Characterization and Comparative Analysis of Complete Chloroplast Genomes of Four Bromus (Poaceae, Bromeae) Species

Bromus (Poaceae Bromeae) is a forage grass with high adaptability and ecological and economic value. Here, we sequenced Bromus ciliatus, Bromus benekenii, Bromus riparius, and Bromus rubens chloroplast genomes and compared them with four previously described species. The genome sizes of Bromus species ranged from 136,934 bp (Bromus vulgaris) to 137,189 bp (Bromus ciliates, Bromus biebersteinii), with a typical quadripartite structure. The studied species had 129 genes, consisting of 83 protein-coding, 38 tRNA-coding, and 8 rRNA-coding genes. The highest GC content was found in the inverted repeat (IR) region (43.85–44.15%), followed by the large single-copy (LSC) region (36.25–36.65%) and the small single-copy (SSC) region (32.21–32.46%). There were 33 high-frequency codons, with those ending in A/U accounting for 90.91%. A total of 350 simple sequence repeats (SSRs) were identified, with single-nucleotide repeats being the most common (61.43%). A total of 228 forward and 141 palindromic repeats were identified. No reverse or complementary repeats were detected. The sequence identities of all sequences were very similar, especially with respect to the protein-coding and inverted repeat regions. Seven highly variable regions were detected, which could be used for molecular marker development. The constructed phylogenetic tree indicates that Bromus is a monophyletic taxon closely related to Triticum. This comparative analysis of the chloroplast genome of Bromus provides a scientific basis for species identification and phylogenetic studies.


Introduction
Bromus (Poaceae Bromeae) is an important forage grass with annual, biennial, and perennial growth [1].It is mainly found in Europe, Asia, America, and Africa, and is highly adaptable to drought, salinity, and cold [2].The majority of Bromus species are utilized as cereal crops, pasture grass, or silage, and some have important medicinal value [2][3][4].This genus contains approximately 250 species [5], and the probability of polyploidy in these species is high, ranging from 2× to 12× [2].Owing to its complex classification and wide geographical scope, no comprehensive classification exists of all Bromus species in the world; however, a number of regional descriptions and identification tables for Bromus plants have been published in different regions [6][7][8].The criteria for Bromus classification remain debatable.Bromus is divided into six groups, five genera, and seven subgenera based on morphology, serology, and cytogenetics; however, reliance on shared morphological features may lead to taxonomic confusion because parallel or convergent evolution is widespread in the family Poaceae [9][10][11].Pillay [12,13] studied the variation in the chloroplast (cp) genome enzyme cleavage sites, constructed a physical map of the cp genome and a simple phylogenetic tree, and confirmed that the cp genome is a useful tool in the study of Bromus phylogeny.A study by Saarela [14] revealed discrepancies between the results of plastid and ribosomal trees, indicating the need for additional data to define Bromus genetic relationships.Previous studies have formed the foundation for identifying and subdividing Bromus.However, the intricate evolutionary relationships remain uncertain.
Chloroplasts are semi-autonomous hereditary organelles formed by eukaryotic cells that phagocytose cyanobacteria [15,16].They are important sites for energy conversion and photosynthesis in plants; are commonly found in algae, land plants, and protists; and synthesize proteins, starches, and fatty acids [17].In most plants, the cp genome is double-stranded and has a typical quadripartite structure, including a large singlecopy (LSC) region, small single-copy (SSC) region, and two copies of inverted repeat (IR) regions [18].The sizes of plant cp genomes range from 107 to 218 kb, with variations primarily caused by contraction and expansion of the IR regions [19].The cp genome is characterized by maternal inheritance, a moderate evolutionary rate [20], relatively stable genome content [21], and a low recombination rate [22,23].In nature, there are a multitude of plant species, and identification of closely related species has always been a challenge [24].With the advancement of sequencing technology, the cp genome has become a widely used molecular marker in systematic studies of diversity, phylogeny, and taxonomic issues, and it has become one of the most useful tools in molecular systematics [25].Cp DNA sequence fragments, such as matK, rbcL, trnH, psbA, rpoC1, rpoB, accD, ycf5, and ndhJ, are commonly used for plant DNA barcoding [18].Pillay [13] analyzed the enzyme cleavage sites of Bromus using phylogenetic information and obtained a simple phylogenetic chart, which provided a preliminary analysis of the genetic relationships of Bromus.Saarela and Nasiri [1,14] confirmed the monophyletic nature of Bromus.However, the ribosomal and cp data were inconsistent for the Bromus major lineage.Yoshihiro et al. [26] performed a comparative analysis of rice, maize, and wheat cp genomes in Poaceae, focusing mainly on non-coding regions, and constructed a grass phylogenetic tree.To date, more than 4671 species of Poaceae plants have been sequenced, including nine species of Bromus plants.Nevertheless, the number of publicly available whole-genome Bromus sequences remains relatively limited, despite the crucial role of the cp genome in classification.
Previous studies have analyzed the classification, genetic relationships, and phylogeny of Bromus based on morphology and molecules; however, few analyses have been conducted on the basis of the cp genome, and no comprehensive and systematic studies of Bromus have been conducted.In this investigation, we conducted sequencing of the cp genomes of four Bromus species and conducted analysis in conjunction with previously published data from four additional sequenced species (B.inermis, B. biebersteinii, B. vulgaris, and B. diandrus), with the goal of gaining insights into the phylogenetic relationships between these taxa.Furthermore, we examined boundary stretching, nucleotide polymorphisms (pi), simple repeats (SSRs), codon usage bias, and the phylogeny of eight Bromus plants.The aim of this study was to identify Bromus species and understand the phylogenetic relationships between these and other Bromus species.

