A method for identifying cold tolerance of pennisetum using 65 core molecular marker set, primer set and gene chip and application thereof

Abstract

The application relates to the technical field of molecular biology, and discloses a cold-tolerance identification method of Pennisetum based on a 65-core molecular marker set, a primer group and a gene chip and application thereof. The application collects core germplasm resources of the Pennisetum through a system, and constructs a GWAS population by using multi-environment (different altitudes) conditions, so that a wide genetic background and phenotype variation are fully covered, and a material foundation for genetic analysis of cold tolerance is laid. In the aspect of cold-tolerance phenotype identification, the application innovatively takes the chlorophyll content of leaves and the percentage of green leaves after natural low-temperature stress in autumn and winter fields as the cold-tolerance evaluation indexes, judges individuals with more than 40 excellent allelic variations as high-cold-tolerance varieties, and establishes a rapid and non-destructive phenotype screening method which can be implemented at the seedling stage.

Description

Technical Field

[0001] This invention relates to the field of molecular biology technology, and discloses a method for identifying the cold resistance of Napier grass based on a set of 65 core molecular markers, primer sets, and gene chips, as well as its application. Background Technology

[0002] As an important economic species that combines the value of high-yield forage and high-efficiency energy plants, Napier grass has shown great application potential in tropical and subtropical regions.

[0003] The optimal growth temperature for Napier grass is 30-35℃. Growth ceases below 10℃, and prolonged low temperatures can lead to the death of the entire plant (including both above-ground and underground parts). Its dry matter yield decreases significantly with increasing latitude, from 80 tons / hectare in tropical regions to approximately 45 tons / hectare at 30°N, and further to 22-30 tons / hectare at 36°N, severely limiting its application in cooler, higher latitude regions.

[0004] Currently, the evaluation of cold hardiness of *Pennisetum alopecuroides* mainly relies on physiological indicators measured under controlled laboratory conditions during the seedling stage. However, these indicators have a weak correlation with actual overwintering performance in the field, making it difficult to accurately reflect the true cold resistance of the plants in the field environment, and thus failing to provide a reliable basis for cold-hardy breeding. Existing field cold-hardiness phenotypic identification methods are not only time-consuming and labor-intensive, but also easily affected by environmental factors, making them unsuitable for efficient screening of large-scale breeding populations, severely delaying the exploration of cold-hardy resources and the breeding process of cold-hardy varieties.

[0005] Therefore, establishing an efficient and accurate early identification technology system for cold hardiness in seedlings is urgently needed. Utilizing genome-wide association analysis (GWAS) to locate SNP loci associated with chlorophyll content and green leaf percentage after low-temperature stress, and developing molecular markers for selection assistance, holds promise for accurate prediction of cold hardiness at the seedling stage. This strategy can significantly reduce reliance on traditional overwintering phenotypic identification, greatly improve screening efficiency (a single technician can complete genotyping of hundreds of samples daily), effectively reduce the risk of misjudgment caused by environmental variations, improve selection accuracy and breeding efficiency, thereby shortening the breeding cycle and accelerating the exploration of cold-hardy Napier grass resources and the breeding of new varieties. Promoting this type of marker-assisted selection technology will provide crucial support for overcoming the bottleneck in cold-hardy Napier grass breeding.

[0006] Furthermore, on the one hand, there are differences in understanding among those skilled in the art; on the other hand, the inventors studied a large number of documents and patents when making this invention, but due to space limitations, not all details and contents were listed in detail. However, this does not mean that the present invention does not possess the features of these prior art. On the contrary, the present invention already possesses all the features of the prior art, and the applicant reserves the right to add relevant prior art to the background art. Summary of the Invention

[0007] This invention relates to the field of molecular biology technology, and discloses a method for identifying the cold resistance of Napier grass based on a set of 65 core molecular markers, primer sets, and gene chips, as well as its application.

