SNP molecular marker related to drought resistance character of leymus chinensis and application

Abstract

The invention discloses a Leymus chinensis drought resistance character related SNP molecular marker and application, the molecular marker is selected from one of SNP1, SNP2, SNP3 and SNP4, a molecular marker combination is formed by combining four markers of SNP1, SNP2, SNP3 and SNP4, the molecular marker combination is C, GC, T and ATG homozygous genotypes according to the sequence of SNP1-SNP4, that is, when the molecular marker combination is CC-GGCC-TT-AATTGG genotype combination, the corresponding Leymus chinensis has the strongest drought resistance. According to the molecular marker related to the drought-resistant character of the leymus chinensis and the combination of the molecular marker, the drought-resistant character of the leymus chinensis can be accurately evaluated at the molecular level, a drought-resistant seed source is accurately and efficiently screened at the seedling stage of the leymus chinensis, and the breeding period of the leymus chinensis is shortened.

Description

Technical Field

[0001] The invention relates to the technical field of plant molecular breeding, and in particular to SNP molecular markers related to drought resistance traits of Leymus chinensis and applications thereof. Background Art

[0002] At present, in order to ensure meat food security, animal husbandry needs a large amount of land for forage production. Increasing the use of arid and semi-arid land in the north is a key measure to increase the scale of animal husbandry industry and supplement the meat food supply of Chinese residents. The selection and cultivation of forage resources with strong drought resistance is an important part of achieving this goal.

[0003] Leymus chinensis is a perennial herbaceous plant of the Poaceae family. It is named because sheep and goats like to eat it. Historically, it is the main dominant species in the arid and semi-arid grassland areas in the north of China, such as Northeast China and Inner Mongolia. Leymus chinensis has a well-developed root system and can absorb water and nutrients deep into the soil. It grows well in arid environments and is a common forage grass in wasteland suitable for grazing. Leymus chinensis is rich in nutrients such as protein, crude fiber, minerals and vitamins, which can provide rich nutrition for cattle and sheep, promote their growth and development, and is widely recognized by animal husbandry producers. However, compared with other competing species in the grassland, the development of seedlings is relatively slow, and it is particularly sensitive to drought stress, which has become the main bottleneck of its application. Therefore, breeding Leymus chinensis resources with outstanding drought resistance can effectively expand the area of ​​wasteland suitable for grazing, increase forage yield, and improve the scale of animal husbandry industry.

[0004] Although drought-resistant germplasm screening has been carried out for Gramineae plants, Gramineae is a very broad and huge plant group defined based on the traditional Linnaean classification system, and drought-resistant screening methods between different species are difficult to be applied to each other. Take rice as an example. Although rice and Leymus chinensis belong to the same Gramineae family, they have many major differences in genetics, physiology and anatomy. In terms of genetics: rice belongs to the genus Oryza of the Gramineae family, is an autologous diploid plant, contains 24 chromosomes (2n=2x=24), has a genome size of about 385Mb, and contains about 40,000 protein-coding genes; while Leymus chinensis belongs to the genus Leymus of the Gramineae family, is an allotetraploid plant, contains 28 chromosomes (2n=4x=28) , the genome size is about 8Gb, containing more than 80,000 protein-coding genes; Physiologically: Rice originated in the middle reaches of the Pearl River in China, with an average annual rainfall of 700-2400mm and an average annual temperature of 14-22°C, while Leymus chinensis originated in the eastern steppe area of ​​Eurasia, with an average annual rainfall of only 120-200mm and an average annual temperature of only -2-6°C. The two have different optimal growth conditions; Anatomically: The leaves of Leymus chinensis seedlings are needle-shaped, with a stomata density of 90-170 / mm 2The length of the stomata is 38 to 42 μm, and it has rhizomes and sand structures, which are typical characteristics of xerophytes. However, the leaves of rice are ribbon-shaped, and the stomatal density is only 11 to 27 per mm. 2 The length of the stomata is only 9 to 16 μm, showing the characteristics of mesophytes. Therefore, the huge difference between the two determines that the drought resistance screening method suitable for rice is not suitable for Leymus chinensis.

