Bunchgrass mitochondrial genome and uses thereof

Abstract

The application provides a buffalo grass mitochondrial genome and use thereof, relates to the technical field of the buffalo grass mitochondrial genome; the nucleotide sequence of the buffalo grass mitochondrial genome is shown in the combined sequence of SEQ.ID.NO.1, SEQ.ID.NO.2, SEQ.ID.NO.3, SEQ.ID.NO.4 and SEQ.ID.NO.5; the complete sequence of the buffalo grass mitochondrial genome is disclosed for the first time; the buffalo grass mitochondrial genome is applied to the phylogenetic research of the poaceae family; the composition characteristics of the genome repeat sequence and the evolutionary characteristics of the migration of the chloroplast gene fragment to the mitochondrion are clarified; and important reference data for revealing the structural variation and the evolution law of the mitochondrial genome of the poaceae family are provided.

Description

Technical Field

[0001] This invention relates to the fields of molecular biology and plant genetics, specifically to the mitochondrial genome of *Buffalo grass* and its uses. Background Technology

[0002] Buffalo grass (Bouteloua dactyloides (Nutt.) Engelm., abbreviated as B. dactyloides), also known as Buchloe dactyloides (Nutt.) Engelm., and commonly called Buffalo Grass, is a perennial herbaceous plant belonging to the Poaceae family. It is naturally distributed in the semi-arid temperate and subtropical regions of central North America. Buffalo grass exhibits extremely strong environmental adaptability. With its soft-textured leaves, low-growing habit, well-developed stolons, and low maintenance costs, and especially its outstanding drought, salt, and poor soil tolerance, it is now widely used in turf establishment, landscaping, and soil stabilization and slope protection projects, making it a resilient grass species with great development potential.

[0003] Current research on buffalo grass, both domestically and internationally, primarily focuses on variety selection, phenotypic identification, seed germination characteristics, germplasm genetic diversity, chromosome ploidy analysis, abiotic stress response, and clonal division growth patterns. Research on molecular genetics is relatively limited, mainly involving genetic diversity analysis using ISSR, RAPD molecular markers, and isoenzyme techniques; germplasm resource evaluation and early sex identification; and optimization and screening of genomic DNA extraction methods. Currently, issues such as insufficient utilization of germplasm resources, limited development of molecular markers, and a lack of diverse research methods hinder the progress of genetic improvement and the discovery of superior genes.

[0004] Therefore, providing the mitochondrial genome of *Buffalo grass* and its applications is an urgent problem to be solved. Summary of the Invention

[0005] Specifically addressing the shortcomings of existing technologies, this invention provides the mitochondrial genome of *Buffalo grass* and its applications. The nucleotide sequence of the *Buffalo grass* mitochondrial genome is shown in SEQ.ID.NO.1. This invention discloses for the first time the complete sequence of the *Buffalo grass* mitochondrial genome and applies it to the phylogenetic study of Poaceae, elucidating the compositional characteristics of its genome repetitive sequences and the evolutionary features of chloroplast gene fragment migration to mitochondria. This provides important reference data for revealing the structural variations and evolutionary patterns of mitochondrial genomes in Poaceae plants.

[0006] The objective of this invention is achieved through the following technical solution: In a first aspect, the present invention provides a bison grass mitochondrial genome, the nucleotide sequence of which is shown as the combined sequence of SEQ.ID.NO.1, SEQ.ID.NO.2, SEQ.ID.NO.3, SEQ.ID.NO.4 and SEQ.ID.NO.5.

[0007] In some specific embodiments of the present invention, the bison grass mitochondrial genome includes tRNA genes, rRNA genes, and protein-coding genes; The protein-coding genes include ATP synthase, NADH dehydrogenase, cytochrome c biosynthetic protein, cytochrome c oxidase, protein transport subunit, maturation enzyme, cytochrome b, ribosomal large subunit protein, and ribosomal small subunit protein.

[0008] In a second aspect, the present invention provides a method for assembling and annotating the mitochondrial genome of *Buffalo grass* as described in the first aspect, the method comprising the following steps: S1. Collect samples, extract the mitochondrial genome of *Buffalo grass*, and sequence it to obtain the raw data of the *Buffalo grass* mitochondrial genome. S2. The raw data of the bison grass mitochondrial genome are screened and assembled to obtain the assembled bison grass mitochondrial genome; then the assembled bison grass mitochondrial genome sequence is annotated and a bison grass mitochondrial genome map is drawn.