[0008] To address the aforementioned technical problems, one objective of this invention is to provide a set of 65 core molecular markers, which includes SNP markers numbered SNP 1-65 as follows:

[0009] The SNP is labeled as base G:A, located at position chrB1_64515476 of the grass;

[0010] The SNP is labeled as base G:A, located at position chrB7_29024501 of the grass;

[0011] The SNP is labeled as base G:A, located at position chrB7_45105115 of the grass;

[0012] The SNP is labeled as base T:A, located at position chrB7_27795665 of the grass;

[0013] The SNP is marked with the base T:C, located at position chrB7_27795662 of the grass;

[0014] The SNP is labeled as base G:A, located at position chrB7_49893610 of the grass;

[0015] The SNP is labeled as base G:A, located at position chrB6_29986440 of the grass;

[0016] The SNP is labeled as base G:A, located at position chrB7_15272734 of the grass;

[0017] The SNP is labeled with the base G:C, located at the chrB7_119411306th position of the grass;

[0018] The SNP is labeled as base G:A, located at position chrB7_50693409 of the grass;

[0019] The SNP is labeled as base G:A, located at position chrB7_50541147 of the grass;

[0020] The SNP is labeled as base G:A, located at position chrA3_96624776 of the grass;

[0021] The SNP is labeled as base G:A, located at position chrA7_45758326 of the grass;

[0022] The SNP is labeled as base G:A, located at position chrB4_41545400 of the grass;

[0023] The SNP is labeled as base G:A, located at position chrB1_43049394 of the grass;

[0024] The SNP is labeled as base G:A, located at position chrB6_133829538 of the grass;

[0025] The SNP is labeled as base G:A, located at position chrA3_79205623 of the grass;

[0026] The SNP is marked with the base T:C, located at the chrB6_186861238th position of the grass;

[0027] The SNP is labeled as base G:A, located at position chrB7_49308097 of the grass;

[0028] The SNP is labeled as base G:A, located at position chrB2_81759859 of the grass;

[0029] The SNP is labeled with base T:A, located at position chrB7_111295430 of the grass;

[0030] The SNP is labeled with the base T:G, located at position chrA5_40602857 of the grass;

[0031] The SNP is labeled as base G:A, located at position chrB7_64811713 of the grass;

[0032] The SNP is labeled as base G:A, located at position chrA1_1132398 of the grass;

[0033] The SNP is labeled as base G:A, located at position chrB2_46430985 of the grass;

[0034] The SNP is labeled with the base G:C, located at the chrB2_74078794th position of the grass;

[0035] The SNP is labeled as base G:A, located at position chrB6_183157373 of the grass;

[0036] The SNP is labeled as base C:A, located at position chrB7_20908754 of the grass;

[0037] The SNP is labeled as base A:G, located at position chrA5_64602902 of the Napier grass;

[0038] The SNP is labeled as base G:A, located at position chrB2_73078206 of the grass;

[0039] The SNP is labeled as base G:A, located at position chrB7_46588580 of the grass;

[0040] The SNP is labeled as base A:G, located at position chrA3_79053484 of the grass;

[0041] The SNP is labeled with the base T:C, located at position chrA3_79012904 of the grass;

[0042] The SNP is labeled as base A:G, located at position chrB4_18322082 of the Napier grass;

[0043] The SNP is labeled as base A:G, located at position chrB7_56383576 of the grass;

[0044] The SNP is labeled as base A:G, located at position chrA3_82339032 of the grass;

[0045] The SNP is labeled with the base C:T, located at position chrB2_81759922 of the grass;

[0046] The SNP is labeled with the base C:T, located at position chrB2_13004238 of the grass;

[0047] The SNP is labeled as base A:G, located at position chrB6_93488911 of the grass;

[0048] The SNP is labeled with the base C:T, located at position chrB2_46167039 of the grass;

[0049] The SNP is labeled with the base C:T, located at the chrB4_117851973rd position of the grass;

