SNP molecular marker combination related to salt-alkali tolerance of elytrigia repens and application thereof

Abstract

The application discloses a SNP molecular marker combination related to salt-alkali tolerance of Puccinellia distans and application thereof. The SNP molecular marker combination is composed of five SNP molecular markers, and the group of SNP molecular markers is located at positions 80176348 of chromosome 6, 490854164 of chromosome 9, 291625249 of chromosome 11 and 64884340 and 65147058 of chromosome 12 of Puccinellia distans. Base polymorphisms CC / CG (GG), TT / TG, AA / TT, AA / GG and TT / CC exist in the above positions, wherein CC-TT-AA-AA-TT is a salt-alkali tolerance genotype, and CG / GG-TG-TT-GG-CC is a salt-alkali sensitive genotype. The SNP molecular marker combination is used for detecting the salt-alkali tolerance of Puccinellia distans, is accurate and reliable, and effectively improves the identification efficiency of salt-alkali tolerance Puccinellia distans germplasm and varieties.

Description

Technical Field

[0001] This invention belongs to the field of forage genetics and breeding technology, specifically relating to a combination of SNP molecular markers related to salt and alkali tolerance in Leymus chinensis and its application. Background Technology

[0002] Elymus dahuricus is a perennial herbaceous plant belonging to the Poaceae family. It possesses characteristics such as salt and alkali tolerance, drought resistance, cold resistance, wind and sand resistance, and resistance to diseases and pests, making it an important ecological grass species in northern my country. Soil salinization is a global environmental and ecological problem. The synergistic effect of salt and alkali stress exacerbates damage to plants, severely affecting plant growth and jeopardizing my country's food security. Selecting, cultivating, and planting salt-tolerant plant varieties is the most economical and effective means of developing and utilizing saline-alkali land, and is of great significance for ensuring food security and improving the ecological environment.

[0003] DNA molecular markers are a class of heritable molecular markers based on genomic DNA sequence polymorphisms. They possess advantages such as abundance and genetic stability, and are widely used in fields such as functional gene mapping and molecular breeding. Based on their technical principles, DNA molecular markers are mainly divided into four categories: techniques based on molecular hybridization (such as RFLP), techniques based on PCR (such as RAPD and SSR), techniques based on PCR combined with enzyme digestion (such as AFLP), and techniques based on single nucleotide polymorphisms (SNPs). SNPs refer to polymorphisms caused by variations in a single nucleotide (A, T, C, G) in the genome. Their numbers are extremely abundant in the genome, and markers developed based on SNPs are the latest third-generation molecular markers. Compared to second-generation markers (such as SSRs), SNP molecular markers have advantages such as high polymorphism, genetic stability, and suitability for automated high-throughput analysis. In agricultural bio-breeding, SNP marker technology can achieve more accurate variety identification and genotyping. When combined with molecular marker-assisted breeding technology, it can enable efficient selection in the early stages of breeding, thereby significantly shortening the breeding cycle, improving selection accuracy and efficiency, and overcoming the limitations of traditional breeding where phenotypic selection is easily affected by the environment. It has broad application prospects in the field of molecular design breeding.

[0004] Currently, the development of molecular markers related to *Leymus chinensis* mainly focuses on traditional molecular markers such as SSR, RAPD, and ISSR. Their applications are largely limited to genetic diversity analysis, germplasm resource identification, or preliminary association with traits such as cold resistance and grain shattering. Molecular markers directly related to salt tolerance, a complex quantitative trait, especially high-density SNP markers based on single nucleotide polymorphisms, have not yet been reported. Existing *Leymus chinensis* molecular markers are insufficient in terms of genome coverage density, polymorphism information content, and the ability to analyze traits controlled by multiple genes. Therefore, developing SNP molecular marker combinations that can rapidly and accurately identify salt tolerance in *Leymus chinensis* is urgently needed for the precise screening of salt-tolerant germplasm resources and for breeding new varieties using marker-assisted breeding techniques. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a combination of SNP molecular markers related to salt tolerance in Leymus chinensis and their applications, so as to provide technical support for the discovery of key genes for salt tolerance in Leymus chinensis and the breeding of new salt-tolerant Leymus chinensis varieties.

[0006] The objective of this invention is achieved through the following technical solution: a combination of SNP molecular markers related to salt and alkali tolerance in *Leymus chinensis*, wherein the combination of SNP molecular markers consists of 5 SNP molecular markers, the names and positions of which are shown in the table below:

[0007]

[0008] Furthermore, the specific primer set used to amplify the SNP molecular marker combination is shown in the table below:

[0009]

[0010] This invention also provides a method for detecting the salt and alkali tolerance of *Leymus chinensis*, comprising the following steps: extracting genomic DNA from the *Leymus chinensis* sample to be tested, performing PCR amplification and sequencing analysis of the genomic DNA using a specific primer set, as shown in the table below:

[0011]

[0012] The five SNP sites Ed_SNP_1, Ed_SNP_2, Ed_SNP_3, Ed_SNP_4, and Ed_SNP_5 all belong to the salt-alkali tolerant polymorphism, indicating strong salt and alkali tolerance in *Leymus chinensis* materials; the five SNP sites Ed_SNP_1, Ed_SNP_2, Ed_SNP_3, Ed_SNP_4, and Ed_SNP_5 all belong to the salt-alkali sensitive polymorphism, indicating weak salt and alkali tolerance in *Leymus chinensis* materials.

[0013] This invention also provides the application of SNP molecular marker combinations related to salt and alkali tolerance in the identification of Leymus chinensis germplasm resources. The Leymus chinensis samples to be tested were analyzed, as shown in the table below:

[0014]

[0015] Five SNP sites, Ed_SNP_1, Ed_SNP_2, Ed_SNP_3, Ed_SNP_4, and Ed_SNP_5, all belong to the salt-alkali tolerant polymorphism, indicating strong salt and alkali tolerance in *Leymus chinensis* materials; while five SNP sites, Ed_SNP_1, Ed_SNP_2, Ed_SNP_3, Ed_SNP_4, and Ed_SNP_5, all belong to the salt-alkali sensitive polymorphism, indicating weak salt and alkali tolerance in *Leymus chinensis* materials.

