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

Abstract

The invention relates to the technical field of molecular biology, and discloses 74 core molecular marker sets for identifying cold resistance of pennisetum alopecuroides, a primer group, a detection reagent, a kit, a gene chip and application thereof. 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 and phenotypic variation is fully covered, and a representative material foundation is laid for cold-resistant genetic analysis. In the aspect of phenotype evaluation, a method for determining the number of reviving and root cutting seedlings after natural overwintering by combining single-root double-stem-node oblique cutting planting is innovatively provided, a set of scientific and stable cold resistance and reproducibility evaluation system capable of being operated in a standardized mode is established, and the reliability and efficiency of phenotype identification are remarkably improved. The detection is not influenced by human and climate environments; the method realizes efficient and accurate early cold resistance identification of pennisetum alopecuroides, greatly improves the breeding screening efficiency and scale, and accelerates the breeding process.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of molecular biology, and discloses a 74-molecular-marker set for identifying cold tolerance of Pennisetum, a primer group, a detection reagent, a kit, a gene chip and application thereof. BACKGROUND

[0002] Pennisetum is originally from Africa and other tropical and subtropical regions. At present, Pennisetum has been widely researched and applied as a high-yield and high-quality forage grass due to its rapid growth, tall plant, high biological yield (the highest yield of gramineous forage grass, with an average annual dry matter yield of 80 tons per hectare), rich nutrition, wide soil adaptability and outstanding disease and pest resistance. The Pennisetum can be dried into hay or converted into silage feed through fermentation process, effectively solving the problem of insufficient forage supply in dry season and winter. In addition, Pennisetum is also an excellent energy plant, with alcohol yield being 3 times that of energy plant switchgrass and heat value being 0.7 times that of coal, which is a good material for producing biofuel, biochar, alcohol, methane and paper.

[0003] However, Pennisetum prefers warm and humid climate conditions and is not resistant to low temperature frost, and cannot naturally overwinter in the north of the Yangtze River, which has become an important bottleneck hindering the wide application of Pennisetum in China. In the prior art, there is no related report on improving the cold resistance of hybrid Pennisetum. The optimum growth temperature of Pennisetum is 30-35℃, and the growth stops when the environmental temperature is lower than 10℃, and the continuous low temperature will cause the death of the whole plant (aboveground and underground tissues). With the increase of latitude, the yield of Pennisetum shows a significant downward trend, from 80 tons of dry matter per hectare per year in tropical regions to 45 tons at 30 degrees north latitude, and finally to 30 tons at 36 degrees north latitude, or even 22 tons, which greatly limits its application in northern regions.

[0004] In terms of cold tolerance evaluation, the current mainly relies on physiological indexes measured in the laboratory under controlled conditions at the seedling stage. For example, a Chinese patent with the application number CN202311217536.4 discloses an evaluation method for cold tolerance of Pennisetum based on principal component analysis. The method evaluates the cold tolerance by combining physiological indexes such as leaf relative water content, proline content, malondialdehyde content and low-temperature semi-lethal temperature, and calculating the comprehensive score D value by principal component analysis after low-temperature stress treatment of Pennisetum seedlings.

[0005] However, the correlation between such indicators and the actual overwintering performance in the field is weak, which is difficult to accurately reflect the true cold tolerance of the plant in the field environment, and thus cannot provide reliable guidance for breeding practice. This method not only consumes time and effort, but also is difficult to apply to large-scale breeding population screening, which seriously delays the breeding process of cold-tolerant Pennisetum varieties. Although researchers usually conduct phenotypic identification through post-overwintering green-up rate investigation and rootless seedling counting, such methods still have limitations such as long data collection period, high labor intensity, and significant environmental interference.

[0006] Therefore, it is particularly urgent to develop an efficient and accurate early identification technology system for cold tolerance. Using genome-wide association analysis (GWAS) to locate SNP sites significantly related to field green-up ability and rootless seedling number, and then establishing a molecular marker that can be used for assisted selection, will help achieve seedling stage prediction of cold tolerance. This strategy can significantly reduce the dependence on traditional overwintering phenotypic identification, greatly improve the screening efficiency - a single technician can complete the genotyping of hundreds of samples per day, effectively avoid misjudgment caused by environmental variation, improve selection accuracy and breeding efficiency, and ultimately shorten the breeding period and accelerate the breeding process of cold-tolerant Pennisetum varieties. The promotion of such molecular marker-assisted selection technology will provide key technical support for breaking through the bottleneck of Pennisetum cold-tolerance breeding, and has important significance for promoting the independent innovation of grass seed industry and sustainable forage supply in China.