[0050] The SNP is labeled with the base C:T, located at the chrA1_163181362nd position of the grass;

[0051] The SNP is labeled with the base C:T, located at position chrA5_42762088 of the grass;

[0052] The SNP is labeled with the base C:T, located at position chrB2_79722191 of the grass;

[0053] The SNP is labeled as base G:A, located at position chrA2_4199397 of the grass;

[0054] The SNP is labeled with the base C:T, located at position chrB7_68554707 of the grass;

[0055] The SNP is labeled with the base C:T, located at position chrB2_38519659 of the grass;

[0056] The SNP is labeled as base A:T, located at position chrA5_6312866 of the grass;

[0057] The SNP is labeled with the base C:T, located at position chrA5_6312867 of the grass;

[0058] The SNP is labeled with the base C:T, located at position chrA7_135412766 of the grass;

[0059] The SNP is labeled with the base C:T, located at the chrB4_23125675th position of the grass;

[0060] The SNP is labeled with the base C:T, located at position chrA5_12051038 of the grass;

[0061] The SNP is labeled with the base C:G, located at position chrB7_6855308 of the grass;

[0062] The SNP is labeled as base A:T, located at position chrB7_46326638 of the grass;

[0063] The SNP is labeled as base A:T, located at position chrB2_13004143 of the grass;

[0064] The SNP is labeled with the base C:T, located at position chrB7_68652993 of the grass;

[0065] The SNP is labeled with the base C:T, located at position chrB2_8958258 of the grass;

[0066] The SNP is marked with the base C:T, and the position is the ScaffoldUN_7865923rd position of the grass.

[0067] The SNP is labeled as base A:T, located at position chrA7_86889323 of the grass;

[0068] The SNP is labeled with the base C:T, located at position chrA7_86889337 of the grass;

[0069] The SNP is labeled with the base C:T, located at the chrA2_117167685th position of the grass;

[0070] The SNP is labeled with the base C:T, located at the chrB6_183157018th position of the grass;

[0071] The SNP is labeled with the base C:G, located at position chrB5_19579799 of the grass;

[0072] The SNP is labeled with the base C:T, located at position chrB4_29160095 of the grass;

[0073] The SNP is labeled with the base C:T, located at position chrB7_46656020 of the *Phragmites australis*.

[0074] The SNP molecular marker set provided by this invention is a specific SNP molecular marker set. Its core is to use 65 core SNP molecular markers as detection targets, and based on this, corresponding primer sets, detection reagents, kits and gene chips have been developed.

[0075] One of the objectives of this invention is to provide a set of 65 core molecular markers as detection targets for the identification of cold resistance in Napier grass. The set of 65 core molecular markers includes the aforementioned set of 65 core molecular markers for the identification of cold resistance in Napier grass.

[0076] According to a preferred embodiment, when more than 40 individuals are marked as dominant haplotypes, it indicates that the individual plants of *Phragmites australis* are highly cold-resistant materials.

[0077] According to a preferred embodiment, when there are 40 or fewer individuals marked as dominant haplotypes, it indicates that the individual *Phragmites australis* is a low-hardiness material.

[0078] According to a preferred embodiment, the Napier grass used for identification is seedling Napier grass.

[0079] According to a preferred embodiment, the application is as follows:

[0080] Identification and improvement of germplasm resources of *Pennisetum affine*; or

[0081] Application in early prediction of cold hardiness of Napier grass seedlings.

[0082] One objective of this invention is to provide a primer set for amplifying target sites for identifying the cold resistance of Napier grass. The primer set comprises SEQ ID NO:1 to SEQ ID NO:124.

[0083] One of the objectives of this invention is to provide a detection reagent comprising the core molecular marker set or the primer set described above.

[0084] One of the objectives of this invention is to provide a kit containing the core molecular marker set or the primer set described above.

