its relatively slow rate of variation, primarily due to insertions, deletions, and point mutations in non-coding regions, serves as an ideal material for phylogenetic analysis [2].Typically, the chloroplast genome is a double-stranded circular configuration, comprising a large single-copy region (LSC) and a small single-copy region (SSC) separated by two inverted repeat regions (IRa and IRb), forming a quadripartite structure [3].
The chloroplast genome is characterized by the integrity of its replication, transcription, and translation systems, its small molecular weight, the conservatism of gene structure and quantity, and the uniformity of gene arrangement.These features make the chloroplast genome a valuable tool for studies in plant systematics, genetics, and evolutionary biology.Chloroplast genome research has been pivotal in elucidating the phylogenetic relationships within the genus Pyrus, including species such as Pyrus pyrifolia cultivar Wonwhang [4], Pyrus ussuriensis Maxim.[5], and Pyrus pashia [6], revealing the complex evolutionary relationships among these species.
Pyrus bretschneideri, commonly known as the Chinese white pear, is one of the most important pear species in China.Notable cultivars of Pyrus bretschneideri include "Dang-shansuli", "Yali", and the "Qixiadaxiangshui" pear.These cultivars or strains are known for their crisp texture, sweetness, and high juice content, making them highly popular among consumers.Current studies on chloroplast genomes of pear species have primarily focused on a limited number of cultivars, leading to an incomplete understanding of the genetic diversity and evolutionary dynamics within the genus.The limited phylogenetic information from commonly used chloroplast regions, such as rpl16, trnH, and matK, results in phylogenetic trees with low resolution and weak bootstrap support [7].Therefore, it is necessary to develop high-resolution genetic markers to promote the species identification, germplasm screening, and phylogeny of Pyrus to further facilitate the utilization and conservation of pear germplasm resources.
The "Qixiadaxiangshui" pear is a high-quality cultivar of pear native to Qixia City, Shandong Province, China.Known for its crisp texture, abundant juice, and rich aroma, it is highly valued locally but remains relatively unknown outside China [8].Reports on the chloroplast genome of the "Qixiadaxiangshui" pear are scarce, limiting our understanding of its genetic makeup and evolutionary background.The lack of molecular genetic information on "Qixiadaxiangshui" not only restricts its research on the classification and genetic diversity of the Pyrus genus but also impedes the application of this excellent cultivar in pear breeding.To address these gaps, our study undertook an in-depth sequencing of the chloroplast genome of the "Qixiadaxiangshui" pear.We analyzed a series of genomic variation hotspots within the chloroplast genomes of the genus Pyrus to thoroughly assess the evolutionary status of the "Qixiadaxiangshui" pear and its phylogenetic relationships with other pear germplasm.Our research aims to provide a scientific basis and new perspectives for genetic breeding and molecular evolution studies of the "Qixiadaxiangshui" pear, contributing to the improvement and optimization of this variety and the conservation and utilization of pear genetic resources.

Materials
Fresh leaves of the "Qixiadaxiangshui" pear (Pyrus bretschneideri Rehd.1) were collected from ten healthy and mature plants grown at the JinNiu Mountain experimental base of the Shandong Institute of Pomology in Tai'an City, Shandong Province, on 5 April 2021.These plants were selected based on their uniform growth conditions and absence of disease or pest infestation to ensure consistency and reliability of the genomic data.The leaves were washed, dried, and stored at −80 • C. The chloroplast genome sequences of closely related species of "Qixiadaxiangshui" pear were retrieved from the NCBI database.Detailed information on the experimental materials is provided in Table S1.

DNA Extraction and Sequencing
DNA from fresh leaves of the "Qixiadaxiangshui" pear was extracted using the Plant DNA Extraction Kit (Vazyme Biotech Co., Ltd., Nanjing, China).The quality of the extracted DNA was verified through agarose gel electrophoresis, and its concentration was measured using a NanoDrop 2000 spectrophotometer (Thermo Scientific TM , Waltham, MA, USA).After verifying the quality of the sample genomic DNA, the DNA was fragmented using an ultrasonic shearing method.The fragmented DNA was then subjected to fragment purification, end repair, 3 ′ -end adenylation, and ligation with sequencing adapters.Fragment size selection was performed using agarose gel electrophoresis, followed by PCR amplification to construct the sequencing library.The constructed library underwent quality control, and libraries that passed quality control were sequenced using the Illumina NovaSeq 6000 platform with a paired-end read length of 150 bp.

