A 74-core molecular marker set for identifying cold tolerance of pennisetum, primer group, detection reagent, kit, gene chip and application thereof

Abstract

The present application relates to the technical field of molecular biology, and discloses a 74-core molecular marker set for cold tolerance identification of Pennisetum, a primer group, a detection reagent, a kit, a gene chip and applications thereof.The present application collects core germplasm resources of Pennisetum systematically, and constructs a GWAS population by using multi-environment (different altitudes) conditions, fully covers extensive genetic and phenotypic variations, and lays a foundation of representative materials for genetic analysis of cold tolerance.In the aspect of phenotype evaluation, the present application innovatively proposes a method of "single root double stem node oblique planting" combined with the determination of the number of root cutting seedlings after natural overwintering, establishes a scientific, stable and standardizable evaluation system for cold tolerance and regeneration, and significantly improves the reliability and efficiency of phenotype identification.The present application is not affected by human and climate environment, realizes efficient and accurate early cold tolerance identification of Pennisetum, greatly improves the efficiency and scale of breeding screening, and accelerates the breeding process.

Description

Technical Field

[0001] This invention relates to the field of molecular biology technology, and discloses a set of 74 core molecular markers, primer sets, detection reagents, kits, gene chips and their applications for identifying the cold resistance of Napier grass. Background Technology

[0002] Due to its rapid growth, tall stature, high biomass, rich nutrients, wide soil adaptability, and outstanding resistance to diseases and pests, Napier grass has been widely researched and promoted as a high-yield, high-quality forage. It can be dried into hay or fermented into silage, effectively alleviating the problem of insufficient forage supply during the dry season and winter. Furthermore, Napier grass is also an excellent energy plant, a superior material for producing biofuels, biochar, alcohol, methane, and paper.

[0003] However, *Pennisetum alopecuroides* prefers warm and humid climates and is intolerant of low temperatures and frost. Currently, there are no reports on technologies to improve the cold resistance of hybrid *Pennisetum alopecuroides*. The optimal growth temperature for *Pennisetum alopecuroides* is 30-35℃; it ceases growth when the ambient temperature drops below 10℃, and sustained low temperatures will cause the entire plant (both above and below ground) to die. With increasing latitude, the yield of *Pennisetum alopecuroides* shows a significant downward trend.

[0004] In evaluating cold hardiness, current methods primarily rely on physiological indicators measured under controlled laboratory conditions during the seedling stage. For example, Chinese patent application CN202311217536.4 discloses a method for evaluating the cold hardiness of Napier grass based on principal component analysis. This method assesses cold hardiness by subjecting Napier grass seedlings to low-temperature stress treatment, combined with physiological indicators such as relative leaf water content, proline content, and malondialdehyde content, as well as parameters such as the half-lethal temperature of low temperature, and uses principal component analysis to calculate a comprehensive score D value.

[0005] However, the correlation between these indicators and actual overwintering performance in the field is weak, making it difficult to accurately reflect the true cold resistance of plants in a field environment, and thus unable to provide reliable guidance for breeding practices. This method is not only time-consuming and labor-intensive, but also difficult to apply to the rapid screening of large-scale breeding populations, seriously delaying the breeding process of cold-resistant Napier grass varieties. Although researchers usually conduct phenotypic identification through methods such as investigating the greening rate after overwintering and counting root-cut seedlings, these methods still have limitations such as long data collection cycles, high labor intensity, and significant susceptibility to environmental interference.

[0006] Therefore, developing an efficient and accurate early identification technology system for cold hardiness is particularly urgent. Utilizing genome-wide association analysis (GWAS) to locate SNP loci significantly associated with field greening ability and the number of root-cut seedlings, and then establishing molecular markers that can be used for selection assistance, holds promise for predicting 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, while effectively avoiding misjudgments caused by environmental variations, improving selection accuracy and breeding efficiency, ultimately shortening the breeding cycle and accelerating the breeding process of new cold-hardy Napier grass varieties. Promoting this type of molecular marker-assisted selection technology will provide key technical support for overcoming the bottleneck in cold-hardy Napier grass breeding. Summary of the Invention

[0007] This invention relates to the field of molecular biology technology, and discloses a set of 74 core molecular markers, primer sets, detection reagents, kits, gene chips and their applications for identifying the cold resistance of Napier grass.

