An est-ssr molecular marker related to cold resistance based on a poa palustris transcriptome sequence and application thereof

Abstract

This invention discloses an EST-SSR molecular marker related to cold resistance developed based on the transcriptome sequence of Kentucky bluegrass and its application, belonging to the field of biotechnology. This invention develops an EST-SSR molecular marker primer set based on transcriptome sequencing data of leaf cold-resistance in Kentucky bluegrass. This primer set can be applied in the fields of species identification, genetic pedigree analysis, genetic map construction, germplasm resource conservation, and assisted breeding of Kentucky bluegrass. Using 29 pairs of SSR primers, genetic diversity analysis was performed on four Kentucky bluegrass species, exhibiting good reproducibility, clear banding, and high polymorphism. It can be widely used in Kentucky bluegrass species identification, high-density genetic linkage map construction, diversity analysis, and molecular-assisted breeding.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to an EST-SSR molecular marker related to cold resistance developed based on the transcriptome sequence of Kentucky bluegrass and its application. Background Technology

[0002] *Poa* L. belongs to the subfamily Poideae of the family Gramineae. It is mainly distributed in temperate and cold regions and is a loosely tufted, low-growing grass. Due to its high nutritional value, palatability, strong adaptability to adversity, rapid regeneration, and high tolerance to grazing, *Poa* has become an important forage and valuable turfgrass resource. Cold-climate *Poa crymophila* is a perennial plant of the genus *Poa* in the family Gramineae. It is an important native grass species of the Qinghai-Tibet Plateau, possessing strong cold resistance, and is a valuable resource for breeding and developing new forage varieties.

[0003] SSR (Simple Sequence Repeat) markers are DNA fragments composed of tandem repeat sequences of 2–6 nucleotides, mostly 100–200 base pairs in length. Their main characteristics include high polymorphism, co-dominant inheritance, and close association with functional genes. Based on their origin, they can be divided into genomic SSRs and expressed sequence tag SSRs (EST-SSRs). Traditional SSR markers are developed from random genomic sequences, and their linkage with functional genes is uncertain. However, SSR or EST-SSR molecular markers developed based on candidate genes have a certain linkage with target traits and have broad application prospects in germplasm resource identification and marker-assisted breeding. In recent years, next-generation sequencing technologies such as transcriptome sequencing (RNA-seq) have provided high-throughput data, offering rapid, reliable, and economical research tools for the identification and development of molecular markers. The genome information of Kentucky bluegrass is unknown, and transcriptome sequencing is the main method for developing specific SSR primers. However, no SSR marker development and application of cold-resistance candidate genes for Kentucky bluegrass using transcriptome sequencing has been found to date.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide an EST-SSR molecular marker related to cold resistance developed based on the transcriptome sequence of Kentucky bluegrass and its application. The primer pair of this EST-SSR molecular marker is derived from its low-temperature resistant transcriptome sequence and has the characteristics of rich polymorphism, stable amplification, and easy band identification, filling the gap in the development of SSR primers based on candidate genes for cold resistance of Kentucky bluegrass.

[0006] In a first aspect, the present invention provides an EST-SSR molecular marker primer set developed based on the transcriptome sequence of Kentucky bluegrass, which includes any one or more pairs of primer pairs of P1, P2, P3, P4, P17, P18, P21, P23, P37, P38, P54, P65, P71, P72, P88, P89, P91, P92, P93, P94, P95, P98, P99, P148, P150, P192, P193, P195 and P197;

[0007] The nucleotide sequence of the P1 primer pair is shown in SEQ ID NO.1-2;

[0008] The nucleotide sequences of the P2 primer pair are shown in SEQ ID NO.3-4;

[0009] The nucleotide sequences of the P3 primer pair are shown in SEQ ID NO.5-6;

[0010] The nucleotide sequences of the P4 primer pair are shown in SEQ ID NO.7-8;

[0011] The nucleotide sequences of the P17 primer pair are shown in SEQ ID NO.9-10;

[0012] The nucleotide sequences of the P18 primer pair are shown in SEQ ID NO.11-12;

[0013] The nucleotide sequences of the P21 primer pair are shown in SEQ ID NO.13-14;

[0014] The nucleotide sequences of the P23 primer pair are shown in SEQ ID NO.15-16;

[0015] The nucleotide sequences of the P37 primer pair are shown in SEQ ID NO.17-18;

