Cold resistance related EST-SSR (expressed sequence tag-simple sequence repeats) molecular marker developed on basis of transcriptome sequence of bluegrass cold region and application of EST-SSR molecular marker

Abstract

The invention discloses a cold-resistance-related EST-SSR molecular marker developed based on a transcriptome sequence of bluegrass in cold land and application, and belongs to the technical field of biology. The EST-SSR molecular marker primer combination is developed on the basis of low-temperature-resistant transcriptome sequencing data of leaves for the cold-field bluegrass, and the primer combination can be applied to the fields of bluegrass species identification, genetic pedigree analysis, genetic map construction, protection of germplasm resources and auxiliary breeding; the four bluegrass species are subjected to genetic diversity analysis by using 29 pairs of SSR primers, and the SSR primers have the characteristics of good repeatability, clear spectrum band, high polymorphism and the like, and can be widely applied to bluegrass species identification, high-density genetic linkage map construction, diversity analysis and molecular assisted breeding.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an EST-SSR molecular marker related to cold resistance developed based on a transcriptome sequence of Poa frostii and its application. Background Art

[0002] Poa L. belongs to the Pooideae subfamily of the Gramineae family. It is mainly distributed in temperate and cold zones and is a sparse, bushy grass. Poa has become an important forage and precious lawn plant resource due to its high nutrition, good palatability, strong adaptation to adversity, rapid regeneration and high grazing resistance. Poa crymophila is a perennial plant of the Poaceae family. It is an important native grass species on the Qinghai-Tibet Plateau. It has strong cold resistance and is a valuable resource for breeding and development of new forage varieties.

[0003] SSR (Simple sequences repeat) markers are DNA fragments composed of tandem repeat sequences with 2 to 6 nucleotides as basic units. The length is mostly 100 to 200 base pairs. The main characteristics are high polymorphism, co-dominant inheritance, and close association with functional genes. According to different sources, they can be divided into genomic SSR and expressed sequence tag (Expressed Sequence Tag) SSR (EST-SSR). Traditional SSR markers are developed from random genomic sequences and have uncertain linkage with functional genes. However, SSR or EST-SSR molecular markers developed based on candidate genes have certain linkage with target traits and have broad application prospects in germplasm resource identification and molecular marker-assisted breeding. In recent years, new generation sequencing technologies such as transcriptome sequencing (RNA-seq) have provided high-throughput data, providing a fast, reliable and economical research tool for the identification and development of molecular markers. The genome information of cold-weather bluegrass is unknown, and transcriptome sequencing is the main method to develop its specific SSR primers. However, the development and application of SSR markers for cold-weather bluegrass cold resistance candidate genes using transcriptome sequencing has not been found so far.

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

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

[0006] In the first aspect, the present invention provides an EST-SSR molecular marker primer combination developed based on the transcriptome sequence of P. frostii, comprising 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] Wherein, the nucleotide sequence of the P1 primer pair is shown in SEQ ID NO.1-2;

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

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

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

[0011] The nucleotide sequence of the P17 primer pair is shown in SEQ ID NOs. 9-10;

[0012] The nucleotide sequence of the P18 primer pair is shown in SEQ ID NOs. 11-12;

[0013] The nucleotide sequence of the P21 primer pair is shown in SEQ ID NOs. 13-14;

[0014] The nucleotide sequence of the P23 primer pair is shown in SEQ ID NOs. 15-16;

[0015] The nucleotide sequence of the P37 primer pair is shown in SEQ ID NOs. 17-18;

[0016] The nucleotide sequence of the P38 primer pair is shown in SEQ ID NOs. 19-20;

[0017] The nucleotide sequence of the P54 primer pair is shown in SEQ ID NOs. 21-22;

[0018] The nucleotide sequence of the P65 primer pair is shown in SEQ ID NOs. 23-24;

[0019] The nucleotide sequence of the P71 primer pair is shown in SEQ ID NOs. 25-26;

[0020] The nucleotide sequence of the P72 primer pair is shown in SEQ ID NOs. 27-28;