[0009] In some specific embodiments of the present invention, the sequencing method in step S1 is as follows: for the extracted bison grass mitochondrial genome, high-throughput sequencing is performed using Illumina NovaSeq 6000 sequencing technology and nanopore single-molecule sequencing technology to obtain the raw data of the bison grass mitochondrial genome.

[0010] In some specific embodiments of the present invention, the Illumina NovaSeq 6000 sequencing technology is used to sequence short-read libraries, and the nanopore single-molecule sequencing technology is used to sequence long-read libraries.

[0011] In some specific embodiments of the present invention, the specific method for screening and assembling the raw data of the bison grass mitochondrial genome in step S2 is as follows: using the mitochondrial genomes of closely related species goosegrass and bermudagrass as reference sequences, mitochondrial-related contigs are screened to obtain a bison grass mitochondrial genome draft. Then, the short-read long-read library and the long-read long-read library of the bison grass mitochondrial genome draft are aligned to the mitochondrial contigs and filtered. After mixing and assembling, the assembled bison grass mitochondrial genome is obtained.

[0012] In some specific embodiments of the present invention, the annotation method in step S2 is as follows: the entire mitochondrial genome of *Pterocarya stenoptera* is annotated using the online software PMGA; for tRNA genes, the software tRNAscan-SE is used for annotation; and for rRNA genes, the software BLASTN is used for annotation.

[0013] Thirdly, the present invention provides the use of the bison grass mitochondrial genome as described in the first aspect in constructing a phylogenetic tree of the Poaceae family.

[0014] In some specific embodiments of the present invention, the method for constructing a phylogenetic tree of the Poaceae family is as follows: the mitochondrial genomes of 40 closely related groups of buffalo grass are downloaded from a gene database, common genes among the species are extracted using the software PhyloSuite, multiple alignments of the common genes among the species are performed using the MAFFT software, and then a phylogenetic tree is constructed using the IQ-TREE software.

[0015] Fourthly, the present invention provides the use of the bison grass mitochondrial genome in the identification of bison grass germplasm as described in the first aspect.

[0016] The beneficial effects achieved by this invention are as follows: This invention discloses for the first time the complete mitochondrial genome sequence of *Buffalo grass*. The *Buffalo grass* mitochondrial genome has a multi-branched structure with a total length of 799,544 bp and a GC content of 44.71%. A total of 57 genes were annotated in the *Buffalo grass* mitochondrial genome, including 23 tRNA genes, 3 rRNA genes, and 31 protein-coding genes. Among the protein-coding genes, a total of 435 RNA editing sites were detected. The *Buffalo grass* mitochondrial genome contains a large number of repetitive sequences and chloroplast migration fragments, including 188 SSR sites, 66 tandem repeats, and 1965 pairs of scattered repeat sequences, with short repeats being the predominant type. At the same time, 31 chloroplast homologous fragments were identified, with a total length of 20,134 bp, accounting for 2.52% of the total length of the mitochondrial genome.

[0017] This invention applies the mitochondrial genome of *Buffalo grass* to the phylogenetic study of Poaceae, elucidating the compositional characteristics of its genome repetitive sequences and the evolutionary features of chloroplast gene fragment migration to mitochondria. It provides important reference data for revealing the structural variation and evolutionary laws of mitochondrial genomes in Poaceae plants, and also lays an important molecular theoretical foundation for the identification of *Buffalo grass* germplasm resources, evaluation of genetic diversity, discovery of stress-resistant genes, and the formulation of improved varieties and ecological protection strategies. Attached Figure Description

[0018] To more clearly illustrate the technical solution of this application, the drawings used in the embodiments will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 A schematic diagram of the original results of the assembly of the bison grass mitochondrial genome; Figure 2A Map of the contig1 mitochondrial genome of *Buffalo grass*; Figure 2B A map of the contig2 mitochondrial genome of *Buffalo grass*; Figure 3 A diagram illustrating the codon bias analysis of the mitochondrial genome of *Buffalo grass*. Figure 4 A graph showing the predicted number of RNA editing sites for each PCG in the bison grass mitochondrial genome; Figure 5 A distribution map of amino acid substitution types resulting from RNA editing in the mitochondrial genome of *Buffalo grass*. Figure 6 A bar chart of microsatellite repeat sequences in the mitochondrial genome of *Buffalo grass*; Figure 7 A diagram showing the migration of the mitochondrial genome sequence of *Buffalo grass*. Figure 8 A diagram showing collinearity analysis of the mitochondrial genome of *Buffalo grass*. Figure 9 Phylogenetic analysis diagram of the mitochondrial genome of *Buffalo grass*. Detailed Implementation

[0020] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0021] The mitochondrial genome is the cytoplasmic genome within higher plant cells, independent of the nuclear genome, and possesses semi-autonomous inheritance characteristics. Plant mitochondrial genomes are typically larger and structurally less stable than chloroplast genomes, and are prone to frequent rearrangements mediated by repetitive sequences. Differences in mitochondrial genomes among different plants are mainly reflected in their total length, gene composition, gene arrangement, GC content, and molecular conformation.