[0016] The beneficial effects of this invention are as follows: This invention uses genome-wide association analysis to identify SNP loci associated with salt tolerance in *Leymus chinensis* by analyzing indicators directly related to seedling emergence time, aboveground relative dry matter weight, and salt tolerance under salt-alkali stress. A set of SNP molecular markers significantly associated with salt tolerance in *Leymus chinensis* is disclosed. These SNP molecular markers are located at positions 80176348 on chromosome 6, 490854164 on chromosome 9, 291625249 on chromosome 11, and 64884340 and 65147058 on chromosome 12. The SNP polymorphisms at these loci are, in descending order: CC-TT-AA-AA-TT indicates a salt-tolerant genotype, and CG / GG-TG-TT-GG-CC indicates a salt-sensitive genotype. This group of molecular markers for single nucleotide polymorphisms is accurate, efficient, reliable, convenient and stable to amplify, and provides objective results. It can be used as an auxiliary selection molecular marker for salt tolerance traits in the breeding of Leymus chinensis, effectively improving the identification efficiency and accuracy of different salt-tolerant Leymus chinensis germplasm / varieties and accelerating the breeding process. Attached Figure Description

[0017] Figure 1 Figures show the growth of Leymus chinensis under different concentrations of salt and alkali stress; where A is a phenotypic graph of Leymus chinensis under different concentrations of salt and alkali stress, B is a bar chart of the germination rate of Leymus chinensis under different concentrations of salt and alkali stress, and C is a bar chart of the aboveground biomass of Leymus chinensis under different concentrations of salt and alkali stress.

[0018] Figure 2 Manhattan plot for genome-wide association analysis of aboveground biomass in *Leymus chinensis* under salt-alkali stress; where the x-axis represents the chromosome location of each SNP; the y-axis represents the negative logarithm of the p-value of each SNP locus under the EMMAX model; AB represents Aboveground Biomass.

[0019] Figure 3Manhattan plot for genome-wide association analysis of emergence time index of Leymus chinensis under salt-alkali stress; where the horizontal axis represents the chromosome position of each SNP; the vertical axis represents the negative logarithm of the P-value of each SNP locus under the EMMAX model; DE represents Date of Emergence.

[0020] Figure 4 The figures are GWAS haplotype analysis box plots of 150 *Leymus chinensis* materials under salt-alkali stress in Example 1; where A is the GWAS haplotype analysis box plot of aboveground biomass of *Leymus chinensis* materials under salt-alkali stress, and B is the GWAS haplotype analysis box plot of emergence time of *Leymus chinensis* materials under salt-alkali stress. Detailed Implementation

[0021] The present invention will now be described in detail with reference to the accompanying drawings.

[0022] Example 1

[0023] 1. Development of SNP molecular markers related to salt tolerance in Leymus chinensis under salt-alkali stress conditions

[0024] 1.1 Constructing Natural Communities:

[0025] Fifty-five Elymus germplasm resources were collected from Shanxi, Hebei, Beijing, Inner Mongolia and other places to form a natural population (see Table 1). This population has a wide range of origins and contains rich genetic variation.