[0008] To address the aforementioned technical problems, one objective of this invention is to provide a set of 74 core molecular markers for identifying the cold resistance of *Phragmites australis*. This set of 74 core molecular markers includes the SNP markers numbered SNP1 to 74 as follows:

[0009] The SNP is labeled as base A:G, located at position chrB2_48486846 of the Napier grass;

[0010] The SNP is labeled with the base C:T, located at position chrA6_67925981 of the grass;

[0011] The SNP is marked with the base G:T, located at position chrB6_81210972 of the grass;

[0012] The SNP is marked with the base T:C, located at position chrB3_86452525 of the grass;

[0013] The SNP is labeled as base A:G, located at position chrA5_94911641 of the grass;

[0014] The SNP is labeled with the base C:T, located at position chrA5_94911645 of the grass;

[0015] The SNP is labeled with the base C:G, located at position chrB6_102842461 of the grass;

[0016] The SNP is labeled as base G:A, located at position chrB6_117864964 of the grass;

[0017] The SNP is labeled as base A:T, located at position chrB6_121632388 of the grass;

[0018] The SNP is labeled as base C:A, located at position chrB6_122619783 of the grass;

[0019] The SNP is labeled with the base T:C, located at position chrB6_142954294 of the grass;

[0020] The SNP is labeled with the base C:T, located at position chrB6_144593054 of the grass;

[0021] The SNP is labeled with base T:A, located at position chrB1_161178990 of the grass;

[0022] The SNP is labeled with the base G:T, located at position chrB2_79195022 of the grass;

[0023] The SNP is labeled as base G:A, located at position chrB7_99782637 of the grass;

[0024] The SNP is labeled with the base C:T, located at position chrB6_139341295 of the grass;

[0025] The SNP is labeled as base G:A, located at position chrA7_16803736 of the grass;

[0026] The SNP is labeled with base T:A, located at position chrB2_28762883 of the grass;

[0027] The SNP is labeled with the base T:A, located at position chrA5_46174486 of the grass;

[0028] The SNP is labeled with the base G:T, located at position chrA4_97584889 of the grass;

[0029] The SNP is labeled with the base C:T, located at position chrB7_122721579 of the grass;

[0030] The SNP is labeled as base G:A, located at position chrB1_158396834 of the grass;

[0031] The SNP is labeled as base G:A, located at position chrA2_48296938 of the grass;

[0032] The SNP is labeled with the base G:T, located at the chrB2_23656109th position of the grass;

[0033] The SNP is labeled as base A:G, located at position chrA1_50010676 of the grass;

[0034] The SNP is labeled as base G:A, located at position chrB6_8254957 of the grass;

[0035] The SNP is labeled with the base G:T, located at position chrA2_15662900 of the grass;

[0036] The SNP is labeled with the base T:G, located at position chrA2_15664750 of the grass;

[0037] The SNP is labeled as base A:G, located at position chrA4_29178270 of the grass;

[0038] The SNP is labeled as base G:A, located at position chrB3_32980416 of the grass;

[0039] The SNP is labeled as base A:G, located at position chrB7_106094743 of the grass;

[0040] The SNP is labeled with base T:A, located at position chrA2_30154555 of the grass;

[0041] The SNP is labeled with the base C:T, located at position chrA2_32222409 of the grass;

[0042] The SNP is labeled with the base C:T, located at position chrA4_38997045 of the grass;

[0043] The SNP is marked with the base T:C, located at the chrA2_59532018th position of the grass;

[0044] The SNP is labeled as base A:T, located at position chrB6_57518900 of the grass;