Chloroplast Genome Assembly and Annotation
High-quality reads were obtained by filtering the raw data with fastp (version 0.20.0).Sequences aligning to a proprietary chloroplast genome database were identified using bowtie2 v2.2.4 in very-sensitive-local mode and were considered as chloroplast DNA (cpDNA) sequences of the sample.The assembly core module utilized SPAdes v3.10.1 [9] with k-mer values of 55, 87, and 121, assembling the chloroplast genome without reference.The assembled genome was quality controlled against the reference sequence Pyrus_bretschneideri_Rehd.1:KY626169.1.Chloroplast genome annotation was performed using two approaches: (1) CDS annotation with Prodigal v2.6.3, rRNA prediction with HMMER v3.1b2, and tRNA prediction with ARAGORN v1.2.38 and (2) gene sequences from closely related species published on NCBI were obtained and aligned against the assembled sequence using BLAST v2.6 to obtain another annotation result.Discrepancies between annotation results were manually reviewed to eliminate erroneous and redundant annotations, define multi-exon boundaries, and finalize the annotation.The chloroplast genome map was generated using OGDRAW v1.3.1 [10].

Comparative Analysis of Chloroplast Genomes
Differences in the four boundary regions of chloroplast genomes between "Qixiadaxiangshui" and nine other pears of the Rosaceae family, including the Pyrus bretschneideri strain Dangshansuli, the Pyrus bretschneideri strain Yali, the Pyrus communis strain Bartlett, the Pyrus calleryana cultivar OPR125, Pyrus hopeiensis, Pyrus phaeocarpa, the Pyrus pyrifolia cultivar Whangkeumbae, the Pyrus pyrifolia cultivar Wonwhang, and Pyrus ussuriensis, were analyzed using the IRscope online tool [12].Multiple sequence alignments of chloroplast genomes from ten pear germplasm were performed using the mVISTA tool [13] in shuffle-LAGAN mode.Nucleotide diversity (Pi) of the chloroplast genomes was calculated with DNAsp, setting the window length to 600 bp and step size to 200 bp.

Structure and Composition of the Chloroplast Genome of the "Qixiadaxiangshui" Pear
After removing adapters and low-quality data, a total of 7.62 Gb of raw data were obtained with 25,428,945 original reads, achieving a Q20 of 98.29%.The assembly and annotation of the chloroplast genome of the "Qixiadaxiangshui" pear revealed a typical quadripartite structure, consisting of a large single-copy (LSC) region, a pair of inverted repeats (IRs), and a small single-copy (SSC) region, with lengths of 87,863 bp, 26,392 bp, and 19,238 bp, respectively (Figure 1).The GC content was 36.58%,indicating a higher AT content of 63.42%.Variations in GC values were observed across the IR, LSC, and SSC regions, with the IR region having the highest GC content (42.64%) compared to the LSC (34.28%) and SSC (30.40%) regions (Table 1).The results reveal a highly conserved quadripartite structure with variable GC content across different regions, indicating its complex genetic architecture and adaptation potential.
regions, with the IR region having the highest GC content (42.64%) compared to the LSC (34.28%) and SSC (30.40%) regions (Table 1).The results reveal a highly conserved quadripartite structure with variable GC content across different regions, indicating its complex genetic architecture and adaptation potential.Table 1.Base composition of chloroplast genome of "Qixiadaxiangshui" pear.

RSCU Analysis of the "Qixiadaxiangshui" Pear Genome
The Relative Synonymous Codon Usage (RSCU) value, which equals the actual frequency of a codon divided by its expected frequency, highlights the codon usage bias and provides insights into the evolutionary dynamics and gene expression regulation within the "Qixiadaxiangshui" pear chloroplast genome.The RSCU analysis of the chloroplast genome revealed a total of 26,088 codons encoding 20 amino acids (excluding stop codons) (Figure 2 and Table 3).Leucine (Leu) had the highest number of codons (2752), accounting for 10.55% of the total, while cysteine (Cys) had the lowest (299, 1.15%).There are six types of codons encoding Leu, with the UUA codon having the highest RSCU value of 1.9272 and the CUG codon having the lowest at 0.39.Excluding the codon AUG (methionine, Met), the remaining 30 preferred synonymous codons (RSCU > 1) all ended with an A/T(U) base.The preferred terminator used was UAA.The RSCU analysis not only demonstrates a preference for certain codons in the encoding of amino acids but also reflects the genetic and evolutionary intricacies of the "Qixiadaxiangshui" pear, with leucine and cysteine codon frequencies highlighting the nuanced regulation of gene expression.and the CUG codon having the lowest at 0.39.Excluding the codon AUG (methionine, Met), the remaining 30 preferred synonymous codons (RSCU > 1) all ended with an A/T(U) base.The preferred terminator used was UAA.The RSCU analysis not only demonstrates a preference for certain codons in the encoding of amino acids but also reflects the genetic and evolutionary intricacies of the "Qixiadaxiangshui" pear, with leucine and cysteine codon frequencies highlighting the nuanced regulation of gene expression.