[0085] One of the objectives of this invention is to provide a gene chip that includes the core molecular marker set or the primer set described above.

[0086] One of the objectives of this invention is to provide a reaction system for the above primer set. The total reaction system for PCR amplification is 25 μL, including 12.5 μL of 2× Taq Pro Master Mix, 0.8 μL each of forward and reverse primers (10 μM), 1.0 μL of template DNA (20-50 ng / μL), and ddH2O to make up to 25 μL.

[0087] One of the objectives of this invention is to provide a method for identifying cold-resistant varieties of Pennisetum alopecuroides, which includes the following steps:

[0088] The genomic DNA of the varieties to be tested was amplified using primers SEQ ID NO:1 to SEQ ID NO:124, and the results were combined with 65 SNP markers and the determined typing results.

[0089] When more than 40 haplotypes were identified as dominant haplotypes, it indicated that the chlorophyll content of *Pennisetum affine* leaves after low-temperature stress was higher than the average chlorophyll content of the population leaves. This result suggests that the individual is a highly cold-hardy resource.

[0090] When there are 40 or fewer haplotypes marked as dominant, it indicates that the leaf chlorophyll content is lower than the average leaf chlorophyll content of the entire population of *Pennisetum affine*. This result suggests that the individual is a low-hardiness resource.

[0091] One of the objectives of this invention is to provide a method for identifying cold-resistant varieties of Pennisetum alopecuroides, which includes the following steps:

[0092] If the chlorophyll content of an individual Napier grass leaf exceeds the average chlorophyll content of the leaves of its population, it is identified as a cold-resistant variety.

[0093] The beneficial effects of this invention are:

[0094] This invention systematically collects core germplasm resources of *Pennisetum alopecuroides* and constructs GWAS populations under multiple environmental conditions (different altitudes), fully covering a wide range of genetic backgrounds and phenotypic variations, thus laying a material foundation for the genetic analysis of cold hardiness. In terms of cold hardiness phenotypic identification, this invention innovatively uses leaf chlorophyll content and green leaf percentage as indicators of cold hardiness evaluation. Individuals with chlorophyll content higher than the population average are identified as highly cold-hardy materials, establishing a rapid and non-destructive phenotypic screening method that can be implemented at the seedling stage.

[0095] Based on high-throughput genotyping and genome-wide association analysis, 65 SNP loci significantly associated with photosynthetic capacity (chlorophyll content and green leaf percentage) of *Pennisetum affine* after field-tested low-temperature stress were successfully identified. Specific molecular marker primers with well-defined product characteristics and flanking sequence information were designed, achieving efficient transformation from genetic loci to a practical molecular marker system. Simultaneously, standardized PCR and genotyping procedures were established, ensuring reproducible operations, accurate results, and rapid, batch detection capabilities, providing reliable technical support for the breeding application of molecular markers.

[0096] The developed molecular markers showed a significant correlation with the chlorophyll content in leaves of *Pennisetum alopecuroides* after low-temperature stress, a phenotype indicating cold tolerance. This marker can be used for early prediction of the plant's cold tolerance potential during the seedling stage. This technology effectively overcomes the limitations of traditional field overwintering identification methods, which are characterized by long cycles and susceptibility to environmental interference. It enables indirect screening of highly cold-tolerant germplasm through genotyping during the seedling stage, significantly improving screening efficiency and accuracy, and substantially shortening the breeding cycle. This provides crucial technical support for the breeding of new cold-tolerant varieties of *Pennisetum alopecuroides*. Attached Figure Description

[0097] Figure 1 Correlation analysis of green leaf percentage and chlorophyll content in field of *Pennisetum comosum* after autumn low temperature stress;

[0098] Figure 2 Correlation analysis of dominant haplotype number and chlorophyll content after low temperature stress;

[0099] Figure 3 Correlation analysis between the number of dominant haplotypes and the percentage of green leaves after low temperature stress;