[0045] The SNP is labeled with the base T:G, located at position chrB6_82497956 of the grass;

[0046] The SNP is labeled as base A:G, located at position chrB6_82497974 of the grass;

[0047] The SNP is labeled with the base T:C, located at position chrA4_91805848 of the grass;

[0048] The SNP is labeled with the base C:T, located at position chrA2_37111926 of the grass;

[0049] The SNP is labeled as base A:T, located at position chrA2_37152867 of the grass;

[0050] The SNP is labeled as base A:T, located at position chrA2_37152874 of the grass;

[0051] The SNP is labeled as base G:A, located at position chrA2_37152896 of the grass;

[0052] The SNP is labeled with the base C:T, located at position chrA3_61140681 of the grass;

[0053] The SNP is labeled as base A:T, located at position chrA1_16460560 of the grass;

[0054] The SNP is labeled as base G:A, located at position chrB5_81211080 of the grass;

[0055] The SNP is labeled with the base C:T, located at position chrB1_176898490 of the grass;

[0056] The SNP is marked with the base T:C, located at position chrB1_9281522 of the grass;

[0057] The SNP is marked with the base T:C, located at position chrB1_9727233 of the grass;

[0058] The SNP is marked with the base T:C, located at position chrA2_28796142 of the grass;

[0059] The SNP is labeled with the base C:T, located at position chrB6_35440206 of the grass;

[0060] The SNP is labeled as base A:G, located at position chrB6_35442142 of the Napier grass;

[0061] The SNP is labeled with the base T:C, located at the chrB6_35502016th position of the Napier grass;

[0062] The SNP is labeled with the base G:T, located at position chrA4_37962206 of the grass;

[0063] The SNP is labeled as base A:G, located at position chrB6_38037456 of the grass;

[0064] The SNP is labeled with the base G:T, located at position chrB6_39080862 of the grass;

[0065] The SNP is labeled with the base C:T, located at position chrB6_39502565 of the grass;

[0066] The SNP is marked with the base T:C, located at position chrB6_50861173 of the grass;

[0067] The SNP is labeled as base G:A, located at position chrB6_50861183 of the grass;

[0068] The SNP is marked with the base T:C, located at the chrB6_51186976th position of the grass;

[0069] The SNP is labeled as base A:G, located at position chrB6_51816917 of the grass;

[0070] The SNP is labeled as base G:A, located at position chrB6_51816998 of the grass;

[0071] The SNP is labeled as base A:G, located at position chrB6_51817831 of the grass;

[0072] The SNP is labeled as base A:G, located at position chrB6_51823957 of the grass;

[0073] The SNP is marked with the base T:C, located at the chrB6_51824188th position of the grass;

[0074] The SNP is marked with the base T:C, located at position chrA7_59005844 of the grass;

[0075] The SNP is labeled with the base G:C, located at position chrA4_59737791 of the grass;

[0076] The SNP is labeled with base T:A, located at position chrB2_111371030 of the grass;

[0077] The SNP is labeled with the base T:A, located at position chrA7_9111403 of the grass;

[0078] The SNP is labeled as base G:A, located at position chrB6_41472864 of the grass;

[0079] The SNP is labeled as base A:G, located at position chrA7_42957102 of the grass;

[0080] The SNP is labeled with the base T:C, located at position chrA7_42958685 of the grass;

[0081] The SNP is labeled as base A:G, located at position chrA7_59005815 of the grass;

[0082] The SNP is labeled as base G:A, located at position chrB7_923607 of the *Phaseolus spp.*.

[0083] The SNP molecular marker set provided by this invention is a specific SNP molecular marker set. Its core is the application of 74 core SNP molecular markers as detection targets, and corresponding primer sets, detection reagents, kits and gene chips have been developed based on this.

[0084] One of the objectives of this invention is to provide a core molecular marker set as a detection target for identifying the cold resistance of Napier grass. The core molecular marker set includes the 74 core molecular markers mentioned above for identifying the cold resistance of Napier grass.