cpSSR Analysis of the "Qixiadaxiangshui" Pear Genome
A total of 209 repeat loci were identified in the genome, including 48 pairs of complex repeat sequences (Table 4).Within these, 129 were located in the large single-copy (LSC) region, 38 in the small single-copy (SSC) region, and 42 in the inverted repeat (IR) regions.The analysis revealed 181 mononucleotide simple sequence repeats (SSRs), including 19 dinucleotide SSRs, 64 trinucleotide SSRs, 5 tetranucleotide SSRs, 1 pentanucleotide SSR, and 2 hexanucleotide SSRs.Among the mononucleotide repeats (Figure 3), sequences of A and T were predominant.Dinucleotide repeats were primarily composed of AT and TA sequences.Trinucleotide repeats were the most varied in type, though they occurred less frequently.Tetranucleotide, pentanucleotide, and hexanucleotide repeats were rare in both type and occurrence.The cpSSR analysis uncovers a significant diversity of simple sequence repeats, especially in the mononucleotide category, predominantly composed of A and T sequences, illustrating the complexity and variability within the genome structure.

cpSSR Analysis of the "Qixiadaxiangshui" Pear Genome
A total of 209 repeat loci were identified in the genome, including 48 pairs of complex repeat sequences (Table 4).Within these, 129 were located in the large single-copy (LSC) region, 38 in the small single-copy (SSC) region, and 42 in the inverted repeat (IR) regions.The analysis revealed 181 mononucleotide simple sequence repeats (SSRs), including 19 dinucleotide SSRs, 64 trinucleotide SSRs, 5 tetranucleotide SSRs, 1 pentanucleotide SSR, and 2 hexanucleotide SSRs.Among the mononucleotide repeats (Figure 3), sequences of A and T were predominant.Dinucleotide repeats were primarily composed of AT and TA sequences.Trinucleotide repeats were the most varied in type, though they occurred less frequently.Tetranucleotide, pentanucleotide, and hexanucleotide repeats were rare in both type and occurrence.The cpSSR analysis uncovers a significant diversity of simple sequence repeats, especially in the mononucleotide category, predominantly composed of A and T sequences, illustrating the complexity and variability within the genome structure.