[0005] SNP molecular markers play an important auxiliary role in plant drought resistance breeding. However, there is a lack of suitable SNP molecular markers in the breeding of drought-resistant Leymus chinensis. The traditional method of establishing SNP molecular markers associated with drought resistance traits has the characteristics of poor accuracy, insufficient sample throughput, and high demand for manpower and material resources, making it difficult to effectively apply them to the breeding of plants with a large number of provenances. China has an extremely rich number of Leymus chinensis provenances. In order to solve the above problems, it is urgent to develop an effective SNP molecular marker specifically associated with the drought resistance traits of Leymus chinensis. Summary of the invention

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

[0007] In view of this, the scheme of the present invention is:

[0008] The first aspect of the present invention is to propose a SNP molecular marker related to drought resistance of Leymus chinensis, wherein the molecular marker is 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 has a 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 a 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 has a T / G polymorphism;

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

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

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

[0015] Further, the genotype of the SNP1 marker is CC and GG, the genotype of the SNP2 marker is GGCC and CCAA, the genotype of the SNP3 marker is TT and GG, and the genotype of the SNP4 marker is AATTGG / GGGGCC;

[0016] When the molecular marker combination is C, GC, T, and ATG homozygous genotypes according to SNP1 to SNP4, that is, CC-GGCC-TT-AATTGG genotype combination, the corresponding Leymus chinensis has the strongest drought resistance;

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

[0018] The third aspect of the present invention is to propose a primer combination for amplifying the molecular marker combination described in the second aspect, wherein the primer combination consists 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 NOs: 5-6;

[0020] The primers for amplifying the molecular marker SNP2 include the nucleotide sequences shown in SEQ ID NOs: 8-9;

[0021] The primers for amplifying the molecular marker SNP3 include the nucleotide sequences shown in SEQ ID NOs: 11-12;

[0022] The primers for amplifying the molecular marker SNP4 include the nucleotide sequences shown in SEQ ID NOs: 14-15.

[0023] Preferably, the primer combination is:

[0024] The nucleotide sequences of the primers for amplifying the molecular marker SNP1 are shown in SEQ ID NOs: 5-7;

[0025] The nucleotide sequences of the primers for amplifying the molecular marker SNP2 are shown in SEQ ID NOs: 8-10;

[0026] The nucleotide sequences of primers for amplifying the molecular marker SNP3 are shown in SEQ ID NOs: 11-13;

[0027] The nucleotide sequences of primers for amplifying the molecular marker SNP4 are shown in SEQ ID NOs: 14-16.

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

[0029] The fifth aspect of the present invention is to provide a method for obtaining the SNP molecular marker described in the first aspect and the molecular marker combination 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 search for SNP sites in the promoter region of genes that were upregulated by drought stress in the total population of Leymus chinensis.

[0032] Predict the loss of function of components caused by SNP mutations;

[0033] Hi-tom was used to analyze the provenance of drought-resistant Leymus chinensis and compare the proportion of different SNPs in the drought-resistant population and the total population;

[0034] Different SNP types were weighted and scored, the association between SNP types and drought resistance traits was analyzed, and SNP molecular markers related to drought resistance traits of Leymus chinensis were obtained.

[0035] The sixth aspect of the present invention is to propose the use of the SNP molecular marker described in the first aspect, the molecular marker combination described in the second aspect, the primer combination described in the third aspect, the detection reagent or kit described in the fourth aspect, and the SNP marker or molecular marker combination obtained by the method described in the fifth aspect in any of the following (1) to (6):

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

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

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

[0039] (4) preparing products for identifying or assisting in identifying the drought resistance of Leymus chinensis;