[0085] According to a preferred embodiment, when more than 39 are marked as dominant haplotypes, it indicates that the number of regrowth seedlings is the dominant individual.

[0086] According to a preferred embodiment, when more than 25 and no more than 39 are marked as dominant haplotypes, it indicates that the number of regrowth seedlings is the average number of individuals.

[0087] According to a preferred embodiment, when there are no more than 25 haplotypes marked as dominant haplotypes, it indicates that the number of regrowth seedlings is inferior.

[0088] In summary, among the 74 molecular markers for cold hardiness in *Phragmites australis*, those with a dominant haplotype number > 39 were considered highly cold hardy, those with a dominant haplotype number < 25 and a dominant haplotype number ≤ 39 were considered moderately cold hardy, and those with a dominant haplotype number ≤ 25 were considered low cold hardy.

[0089] According to a preferred embodiment, the application is as follows:

[0090] Identification and improvement of germplasm resources of *Pennisetum affine*; or

[0091] Application in early prediction of cold hardiness of Napier grass seedlings.

[0092] One objective of this invention is to provide a primer set for detecting target sites and identifying the cold resistance of Napier grass. The primer set contains SEQ ID NO:1 to SEQ ID NO:148.

[0093] One of the objectives of this invention is to provide a reaction system for the aforementioned primer set. The total reaction system for PCR amplification is 20 μL, comprising 2.0 µL of 10×PCR buffer (containing Mg2+); 0.4 µL of dNTP mix (2.5 mM each); 0.5 µL of forward primer (10 µM); 0.5 µL of reverse primer (10 µM); 0.2 µL of Taq DNA polymerase (5 U / µL); 1 µL of template DNA (concentration 20~50 ng / µL); and the remainder is added to 20 µL with sterile water (ddH2O).

[0094] 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.

[0095] One of the objectives of this invention is to provide a kit containing the core molecular marker set or the primer set described above.

[0096] 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.

[0097] 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:

[0098] The genomic DNA of the varieties to be tested was amplified using primers SEQ ID NO:1 to SEQ ID NO:148, and the results were combined with 74 SNP markers and the determined typing results.

[0099] When more than 39 haplotypes were identified as dominant, it indicated that the number of regreened, root-cut seedlings was higher than the average number of regreened, root-cut seedlings in the population of *Pennisetum purpureum*. This result suggests that the individual was a highly cold-hardy variety.

[0100] According to a preferred embodiment, when more than 25 but no more than 39 are marked as dominant haplotypes, it indicates that the number of regrowth and root-pruning seedlings is on par with the average number of regrowth and root-pruning seedlings in the population of *Pennisetum purpureum*. This result indicates that the individual is a moderately cold-hardy variety.

[0101] According to a preferred embodiment, when no more than 25 haplotypes are identified as dominant haplotypes, it indicates that the number of regrowth seedlings from an individual *Pennisetum alopecuroides* is lower than the average number of regrowth seedlings from the population. This result indicates that the individual is a low-hardiness variety.

[0102] Among the 74 cold-resistance molecular markers of *Phragmites australis*:

[0103] Highly cold-resistant type: Dominant haplotype number > 39;

[0104] Medium cold-resistant type: 25 < dominant haplotype number ≤ 39;

[0105] Low cold resistance type: Dominant haplotype number ≤ 25.

[0106] 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:

[0107] If the number of revived and root-cut seedlings of an individual *Pennisetum purpureum* exceeds the average number of revived and root-cut seedlings in its population, it is identified as a cold-resistant variety.

[0108] The beneficial effects of this invention are:

[0109] 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 and phenotypic variations, thus laying a representative material foundation for the genetic analysis of cold hardiness. Regarding phenotypic evaluation, a method combining single-root double-segment oblique planting with the determination of the number of seedlings after natural overwintering and root pruning is proposed. This establishes a scientific, stable, and standardized evaluation system for cold hardiness and regeneration capacity, significantly improving the reliability and efficiency of phenotypic identification.