[0022] Example 1: Extraction and sequencing of the mitochondrial genome of *Buffalo grass*

[0023] The experimental material was buffalo grass leaves. On December 5, 2025, fresh green buffalo grass leaves were picked in Greenhouse No. 8 of the Shandong Provincial Forestry and Grassland Germplasm Resources Center in Jinan City (36.627497N, 117.166424E, altitude 209.9 m) and briefly stored in liquid nitrogen. After returning to the laboratory, they were stored in a -80℃ freezer.

[0024] Mitochondrial genomic DNA from *Buffalo grass* was isolated and purified using the TIANGEN genomic DNA extraction kit. Library construction and high-throughput sequencing were then performed using the Illumina NovaSeq 6000 sequencing platform and Oxford Nanopore Technologies (ONT) nanopore single-molecule sequencing technology, respectively. Raw sequencing data, including short-reads and long-reads, were obtained, and the quality of the raw data was systematically tested and evaluated.

[0025] Example 2: Assembly and annotation of the mitochondrial genome of *Buffalo grass*

[0026] First, based on long-read data, GFA format results were obtained by assembling data using Flye (v2.9.2-b1786) with default parameters. After constructing the contigs library, the mitochondrial genomes of closely related species *Eleusine indica* (NC_040989.1) and *Cynodon dactylon* (PP493251.1) were used as query sequences. The BLASTn program (-evalue1e-5 -outfmt 6 -max_hsps 10 -word_size 7 -task) was then used to generate the results. Mitochondrial-related contigs were identified using blastn-short, and a draft mitochondrial genome was obtained by visualization screening using Bandage (v0.8.1). Subsequently, minimap2 (v2.26-r1175) was used to align long-reads and short-reads data to mitochondrial contigs and filter out the corresponding reads. Finally, the complete mitochondrial genome was obtained by mixing and assembling the data using the default parameters of Unicycler, and the visualization verification was completed again using Bandage (v0.8.1).

[0027] Mitochondrial genome annotation was performed using PMGA (http: / / www.1kmpg.cn / pmga / ), a dedicated annotation tool for mitochondrial genomes. 319 mitochondrial genomes were selected as the reference database. This tool can efficiently annotate splicing sites and trans-spliced ​​genes. tRNA genes were annotated using tRNAscan-SE (v.2.0.11), and rRNA genes were annotated using BLASTN (v2.16.0). The annotation results were manually checked and errors corrected using Apollo (v1.11.8). Finally, the visualization of the mitochondrial genome map was constructed using OGDRAW software.

[0028] A schematic diagram of the original results assembled based on long-reads data, as shown in the visualization analysis. Figure 1 The mitochondrial assembly of *Buffalo grass* contained two contigs (Table 1), and these contigs directly formed self-circles, indicating that the main structure of the *Buffalo grass* mitochondrial genome is a multi-branched structure. The total length of the *Buffalo grass* mitochondrial genome is 799,544 bp, and the GC content is 44.71%.

[0029] Table 1

[0030]

[0031] The mitochondrial genome of *Buffalo grass* was annotated, identifying 31 unique protein-coding genes (including 2 multicopy genes), comprising 24 core genes and 7 non-core genes. Additionally, 23 tRNA genes (7 of which were multicopy) and 3 rRNA genes were annotated. The core genes include 5 ATP synthases; 9 NADH dehydrogenases; 4 Cytochrome c biogenesis proteins; 3 Cytochrome c oxidases; 1 protein transport subunit; 1 maturation enzyme; and 1 Cytochrome b. The non-core genes include 1 Ribosomal protein large subunit and 6 Ribosomal protein small subunits. Figure 2A , Figure 2B (Table 2).

[0032] Table 2. Genes encoding the mitochondrial genome of *Buffalo grass*

[0033]

[0034] Note: The number in parentheses represents the number of copies of the gene, such as (×2) which means there are two copies.