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

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

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

[0043] The eighth aspect of the present invention provides a method for genetic improvement of sheepgrass, comprising the steps of successive breeding of homozygous individuals of the CC genotype of the SNP1 molecular marker, and eliminating individuals of all other genotypes at the site; or successive breeding of homozygous individuals of the GGCC genotype of the SNP2 molecular marker, and eliminating individuals of all other genotypes at the site; or successive breeding of homozygous individuals of the TT genotype of the SNP3 molecular marker, and eliminating individuals of all other genotypes at the site; or successive breeding of homozygous individuals of the AATTGG genotype of the SNP4 molecular marker, and eliminating individuals of all other genotypes at the site; preferably, the method comprises the step of successive breeding of individuals of the CC-GGCC-TT-AATTGG genotype combination of the above-mentioned molecular marker combination, and eliminating individuals of all other genotype combinations of the 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 the drought resistance traits of Leymus chinensis provided by the present 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 Leymus chinensis seedling stage, and shorten the Leymus chinensis breeding cycle.

[0046] The method for identifying or screening Leymus chinensis with strong drought resistance provided by the present 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 genetic improvement method of Leymus chinensis provided by the invention has a wide application range, broad application prospects, and can achieve excellent economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 Schematic diagram of primers used for amplifying molecular markers of the present invention.

[0049] Figure 2 It is the relative expression level of the drought resistance related gene for which the molecular marker is designed in Example 1 of the present invention.

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

[0051] Figure 4This is a molecular marker gel map of drought resistance-related gene elements of some Leymus chinensis sources in Example 3 of the present invention. DETAILED DESCRIPTION

[0052] The technical solution of the present invention will be clearly and completely described below in conjunction with the preferred embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0053] The word “exemplary” is used exclusively herein to mean “serving as an example, example, or illustration.” Any embodiment described herein as “exemplary” is not necessarily to be construed as preferred or advantageous over other embodiments.

[0054] In order to find an indicator that can accurately identify the drought resistance of Leymus chinensis, the inventors conducted a large number of studies and found that after drought stress, the genes with the most significant up-regulated expression levels are often associated with the drought resistance of plants. Among them, the homologous genes of Lc3Ns024690 and Lc3Xm072595 are both AT3G57520.1 of Arabidopsis thaliana, encoding a raffinose-specific α-galactosidase that can catalyze the decomposition of raffinose into α-galactose and sucrose, and affect the drought resistance of plants by affecting sugar metabolism and osmotic pressure; the homologous gene of Lc6Ns028159 is AT3G50980.1 of Arabidopsis thaliana, encoding a DEHYDRIN XERO 1 protein, which is involved in plant dehydration and drying; the homologous gene of Lc1Xm058728 is AT3G08860.1 of Arabidopsis thaliana, which encodes a protein with β-alanine aminotransferase activity, which affects the plant's drought resistance by affecting the synthesis of alanine, an anti-drought substance. The upregulation of the expression of these drought-resistant related genes is often considered to be the plant's resistance to adverse stress. The higher the expression of drought-resistant related genes, the stronger the plant's drought resistance.

[0055] Secondly, the elements on the promoter affect the expression of the genes in which they are located. Mutations in elements that respond to drought stress will result in the expression of plant drought-resistant genes being blocked. The promoter of Lc3Ns024690 contains the ACCAGC element, which responds to the anaerobic environment formed by stomatal closure caused by drought; Lc1Xm058728 also contains the cis-regulatory element GTTTCG that responds to anaerobic induction; the "common cis-acting element ATTGG in the promoter and enhancer region" on the promoter of Lc3Xm072595 and the "core promoter element TTTATGGGCGA around transcription start -30" on the promoter of Lc6Ns028159 promote gene expression. Mutations in these elements will reduce the ability of plants to respond to drought, thereby affecting the drought tolerance of plants.

[0056] The traits related to the adaptation of Leymus chinensis to drought, such as the survival rate and the decrease in relative water content after drought stress, are the most intuitive reflection of the degree of adaptation of Leymus chinensis to drought stress.