[0026] Table 1 Information on 55 Leymus chinensis germplasm resources

[0027] Material Number Material Name Habitat GPS coordinates Collection location 6023 Leymus chinensis Low-lying areas along the road 39.225421°N, 112.375948°E Shanxi-Shuocheng District Hubei Line 6024 Leymus chinensis riverside 39.098224°N, 112.326627°E Shanxi-Ningwu County Hubei Line 6025 Leymus chinensis roadside path 39.099552°N, 112.326054°E Shanxi-Ningwu County·Hubei Line 6026 Leymus chinensis Vacant land next to farmland 38.806127°N.113.234916°E Shanxi-Wutai County·Doudong Line 6028 Leymus chinensis roadside slopes 39.414907°N, 113.852250°E Shanxi-Hunyuan County·Zhengyang Line 6029 Leymus chinensis roadside 39.634214°N.114.268756°E Shanxi-Guangling County 2582 Leymus chinensis farmland edge 39.889419°N.114.886520°E Hebei - Yu County, County Road 212 2583 Leymus chinensis roadside farmland 40.079015°N, 115.080394°E Hebei - Zhuolu County, National Highway 109 2584 Leymus chinensis At the bottom of the hillside 40.182710°N, 115.513198°E Hebei - Huailai County, Provincial Highway 241 2585 Leymus chinensis National Highway 39.988540°N 115.428232°E Hebei-Zhuolu County·Xiaolongmen National Forest Park 2586 Leymus chinensis Orchard land 40.135289°N, 115.841517°E Beijing - Mentougou District, Dazhen Road 2587 Leymus chinensis abandoned land 40.387546°N, 116.079559°E Beijing - Yanqing District, Xilian Road 2588 Leymus chinensis riverside 41.293211°N, 116.756721°E Hebei-Fengning Manchu Autonomous County 2589 Leymus chinensis roadside and riverbank 41.633737°N.117.061743°E Hebei - Longhua County, National Highway 111 2590 Leymus chinensis roadside 42 023462°N, 117.662893°E Hebei-Weichang Manchu and Mongolian Autonomous County · Tianhexing Wood Industry Co., Ltd. Weichang 7071 Leymus chinensis Dry ditch bottom by the roadside 42.170755°N 117.560278°E Hebei-Weichang Manchu and Mongolian Autonomous County, Haliha Township People's Government 7072 Leymus chinensis roadside 42.339805°N, 117.466657°E Hebei-Weichang Manchu and Mongolian Autonomous County, Hebei Saiyouhanba National Forest Park 7073 Leymus chinensis roadside 42.547839°N, 117.231591°E Ulanbutong Scenic Area, Keshiketeng Banner, Inner Mongolia 7074 Leymus chinensis roadside 43.484347°N, 117.787069°E Inner Mongolia - Keshiketeng Banner, Keshiketeng Banner Reshui Forest Farm 7075 Leymus chinensis roadside 43.532785°N.118.592717°E Inner Mongolia - Bairin Right Banner, National Highway 303 7076 Leymus chinensis roadside 43.899308°N, 119.640051°E Inner Mongolia - Balinzuo Banner · 303 National Highway 7077 Leymus chinensis roadside 43.848476°N, 120.238296°E Inner Mongolia - Arukorqin Banner - National Highway 303 0442 Leymus chinensis roadside ditches 45.747720°N, 120.463280°E Toschejunzhalage, Right Middle Banner, Horqin, Inner Mongolia 7080 Leymus chinensis roadside 45.509563°N, 120.608828°E Chagantalahai, Right Middle Banner, Horqin, Inner Mongolia 7079 Leymus chinensis roadside 45.226365°N, 121.104088°E Inner Mongolia - Horqin Right Middle Banner, Township Road 024 0443 Leymus chinensis meadow grassland 45.798165°N, 120.362513°E Inner Mongolia - Horqin Right Middle Banner, Haleledaban 0444 Leymus chinensis roadside grass 46.429513°N 120,380774°E Inner Mongolia - Horqin Right Front Banner, National Highway 302 0447 Leymus chinensis roadside 47.359513°N.120.481703°E Arxan National Forest Park, Arxan City, Inner Mongolia 0445 Leymus chinensis roadside 47.281852°N, 120.415159°E Arxan National Forest Park, Arxan City, Inner Mongolia 0448 Leymus chinensis roadside slope bottom 47.324287°N, 119.732432°E Inner Mongolia - Arxan City, National Highway 331 2531 Leymus chinensis On the small hillside by the roadside 48.268863°N, 119.747432°E Inner Mongolia-Ewenki Autonomous Banner, Provincial Highway 202 0450 Leymus chinensis roadside 48.099793°N.119.731957°E Inner Mongolia - Xin Barag Left Banner, Provincial Highway 202 0449 Leymus chinensis roadside 48.076117°N, 119.651666°E Inner Mongolia - Xin Barag Left Banner, Provincial Highway 202 2533 Leymus chinensis Roadside ditch bottom 48.968600°N, 119.358279°E Inner Mongolia - Evenki Autonomous Banner - National Highway 332 2532 Leymus chinensis roadside 49.203690°N, 119.703389°E Hailar National Forest Park, Hailar District, Inner Mongolia 2534 Leymus chinensis roadside 48.849500°N, 118.891061°E Inner Mongolia - Chenbalhu Banner - National Highway 332 2537 Leymus chinensis roadside 47.346873°N, 119 596556°E Inner Mongolia - Arxan City, border highway 2535 Leymus chinensis roadside 48.048979°N, 118, 471818°E Inner Mongolia - Xin Barag Left Banner - National Highway 331 0111 Leymus chinensis Side of the small road (dirt road) 46.562515°N, 119.870754°E Inner Mongolia - East Ujimqin Banner, Hustubalari 0112 Leymus chinensis roadside slope bottom 46.580985°N, 119.390562°E Inner Mongolia - East Ujimqin Banner 331 A'er Line and 502 County 0114 Leymus chinensis roadside 45.974897°N, 119.054192°E Inner Mongolia - East Ujimqin Banner, Ulagai Management Area 0115 Leymus chinensis roadside slope bottom 45.458519°N, 118.845180°E Inner Mongolia - East Ujimqin Banner, Provincial Highway 206 0119 Leymus chinensis roadside 44.416411°N, 116.937589°E Inner Mongolia - West Ujimqin Banner - National Highway 207 0117 Leymus chinensis roadside 44.673301°N, 117.896861°E Inner Mongolia - West Ujimqin Banner - National Highway 207 0120 Leymus chinensis Typical grassland edge 43.452774 N, 116.127534°E Inner Mongolia - Xilinhot City, Huitengxile Natural Botanical Garden 8561 Leymus chinensis Typical grassland by the roadside 43.419761°N, 116.095946°E Huitengxile Natural Botanical Garden (West Gate), Xilinhot City, Inner Mongolia 8563 Leymus chinensis roadside 43.198331°N, 116.139095°E Inner Mongolia - Abaga Banner - National Highway 207 8564 Leymus chinensis roadside 42.181710°N, 116 423090°E Inner Mongolia - Duolun County, National Highway 510 8565 Leymus chinensis roadside slope 42.208887°N, 116.630493°E Inner Mongolia - Duolun County, Inner Mongolia Duolun Luanhe River Source National Wetland Park 8566 Leymus chinensis Lakeside slope bottom 42.199102°N, 116.638806°E Inner Mongolia - Duolun County, Inner Mongolia - Luanhe River Source National Wetland Park 8567 Leymus chinensis meadow grassland 42.198513°N, 116.638106°E Inner Mongolia - Duolun County, Inner Mongolia - Luanhe River Source National Wetland Park 8568 Leymus chinensis Lakeside 41.596925°N, 116.034551°E Hebei - Fengning Manchu Autonomous County, National Highway 239 8570 Leymus chinensis Dry riverbed sandy land 41.558091°N, 116.043298°E Hebei-Fengning Manchu Autonomous County·G95 Capital Ring Expressway SXHYC Leymus chinensis roadside slopes 39.192910°N, 113.36779°E Shanxi-Huayan Village S1 Leymus chinensis roadside 49.161856°N, 120.220205°E Inner Mongolia-Hulunbuir - National Highway 301 S2 Leymus chinensis meadow grassland 49.111658°N, 120.519351°E Inner Mongolia-Hulunbuir

[0028] 1.2 Experimental Design:

[0029] Since the seed germination period and seedling stage are the periods when plants are least resistant to adverse conditions and most sensitive to the external environment, the seed germination period and seedling stage are selected as the developmental periods for evaluating salt and alkali tolerance phenotypes.