[0016] The nucleotide sequences of the P38 primer pair are shown in SEQ ID NO.19-20;

[0017] The nucleotide sequences of the P54 primer pair are shown in SEQ ID NO.21-22;

[0018] The nucleotide sequences of the P65 primer pair are shown in SEQ ID NO.23-24;

[0019] The nucleotide sequences of the P71 primer pair are shown in SEQ ID NO.25-26;

[0020] The nucleotide sequences of the P72 primer pair are shown in SEQ ID NO.27-28;

[0021] The nucleotide sequences of the P88 primer pair are shown in SEQ ID NO.29-30;

[0022] The nucleotide sequences of the P89 primer pair are shown in SEQ ID NO.31-32;

[0023] The nucleotide sequences of the P91 primer pair are shown in SEQ ID NO.33-34;

[0024] The nucleotide sequences of the P92 primer pair are shown in SEQ ID NO.35-36;

[0025] The nucleotide sequences of the P93 primer pair are shown in SEQ ID NO.37-38;

[0026] The nucleotide sequences of the P94 primer pair are shown in SEQ ID NO.39-40;

[0027] The nucleotide sequences of the P95 primer pair are shown in SEQ ID NO.41-42;

[0028] The nucleotide sequences of the P98 primer pair are shown in SEQ ID NO.43-44;

[0029] The nucleotide sequences of the P99 primer pair are shown in SEQ ID NO.45-46;

[0030] The nucleotide sequences of the P148 primer pair are shown in SEQ ID NO.47-48;

[0031] The nucleotide sequences of the P150 primer pair are shown in SEQ ID NO.49-50;

[0032] The nucleotide sequences of the P192 primer pair are shown in SEQ ID NO.51-52;

[0033] The nucleotide sequences of the P193 primer pair are shown in SEQ ID NO.53-54;

[0034] The nucleotide sequences of the P195 primer pair are shown in SEQ ID NO.55-56;

[0035] The nucleotide sequences of the P197 primer pair are shown in SEQ ID NO.57-58.

[0036] Secondly, the present invention provides a kit containing the above-mentioned EST-SSR molecular marker primer combination.

[0037] In some embodiments, the kit may further include at least one of DNA extraction reagent, DNA amplification reagent, and electrophoresis reagent.

[0038] Thirdly, this invention provides the application of EST-SSR molecular marker primer combinations or kits in the construction of Kentucky bluegrass genetic maps or the analysis of genetic diversity.

[0039] Fourthly, this invention provides the application of EST-SSR molecular marker primer combinations or kits in molecular marker-assisted breeding or germplasm resource conservation of Kentucky bluegrass.

[0040] Fifthly, this invention provides the application of EST-SSR molecular marker primer combinations or kits in the identification of Kentucky bluegrass germplasm.

[0041] In some embodiments, the above-mentioned Kentucky bluegrass germplasm includes: Kentucky bluegrass of Qinghai cold region, Kentucky bluegrass of Qinghai flat stem, Kentucky bluegrass of Qinghai grassland, and Kentucky bluegrass of China.

[0042] This invention designs SSR-related primers based on Kentucky bluegrass transcriptome data and performs genetic diversity analysis, providing specific polymorphic SSR markers for Kentucky bluegrass research. These markers can provide important reference value for subsequent evolutionary research and germplasm resource conservation of Kentucky bluegrass. In addition, based on the individual-specific phenotypic characteristics of the samples, specific SSR markers associated with superior Kentucky bluegrass phenotypes can be constructed in conjunction with corresponding transcriptome data annotation, providing important guidance for the breeding of superior Kentucky bluegrass varieties.

[0043] Meanwhile, this invention also utilized some primers from the aforementioned EST-SSR marker primer combination to conduct germplasm identification of different Kentucky bluegrass varieties. The results showed that any one or more primer pairs from the aforementioned EST-SSR marker primer combination could identify and distinguish varieties such as *Poa qinghaiensis*, *Poa qinghaiensis*, *Poa qinghaiensis*, and *Poa huahuiensis*. To demonstrate this effect, this invention also provides a method and results for identifying the aforementioned four Kentucky bluegrass varieties using any two primer pairs (i.e., primer pair P37 and primer pair P148):

[0044] A method for identifying different cold-resistant Kentucky bluegrass species, comprising:

[0045] Using the DNA of the Kentucky bluegrass to be tested as a template, PCR amplification was performed using the above-mentioned EST-SSR marker primer combination to obtain the amplification product;

[0046] The amplification products are subjected to electrophoresis to obtain information on the number of amplified bands and the size of the amplified fragments.