[0057] Based on the above research, the survival rate and relative water content decrease slope after drought stress are preferably selected as indicators for identifying drought resistance of Leymus chinensis, and SNP markers significantly correlated 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 the drought resistance of sheep fescue are provided, and the molecular markers include SNP1, SNP2, SNP3 and SNP4, specifically: the molecular marker SNP1 is located at the 220th base of the nucleotide sequence shown in SEQ ID NO: 1, and has a C / G polymorphism; the molecular marker SNP2 is located at the 380th and 381st bases of the nucleotide sequence shown in SEQ ID NO: 2, and has a GC / CA polymorphism; the molecular marker SNP3 is located at the 277th base of the nucleotide sequence shown in SEQ ID NO: 3, and has a T / G polymorphism; the molecular marker SNP4 is located at the 394th, 395th and 396th bases of the nucleotide sequence shown in SEQ ID NO: 4, and has an 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 can catalyze the decomposition of raffinose into α-galactose and sucrose, and affect the plant's ability to resist early morning drought by affecting sugar metabolism and osmotic pressure; the homologous gene of Lc6Ns028159 is AT3G50980.1 of Arabidopsis thaliana, encoding a DEHYDRIN XERO 1 protein that is involved in the dehydration and drying of plants; the homologous gene of Lc1Xm058728 is AT3G08860.1 of Arabidopsis thaliana, encoding a protein with β-alanine aminotransferase activity, and affects the plant's ability to resist early morning drought by affecting the synthesis of the anti-early morning 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 relative water content decrease slope.

[0061] The relative water 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 found that under drought conditions, plants reduce intracellular osmotic potential by accumulating small molecular weight organic osmotic regulating substances in their bodies, providing plants with water absorption capacity under low water potential, thereby stabilizing membrane structure and macromolecular conformation and improving plants' ability to adapt to adversity. Among them, carbohydrate metabolism is an important osmotic regulating mechanism in higher plants. By decomposing large molecular weight sugars to form more small molecular weight sugars, it can reduce intracellular osmotic potential, thereby improving the water retention capacity of plants, which is directly related to the resistance of plants to drought.

[0063] Plants can increase their drought resistance by enhancing the synthesis of alanine. The principle is that alanine can enable crops to maintain a good growth trend under drought conditions and thus resist drought. In addition, alanine can also promote the synthesis of endogenous hormone polyamines in crops and improve their drought resistance.

[0064] Dehydrin protects cells from dehydration by maintaining the stability and function of certain proteins in extreme environments, thereby reducing damage to plants after severe water loss. When plants are subjected to severe drought stress, upregulating the expression of dehydrin will increase the survival rate of plants after drought stress.

[0065] The relative water content change of plants after drought stress and the survival rate after the drought stress is stopped are related to sugar metabolism, alanine metabolism and dehydrin metabolism under drought stress. Therefore, the relative water content change and survival rate after drought stress are preferably selected as the identification index of the drought resistance of Leymus chinensis in the present invention.

[0066] As a preferred embodiment, the genotypes of the SNP1 marker are CC, GG and CG. After the genotype of the SNP1 marker CC is subjected to the anaerobic environment formed by drought stress, the expression level of the drought-resistant related gene Lc1Xm058728 is theoretically significantly higher than that of the GG and CG genotypes. Among them, the CC genotype is the homozygous type of the SNP1 marker C of the leymus chinensis, the GG genotype is the homozygous type of the SNP1 marker G of the leymus chinensis, and the CG genotype is the heterozygous type of the SNP1 marker C and G of the leymus chinensis.

[0067] As a preferred embodiment, the genotypes of the SNP2 marker are GGCC, CCAA and GCCA. When the sheepgrass with the SNP2 marker genotype of GGCC is subjected to an anaerobic environment formed by drought stress, theoretically, the expression level of the drought-resistance-related gene Lc3Ns024690 is significantly higher than that of the CCAA and GCCA genotypes.