Identification of Repeat Sequences and SSR Sequences
Different repetition types with a minimum repeat length of 30 and a Hamming distance of 3 [31], including complementary, palindromic, forward, and reverse types, were identified by REPuter tools.MISA software v2.1 was used for the SSRs detected [32], with parameter configurations delineating the unit size (in nucleotides) and minimum repeat: 1-10, 2-6, 3-4, 4-3, 5-3, and 6-3.Finally, the minimum interspace between two SSRs was set as 100 base pairs.

Comparative Genome Analysis
Variations among all Bromus cp genomes were compared using mVISTA [33], with B. inermis as the reference sequence.Additionally, IR-Scope was used for the comparison and visual representation of the connections and boundaries within the IR regions [34].The cp genomes of all eight Bromus species were aligned using MAFFT v7.313 [35] with default settings.Nucleotide diversity (Pi) rates, indicating sequence divergence among Bromus species, were then calculated using DnaSP v6.12 [36].

Phylogenetic Analysis
For the phylogenetic analysis, we employed the entire cp genomes and common protein-coding genes of the four recently sequenced Bromus species, along with sequences from four Bromus species and two outgroup species (Oryza sativa (NC_008155), Brachypodium distachyon (NC_011032)) previously documented in the NCBI database (Table S1), as well as 27 species from 10 genera in Poaceae.The sequences were aligned by MAFFT v7.313 with default parameters [35] and used for constructing trees via both the maximum likelihood (ML) and Bayesian inference (BI) methods [37].The shared protein-coding genes were analyzed using the GTR + F + I + G4 substitution model for both the maximum likelihood (ML) and Bayesian inference (BI) methods.The ML analysis was conducted with RAxML v8.2.11 based on a non-parametric bootstrap approach with 1000 replicates [38], while the Bayesian inference analysis was conducted with MrBayes v3.2.6 software [39].