[0100] Figure 4 Haplotype analysis diagram of the first part of 65 cold-resistant SNP sites of Pennisetum arvense;

[0101] Figure 5 Haplotype analysis diagram of the second part of 65 cold-resistant SNP sites of Pennisetum arvense;

[0102] Figure 6 Haplotype analysis diagram of the third part of 65 cold-resistant SNP sites of Pennisetum arvense;

[0103] Figure 7 Haplotype analysis diagram of the fourth part of 65 cold-resistant SNP sites of Pennisetum arvense;

[0104] Figure 8 Haplotype analysis diagram of the fifth part of 65 cold-resistant SNP sites of Pennisetum arvense;

[0105] Figure 9 Haplotype analysis diagram of the sixth part of 65 cold-resistant SNP sites of Pennisetum arvense;

[0106] Figure 10 Haplotype analysis diagram of the seventh part of 65 cold-resistant SNP sites of Pennisetum arvense;

[0107] Figure 11 Haplotype analysis diagram of part eight of 65 cold-resistant SNP sites in Pennisetum arvense;

[0108] Figure 12 Haplotype analysis diagram of the ninth part of 65 cold-resistant SNP sites of Pennisetum arvense;

[0109] Figure 13 Haplotype analysis diagram of part 10 of 65 cold-resistant SNP sites in Pennisetum arvense;

[0110] Figure 14 Haplotype analysis diagram of part eleven of 65 cold-resistant SNP sites in Pennisetum arvense;

[0111] Figure 15 Haplotype analysis diagram of the twelfth part of 65 cold-resistant SNP sites of Pennisetum arvense;

[0112] Figure 16 Haplotype analysis diagram of part thirteen of 65 cold-resistant SNP sites in Pennisetum arvense;

[0113] Figure 17 Haplotype analysis diagram of the fourteenth part of 65 cold-resistant SNP sites of Pennisetum arvense;

[0114] Figure 18 Haplotype analysis diagram of the fifteenth part of 65 cold-resistant SNP sites of Pennisetum arvense;

[0115] Figure 19 Haplotype analysis diagram of the sixteenth part of 65 cold-resistant SNP sites of Pennisetum arvense;

[0116] Figure 20 Haplotype analysis diagram of the seventeenth part of 65 cold-resistant SNP sites of Pennisetum arvense;

[0117] Figure 21 Haplotype analysis diagram of the eighteenth part of 65 cold-resistant SNP sites in Pennisetum arvense;

[0118] Figure 22 Haplotype analysis diagram of the nineteenth part of 65 cold-resistant SNP sites of Pennisetum arvense;

[0119] Figure 23 Haplotype analysis diagram of the twentieth part of 65 cold-resistant SNP sites of Pennisetum arvense;

[0120] Figure 24 Haplotype analysis diagram of part 21 of 65 cold-resistant SNP sites of Pennisetum arvense;

[0121] Figure 25Haplotype analysis diagram of part 22 of 65 cold-resistant SNP sites of Pennisetum arvense;

[0122] Figure 26 This study investigated the field phenotypes of different chlorophyll contents in *Pennisetum affine* materials after autumn low-temperature stress. Detailed Implementation

[0123] In the description of this invention, terminology is used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. In the description of this invention, "a plurality of" means two or more, unless otherwise explicitly defined.

[0124] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials, reagents or instruments used, unless otherwise specified by the manufacturer, are all commercially available reagents and materials; the conditions not specified in the examples are all carried out according to conventional conditions or conditions recommended by the manufacturer. At the same time, the present invention does not limit the source of the raw materials used. Unless otherwise specified, the raw materials used in the present invention are all commercially available products in this technical field.