Analysis of IR Boundary Features
The comparison of the inverted repeat (IR), large single-copy (LSC), and small singlecopy (SSC) boundaries of the chloroplast genomes of ten pear germplasm, including the "Qixiadaxiangshui" pear (Pyrus bretschneideri Rehd.1), is illustrated in Figure 4.The length of the LSC region in the chloroplast genomes of these ten pear plants ranges from 87,863 bp to 88,200 bp, the IR region from 26,386 bp to 26,411 bp, and the SSC region from 19,201 bp to 19,251 bp.The sizes of the IR, SSC, and LSC regions are similar across the ten pears, with the genes on both sides of the JLB, JSB, JSA, and JLA boundaries being consistent, indicating a high degree of conservation.At the LSC/IRb boundary (JLB), the rps19 gene in the LSC region of Bartlett pear (Pyrus communis strain Bartlett) extends into the IRb region by 134 bp, while in the other nine pears, the extension is 120 bp.The SSC/IRb boundary (JSB) shows that the boundary is within the ndhF gene for all ten germplasm, with the ndhF gene of the Bartlett pear extending into the IRb region by 14 bp, which is 2 bp more than the other nine pears.The JSA (SSC/IRa) boundary is located within the ycf1 gene for all ten germplasm, with the ycf1 gene of Bartlett extending into the Ira region by 1076 bp, whereas the extension in the other nine pears is 1094 bp.The LSC/Ira (JLA) boundary between the rpl2 and trnH genes shows that the rpl2 gene is entirely within the Ira region, and the trnH gene contracts towards the LSC region by 29 to 104 bp, with little variation in range.
indicating a high degree of conservation.At the LSC/IRb boundary (JLB), the rps19 gene in the LSC region of Bartlett pear (Pyrus communis strain Bartlett) extends into the IRb region by 134 bp, while in the other nine pears, the extension is 120 bp.The SSC/IRb boundary (JSB) shows that the boundary is within the ndhF gene for all ten germplasm, with the ndhF gene of the Bartlett pear extending into the IRb region by 14 bp, which is 2 bp more than the other nine pears.The JSA (SSC/IRa) boundary is located within the ycf1 gene for all ten germplasm, with the ycf1 gene of Bartlett extending into the Ira region by 1076 bp, whereas the extension in the other nine pears is 1094 bp.The LSC/Ira (JLA) boundary between the rpl2 and trnH genes shows that the rpl2 gene is entirely within the Ira region, and the trnH gene contracts towards the LSC region by 29 to 104 bp, with little variation in range.The expansion, contraction, or even complete absence of the IR region is a common evolutionary phenomenon in chloroplast genomes, with the expansion in some species leading to the duplication of the rps19 and ycf1 genes within this region [17].For the "Qixiadaxiangshui" pear, Pyrus hopeiensis, and Pyrus ussuriensis, a copy of the ycf1 gene occurs in the IRb region at the JSB boundary, with an incomplete copy in Pyrus hopeiensis and the absence of the ycf1 gene copy in other pears; similarly, an incomplete copy of the rps19 gene is found in the Ira region for Pyrus phaeocarpa and Pyrus hopeiensis, while other species lack this copy.The analysis of IR boundary features demonstrates a remarkable conservation in the structure and size of the LSC, SSC, and IR regions, alongside consistent gene positioning at key boundaries.This conservation reflects the evolutionary stability of these regions, despite the variability in gene extension and contraction at the boundaries, indicative of the dynamic evolutionary processes influencing chloroplast genome architecture.

Comparative Analysis of Chloroplast Genomes among Pear Germplasm
Using the Pyrus bretschneideri strain Yali as a reference, a comprehensive sequence comparison of ten pear germplasm, including the "Qixiadaxiangshui" pear, Pyrus bretschneideri strain Yali, Pyrus pyrifolia cultivar Whangkeumbae, and Pyrus ussuriensis, was conducted using the mVISTA online tool.This comparison aimed to discern the differences in the chloroplast genome sequences among these ten pears.The results indicated a high degree of similarity in the chloroplast genome sequences across the ten pear germplasm.Notably, some variable hotspots were primarily located in non-coding regions (Figure 5), including intergenic spaces such as rps16-trnQ-UUG, trnR-UCU-atpA, trnT-GGU-psbD, ndhC-trnM-CAU, trnM-CAU-atpE, and accD-psal.Additionally, a comparison of the chloroplast genomes of these ten pears was performed using the MAFFT v7.520 software (Figure 6), with the nucleotide diversity (Pi) calculated using DNAsp v6.1 software to analyze the level of genomic variation among the Pyrus.The results showed that Pi values ranged from 0.00033 to 0.01426, with an average value of 0.001297.Intergenic regions with Pi values greater than 0.005 included ndhC-trnM-CAU-atpE and trnR-UCU-atpA.The intergenic region ndhC-trnM-CAU-atpE had the highest Pi value of 0.01426, containing 31 variable sites.Following this, the trnR-UCU-atpA region's Pi value reached 0.00985, encompassing 17 variable sites.

Phylogenetic Analysis of Rosaceae Plants
A phylogenetic tree of the complete chloroplast genomes of 28 Rosaceae species and Castanea mollissima was constructed using multiple sequence alignments and the maximum likelihood method (Figure 7).The results revealed that the tested Rosaceae species were divided into three clades: one comprising the genera Rubus, Rosa, and Fragaria, which received 100% support; another consisting of the genera Prunus, also with 100% support; and a third clade formed by the genera Malus, Crataegus, and Pyrus, again with 100% support.Within the genus Pyrus, the "Qixiadaxiangshui" pear clustered closely with the Dangshansuli, Wonwhang, and Yali, all supported with a 100% bootstrap value.The phylogenetic analysis highlights the genetic divergence and common ancestry within the Rosaceae family.The high bootstrap support values confirm the robustness of our phylogenetic tree.The close clustering of the "Qixiadaxiangshui" pear with Dangshansuli, Wonwhang, and Yali suggests these cultivars share a recent common ancestor, indicating conserved genetic traits advantageous for breeding programs.By comparing the results obtained from the mVISTA online tool and MAFFT v7.520 software, several key observations were made.Both methods consistently identified high levels of similarity in the chloroplast genome sequences across the ten pear germplasm and highlighted variable hotspots predominantly located in non-coding regions.While mVISTA identified these variable hotspots, the nucleotide diversity analysis of MAFFT quantified these differences with precise Pi values, revealing the extent of variability within regions like ndhC-trnM-CAU-atpE and trnR-UCU-atpA.This dual approach underscores the genetic diversity within the genus Pyrus, offering both a visual and quantitative understanding of the evolutionary dynamics of these pears.

