Leymus mollis drought resistance trait related snp molecular marker and application

Abstract

The application discloses a SNP molecular marker related to drought resistance of Leymus chinensis and application, the molecular marker is selected from one of SNP1, SNP2, SNP3 and SNP4, and the molecular marker combination is combined by the four markers of SNP1, SNP2, SNP3 and SNP4, the C, GC, T, ATG homozygous genotype of the molecular marker combination is in order as SNP1-SNP4, that is, the CC-GGCC-TT-AATTGG genotype combination, and the corresponding Leymus chinensis has the strongest drought resistance. Through the molecular marker related to drought resistance of Leymus chinensis and the combination provided by the application, the drought resistance of Leymus chinensis can be accurately evaluated at the molecular level, drought-resistant seed sources can be accurately and efficiently screened at the seedling stage of Leymus chinensis, and the breeding cycle of Leymus chinensis is shortened.

Description

Technical Field

[0001] This invention relates to the field of plant molecular breeding technology, specifically to SNP molecular markers related to drought resistance traits in Leymus chinensis and their applications. Background Technology

[0002] Currently, to ensure meat and grain security, the livestock industry requires a large amount of land for forage production. Increasing the utilization of arid and semi-arid land in the north is a key measure to expand the scale of the livestock industry and supplement the meat and grain supply for Chinese residents. Selecting and cultivating forage resources with strong drought resistance is an important link in achieving this goal.

[0003] Leymus chinensis, a perennial herbaceous plant belonging to the Poaceae family, is named for its grazing habits among sheep and goats. Historically, it was a dominant species in the arid and semi-arid grasslands of Northeast China and Inner Mongolia. With its extensive root system, Leymus chinensis can deeply penetrate the soil to absorb water and nutrients, thriving in arid environments and making it a common forage grass in suitable grazing wastelands. Rich in protein, crude fiber, minerals, and vitamins, Leymus chinensis provides ample nutrition for cattle and sheep, promoting their growth and development, and is widely recognized by livestock producers. However, compared to other competing grassland species, its seedling development is relatively slow, and it is particularly sensitive to drought stress, which is a major bottleneck in its application. Therefore, breeding drought-resistant Leymus chinensis resources can effectively expand the area of ​​suitable grazing wastelands, increase forage yield, and expand the scale of the livestock industry.

[0004] Although drought-resistant germplasm screening is currently underway for grasses, the Poaceae family is a very broad and vast plant group defined by the traditional Linnaeus classification system, making it difficult to apply drought-resistant screening methods across different species. Taking rice as an example, although rice and Leymus chinensis belong to the same Poaceae family, they differ significantly in genetics, physiology, and anatomy. Genetically, rice belongs to the genus *Oryza* and is an autodiploid plant with 24 chromosomes (2n = 2x = 24), a genome size of approximately 385 Mb, and about 40,000 protein-coding genes; while Leymus chinensis belongs to the genus *Leymus* and is an allotetraploid plant with 28 chromosomes (2n = 4x = 28). The genome size is approximately 8Gb, containing over 80,000 genes encoding proteins. Physiologically, rice originated in the middle reaches of the Pearl River in China, with an average annual rainfall of 700-2400 mm and an average annual temperature of 14-22℃, while Leymus chinensis originated in the eastern Eurasian steppe region, with an average annual rainfall of only 120-200 mm and an average annual temperature of only -2-6℃. Their optimal growth conditions differ significantly. Anatomically, Leymus chinensis seedlings have needle-like leaves with a stomatal density as high as 90-170 stomata / mm². 2Its stomata are 38–42 μm long, and it has rhizomes and sand-like structures, exhibiting typical characteristics of xerophytes; while rice leaves are strap-shaped, with a stomatal density of only 11–27 per mm. 2 Its stomata are only 9–16 μm long, exhibiting characteristics of mesophytes. Therefore, the significant differences between the two determine that drought-resistant screening methods suitable for rice are not suitable for Leymus chinensis.

[0005] SNP molecular markers play an important auxiliary role in plant drought resistance breeding. However, suitable SNP molecular markers are lacking in the breeding of drought-resistant Leymus chinensis. Traditional methods for establishing SNP molecular markers associated with drought resistance traits suffer from poor accuracy, insufficient sample throughput, and high demands on human and material resources, making them difficult to apply effectively to the breeding of plants with large germplasm resources. China has extremely rich Leymus chinensis germplasm resources. To solve the above problems, there is an urgent need to develop an effective SNP molecular marker specifically associated with drought resistance traits in Leymus chinensis. Summary of the Invention

[0006] This invention provides SNP molecular markers related to drought resistance traits in Leymus chinensis and their applications, providing a reference for breeding drought-resistant Leymus chinensis.

[0007] In view of this, the solution of the present invention is as follows:

[0008] The first aspect of this invention is to propose SNP molecular markers related to drought resistance traits in Leymus chinensis, wherein the molecular markers are selected from one of SNP1, SNP2, SNP3, and SNP4; wherein:

[0009] The molecular marker SNP1 is located at the 220th base of the nucleotide sequence shown in SEQ ID NO: 1 and exhibits C / G polymorphism.

[0010] The molecular marker SNP2 is located at bases 380 and 381 of the nucleotide sequence shown in SEQ ID NO: 2, and has GC / CA polymorphism;

[0011] The molecular marker SNP3 is located at the 277th base of the nucleotide sequence shown in SEQ ID NO: 3 and exhibits T / G polymorphism.

[0012] The molecular marker SNP4 is located at bases 394, 395, and 396 of the nucleotide sequence shown in SEQ ID NO: 4, and exhibits ATG / GGC polymorphism.

[0013] Furthermore, the drought resistance traits include survival rate after drought stress and physiological and biochemical traits; the physiological and biochemical traits are the slope of the decrease in relative water content of leaves.

[0014] A second aspect of the present invention is to propose a molecular marker combination related to drought resistance traits of Leymus chinensis, wherein the molecular marker combination is composed of two or more SNP1, SNP2, SNP3 and SNP4.

[0015] Furthermore, the genotypes of the SNP1 marker are CC and GG, the genotypes of the SNP2 marker are GGCC and CCAA, the genotypes of the SNP3 marker are TT and GG, and the genotype of the SNP4 marker is AATTGG / GGGGCC.

[0016] When the molecular marker combination is a homozygous genotype of C, GC, T, ATG in the order of SNP1 to SNP4, that is, the CC-GGCC-TT-AATTGG genotype combination, the corresponding sheepgrass has the strongest drought resistance.

[0017] The haplotype molecular markers, when the SNP1 to SNP4 are homozygous genotypes GG, CCAA, GG, and GGGGCC respectively, that is, when the genotype combination is GG-CCAA-GG-GGGGCC, the corresponding sheepgrass has the weakest drought resistance.

[0018] A third aspect of the invention is to provide a primer set for amplifying the molecular marker combination described in the second aspect, the primer set consisting of primers for amplifying SNP1 to SNP4 respectively, wherein:

[0019] The primers for amplifying the molecular marker SNP1 include the nucleotide sequences shown in SEQ ID NO: 5-6;

[0020] The primers for amplifying the molecular marker SNP2 include nucleotide sequences as shown in SEQ ID NO: 8-9;

[0021] The primers for amplifying the molecular marker SNP3 include the nucleotide sequences shown in SEQ ID NO: 11-12;

[0022] The primers for amplifying the molecular marker SNP4 include the nucleotide sequences shown in SEQ ID NO: 14-15.