Genomic Features of Bromus
The cp genome of the genus demonstrates the typical quadripartite structure (Figure 1 S2).The GC contents of the newly sequenced cp genomes vary between 38.31% and 38.38%, encompassing the GC content of all recognized Bromus species (Table S2).The highest GC content was found in the IR region (43.85-44.15%),followed by the LSC region (36.25-36.65%)and the SSC region (32.21-32.46%).In all, 64 different codons, which encode for 21 amino acids, were detected in all Bromus sequences (Figure 2).
The total number of codons in the new sequences was found to be 19,696-19,872.AUU was the most abundant (821-838) and UGA was the least (16-25) (Figure 2).Within the Bromus cp genome, the relative synonymous codon usage (RSCU) values ranged from 0.09 to 2.09 for all codons, with 33 codons exhibiting a high frequency, possessing RSCU values exceeding 1.00.Of the codons exhibiting RSCU values greater than 1, 90.91% concluded with A/U bases, while 9.09% concluded with C/G bases.Codon UUA, which encodes phenylalanine (Phe), had the highest relative probability of use, with an RSCU of 2.09.The RSCU values of the remaining 32 codons ranged from 1.01 to 1.91.This analysis revealed a preference for codons ending in A/U in the Bromus cp genome.

Repeat Analysis
In total, 350 SSRs were identified across eight genomes of members of the Bromus genus (Figure 3).The majority of the SSRs consisted of A/T pairs rather than C/G pairs.The quantity of SSRs in Bromus varied between 41 and 49.Single-nucleotide repeats constituted the most prevalent type of repetition (61.43%), succeeded by tetranucleotide repeats (25.14%) and trinucleotide repeats (6.9%).Notably, the Bromus cp genome lacks dinucleotide repeats.Among the studied species, only B. ciliatus, B. riparius, B. rubens, and B. inermis had a single hexanucleotide repeat sequence, whereas B. diandrus had two hexanucleotide repeat sequences.The AATTAG/AATTCT repeats were detected in B. ciliatus and B. rubens, whereas the AATCCT/AGGATT repeats were detected in both B. riparius and B. inermis.In addition, B. diandrus contained both the AAAAAT/ATTTTTT and ACATCT/AGATGT repeats (Table S3).peats (25.14%) and trinucleotide repeats (6.9%).Notably, the Bromus cp genome lacks dinucleotide repeats.Among the studied species, only B. ciliatus, B. riparius, B. rubens, and B. inermis had a single hexanucleotide repeat sequence, whereas B. diandrus had two hexanucleotide repeat sequences.The AATTAG/AATTCT repeats were detected in B. ciliatus and B. rubens, whereas the AATCCT/AGGATT repeats were detected in both B. riparius and B. inermis.In addition, B. diandrus contained both the AAAAAT/ATTTTTT and ACATCT/AGATGT repeats (Table S3).Long repetitive sequences are repetitive sequences ≥30 bp in length, which are favorable for genome rearrangement and increase population genetic diversity.A total of 369 repetitive sequences were detected in Bromus, including 228 forward (F) and 141 palindromic (P) repetitive sequences; no reverse (R) or complementary (C) repetitive sequences were detected (Figure 4).The lowest number of repeats was found in B. biebersteinii (43), and the highest was found in B. diandrus (49).B. rubens and B. diandrus had a higher number of repeats (at 41-45 bp) than those in the other six species.B. biebersteinii, B. vulgaris, and B. diandrus had a higher number of repetitive sequences (46-50 bp) that were repeated five times, whereas the other species showed repeats of only three times.Long repetitive sequences are repetitive sequences ≥30 bp in length, which are favorable for genome rearrangement and increase population genetic diversity.A total of 369 repetitive sequences were detected in Bromus, including 228 forward (F) and 141 palindromic (P) repetitive sequences; no reverse (R) or complementary (C) repetitive sequences were detected (Figure 4).The lowest number of repeats was found in B. biebersteinii (43), and the highest was found in B. diandrus (49).B. rubens and B. diandrus had a higher number of repeats (at 41-45 bp) than those in the other six species.B. biebersteinii, B. vulgaris, and B. diandrus had a higher number of repetitive sequences (46-50 bp) that were repeated five times, whereas the other species showed repeats of only three times.