Phylogenetic Analysis of Rosaceae Plants
A phylogenetic tree of the complete chloroplast genomes of 28 Rosaceae species and Castanea mollissima was constructed using multiple sequence alignments and the maximum likelihood method (Figure 7).The results revealed that the tested Rosaceae species were divided into three clades: one comprising the genera Rubus, Rosa, and Fragaria, which received 100% support; another consisting of the genera Prunus, also with 100% support; and a third clade formed by the genera Malus, Crataegus, and Pyrus, again with 100% support.Within the genus Pyrus, the "Qixiadaxiangshui" pear clustered closely with the Dangshansuli, Wonwhang, and Yali, all supported with a 100% bootstrap value.The phylogenetic analysis highlights the genetic divergence and common ancestry within the Rosaceae family.The high bootstrap support values confirm the robustness of our phylogenetic tree.The close clustering of the "Qixiadaxiangshui" pear with Dangshansuli, Wonwhang, and Yali suggests these cultivars share a recent common ancestor, indicating conserved genetic traits advantageous for breeding programs.

Discussion
Pears are ranked as the third most significant fruit in China and are revered as the "forebear of all fruits" holding substantial economic value.In this study, we sequenced, assembled, and annotated the chloroplast genome of the "Qixiadaxiangshui" pear (Pyrus bretschneideri Rehd.1), employing bioinformatics analyses.Our findings reveal that the chloroplast genome structure of the "Qixiadaxiangshui" pear is a typical circular quadripartite structure, similar to most angiosperms.Previous studies have established that the size of chloroplast genomes in angiosperms generally ranges from 120 to 160 kb [18,19].In this work, the length of the "Qixiadaxiangshui" pear chloroplast genome is 159,885 bp, which falls within this typical range and is consistent with the sizes reported for other Pyrus species [7].The slight variations in chloroplast genome size are primarily attributed to the expansion and contraction of the IR boundary, a common phenomenon observed in angiosperm chloroplast genomes [20,21].Comparative analysis of structure boundaries among the "Qixiadaxiangshui" pear and other pear cultivars revealed minor variations in the IR/SC boundary positions, consistent with observations in previous studies on the Pyrus genus.Notably, expansions of the IR boundary were observed in the rps19 gene of the Bartlett pear and the ndhF gene of other Pyrus germplasm, similar to expansions found in other plant genera such as Ulmus [22], Manglietia [23], and Physalis [24].These findings suggest a stable chloroplast genome structure within the "Qixiadaxiangshui" pear, although further studies using biological replicates from multiple individuals are necessary to confirm these initial observations and uncover other structural variations.
Repetitive sequences and SSRs are widely distributed in chloroplast genomes and are closely related to genome rearrangement and recombination [25].These elements serve as important molecular markers and are extensively used in plant population genetics and phylogeny [26][27][28].In this study, we identified 209 SSRs in the chloroplast genome of the "Qixiadaxiangshui" pear, predominantly consisting of mononucleotide repeats composed of A/T bases.Most of these SSRs were located in the LSC region.These findings are consistent with SSR analyses of other Pyrus chloroplast genomes [4][5][6], highlighting the conserved nature of these sequences within the genus.The detected long repeats and SSRs provide valuable molecular marker information, which can be utilized to reveal