[0047] The variety of Kentucky bluegrass to be tested is determined based on the difference in the number of bands and / or the size of the amplified fragments.

[0048] Among them, the Kentucky bluegrass to be tested included Kentucky bluegrass of Qinghai cold region, Kentucky bluegrass of Qinghai flat stem, Kentucky bluegrass of Qinghai grassland, and Kentucky bluegrass of China.

[0049] In some embodiments, P37 and P148 of the above-described EST-SSR marker primer combination are used to identify the varieties of Kentucky bluegrass.

[0050] Among them, the PCR amplification of primer P37 showed that the 160bp bands were found in *Poa chinensis*, *Poa chinensis*, and *Poa huahui*.

[0051] PCR amplification was performed using primer pair P148. The following results were obtained: *Poa chinensis* with bands of 80bp, 100bp, 145bp, 175bp, and 280bp; *Poa chinensis* with bands of 80bp and 145bp; *Poa chinensis* with bands of 65bp, 80bp, 100bp, 175bp, and 190bp; and *Poa chinensis* with bands of 70bp and 75bp.

[0052] In some embodiments, the reaction system for PCR amplification is as follows: 2 μL of 25 ng / μL template DNA, 0.5 μL each of 10 μmol / μL forward and reverse primers, 0.1 μL of 2.5 U / μL Golden DNA Polymerase, 10 μL of 2×Reaction Mix, and 6.9 μL of dd H2O.

[0053] In some embodiments, the PCR amplification program is as follows: 94℃ pre-denaturation for 3 min, 94℃ denaturation for 30 s, 60℃~55℃ annealing for 30 s, 72℃ extension for 50 s, for a total of 5 cycles, with the annealing temperature decreasing by 1℃ in each cycle; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 50 s, for a total of 29 cycles; final extension for 7 min, and storage at 4℃.

[0054] The present invention has the following beneficial effects:

[0055] This invention targets Kentucky bluegrass in cold-climate regions and develops an EST-SSR molecular marker primer combination based on transcriptome sequencing data of leaf cold tolerance. This primer combination can be applied in the fields of species identification, genetic pedigree analysis, genetic map construction, germplasm resource conservation, and assisted breeding of Kentucky bluegrass. Using 29 pairs of SSR primers, genetic diversity analysis was performed on four Kentucky bluegrass species, which showed good reproducibility, clear banding, and high polymorphism. It can be widely used in species identification, high-density genetic linkage map construction, diversity analysis, and molecular-assisted breeding of Kentucky bluegrass. Attached Figure Description

[0056] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0057] Figure 1 The results of the cold resistance evaluation of four Kentucky bluegrass species were based on the analysis of seven indicators: proline (Pro), soluble sugar (SS), abscisic acid (MDA), chlorophyll (Chl), fresh weight (FW), and superoxide dismutase (SOD) and peroxidase activity (POD). The results were obtained by clustering the four Kentucky bluegrass species, namely, *Poa qinghaiensis* (BJ), *Poa qinghaiensis* (CD), *Poa huahuiensis* (HH), and *Poa qinghaiensis* (LD), after a comprehensive evaluation of their cold resistance using the membership function method.

[0058] Figure 2 The results of PCR amplification using primers P37 and P148 in 40 individual plants of four Kentucky bluegrass species are shown. Different codes represent the genotypes of the individuals. B1–B10 represent the 10 genotypes of BJ (flat-stemmed Kentucky bluegrass), C1–C10 represent the 10 genotypes of CD (grassland bluegrass), H1–H10 represent the 10 genotypes of HH (Chinese grey bluegrass), and L1–L10 represent the 10 genotypes of LD (coolland bluegrass).

[0059] Figure 3 Principal component analysis and cluster analysis results of 29 EST-SSR markers for four different populations of the genus *Poa* are presented. A represents the principal coordinate analysis of 29 EST-SSR markers for four different populations of the genus *Poa*; B represents the cluster analysis of 29 EST-SSR markers for four different populations of the genus *Poa*. Different codes represent individual genotypes: B1–B10 represent the 10 genotypes of BJ (flat-stemmed *Poa*), C1–C10 represent the 10 genotypes of CD (*Poa grass*), H1–H10 represent the 10 genotypes of HH (*Poa huahui*), and L1–L10 represent the 10 genotypes of LD (*Poa d'icefield*).