[0110] Based on high-throughput genotyping and genome-wide association analysis, this study, after rigorous screening and haplotype verification, successfully identified 74 SNP loci significantly associated with cold tolerance. Furthermore, using Primer3, specific molecular marker primers with well-defined product characteristics (including size, Tm value, GC content, etc.) and flanking sequence information were designed, achieving efficient transformation from GWAS results to a practical molecular marker system. Simultaneously, a standardized PCR reaction system and genotyping workflow were established, including amplification, electrophoresis detection, purification, and sequencing, ensuring reproducible operations and reliable results. This enables rapid and batch identification of sample genotypes, greatly promoting the application of molecular markers in practical breeding.

[0111] The developed molecular markers showed a significant correlation with field cold-hardiness phenotypes, and a highly significant positive correlation (r=0.7952) existed between the number of dominant haplotypes and the number of seedlings that had undergone root cutting after regeneration. This fully validated the accuracy and application value of the marker system in predicting cold hardiness. This invention effectively overcomes the limitations of traditional field overwintering identification methods, which are characterized by long cycles, high workload, and susceptibility to environmental interference. It enables efficient and accurate genotyping and selection during the seedling stage, significantly improving breeding efficiency and shortening the breeding cycle. This provides solid technical support and promising prospects for the breeding of new cold-hardy varieties of *Pennisetum alopecuroides*. Attached Figure Description

[0112] Figure 1 The number of root-cut seedlings that regreened and sprouted in the field of *Pennisetum purpureum* was determined by differences in the number of seedlings.

[0113] Figure 2 A partial haplotype analysis diagram of 74 cold-resistant SNP sites in Pennisetum var. chinensis;

[0114] Figure 3 A partial haplotype analysis diagram of 74 cold-resistant SNP sites in Pennisetum var. chinensis;

[0115] Figure 4 A partial haplotype analysis diagram of 74 cold-resistant SNP sites in Pennisetum var. chinensis;

[0116] Figure 5 A partial haplotype analysis diagram of 74 cold-resistant SNP sites in Pennisetum var. chinensis;

[0117] Figure 6 A partial haplotype analysis diagram of 74 cold-resistant SNP sites in Pennisetum var. chinensis;

[0118] Figure 7 A partial haplotype analysis diagram of 74 cold-resistant SNP sites in Pennisetum var. chinensis;

[0119] Figure 8 A partial haplotype analysis diagram of 74 cold-resistant SNP sites in Pennisetum var. chinensis;

[0120] Figure 9 A partial haplotype analysis diagram of 74 cold-resistant SNP sites in Pennisetum var. chinensis;

[0121] Figure 10 A partial haplotype analysis diagram of 74 cold-resistant SNP sites in Pennisetum var. chinensis;

[0122] Figure 11 A partial haplotype analysis diagram of 74 cold-resistant SNP sites in Pennisetum var. chinensis;

[0123] Figure 12 A partial haplotype analysis diagram of 74 cold-resistant SNP sites in Pennisetum var. chinensis;

[0124] Figure 13 A partial haplotype analysis diagram of 74 cold-resistant SNP sites in Pennisetum var. chinensis;

[0125] Figure 14 A partial haplotype analysis diagram of 74 cold-resistant SNP sites in Pennisetum var. chinensis;

[0126] Figure 15 The graph shows the correlation between the number of dominant haplotypes and the mean number of seedlings that have sprouted and had their roots cut back at multiple locations. Detailed Implementation

[0127] 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.

[0128] 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.