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

[0069] As a preferred embodiment, the genotypes of the SNP4 marker are AATTGG, GGGGCC and ATGGGC, and the expression level of the drought resistance-related gene Lc6Ns028159 of Leymus chinensis whose genotypes are AATTGG is significantly higher than those of the GGGGCC and ATGGGC genotypes.

[0070] In order to eliminate the mis-selection and mis-elimination of excellent genes of Leymus chinensis caused by 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 many methods for genotyping, such as resequencing, direct sequencing, time-of-flight mass spectrometry, chip technology, etc. In the present invention, it is preferred to use a resequencing method (using Hi-tom to resequence the selected germplasm) to genotype the SNP.

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

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

[0074] When the molecular marker combination is G, CA, G, and GGC homozygous genotypes according to SNP1 to SNP4 (i.e., 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 C, GC, T, ATG homozygous genotypes (i.e. CC-GGCC-TT-AATTGG genotype combination) according to SNP1 to SNP4, the corresponding relative water content of the leaves decreases the slowest and the survival rate is the highest after the leymus chinensis is subjected to drought stress;

[0076] When the molecular marker combination is G, CA, G, and GGC homozygous genotypes according to SNP1 to SNP4 (i.e., GG-CCAA-GG-GGGGCC genotype combination), the corresponding sheep grass has the fastest decrease in leaf relative water content and the lowest survival rate after being subjected to drought stress.

[0077] In a third embodiment, primers for amplifying molecular markers related to drought resistance of sheep fescue are provided, wherein the primers for amplifying molecular marker SNP1 are Lc1Xm058728-currency-up and Lc1Xm058728-existence-low, Lc1Xm058728-not have-low; the primers for amplifying molecular marker SNP2 are Lc3Ns024690-currency-low and Lc3Ns024690-existence-up, Lc3Ns024690-not have-up; the primers for amplifying molecular marker SNP3 are Lc3Xm072595-currency-low and Lc3Xm072595-existence-up, Lc3Xm072595-not have-up have-up; the primers for amplifying the molecular marker SNP4 are Lc6Ns028159-currency-low, Lc6Ns028159-existence-up, and Lc6Ns028159-not have-up. The primers used to amplify the molecular markers are shown in Tables 1 and Figure 1 shown.

[0078] Table 1:

[0079]

[0080] In the fourth embodiment, a primer combination for amplifying the molecular marker combination described in the second aspect is provided, wherein the primer combination is composed of the above-mentioned primers for amplifying SNP1 to SNP4, respectively, and the primer nucleotide sequences for amplifying the molecular markers SNP1, SNP2, SNP3, and SNP4 are shown in SEQ ID NOs: 5-7, 8-10, 11-13, and 14-16, respectively. Some of the primers can be selected for the detection of multiple SNP molecular markers to screen drought-resistant Leymus chinensis. Including selecting "currency" and "existence" and the composed primer pair to detect whether there is a mutation at the corresponding site, and adding "not have" because the mutation type of the corresponding site is detected to be a homozygous mutation or a heterozygous mutation.

[0081] In the fifth embodiment, a detection reagent or kit containing the above detection molecular marker primer or primer combination is provided. Preferably, the detection reagent or kit also includes a PCR amplification reagent, and the PCR amplification reagent includes a PCR buffer, dNTP and DNA polymerase.

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

[0083] 1. Configure the following PCR reaction system

[0084]

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

[0086]

[0087] 3. Run the PCR product on 1% agarose gel and observe the amplified bands.

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

[0089] Step 1. Mining SNPs associated with drought resistance

[0090] The three-leaf-stage Leymus chinensis was divided into an experimental group where watering was stopped and a control group where water was supplemented every 3 days. Leaves of the treatment group and the control group were harvested on the 9th day, and RNA was extracted using an RNA extraction kit. The RNA samples were sent to Novogene for RNA-Seq analysis. Through differential expression analysis, genes with large up-regulation times under drought stress were identified.