[0023] Preferably, the primer combination is as follows:

[0024] The primer nucleotide sequences for amplifying the molecular marker SNP1 are shown in SEQ ID NO: 5-7;

[0025] The primer nucleotide sequences for amplifying the molecular marker SNP2 are shown in SEQ ID NO: 8-10;

[0026] The primer nucleotide sequences for amplifying the molecular marker SNP3 are shown in SEQ ID NO: 11-13;

[0027] The primer nucleotide sequences for amplifying the molecular marker SNP4 are shown in SEQ ID NO: 14-16.

[0028] A fourth aspect of the invention is to provide a detection reagent or kit comprising the primer combination described in the third aspect.

[0029] A fifth aspect of the present invention is to provide a method for obtaining the SNP molecular markers described in the first aspect and the molecular marker combinations described in the second aspect, the method comprising the following steps:

[0030] Genes upregulated by drought stress were identified based on RNA-Seq.

[0031] Hi-tom was used to identify SNP sites in the promoter regions of genes upregulated under drought stress in the overall population of Leymus chinensis.

[0032] Predicting component function loss due to SNP mutations;

[0033] Hi-tom was used to analyze drought-resistant Leymus chinensis germplasm and compare the proportions of different SNPs in the drought-resistant population and the total population.

[0034] We weighted and scored different SNP types, analyzed the association between SNP types and drought resistance traits, and obtained SNP molecular markers related to drought resistance traits in Leymus chinensis.

[0035] A sixth aspect of the present invention is to provide the use of the SNP molecular markers described in the first aspect, the molecular marker combinations described in the second aspect, the primer combinations described in the third aspect, the detection reagents or kits described in the fourth aspect, and the SNP markers or molecular marker combinations obtained by the method described in the fifth aspect in any of the following aspects (1) to (6):

[0036] (1) To identify or assist in identifying the drought resistance of Leymus chinensis;

[0037] (2) Screening or assisting in the screening of new varieties of sheepgrass with strong drought resistance;

[0038] (3) Molecular marker-assisted breeding of Leymus chinensis;

[0039] (4) Prepare products for identification or auxiliary identification of the drought resistance of Leymus chinensis;

[0040] (5) Prepare products for screening or assisting in the screening of new varieties of Leymus chinensis with strong drought resistance;

[0041] (6) Prepare products for molecular marker-assisted breeding of Leymus chinensis.

[0042] A seventh aspect of the present invention provides a method for identifying or screening the drought resistance of Leymus chinensis, the method comprising the steps of detecting the polymorphism or genotype of the SNP molecular markers of Leymus chinensis as described in the first aspect, or detecting the genotype of the combination of molecular markers as described in the second aspect.

[0043] An eighth aspect of the present invention provides a method for genetic improvement of Leymus chinensis, the method comprising the steps of successively breeding homozygous individuals with the CC genotype of the SNP1 molecular marker and eliminating individuals with all other genotypes at that locus; or successively breeding homozygous individuals with the GGCC genotype of the SNP2 molecular marker and eliminating individuals with all other genotypes at that locus; or successively breeding homozygous individuals with the TT genotype of the SNP3 molecular marker and eliminating individuals with all other genotypes at that locus; or successively breeding homozygous individuals with the AATTGG genotype of the SNP4 molecular marker and eliminating individuals with all other genotypes at that locus; preferably, the method comprises the step of successively breeding individuals with the CC-GGCC-TT-AATTGG genotype combination of the above molecular marker combinations and eliminating individuals with all other genotype combinations of that marker combination.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] The molecular markers and combinations thereof related to drought resistance traits of Leymus chinensis provided by this invention can accurately evaluate the drought resistance traits of Leymus chinensis at the molecular level, accurately and efficiently screen drought-resistant tree species during the seedling stage of Leymus chinensis, and shorten the breeding cycle of Leymus chinensis.

[0046] The method for identifying or screening drought-resistant Leymus chinensis provided by this invention can predict the drought resistance of Leymus chinensis early, quickly, at low cost and effectively, and provides an effective means for molecular marker-assisted selection breeding of Leymus chinensis.

[0047] The molecular marker or sheepgrass genetic improvement method provided by this invention has a wide range of applications, broad application prospects, and can achieve excellent economic value. Attached Figure Description

[0048] Figure 1 This is a schematic diagram of the primers used in this invention for amplifying molecular markers.

[0049] Figure 2 The relative expression levels of drought-related genes designed with molecular markers in Example 1 of this invention.

[0050] Figure 3 This is a genotype effect diagram showing the survival rate of the most drought-resistant sheepgrass genotype combination compared to other genotype combinations in Example 2 of the present invention.

[0051] Figure 4This is a gel image of molecular markers for drought-resistant gene elements from some of the Leymus chinensis germplasm sources in Example 3 of this invention. Detailed Implementation

[0052] The technical solution of the present invention will now be clearly and completely described in conjunction with preferred embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0053] The term “exemplary” as used herein means “serving as an example, embodiment, or illustration.” Any embodiment illustrated herein as “exemplary” is not necessarily to be construed as superior to or better than other embodiments.

[0054] To find an accurate indicator for identifying drought resistance in Leymus chinensis, the inventors conducted extensive research. The results showed that genes with the most significant upregulation after drought stress are often associated with drought resistance in plants. Specifically, the homologs of Lc3Ns024690 and Lc3Xm072595 are both Arabidopsis thaliana AT3G57520.1, encoding a raffinose-specific α-galactosidase that catalyzes the breakdown of raffinose into α-galactose and sucrose, thus affecting the plant's drought resistance by influencing sugar metabolism and osmotic pressure. The homolog of Lc6Ns028159 is Arabidopsis thaliana AT3G50980.1, encoding a DEHYDRIN XERO... One protein is involved in plant dehydration and drying. The homolog of Lc1Xm058728 is AT3G08860.1 from Arabidopsis thaliana, encoding a protein with β-alanine aminotransferase activity. This protein affects the plant's drought resistance by influencing the synthesis of alanine, a drought-resistant substance. Upregulation of these drought-related genes is often considered a plant's defense against abiotic stress; higher expression levels of these genes correlate with stronger drought resistance.

[0055] Secondly, promoter elements affect the expression of their respective genes; mutations in drought-responsive elements will inhibit the expression of drought-resistant genes in plants. The Lc3Ns024690 promoter contains the ACCAGC element, which responds to the anaerobic environment created by stomatal closure due to drought; Lc1Xm058728 also contains the cis-regulatory element GTTTCG, which responds to anaerobic induction; the "common cis-regulatory element ATTGG in promoter and enhancer regions" in the Lc3Xm072595 promoter and the "core promoter element TTTATGGGCGA around transcription initiation-30" in the Lc6Ns028159 promoter promote gene expression. Mutations in these elements will reduce the plant's ability to respond to drought, thus affecting its drought tolerance.