[0035] Example 3: Analysis of codon bias and RNA editing sites in the mitochondrial genome of *Buffalo grass*

[0036] Codon usage preference is an important feature of eukaryotic genome evolution, formed by the combined effects of natural selection, mutation pressure, and gene expression regulation, and is the result of long-term adaptive evolution of species.

[0037] Amino acids generally correspond to one or more codons. For example, methionine (Met) corresponds to AUG, aspartic acid (Asn) corresponds to AAU and AAC, the stop codon (End) corresponds to UAA, UAG, and UGA, and leucine (Leu) corresponds to UUA, CUU, UUG, CUA, CUC, and CUG, etc. Different plants exhibit significant preferences in codon usage, a result of long-term evolutionary selection. Codon usage patterns differ significantly among species and genomes, typically measured by relative synonymous codon usage (RSCU). An RSCU > 1 indicates a higher-than-expected usage frequency, while a RSCU < 1 indicates a lower-than-expected usage frequency.

[0038] Codon bias analysis was performed on protein-coding genes (PCGs) in the mitochondrial genome using Phylosuite software (v1.1.16) and Mega software (v7.0), and RSCU values ​​were calculated.

[0039] Using the sequences of all PCGs encoded by the bison grass mitochondrial genome as input, Deepred-mt was used to identify C-to-U RNA editing events in PCGs from the bison grass mitochondrial genome, with a cutoff value of 0.9 as the criterion. This tool, based on a convolutional neural network (CNN) model, retains all results with probability values ​​greater than 0.9, resulting in higher accuracy.

[0040] Analysis of relative synonymous codon usage in the mitochondrial genome of *Buffalo grass* (…) Figure 3(Table 3) revealed that the RSCU values ​​for start codons AUG and tryptophan UGG were both 1, indicating no bias. Furthermore, among all amino acids, glutamate (Gln) showed the strongest bias towards the CAA codon, with the highest RSCU value among mitochondrial PCGs at 1.58. This was followed by alanine (Ala) with an RSCU value of 1.56. Glutamate (Gln) showed the weakest bias towards the CAG codon, with the lowest RSCU value among mitochondrial PCGs at 0.42. This was followed by histidine (His) with an RSCU value of 0.47.

[0041] Table 3. Relative synonymous codon usage for each amino acid in the mitochondrial genome of *Buffalo grass*.

[0042]

[0043]

[0044] CU-type RNA editing is prevalent in the mitochondria of higher plants, which can alter amino acids, generate start and stop codons, and enhance protein conservation and gene expression efficiency. By identifying RNA editing events in PCGs from bison grass mitochondria, a total of 435 potential RNA editing sites were identified. Figure 4 The most frequently identified RNA editing sites were found in the ccmC gene (35 sites), followed by the ccmB gene (31 sites), and then the nad2 gene (30 sites). The gene with the fewest editing sites was rps7 (2 sites), followed by atp8 and rps1 (3 sites each).

[0045] After statistically analyzing RNA editing events in the mitochondrial genes of *Buffalo grass*, the distribution of amino acid substitution types after editing was also analyzed. The results showed a clear preference for amino acid substitution types resulting from RNA editing. Figure 5 Of the various types of RNA editing, Ser→Leu, Pro→Leu, and Ser→Phe were the most prevalent, representing the largest proportion of replacement events. Other replacement types, such as Arg→Cys, Pro→Ser, and His→Tyr, occurred less frequently. Overall, RNA editing primarily tends to convert hydrophilic amino acids such as serine (Ser) and proline (Pro) into hydrophobic amino acids such as leucine (Leu) and phenylalanine (Phe), which aligns with the functional characteristics of RNA editing, namely increasing protein hydrophobicity and promoting correct protein folding.

[0046] Example 4: Analysis of Mitochondrial Genome Repetitive Sequences and Sequence Transfer in Buffalo Grass

[0047] The plant mitochondrial genome is rich in various repetitive sequences, including simple repetitive sequences, tandem repeats, inverted repeats, and scattered repeats. Repetitive sequences can promote homologous recombination and are a key factor driving structural variation and evolution of the mitochondrial genome.

[0048] Repeat sequences, including microsatellite repeats, tandem repeats, and sporadic repeats, were identified using MISA (v2.1) (https: / / webblast.ipk-gatersleben.de / misa / ), TRF (v4.09) (https: / / tandem.bu.edu / trf / trf.unix.help.html), and Rousfinder software, respectively. The results were visualized using Excel (2021) software and the Circos package (v0.69.9).