[0129] As a typical warm-season C4 plant, the poor cold tolerance of Pennisetum alopecuroides is a major factor limiting its cultivation. Therefore, accurate assessment of cold tolerance is crucial for breeding and cultivation. The number of newly sprouted, root-cut seedlings serves as a core indicator for assessing cold tolerance due to the profound intrinsic link between its biological characteristics and cold-resistance mechanisms. The above-ground parts of Pennisetum alopecuroides completely die under frost; its cold tolerance depends entirely on the ability of its underground rhizomes, tillers, and roots to withstand low-temperature stress and maintain vitality. Each spring, the newly sprouted, root-cut seedlings originate directly from a successfully overwintered underground bud. Therefore, the number of newly sprouted seedlings directly and quantitatively reflects the survival rate of the underground bud, becoming a reliable basis for measuring overwintering survival ability.

[0130] Behind this phenomenon lies a key physiological and ecological mechanism. Cold-hardy plants efficiently redirect nutrients to their underground parts in autumn, accumulating abundant non-structural carbohydrates such as starch and soluble sugars. These substances not only enhance freeze resistance by lowering cell freezing points and stabilizing membrane structures, but also provide an energy source for spring regrowth. The large number and robust growth of regrowth seedlings indicate that the underground tissues not only effectively avoid low-temperature damage but also possess sufficient nutrient reserves to support regeneration. Furthermore, cold-hardiness studies typically employ uniform mowing treatment. This method eliminates errors caused by above-ground residues, forcing all plants to regenerate from the same starting point using underground organs. This amplifies the differences in cold hardiness under different genotypes or management practices, making the number of regrowth seedlings a clear and sensitive discriminative indicator.

[0131] Therefore, the number of seedlings that turn green again after root cutting is not an isolated trait, but a comprehensive reflection of the cold resistance of Pennisetum acutum.

[0132] This invention constructs four GWAS populations of *Pennisetum alopecuroides* at different altitudes, and proposes a method for developing and utilizing molecular markers for cold resistance in *Pennisetum alopecuroides* based on GWAS screening using three population screening markers and one population validation marker. The method includes the following steps: germplasm resource collection and GWAS population construction; using the number of seedlings with roots cut back after greening as an indicator of cold resistance; high-throughput sequencing and SNP screening; molecular marker primer design for candidate SNP sites; PCR amplification and genotyping.

[0133] Example 1

[0134] 1. Sampling

[0135] This invention utilizes four GWAS (Georgia macrophylla) populations constructed at different altitudes to evaluate the cold hardiness of *Pennisetum comosum*. The populations are propagated using vegetative propagation. *Pennisetum comosum* stems with 2-5 nodes from the base are selected, and each point is propagated using a single stem, retaining two nodes. The stems are planted at a 45° angle, ensuring the lower nodes are buried in the soil to allow for normal growth and tillering. The plants are uniformly harvested in autumn for natural overwintering, with a harvesting height of 12-15 cm. Figure 1 As shown, the number of seedlings that turn green again the following year after overwintering is used as an important indicator for evaluating the cold resistance and regeneration ability of Pennisetum acutum.

[0136] For the sampling of *Pennisetum affine*, fresh tissue from the middle of the leaf of each sample was taken for DNA extraction using the TIANGEN High-Efficiency Plant Genomic DNA Extraction Kit.

[0137] 2. Resequencing

[0138] The GWAS population was resequencing at a depth of 10×. The raw data underwent quality control, including adapter removal and low-quality read removal. Referring to the *Phragmites australis* genome size (2.07 GB), variant detection was performed using analysis software capable of handling high-throughput sequencing data and detecting genomic variations. SNP sites were filtered using software capable of analyzing genomic variant data (--maf 0.05--max-missing 0.8).

[0139] 3. Analysis

[0140] Association analysis of high-quality SNP loci with the cold-hardy trait of *Pennisetum purpureum* seedlings was conducted using a mixed linear model in Gemma software, revealing significantly associated SNP loci (P<10). -4 Furthermore, through rigorous haplotype screening, different haplotypes at the variant sites must exhibit highly significant phenotypic differences in the population, such as... Figure 2-14 As shown, a total of 74 important candidate sites were obtained.