Comparative Analysis of the Bromus Chloroplast Genome
Regions of significant variability within cp genomes serve as valuable tools for discerning closely related species and conducting molecular evolution investigations [40].Using B. inermis as a benchmark, mVISTA was employed to analyze the cp genomes of seven Bromus species, incorporating both newly sequenced genomes and those retrieved from the NCBI repository, in order to explore sequence diversity (Figure 5).The cp genome is homologous within Bromus, and is conserved across species with high covariance;

Comparative Analysis of the Bromus Chloroplast Genome
Regions of significant variability within cp genomes serve as valuable tools for discerning closely related species and conducting molecular evolution investigations [40].Using B. inermis as a benchmark, mVISTA was employed to analyze the cp genomes of seven Bromus species, incorporating both newly sequenced genomes and those retrieved from the NCBI repository, in order to explore sequence diversity (Figure 5).The cp genome is homologous within Bromus, and is conserved across species with high covariance; however, there are some differences.The mutation rate varied in different regions, which reflects the evolution of Bromus cps.There were differences in cp genome lengths between species, with B. vulgaris being the shortest (136,934 bp) and B. ciliatus and B. biebersteinii being slightly longer (137,189 bp).Overall, the cp genomes of the other seven Bromus species displayed notable structural similarity and maintained a high level of conservation regarding both size and gene sequences.However, the number of some conserved non-coding region (CNS) variants, including rpoC2-rps2, ndhC-trnV-UAC, psbM-petN, trnF-GAA-ndhJ, psbE-petG, and rpl16, was higher than those in other regions.In the non-coding region between rpoC2 and rps2, B.  The intergenic spacer (IR) region of the cp genome is considered highly conserved; however, its boundary region can contract or expand, causing the cp genome to change in length [41].Therefore, we investigated the structural features of the LSC, SSC, and IR regions in the cp genomes of the eight Bromus species.Specifically, we focused on the LSC/IRb junction (JLB), positioned between the rpl22 gene within the LSC region and the rps19 gene within the IRb region (Figure 6).It is noteworthy that the rps19 gene spans from the IRb into the LSC, typically ranging from 33 to 39 bp in length.The boundary known as JLA, which separates the LSC and IRa regions, is positioned between the psbA gene in the LSC and the rps19 gene in the IRa.The rps19 gene crosses from the IRa into the LSC, covering a distance of 34-40 bp.Furthermore, the ndhF gene is positioned within a range of 26 to 85 bp from the boundary of IRb and SSC (JSB).In all Bromus species, the ndhH gene extends across the junction (JSA) between the SSC and IRa regions, with lengths varying from 861 to 975 bp in the SSC region and 207 to 321 bp in the IRa region.The variations in the lengths of these regions primarily manifest in the positions of rps19, rpl22, ndhF, ndhH, and psbA, indicating dynamic expansions and contractions in the inverted repeat regions.The eight Bromus species were identical to the others.The most conserved region was the IR region, with only a few diversity hotspots.The bulk of the areas exhibiting high genetic diversity were situated within the LSC and SSC regions (Figure 7).The peak Pi value within the notably variable region reached 0.01994, while the nadir was noted at 0.01006.Twenty-two highly mutated regions with Pi values greater than 0.01 were detected, namely, trnC-GCA, trnT-GGU, rbcL-psal, trnL-UGA-ccsA, rbcL-psal, psbA-matK, and trnD-GUC, trnT-UGU-trnL-UAA, trnL-UAA-trnF-GAA, trnL-UAA-trnF-GAA, rpoC2-rps2, rbcL-psal, trnD-GUC-psbM, trnC-GCA, petN-trnC-GCA, trnD-GUC-psbM, trnT-UGU-trnL-UAA, rpoC2-rps2, rpoC2-rps2, and three ndhF.These regions exhibit high levels of mutation, making them ideal for phylogenetic analyses of Bromus species and the development of molecular markers for plant identification.

