Pennisetum alopecuroides cold resistance identification method based on 65 core molecular marker sets, primer group and gene chip and application of pennisetum alopecuroides cold resistance identification method

Abstract

The invention relates to the technical field of molecular biology, and discloses a pennisetum alopecuroides cold resistance identification method based on 65 core molecular marker sets, a primer group and a gene chip and application of the pennisetum alopecuroides cold resistance identification method. According to the method, core germplasm resources of pennisetum alopecuroides are systematically collected, and the GWAS population is constructed by utilizing multi-environment (different altitudes) conditions, so that wide genetic background and phenotypic variation are fully covered, and a material foundation is laid for cold-resistant genetic analysis. In the aspect of cold-resistant phenotype identification, leaf chlorophyll content and green leaf percentage after natural low-temperature stress in autumn and winter fields are innovatively taken as cold-resistant evaluation indexes, and individuals with excellent allelic variation number greater than 40 of a pennisetum alopecuroides material are judged as high-cold-resistant varieties; a rapid and lossless phenotype screening method which can be implemented in the seedling stage is established.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular biology, and discloses a cold tolerance identification method for Pennisetum based on a 65-core molecular marker set, a primer set and a gene chip, and application thereof. BACKGROUND

[0002] As an important economic species with high forage yield and high-efficiency energy plant value, Pennisetum exhibits great application potential in tropical and subtropical regions.

[0003] The optimum growth temperature of Pennisetum is 30-35℃, and the growth stagnates when the temperature is lower than 10℃, and the whole plant (including the aboveground and underground parts) can die under sustained low temperature. With the increase of latitude, the dry matter yield of Pennisetum significantly decreases, from 80 tons / ha in tropical regions to about 45 tons / ha at 30 degrees north latitude, and further to 22-30 tons / ha at 36 degrees north latitude, which greatly limits the application of Pennisetum in cold regions at high latitudes.

[0004] At present, the evaluation of cold tolerance of Pennisetum mainly depends on physiological indexes measured in the laboratory under controlled conditions at the seedling stage. However, these indexes have weak correlation with the actual overwintering performance in the field, and it is difficult to accurately reflect the real cold resistance of the plant in the field environment, so they cannot provide reliable basis for cold tolerance breeding. The existing field cold tolerance phenotype identification not only has long operation period and high labor intensity, but also is easily disturbed by the environment, and is difficult to be applied to the efficient screening of large-scale breeding populations, which seriously delays the process of cold tolerance resource excavation and cold tolerance variety breeding.

[0005] Therefore, it is urgent to establish an efficient and accurate early identification technology system for cold tolerance at the seedling stage. The SNP sites related to the chlorophyll content and the percentage of green leaves after low-temperature stress of leaves are located by using whole genome association analysis (GWAS), and molecular markers that can be used for assisted selection are developed, which is expected to realize accurate prediction of cold tolerance at the seedling stage. This strategy can significantly reduce the dependence on traditional overwintering phenotype identification, greatly improve the screening efficiency, that is, a single technician can complete the genotype detection of hundreds of samples per day, effectively reduce the misjudgment risk caused by environmental variation, improve the accuracy and efficiency of selection, shorten the breeding period, and accelerate the process of cold tolerance resource excavation and cold tolerance variety breeding of Pennisetum. The popularization of such molecular marker assisted selection technology will provide key support for breaking through the bottleneck of cold tolerance breeding of Pennisetum.

[0006] In addition, on the one hand, there are differences in the understanding of those skilled in the art; on the other hand, the inventors have studied a large number of literatures and patents when making the present application, but due to the limited space, all the details and contents are not listed in detail, which does not mean that the present application does not have these characteristics of the prior art, on the contrary, the present application has all the characteristics of the prior art, and the applicant reserves the right to add relevant prior art in the background. SUMMARY

[0007] The application relates to the technical field of molecular biology, and discloses a cold tolerance identification method for Pennisetum based on a 65-core molecular marker set, a primer set and a gene chip and application thereof.