Discussion
Pears are ranked as the third most significant fruit in China and are revered as the "forebear of all fruits" holding substantial economic value.In this study, we sequenced, assembled, and annotated the chloroplast genome of the "Qixiadaxiangshui" pear (Pyrus bretschneideri Rehd.1), employing bioinformatics analyses.Our findings reveal that the chloroplast genome structure of the "Qixiadaxiangshui" pear is a typical circular quadripartite structure, similar to most angiosperms.Previous studies have established that the size of chloroplast genomes in angiosperms generally ranges from 120 to 160 kb [18,19].In this work, the length of the "Qixiadaxiangshui" pear chloroplast genome is 159,885 bp, which falls within this typical range and is consistent with the sizes reported for other Pyrus species [7].The slight variations in chloroplast genome size are primarily attributed to the expansion and contraction of the IR boundary, a common phenomenon observed in angiosperm chloroplast genomes [20,21].Comparative analysis of structure boundaries among the "Qixiadaxiangshui" pear and other pear cultivars revealed minor variations in the IR/SC boundary positions, consistent with observations in previous studies on the Pyrus genus.Notably, expansions of the IR boundary were observed in the rps19 gene of the Bartlett pear and the ndhF gene of other Pyrus germplasm, similar to expansions found in other plant genera such as Ulmus [22], Manglietia [23], and Physalis [24].These findings suggest a stable chloroplast genome structure within the "Qixiadaxiangshui" pear, although further studies using biological replicates from multiple individuals are necessary to confirm these initial observations and uncover other structural variations.
Repetitive sequences and SSRs are widely distributed in chloroplast genomes and are closely related to genome rearrangement and recombination [25].These elements serve as important molecular markers and are extensively used in plant population genetics and phylogeny [26][27][28].In this study, we identified 209 SSRs in the chloroplast genome of the "Qixiadaxiangshui" pear, predominantly consisting of mononucleotide repeats composed of A/T bases.Most of these SSRs were located in the LSC region.These findings are consistent with SSR analyses of other Pyrus chloroplast genomes [4][5][6], highlighting the conserved nature of these sequences within the genus.The detected long repeats and SSRs provide valuable molecular marker information, which can be utilized to reveal population-level polymorphisms and phylogenetic relationships of the Pyrus species in future research.
Mutation, genetic drift, and natural selection are key factors influencing codon preference [7].RSCU is a crucial evolutionary feature of the genome, significantly impacting gene expression, and can provide insights into the evolutionary processes of organisms [29,30].The codon analysis for the "Qixiadaxiangshui" pear revealed that 96.77% of the 31 codons with RSCU values greater than 1 ended with A/T(U) bases.This indicates a strong preference for A/T-ending codons in the chloroplast genes of the "Qixiadaxiangshui" pear, consistent with previous observations in the chloroplast genomes of other angiosperms [31,32].Knight et al. [33] developed a model suggesting that the GC base composition of the genome drives codon usage.However, some researchers argue that genome-wide codon bias is determined by non-random mutations and the selective pressure for protein translation efficiency [34,35].Therefore, it is speculated that the preference for A/T-ending codons in the chloroplast genome of the "Qixiadaxiangshui" pear is likely due to a combination of mutation biases favoring A/T bases and selection pressure.This finding enhances our understanding of the genetic structure of the "Qixiadaxiangshui" pear and provides a reference for future technical research on chloroplast genetic engineering aimed at improving desirable traits in pear cultivars.
The comparison of the entire chloroplast genome sequences between the "Qixiadaxiangshui" pear and other Pyrus cultivars demonstrated high similarity, with coding regions being more conserved than non-coding regions, a pattern consistent with other angiosperms [36,37].Our nucleotide diversity analysis identified several mutation hotspots with precise Pi values, such as ndhC-trnM-CAU and trnR-UCU-atpA.These regions are known to undergo faster nucleotide substitution rates at the species level, providing important references for the development of DNA barcodes [38].The identified mutation hotspots and the sequenced chloroplast genome in our study offer valuable molecular marker tools, which can provide a wealth of informative sites for the phylogeny and molecular identification of the genus Pyrus.This information not only enhances our understanding of the genetic diversity within Pyrus but also supports the development of efficient strategies for pear breeding and conservation.
To further clarify the phylogenetic position of the "Qixiadaxiangshui" pear within the Rosaceae family, a phylogenetic tree constructed based on the complete chloroplast genomes of the Rosaceae species divided the species into three branches, with the "Qixiadaxiangshui" pear clustering with plants of the genera Crataegus and Malus on the same branch.The close clustering of the "Qixiadaxiangshui" pear with Dangshansuli, Wonwhang, and Yali suggests these cultivars share a recent common ancestor, indicating conserved genetic traits advantageous for breeding programs.However, the current study is limited by the sample size and the scope of genomic regions analyzed, necessitating further research with larger datasets and additional genomic regions to validate and expand upon these findings.