[0030] A salt solution with a molar ratio of NaCl and NaHCO3 of 3:1 was used to simulate salt-alkali stress. Vermiculite and volcanic rock (weight ratio 2:1) were used as the culture medium, and 1 / 2 Hoagland nutrient solution was used as the nutrient substrate (quantitative watering every 2 weeks). Nine plump, healthy *Elymus sibiricum* seeds were planted in each seedling pot and cultured for 7 weeks. The germination rate and aboveground biomass of three randomly selected *Elymus sibiricum* samples under different salt-alkali stress treatments were measured, and the data were analyzed to determine the optimal salt-alkali stress concentration for detecting the salt-alkali tolerance phenotype of *Elymus sibiricum*. Figure 1It was found that, compared with the control, salt-alkali stress significantly affected the emergence and growth of the plants. Under a salt-alkali stress concentration of 100 mmol / L, the germination rate, plant height, and aboveground biomass of *Elymus sibiricum* were approximately half that of the control (0 mmol / L). Below this concentration, it was impossible to effectively distinguish the salt-alkali tolerance phenotypes of different plants; above this concentration, it was impossible to obtain enough plants for subsequent experiments. Therefore, under the above culture medium and 1 / 2 Hoagland nutrient solution conditions, the optimal stress condition for detecting the salt-alkali tolerance phenotype of *Elymus sibiricum* was determined to be a 100 mmol / L NaCl + NaHCO3 solution (molar ratio of NaCl to NaHCO3: 3:1).

[0031] The salt and alkali tolerance detection system for *Leymus chinensis* constructed above was used to evaluate the salt and alkali tolerance of *Leymus chinensis* materials. The salt and alkali tolerance evaluation tests showed significant differences in salt and alkali tolerance among different *Leymus chinensis* germplasm materials (see Tables 2.1 and 2.2), making them high-quality materials for genome-wide association analysis of salt and alkali tolerance traits in *Leymus chinensis*.

[0032] Table 2.1 Evaluation index values ​​of salt and alkali tolerance of Leymus chinensis germplasm materials under salt and alkali stress treatment with 0 mmol / L control and 100 mmol / L NaCl:NaHCO3 (3:1) solution.

[0033] Material Number Germination rate (%) Germination rate (%) Relative germination rate (%) Control plant height (cm) Plant height after treatment (cm) Relative plant height (%) Control stem diameter (mm) Treatment stem diameter (mm) Relative stem diameter (%) 7074 72.00% 17.28% 24.01% 29.23 27.49 94.05% 1.50 2.90 193.10% 0443 85.33% 44.44% 52.08% 24.24 19.62 80.94% 1.81 1.78 98.47% 6025 53.33% 14.81% 27.78% 22.87 16.42 71.77% 1.74 1.25 71.88% 6023 72.00% 25.93% 36.01% 24.67 22.95 93.05% 1.54 1.66 107.88% 6028 77.33% 73.46% 94.99% 30.14 27.09 89.88% 1.28 1.48 115.60% 8564 22.67% 18.52% 81.70% 31.98 26.13 81.72% 1.49 1.39 93.40% 7073 57.33% 11.07% 19.32% 30.26 26.20 86.58% 1.51 1.39 92.01% 7071 37.33% 11.73% 31.42% 27.23 23.11 84.86% 1.64 1.28 78.29% 2584 85.33% 72.84% 85.36% 28.23 23.81 84.34% 1.57 1.30 82.83% 7079 76.00% 70.99% 93.40% 35.67 29.95 83.97% 1.38 1.27 92.19% 8561 64.00% 36.42% 56.91% 31.66 28.35 89.56% 1.68 1.90 112.65% 0442 56.00% 47.53% 84.88% 20.93 15.74 75.23% 2.34 1.43 61.10% 6029 94.67% 80.25% 84.77% 33.88 27.93 82.44% 1.70 1.68 99.35% 8565 92.00% 83.33% 90.58% 30.13 26.71 88.67% 1.25 0.91 72.64% 6026 70.67% 80.25% 113.56% 32.59 23.80 73.03% 1.09 0.84 76.54% 2583 54.67% 48.15% 88.08% 25.71 21.16 82.28% 1.35 0.63 46.59% 7075 57.33% 14.81% 25.84% 29.69 27.42 92.33% 1.74 1.33 76.40% 0120 68.00% 55.56% 81.70% 28.91 24.51 84.78% 1.23 1.09 88.66% SXHYC 76.00% 59.88% 78.78% 29.89 27.75 92.82% 1.48 1.15 77.55% 2589 60.00% 20.99% 34.98% 31.59 21.45 67.91% 1.65 1.93 116.90% 8570 69.33% 54.32% 78.35% 31.91 30.15 94.48% 1.30 1.03 79.31% 2585 81.33% 69.75% 85.76% 23.62 28.85 122.15% 0.89 1.38 154.60% 0450 49.33% 15.43% 31.28% 31.01 23.68 76.36% 0.62 1.08 172.68% 8566 50.67% 43.83% 86.50% 35.37 24.35 68.83% 0.85 1.47 173.24% 2586 80.00% 57.41% 71.76% 24.42 19.77 80.96% 0.71 3.90 550.20% 2534 58.67% 41.36% 70.50% 19.35 25.06 129.55% 0.87 0.92 105.70% 7080 61.33% 38.89% 63.41% 18.95 14.12 74.52% 0.56 0.37 65.88% 6024 84.00% 70.99% 84.51% 21.27 19.74 92.80% 0.81 0.90 110.61% 0449 22.67% 12.96% 57.19% 25.13 18.96 75.45% 0.87 0.99 113.73% 7077 2.67% 21.07% 790.28% 6.08 13.68 224.93% 0.28 0.92 333.94% 7072 50.67% 26.54% 52.39% 25.47 25.26 99.18% 0.88 1.45 164.08% 0444 70.67% 27.16% 38.43% 22.43 18.98 84.62% 0.67 1.23 183.56% 0115 92.00% 82.10% 89.24% 24.60 26.86 109.21% 0.57 1.06 186.40% 2588 56.00% 48.77% 87.08% 23.98 23.72 98.92% 0.99 0.98 98.85% 2533 86.67% 38.27% 44.16% 31.85 19.50 61.22% 0.55 0.93 168.53% 0447 85.33% 45.68% 53.53% 22.50 18.66 82.95% 0.86 1.06 124.04% 0114 93.33% 31.48% 33.73% 24.03 20.87 86.82% 1.26 1.63 129.69% 0112 84.00% 42.59% 50.71% 24.52 20.18 82.31% 0.68 0.92 135.85% 2537 74.67% 18.52% 24.80% 19.57 14.39 73.54% 0.68 1.21 177.25% 2531 46.67% 33.95% 72.75% 38.31 23.86 62.29% 0.74 1.04 140.04% 2536 70.67% 48.15% 68.13% 26.56 22.58 85.00% 0.80 1.23 153.42% 2532 69.33% 11.04% 15.92% 23.45 25.10 107.04% 1.12 0.70 62.89% 2587 57.33% 39.51% 68.91% 16.13 13.59 84.24% 0.97 0.78 80.02% 8568 46.67% 48.15% 103.17% 30.13 20.33 67.48% 1.25 0.82 65.59% 0117 60.00% 56.17% 93.62% 28.94 25.35 87.61% 1.11 1.03 92.75% 0448 38.67% 11.04% 28.54% 22.59 19.50 86.31% 1.05 0.86 81.75% 2590 42.67% 24.07% 56.42% 18.85 18.88 100.15% 1.39 0.94 67.70% 0111 76.00% 11.07% 14.57% 24.89 18.90 75.92% 1.07 0.79 74.42% 8563 94.67% 20.37% 21.52% 28.11 22.90 81.45% 1.49 1.00 67.22% 2535 90.67% 71.60% 78.98% 22.74 22.62 99.47% 0.93 0.99 106.90% S2 85.33% 82.72% 96.93% 27.61 23.22 84.11% 0.92 1.46 157.89% S1 86.67% 88.27% 101.85% 27.34 24.75 90.51% 1.23 1.38 112.05% 0445 18.67% 44.44% 238.10% 28.74 24.77 86.17% 1.06 0.75 70.13% 7076 0.00% 35.80% 0.00% 0.00 23.13 0.00% 0.00 1.02 0.00% 0119 81.33% 78.40% 96.39% 28.74 24.46 85.11% 1.33 1.01 75.98%