[0008] In view of the above technical problems, one of the purposes of the application is to provide a 65-core molecular marker set, which comprises SNP markers numbered as SNP 1-65 as follows: The SNP marker is base G:A, and the position is chrB1_64515476 of the Pennisetum; The SNP marker is base G:A, and the position is chrB7_29024501 of the Pennisetum; The SNP marker is base G:A, and the position is chrB7_45105115 of the Pennisetum; The SNP marker is base T:A, and the position is chrB7_27795665 of the Pennisetum; The SNP marker is base T:C, and the position is chrB7_27795662 of the Pennisetum; The SNP marker is base G:A, and the position is chrB7_49893610 of the Pennisetum; The SNP marker is base G:A, and the position is chrB6_29986440 of the Pennisetum; The SNP marker is base G:A, and the position is chrB7_15272734 of the Pennisetum; The SNP marker is base G:C, and the position is chrB7_119411306 of the Pennisetum; The SNP marker is base G:A, and the position is chrB7_50693409 of the Pennisetum; The SNP marker is base G:A, and the position is chrB7_50541147 of the Pennisetum; The SNP marker is base G:A, and the position is chrA3_96624776 of the Pennisetum; The SNP marker is base G:A, and the position is chrA7_45758326 of the Pennisetum; The SNP marker is base G:A, and the position is chrB4_41545400 of the Pennisetum; The SNP marker is base G:A, and the position is chrB1_43049394 of the Pennisetum; The SNP marker is base G:A, and the position is chrB6_133829538 of the Pennisetum; SNP marker is base G:A, position is chrA3_79205623 of Pennisetum ciliare; SNP marker is base T:C, position is chrB6_186861238 of Pennisetum ciliare; SNP marker is base G:A, position is chrB7_49308097 of Pennisetum ciliare; SNP marker is base G:A, position is chrB2_81759859 of Pennisetum ciliare; SNP marker is base T:A, position is chrB7_111295430 of Pennisetum ciliare; SNP marker is base T:G, position is chrA5_40602857 of Pennisetum ciliare; SNP marker is base G:A, position is chrB7_64811713 of Pennisetum ciliare; SNP marker is base G:A, position is chrA1_1132398 of Pennisetum ciliare; SNP marker is base G:A, position is chrB2_46430985 of Pennisetum ciliare; SNP marker is base G:C, position is chrB2_74078794 of Pennisetum ciliare; SNP marker is base G:A, position is chrB6_183157373 of Pennisetum ciliare; SNP marker is base C:A, position is chrB7_20908754 of Pennisetum ciliare; SNP marker is base A:G, position is chrA5_64602902 of Pennisetum ciliare; SNP marker is base G:A, position is chrB2_73078206 of Pennisetum ciliare; SNP marker is base G:A, position is chrB7_46588580 of Pennisetum ciliare; SNP marker is base A:G, position is chrA3_79053484 of Pennisetum ciliare; SNP marker is base T:C, position is chrA3_79012904 of Pennisetum ciliare; SNP marker is base A:G, position is chrB4_18322082 of Pennisetum ciliare; SNP marker is base A:G, position is chrB7_56383576 of Pennisetum ciliare; SNP marker is base A:G, position is chrA3_82339032 of Pennisetum ciliare; SNP marker is base C:T, position is chrB2_81759922 of Pennisetum; SNP marker is base C:T, position is chrB2_13004238 of Pennisetum; SNP marker is base A:G, position is chrB6_93488911 of Pennisetum; SNP marker is base C:T, position is chrB2_46167039 of Pennisetum; SNP marker is base C:T, position is chrB4_117851973 of Pennisetum; SNP marker is base C:T, position is chrA1_163181362 of Pennisetum; SNP marker is base C:T, position is chrA5_42762088 of Pennisetum; SNP marker is base C:T, position is chrB2_79722191 of Pennisetum; SNP marker is base G:A, position is chrA2_4199397 of Pennisetum; SNP marker is base C:T, position is chrB7_68554707 of Pennisetum; SNP marker is base C:T, position is chrB2_38519659 of Pennisetum; SNP marker is base A:T, position is chrA5_6312866 of Pennisetum; SNP marker is base C:T, position is chrA5_6312867 of Pennisetum; SNP marker is base C:T, position is chrA7_135412766 of Pennisetum; SNP marker is base C:T, position is chrB4_23125675 of Pennisetum; SNP marker is base C:T, position is chrA5_12051038 of Pennisetum; SNP marker is base C:G, position is chrB7_6855308 of Pennisetum; SNP marker is base A:T, position is chrB7_46326638 of Pennisetum; SNP marker is base A:T, position is chrB2_13004143 of Pennisetum; SNP marker is base C:T, position is chrB7_68652993 of Pennisetum; The SNP marker is base C:T, and the position is Scaffold UN_7865923 of Pennisetum. The SNP marker is base C:T, and the position is Scaffold UN_7865923 of Pennisetum. The SNP marker is base C:T, and the position is Scaffold UN_7865923 of Pennisetum. The SNP marker is base C:T, and the position is Scaffold UN_7865923 of Pennisetum. The SNP marker is base C:T, and the position is Scaffold UN_7865923 of Pennisetum. The SNP marker is base C:T, and the position is Scaffold UN_7865923 of Pennisetum. The SNP marker is base C:T, and the position is Scaffold UN_7865923 of Pennisetum. The SNP marker is base C:T, and the position is Scaffold UN_7865923 of Pennisetum. The SNP marker is base C:T, and the position is Scaffold UN_7865923 of Pennisetum.