[0049] Based on next-generation sequencing data, the nematoplast genome was assembled using GetOrganelle software (v1.7.7.0), the chloroplast genome was annotated using CPGAVAS2, and the annotation results were corrected using CPGView software. Homologous fragments were analyzed using BLASTn software (v2.16.0), and the results were visualized using the Circos package (v0.69.9).

[0050] Simple Sequence Repeat (SSR) is a special type of tandem repeat sequence; a total of 188 SSRs have been found in the mitochondrial genome of *Buffalo grass*. Figure 6 Of these, 88 were located in Chromosome 1 and 100 in Chromosome 2. Tetrameric SSRs accounted for 45.21% of all SSRs, totaling 85. Adenine (A) monomeric repeat sequences accounted for 54.55% of the 22 monomeric SSRs, totaling 12.

[0051] In addition, 66 tandem repeats with a matching degree greater than 81% and a length between 5 and 36 bp were found in the mitochondrial genome, of which 35 were located in Chromosome 1 and 31 in Chromosome 2. Simultaneously, 1965 sets of dispersed repeats with a length greater than or equal to 30 bp were observed, including 173 palindromic repeats and 1792 forward repeats. The longest dispersed repeat unit was R1, with a length of 931 bp.

[0052] During mitochondrial evolution, chloroplast fragments migrated into the mitochondrial genome, and their length and sequence similarity varied across species. In *Buffalo grass*, a total of 31 fragments were found to be homologous to both the mitochondrial and chloroplast genomes, with a total length of 20134 bp, accounting for 2.52% of the total mitochondrial genome length. Figure 7 The longest of these was MTPT15 (6603 bp). Furthermore, annotation identified six protein-coding genes (psaB, psbK, rpl2, rpl23, rpoC1, ycf2) and nine tRNA genes (trnC-GCA, trnF-GAA, trnH-GUG, trnI-CAU, trnM-CAU, trnN-GUU, trnP-UGG, trnS-GGA, trnW-CCA) on homologous fragments.

[0053] Example 5: Mitochondrial genome collinearity and phylogenetic analysis of Buffalo grass

[0054] During mitochondrial evolution, chloroplast DNA fragments frequently migrate into the mitochondrial genome, forming mitochondrial chloroplast homologous segments (MTPTs). The length, number, and sequence similarity of these MTPTs vary significantly among species. This study and analyses of closely related species both detected this chloroplast-to-mitochondrial sequence migration.

[0055] Sequence homology alignment was performed using the BLASTn program to identify collinear blocks, with parameters set to -evalue 1e-5, -word_size 9, -gapopen 5, -gapextend 2, -reward 2, and -penalty -3. Collinear blocks with a length greater than or equal to 500 bp were selected for further analysis. Based on the alignment results, pairwise genome collinearity analysis was performed using MCscanX software to generate multicollinearity maps and identify conserved collinear regions.

[0056] Based on the degree of phylogenetic relationship among species, closely related taxa were screened, and 40 mitochondrial genomes were downloaded from the GenBank database (Table 4). Common genes among species were extracted using PhyloSuite software (v1.1.16), and multiple alignments of target gene sequences were performed using MAFFT software (v7.525). Subsequently, a phylogenetic tree was constructed using the maximum likelihood method (ML) with the parameter set to "--alrt 1000 -B 1000" to assess branch reliability. Finally, the phylogenetic tree was visualized using ITOL software (v7), generating an intuitive evolutionary relationship map.

[0057] Table 4. Plant species information from the 40 downloaded mitochondrial genomes.

[0058]

[0059]

[0060] Collinearity analysis results showed that there were numerous homologous segments in the mitochondrial genomes of *Buffalo grass* and closely related species. Figure 8 The presence of homologous segments indicates a shared evolutionary origin. However, the significant differences in the order of these homologous segments across species suggest that frequent rearrangements occurred in the bison grass mitochondrial genome during evolution, disrupting the original collinearity. This result also confirms the typical characteristics of non-conservative mitochondrial genome structure and susceptibility to rearrangements in higher plants.