[0060] Figure 4The results of the STRUCTURE analysis of 29 EST-SSR markers for four different populations of the genus *Poa* are shown. In Figure C, A represents the average LnP(D) value after 20 runs at each K value; B represents the optimal hierarchical structure for K=3 determined using the largest ΔK value; C represents the three main groups of 40 *Poa* genotypes at K=3. In Figure C, the vertical axis represents the membership coefficient of each genotype, and different codes represent the genotypes of each individual. Specifically, 1–10 represent the 10 genotypes (B1 to B10) of BJ (flat-stemmed *Poa*), 11–20 represent the 10 genotypes (C1 to C10) of CD (*Poa grass*), 21–30 represent the 10 genotypes (H1 to H10) of HH (*Poa huahui*), and 31–40 represent the 10 genotypes (L1 to L10) of LD (*Poa d'icefield*). Different colors represent different populations. Detailed Implementation

[0061] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0062] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be used in this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0063] Example 1

[0064] This example describes the development of molecular markers linked to candidate genes for cold resistance in Kentucky bluegrass, as detailed below:

[0065] In the early stages of this experiment, transcriptome sequencing of cold-resistance genes in Kentucky bluegrass leaves was performed. Based on the transcriptome sequencing data, 200 EST-SSR primers for cold resistance candidate genes were designed and synthesized using gene function injection. SSR loci were identified and located using Microsatellite (MISA) software, and SSR primers were designed using Primer 3-v2.3.4 software. The repeating units selected for the primer design sites were dinucleotides, trinucleotides, tetranucleotides, pentanucleotides, and hexanucleotides, with minimum repeat counts of 9, 8, 7, 6, and 5, respectively. Sequences with a distance greater than 50 bp from both ends of the original sequence were used in the primer design. Primer lengths were controlled between 18 and 22 bp, with expected product lengths of 50–350 bp. A total of 106 primer pairs were ultimately screened. Using DNA from four Kentucky bluegrass species as templates, PCR amplification was performed using the 106 SSR primer pairs, and 29 primer pairs with clear bands and good polymorphism were finally selected.

[0066] Main steps:

[0067] (1) Source of materials: Seeds of Kentucky bluegrass (LD), Kentucky bluegrass (BJ), Kentucky bluegrass (CD), and Kentucky bluegrass (HH) were provided by the Qinghai Academy of Animal Science and Veterinary Medicine. Among them, Kentucky bluegrass, Kentucky bluegrass, and Kentucky bluegrass are cultivated varieties, while Kentucky bluegrass is a germplasm material.

[0068] (2) Obtaining SSR polymorphic sites: Total RNA was extracted from young leaves of Kentucky bluegrass under low-temperature treatment, and a transcriptome sequencing library was constructed. High-throughput sequencing was performed using an Illumina next-generation sequencer. After rigorous filtering, the sequencing data were assembled, yielding 53,893 unigenes. 12,972 SSR sites were successfully identified, representing an SSR site frequency of 24.07%. These SSR sites were widely distributed across 10,766 unigenes, accounting for 19.98% of all sequences. Among them, 2,367 sequences contained multiple SSRs, and 433 SSRs were present during compound formation. The identified SSR sequences were diverse, covering repeat sequences from single nucleotides to hexanucleotides, with significant differences in the number of each type. Trinucleotide repeat sequences were the most abundant, accounting for 41.84% of all repeat sequences (5,427 in total).

[0069] (2) Based on gene function annotation, 200 candidate genes closely related to cold resistance were screened from 46,542 Kentucky bluegrass Unigenes. SSR primers were designed using Primer3-v2.3.4 software. The repeating units selected for the primer design sites were dinucleotides, trinucleotides, tetranucleotides, pentanucleotides, and hexanucleotides, with minimum repeat counts of 9, 7, 6, 5, and 4, respectively. Sequences with a distance greater than 50 bp from both ends of the sequence were used for primer design. Primer lengths were controlled between 17 and 24 bp, with an expected product length of 100–300 bp. Finally, 106 primer pairs were selected for amplification.