[0009] The SNP molecular marker set provided by the application is a specific SNP molecular marker set, and the core is to use 65 core SNP molecular marker sets as detection targets, and a corresponding primer group, detection reagent, kit and gene chip are developed based on the detection targets.

[0010] One of the purposes of the application is to provide the application of the 65 core molecular marker set as a detection target in identifying the cold tolerance of Pennisetum, and the 65 core molecular marker set includes the 65 core molecular marker set for identifying the cold tolerance of Pennisetum.

[0011] According to a preferred embodiment, when more than 40 markers are advantage haplotypes, the Pennisetum individual is a high cold-tolerant material.

[0012] According to a preferred embodiment, when less than or equal to 40 markers are advantage haplotypes, the Pennisetum individual is a low cold-tolerant material.

[0013] According to a preferred embodiment, the Pennisetum used for identification is a seedling stage Pennisetum.

[0014] According to a preferred embodiment, the application is: Pennisetum germplasm identification and improvement; or Application in early prediction of cold tolerance of Pennisetum seedling stage.

[0015] One of the purposes of the present application is also to provide a primer set for amplifying the cold tolerance identification target point of Pennisetum.

[0016] One of the purposes of the present application is also to provide a detection reagent comprising the core molecular marker set in the above application or the primer set.

[0017] One of the purposes of the present application is also to provide a kit comprising the core molecular marker set in the above application or the primer set.

[0018] One of the purposes of the present application is also to provide a gene chip comprising the core molecular marker set in the above application or the primer set.

[0019] One of the purposes of the present application is also to provide a reaction system of the primer set, and the total reaction system of PCR amplification is 25 μL, including 2× Taq Pro Master Mix 12.5 μL, 0.8 μL of forward and reverse primers (10 μM) respectively, 1.0 μL of template DNA (20-50 ng / μL), and ddH2O to make up to 25 μL.

[0020] One of the purposes of the present application is also to provide a method for identifying a cold-tolerant variety of Pennisetum, comprising the following steps: The genomic DNA of the to-be-detected variety is amplified by using the primers of SEQ ID NO:1-SEQ ID NO:128, and 65 SNP markers and the determined typing results are combined.