[0034] Table 2.2 Evaluation index values ​​of salt and alkali tolerance of Leymus chinensis germplasm materials under salt and alkali stress treatment with 0 mmol / L control and 100 mmol / L NaCl:NaHCO3 (3:1) solution.

[0035] Material Number Compare the number of tillers (individuals) Number of tillers processed (individuals) Relative tillering number (%) Fresh weight of aboveground parts (g / plant) as control Fresh weight of treated aboveground parts (g / plant) Relative fresh weight above ground (%) 7074 1.40 2.11 150.79% 0.2915 0.3849 132.06% 0443 1.80 1.83 101.56% 0.3401 0.2211 65.01% 6025 1.40 1.50 107.14% 0.3190 0.1773 55.59% 6023 1.80 1.82 101.08% 0.3303 0.3031 91.79% 6028 1.60 1.40 87.78% 0.3001 0.2531 84.33% 8564 1.73 2.00 115.38% 0.3751 0.3156 84.12% 7073 1.47 1.67 113.64% 0.3446 0.2538 73.64% 7071 1.80 1.33 74.07% 0.2977 0.2633 88.45% 2584 1.47 1.41 96.37% 0.3133 0.1802 57.53% 7079 1.47 2.01 137.14% 0.3172 0.3180 100.25% 8561 1.33 1.59 119.31% 0.3712 0.2998 80.76% 0442 2.33 1.40 59.80% 0.3692 0.1169 31.66% 6029 1.13 1.34 118.26% 0.3552 0.3449 97.09% 8565 1.13 1.11 97.57% 0.1758 0.1842 104.74% 6026 1.00 1.22 121.96% 0.2619 0.1886 72.00% 2583 1.60 1.19 74.31% 0.3388 0.2035 60.07% 7075 1.40 1.83 130.95% 0.2858 0.2481 86.81% 0120 1.20 1.24 103.47% 0.2431 0.2188 90.00% SXHYC 1.27 1.45 114.34% 0.3219 0.3637 112.99% 2589 1.47 2.34 159.72% 0.3160 0.4258 134.74% 8570 2.00 1.53 76.39% 0.4327 0.3103 71.71% 2585 2.07 1.87 90.25% 0.3066 0.2820 91.97% 0450 1.13 1.42 125.00% 0.3287 0.2486 75.63% 8566 1.33 1.90 142.26% 0.2935 0.3344 113.94% 2586 1.33 1.42 106.39% 0.2678 0.1962 73.26% 2534 1.13 1.68 147.88% 0.2585 0.3059 118.32% 7080 1.07 1.14 106.60% 0.2598 0.1360 52.36% 6024 1.80 1.56 86.49% 0.2696 0.3153 116.94% 0449 1.87 2.17 116.07% 0.3538 0.2192 61.96% 7077 0.67 1.83 275.00% 0.0627 0.1144 182.50% 7072 1.60 2.17 135.42% 0.3396 0.4697 138.31% 0444 1.27 1.19 93.64% 0.2105 0.2051 97.46% 0115 1.00 1.33 133.44% 0.1983 0.2149 108.33% 2588 1.07 1.13 106.25% 0.2123 0.2077 97.86% 2533 1.00 1.17 116.85% 0.1984 0.1912 96.35% 0447 1.07 1.57 147.02% 0.1417 0.1393 98.30% 0114 1.00 1.35 135.00% 0.2898 0.2071 71.44% 0112 1.07 1.11 104.51% 0.2271 0.1803 79.40% 2537 1.13 1.44 127.45% 0.1455 0.1098 75.44% 2531 1.20 1.59 132.87% 0.2314 0.2352 101.63% 2536 1.13 1.35 119.28% 0.2273 0.1910 84.01% 2532 1.42 1.00 70.59% 0.1818 0.1081 59.47% 2587 1.20 1.00 83.33% 0.1282 0.0663 51.73% 8568 1.13 1.00 88.24% 0.1758 0.1157 65.82% 0117 1.07 1.02 95.83% 0.3676 0.2165 58.88% 0448 1.07 1.00 93.75% 0.1958 0.1439 73.48% 2590 1.40 1.17 83.33% 0.2202 0.1240 56.31% 0111 1.00 1.00 100.00% 0.2147 0.0936 43.60% 8563 1.20 1.11 92.59% 0.3004 0.1607 53.51% 2535 1.00 1.00 100.00% 0.1752 0.1729 98.72% S2 1.00 1.88 187.76% 0.2007 0.3173 158.09% S1 1.00 1.58 157.55% 0.2522 0.3192 126.56% 0445 1.00 1.00 100.00% 0.2106 0.1653 78.48% 7076 0.00 1.04 0.00% 0.0000 0.2023 0.00% 0119 1.27 1.03 81.68% 0.2793 0.2154 77.13%