[0070] (3) DNA extraction: Genomic DNA was extracted from fresh leaf tissue samples collected from 40 individual Kentucky bluegrass plants using an adsorption column method with a plant genomic DNA extraction kit (Tiangen Biotech Co., Ltd., Beijing). The DNA quality was determined using a NanoDrop ND1000 spectrophotometer, and DNA meeting the requirements was diluted to 25 ng / μL and stored at -20℃.

[0071] (4) PCR amplification was performed using the 106 primer pairs described above to obtain PCR products;

[0072] (5) The PCR amplification system includes the following components: 2 μL (25 ng / μL) template DNA, 0.5 μL (10 μmol / μL) each of upstream and downstream primers, 0.1 μL (2.5 U / μL) Golden DNA Polymerase (Tiangen Biotech Co., Ltd., Beijing), 10 μL 2×Reaction Mix (Tiangen Biotech Co., Ltd., Beijing), and 6.9 μL dd H2O.

[0073] (6) The PCR amplification program was as follows: 94℃ pre-denaturation for 3 min, 94℃ denaturation for 30 s, 60℃~55℃ annealing for 30 s, 72℃ extension for 50 s, for a total of 5 cycles, with the annealing temperature decreasing by 1℃ in each cycle; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 50 s, for a total of 29 cycles; and finally extension for 7 min, and storage at 4℃.

[0074] (7) The obtained PCR products were subjected to acrylamide gel electrophoresis, staining and development, and finally 29 pairs of SSR marker primers with clear bands, high polymorphism and good reproducibility were obtained.

[0075] Table 1 EST-SSR Primer Information

[0076]

[0077]

[0078]

[0079] Example 2

[0080] This embodiment describes a method for identifying different cold-resistant Kentucky bluegrass species, as detailed below:

[0081] Previous principal component analysis based on seven indicators—proline, soluble sugar, abscisic acid, chlorophyll, fresh weight, and superoxide dismutase and peroxidase activities—was used to comprehensively evaluate the cold resistance of four Kentucky bluegrass species using the membership function method, followed by cluster analysis. The results are as follows: Figure 1 As shown. Four Kentucky bluegrass species with different cold resistance were identified using the P37 and P148 markers. The materials were from the same source as in Example 1.

[0082] The main steps are as follows:

[0083] (1) DNA extraction, the extraction method is the same as in Example 1;

[0084] (2) Using the genomic DNA of the Kentucky bluegrass material to be identified as a template, PCR amplification was performed using the primers P37 and P148. The primer information is shown in Table 1. The PCR products were obtained.

[0085] (3) The PCR amplification system includes the following components: 2 μL (25 ng / μL) template DNA, 0.5 μL (10 μmol / μL) each of upstream and downstream primers, 0.1 μL (2.5 U / μL) Golden DNA Polymerase (Tiangen Biotech Co., Ltd., Beijing), 10 μL 2×Reaction Mix (Tiangen Biotech Co., Ltd., Beijing), and 6.9 μL dd H2O.

[0086] (4) The PCR amplification program includes 5 cycles of 94℃ pre-denaturation for 3 min, 94℃ denaturation for 30 s, 60℃~55℃ annealing for 30 s, and 72℃ extension for 50 s, with the annealing temperature decreasing by 1℃ in each cycle; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, and 72℃ extension for 50 s, for a total of 29 cycles; and a final extension for 7 min, followed by storage at 4℃.

[0087] (5) The obtained PCR products were subjected to acrylamide gel electrophoresis, staining and development;

[0088] (6) Analysis of the amplified electrophoresis bands, the results are as follows: Figure 2As shown: PCR amplification using primer pair P37 revealed three species with a specific characteristic band of 160 bp: *Poa qinghaiensis*, *Poa qinghaiensis*, and *Poa huarensis*. PCR amplification using primer pair P148 revealed *Poa qinghaiensis* with bands of 80 bp, 100 bp, 145 bp, 175 bp, and 280 bp; *Poa qinghaiensis* with bands of 80 bp and 145 bp; *Poa huarensis* with bands of 65 bp, 80 bp, 100 bp, 175 bp, and 190 bp; and *Poa qinghaiensis* with bands of 70 bp and 75 bp. Therefore, these characteristic band combinations can be used to identify different cold-resistant *Poa* species.