[0021] When more than 40 markers are the dominant haplotype, it indicates that the chlorophyll content of the leaf of the individual is higher than the average level of the chlorophyll content of the leaves of the population after low-temperature stress. The result shows that the individual is a high-cold-tolerance resource.

[0022] When less than or equal to 40 markers are the dominant haplotype, it indicates that the chlorophyll content of the leaf is lower than the average level of the chlorophyll content of the leaves of the population. The result shows that the individual is a low-cold-tolerance resource.

[0023] One of the purposes of the present application is also to provide a method for identifying a cold-tolerant variety of Pennisetum, comprising the following steps: If the chlorophyll content of the leaf of the individual Pennisetum is higher than the average level of the chlorophyll content of the leaves of the population, the individual is identified as a cold-tolerant variety.

[0024] The present application has the following beneficial effects: The present application collects the core germplasm resources of Pennisetum by system, and uses the multi-environment (different altitudes) conditions to construct GWAS population, fully covers the wide genetic background and phenotype variation, and lays the material foundation for the genetic analysis of cold tolerance. In the aspect of cold tolerance phenotype identification, the present application innovatively takes the leaf chlorophyll content and the percentage of green leaves as the evaluation indexes of cold tolerance, determines the individual with the chlorophyll content higher than the average level of the population as the high cold-tolerant material, and establishes a rapid and non-destructive phenotype screening method which can be implemented at the seedling stage.

[0025] Based on the high-throughput genotyping and whole genome association analysis, 65 SNP sites significantly related to the photosynthetic capacity (chlorophyll content and percentage of green leaves) of Pennisetum under low-temperature stress in the field are successfully identified, and specific molecular marker primers with clear product characteristics and flanking sequence information are designed, so that the efficient transformation from genetic sites to practical molecular marker system is realized. At the same time, the standardized PCR and genotyping process is established, so that the operation is repeatable and the result is accurate, and the rapid and batch detection capacity is provided, which provides reliable technical support for the breeding application of molecular markers.

[0026] The developed molecular markers are significantly related to the cold tolerance phenotype of the chlorophyll content of Pennisetum leaves after low-temperature stress, and can be used for early prediction of the cold tolerance potential of the plant at the seedling stage. The technology effectively overcomes the limitations of long identification period and environmental interference in traditional field overwintering identification, realizes the indirect screening of high cold-tolerant germplasm at the seedling stage through genotyping, greatly improves the screening efficiency and accuracy, significantly shortens the breeding period, and provides key technical support for the breeding of new cold-tolerant varieties of Pennisetum. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 For correlation analysis of the percentage of green leaves and the chlorophyll content of Pennisetum in the field after low-temperature stress in autumn; Figure 2 For correlation analysis of the number of dominant haplotypes and the chlorophyll content after low-temperature stress; Figure 3 For correlation analysis of the number of dominant haplotypes and the percentage of green leaves after low-temperature stress; Figure 4 For haplotype analysis diagram of the first part of 65 cold-tolerant SNP sites of Pennisetum; Figure 5 For haplotype analysis diagram of the second part of 65 cold-tolerant SNP sites of Pennisetum; Figure 6 For haplotype analysis diagram of the third part of 65 cold-tolerant SNP sites of Pennisetum; Figure 7 For haplotype analysis diagram of the fourth part of 65 cold-tolerant SNP sites of Pennisetum; Figure 8Haplotype analysis diagram for the fifth part of 65 cold tolerance SNP loci of Pennisetum Figure 9 Haplotype analysis diagram for the sixth part of 65 cold tolerance SNP loci of Pennisetum Figure 10 Haplotype analysis diagram for the seventh part of 65 cold tolerance SNP loci of Pennisetum Figure 11 Haplotype analysis diagram for the eighth part of 65 cold tolerance SNP loci of Pennisetum Figure 12 Haplotype analysis diagram for the ninth part of 65 cold tolerance SNP loci of Pennisetum Figure 13 Haplotype analysis diagram for the tenth part of 65 cold tolerance SNP loci of Pennisetum Figure 14 Haplotype analysis diagram for the eleventh part of 65 cold tolerance SNP loci of Pennisetum Figure 15 Haplotype analysis diagram for the twelfth part of 65 cold tolerance SNP loci of Pennisetum Figure 16 Haplotype analysis diagram for the thirteenth part of 65 cold tolerance SNP loci of Pennisetum Figure 17 Haplotype analysis diagram for the fourteenth part of 65 cold tolerance SNP loci of Pennisetum Figure 18 Haplotype analysis diagram for the fifteenth part of 65 cold tolerance SNP loci of Pennisetum Figure 19 Haplotype analysis diagram for the sixteenth part of 65 cold tolerance SNP loci of Pennisetum Figure 20 Haplotype analysis diagram for the seventeenth part of 65 cold tolerance SNP loci of Pennisetum Figure 21 Haplotype analysis diagram for the eighteenth part of 65 cold tolerance SNP loci of Pennisetum Figure 22 Haplotype analysis diagram for the nineteenth part of 65 cold tolerance SNP loci of Pennisetum Figure 23 Haplotype analysis diagram for the twentieth part of 65 cold tolerance SNP loci of Pennisetum Figure 24 Haplotype analysis diagram for the twenty-first part of 65 cold tolerance SNP loci of Pennisetum Figure 25 Haplotype analysis diagram for the twenty-second part of 65 cold tolerance SNP loci of Pennisetum Figure 26 Field phenotypes of different chlorophyll contents of Pennisetum materials after low temperature stress in autumn DETAILED DESCRIPTION