[0056] Traits related to Leymus chinensis' drought adaptation, such as survival rate and relative water content decrease after drought stress, are the most direct reflection of Leymus chinensis' adaptability to drought stress.

[0057] Based on the above research, the survival rate and the slope of relative water content decrease after drought stress are preferred as indicators for identifying the drought resistance of Leymus chinensis in this invention. SNP markers that are significantly associated with the drought resistance of Leymus chinensis are obtained by RNA-Seq and Hi-tom association analysis.

[0058] In the first embodiment, molecular markers related to drought resistance traits of Leymus chinensis are provided, including SNP1, SNP2, SNP3, and SNP4. Specifically, molecular marker SNP1 is located at base 220 of the nucleotide sequence shown in SEQ ID NO: 1 and exhibits C / G polymorphism; molecular marker SNP2 is located at bases 380 and 381 of the nucleotide sequence shown in SEQ ID NO: 2 and exhibits GC / CA polymorphism; molecular marker SNP3 is located at base 277 of the nucleotide sequence shown in SEQ ID NO: 3 and exhibits T / G polymorphism; and molecular marker SNP4 is located at bases 394, 395, and 396 of the nucleotide sequence shown in SEQ ID NO: 4 and exhibits ATG / GGC polymorphism.

[0059] In the above embodiments, the homologous genes of Lc3Ns024690 and Lc3Xm072595 are both AT3G57520.1 of Arabidopsis thaliana, encoding a raffinose-specific α-galactosidase that catalyzes the breakdown of raffinose into α-galactose and sucrose, thus affecting the plant's drought resistance by influencing sugar metabolism and osmotic pressure; the homologous gene of Lc6Ns028159 is AT3G50980.1 of Arabidopsis thaliana, encoding a DEHYDRIN XERO 1 protein, which participates in plant dehydration; the homologous gene of Lc1Xm058728 is AT3G08860.1 of Arabidopsis thaliana, encoding a protein with β-alanine aminotransferase activity, which affects the plant's drought resistance by influencing the synthesis of the drought-resistant substance alanine.

[0060] In a further preferred embodiment, the drought resistance trait includes the survival rate after drought stress treatment, and the physiological and biochemical trait is the slope of the relative water content decrease.

[0061] The relative moisture content (RWC) is calculated as follows: RWC = (FW - DW / TW - DW) * 100, where FW is fresh weight, TW is water-saturated weight, and DW is dry weight.

[0062] The inventors discovered that under drought conditions, plants lower their intracellular osmotic potential by accumulating small-molecule organic osmotic regulators, thereby increasing their water absorption capacity under low water potential. This stabilizes membrane structure and macromolecular conformation, enhancing their adaptability to adverse conditions. Among these mechanisms, carbohydrate metabolism is a crucial osmotic regulation mechanism in higher plants. By breaking down large-molecule sugars into more small-molecule sugars, the intracellular osmotic potential is lowered, thus increasing the plant's water retention capacity and directly contributing to its drought resistance.

[0063] Plants can increase their drought resistance by enhancing alanine synthesis. The principle behind this is that alanine helps crops maintain good growth under drought conditions, thus resisting drought. Furthermore, alanine can promote the synthesis of endogenous polyamine hormones in crops, improving their ability to withstand drought stress.

[0064] Dehydrating proteins protect cells from dehydration damage by maintaining the stability and function of certain proteins under extreme conditions, thereby reducing plant damage after severe water loss. Upregulating the expression of dehydrating proteins after severe drought stress improves plant survival rates.

[0065] The changes in relative water content of plants after exposure to drought stress, and their survival rate after the cessation of drought stress, are related to sugar metabolism, alanine metabolism, and dehydrated protein metabolism under drought stress. Therefore, in this invention, the changes in relative water content and survival rate after exposure to drought stress are preferably selected as indicators for evaluating the drought resistance of Leymus chinensis.

[0066] In a preferred embodiment, the SNP1 marker genotypes are CC, GG, and CG. Theoretically, the expression level of the drought-resistant gene Lc1Xm058728 in Leymus chinensis with the SNP1 marker genotype CC is significantly higher than that in the GG and CG genotypes after being subjected to an anaerobic environment created by drought stress. Specifically, the CC genotype is homozygous for the SNP1 marker C in Leymus chinensis, the GG genotype is homozygous for the SNP1 marker G in Leymus chinensis, and the CG genotype is heterozygous for both the SNP1 markers C and G in Leymus chinensis.

[0067] In a preferred embodiment, the SNP2 marker genotypes are GGCC, CCAA, and GCCA. Theoretically, the expression level of the drought-resistant gene Lc3Ns024690 in Leymus chinensis with the SNP2 marker genotype GGCC is significantly higher than that in Leymus chinensis with the CCAA and GCCA genotypes after being subjected to an anaerobic environment formed by drought stress.

[0068] In a preferred embodiment, the SNP3 marker genotypes are TT, GG, and TG. The expression level of the drought-related gene Lc3Xm072595 in Leymus chinensis with the SNP3 marker genotype TT is significantly higher than that in the GG and TG genotypes.

[0069] In a preferred embodiment, the SNP4 marker genotypes are AATTGG, GGGGCC, and ATGGGC, and the expression level of the drought-related gene Lc6Ns028159 in Leymus chinensis with the SNP3 marker genotype AATTGG is significantly higher than that in the GGGGCC and ATGGGC genotypes.

[0070] To eliminate the possibility of misselection and misjudgment of superior genes in Leymus chinensis due to external environmental factors during phenotypic selection, it is necessary to perform genotyping on SNP loci to enhance the accuracy of target selection.

[0071] There are various methods for genotyping, such as resequencing, direct sequencing, time-of-flight mass spectrometry, and microarray technology. In this invention, resequencing (using Hi-tom to resequently sequence the selected germplasm) is preferred for genotyping the SNP.

[0072] In the second embodiment, a molecular marker combination related to drought resistance traits of Leymus chinensis is provided, wherein the molecular marker combination is composed of two or more markers from SNP1, SNP2, SNP3 and SNP4, preferably composed of four markers from SNP1, SNP2, SNP3 and SNP4.

[0073] As a preferred embodiment, when the molecular marker combination is a homozygous genotype of C, GC, T, ATG in the order of SNP1 to SNP4 (i.e., the CC-GGCC-TT-AATTGG genotype combination), the corresponding sheepgrass has the strongest drought resistance.

[0074] When the molecular marker combination is a homozygous genotype of G, CA, G, GGC in sequence from SNP1 to SNP4 (i.e., the GG-CCAA-GG-GGGGCC genotype combination), the corresponding Leymus chinensis has the weakest drought resistance.

[0075] In a further preferred embodiment, when the molecular marker combination is a homozygous genotype of C, GC, T, ATG in the order of SNP1 to SNP4 (i.e., the CC-GGCC-TT-AATTGG genotype combination), the corresponding sheepgrass experiences the slowest decrease in relative leaf water content and the highest survival rate after being subjected to drought stress.

[0076] When the molecular marker combination is a homozygous genotype of G, CA, G, GGC in sequence from SNP1 to SNP4 (i.e., the GG-CCAA-GG-GGGGCC genotype combination), the corresponding Leymus chinensis exhibits the fastest decrease in relative leaf water content and the lowest survival rate after being subjected to drought stress.