[0007] 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 art. SUMMARY

[0008] The present application relates to the field of molecular biology, and discloses a 74-core molecular marker set for cold tolerance identification of Pennisetum, a primer set, a detection reagent, a kit, a gene chip and applications thereof.

[0009] To solve the above technical problems, one of the purposes of the present application is to provide a 74-core molecular marker set for cold tolerance identification of Pennisetum, which comprises SNP markers numbered as SNP1-74 as follows: The SNP marker is base A:G, and the position is chrB2_48486846 of Pennisetum; The SNP marker is base C:T, and the position is chrA6_67925981 of Pennisetum; The SNP marker is base G:T, and the position is chrB6_81210972 of Pennisetum; SNP marker is base T:C, position is chrB3_86452525 of Pennisetum ciliare; SNP marker is base A:G, position is chrA5_94911641 of Pennisetum ciliare; SNP marker is base C:T, position is chrA5_94911645 of Pennisetum ciliare; SNP marker is base C:G, position is chrB6_102842461 of Pennisetum ciliare; SNP marker is base G:A, position is chrB6_117864964 of Pennisetum ciliare; SNP marker is base A:T, position is chrB6_121632388 of Pennisetum ciliare; SNP marker is base C:A, position is chrB6_122619783 of Pennisetum ciliare; SNP marker is base T:C, position is chrB6_142954294 of Pennisetum ciliare; SNP marker is base C:T, position is chrB6_144593054 of Pennisetum ciliare; SNP marker is base T:A, position is chrB1_161178990 of Pennisetum ciliare; SNP marker is base G:T, position is chrB2_79195022 of Pennisetum ciliare; SNP marker is base G:A, position is chrB7_99782637 of Pennisetum ciliare; SNP marker is base C:T, position is chrB6_139341295 of Pennisetum ciliare; SNP marker is base G:A, position is chrA7_16803736 of Pennisetum ciliare; SNP marker is base T:A, position is chrB2_28762883 of Pennisetum ciliare; SNP marker is base T:A, position is chrA5_46174486 of Pennisetum ciliare; SNP marker is base G:T, position is chrA4_97584889 of Pennisetum ciliare; SNP marker is base C:T, position is chrB7_122721579 of Pennisetum ciliare; SNP marker is base G:A, position is chrB1_158396834 of Pennisetum ciliare; SNP marker is base G:A, position is chrA2_48296938 of Pennisetum ciliare; SNP marker is base G:T, position is chrB2_23656109 of Pennisetum ciliare; SNP marker is base A:G, position is chrAl_50010676 of Pennisetum ciliare; SNP marker is base G:A, position is chrB6_8254957 of Pennisetum ciliare; SNP marker is base G:T, position is chrA2_15662900 of Pennisetum ciliare; SNP marker is base T:G, position is chrA2_15664750 of Pennisetum ciliare; SNP marker is base A:G, position is chrA4_29178270 of Pennisetum ciliare; SNP marker is base G:A, position is chrB3_32980416 of Pennisetum ciliare; SNP marker is base A:G, position is chrB7_106094743 of Pennisetum ciliare; SNP marker is base T:A, position is chrA2_30154555 of Pennisetum ciliare; SNP marker is base C:T, position is chrA2_32222409 of Pennisetum ciliare; SNP marker is base C:T, position is chrA4_38997045 of Pennisetum ciliare; SNP marker is base T:C, position is chrA2_59532018 of Pennisetum ciliare; SNP marker is base A:T, position is chrB6_57518900 of Pennisetum ciliare; SNP marker is base T:G, position is chrB6_82497956 of Pennisetum ciliare; SNP marker is base A:G, position is chrB6_82497974 of Pennisetum ciliare; SNP marker is base T:C, position is chrA4_91805848 of Pennisetum ciliare; SNP marker is base C:T, position is chrA2_37111926 of Pennisetum ciliare; SNP marker is base A:T, position is chrA2_37152867 of Pennisetum ciliare; SNP marker is base A:T, position is chrA2_37152874 of Pennisetum ciliare; SNP marker is base G:A, position is chrA2_37152896 of Pennisetum ciliare; SNP marker is base C:T, position is chrA3_61140681 of switchgrass; SNP marker is base A:T, position is chrAl_16460560 of switchgrass; SNP marker is base G:A, position is chrB5_81211080 of switchgrass; SNP marker is base C:T, position is chrBl_176898490 of switchgrass; SNP marker is base T:C, position is chrBl_9281522 of switchgrass; SNP marker is base T:C, position is chrBl_9727233 of switchgrass; SNP marker is base T:C, position is chrA2_28796142 of switchgrass; SNP marker is base C:T, position is chrB6_35440206 of switchgrass; SNP marker is base A:G, position is chrB6_35442142 of switchgrass; SNP marker is base T:C, position is chrB6_35502016 of switchgrass; SNP marker is base G:T, position is chrA4_37962206 of switchgrass; SNP marker is base A:G, position is chrB6_38037456 of switchgrass; SNP marker is base G:T, position is chrB6_39080862 of switchgrass; SNP marker is base C:T, position is chrB6_39502565 of switchgrass; SNP marker is base T:C, position is chrB6_50861173 of switchgrass; SNP marker is base G:A, position is chrB6_50861183 of switchgrass; SNP marker is base T:C, position is chrB6_51186976 of switchgrass; SNP marker is base A:G, position is chrB6_51816917 of switchgrass; SNP marker is base G:A, position is chrB6_51816998 of switchgrass; SNP marker is base A:G, position is chrB6_51817831 of switchgrass; The SNP marker is base A:G, and the position is chrB6_51823957 of Pennisetum. The SNP marker is base T:C, and the position is chrB6_51824188 of Pennisetum. The SNP marker is base T:C, and the position is chrA7_59005844 of Pennisetum. The SNP marker is base G:C, and the position is chrA4_59737791 of Pennisetum. The SNP marker is base T:A, and the position is chrB2_111371030 of Pennisetum. The SNP marker is base T:A, and the position is chrA7_9111403 of Pennisetum. The SNP marker is base G:A, and the position is chrB6_41472864 of Pennisetum. The SNP marker is base A:G, and the position is chrA7_42957102 of Pennisetum. The SNP marker is base T:C, and the position is chrA7_42958685 of Pennisetum. The SNP marker is base A:G, and the position is chrA7_59005815 of Pennisetum. The SNP marker is base G:A, and the position is chrB7_923607 of Pennisetum.