[0028] In the description of the present application, the terms are used only for descriptive purposes, and cannot be construed to indicate or imply relative importance or imply the number of indicated technical features. In the description of the present application, the meaning of "a plurality of" is two or more, unless otherwise specifically limited.

[0029] Unless otherwise specified, the test methods used in the following examples are conventional methods; the materials, reagents or instruments used are commercially available reagents and materials; if the specific conditions are not specified in the examples, the conventional conditions or the conditions recommended by the manufacturer are used; and the present application does not limit the source of the raw materials used, unless otherwise specified, the raw materials used in the present application are ordinary commercially available products in the technical field.

[0030] Pennisetum is a warm C4 plant, which has poor cold tolerance, which restricts its planting in high latitude and high altitude areas. Therefore, building a scientific cold tolerance evaluation system has important value for cold tolerance resource screening and variety breeding. Chlorophyll content and green leaf percentage after autumn low temperature stress have become important physiological and morphological indicators for evaluating the cold tolerance of Pennisetum. The effectiveness of this method is based on the physiological nature of low temperature damage. In the autumn gradual cold environment, although the plant has not died, low temperature will directly damage the photosynthetic structure, accelerate the degradation of chlorophyll and cause tissue chlorosis and necrosis. Whether the plant can maintain physiological activity and store energy for wintering mainly depends on whether the photosynthetic system can maintain functional integrity at low temperature and whether the cell membrane can maintain stability. Chlorophyll content directly reflects the damage degree of chloroplast structure and function, and green leaf percentage reflects the ability of the plant to resist low temperature damage and maintain tissue survival at the organ level. Therefore, chlorophyll content can be used as an early and sensitive physiological indicator of cold tolerance, and green leaf percentage is a direct and overall evaluation of the survival state under low temperature. The combination of the two can provide a systematic and quantifiable evaluation basis for cold tolerance.