[0091] The Arabidopsis homologous genes of these genes were searched on the Arabidopsis TAIR website, and the homologous genes Lc3Ns024690, Lc3Xm072595, Lc6Ns028159, and Lc1Xm058728, which were clearly annotated to affect drought resistance, were selected as candidate genes for discovering molecular markers for drought resistance;

[0092] Using the genome of Leymus_chinensis_Lc6-5 version (China National Genomics Data Center, BioProject No. 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, search for the 600 bp sequence upstream of the promoter of Lc3Ns024690, Lc3Xm072595, Lc6Ns028159 and Lc1Xm058728 genes;

[0093] The EDTA method was used to extract the mixed DNA of the first true leaf of 600 Leymus chinensis germination seedlings from different sources. The true leaf of each source was about 1 cm long. According to the Hi-tom technical requirements, sequencing primers were designed. Each pair of primers amplified a 300 bp fragment. Therefore, in order to amplify the promoter 0 bp to -600 bp sequence, two pairs of primers were designed for each gene.

[0094] Hi-tom analysis of the sequence found SNP sites that may affect the plant's drought resistance. Usually, these SNPs cause the loss of key drought resistance elements on the gene.

[0095] Step 2. Determination of drought resistance phenotypic traits of Leymus chinensis

[0096] Sow the seeds of Leymus chinensis on the surface of peat soil, put them in the incubator at 21:00 every day, and put them in the incubator at 9:00 the next day. When the surface of the peat soil is dry, add tap water. When the seeds germinate, place them in the incubator all day.

[0097] Add tap water to peat soil and vermiculite to make them saturated with water, then mix them evenly in a 1:1 ratio. Fill the evenly mixed culture soil into a 128-hole plug tray, select seedlings with uniform growth and undamaged roots, and plant them in the plug tray, with one plant planted in one planting slot;

[0098] After the seedlings were planted, the plug tray was immersed in 4g / 5L universal balanced fertilizer for 5min, and then the 128-hole plug tray was placed on the empty 32-hole plug tray, and the new plug tray was placed on the tray, so that the planting trough containing the plants was suspended in the air to ensure that the experimental results were not affected by water accumulation;

[0099] After planting, place the plug tray in the culture room for cultivation. Every 3 days, immerse the 128-hole plug tray in a culture solution containing 4g / 5L general balanced fertilizer for 1 hour to supplement water and fertilizer.

[0100] After 15 days of planting, one small unit of each variety arranged in a field pattern continued to be watered as a control group without drought treatment. It was immersed in tap water every 3 days, while other plants in the plug tray were subjected to drought treatment. During the drought treatment, the parts above the pulvinus of the plants treated with different drought days were harvested, and the fresh weight was measured with a one-thousandth balance. After being immersed in distilled water for 12 hours, the water absorption saturation weight was measured, and then the leaves were placed in a 60°C oven for 12 hours, and the dry weight was measured to calculate the relative water content of the leaves, and the relative water content of the leaves = (fresh weight - dry weight / water absorption saturation weight - dry weight) * 100%;

[0101] After the drought treatment, all planting troughs were immersed in a culture solution containing 4g / 5L general balanced fertilizer for 1 hour to replenish water and fertilizer every 3 days. After the 15th day of water replenishment, the number of plants with green leaves re-pulled from each variety in the drought treatment group was counted and recorded as the number of surviving plants.

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

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

[0104] Molecular marker primers are designed around SNPs that may affect drought resistance, where the base difference of SNPs leads to one to two base differences at the 3' end of different molecular marker primers;

[0105] Hi-tom sequencing was performed on the DNA regions where the mutation elements of 61 drought-tolerant Leymus chinensis provenances were located, and the proportion of elements and mutations in 600 Leymus chinensis provenances and 61 total drought-resistant Leymus chinensis provenances was compared, and weight scores were given to different molecular marker types;

[0106] Molecular markers were used to detect sheepgrass from different sources, and the drought resistance phenotypic traits of these sheepgrasses were compared to further test the accuracy of the molecular markers.