[0021] The nucleotide sequence of the P88 primer pair is shown in SEQ ID NOs. 29-30;

[0022] The nucleotide sequence of the P89 primer pair is shown in SEQ ID NOs. 31-32;

[0023] The nucleotide sequence of the P91 primer pair is shown in SEQ ID NOs. 33-34;

[0024] The nucleotide sequence of the P92 primer pair is shown in SEQ ID NOs. 35-36;

[0025] The nucleotide sequence of the P93 primer pair is shown in SEQ ID NOs. 37-38;

[0026] The nucleotide sequence of the P94 primer pair is shown in SEQ ID NOs. 39-40;

[0027] The nucleotide sequence of the P95 primer pair is shown in SEQ ID NOs. 41-42;

[0028] The nucleotide sequence of the P98 primer pair is shown in SEQ ID NOs. 43-44;

[0029] The nucleotide sequence of the P99 primer pair is shown in SEQ ID NOs. 45-46;

[0030] The nucleotide sequence of the P148 primer pair is shown in SEQ ID NOs. 47-48;

[0031] The nucleotide sequence of the P150 primer pair is shown in SEQ ID NOs. 49-50;

[0032] The nucleotide sequence of the P192 primer pair is shown in SEQ ID NOs. 51-52;

[0033] The nucleotide sequence of the P193 primer pair is shown in SEQ ID NOs. 53-54;

[0034] The nucleotide sequence of the P195 primer pair is shown in SEQ ID NOs. 55-56;

[0035] The nucleotide sequence of the P197 primer pair is shown in SEQ ID NOs. 57-58.

[0036] In a second aspect, the present invention provides a kit comprising the above-mentioned EST-SSR molecular marker primer combination.

[0037] In some embodiments, the above-mentioned kit also includes at least one of a DNA extraction reagent, a DNA amplification reagent and an electrophoresis reagent.

[0038] In a third aspect, the present invention provides the use of an EST-SSR molecular marker primer combination or a kit in the construction of a genetic map of Poa pratensis or the analysis of genetic diversity.

[0039] In a fourth aspect, the present invention provides the use of an EST-SSR molecular marker primer combination or a kit in molecular marker-assisted breeding of bluegrass or protection of germplasm resources.

[0040] In a fifth aspect, the present invention provides the use of an EST-SSR molecular marker primer combination or a kit in the identification of Poa annua germplasm.

[0041] In some embodiments, the above-mentioned bluegrass germplasm includes: Qinghai cold-land bluegrass, Qinghai flat-stem bluegrass, Qinghai meadow bluegrass and Chinese gray bluegrass.

[0042] Based on the transcriptome data of bluegrass, the present invention designs SSR-related primers and performs genetic diversity analysis on them, providing specific polymorphic SSR markers for bluegrass species research. These markers can provide important reference value for subsequent evolutionary research and germplasm resource protection of bluegrass; in addition, based on the specific phenotypic characteristics of the sample individuals, the corresponding transcriptome data annotations can be combined to construct specific SSR markers associated with the excellent phenotypes of bluegrass, providing important guiding significance for the breeding of bluegrass varieties.

[0043] At the same time, the present invention also uses some primers in the above EST-SSR marker primer combination to identify the germplasm of different bluegrass varieties. The results show that any one or more pairs of primers in the above EST-SSR marker primer combination can identify and distinguish varieties such as Qinghai cold-land bluegrass, Qinghai flat-stemmed bluegrass, Qinghai meadow bluegrass and Chinese gray bluegrass. In order to prove this effect, the present invention also provides a method and result for identifying the above four varieties of bluegrass using any two of the above primer pairs (i.e., P37 primer pair and P148 primer pair):

[0044] A method for identifying bluegrass species with different cold resistance, comprising:

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

[0046] Perform electrophoresis detection on the amplified product to obtain the number of amplified bands and the size of the amplified fragment;

[0047] According to the number of bands and / or the difference in the size of the amplified fragments, the variety of the tested bluegrass is determined;

[0048] Among them, the early grasses to be tested include Qinghai cold-land early grass, Qinghai flat-stem early grass, Qinghai meadow early grass and Chinese gray early grass.