[0077] In the third embodiment, primers for amplifying molecular markers related to drought resistance traits in Leymus chinensis are provided. Specifically, the primers for amplifying molecular marker SNP1 are Lc1Xm058728-currency-up, Lc1Xm058728-existence-low, and Lc1Xm058728-not have-low; the primers for amplifying molecular marker SNP2 are Lc3Ns024690-currency-low, Lc3Ns024690-existence-up, and Lc3Ns024690-not have-up; and the primers for amplifying molecular marker SNP3 are Lc3Xm072595-currency-low, Lc3Xm072595-existence-up, and Lc3Xm072595-not The primers for amplifying the molecular marker SNP4 are Lc6Ns028159-currency-low, Lc6Ns028159-existence-up, and Lc6Ns028159-not have-up. The primers used for amplifying the molecular marker are shown in Table 1. Figure 1 As shown.

[0078] Table 1:

[0079]

[0080] In the fourth embodiment, a primer set for amplifying the molecular marker combination described in the second aspect is provided. This primer set consists of the primers described above for amplifying SNP1 to SNP4, respectively. The nucleotide sequences of the primers for amplifying molecular markers SNP1, SNP2, SNP3, and SNP4 are shown in SEQ ID NO: 5-7, 8-10, 11-13, and 14-16, respectively. Several primers can be selected for the detection of multiple SNP molecular markers to screen for drought-resistant *Leymus chinensis*. This includes selecting "currency" and "existence" with the primer pairs to detect whether mutations exist at the corresponding sites, and adding "not have" to detect whether the mutation type at the corresponding site is homozygous or heterozygous.

[0081] In a fifth embodiment, a detection reagent or kit containing the aforementioned detection molecular marker primers or primer combinations is provided. Preferably, the detection reagent or kit further includes PCR amplification reagents, which include PCR buffer, dNTPs, and DNA polymerase.

[0082] Preferably, the detection reagent or kit is used according to the experimental procedure:

[0083] 1. Configure the following PCR reaction system.

[0084]

[0085] 2. Place the reaction system in a PCR instrument for the reaction. PCR reaction conditions:

[0086]

[0087] 3. Electrophoresis the PCR products on a 1% agarose gel and observe the amplified bands.

[0088] In the sixth embodiment, a method for obtaining the above-mentioned SNP molecular marker is provided, the method comprising the following steps:

[0089] Step 1. Discover SNPs related to drought resistance

[0090] Leymus chinensis at the three-leaf stage was divided into an experimental group where watering was stopped and a control group where water was replenished every 3 days. On the 9th day, leaves from both the treatment group and the control group were harvested, and RNA was extracted using an RNA extraction kit. The RNA samples were sent to Novogene for RNA-Seq analysis. Differential expression analysis identified genes that were upregulated by a large fold under drought stress.

[0091] Search for Arabidopsis homologs of these genes on the Arabidopsis TAIR website, and identify Lc3Ns024690, Lc3Xm072595, Lc6Ns028159 and Lc1Xm058728, which are clearly labeled as candidate genes for discovering drought resistance molecular markers.

[0092] Using the Leymus_chinensis_Lc6-5 version of the genome (China National Genomics Data Center, BioProject number PRJCA010499, reference "Tong Li, et al. Genome evolution and initial breeding of the Triticeae grass Leymus chinensis dominating the Eurasian Steppe. PNAS. 2023, 120(44)"), and the obtained CDS and pep fasta files, we searched for 600 bp sequences upstream of the promoters of the Lc3Ns024690, Lc3Xm072595, Lc6Ns028159 and Lc1Xm058728 genes;

[0093] DNA was extracted from the first true leaf of 600 different seedlings of Leymus chinensis using the EDTA method. A true leaf leaf of about 1 cm in length was taken from each seedling. Sequencing primers were designed according to the requirements of Hi-tom technology. Each pair of primers amplified a 300 bp fragment. Therefore, in order to amplify the promoter sequence from 0 bp to -600 bp, two pairs of primers were designed for each gene.

[0094] Hi-tom analysis of the sequence identified SNP sites that may affect plant drought resistance. Typically, these SNPs cause the loss of key drought resistance elements in genes.

[0095] Step 2. Determination of drought resistance phenotypes in Leymus chinensis

[0096] Sow sheepgrass seeds on the surface of peat moss, place them in an incubator at 9 pm every day, and place them in a culture room at 9 am the next day. Add tap water when the surface of the peat moss dries. Once the seeds have germinated, place them in the culture room all day.

[0097] Add tap water to peat moss and vermiculite until they are saturated, then mix them evenly in a 1:1 ratio. Fill the 128-cell seedling trays with the mixed potting soil. Select seedlings that are growing uniformly and have no root damage and plant them in the seedling trays, with one plant per seedling.

[0098] After the seedlings were planted, the seedling trays were immersed in a solution of 4g / 5L of general-purpose balanced fertilizer for 5 minutes. Then, the 128-well seedling trays were placed over the empty 32-well seedling trays. The new seedling trays were placed on the trays so that the planting troughs containing the plants were suspended in the air to ensure that the experimental results were not affected by water accumulation.

[0099] After planting, place the seedling trays in the culture room for cultivation. Every 3 days, immerse the 128-cell seedling trays in a culture solution containing 4g / 5L of general-purpose balanced fertilizer for 1 hour to replenish water and fertilizer.

[0100] Fifteen days after transplanting, each variety's small units arranged in a grid pattern continued to be watered as a control group without drought treatment. Every three days, these units were immersed in tap water, while other plants in the seed trays underwent drought treatment. During the drought treatment, the leaf sheaths and above of the plants with different drought durations were harvested, and their fresh weight was measured using a 0.1% balance. After soaking in distilled water for 12 hours, their water-saturated weight was measured. Then, the leaves were placed in a 60℃ oven for 12 hours, and their dry weight was measured. The relative water content of the leaves was calculated as: Relative water content of leaves = (fresh weight - dry weight / water-saturated weight - dry weight) * 100%.

[0101] After the drought treatment, all planting troughs were immersed in a nutrient solution containing 4g / 5L of general-purpose balanced fertilizer for 1 hour to replenish water and fertilizer. This replenishment was done every 3 days. On the 15th day after replenishment, the number of plants in the drought treatment group that re-emerged green leaves was counted, and this number was recorded as the survival number of the plants.

[0102] This step evaluated 600 Leymus chinensis germplasm sources, of which 61 were drought-resistant and survived after the drought stress ended.

[0103] Step 3. Obtain SNP molecular markers associated with drought resistance traits in Leymus chinensis.

[0104] Molecular marker primers were designed around SNPs that may affect drought resistance. The base differences of SNPs lead to one or two differences in the 3' end of different molecular marker primers.

[0105] Hi-tom sequencing was performed on the DNA regions containing mutant elements from 61 drought-resistant Leymus chinensis germplasm sources. The proportions of elements and mutations in 600 Leymus chinensis germplasm sources and 61 total drought-resistant Leymus chinensis germplasm sources were compared, and different molecular marker types were weighted and scored.

[0106] Molecular markers were used to detect different strains of Leymus chinensis, and the drought resistance phenotypes of these Leymus chinensis were compared to further test the accuracy of the molecular markers.