[0036] Note: Relative values ​​(%) of each indicator = (Average value of indicator in the treatment group / Average value of indicator in the control group) × 100%

[0037] 1.3 Identification of salt-alkali tolerance traits in Leymus chinensis:

[0038] Under saline-alkali stress in a 100 mmol / L NaCl + NaHCO3 (3:1) solution, 30 plants from each of the above 55 *Leymus chinensis* germplasm materials were used. The salt tolerance phenotype of individual plants was assessed by measuring emergence time, plant height, stem diameter, number of tillers, and aboveground fresh weight at 7 weeks of age. The genetic diversity of the tested individuals was analyzed (see Table 3). The results showed that the genetic diversity index of the test population composed of *Leymus chinensis* individuals ranged from 0.83 to 1.96, indicating significant differences among individuals and rich genetic diversity, meeting the criteria for a high-quality natural population with genome-wide association. Furthermore, all indicators related to salt tolerance showed varying degrees of variation, ranging from 24.16% to 61.19%. Aboveground biomass had the highest coefficient of variation, indicating significant differences among individuals and suggesting it can be used as a primary indicator for assessing salt tolerance. The coefficient of variation for emergence time was the second most significant, indicating that there were significant differences in the impact of salt and alkali stress on different individuals, affecting germination. This index can be used as an auxiliary indicator for salt and alkali tolerance assessment. Since plant height, stem diameter, and tiller number are closely related to aboveground biomass, and their genetic diversity index and coefficient of variation are smaller than those of aboveground biomass and emergence time, they are only used as reference indicators for salt and alkali tolerance assessment and will not be further analyzed.

[0039] Table 3 Phenotypic traits of Leymus chinensis under salt-alkali stress with 100 mmol / L NaCl:NaHCO3 (3:1) solution

[0040] Results of variation analysis and genetic diversity analysis

[0041]

[0042] The salt tolerance of individual plants of Leymus chinensis was comprehensively evaluated using the fuzzy membership function method. 150 individual plants with large differences in salt tolerance phenotypes and rich phenotypic diversity were selected. The values ​​of salt tolerance-related indicators were counted (see Tables 4.1-4.6), and their genomes were resequencing to determine the genotype of the samples.

[0043] Table 4.1 Salt tolerance phenotypic values ​​of individual plants of *Leymus chinensis* (1-29 plants) under salt-alkali stress of 100 mmol / L NaCl:NaHCO3 (3:1).

[0044]

[0045] Table 4.2 Salt tolerance phenotypic values ​​of individual plants of *Leymus chinensis* (30-58) under 100 mmol / L NaCl:NaHCO3 (3:1) salt-alkali stress.

[0046]

[0047] Table 4.3 Salt tolerance phenotypic values ​​of individual plants of *Leymus chinensis* (plants 59-86) under salt-alkali stress with 100 mmol / L NaCl:NaHCO3 (3:1).

[0048]

[0049] Table 4.4 Salt tolerance phenotypic values ​​of individual plants of *Leymus chinensis* (87-114) under salt-alkali stress with 100 mmol / L NaCl:NaHCO3 (3:1).

[0050]

[0051] Table 4.5 Salt tolerance phenotypic values ​​of individual plants of *Leymus chinensis* (115-142) under 100 mmol / L NaCl:NaHCO3 (3:1) salt-alkali stress.

[0052]

[0053] Table 4.6 Salt tolerance phenotypic values ​​of individual plants of *Leymus chinensis* (143-150) under salt-alkali stress with 100 mmol / L NaCl:NaHCO3 (3:1).

[0054]

[0055] 1.4 Genome-wide association analysis of salt-alkali tolerance-related traits in *Leymus chinensis*:

[0056] Using salt tolerance traits as correlation indicators, an association analysis of salt tolerance traits in the *Leymus chinensis* test population was conducted using EMMAX based on a mixed linear model (MLM). To control for false positives, the threshold was adjusted using Bonferroni correction: 1 / 0.05 was divided by the number of independent markers (calculated using plink), where independent markers were those that were not linked to each other, representing the calculated linkage between markers. The association value and the calculated threshold were both logarithmic. 10 To visualize locations, draw a Manhattan plot. Figure 2 and Figure 3 The vertical axis represents the negative logarithm of the P-value of each SNP site in the EMMAX model, with the base 10 being the negative logarithm. Sites with a negative logarithm greater than 7.5 are considered as associated SNP sites.

[0057] The results showed that polymorphic sites were found at positions 80176348 on chromosome 6, 490854164 on chromosome 9, 291625249 on chromosome 11, and 64884340 and 65147058 on chromosome 12 of *Elymus sibiricum*. These SNP sites were significantly associated with the aboveground biomass and emergence time of *Elymus sibiricum* after salt and alkali stress.

[0058] The nucleotide sequence of chromosome 6 (Chr6) with a 500bp interval around position 80176348 is shown in SEQ ID NO. 1. The 'r' at position 501 in SEQ ID NO. 1 is a polymorphic site; 'r' at position C indicates the CC genotype, 'r' at both C and G indicates the CG genotype, and 'r' at position G indicates the GG genotype. The nucleotide sequence of chromosome 9 (Chr9) with a 500bp interval around position 490854164 is shown in SEQ ID NO. 2. The 'r' at position 501 in SEQ ID NO. 2 is a polymorphic site; 'r' at position T indicates the TT genotype, and 'r' at both T and G indicates the TG genotype. The nucleotide sequence of chromosome 11 (Chr11) with a 500bp interval around position 291625249 is shown in SEQ ID NO. 3. In SEQ ID NO. 3, the r at position 501 is a polymorphic site; when r is base A, the genotype is AA, and when r is base T, the genotype is TT. The nucleotide sequence of 500 bp upstream and downstream of position 64884340 on chromosome 12 (Chr12) is shown in SEQ ID NO. 4. The r at position 501 in SEQ ID NO. 4 is a polymorphic site; when r is base A, the genotype is AA, and when r is base G, the genotype is GG. The nucleotide sequence of 500 bp upstream and downstream of position 65147058 on chromosome 12 (Chr12) is shown in SEQ ID NO. 5. The r at position 501 in SEQ ID NO. 5 is a polymorphic site; when r is base T, the genotype is TT, and when r is base C, the genotype is CC.