[0049] In some embodiments, P37 and P148 in the above EST-SSR marker primer combination are used to identify the variety of the tested bluegrass;

[0050] Among them, the 160bp bands of P37 primer pair were Qinghai flat stem bluegrass, Qinghai bluegrass bluegrass and Chinese gray bluegrass bluegrass;

[0051] PCR amplification was performed using the P148 primer pair. The species with 80bp, 100bp, 145bp, 175bp and 280bp bands was Qinghai flat-stem bluegrass, the species with 80bp and 145bp bands was Qinghai meadow bluegrass, the species with 65bp, 80bp, 100bp, 175bp and 190bp bands was Chinese gray bluegrass, and the species with 70bp and 75bp bands was Qinghai cold-land bluegrass.

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

[0053] In some embodiments, the amplification program for PCR amplification is: pre-denaturation at 94°C for 3 minutes, denaturation at 94°C for 30 seconds, annealing at 60°C to 55°C for 30 seconds, and extension at 72°C for 50 seconds, for a total of 5 cycles, with the annealing temperature decreasing by 1°C each cycle; denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 50 seconds, for a total of 29 cycles; and finally extension for 7 minutes and storage at 4°C.

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

[0055] The present invention develops an EST-SSR molecular marker primer combination for cold-weather bluegrass based on transcriptome sequencing data of leaf low-temperature resistance. The primer combination can be applied to the fields of bluegrass species identification, genetic pedigree analysis, genetic map construction, protection of germplasm resources and assisted breeding. 29 pairs of SSR primers are used to perform genetic diversity analysis on four bluegrass species, which has the characteristics of good repeatability, clear spectrum bands, high polymorphism, etc., and can be widely used in bluegrass species identification, high-density genetic linkage map construction, diversity analysis and molecular-assisted breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0057] Figure 1 The results of cold resistance evaluation of four cold-weather bluegrass species are based on the analysis of seven indicators, namely proline (Pro), soluble sugar (SS), abscisic acid (MDA), chlorophyll (Chl), fresh weight (FW), superoxide dismutase (SOD) and peroxidase activity (POD). The cold resistance of four bluegrass species, namely, Qinghai flat-stemmed bluegrass (BJ), Qinghai meadow bluegrass (CD), Chinese gray bluegrass (HH) and Qinghai cold-weather bluegrass (LD), was comprehensively evaluated by using the membership function method and then clustered.

[0058] Figure 2 The results of PCR amplification of 40 individual plants of four bluegrass species using primers P37 and P148. Different codes represent the genotypes of the individuals. B1-B10 represent 10 genotypes of BJ (Poa japonica), C1-C10 represent 10 genotypes of CD (Poa pratensis), H1-H10 represent 10 genotypes of HH (Poa chinensis), and L1-L10 represent 10 genotypes of LD (Poa frutescens).

[0059] Figure 3 The results of principal component analysis and cluster analysis of 29 EST-SSR markers on 4 different populations of the genus Poa; A is the principal coordinate analysis of 29 EST-SSR markers on 4 different populations of the genus Poa; B is the cluster analysis of 29 EST-SSR markers on 4 different populations of the genus Poa. Different codes represent the genotypes of individuals, B1~B10 represent 10 genotypes of BJ (flat-stemmed bluegrass), C1~C10 represent 10 genotypes of CD (meadow bluegrass), H1~H10 represent 10 genotypes of HH (Chinese bluegray bluegrass), L1~L10 represent 10 genotypes of LD (coldland bluegrass);