[0010] The SNP molecular marker set provided by the application is a specific SNP molecular marker set, the core of which is to use 74 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.

[0011] One of the purposes of the application is to provide the application of the core molecular marker set as a detection target in identifying the cold tolerance of Pennisetum, and the core molecular marker set includes the 74 core molecular marker sets for identifying the cold tolerance of Pennisetum.

[0012] According to a preferred embodiment, when more than 39 markers are advantage haplotypes, it is indicated that the number of green-up cut root seedlings is an advantage individual.

[0013] According to a preferred embodiment, when more than 25 and not more than 39 markers are advantage haplotypes, it is indicated that the number of green-up cut root seedlings is an average individual.

[0014] According to a preferred embodiment, when not more than 25 markers are advantage haplotypes, it is indicated that the number of green-up cut root seedlings is a disadvantage individual.

[0015] Summarized as: in 74 cold-tolerant molecular markers of wolf tail grass, the number of individual dominant haplotype is greater than 39, which is high cold-tolerant type, 25 is less than or equal to the number of individual dominant haplotype, which is medium cold-tolerant type, and the number of individual dominant haplotype is less than or equal to 25, which is low cold-tolerant type.

[0016] According to a preferred embodiment, the application is: Wolf tail grass germplasm identification and improvement; or The application in early prediction of wolf tail grass seedling stage cold tolerance.

[0017] One of the purposes of the application is also to provide a primer set for detecting target points to identify the cold tolerance of wolf tail grass. The primer set comprises SEQ ID NO: 1 to SEQ ID NO: 148.

[0018] One of the purposes of the application is also to provide a reaction system of the above-mentioned primer set, and the total reaction system of PCR amplification is 20 μL, which comprises 10×PCR buffer (10×PCR buffer containing Mg2+) 2.0 μL; dNTP mixture (dNTP mixture, 2.5 mM each) 0.4 μL; forward primer (forward primer, 10 μM) 0.5 μL; reverse primer (reverse primer, 10 μM) 0.5 μL; Taq DNA polymerase (Taq DNA polymerase, 5 U / μL) 0.2 μL; template DNA 1 μL (concentration 20-50 ng / μL); the rest is sterilized water (ddH2O) to 20 μL.