Phylogenetic Analysis
For clarification of the affinities and phylogenetic positions of Bromus during the evolutionary process (Figure 8), we downloaded the cp genome sequences of 27 species from 10 genera from the NCBI database.A phylogenetic tree was constructed using O. sativa and B. distachyon as outgroups.The topology of the tree revealed that the 8 Bromus species clustered in different branches from the 21 species of the other seven Poaceae.In the Bromus branch, B. catharticus first diverged to form a branch independent of the other Bromus species, followed by B. diandrus and B. vulgaris.The remaining six species were shown to be closely related to each other.Among these, B. inermis and B. riparius, as well as B. biebersteinii and B. benekenii, clustered together and were the most closely related.

Phylogenetic Analysis
For clarification of the affinities and phylogenetic positions of Bromus during the evolutionary process (Figure 8), we downloaded the cp genome sequences of 27 species from 10 genera from the NCBI database.A phylogenetic tree was constructed using O. sativa and B. distachyon as outgroups.The topology of the tree revealed that the 8 Bromus species clustered in different branches from the 21 species of the other seven Poaceae.In the Bromus branch, B. catharticus first diverged to form a branch independent of the other Bromus species, followed by B. diandrus and B. vulgaris.The remaining six species were shown to be closely related to each other.Among these, B. inermis and B. riparius, as well as B. biebersteinii and B. benekenii, clustered together and were the most closely related.

Discussion
In this investigation, we sequenced, annotated, compared, and analyzed the complete cp genomes of B. ciliates, B. benekenii, B. riparius, and B. rubens, alongside four previously reported Bromus cp genomes.The cp genomes of all eight Bromus species had a typical quadripartite structure consisting of LSC, SSC, IRa, and IRb, with full genome lengths ranging from 137,038 bp (B.benekenii) to 137,189 bp (B.ciliatus) (Table S3 and Figure 1),