[0125] Napier grass is a warm-season C4 plant with poor cold tolerance, which limits its widespread cultivation in high-latitude and high-altitude regions. Therefore, constructing a scientific cold tolerance evaluation system is of great value for screening cold-resistant resources and breeding varieties. Chlorophyll content and green leaf percentage after autumn low-temperature stress have become important physiological and morphological indicators for evaluating the cold tolerance of Napier grass. The effectiveness of this method is based on the physiological nature of low-temperature damage. In the gradually cooling autumn environment, although the plant does not die, low temperatures directly damage photosynthetic structures, accelerate chlorophyll degradation, and cause tissue chlorosis and necrosis. Whether the plant can maintain physiological activity and store energy for overwintering depends mainly on whether its photosynthetic system can maintain functional integrity and cell membrane stability at low temperatures. Chlorophyll content directly reflects the degree of damage to chloroplast structure and function, while the green leaf percentage comprehensively reflects the plant's ability to resist low-temperature damage and maintain tissue survival at the organ level. Therefore, chlorophyll content can serve as an early and sensitive physiological indicator of cold resistance, while the percentage of green leaves is a direct and overall evaluation of low-temperature survival status. The combination of the two can provide a systematic and quantifiable evaluation basis for cold resistance.

[0126] From a physiological perspective, chlorophyll content and green leaf percentage, as key indicators of photosynthesis, directly affect the synthesis of photosynthetic assimilates in autumn and their transport to underground organs. Individual plants with higher chlorophyll content and green leaf percentages typically accumulate more non-structural carbohydrates such as starch and soluble sugars. These substances not only help maintain cellular osmotic balance and membrane stability, but also often exhibit stronger cold resistance. In conclusion, 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 resistance of *Pennisetum alopecuroides*.

[0127] This invention constructs four *Pennisetum affine* GWAS populations at different altitudes, and each population is planted using a standardized single-root, double-stalk oblique planting method to ensure normal plant growth and tillering. For example... Figure 26 As shown, in autumn and winter, as the temperature decreases, the chlorophyll content (using the CL-01 chlorophyll content meter Hansatech) or the percentage of green leaves (by counting the proportion of green leaves to the total number of leaves in the Napier grass; leaves with more than or equal to 50% turning yellow are considered yellow leaves) are measured as key indicators for evaluating the cold resistance of Napier grass.

[0128] 1. Correlation studies of 65 core molecular marker sets

[0129] Fresh tissue was 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 with an average sequencing depth of 10× was performed on the constructed GWAS population. Strict quality control was applied to the raw data to remove adapters and low-quality reads. Based on the published *Pennisetum purpureus* reference genome (2.07 GB), variant detection was performed using biological software, and SNP sites were screened using biological software (parameters set to: --maf 0.05 --max-missing 0.8). The filtered high-quality SNP dataset was then compared with field-measured chlorophyll content and green leaf percentage data for two cold-hardy phenotypes to perform genome-wide association analysis. The analysis used a mixed linear model from biological software, with a selection criterion of P < 10. -6 Based on this, haplotype analysis was performed on significantly associated sites, ultimately identifying 65 core SNP markers with highly significant phenotypic differences among different haplotypes and closely related to cold resistance.

[0130] Based on the field phenotypic data collected from the samples, the results are as follows: Figure 1 As shown, the chlorophyll content and green leaf percentage of Napier grass under low temperature stress in the field showed a significant positive correlation, with a Pearson correlation coefficient r=0.714.

[0131] The results are as follows Figure 2The number of dominant haplotypes was shown to be significantly positively correlated with chlorophyll content under low-temperature stress, with a Pearson correlation coefficient r = 0.808. The results are as follows... Figure 3 The number of dominant haplotypes was significantly positively correlated with the percentage of green leaves under low-temperature stress, with a Pearson correlation coefficient r = 0.816. These results indicate that chlorophyll content and the percentage of green leaves are important indicators for evaluating the cold tolerance of *Pennisetum alopecuroides*.