[0019] One of the purposes of the application is also to provide a detection reagent, which comprises the core molecular marker set in the above-mentioned application or the primer set.

[0020] One of the purposes of the application is also to provide a kit, which comprises the core molecular marker set in the above-mentioned application or the primer set.

[0021] One of the purposes of the application is also to provide a gene chip, which comprises the core molecular marker set in the above-mentioned application or the primer set.

[0022] One of the purposes of the application is also to provide a method for identifying cold-tolerant varieties of wolf tail grass, which comprises the following steps: The genomic DNA of the variety to be detected is amplified by using the primer pair of SEQ ID NO: 1 to SEQ ID NO: 148, and the combination of 74 SNP markers and the determined typing results.

[0023] When more than 39 markers are advantage haplotypes, it indicates that the number of regrowth stump sprouts of the individual is higher than the average level of the population. This result indicates that the individual is a high cold-tolerant variety.

[0024] According to a preferred embodiment, when more than 25 and not more than 39 markers are advantage haplotypes, it indicates that the number of regrowth stump sprouts of the individual is equal to the average level of the population. This result indicates that the individual is a medium cold-tolerant variety.

[0025] According to a preferred embodiment, when not more than 25 markers are advantage haplotypes, it indicates that the number of regrowth stump sprouts of the individual is lower than the average level of the population. This result indicates that the individual is a low cold-tolerant variety.

[0026] Among the 74 cold-tolerant molecular markers of switchgrass: High cold-tolerant type: the number of advantage haplotypes > 39; Medium cold-tolerant type: 25 < the number of advantage haplotypes ≤ 39; Low cold-tolerant type: the number of advantage haplotypes ≤ 25.

[0027] One of the purposes of the present application is also to provide a method for identifying a cold-tolerant variety of switchgrass, comprising the following steps: If the number of regrowth stump sprouts of the individual after overwintering is more than the average number of regrowth stump sprouts of the population, the individual is identified as a cold-tolerant variety.

[0028] The beneficial effects of the present application are: The present application collects switchgrass core germplasm resources systematically, and uses multi-environment (different altitudes) conditions to construct GWAS populations, which fully covers a wide range of genetic and phenotypic variations, and lays a representative material foundation for genetic analysis of cold tolerance. In terms of phenotype evaluation, a method of single-root double-stem-node oblique planting combined with determination of the number of regrowth stump sprouts after natural overwintering is proposed, and a scientific, stable and standardizable evaluation system for cold tolerance and regenerative ability is established, which significantly improves the reliability and efficiency of phenotype identification.

[0029] Based on high-throughput genotyping and genome-wide association analysis, 74 SNP sites significantly related to cold tolerance are successfully identified through strict screening and haplotype verification, and specific molecular marker primers with clear product characteristics (including size, Tm value, GC content, etc.) and flanking sequence information are designed using Primer3, realizing efficient conversion from GWAS results to practical molecular marker system. At the same time, a standardized PCR reaction system and genotyping process are established, including amplification, electrophoresis detection, purification and sequencing, which ensures that the operation is repeatable and the results are reliable, and can quickly and batch identify sample genotypes, greatly promoting the application of molecular markers in actual breeding.