[0031] From the physiological mechanism, chlorophyll content and green leaf percentage, as key indicators of photosynthesis, directly affect the synthesis of autumn photosynthetic products and their transport to underground organs. Higher chlorophyll content and green leaf percentage in individual plants usually accumulate more non-structural carbohydrates such as starch and soluble sugars, which help maintain cell osmotic balance and membrane structure stability. Therefore, they often show stronger cold resistance. In summary, chlorophyll content and green leaf percentage, as key parameters reflecting the photosynthetic physiological state under field low temperature stress, are important indicators for evaluating the cold tolerance of Pennisetum.

[0032] The present application constructs four Pennisetum GWAS populations at different altitudes, and uses single-root double-stem-node oblique insertion method for standardized planting at each point to ensure normal growth and tillering of the plants. For example, Figure 26The chlorophyll content (using CL-01 chlorophyll content meter Hansatech) or the percentage of green leaves (by counting the number of green leaves of Pennisetum purpureum accounting for the total number of leaves, and the leaves with more than 50% yellowing were yellow leaves) was determined as the key indicator for evaluating the cold tolerance of Pennisetum purpureum.

[0033] 1. Correlation research of 65 core molecular markers Fresh tissues were collected from the middle leaves of each sample, and high-quality genomic DNA was obtained using the TIANGEN high-efficiency plant genomic DNA extraction kit. Subsequently, whole-genome resequencing was performed on the constructed GWAS population with an average sequencing depth of 10x; strict quality control was performed on the raw data to remove adapters and low-quality reads. Using the published Pennisetum purpureum reference genome (2.07 GB) as the alignment basis, biological software was used for variant detection, and biological software was used to screen SNP sites (parameter settings: --maf 0.05--max-missing 0.8). The filtered high-quality SNP data set was subjected to whole-genome association analysis with the two cold tolerance phenotype data of chlorophyll content and green leaf percentage determined in the field; the analysis used a mixed linear model of biological software, and the screening standard was P<10 -6 On this basis, haplotype analysis was performed on the significantly associated sites, and finally 65 core SNP markers were identified, which were closely related to cold tolerance and had extremely significant differences in phenotypes between different haplotypes.

[0034] Combined with the collected data of the field phenotype traits of the samples, the results are shown in Figure 1 The chlorophyll content and the percentage of green leaves under low temperature stress in the field of Pennisetum purpureum showed a significant positive correlation, with a Pearson correlation coefficient r=0.714.

[0035] The results are shown in Figure 2 The number of dominant haplotypes showed a significant positive correlation with the chlorophyll content under low temperature stress, with a Pearson correlation coefficient r=0.808. The results are shown in Figure 3 The number of dominant haplotypes showed a significant positive correlation with the percentage of green leaves under low temperature stress, with a Pearson correlation coefficient r=0.816. The correlation results show that chlorophyll content and the percentage of green leaves are important indicators for evaluating the cold tolerance of Pennisetum purpureum.

[0036] Figures 4-25Table 1 shows the information of 65 cold tolerance significantly associated SNP markers of Pennisetum alopecuroides. The information includes chromosome information where the SNP is located, SNP specific location information, haplotype type, cold tolerance haplotype, cold-sensitive haplotype reference allele, variant allele, location of the variant, and primer pair of the sequence where the amplification site is located. NON SYNONYMOUS CODING indicates that the variation occurring in the protein coding region results in the change of amino acid sequence. SYNONYMOUS CODING indicates that the variation is performed by different codons of the same amino acid. UPSTREAM indicates the variation in the non-coding region upstream of the gene. UTR 3 PRIME indicates the non-coding sequence region of the messenger RNA (mRNA) immediately after the coding region (CDS), after the stop codon to the 3' end of the mRNA. The 65 sites are closely related to 59 genes, 1 site is located in the 3' untranslated region, 40 sites are located in the upstream regulatory region of the promoter, 8 sites are located in the exon region, and the base variation of 16 sites causes synonymous coding variation.

[0037] 2. Function verification Again, 4 Pennisetum alopecuroides GWAS populations were constructed at different altitudes to verify the effectiveness of the above molecular marker set.