Conclusions
In this study, the complete chloroplast genome of the "Qixiadaxiangshui" pear (Pyrus bretschneideri Rehd.1) was first reported, enriching the genetic resources and laying the foundation for exploring its genetic background and resource utilization.Specifically, its structure, gene composition, GC content, and codon bias were similar to those of typical angiosperms.The chloroplast genomes of the "Qixiadaxiangshui" pear shared common characteristics with other pear species, such as size, structure, gene composition, and low sequence variation, demonstrating that the chloroplast genome of Pyrus is relatively conservative.Additionally, several mutation hotspots, such as ndhC-trnM-CAU and trnR-UCU-atpA, were identified, which can be applied in molecular identification and phylogenetic studies of Pyrus.The phylogenetic results exhibited the closest genetic relationship between the "Qixiadaxiangshui" pear and other cultivars like Dangshansuli, Wonwhang, and Yali, suggesting a recent common ancestor.In summary, these results contribute to a better understanding of the phylogeny and genetic improvement of Pyrus germplasm resources.However, further research with larger datasets and additional genomic regions is necessary to validate and expand upon these findings.

Figure 1 .
Figure 1.Chloroplast genome map of "Qixiadaxiangshui" Pear.On the bottom left, the genes corresponding to the different functional groups are depicted with distinctive colors.The outer side of the circle represents positive coding genes, the inner side of the circle represents reverse coding genes, and the inner gray circle represents GC content.IRa/IRb, LSC, and SSC regions are shown by black lines surrounding the dark grey area.

Figure 1 .
Figure 1.Chloroplast genome map of "Qixiadaxiangshui" Pear.On the bottom left, the genes corresponding to the different functional groups are depicted with distinctive colors.The outer side of the circle represents positive coding genes, the inner side of the circle represents reverse coding genes, and the inner gray circle represents GC content.IRa/IRb, LSC, and SSC regions are shown by black lines surrounding the dark grey area.

Figure 2 .
Figure 2. Total RSCU values of amino acids in chloroplast genome of "Qixiadaxiangshui" pear.The numbers on the bar chart represent the proportion of amino acid codons in the total codons (%).

Figure 2 .
Figure 2. Total RSCU values of amino acids in chloroplast genome of "Qixiadaxiangshui" pear.The numbers on the bar chart represent the proportion of amino acid codons in the total codons (%).

Figure 3 .
Figure 3. Statistical chart of SSR quantity by type.The horizontal axis represents the number of repetitions, while the vertical axis represents the number of repetitions of the same type.

Figure 3 .
Figure 3. Statistical chart of SSR quantity by type.The horizontal axis represents the number of repetitions, while the vertical axis represents the number of repetitions of the same type.

Figure 4 .
Figure 4. Whole genome boundary map of chloroplast of "Qixiadaxiangshui" pear and nine pear plants.The numbers with the arrows represent the distance in bp between each junction.Note that

Figure 4 .
Figure 4. Whole genome boundary map of chloroplast of "Qixiadaxiangshui" pear and nine pear plants.The numbers with the arrows represent the distance in bp between each junction.Note that the distances are not drawn to scale and that the cp genomes follow the typical quadripartite structure with one LSC, one SSC, and two IR regions.

Horticulturae 2024 , 15 Figure 5 .
Figure 5.Comparison of chloroplast genes among ten pear genera.The gray scissors above indicate the direction of the gene.The dark blue region represents exons, the light blue region represents untranslated regions (UTRs), and the pink region represents non-coding sequences (CNS).The yaxis represents consistency from 50% to 100%.

Figure 5 .
Figure 5.Comparison of chloroplast genes among ten pear genera.The gray scissors above indicate the direction of the gene.The dark blue region represents exons, the light blue region represents untranslated regions (UTRs), and the pink region represents non-coding sequences (CNS).The y-axis represents consistency from 50% to 100%.

Figure 6 .
Figure 6.Chloroplast genome nucleotide diversity of ten cultivars in the Pyrus genus.

Figure 7 .
Figure 7. Phylogenetic analysis of chloroplast genomes based on 29 species from nine genera.

Figure 7 .
Figure 7. Phylogenetic analysis of chloroplast genomes based on 29 species from nine genera.

Table 4 .
Distribution analysis of SSRs.