[0030] The developed molecular marker is significantly correlated with the cold tolerance phenotype in the field, and there is a significant positive correlation between the number of advantageous haplotypes and the number of regreening root cutting seedlings (r=0.7952), which fully verifies the accuracy and application value of the marker system in cold tolerance prediction. The present application effectively overcomes the limitations of long cycle, high work intensity and easy environmental interference in traditional field overwintering identification, and can realize efficient and accurate genotyping and selection at the seedling stage, greatly improving the breeding efficiency and shortening the breeding period, providing a solid technical support and promotion prospect for the breeding of new cold-tolerant varieties of wolf tail grass. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 The number of regreening root cutting seedlings of wolf tail grass in the field is different; Figure 2 Part of the haplotype analysis diagram of 74 cold-tolerant SNP sites of wolf tail grass; Figure 3 Part of the haplotype analysis diagram of 74 cold-tolerant SNP sites of wolf tail grass; Figure 4 Part of the haplotype analysis diagram of 74 cold-tolerant SNP sites of wolf tail grass; Figure 5 Part of the haplotype analysis diagram of 74 cold-tolerant SNP sites of wolf tail grass; Figure 6 Part of the haplotype analysis diagram of 74 cold-tolerant SNP sites of wolf tail grass; Figure 7 Part of the haplotype analysis diagram of 74 cold-tolerant SNP sites of wolf tail grass; Figure 8 Part of the haplotype analysis diagram of 74 cold-tolerant SNP sites of wolf tail grass; Figure 9 Part of the haplotype analysis diagram of 74 cold-tolerant SNP sites of wolf tail grass; Figure 10 Part of the haplotype analysis diagram of 74 cold-tolerant SNP sites of wolf tail grass; Figure 11 Part of the haplotype analysis diagram of 74 cold-tolerant SNP sites of wolf tail grass; Figure 12 Part of the haplotype analysis diagram of 74 cold-tolerant SNP sites of wolf tail grass; Figure 13 Part of the haplotype analysis diagram of 74 cold-tolerant SNP sites of wolf tail grass; Figure 14 Part of the haplotype analysis diagram of 74 cold-tolerant SNP sites of wolf tail grass; Figure 15 Part of the haplotype analysis diagram of 74 cold-tolerant SNP sites of wolf tail grass; DETAILED DESCRIPTION

[0032] In the description of the application, the terms are used only for descriptive purposes, and cannot be construed to indicate or imply relative importance or imply the number of technical features indicated. In the description of the application, the meaning of "a plurality" is two or more, unless otherwise explicitly specified.

[0033] Unless otherwise specified, the test methods used in the following examples are conventional methods; the materials, reagents or instruments used, if not specified by the manufacturer, are reagents and materials available from commercial channels; if the specific conditions are not specified in the examples, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer, and at the same time, the source of the raw materials used by the present application is not limited, and if not specified, the raw materials used in the present application are ordinary commercially available products in the technical field.

[0034] As a typical warm-type C4 plant, the poor cold tolerance of Pennisetum villosum is the main factor limiting the expansion of its planting range, so accurate evaluation of cold tolerance is crucial for breeding and cultivation. The number of green shoots of cut root seedlings is the core index for evaluating cold tolerance because of its deep internal connection between biological characteristics and cold resistance mechanism. The aboveground part of P. villosum will completely die under frost, and its cold tolerance completely depends on whether the underground rhizomes, tillering nodes and root systems can resist low temperature stress and maintain vitality. Each green shoot that returns in spring directly comes from a successful overwintering underground bud point, so the number of green shoots directly and quantitatively reflects the survival rate of underground bud points, and becomes a reliable basis for measuring overwintering survival ability.

[0035] This appearance contains key physiological and ecological mechanisms. Plants with strong cold tolerance can efficiently return nutrients to the underground part in autumn, accumulating rich non-structural carbohydrates such as starch and soluble sugar, which not only enhance the frost resistance by reducing the cell freezing point and stabilizing the membrane structure, but also provide energy sources for spring green shoots. The number of green shoots is large and the growth is healthy, indicating that the underground tissue not only effectively avoids low temperature damage, but also has sufficient nutrient reserves to support regeneration. In addition, uniform mowing treatment is usually used in cold tolerance research, which eliminates the error caused by the remaining aboveground part, forces all plants to rely on underground organs for regeneration from the same starting point, and thus magnifies the differences in cold tolerance of different genotypes or management measures, making the number of green shoots a clear and sensitive index for discrimination.

[0036] Therefore, the number of green shoots of cut root seedlings is not an isolated trait, but a comprehensive manifestation of the cold tolerance of P. villosum.

[0037] The application is based on four Pennisetum alopecuroides GWAS populations constructed at different altitudes, and a Pennisetum alopecuroides cold tolerance molecular marker development and utilization method based on GWAS screening is proposed, which comprises the following steps: germplasm resource collection, GWAS population construction; green-up root cutting seedling number as cold tolerance evaluation index; high-throughput sequencing and SNP screening; candidate SNP site molecular marker primer design; PCR amplification and genotyping.