[0107] In the seventh embodiment, the application of the above-described SNP molecular markers, molecular marker combinations, primers, primer combinations, detection reagents, kits, or SNP markers and combinations obtained by the above methods in any of the following aspects (1) to (6):

[0108] (1) To identify or assist in identifying the drought resistance of Leymus chinensis;

[0109] (2) Screening or assisting in the screening of new varieties of sheepgrass with strong drought resistance;

[0110] (3) Molecular marker-assisted breeding of Leymus chinensis;

[0111] (4) Prepare products for identification or auxiliary identification of the drought resistance of Leymus chinensis;

[0112] (5) Prepare products for screening or assisting in the screening of new varieties of Leymus chinensis with strong drought resistance;

[0113] (6) Prepare products for molecular marker-assisted breeding of Leymus chinensis.

[0114] In the eighth embodiment, a method for identifying or screening plants with strong drought resistance is provided, the method comprising the steps of detecting the polymorphism or genotype of the above-mentioned SNP molecular markers of Leymus chinensis, or detecting the genotype of combinations of molecular markers.

[0115] The drought resistance of the sheepgrass is preferably assessed by the slope of the relative water content decrease and the survival rate.

[0116] Preferably, the method for identifying or screening drought-resistant sheepgrass includes the following steps:

[0117] Step 1: Extract genomic DNA from the Leymus chinensis plant to be tested;

[0118] Step II: Using the extracted genomic DNA as a template, perform PCR amplification;

[0119] Step III: Determine the polymorphism or genotype of the above SNP molecular markers in the test herb *Leymus chinensis*.

[0120] Step IV: Determine the drought resistance of the tested Leymus chinensis based on the genotype test results.

[0121] In step II, the primers used for PCR amplification are molecular markers SNP1, SNP2, SNP3, and SNP4.

[0122] In step III, the drought resistance of Leymus chinensis can be identified by determining the polymorphism or genotype of any one of the molecular markers SNP1 to SNP4, or by determining the genotype of the combination of molecular markers. Preferably, the drought resistance of Leymus chinensis can be identified by determining the genotype of the combination of molecular markers.

[0123] Methods for determining molecular marker polymorphisms or genotypes include, but are not limited to, sequencing.

[0124] In step IV:

[0125] If the genotype of the SNP1 site of the tested Leymus chinensis is CC, then theoretically, the expression level of the drought-resistant gene Lc1Xm058728 will be high after the Leymus chinensis is subjected to an anaerobic environment formed by drought stress; if the genotype of the SNP1 site of the tested Leymus chinensis is GG or CG, then theoretically, the expression level of the drought-resistant gene Lc1Xm058728 will be low after the Leymus chinensis is subjected to an anaerobic environment formed by drought stress.

[0126] If the genotype of the SNP2 site of the tested Leymus chinensis is GGCC, then theoretically, the expression level of the drought-resistant gene Lc3Ns024690 will be high after the Leymus chinensis is subjected to an anaerobic environment formed by drought stress; if the genotype of the SNP2 site of the tested Leymus chinensis is CCAA and GCCA, then theoretically, the expression level of the drought-resistant gene Lc3Ns024690 will be low after the Leymus chinensis is subjected to an anaerobic environment formed by drought stress.

[0127] If the genotype of the SNP3 locus of the grass to be tested is TT, the expression level of the drought resistance-related gene Lc3Xm072595 of the grass is high; if the genotype of the SNP3 locus of the grass to be tested is GG or TG, the expression level of the drought resistance-related gene Lc3Xm072595 of the grass is low.

[0128] If the genotype of the SNP4 locus of the test herb is AATTGG, the expression level of the drought resistance-related gene Lc6Ns028159 of the herb is high; if the genotype of the SNP4 locus of the test herb is GGGGCC and ATGGGC, the expression level of the drought resistance-related gene Lc6Ns028159 of the herb is relatively low.

[0129] If the genotype combination of the four SNP1, SNP2, SNP3, and SNP4 loci of the tested Leymus chinensis is CC-GGCC-TT-AATTGG, it will experience a slow decline in water content under drought stress, a high survival rate after rehydration, and will exhibit the strongest drought resistance. Conversely, if the genotype combination of the four SNP1, SNP2, SNP3, and SNP4 loci is GG-CCAA-GG-GGGGCC, it will experience a rapid decline in water content under drought stress, a low survival rate after rehydration, and will exhibit the weakest drought resistance.

[0130] In the ninth embodiment, a method for genetic improvement of Leymus chinensis is provided, the method comprising the steps of successive generation selection of homozygous individuals with the CC genotype of the above-mentioned SNP1 molecular marker, and elimination of all other individuals with the same genotype at that locus;

[0131] Or it may include the steps of selecting homozygous individuals of the GGCC genotype of the above-mentioned SNP2 molecular marker and eliminating individuals of all other genotypes at that locus;

[0132] Or it may include the steps of selecting homozygous individuals with the TT genotype of the above SNP3 molecular marker and eliminating individuals with all other genotypes at that locus;

[0133] Or it may include the steps of selecting homozygous individuals with the AATTGG genotype of the above SNP4 molecular marker and eliminating individuals with all other genotypes at that locus;

[0134] This may include the step of selecting individuals with the AA-TT-CC genotype combination of the above molecular marker combination through successive generations, and eliminating individuals with all other genotype combinations of that marker combination.

[0135] Preferably, the method includes the steps of subbreeding individuals with the CC-GGCC-TT-AATTGG genotype combination of the above molecular marker combination, and eliminating individuals with all other genotype combinations of the marker combination.

[0136] To make the experimental methods, technical solutions, advantages, and disadvantages in the embodiments of the present invention clearer, the specific experimental steps in the embodiments of the present invention will be described in detail and completely below. For reagents or instruments whose manufacturers are not specified in the embodiments, conventional products can be purchased from the market. For those not specified with specific conditions, conventional conditions or manufacturer-recommended conditions can be followed.

[0137] Example 1: Obtaining SNP molecular markers associated with drought resistance traits in Leymus chinensis individuals

[0138] 1. Experimental group

[0139] 150 seed sources of Leymus chinensis were provided by the research group of Liu Gongshe at the Institute of Botany, Chinese Academy of Sciences.

[0140] Fifty seed sources of sheepgrass were provided by the research team of Wu Zinian from the Grassland Institute of the Chinese Academy of Agricultural Sciences.

[0141] 400 seed sources of sheepgrass were collected in West Ujimqin.

[0142] 2. DNA extraction

[0143] DNA was extracted from 24 different individuals of Leymus chinensis using the EDTA method. The thoroughly ground samples were placed in EDTA extraction buffer and treated at 60°C for 10 min. After centrifugation, the supernatant was collected, and RNase was added for 15 min. Then, 1 / 3 volume of chloroform was added, and the mixture was vortexed for 1 min. After centrifugation, the supernatant was collected, and an equal volume of isopropanol was added. The precipitate was collected in a centrifuge tube, and 3 volumes of 70% ethanol were added. After vortexing for 1 min, the precipitate was collected. After the liquid in the precipitate had completely evaporated, distilled water was added, and the mixture was vortexed for 1 min. After centrifugation, the supernatant was collected.