Discussion
In this investigation, we sequenced, annotated, compared, and analyzed the complete cp genomes of B. ciliates, B. benekenii, B. riparius, and B. rubens, alongside four previously reported Bromus cp genomes.The cp genomes of all eight Bromus species had a typical quadripartite structure consisting of LSC, SSC, IRa, and IRb, with full genome lengths ranging from 137,038 bp (B.benekenii) to 137,189 bp (B.ciliatus) (Table S3 and Figure 1), which is in agreement with published data on Bromus species [12,42,43].Compared to cattail (Typha orientalis presl) and tobacco (Nicoticular tabacum L.), the genes accD, ycf1, and ycf2 have undergone progressive degradation and eventual loss in the Bromus cp genome [44].This phenomenon has also occurred in other Poaceae species, such as Cynodon dactylon [45,46].A possible reason for the elimination of accD, ycf1, and ycf2 may be their lack of a significant advantage for survival or reproduction, followed by natural selection.Alternatively, this may be attributed to shifted-code mutations and gene deletions caused by insertions and deletions of non-triplet bases [44].We detected a four-copy tRNA gene (trnM-CAU) in the Bromus cp genome.Sutton [47] found that an increase in boron toxicity tolerance in barley was due to an increase in the copy number of boron transporter proteins.Würschum [48] identified the role of copy number variation in Ppd-B1 and Vrn-A1 in global wheat adaptation.Copy number variations may lead to the generation of new gene functions, changes in gene expression levels, and reorganization of gene interaction networks, which may have an impact on the structure, function, and adaptation of polyploid plants, which played key roles in the evolution, adaptation, and gene function of many species [49].RSCU values >1 indicate a preference for using the codon, whereas RSCU values <1 indicate the opposite [50].Codon preference exists in most plant genomes, and natural selection and mutation pressures are the main factors that affect this preference [51].In the present study, we identified 264 RSCUs value > 1 and 248 RSCU values < 1 in the cp genomes of Bromus.Among the codons with RSCU values greater than >1, 90.91% of them ended with base A/U and 9.09% with C/G.This codon preference for the use of A/U endings is similar to that of found in other Poaceae species [52].
SSRs have been widely used as important genetic markers in genetic, polymorphic, and evolutionary studies on plant populations [53,54].In total, 350 SSR sequences were detected in the eight Bromus cp genomes (Figure 3).These sequences demonstrated the highest A/T content at 58.6% and the greatest number of repeats, aligning with findings reported in other plant studies [55][56][57].The repeat units AAAAAT/ATTTTTT and ACATCT/AGATGT, unique to B. diandrus, and the repeat unit AAAAT/ATTTTT, unique to B. ciliatus, discovered in this study can serve as molecular markers to differentiate between the two species.These markers can enhance our understanding of the population structure and genetic diversity of these species, and they will be crucial for molecular breeding and genetic engineering [58].
The expansion and contraction of IR regions along with single-copy boundaries are pivotal in plant cp genome evolution [59].Comparative sequence analysis coupled with IR boundary examination revealed highly consistent gene arrangement lengths and orders across the cp genomes of these eight plant species.Nonetheless, discrepancies arose in boundary gene types and positions, with the most significant variations occurring in the SSC/IR boundaries.These differential sequences provide new potential resources for identifying and studying Bromus species.Additionally, comparative sequence analysis revealed that the CNS region had greater variation than other regions, which is consistent with findings in other Bromus species [60,61].The smallest reverse IR region was identified in B. benekenii (19,410 bp), and the largest reverse IR region was found in B. rubens (21,706 bp).This difference could be attributed to the expansion and contraction of the IR region, along with the impact of the single-copy spacer region, resulting in variations in the overall length of the cp genome [62].The ndhH gene was also found at both ends of the JSA boundary, consistent with previously reported findings in other Poaceae species [46,[63][64][65].The different locations of this gene at the JSA boundary may be attributed to the intramolecular recombination during early evolution [66].
The findings of our phylogenetic analyses supplement insights from earlier investigations, indicating the potential utility of the cp genome in elucidating inter-species relationships within the genus.In most studies, Bromeae and Triticeae are sister groups; however, studies based on the whole Bromeae plastome are nested in Triticeae, making Triticeae paraphyletic [67,68].The results of this study support Bromus and Triticum as sister groups.B. inermis, B. benekenii, and B. riparius, which all belong to Bromopsis, constitute several independent lineages and are not clustered into a single clade, which is in agreement with the results of Saarela and Pillay [13,14].Nasiri [1] found that Bromeae sect.Genea is not monophyletic in plastid trees.In the present study, B. rubens and B. diandrus, both belonging to B. sect.Genea, were not clustered into a single lineage but were sister groups.The cp DNA evolutionary tree constructed by Pillay [13] showed that Ceratochloa is uniquely characterized and separated from other genera, including Bromus.In summary, B. catharticus belongs to Ceratochloa, forming a separate clade.This may be because it emerged in the early Pliocene, earlier than the origin of other Bromus subgenera.

Conclusions
In the examination of four recently sequenced and four previously documented Bromus cp genomes, it was observed that these genomes exhibit a high level of conservation and share a similar overall structure across the genus.Moreover, our investigation highlighted the prevalence of A/T bases in SSR sequences and revealed distinctive repeat units.High similarity in the cp genome sequences was observed among Bromus species, particularly in the coding and IR regions.Seven highly variable CNS regions were pinpointed as potential novel genetic markers for DNA barcoding and molecular phylogenetic analyses.Through a comparative examination of IR boundaries, unique genomic placements for rpl22, rps19, ndhH, and psbA were identified, attributed to IR contraction and expansion events in Bromus species.Notably, the establishment of Bromus as a monophyletic taxon closely related to Triticum was highlighted.These discoveries offer valuable insights into both the identification and phylogenetic classification of Bromus species.

Supplementary Materials:
The following supporting information can be downloaded at https: //www.mdpi.com/article/10.3390/genes15060815/s1,Table S1: GenBank accession numbers of chloroplast genomes; Table S2: Summary of chloroplast genome characteristics in Bromus species; Table S3: Numbers of identified SSRs motifs in the 8 Bromus chloroplast genomes.