[0038] Example 1 1. Sampling The application is based on four Pennisetum alopecuroides GWAS populations constructed at different altitudes, and a Pennisetum alopecuroides cold tolerance molecular marker development and utilization method based on GWAS screening is proposed, which comprises the following steps: germplasm resource collection, GWAS population construction; green-up root cutting seedling number as cold tolerance evaluation index; high-throughput sequencing and SNP screening; candidate SNP site molecular marker primer design; PCR amplification and genotyping. Figure 1 As shown in the figure, the number of root cutting seedlings after the next year of overwintering is used as an important index for evaluating the cold tolerance and regenerative ability of Pennisetum alopecuroides.

[0039] Pennisetum alopecuroides samples are sampled, and fresh tissue in the middle of the leaves of each sample is used for DNA extraction, and a TIANGEN high-efficiency plant genomic DNA extraction kit is used.

[0040] 2. Re-sequencing The GWAS population is re-sequenced, the sequencing depth is 10x, and the original data is quality controlled, including removing adapters and low-quality reads. Referring to the size of the Pennisetum alopecuroides genome (2.07 GB), the analysis software capable of processing high-throughput sequencing data and detecting variations in the genome is used for variation detection. The software capable of analyzing genomic variation data is used to filter SNP sites (--maf 0.05--max-missing 0.8).

[0041] 3. Analysis High-quality SNP sites and Pennisetum alopecuroides cold tolerance traits are analyzed using the mixed linear model of Gemma software, and significant associated SNP sites (P<10 -4 ) are obtained, and through further strict haplotype screening, the different haplotypes of the variation sites in the population need to have extremely significant differences in phenotype, as shown in the figure, a total of 74 important candidate sites are obtained. Figures 2-14

[0042] ​Table 1 shows the information of 74 cold tolerance significantly associated SNP markers of Pennisetum. The information contains 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 P_value. INTERGENIC indicates a variation in the non-coding region between genes. NON_SYNONYMOUS_CODING indicates a variation that occurs in the protein coding region, resulting in a change in the amino acid sequence. STOP_GAINED indicates that the variation causes the premature appearance of the original stop codon, causing the protein synthesis to terminate prematurely. UPSTREAM indicates a variation in the non-coding region upstream of the gene. DOWNSTREAM indicates a variation in the non-coding region downstream of the gene.

[0043] 4. Function verification Based on the 74 important cold tolerance candidate SNP sites of Pennisetum, molecular marker primers were designed using primer3 software. Tables 2 and 3 show the information of the molecular marker primers of the cold tolerance SNP sites of Pennisetum, which contains the SNP name, upstream primer sequence, downstream primer sequence, product size, and upstream and downstream primer length, Tm value and GC content.

[0044] Using each material DNA as the amplification template, a 20 µL reaction system was used: 10×PCR buffer (10×PCR Buffer, containing Mg 2+ ) 2.0 µL; dNTP mixture (dNTP Mix, 2.5 mM each) 0.4 µL; Forward primer (Forward Primer, 10 µM) 0.5 µL; Reverse primer (Reverse Primer, 10 µM) 0.5 µL; Taq DNA polymerase (Taq DNA Polymerase, 5 U / µL) 0.2 µL; Template DNA 1 µL (concentration 20~50 ng / µL); The rest is sterilized water (ddH2O) to 20 µL.

[0045] The PCR reaction program is as follows: Pre-denaturation 95℃ 3 min; Enter cycle (30-35 times): denaturation 95℃ 30 s, annealing 55-60℃ 30 s (adjust according to primer Tm value), extension 72℃ 30~60 s; Final extension 72℃ 5 min, final incubation at 4℃.

[0046] 5-8 μL of the product in the PCR reaction system was added into a 1.5% agarose gel for electrophoresis detection. The target band was cut off using a gel cutting method, and the product was purified using a DNA gel extraction kit. Subsequently, the purified PCR product was sent for sequencing. The nucleotide sequence of the amplified product was determined using first-generation sequencing.

[0047] In combination with the collected data on the field phenotype traits of the samples, the results are shown in Table 1. Figure 15 As shown in Table 1, the number of dominant haplotypes was positively correlated with the number of regrowth stump sprouts, with a Pearson correlation coefficient r = 0.7952.