[0089] Example 3

[0090] This example demonstrates the application of 29 markers to genetic diversity analysis of materials from four Kentucky bluegrass species, as detailed below:

[0091] The main steps are as follows:

[0092] (1) DNA extraction, the extraction method is the same as in Example 1;

[0093] (2) Using the genomic DNA of the Kentucky bluegrass material to be identified as a template, PCR amplification was performed using the 29 primer pairs. The primer information is shown in Table 1. The PCR products were obtained.

[0094] (3) The PCR amplification system and PCR amplification procedure are the same as in Example 1;

[0095] (4) The obtained PCR products were subjected to acrylamide gel electrophoresis, staining and development;

[0096] (5) The amplified bands of 29 polymorphic EST-SSR primers were statistically analyzed using an "0, 1" matrix in Excel 2019. Genetic distance and other correlation analyses were performed using GenAlEx and POPGENE (v1.31) software. Individuals were further subdivided into different subpopulations using STRUCTURE-v2.3.2 software, and cluster analysis was performed. In addition, principal component analysis (PCoA) was performed based on the Jaccard genetic similarity matrix using the DCENTER module in NTSYS (v2.10) software.

[0097] (6) Polymorphism analysis is shown in Table 2:

[0098] Table 2. Polymorphism analysis of 29 EST-SSR primer pairs in four species of the genus *Poa*.

[0099]

[0100]

[0101] As shown in Table 2 above, the 29 polymorphic primers amplified a total of 425 bands, with an average of 14.7 bands per primer. The number of amplified bands ranged from 6 (P17 and P72) to 25 (P193), with an average of 14.7 (Table 4-5). The average percentage of polymorphic bands ranged from 56.52% to 100%, with an average percentage of 80.56%. The expected heterozygosity (He) ranged from 0.77 to 0.95, with an average of 0.90. The observed heterozygosity (Ho) ranged from 0.78 to 1.00, with an average of 0.98. The polymorphism information content (PIC) value ranged from 0.22 (P3 and P93) to 0.42 (P18 and P150), with an average of 0.35.

[0102] (7) Genetic diversity information of genus populations is shown in Table 3:

[0103] Table 3. Genetic diversity information of four Poa populations

[0104]

[0105]

[0106] As shown in Table 3 above, the genetic diversity of the Kentucky bluegrass population was the highest (Na = 1.23, Ne = 1.30, I = 0.26, H = 0.17, %P = 48.71%), followed by the Kentucky bluegrass population (Na = 0.73, Ne = 1.17, I = 0.14, H = 0.09, %P = 23.29%) and the flat-stemmed bluegrass population (Na = 0.76, Ne = 1.13, I = 0.11, H = 0.03, %P = 18.59%), while the genetic diversity of the Kentucky bluegrass population was the lowest (Na = 0.72, Ne = 1.09, I = 0.08, H = 0.05, %P = 13.88%).

[0107] (8) The results of molecular variance analysis (AMONA) of different populations are shown in Table 4:

[0108] Table 4. Analysis of molecular variance (AMONA) of four different populations of Kentucky bluegrass.

[0109] source Degrees of freedom variance Mean variance Variant components Total variation Between populations 3 2219.6 739.87 71.92 78％ Within the population 36 744 20.67 20.67 22％ total 39 2963.6 92.59 100％

[0110] Table 4 above shows that variation among different populations accounts for 87% of the total variation, while genetic variation within a population accounts for only 22%.

[0111] (9) The results of the analysis of Nei's genetic distance (lower left triangular matrix) and genetic consistency (upper right triangular matrix) of different populations are shown in Table 5:

[0112] Table 5. Nei's genetic distance and genetic uniformity analysis of four different Kentucky bluegrass populations.

[0113] population Kentucky bluegrass Kentucky bluegrass Kentucky bluegrass Kentucky bluegrass Kentucky bluegrass **** 0.79 0.63 0.59 Kentucky bluegrass 0.23 **** 0.61 0.65 Kentucky bluegrass 0.46 0.49 **** 0.57 Kentucky bluegrass 0.53 0.44 0.57 ****

[0114] As shown in Table 5 above, the Nei's genetic distance among the four populations of the genus *Poa* ranges from 0.23 (*Poa flattened-stem* and *Poa przewalskii*) to 0.57 (*Poa schrenckii* and *Poa coollandii*), while the genetic uniformity ranges from 0.57 (*Poa schrenckii* and *Poa coollandii*) to 0.79 (*Poa flattened-stem* and *Poa przewalskii*).