[0144] 3. Survival rate detection after drought stress

[0145] After 15 days of drought treatment, all drought-stressed Leymus chinensis individuals were immersed in a culture solution containing 4g / 5L of general-purpose balanced fertilizer for 1 hour to replenish water and fertilizer. This was repeated every 3 days. After another 15 days, the survival rate of the different Leymus chinensis individuals was recorded.

[0146] 4. Obtaining SNP molecular markers associated with drought resistance traits in Leymus chinensis

[0147] (1) Identify drought-resistant related SNPs

[0148] The same species of Leymus chinensis at the three-leaf stage was divided into an experimental group where watering was stopped and a control group where watering was replenished every 3 days. Leaves from both groups were harvested on day 9, and RNA was extracted using an RNA extraction kit. The RNA samples were sent to Novogene for RNA-Seq analysis. Differential expression analysis identified genes with high upregulation under drought stress. Arabidopsis homologs of these genes were searched on the Arabidopsis TAIR website. The homologs Lc3Ns024690, Lc3Xm072595, Lc6Ns028159, and Lc1Xm058728, which clearly indicate their impact on drought resistance, were identified as candidate genes for discovering drought resistance molecular markers. Figure 2 (As shown in the figure). The relevant functional predictions are shown in Table 2.

[0149] Table 2: Candidate gene function prediction

[0150]

[0151] Using the Leymus chinensis Lc6-5 genome and the obtained CDS and PEP FASTA files, we searched for 600 bp sequences upstream of the promoters of the Lc3Ns024690, Lc3Xm072595, Lc6Ns028159, and Lc1Xm058728 genes. DNA was extracted from the first true leaf of 600 different Leymus chinensis seedlings from various provenances using EDTA. Approximately 1 cm of true leaf was taken from each provenance. Sequencing primers were designed according to Hi-Tom technology requirements, amplifying 300 bp fragments with each primer pair. Therefore, to amplify the promoter sequence from 0 bp to -600 bp, two primer pairs were designed for each gene. Hi-Tom analysis was performed on these sequences, identifying SNP sites that may affect the plant's drought resistance. In this step, the Hi-Tom filtering threshold was set to 0.1%, and the data volume for each sample was set to 10,000.

[0152] (2) Weighted scoring

[0153] Hi-tom sequencing was performed on the DNA regions containing mutant elements from 61 drought-resistant Leymus chinensis germplasm sources. The proportions of elements and mutations in 600 Leymus chinensis germplasm sources and 61 drought-resistant Leymus chinensis germplasm sources were compared. The presence of drought-resistant related elements in all 600 Leymus chinensis and 61 drought-resistant Leymus chinensis germplasm sources used in the experiment was detected using Hi-tom sequencing. The results are shown in Table 3. Different molecular marker types were weighted and scored, with the value being the percentage of DNA sequences containing elements in the 61 drought-resistant Leymus chinensis germplasm sources / the percentage of DNA sequences containing elements in the 600 total Leymus chinensis germplasm sources. The higher the ratio, the more it reflects the weight of that category in drought resistance assessment. When only drought-resistant related elements were detected, the value was +; when both drought-resistant and mutant elements were detected, the value was 0; when only mutant elements were detected, the value was -. The higher the value, the stronger the drought resistance of the plant. The results are shown in Table 4, where positive numbers indicate drought resistance and negative numbers indicate lack of drought resistance.

[0154] Table 3: Comparison of the proportion of elements and mutations in common drought-resistant sheepgrass and drought-resistant sheepgrass

[0155]

[0156] Table 4: Weighted scoring results for different molecular marker types

[0157]

[0158]

[0159] (3) DNA extraction and PCR amplification

[0160] Gene fragments Lc3Ns024690, Lc3Xm072595, Lc6Ns028159, and Lc1Xm058728 from 24 different Leymus chinensis individuals were amplified using marker-specific primers. The amplification system consisted of 15 μL of 100 ng DNA, 1 μL of 10 μM / L upstream and downstream primers, annealing at 53°C for 30 s, extension at 72°C for 55 s, for 34 cycles. The amplification of the bands was recorded, and the drought resistance of the 24 different Leymus chinensis individuals was scored based on this data.

[0161] (4) Regression detection

[0162] Comparative analysis revealed that among 24 different Leymus chinensis individuals, those with higher molecular marker scores were more likely to survive drought stress, showing a positive correlation between the two (Table 5). This indicates that the molecular marker method effectively predicted the drought resistance of different Leymus chinensis individuals.

[0163] Table 5: Drought resistance scores of 24 different Leymus chinensis individuals

[0164]

[0165]

[0166] Example 2: Correlation between SNP molecular markers and drought resistance

[0167] 1. High-throughput experimental system for evaluating drought resistance of different Leymus chinensis germplasm sources

[0168] (1) Seed germination

[0169] Sow 0.6g of seeds on the surface of peat moss. Place the petri dishes in a 16℃, dark incubator at 21:00 daily, cover, and then at 9:00 the next day, place them in a 22℃, 16h light / 8h dark incubator with the petri dish lids off. Maintain 55% humidity and 100μmol / (m²) light intensity in the incubator. 2 •s). Add tap water when the peat surface is dry. Once germinating seeds appear, keep the peat moss in the growing room all day.

[0170] (2) Seedling planting and cultivation

[0171] When the seedlings grow to about 4cm in length, add tap water to the potting soil and carefully loosen the soil to separate the seedling roots from the soil. Select seedlings with uniform growth and undamaged roots and plant them in plug trays, one plant per planting trough. Plant five different varieties of Leymus chinensis in one plug tray. Plant the same variety of Leymus chinensis in every four adjacent planting troughs, forming a grid-shaped arrangement. Plant these grid-shaped units sequentially in the plug trays. Each variety contains 6 or 7 grid-shaped units in one plug tray, for a total of 20 different seed sources of Leymus chinensis. After planting, place the plug trays in a cultivation room. Every three days, immerse the plug trays in a nutrient solution containing 4g / 5L of general-purpose balanced fertilizer for 1 hour to replenish water and fertilizer. Each seed source should have at least 12 grid-shaped units planted, distributed in at least two different plug trays.

[0172] (3) Relative moisture content detection

[0173] On days 0, 9, and 15 of the drought, the leaf sheaths of five plants from different drought-treated varieties were harvested. The fresh weight (FW) was measured using a 0.1 g balance. The samples were then placed in 5 ml pipette tips, sealed with aluminum foil, and soaked in distilled water for 12 hours. After centrifugation at 2000 rpm for 1 minute, the water adhering to the leaves was removed, and the water-saturated weight (TW) was measured. The leaves were then placed in a 60°C oven for 12 hours, and the dry weight (DW) was measured.

[0174] According to the relative moisture content calculation formula, RWC = (FW - DW / TW - DW) * 100, the decrease in relative moisture content is calculated. Seed sources of Leymus chinensis with a smaller decrease in relative moisture content are considered to have strong drought resistance. The decrease is calculated as the slope of moisture loss over time.