[0048] Further, based on the verification of the results, the sample phenotype and the corresponding analysis results of the sequence sites showed that among the 74 cold-tolerant core SNP sites of Pennisetum villosum, in the single plants in which more than 39 sites were dominant haplotypes, 91.27% of the individuals had a number of regrowth stump sprouts higher than the average number of regrowth stump sprouts of the population (11.98); in the single plants in which more than 25 sites were dominant haplotypes, 78.95% of the individuals had a number of regrowth stump sprouts higher than the average number of regrowth stump sprouts of the population; and in the single plants in which 25 or fewer sites were dominant haplotypes, 88.50% of the individuals had a number of regrowth stump sprouts lower than the average number of regrowth stump sprouts of the population.

[0049] Table 1

[0050] Table 2

[0051] Table 3

[0052] 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 74 core molecular markers for identifying the cold resistance of *Phragmites australis*, characterized in that, The 74 core molecular marker set includes the following SNP markers numbered SNP1 to 74: The SNP is labeled with the base A:G and is located at the chrB2_48486846th position of the grass; The SNP is labeled with the base C:T and is located at the chrA6_67925981st position of the grass; The SNP is labeled with the base G:T and is located at the chrB6_81210972nd position of the grass; The SNP is labeled with the base T:C and is located at the chrB3_86452525th position of the Napier grass. The SNP is labeled with the base A:G and is located at the chrA5_94911641st position of the grass. The SNP is labeled with the base C:T and is located at position chrA5_94911645 of the grass; The SNP is labeled with the base C:G and is located at the chrB6_102842461st position of the grass; The SNP is labeled with the base G:A, and its position is the chrB6_117864964th position of the grass. The SNP is labeled with the base A:T and is located at the chrB6_121632388th position of the grass. The SNP is labeled with the base C:A, and its position is the chrB6_122619783rd position of the grass. The SNP is labeled with the base T:C and is located at the chrB6_142954294th position of Pennisetum var. chinensis. The SNP is labeled with the base C:T and is located at the chrB6_144593054th position of Pennisetum var. mongolicum. The SNP is labeled with base T:A, and its position is the chrB1_161178990th position of the grass; The SNP is labeled with the base G:T and is located at the chrB2_79195022nd position of the Napier grass. The SNP is labeled with the base G:A, and its position is the chrB7_99782637th position of the grass. The SNP is labeled with the base C:T and is located at the chrB6_139341295th position of the grass. The SNP is labeled with the base G:A, and its position is the chrA7_16803736th position of the grass. The SNP is labeled with base T:A, and its position is the chrB2_28762883rd position of the grass. The SNP is labeled with the base T:A, and its position is the chrA5_46174486th position of the grass. The SNP is labeled with the base G:T and is located at the chrA4_97584889th position of Pennisetum var. chinensis. The SNP is labeled with the base C:T and is located at the chrB7_122721579th position of the grass. The SNP is labeled with the base G:A, and its position is the chrB1_158396834th position of the grass; The SNP is labeled with the base G:A, and its position is the chrA2_48296938th position of the grass. The SNP is labeled with the base G:T and is located at the chrB2_23656109th position of Pennisetum var. chinensis. The SNP is labeled with the base A:G and is located at the chrA1_50010676th position of the foxtail grass. The SNP is labeled with the base G:A, and its position is the chrB6_8254957th position of the Napier grass. The SNP is labeled with the base G:T and is located at the chrA2_15662900th position of the Napier grass. The SNP is labeled with the base T:G and is located at the chrA2_15664750th position of the Napier grass. The SNP is labeled with the base A:G and is located at the chrA4_29178270th position of the grass. The SNP is labeled with the base G:A, and its position is the chrB3_32980416th position of the Napier grass. The SNP is labeled with base A:G and is located at position chrB7_106094743 of the grass; The SNP is labeled with the base T:A, and its position is the chrA2_30154555th position of the grass. The SNP is labeled with the base C:T and is located at the chrA2_32222409th position of the grass. The SNP is labeled with the base C:T and is located at the chrA4_38997045th position of the grass; The SNP is labeled with the base T:C and is located at the chrA2_59532018th position of the Napier grass. The SNP is labeled with the base A:T and is located at the chrB6_57518900th position of the grass. The SNP is labeled with the base T:G and is located at the chrB6_82497956th position of the grass; The SNP is labeled with the base A:G and is located at the chrB6_82497974th position of Pennisetum var. chinensis. The SNP is labeled with the base T:C and is located at position chrA4_91805848 of the grass; The SNP is labeled with the base C:T and is located at the chrA2_37111926th position of the grass. The SNP is labeled with the base A:T and is located at the chrA2_37152867th position of the grass. The SNP is labeled with the base A:T and is located at the chrA2_37152874th position of the grass. The SNP is labeled with the base G:A, and its position is the chrA2_37152896th position of the grass. The SNP is labeled with the base C:T and is located at the chrA3_61140681st position of the grass. The SNP is labeled with the base A:T and is located at the chrA1_16460560th position of the Napier grass. The SNP is labeled with the base G:A, and its position is the chrB5_81211080th position of the grass. The SNP is labeled with the base C:T and is located at the chrB1_176898490th position of the grass. The SNP is labeled with the base T:C and is located at the chrB1_9281522nd position of the Napier grass. The SNP is labeled with the base T:C and is located at the chrB1_9727233rd position of the foxtail grass. The SNP is labeled with the base T:C and is located at the chrA2_28796142nd position of Pennisetum var. chinensis. The SNP is labeled with the base C:T and is located at the chrB6_35440206th position of the Napier grass. The SNP is labeled with the base A:G and is located at the chrB6_35442142nd position of the grass. The SNP is labeled with the base T:C and is located at the chrB6_35502016th position of the grass; The SNP is labeled with the base G:T and is located at the chrA4_37962206th position of the Napier grass. The SNP is labeled with base A:G and is located at position chrB6_38037456 of the grass; The SNP is labeled with the base G:T and is located at the chrB6_39080862nd position of the Napier grass. The SNP is labeled with the base C:T and is located at the chrB6_39502565th position of the grass. The SNP is labeled with the base T:C and is located at the chrB6_50861173rd position of the grass. The SNP is labeled with the base G:A, and its position is the chrB6_50861183rd position of the Napier grass. The SNP is labeled with the base T:C and is located at the chrB6_51186976th position of the grass; The SNP is labeled with the base A:G and is located at the chrB6_51816917th position of the grass; The SNP is labeled with the base G:A, and its position is the chrB6_51816998th position of the Napier grass. The SNP is labeled with the base A:G and is located at the chrB6_51817831st position of the grass; The SNP is labeled with base A:G and is located at position chrB6_51823957 of the grass; The SNP is labeled with the base T:C and is located at the chrB6_51824188th position of the Napier grass. The SNP is labeled with the base T:C and is located at position chrA7_59005844 of the grass; The SNP is labeled with the base G:C and is located at the chrA4_59737791st position of the grass. The SNP is labeled with base T:A, and its position is the chrB2_111371030th position of Pennisetum var. chinensis. The SNP is labeled with the base T:A, and its position is the chrA7_9111403rd position of the grass. The SNP is labeled with the base G:A, and its position is the chrB6_41472864th position of the foxtail grass. The SNP is labeled with the base A:G and is located at the chrA7_42957102nd position of the grass; The SNP is labeled with the base T:C and is located at the chrA7_42958685th position of the grass. The SNP is labeled with the base A:G and is located at the chrA7_59005815th position of the grass; The SNP is labeled with base G:A, located at position chrB7_923607 of the Napier grass.