[0115] (10) The results of principal component analysis (PCoA) are shown in [the table]. Figure 3 A. The two principal components explain 87.3% of the genetic variation in 40 individual plants of the genus *Poa*, with the first principal component accounting for 48.82% and the second principal component accounting for 38.48%. *Poa przewalskii* and *Poa flattened stem*, which are less cold-hardy, were grouped together; *Poa coollandensis*, which is more cold-hardy, was grouped together; and *Poa huali* was grouped together.

[0116] (11) The results of the UPGMA unweighted group average cluster analysis are shown in […]. Figure 3 B. At a genetic similarity coefficient of 0.70, the four Kentucky bluegrass populations were divided into three major groups. The first group included all individual plants of Kentucky bluegrass (CD) and Kentucky bluegrass flat-stemmed (BJ), the second group included all individual plants of Kentucky bluegrass (HH), and the third group included all individual plants of Kentucky bluegrass coolland (LD).

[0117] (12) The results of the STRUCTURE analysis are shown in [the table]. Figure 4 When the optimal K value is 3, the optimal number of groups is 3. The first group is green, containing the less cold-resistant CD and BJ populations; the second group is blue, representing the HH population; and the third group is red, representing the LD population. Figure 4 B and Figure 4 C). The genetic background of each individual plant can be determined based on the color proportions; BJ and CD have a close genetic relationship (K=3).

[0118] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An EST-SSR molecular marker primer combination developed based on the transcriptome sequence of Poa palustris, characterized in that, The EST-SSR molecular marker primer combination is the P37 and P148 primer pair; The nucleotide sequences of primer pair P37 are shown in SEQ ID NO.17-18; the nucleotide sequences of primer pair P148 are shown in SEQ ID NO.47-48.

2. A primer set of EST-SSR molecular markers developed based on the transcriptome sequence of *Poa glabra*, characterized in that, The EST-SSR molecular marker primer combination is the primer pair of P1, P2, P3, P4, P17, P18, P21, P23, P37, P38, P54, P65, P71, P72, P88, P89, P91, P92, P93, P94, P95, P98, P99, P148, P150, P192, P193, P195 and P197; The nucleotide sequence of the P1 primer pair is shown in SEQ ID NO.1-2; The nucleotide sequences of the P2 primer pair are shown in SEQ ID NO.3-4; The nucleotide sequences of the P3 primer pair are shown in SEQ ID NO.5-6; The nucleotide sequences of the P4 primer pair are shown in SEQ ID NO.7-8; The nucleotide sequences of the P17 primer pair are shown in SEQ ID NO.9-10; The nucleotide sequences of the P18 primer pair are shown in SEQ ID NO.11-12; The nucleotide sequences of the P21 primer pair are shown in SEQ ID NO.13-14; The nucleotide sequences of the P23 primer pair are shown in SEQ ID NO.15-16; The nucleotide sequences of the P37 primer pair are shown in SEQ ID NO.17-18; The nucleotide sequences of the P38 primer pair are shown in SEQ ID NO.19-20; The nucleotide sequences of the P54 primer pair are shown in SEQ ID NO.21-22; The nucleotide sequences of the P65 primer pair are shown in SEQ ID NO.23-24; The nucleotide sequences of the P71 primer pair are shown in SEQ ID NO.25-26; The nucleotide sequences of the P72 primer pair are shown in SEQ ID NO.27-28; The nucleotide sequences of the P88 primer pair are shown in SEQ ID NO.29-30; The nucleotide sequences of the P89 primer pair are shown in SEQ ID NO.31-32; The nucleotide sequences of the P91 primer pair are shown in SEQ ID NO.33-34; The nucleotide sequences of the P92 primer pair are shown in SEQ ID NO.35-36; The nucleotide sequences of the P93 primer pair are shown in SEQ ID NO.37-38; The nucleotide sequences of the P94 primer pair are shown in SEQ ID NO.39-40; The nucleotide sequences of the P95 primer pair are shown in SEQ ID NO.41-42; The nucleotide sequences of the P98 primer pair are shown in SEQ ID NO.43-44; The nucleotide sequences of the P99 primer pair are shown in SEQ ID NO.45-46; The nucleotide sequences of the P148 primer pair are shown in SEQ ID NO.47-48; The nucleotide sequences of the P150 primer pair are shown in SEQ ID NO.49-50; The nucleotide sequences of the P192 primer pair are shown in SEQ ID NO.51-52; The nucleotide sequences of the P193 primer pair are shown in SEQ ID NO.53-54; The nucleotide sequences of the P195 primer pair are shown in SEQ ID NO.55-56; The nucleotide sequences of the P197 primer pair are shown in SEQ ID NO.57-58.