[0175] (4) Survival rate detection after drought stress

[0176] After 15 days of drought treatment, all planting troughs were immersed in a nutrient solution containing 4g / 5L of general-purpose balanced fertilizer for 1 hour to replenish water and fertilizer. This replenishment was repeated every 3 days. On the 15th day after replenishment, the number of plants in each variety that sprouted new green leaves in the drought-treated group was counted; this number was recorded as the survival rate of the plants. Seedlings with high survival rates were considered to have strong drought resistance, and the drought tolerance of the seedlings was ranked.

[0177] 2. Drought resistance score

[0178] DNA was extracted from different seed sources of Leymus chinensis using EDTA. A 2ml sample of Leymus chinensis leaf was extracted using 300µl of extraction buffer, followed by protein removal with 100µl of chloroform and washing with 70% ethanol. Gene fragments Lc3Ns024690, Lc3Xm072595, Lc6Ns028159, and Lc1Xm058728 were amplified using marker-specific primers. The amplification system consisted of 15µl of 100ng DNA, 1µl of 10µM / L upstream and downstream primers, annealing at 53℃ for 30s, extension at 72℃ for 55s, for 34 cycles. The amplification of the bands was recorded, and the drought tolerance of Leymus chinensis was scored.

[0179] 3. Regression Detection

[0180] The relationship between molecular marker scores and drought tolerance of Leymus chinensis was compared. The comparison revealed that Leymus chinensis germplasm with higher molecular marker scores exhibited stronger drought resistance and higher survival rates under drought stress; the two showed a positive correlation, with a coefficient of determination of 0.4727 for the regression equation. Figure 3 As shown in Table 6, this indicates that the molecular marker method effectively predicted the drought resistance of different Leymus chinensis germplasm sources. Leymus chinensis with a survival rate of 0% often exhibited a water reduction slope greater than -40% (Table 6). Therefore, the survival rate of Leymus chinensis after drought directly reflects its drought resistance level. The water reduction slope can further assess the drought resistance of Leymus chinensis; the most drought-resistant genotype combination CC-GGCC-TT-AATTGG showed a higher survival rate after drought and a smaller water reduction slope during drought stress compared to other genotype combinations (Table 6).

[0181] Table 6: Results of drought resistance testing for 20 different Leymus chinensis germplasms

[0182]

[0183] Example 3: Testing the drought resistance of eight species of sheepgrass

[0184] 1. The drought resistance of eight species of sheepgrass was tested according to the method in Example 2.

[0185] In the step "Seedling Cultivation", the petri dishes were placed in an incubator at 16℃ and in darkness at 21:00 each day, with the petri dish lids covered. The following day at 9:00, they were placed in a cultivation room at 22℃ with 16 hours of light and 8 hours of darkness, with the petri dish lids opened. The humidity in the cultivation room was 55%, and the light intensity was 100 μmol / (m²). 2 ·s). Compared with constant temperature treatment, seedlings obtained by variable temperature treatment are stronger, have less mold during seed germination, and have a higher germination rate.

[0186] In the "Seedling Planting and Cultivation" step, different varieties of Leymus chinensis are planted in a single seed tray. Every four adjacent planting troughs contain the same variety of Leymus chinensis, forming a grid-like arrangement. These grid-like units are planted sequentially in the seed trays, with each variety containing several such units within a single tray. This grid-like arrangement reduces the variation in drought resistance among different Leymus chinensis varieties due to differences in their location within the seed trays, thus minimizing randomness.

[0187] In the step "Seedling Planting and Cultivation", the middle seedling tray is a seedling tray with 128 planting slots, arranged in rows of 8, for a total of 16 rows. Each planting slot is 3cm wide at the top, 1.5cm wide at the bottom, and 4cm deep. The new seedling tray is a seedling tray with 32 planting slots, arranged in rows of 4, for a total of 8 rows. Place the seedling tray with 128 planting slots on top of the empty seedling tray with 32 planting slots. Place a tray under both seedling trays to suspend the potting soil that holds the plants in the air, so that the experimental results are not affected by water accumulation.

[0188] In the step "Relative Moisture Content Detection," the leaves above the leaf sheath of plants treated for different drought durations were harvested on days 0, 9, and 15 of the drought, and the relative moisture content of the leaves was calculated. Day 0 represents the initial, untreated state, and its sufficient moisture content reflects the health of the Leymus chinensis used in the experiment, indicating that the untreated Leymus chinensis did not wilt due to disease or other factors. Day 9 is the point at which more than half of the Leymus chinensis showed visible wilting; this point is used to distinguish the "drought resistance" of different Leymus chinensis species, i.e., their ability to retain moisture during drought. Day 15 is the point at which all Leymus chinensis wilted and the above-ground parts showed signs of drying; this point is used to assess the plant's "drought tolerance," i.e., its ability to withstand dehydration and drought.

[0189] In the step "Relative Moisture Content Detection", the method for measuring the water saturation weight (TW) is as follows: Carefully insert the sheepgrass into the 5ml syringe tip, trying not to damage the sheepgrass. Seal the end of the syringe tip with aluminum foil, and then soak it in water for 12 hours. After the plant has absorbed enough water, place it in a centrifuge with the thin end of the syringe tip facing down and rotate it at 1000 RPM for 2 minutes to completely remove the water from the surface of the sheepgrass leaves. The weight of the sheepgrass is then measured as the water saturation weight (TW).

[0190] 2. Drought resistance score

[0191] DNA was extracted from eight different seed sources of Leymus chinensis using the EDTA method. Gene fragments Lc3Ns024690, Lc3Xm072595, Lc6Ns028159, and Lc1Xm058728 were amplified using marker-specific primers. The amplification system consisted of 15 μL of 100 ng of DNA, 1 μL of 10 μM / L upstream and downstream primers, annealing at 53°C for 30 s, extension at 72°C for 55 s, for 34 cycles. The amplification results were recorded, and the drought resistance of Leymus chinensis was scored. A gel image of some drought-resistant gene elements from Leymus chinensis seed sources is shown below. Figure 4 As shown, where: M: Marker, 1: Detects SNP G in Lc1Xm058728, 2: Detects SNP C in Lc1Xm058728, 3: Detects SNP GGC in Lc6Ns028159, 4: Detects SNP ATG in Lc6Ns028159, 5: Detects SNP CA in Lc3Ns024690, 6: Detects SNP GC in Lc3Ns024690, 7: Detects SNP G in Lc3Xm072595, 8: Detects SNP T in Lc3Xm072595.

[0192] 3. Regression Detection

[0193] The relationship between molecular marker scores and drought tolerance of Leymus chinensis was compared. The comparison revealed that the two seed sources with the highest molecular marker scores, Leymus chinensis 16 and 3, had the highest survival rates after drought stress, while the two seed sources with the lowest marker scores, Leymus chinensis 4 and 24, had the lowest survival rates after drought stress (Table 7). This indicates that the molecular marker effectively predicted the drought tolerance of Leymus chinensis.