[0061] This study conducted a phylogenetic analysis of 40 species in the order Poales based on 21 conserved mitochondrial protein-coding gene sequences, and constructed a phylogenetic tree using two species from the family Cyperaceae as outgroups. Figure 9 The results showed that all Poaceae species formed a monophyletic clade with high support, clearly differentiating themselves from outgroups. Buffalo grass and goosegrass formed a monophyletic clade with a node support of 89, indicating a close phylogenetic relationship. This clade further clustered with the *Cynodon* hybrid *Cynodon dactylon × Cynodon transvaalensis* and *Cynodon dactylon*, with a support of 100. Furthermore, it clustered with *Sporobolus alterniflorus* and *Zoysia macrostachya* in the same clade, with high node support, indicating a close phylogenetic relationship. Simultaneously, the topological structure of the phylogenetic tree highly matched the phylogenetic framework of Poaceae in the APG classification system, verifying the taxonomic position of buffalo grass within Poaceae and providing reliable molecular evidence for further research on the evolutionary origin and phylogenetic relationships of this species.

[0062] The above specific embodiments further illustrate the purpose, technical solution and beneficial effects of this application. It should be understood that the above are only specific embodiments of this application and are not intended to limit the scope of protection of this application. Any modifications, equivalent substitutions, improvements, etc., made on the basis of the technical solution of this application should be included within the scope of protection of this application.

Claims

1. The mitochondrial genome of *Buffalo grass*, characterized by: The nucleotide sequence of the bison grass mitochondrial genome is a combined sequence of SEQ.ID.NO.1, SEQ.ID.NO.2, SEQ.ID.NO.3, SEQ.ID.NO.4, and SEQ.ID.NO.

5.

2. The bison grass mitochondrial genome according to claim 1, characterized in that, The bison grass mitochondrial genome includes tRNA genes, rRNA genes, and protein-coding genes; The protein-coding genes include ATP synthase, NADH dehydrogenase, cytochrome c biosynthetic protein, cytochrome c oxidase, protein transport subunit, maturation enzyme, cytochrome b, ribosomal large subunit protein, and ribosomal small subunit protein.

3. A method for assembling and annotating the mitochondrial genome of *Buffalo grass* as described in claim 1, characterized in that, The method includes the following steps: S1. Collect samples, extract the mitochondrial genome of *Buffalo grass*, and sequence it to obtain the raw data of the *Buffalo grass* mitochondrial genome. S2. The raw data of the bison grass mitochondrial genome are screened and assembled to obtain the assembled bison grass mitochondrial genome; then the assembled bison grass mitochondrial genome sequence is annotated and a bison grass mitochondrial genome map is drawn.

4. The method for assembling and annotating the mitochondrial genome of *Buffalo grass* according to claim 3, characterized in that, The specific sequencing method described in step S1 is as follows: for the extracted bison grass mitochondrial genome, high-throughput sequencing is performed using Illumina NovaSeq 6000 sequencing technology and nanopore single-molecule sequencing technology to obtain the raw data of the bison grass mitochondrial genome.

5. The method for assembling and annotating the mitochondrial genome of *Buffalo grass* according to claim 4, characterized in that, Short-read libraries were sequenced using the Illumina NovaSeq 6000 sequencing technology, and long-read libraries were sequenced using the nanopore single-molecule sequencing technology.

6. The method for assembling and annotating the mitochondrial genome of *Buffalo grass* according to claim 3, characterized in that, The specific method for screening and assembling the raw data of the bison grass mitochondrial genome in step S2 is as follows: using the mitochondrial genomes of closely related species goosegrass and bermudagrass as reference sequences, mitochondrial-related contigs are screened to obtain a bison grass mitochondrial genome draft. Then, the short-read long-read library and the long-read long-read library of the bison grass mitochondrial genome draft are aligned to the mitochondrial contigs and filtered. After mixing and assembling, the assembled bison grass mitochondrial genome is obtained.

7. The method for assembling and annotating the mitochondrial genome of *Buffalo grass* according to claim 3, characterized in that, The specific annotation method described in step S2 is as follows: the online software PMGA is used to perform overall annotation of the mitochondrial genome of *Pterocarya stenoptera*. For tRNA genes, the software tRNAscan-SE is used for annotation, and for rRNA genes, the software BLASTN is used for annotation.

8. The use of the bison grass mitochondrial genome as described in claim 1 in constructing a phylogenetic tree of the Poaceae family.

9. The use according to claim 8, characterized in that, The method for constructing the phylogenetic tree of the Poaceae family is as follows: the mitochondrial genomes of 40 closely related groups of buffalo grass are downloaded from a gene database, common genes among the species are extracted using the software PhyloSuite, multiple alignments of the common genes among the species are performed using the MAFFT software, and then the phylogenetic tree is constructed using the IQ-TREE software.

10. The use of the bison grass mitochondrial genome as described in claim 1 in the identification of bison grass germplasm.

Images