[0060] Figure 4Figure 2 shows the results of STRUCTURE analysis of 4 different populations of Poa using 29 EST-SSR markers; A is the average LnP(D) value of 20 runs at each K value; B is the optimal hierarchy with K=3 determined using the largest ΔK value; C is the three main groups of 40 Poa genotypes when K=3; in Figure C, the ordinate represents the membership coefficient of each genotype, and different codes represent the genotype of each individual, where 1-10 represent the 10 genotypes (B1 to B10) of BJ (Poa japonica), 11-20 represent the 10 genotypes (C1 to C10) of CD (Poa pratensis), 21-30 represent the 10 genotypes (H1 to H10) of HH (Poa sinomenina), 31-40 represent the 10 genotypes (L1 to L10) of LD (Poa sylvatica), and different colors represent different populations. DETAILED DESCRIPTION

[0061] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0062] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein may be applied to the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0063] Example 1

[0064] This example is the development of molecular markers linked to cold resistance candidate genes of Poa frostii, as follows:

[0065] In the early stage of this experiment, transcriptome sequencing of cold-tolerant leaves of Poa annua was carried out. Based on the transcriptome sequencing data, 200 EST-SSR primers for cold-tolerant candidate genes were mined and designed and synthesized according to gene function. The SSR loci were identified and located by Microsatellite (MISA) software, and SSR primers were designed by primer3-v2.3.4 software. The repeating units of the selected loci were dinucleotides, trinucleotides, tetranucleotides, pentanucleotides and hexanucleotides, and the minimum number of repeats was 9, 8, 7, 6, and 5 times, respectively. The sequences with a distance of more than 50 bp from both ends of the loci were used to design primers. The primer length was controlled at 18-22 bp, and the expected product length was 50-350 bp. Finally, 106 pairs of primers were screened. DNA of four Poa annua species was used as a template, and PCR amplification was performed using the above 106 pairs of SSR primers, and 29 pairs of primers with clear bands and good polymorphism were finally selected.

[0066] Main steps:

[0067] (1) Source of materials: Seeds of Qinghai coldland bluegrass (LD), Qinghai flat-stemmed bluegrass (BJ), Qinghai meadow bluegrass (CD) and Chinese gray bluegrass (HH) were provided by Qinghai Academy of Animal Husbandry and Veterinary Medicine. Among them, Qinghai coldland bluegrass, Qinghai flat-stemmed bluegrass and Qinghai meadow bluegrass are all cultivated varieties, and Chinese gray bluegrass bluegrass is the germplasm material.

[0068] (2) Obtaining SSR polymorphic sites: Total RNA was extracted from young leaves of cold-land bluegrass under low temperature treatment, and a transcriptome sequencing library was constructed. High-throughput sequencing was performed using an Illumina second-generation sequencer. The sequencing data was assembled after strict filtering, and 53,893 Unigenes were obtained. 12,972 SSR sites were successfully identified, and the frequency of SSR sites was 24.07%. These SSR sites were widely distributed in 10,766 Unigenes, accounting for 19.98% of all sequences. Among them, 2,367 sequences contained multiple SSRs, and 433 SSRs were present during the compound formation process. The identified SSR sequence types were rich, covering repeat sequences from mononucleotides to hexanucleotides, and the number of each type showed significant differences. Among them, trinucleotide repeat sequences were the most abundant, accounting for 41.84% of all repeat sequences (a total of 5,427).

[0069] (2) Based on gene function annotation, 200 candidate genes closely related to cold resistance were screened from 46,542 cold-weather bluegrass unigenes. SSR primers were designed using primer3-v2.3.4 software. The repeating units of the selected sites were dinucleotides, trinucleotides, tetranucleotides, pentanucleotides and hexanucleotides, with a minimum number of repeats of 9, 7, 6, 5 and 4 times, respectively. The sequences with a distance of more than 50 bp from both ends of the site were used to design primers. The primer length was controlled at 17-24 bp, and the expected product length was 100-300 bp. Finally, 106 pairs of primers were selected for amplification.

[0070] (3) DNA extraction: Genomic DNA was extracted from fresh leaf tissue samples collected from 40 individual plants of Poa annua using the plant genomic DNA extraction kit (Tiangen Biochemical Technology Co., Ltd., Beijing) using the adsorption column method. The DNA quality was measured using a NanoDrop ND1000 spectrophotometer, and the DNA that met the requirements was diluted to 25 ng / μL and stored at -20°C.