3. A reagent kit, characterized in that, Contains the EST-SSR molecular marker primer combination as described in claim 1 or 2.

4. The application of the EST-SSR molecular marker primer combination as described in claim 2 or the kit as described in claim 3 in the construction of the genetic map of *Poa annua* or the analysis of genetic diversity, characterized in that... The Kentucky bluegrass germplasm was selected from Kentucky bluegrass of Qinghai cold region, Kentucky bluegrass of Qinghai flat stem, Kentucky bluegrass of Qinghai grassland, and Kentucky bluegrass of China.

5. The application of the EST-SSR molecular marker primer combination as described in claim 2 or the kit as described in claim 3 in the conservation of Kentucky bluegrass germplasm resources, characterized in that, The Kentucky bluegrass germplasm was selected from Kentucky bluegrass of Qinghai cold region, Kentucky bluegrass of Qinghai flat stem, Kentucky bluegrass of Qinghai grassland, and Kentucky bluegrass of China.

6. The application of the EST-SSR molecular marker primer combination as described in claim 1 or the kit as described in claim 3 in the identification of Kentucky bluegrass germplasm, characterized in that, The Kentucky bluegrass germplasm was selected from Kentucky bluegrass of Qinghai cold region, Kentucky bluegrass of Qinghai flat stem, Kentucky bluegrass of Qinghai grassland, and Kentucky bluegrass of China.

7. A method for identifying different cold-resistant Kentucky bluegrass varieties, characterized in that, include: Using the DNA of the target Kentucky bluegrass as a template, the EST described in claim 1 was used... PCR amplification was performed using an SSR-labeled primer combination to obtain the amplification product; The amplification products are subjected to electrophoresis to obtain information on the number of amplified bands and the size of the amplified fragments. The variety of Kentucky bluegrass to be tested is determined based on the difference in the number of bands and / or the size of the amplified fragments. Among them, the Kentucky bluegrass to be tested was selected from Kentucky bluegrass of Qinghai cold region, Kentucky bluegrass of Qinghai flat stem, Kentucky bluegrass of Qinghai grassland and Kentucky bluegrass of China.

8. The identification method according to claim 7, characterized in that, Using the EST The P37 primer pair and P148 primer pair in the SSR marker primer combination were used to identify the varieties of Kentucky bluegrass. PCR amplification was performed using the P37 primer pair: the 160 bp bands were found in *Poa qinghaiensis*, *Poa qinghaiensis*, and *Poa huahuiensis*. PCR amplification was performed using the P148 primer pair: the sample containing bands of 80 bp, 100 bp, 145 bp, 175 bp, and 280 bp was identified as *Poa chinensis* var. *qinghaiensis*; the sample containing bands of 80 bp and 145 bp was identified as *Poa chinensis* var. *qinghaiensis*; the sample containing bands of 65 bp, 80 bp, 100 bp, 175 bp, and 190 bp was identified as *Poa chinensis* var. *huaensis*; and the sample containing bands of 70 bp and 75 bp was identified as *Poa chinensis* var. *qinghai ...

9. The identification method according to claim 7, characterized in that, The reaction system for PCR amplification was as follows: 2 μL of 25 ng / μL template DNA, 0.5 μL each of 10 μmol / μL forward and reverse primers, 0.1 μL of 2.5 U / μL Golden DNA Polymerase, 10 μL of 2×Reaction Mix, and 6.9 μL of dd H2O.

10. The identification method according to claim 7, characterized in that, The PCR amplification program was as follows: 94℃ pre-denaturation for 3 min, 94℃ denaturation for 30 s, 60℃~55℃ annealing for 30 s, 72℃ extension for 50 s, for a total of 5 cycles, with the annealing temperature decreasing by 1℃ in each cycle; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 50 s, for a total of 29 cycles; final extension for 7 min, and storage at 4℃.

Images