2. The application of a core molecular marker set as a detection target in identifying the cold resistance of *Phragmites australis*, characterized in that... The core molecular marker set includes the 74 core molecular markers for identifying the cold resistance of Pennisetum as described in claim 1.

3. The application according to claim 2, characterized in that, When more than 39 haplotypes are marked as dominant, it indicates that the individual is a highly cold-resistant variety.

4. The application according to claim 2, characterized in that, When more than 25 but no more than 39 individuals are marked as dominant haplotypes, it indicates that the individual is a moderately cold-resistant variety.

5. The application according to claim 2, characterized in that, When there are no more than 25 individuals marked as dominant haplotypes, it indicates that the individual is a low-hardiness variety.

6. The application 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 detecting targets to identify the cold resistance of Pennisetum acutum, characterized in that, The primer set contains SEQ ID NO:1 to SEQ ID NO:

148.

8. A detection reagent, characterized in that, It includes the 74 core molecular markers set for identifying the cold resistance of Napier grass as described in claim 1 or the primer set as described in claim 6.

9. A reagent kit, characterized in that, It includes the 74 core molecular markers set for identifying the cold resistance of Napier grass as described in claim 1 or the primer set as described in claim 6.

10. A gene chip, characterized in that, It includes the 74 core molecular markers set for identifying the cold resistance of Napier grass as described in claim 1 or the primer set as described in claim 6.

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