[0038] Table 2 shows the information of the cold tolerance SNP site molecular marker primers of Pennisetum alopecuroides. The information includes SNP name, Tm value and GC content of the upstream and downstream primer sequences shown in Table 1.

[0039] Using the DNA of each material as the amplification template, a 25 μL reaction system was used: 2× Taq Pro Master Mix 12.5 μL, 0.8 μL of forward and reverse primers (10 μM) each, 1.0 μL of template DNA (20–50 ng / μL), and ddH2O to make up to 25 μL.

[0040] The PCR reaction program is as follows: 95℃ pre-denaturation for 5 minutes; Then 35 cycles of amplification, including 95℃ denaturation for 30 seconds, 58–62℃ annealing for 30 seconds (adjusted according to the Tm value of the primer), and 72℃ extension for 45 seconds; Final 72℃ extension for 7 minutes.

[0041] 3–5 μL of the product in the PCR reaction system was added to a 1.5% agarose gel for electrophoresis detection. The gel cutting method was used to cut the target band and the DNA gel recovery kit was used to purify the product. After purification, Sanger sequencing was used for genotype confirmation.

[0042] Based on the verification of the results, the sample phenotype and the corresponding analysis results of the sequence sites show that: among the 65 cold-tolerant core SNP sites of Pennisetum ciliare, when the excellent allelic variation number of 65 sites is less than or equal to 40, there is a 95.56% probability that the leaf chlorophyll content is lower than the average value after field low temperature stress; when the excellent allelic variation number is greater than 40, there is a 82.96% probability that the leaf chlorophyll content is higher than the average value (26.81 SPAD) after field stress. When the excellent allelic variation number of 65 sites is less than or equal to 40, there is a 80.64% probability that the percentage of green leaves is lower than the average value after field low temperature stress; when the excellent allelic variation number is greater than 40, there is a 89.26% probability that the leaf chlorophyll content is higher than the average value (0.444) after field stress.

[0043] Table 1

[0044] Table 2

[0045] It should be noted that the above specific embodiments are exemplary, and those skilled in the art can think of various solutions under the inspiration of the disclosure of the present application, and these solutions also belong to the disclosed range of the present application and fall within the protection scope of the present application. Those skilled in the art should understand that the specification and drawings of the present application are illustrative and do not constitute a limitation on the claims. The protection scope of the present application is defined by the claims and their equivalents.