Figure 1 .
Figure 1.Circular diagram of the Bromus chloroplast genome.Genes depicted within the circumference are transcribed in a clockwise direction, while those situated outside are transcribed counterclockwise.Genes affiliated with distinct functional categories are colorized and labeled accordingly in the accompanying key (LSC: large single-copy, SSC: small single-copy, IR: inverted repeat).

Figure 1 .
Figure 1.Circular diagram of the Bromus chloroplast genome.Genes depicted within the circumference are transcribed in a clockwise direction, while those situated outside are transcribed counterclockwise.Genes affiliated with distinct functional categories are colorized and labeled accordingly in the accompanying key (LSC: large single-copy, SSC: small single-copy, IR: inverted repeat).

Figure 3 .
Figure 3. Analysis of diverse SSR variants across eight Bromus chloroplast genomes.

Figure 3 .
Figure 3. Analysis of diverse SSR variants across eight Bromus chloroplast genomes.

Figure 4 .
Figure 4. Tandem repeats sequences in the eight Bromus chloroplast genomes.(a) Total number of repeat types, F: forward repetitive sequences, P: palindromic repetitive sequences.(b) Number of repeats by length.

Figure 4 .
Figure 4. Tandem repeats sequences in the eight Bromus chloroplast genomes.(a) Total number of repeat types, F: forward repetitive sequences, P: palindromic repetitive sequences.(b) Number of repeats by length.

Figure 5 .
Figure 5. Eight chloroplast genomes were aligned globally using B. inermis as a reference.Each coordinate represents a region of the chloroplast genome.The vertical axis represents the percentage of the aligned region sequence similarity.

Figure 5 .
Figure 5. Eight chloroplast genomes were aligned globally using B. inermis as a reference.Each coordinate represents a region of the chloroplast genome.The vertical axis represents the percentage of the aligned region sequence similarity.

Figure 5 .
Figure 5. Eight chloroplast genomes were aligned globally using B. inermis as a reference.Each coordinate represents a region of the chloroplast genome.The vertical axis represents the percentage of the aligned region sequence similarity.

Figure 6 .
Figure 6.Comparison between LSC, IR, and SSC junction boundaries in the cp genomes of eight Bromus species.JLB, JSB, JSA, and JLA are representative of LSC/IRb, SSC/IRa, LSC/IRa, and LSC/IRa, respectively.

Figure 6 .
Figure 6.Comparison between LSC, IR, and SSC junction boundaries in the cp genomes of eight Bromus species.JLB, JSB, JSA, and JLA are representative of LSC/IRb, SSC/IRa, LSC/IRa, and LSC/IRa, respectively.

Figure 7 .
Figure 7.Nucleotide diversity (Pi) values of eight Bromus species shown as a line chart.On the Xaxis, locations of cp genomes are listed.The values of Pi are shown on the Y-axis.

Figure 7 .
Figure 7.Nucleotide diversity (Pi) values of eight Bromus species shown as a line chart.On the X-axis, locations of cp genomes are listed.The values of Pi are shown on the Y-axis.

Genes 2024 , 14 Figure 8 .
Figure 8. Phylogenetic tree of Bromus with 21 other representative Poaceae Barnhart species.B. distachyon and O. sativa were selected as the outgroups.The maximum likelihood (ML) and Bayesian inference (BI) methods were used to construct the tree based on shared protein-coding genes.Maximum likelihood bootstrap support values and Bayesian posterior probabilities are shown for each node.

Figure 8 .
Figure 8. Phylogenetic tree of Bromus with 21 other representative Poaceae Barnhart species.B. distachyon and O. sativa were selected as the outgroups.The maximum likelihood (ML) and Bayesian inference (BI) methods were used to construct the tree based on shared protein-coding genes.Maximum likelihood bootstrap support values and Bayesian posterior probabilities are shown for each node.

Table 1 .
Gene composition in the Bromus chloroplast genome.

Table 1 .
Gene composition in the Bromus chloroplast genome.