[0071] (4) performing PCR amplification using the 106 primer pairs to obtain a PCR product;

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

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

[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 repeatability were obtained.

[0075] Table 1 EST-SSR primer information

[0076]

[0077]

[0078]

[0079] Example 2

[0080] This embodiment is a method for identifying bluegrass species with different cold resistance, which is as follows:

[0081] Previously, based on principal component analysis of seven indicators, including proline, soluble sugar, abscisic acid, chlorophyll, fresh weight, and superoxide dismutase and peroxidase activities, the cold resistance of four bluegrass species was comprehensively evaluated and clustered by using the membership function method. The results are as follows Figure 1 Four bluegrass species with different cold resistance were identified using P37 and P148 markers, and the sources of the materials were the same as those 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 bluegrass material to be identified as a template, PCR amplification was performed using the primer pair P37 and P148, the primer information is shown in Table 1, and a PCR product was obtained;

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

[0086] (4) The PCR amplification procedure included 5 cycles of pre-denaturation at 94°C for 3 min, denaturation at 94°C for 30 s, annealing at 60°C–55°C for 30 s, and extension at 72°C for 50 s, with the annealing temperature decreasing by 1°C each cycle; 29 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 50 s; and a final extension of 7 min and storage at 4°C.

[0087] (5) performing acrylamide gel electrophoresis, staining and developing the obtained PCR products;

[0088] (6) Analyze the amplified electrophoresis bands. The results are as follows: Figure 2As shown: primer pair P37 was used for PCR amplification, and the three species with specific characteristic bands of 160bp were Qinghai flat-stem bluegrass, Qinghai meadow bluegrass and Chinese gray bluegrass. Primer pair P148 was used for PCR amplification, and the species with bands of 80bp, 100bp, 145bp, 175bp and 280bp were Qinghai flat-stem bluegrass, the species with bands of 80bp and 145bp were Qinghai meadow bluegrass, the species with bands of 65bp, 80bp, 100bp, 175bp and 190bp were Chinese gray bluegrass, and the species with bands of 70bp and 75bp were Qinghai cold-land bluegrass. Therefore, bluegrass with different cold resistance can be identified by using these characteristic band combinations.

[0089] Example 3

[0090] This example is an analysis of genetic diversity of 4 bluegrass species using 29 markers, as follows:

[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 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, and a PCR product was obtained;

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

[0095] (4) performing acrylamide gel electrophoresis, staining and developing the obtained PCR products;

[0096] (5) The 29 pairs of polymorphic EST-SSR primer amplification bands were statistically analyzed using the "0, 1" matrix using EXCEL 2019. The software GenAlEx and POPGENE (v1.31) were used for genetic distance and other related analyses. The STRUCTURE-v2.3.2 software was used to subdivide the individuals into different subgroups and perform cluster analysis. In addition, based on the Jaccard genetic similarity matrix, the DCENTER module in the NTSYS (v2.10) software was used to perform principal component analysis (PCoA).

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

[0098] Table 2 Polymorphism analysis of 29 pairs of EST-SSR primers in four Poa species

[0099]

[0100]

[0101] As shown in Table 2 above, 29 polymorphic primers amplified a total of 425 bands, with an average of 14.7 bands amplified by each primer. The number of amplified bands ranged from 6 (P17 and P72) to 25 (P193), with an average of 14.7 bands (Table 4-5). The average percentage of polymorphic bands ranged from 56.52% to 100%, and the average percentage of polymorphic bands was 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) values ​​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 cold-weather bluegrass population is the highest (Na=1.23, Ne=1.30, I=0.26, H=0.17, %P=48.71%), followed by the meadow 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%), and the genetic diversity of the Chinese gray bluegrass population is 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 AMONA analysis of four different populations of Poa annua

[0109] source Degrees of Freedom Variance and Average variance Variation 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] As shown in Table 4 above, the variation between different populations accounts for 87% of the total variation, while the genetic variation within a population accounts for only 22%.