Claims

1. A set of 65 core molecular markers for cold tolerance identification of Pennisetum spicatum, characterized in that, The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The SNP marker is base G:A, and the position is chrBl_64515476 of Pennisetum. The The SNP is labeled with the base G:C and is located at the chrB2_74078794th position of Pennisetum var. chinensis. The SNP is labeled with the base G:A, and its position is the chrB6_183157373rd position of the foxtail grass. The SNP is labeled with the base C:A, and its position is the chrB7_20908754th position of the grass. The SNP is labeled with the base A:G and is located at the chrA5_64602902nd position of the Napier grass. The SNP is labeled with the base G:A, and its position is the chrB2_73078206th position of the Napier grass. The SNP is labeled with the base G:A, and its position is the chrB7_46588580th position of the Napier grass. The SNP is labeled with the base A:G and is located at the chrA3_79053484th position of Pennisetum var. chinensis. The SNP is labeled with the base T:C and is located at the chrA3_79012904th position of the grass; The SNP is labeled with the base A:G and is located at the chrB4_18322082nd position of the grass. The SNP is labeled with the base A:G and is located at the chrB7_56383576th position of Pennisetum var. mongolicum. The SNP is labeled with the base A:G and is located at the chrA3_82339032nd position of the grass. The SNP is labeled with the base C:T and is located at the chrB2_81759922nd position of the grass. The SNP is labeled with the base C:T and is located at the chrB2_13004238th position of the Napier grass. The SNP is labeled with base A:G and is located at position chrB6_93488911 of the Napier grass. The SNP is labeled with the base C:T and is located at the chrB2_46167039th position of the grass; The SNP is labeled with the base C:T and is located at the chrB4_117851973rd position of Pennisetum var. chinensis. The SNP is labeled with the base C:T and is located at the chrA1_163181362nd position of the grass; The SNP is labeled with the base C:T and is located at position chrA5_42762088 of the grass; The SNP is labeled with the base C:T and is located at the chrB2_79722191st position of the grass. The SNP is labeled with the base G:A, and its position is the chrA2_4199397th position of the grass. The SNP is labeled with the base C:T and is located at the chrB7_68554707th position of the grass; The SNP is labeled with the base C:T and is located at the chrB2_38519659th position of the grass. The SNP is labeled with the base A:T and is located at the chrA5_6312866th position of the grass; The SNP is labeled with the base C:T and is located at position chrA5_6312867 of the grass; The SNP is labeled with the base C:T and is located at the chrA7_135412766th position of the grass; The SNP is labeled with the base C:T and is located at the chrB4_23125675th position of the foxtail grass. The SNP is labeled with the base C:T and is located at the chrA5_12051038th position of the Napier grass. The SNP is labeled with the base C:G and is located at the chrB7_6855308th position of Pennisetum var. mongolicum. The SNP is labeled with the base A:T and is located at the chrB7_46326638th position of the grass; The SNP is labeled with the base A:T and is located at the chrB2_13004143rd position of the grass; The SNP is labeled with the base C:T and is located at the chrB7_68652993rd position of the grass. The SNP is labeled with the base C:T and is located at the chrB2_8958258th position of the grass. The SNP is labeled with the base C:T and is located at the ScaffoldUN_7865923rd position of Pennisetum spp.; The SNP is labeled with the base A:T and is located at the chrA7_86889323rd position of the grass. The SNP is labeled with the base C:T and is located at position chrA7_86889337 of the grass; The SNP is labeled with the base C:T and is located at the chrA2_117167685th position of the grass; The SNP is labeled with the base C:T and is located at the chrB6_183157018th position of the grass; The SNP is labeled with the base C:G and is located at the chrB5_19579799th position of the grass; The SNP is labeled with the base C:T and is located at the chrB4_29160095th position of the grass; The SNP is labeled with the base C:T and is located at position chrB7_46656020 of the Napier grass.

2. The use of the 65 core molecular marker set as a detection target in identifying cold tolerance of Pennisetum for the purpose of breeding, characterized in that, The 65 core molecular marker set includes the 65 core molecular marker set for identifying the cold resistance of Napier grass as described in claim 1.

3. Use according to claim 2, characterized in that, When more than 40 individuals of *Pennisetum affine* are marked as dominant haplotypes, it indicates that the individual is a highly cold-resistant material.

4. Use according to claim 2, characterized in that, When there are 40 or fewer haplotypes marked as dominant, it indicates that the individual is a low-hardiness Napier grass material.

5. Use according to claim 2, characterized in that, The *Pennisetum alopecuroides* used for identification was seedling stage.

6. Use according to claim 2, characterized in that, The application is as follows: Identification and improvement of germplasm resources of *Pennisetum affine*; or Application in early prediction of cold hardiness of Napier grass seedlings.

7. A primer set for amplifying a target point for identifying cold tolerance of Pennisetum, characterized in that, The primer set contains SEQ ID NO:1 to SEQ ID NO:

128.

8. An assay reagent, characterized in that, It includes the set of 65 core molecular markers for identifying the cold resistance of Napier grass as described in claim 1 or the primer set as described in claim 7.

9. A kit characterized in that, It includes the set of 65 core molecular markers for identifying the cold resistance of Napier grass as described in claim 1 or the primer set as described in claim 7.

10. A gene chip, characterized by It includes the set of 65 core molecular markers for identifying the cold resistance of Napier grass as described in claim 1 or the primer set as described in claim 7.

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