[0194] Table 7: Results of drought resistance testing of eight different Leymus chinensis seed sources

[0195]

[0196] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. The application of the detection reagent, characterized in that, The detection reagent is used to detect SNP molecular markers related to drought resistance traits in Leymus chinensis, and the application is any one of the following: (1) To identify or assist in identifying the drought resistance of Leymus chinensis; (2) Screening or assisting in the screening of new varieties of sheepgrass with strong drought resistance; (3) Molecular marker-assisted breeding for drought resistance in Leymus chinensis; (4) Prepare products for identification or auxiliary identification of the drought resistance of Leymus chinensis; (5) Prepare products for screening or assisting in the screening of new varieties of Leymus chinensis with strong drought resistance; (6) Prepare products for molecular marker-assisted breeding of drought-resistant traits in Leymus chinensis; The molecular marker is selected from one of SNP1, SNP2, SNP3, and SNP4; wherein: The molecular marker SNP1 is located at the 220th base of the nucleotide sequence shown in SEQ ID NO: 1 and has C / G polymorphism. The CC genotype of SNP1 in Leymus chinensis has better drought resistance than the GG and GC genotypes. The molecular marker SNP2 is located at bases 380 and 381 of the nucleotide sequence shown in SEQ ID NO: 2, and has GC / CA polymorphism. The GGCC genotype of SNP2 is more drought resistant than the CCAA and GCCA genotypes in Leymus chinensis. The molecular marker SNP3 is located at the 277th base of the nucleotide sequence shown in SEQ ID NO: 3, and has T / G polymorphism. The TT genotype of SNP3 in Leymus chinensis has better drought resistance than the GG and TG genotypes. The molecular marker SNP4 is located at bases 394, 395, and 396 of the nucleotide sequence shown in SEQ ID NO: 4, and has ATG / GGC polymorphism. The AATTGG genotype of SNP4 in Leymus chinensis has better drought resistance than the GGGGCC and ATGGGC genotypes.

2. The application according to claim 1, characterized in that, The detection reagent is used to detect two or more of the molecular markers SNP1, SNP2, SNP3, and SNP4.

3. The application according to claim 1, characterized in that, The detection reagent is used to detect molecular marker combinations SNP1, SNP2, SNP3 and SNP4; when the molecular marker combination is a homozygous genotype of C, GC, T and ATG in the order of SNP1 to SNP4, that is, the CC-GGCC-TT-AATTGG genotype combination, the corresponding sheepgrass has the strongest drought resistance. The molecular marker combination, with SNP1 to SNP4 being homozygous genotypes of GG, CCAA, GG, and GGGGCC respectively, i.e., the GG-CCAA-GG-GGGGCC genotype combination, corresponds to the weakest drought resistance in Leymus chinensis.

4. The application according to claim 1, characterized in that, The drought resistance traits include survival rate after drought stress and physiological and biochemical traits; the physiological and biochemical traits are the slope of the decrease in relative water content of leaves.

5. A primer set for amplifying molecular marker combinations, characterized in that, The primer combination consists of primers for amplifying molecular markers SNP1 to SNP4, respectively, wherein: The nucleotide sequences of the primers used to amplify the molecular marker SNP1 are shown in SEQ ID NO: 5-7; The nucleotide sequences of the primers used to amplify the molecular marker SNP2 are shown in SEQ ID NO: 8-10; The nucleotide sequences of the primers used to amplify the molecular marker SNP3 are shown in SEQ ID NO: 11-13; The nucleotide sequences of the primers used to amplify the molecular marker SNP4 are shown in SEQ ID NO: 14-16; The molecular marker SNP1 is located at the 220th base of the nucleotide sequence shown in SEQ ID NO: 1 and exhibits C / G polymorphism. The molecular marker SNP2 is located at bases 380 and 381 of the nucleotide sequence shown in SEQ ID NO: 2, and has GC / CA polymorphism; The molecular marker SNP3 is located at the 277th base of the nucleotide sequence shown in SEQ ID NO: 3 and exhibits T / G polymorphism. The molecular marker SNP4 is located at bases 394, 395, and 396 of the nucleotide sequence shown in SEQ ID NO: 4, and exhibits ATG / GGC polymorphism.

6. A detection reagent or kit comprising the primer combination of claim 5.

7. The use of the primer combination of claim 5, the detection reagent or kit of claim 6, in any one of the following (1) to (6): (1) To identify or assist in identifying the drought resistance of Leymus chinensis; (2) Screening or assisting in the screening of new varieties of sheepgrass with strong drought resistance; (3) Molecular marker-assisted breeding for drought resistance in Leymus chinensis; (4) Prepare products for identification or auxiliary identification of the drought resistance of Leymus chinensis; (5) Prepare products for screening or assisting in the screening of new varieties of Leymus chinensis with strong drought resistance; (6) Prepare products for molecular marker-assisted breeding of drought-resistant traits of Leymus chinensis.

8. A method for identifying or screening the drought resistance of Leymus chinensis, characterized in that, The method includes the steps of detecting the polymorphism or genotype of Leymus chinensis SNP molecular markers, or detecting the genotype of Leymus chinensis SNP molecular marker combinations. The molecular marker SNP1 is located at the 220th base of the nucleotide sequence shown in SEQ ID NO: 1 and has C / G polymorphism. The CC genotype of SNP1 in Leymus chinensis has better drought resistance than the GG and GC genotypes. The molecular marker SNP2 is located at bases 380 and 381 of the nucleotide sequence shown in SEQ ID NO: 2, and has GC / CA polymorphism. The GGCC genotype of SNP2 is more drought resistant than the CCAA and GCCA genotypes in Leymus chinensis. The molecular marker SNP3 is located at the 277th base of the nucleotide sequence shown in SEQ ID NO: 3, and has T / G polymorphism. The TT genotype of SNP3 in Leymus chinensis has better drought resistance than the GG and TG genotypes. The molecular marker SNP4 is located at bases 394, 395, and 396 of the nucleotide sequence shown in SEQ ID NO: 4, and has ATG / GGC polymorphism. The AATTGG genotype of SNP4 in Leymus chinensis has better drought resistance than the GGGGCC and ATGGGC genotypes.

9. A method for genetic improvement of Leymus chinensis, characterized in that, The method includes the steps of successively breeding homozygous individuals with the CC genotype of molecular marker SNP1 and eliminating individuals with all other SNP1 genotypes; or successively breeding homozygous individuals with the GGCC genotype of molecular marker SNP2 and eliminating individuals with all other SNP2 genotypes; or successively breeding homozygous individuals with the TT genotype of molecular marker SNP3 and eliminating individuals with all other SNP3 genotypes; or successively breeding homozygous individuals with the AATTGG genotype of molecular marker SNP4 and eliminating individuals with all other SNP4 genotypes. The molecular marker SNP1 is located at the 220th base of the nucleotide sequence shown in SEQ ID NO: 1 and exhibits C / G polymorphism. The molecular marker SNP2 is located at bases 380 and 381 of the nucleotide sequence shown in SEQ ID NO: 2, and has GC / CA polymorphism; The molecular marker SNP3 is located at the 277th base of the nucleotide sequence shown in SEQ ID NO: 3 and exhibits T / G polymorphism. The molecular marker SNP4 is located at bases 394, 395, and 396 of the nucleotide sequence shown in SEQ ID NO: 4, and exhibits ATG / GGC polymorphism.

10. The method according to claim 9; characterized in that, The method includes selecting individuals with the molecular marker combination SNP1~SNP4 having the genotype combination CC-GGCC-TT-AATTGG, and eliminating individuals with all other genotype combinations of that marker combination.

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