[0132] Figure 4-25 Table 1 shows the SNP marker information for 65 SNPs significantly associated with cold hardiness in *Pennisetum purpureus*. The information includes the chromosome information of the SNP, the specific location of the SNP, the haplotype type, the reference allele for the cold-hardy haplotype and the cold-sensitive haplotype, the variant allele, the location of the variant, and the primer pair for the amplification site sequence. NON_SYNONYMOUS_CODING indicates a variant occurring in the protein-coding region, resulting in a change in the amino acid sequence. SYNONYMOUS_CODING indicates different codons encoding the same amino acid. UPSTREAM indicates a variant located in a non-coding region upstream of the gene. UTR_3_PRIME indicates a non-coding sequence region in messenger RNA (mRNA) immediately following the coding region (CDS), after the stop codon, and extending to the 3' end of the mRNA. 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 located in the exon region, 8 sites have base variations that cause synonymous coding variations, and 16 sites have base variations that cause non-synonymous coding variations.

[0133] 2. Functional verification

[0134] Four more GWAS populations of *Phragmites australis* were constructed at different altitudes to verify the effectiveness of the aforementioned molecular marker set.

[0135] Table 2 shows the molecular marker primer information for the cold-resistant SNP sites of Napier grass. The information includes the SNP name, the Tm value of the upstream and downstream primer sequences shown in Table 1, and the GC content.

[0136] Using DNA from each material as amplification template, a 25 µL reaction system was employed.

[0137] 12.5 μL of 2× Taq Pro Master Mix, 0.8 μL each of forward and reverse primers (10 μM), 1.0 μL of template DNA (20–50 ng / μL), and ddH2O to bring the total volume to 25 μL.

[0138] The PCR reaction procedure is as follows:

[0139] Pre-denaturation at 95℃ for 5 minutes;

[0140] This was followed by 35 cycles of amplification, including denaturation at 95°C for 30 seconds, annealing at 58–62°C for 30 seconds (adjusted according to primer Tm value), and extension at 72°C for 45 seconds.

[0141] Finally, maintain a temperature of 72°C for 7 minutes.

[0142] Add 3–5 µL of the PCR product to a 1.5% agarose gel for electrophoresis. The target band is excised using a gel excision method and purified using a DNA Gel Extraction Kit. Genotyping is then confirmed using Sanger sequencing after purification.

[0143] Based on the verification of the results, the phenotypic and sequence locus correspondence analysis of the samples showed that: among the 65 core SNP loci of *Pennisetum purpureus* with cold hardiness, when the number of superior allelic variants was less than or equal to 40, the chlorophyll content of leaves after field low-temperature stress had a 95.56% probability of being lower than the average; when the number of superior allelic variants was greater than 40, the chlorophyll content of leaves after field stress had an 82.96% probability of being higher than the average (26.81 SPAD). When the number of superior allelic variants was less than or equal to 40, the percentage of green leaves after field low-temperature stress had an 80.64% probability of being lower than the average; when the number of superior allelic variants was greater than 40, the chlorophyll content of leaves after field stress had an 89.26% probability of being higher than the average (0.444).

[0144] Table 1

[0145]

[0146] Table 2

[0147]

[0148] It should be noted that the specific embodiments described above are exemplary, and those skilled in the art can devise various solutions inspired by the disclosure of this invention. These solutions all fall within the scope of this invention and its protection. Those skilled in the art should understand that this specification and its accompanying drawings are illustrative and not intended to limit the scope of the claims. The scope of protection of this invention is defined by the claims and their equivalents.

Claims

1. A primer set for amplifying target sites for identifying the cold resistance of Pennisetum acutum, characterized in that, The nucleotide sequences of the primer set are shown in SEQ ID NO:1~SEQ ID NO:

124.

2. A detection reagent, characterized in that, It includes the primer set as described in claim 1.

3. A reagent kit, characterized in that, It includes the primer set as described in claim 1.

4. A gene chip, characterized in that, It includes the primer set as described in claim 1.

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