[0141] Table 1 shows the SNP marker information for 74 SNPs significantly associated with cold hardiness in *Pennisetum purpureus*. The information includes the chromosome location 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 p-value. INTERGENIC indicates a variant located in a non-coding region between genes. NON_SYNONYMOUS_CODING indicates a variant occurring in a protein-coding region, resulting in a change in the amino acid sequence. STOP_GAINED indicates a variant causing the premature appearance of the stop codon, leading to premature termination of protein synthesis. UPSTREAM indicates a variant located in a non-coding region upstream of a gene. DOWNSTREAM indicates a variant located in a non-coding region downstream of a gene.

[0142] 4. Functional verification

[0143] Based on 74 important candidate SNP sites for cold resistance in *Phragmites australis*, molecular marker primers were designed using Primer3 software. Tables 2 and 3 show the molecular marker primer information for the cold resistance SNP sites in *Phragmites australis*, including SNP name, upstream primer sequence, downstream primer sequence, product size, upstream and downstream primer length, Tm value, and GC content.

[0144] Using DNA from each material as amplification template, a 20 µL reaction system was employed.

[0145] 10×PCR Buffer (containing Mg) 2+ 2.0 µL;

[0146] 0.4 µL of dNTP mixture (2.5 mM each);

[0147] 0.5 µL of forward primer (10 µM);

[0148] 0.5 µL of reverse primer (10 µM);

[0149] Taq DNA polymerase (5 U / µL) 0.2 µL;

[0150] Template DNA 1 µL (concentration 20~50 ng / µL);

[0151] Add sterile water (ddH2O) to the remaining volume to a final volume of 20 µL.

[0152] The PCR reaction procedure is as follows:

[0153] Pre-denaturation at 95℃ for 3 min;

[0154] Enter the cycle (30-35 times): denaturation at 95℃ for 30 s, annealing at 55-60℃ for 30 s (adjust according to primer Tm value), extension at 72℃ for 30-60 s;

[0155] Finally, extend the heat to 72℃ for 5 minutes, and then maintain the temperature at 4℃.

[0156] 5–8 µL of the PCR product was added to a 1.5% agarose gel for electrophoresis. The target band was excised using a gel excision method and purified using a DNA Gel Extraction Kit. The purified PCR product was then sequenced. The nucleotide sequence of the amplified product was determined using first-generation sequencing.

[0157] Based on the field phenotypic data collected from the samples, the results are as follows: Figure 15 As shown, the number of dominant haplotypes is significantly positively correlated with the number of seedlings that have undergone root cutting and regeneration, with a Pearson correlation coefficient of r=0.7952.

[0158] Furthermore, based on the verification of the results, the corresponding analysis of the sample phenotypes and their sequence loci showed that: among the 74 core SNP loci of *Pennisetum purpureus* for cold hardiness, in individual plants with more than 39 dominant haplotypes, 91.27% of the individuals had a higher number of regreening and root-cut seedlings than the average number of regreening and root-cut seedlings in the population (11.98); in individual plants with more than 25 dominant haplotypes, 78.95% of the individuals had a higher number of regreening and root-cut seedlings than the average number of regreening and root-cut seedlings in the population; and in individual plants with 25 or fewer dominant haplotypes, 88.50% of the individuals had a lower number of regreening and root-cut seedlings than the average number of regreening and root-cut seedlings in the population.

[0159] Table 1

[0160]

[0161] Table 2

[0162]

[0163] Table 3

[0164]

[0165] 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 identifying the cold resistance of Pennisetum acutum, characterized in that, The nucleotide sequences of the primer set are SEQ ID NO:1~SEQ ID NO:

148.

2. A detection reagent, characterized in that, It includes a primer set, the nucleotide sequences of which are SEQ ID NO:1 to SEQ ID NO:

148.

3. A reagent kit, characterized in that, It includes a primer set, the nucleotide sequences of which are SEQ ID NO:1 to SEQ ID NO:

148.

4. A gene chip, characterized in that, It includes a primer set, the nucleotide sequences of which are SEQ ID NO:1 to SEQ ID NO:148.

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