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

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

[0113] Population Poa serrata Kentucky bluegrass Poa chinensis Poa frostii Poa serrata **** 0.79 0.63 0.59 Kentucky bluegrass 0.23 **** 0.61 0.65 Poa chinensis 0.46 0.49 **** 0.57 Poa frostii 0.53 0.44 0.57 ****

[0114] From Table 5 above, we can see that the Nei's genetic distance of the four populations of the genus Poa ranges from 0.23 (Poa serrata and Poa pratensis) to 0.57 (Poa serrata and Poa pratensis), and the genetic consistency ranges from 0.57 (Poa serrata and Poa pratensis) to 0.79 (Poa serrata and Poa pratensis).

[0115] (10) The results of principal component analysis (PCoA) are shown in Figure 3 A. The two main principal components can explain 87.3% of the genetic variation in 40 individual plants of the genus Poa, of which the first principal component accounts for 48.82% and the second principal component accounts for 38.48%. The poor cold-resistant Poa pratensis and Poa scutellariae were grouped into one group, the cold-resistant Poa scutellariae was grouped into one group, and the Chinese gray Poa was grouped into one group.

[0116] (11) The results of the UPGMA unweighted group average cluster analysis are shown in Figure 3 B, When the genetic similarity coefficient was 0.70, the four bluegrass populations were divided into three major categories. The first category included all the plants of meadow bluegrass (CD) and flat bluegrass (BJ), the second category included all the plants of Chinese bluegrass (HH), and the third category included all the plants of coldland bluegrass (LD).

[0117] (12) STRUCTURE analysis results are shown in Figure 4 , when the optimal K value is 3, the optimal number of groups is 3. The first group is green, including CD and BJ populations with weaker cold resistance; 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 plant can be obtained based on the color ratio. BJ and CD have a close genetic relationship (K=3).

[0118] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

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

2. A kit, characterized in that: Contains the EST-SSR molecular marker primer combination according to claim 1.

3. Use of the EST-SSR molecular marker primer combination according to claim 1 or the kit according to claim 2 in the construction of a genetic map of bluegrass or the analysis of genetic diversity.

4. Use of the EST-SSR molecular marker primer combination according to claim 1 or the kit according to claim 2 in bluegrass molecular marker-assisted breeding or germplasm resource protection.

5. Use of the EST-SSR molecular marker primer combination according to claim 1 or the kit according to claim 2 in identifying bluegrass germplasm.

6. The use according to claim 5, characterized in that: The bluegrass germplasm includes: Qinghai cold-land bluegrass, Qinghai flat-stem bluegrass, Qinghai meadow bluegrass and Chinese gray bluegrass.

7. A method for identifying bluegrass varieties with different cold resistance, characterized in that: include: Using the DNA of the bluegrass to be tested as a template, PCR amplification is performed using the EST SSR marker primer combination of claim 1 to obtain an amplified product; Perform electrophoresis on the amplified product to obtain the number of amplified bands and the size of the amplified fragment; According to the number of bands and / or the difference in the size of the amplified fragments, the variety of the tested bluegrass is determined; Among them, the early grasses to be tested include Qinghai cold-land early grass, Qinghai flat-stem early grass, Qinghai meadow early grass and Chinese gray early grass.

8. The identification method according to claim 7, characterized in that: Using the P37 primer pair and the P148 primer pair in the EST SSR marker primer combination to identify the variety of the bluegrass to be tested; PCR amplification was performed using the P37 primer pair: the 160 bp bands were Qinghai flat-stem bluegrass, Qinghai bluegrass and Chinese gray bluegrass; PCR amplification was performed using the P148 primer pair: the one with 80bp, 100bp, 145bp, 175bp and 280bp bands was Qinghai flat-stem bluegrass, the one with 80bp and 145bp bands was Qinghai meadow bluegrass, the one with 65bp, 80bp, 100bp, 175bp and 190bp bands was Chinese gray bluegrass, and the one with 70bp and 75bp bands was Qinghai cold-land bluegrass.

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

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

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