[0059] 1.5 SNP molecular marker development and primer design:

[0060] PCR amplification primer pairs were designed based on associated SNP sites. Each primer pair contains one specific upstream primer and one specific downstream primer. Primer information is shown in Table 5.

[0061] Table 5 Primer pair information for amplifying SNP molecular markers

[0062]

[0063] 1.5.1 DNA quality control: Test the purity and integrity of 150 DNA samples listed in Tables 4.1-4.6.

[0064] a) Basic purity test: The purity and concentration of DNA samples were determined using a Nanodrop 2000 spectrophotometer. 260 / 280: 1.8-2.1; 260 / 230 >= 1.0.

[0065] DNA integrity: DNA integrity was determined by 1% agarose gel electrophoresis, with the main DNA band clearly visible.

[0066] 1.5.2 DNA dilution: DNA was diluted in samples that passed quality inspection (clear and bright bands without tails in agarose gel electrophoresis) to a concentration range of 5-50 ng.

[0067] 1.5.3 The PCR reaction system is shown in Table 6.

[0068] Table 6 PCR Reaction System

[0069]

[0070] 1.5.4 The PCR reaction procedure is shown in Table 7.

[0071] Table 7 PCR reaction procedure

[0072] .

[0073] 1.6 Haplotype Analysis:

[0074] The amplified PCR products were sequenced to detect the genotypes of 150 *Leymus chinensis* materials at the SNP molecular marker sites. Haplotype analysis was performed combining the SNP markers with phenotypic data on aboveground biomass and emergence time of the 150 tested materials. The results are as follows: Figure 4 As shown in the figure. SNP genotyping is divided into two groups: darker genotypes are CC-TT-AA-AA-TT, and lighter genotypes are CG / GG-TG-TT-GG-CC. The CC-TT-AA-AA-TT genotype of *Leymus chinensis* has an earlier emergence time and higher aboveground biomass, indicating strong salt tolerance; the CG / GG-TG-TT-GG-CC genotype has poor salt tolerance, with a later emergence time and lower aboveground biomass; or although the CG / GG-TG-TT-GG-CC genotype has an earlier emergence time, its aboveground biomass is significantly lower than that of the CC-TT-AA-AA-TT genotype compared to plants emerging at the same time.

[0075] Example 2

[0076] The efficiency of SNP markers was validated using 64 *Leymus chinensis* materials listed in Table 8. Aboveground biomass and emergence time under salt-alkali stress were measured, and the DNA of the tested *Leymus chinensis* materials was detected and grouped according to genotype. Specific results are shown in Table 8, where CC-TT-AA-AA-TT is the salt-tolerant genotype, and CG / GG-TG-TT-GG-CC is the salt-sensitive genotype.

[0077] The efficiency of the SNP marker was validated based on 64 materials in Table 8. Among them, 15 samples were of the CC-TT-AA-AA-TT type, with an average aboveground biomass of 0.5953 g and an average emergence time of 7.07 days; 22 samples were of the CG / GG-TG-TT-GG-CC type, with an average aboveground biomass of 0.1224 g and an average emergence time of 14.59 days; and 27 samples were of other types, with an average aboveground biomass of 0.3127 g and an average emergence time of 13.04 days. The accuracy of this marker in selecting salt-tolerant materials was 93.75%.

[0078] Table 8. Salt-alkali tolerance phenotypes and genotypes of 64 *Leymus chinensis* materials

[0079] Material Number Aboveground biomass (g) Emergence time (d) Ed_SNP_1 Ed_SNP_2 Ed_SNP_3 Ed_SNP_4 Ed_SNP_5 Indicator Type S2-100-5-4 0.8636 7 CC TT AA AA TT high sxhyc-100-3-1 0.7598 5 CC TT AA AA TT high S2-100-3-9 0.7527 7 CC TT AA AA TT high sxhyc-100-7-6 0.6855 6 CC TT AA AA TT high 2585-100-2-4 0.6079 6 CC TT AA AA TT high 2589-100-2-5 0.5933 13 CC TT AA AA TT high 6028-100-5-2 0.5911 7 CC TT AA AA TT high 6024-100-4-1 0.5787 6 CC TT AA AA TT high 6029-100-4-5 0.5732 8 CC TT AA AA TT high 2588-100-4-7 0.5587 5 CC TT AA AA TT high S1-100-3-5 0.5111 5 CC TT AA AA TT high 6028-100-2-4 0.4839 8 CC TT AA AA TT high 2584-100-5-8 0.4751 7 CC TT AA AA TT high 7079-100-7-7 0.4536 10 CC TT AA AA TT High 6029-100-3-5 0.4416 6 CC TT AA AA TT High 2533-100-3-2 0.4187 9 CC TG TT AA TT Medium 0114-100-3-2 0.4117 12 CC TT TT AA TT Medium 2589-100-3-2 0.4107 15 CC TG AA AA TT Medium 2585-100-2-8 0.4052 9 CG TT AA AA TT Medium 6023-100-8-2 0.404 18 CC TG AA AA TT Medium S1-100-2-5 0.4038 6 CG TT AA AA TT Medium 2584-100-6-7 0.3813 9 CG TT TT AA TT Medium 8566-100-2-7 0.3397 13 CC TT TT AA CC Medium 6024-100-3-7 0.3345 8 CG TG TT AA TT Medium 8570-100-2-5 0.3242 18 CC TG TT GG CC Medium 2588-100-2-5 0.3226 15 CC TG AA AA TT Medium 0117-100-3-5 0.3219 8 CC TT TT AA TT Medium 2583-100-6-5 0.3202 16 CC TG AA AA TT Medium 6026-100-5-9 0.2991 15 CC TG AA AA TT Medium 2586-100-4-5 0.2961 18 CC TT AA AA TT Medium 2588_100_3_5 0.2841 7 CC TT TT AA TT Medium 0450-100-1-2 0.2773 9 CG TG TT GG CC Medium 0443-100-2-8 0.2744 8 CC TG TT AA CC Medium 7075-100-5-4 0.2669 20 CC TT TT [[ID=1 ​ ​ 7074-100-2-7 0.2667 11 ​ ​ ​ ​ ​ ​ 6025-100-5-2 0.2663 18 ​ ​ ​ ​ ​ ​ 8561-100-8-3 0.2582 14 ​ ​ ​ ​ ​ ​ 2583-100-7-4 0.2506 19 ​ ​ ​ ​ ​ ​ 8565-100-3-5 0.242 8 ​ ​ ​ ​ ​ ​ 7072-100-3-9 0.2375 10 ​ ​ ​ ​ ​ ​ 0114-100-3-7 0.2136 19 ​ ​ ​ ​ ​ ​ 0112-100-1-8 0.2111 20 ​ ​ ​ ​ ​ ​ 0115-100-8-6 0.1921 10 ​ ​ ​ ​ ​ ​ 0112-100-2-1 0.1887 14 ​ ​ ​ ​ ​ ​ 0442-100-7-8 0.1874 17 ​ ​ ​ ​ ​ ​ 7080-100-3-6 0.1855 10 ​ ​ ​ ​ ​ ​ 8564-100-2-9 0.165 18 ​ ​ ​ ​ ​ ​ 2535-100-2-3 0.1624 9 ​ ​ ​ ​ ​ ​ 2531-100-3-4 0.1622 20 ​ ​ ​ ​ ​ ​ 0115-100-3-1 0.1609 19 ​ ​ ​ ​ ​ ​ 8563-100-2-4 0.1436 21 ​ ​ ​ ​ ​ ​ 8568-100-5-1 0.1304 19 ​ ​ ​ ​ ​ ​ 0120-100-1-5 0.1275 20 ​ ​ ​ ​ ​ ​ 7076-100-2-3 0.1249 7 ​ ​ ​ ​ ​ ​ 0444-100-6-6 0.1219 14 ​ ​ ​ ​ ​ ​ 0119-100-3-2 0.1064 14 ​ ​ ​ ​ ​ ​ 0449-100-6-6 0.0999 21 ​ ​ ​ ​ ​ ​ 0444-100-2-3 0.0992 15 ​ ​ ​ ​ ​ ​ 0445-100-7-9 0.0726 15 ​ ​ ​ ​ ​ ​ 0443-100-1-7 0.0658 11 ​ ​ ​ ​ ​ ​ 2587-100-3-1 0.0635 7 ​ ​ ​ ​ ​ ​ 2531-100-3-3 0.0628 17 ​ ​ ​ ​ ​ ​ 2587-100-2-1 0.0504 8 ​ ​ ​ ​ ​ ​ 2537-100-4-3 0.0201 15 ​ ​ ​ ​ ​ ​

[0080] The above results demonstrate that the SNP molecular markers provided by this invention can be applied to marker-assisted selection in the genetic breeding of crested wheatgrass for salt and alkali tolerance, thereby improving the accuracy of selection.

[0081] In summary, this invention obtained a set of SNP molecular markers related to the salt tolerance trait of *Leymus chinensis*. These SNP molecular markers are located at positions 80176348 on chromosome 6, 490854164 on chromosome 9, 291625249 on chromosome 11, and 64884340 and 65147058 on chromosome 12. These sites exhibit base polymorphisms CC / CG(GG), TT / TG, AA / TT, AA / GG, and TT / CC. Among these, CC-TT-AA-AA-TT represents the salt tolerance genotype, and CG / GG-TG-TT-GG-CC represents the salt sensitivity genotype (see Table 9).

[0082] Table 9. SNP molecular marker information related to salt and alkali tolerance of Leymus chinensis

[0083]

[0084] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for detecting the salt-alkaline tolerance of Puccinellia, characterized by The method comprises the following steps: extracting genomic DNA of a sample of the test Puccinellia material, and performing PCR amplification and sequencing analysis on the genomic DNA of the test Puccinellia material by using specific primers, as shown in the following table: ； The Ed_SNP_1 is amplified by using the specific primers shown in SEQ ID NO. 6 and SEQ ID NO. 7; the Ed_SNP_2 is amplified by using the specific primers shown in SEQ ID NO. 8 and SEQ ID NO. 9; the Ed_SNP_3 is amplified by using the specific primers shown in SEQ ID NO. 10 and SEQ ID NO. 11; the Ed_SNP_4 is amplified by using the specific primers shown in SEQ ID NO. 12 and SEQ ID NO. 13; and the Ed_SNP_5 is amplified by using the specific primers shown in SEQ ID NO. 14 and SEQ ID NO.

15. The Puccinellia material with the salt-tolerant polymorphism of the five SNP sites Ed_SNP_1, Ed_SNP_2, Ed_SNP_3, Ed_SNP_4 and Ed_SNP_5 has strong salt-tolerance, and the Puccinellia material with the salt-sensitive polymorphism of the five SNP sites Ed_SNP_1, Ed_SNP_2, Ed_SNP_3, Ed_SNP_4 and Ed_SNP_5 has weak salt-tolerance.

2. The application of the SNP molecular marker combination related to the salt-tolerant alkaline of Puccinellia distans in the identification of Puccinellia distans germplasm resources, characterized in that: The test Puccinellia material is detected, as shown in the following table: ； The Ed_SNP_1 is amplified by using the specific primers shown in SEQ ID NO. 6 and SEQ ID NO. 7; the Ed_SNP_2 is amplified by using the specific primers shown in SEQ ID NO. 8 and SEQ ID NO. 9; the Ed_SNP_3 is amplified by using the specific primers shown in SEQ ID NO. 10 and SEQ ID NO. 11; the Ed_SNP_4 is amplified by using the specific primers shown in SEQ ID NO. 12 and SEQ ID NO. 13; and the Ed_SNP_5 is amplified by using the specific primers shown in SEQ ID NO. 14 and SEQ ID NO.

15. The Puccinellia material with the salt-tolerant polymorphism of the five SNP sites Ed_SNP_1, Ed_SNP_2, Ed_SNP_3, Ed_SNP_4 and Ed_SNP_5 has strong salt-tolerance, and the Puccinellia material with the salt-sensitive polymorphism of the five SNP sites Ed_SNP_1, Ed_SNP_2, Ed_SNP_3, Ed_SNP_4 and Ed_SNP_5 has weak salt-tolerance.

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