[0107] In a seventh embodiment, the use of the above-mentioned SNP molecular marker, molecular marker combination, primer, primer combination, detection reagent, kit or SNP marker obtained by the above-mentioned method and the combination thereof in any of the following aspects (1) to (6) is provided:

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

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

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

[0111] (4) preparing products for identifying or assisting in identifying the drought resistance of Leymus chinensis;

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

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

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

[0115] Wherein, the drought resistance of the Leymus chinensis is preferably identified by the relative water content decrease slope and the survival rate.

[0116] Preferably, the method for identifying or screening Leymus chinensis with strong drought resistance comprises the following steps:

[0117] Step I, extracting genomic DNA of the tested Leymus chinensis;

[0118] Step II, using the genomic DNA extracted above as a template, performing PCR amplification;

[0119] Step III, determining the polymorphism or genotype of the SNP molecular marker of the tested Leymus chinensis;

[0120] Step IV, determining the drought resistance of the tested Leymus chinensis according to the genotype detection result.

[0121] Wherein, 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 a combination of molecular markers. Preferably, the drought resistance of Leymus chinensis is identified by determining the genotype of a combination of molecular markers.

[0123] Among them, methods for determining molecular marker polymorphism or genotype 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 resistance-related gene Lc1Xm058728 is 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 and CG, then theoretically, the expression level of the drought resistance-related gene Lc1Xm058728 is low after the Leymus chinensis is subjected to an anaerobic environment formed by drought stress;

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

[0127] If the genotype of the SNP3 site of the tested Leymus chinensis is TT, the expression level of the drought resistance-related gene Lc3Xm072595 of the Leymus chinensis is high; if the genotype of the SNP3 site of the tested Leymus chinensis is GG and TG, the expression level of the drought resistance-related gene Lc3Xm072595 of the Leymus chinensis is low;

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

[0129] If the genotype combination of SNP1, SNP2, SNP3, and SNP4 of the tested sheep fescue is CC-GGCC-TT-AATTGG, its water content decreases slowly when subjected to drought stress, and its survival rate is high after rehydration, and its drought resistance is the strongest; if the genotype combination of SNP1, SNP2, SNP3, and SNP4 of the tested sheep fescue is GG-CCAA-GG-GGGGCC, its water content decreases quickly when subjected to drought stress, and its survival rate is low after rehydration, and its drought resistance is the weakest.

[0130] In the ninth embodiment, a method for genetic improvement of Leymus chinensis is provided, the method comprising the steps of selecting homozygous individuals of CC genotype of the above SNP1 molecular marker in succession and eliminating individuals of all other genotypes at the site;

[0131] Or the step includes the step of selecting homozygous individuals of the GGCC genotype of the above-mentioned SNP2 molecular marker through successive generations and eliminating individuals of all other genotypes at the site;

[0132] Or the step includes the step of selecting homozygous individuals of the TT genotype of the above-mentioned SNP3 molecular marker through successive generations and eliminating individuals of all other genotypes at the site;

[0133] Or the step includes the step of selecting homozygous individuals of the AATTGG genotype of the above-mentioned SNP4 molecular marker through successive generations and eliminating individuals of all other genotypes at the site;

[0134] Or it includes the step of sub-breeding individuals with the AA-TT-CC genotype combination of the above molecular marker combination and eliminating individuals with all other genotype combinations of the marker combination.

[0135] Preferably, the method comprises the steps of sub-breeding 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] In order to make the experimental methods, technical solutions and advantages and disadvantages in the embodiments of the present invention clearer, the specific experimental steps in the embodiments of the present invention are described in detail and completely below. If the manufacturer of the reagent or instrument is not indicated in the embodiments, conventional products can be purchased from the market. If the specific conditions are not indicated, they can be carried out according to conventional conditions or conditions recommended by the manufacturer.

[0137] Example 1 Obtaining SNP molecular markers related to drought resistance traits of Leymus chinensis individuals

[0138] 1. Experimental population

[0139] 150 samples of Leymus chinensis seeds were provided by Liu Gongshe's research group at the Institute of Botany, Chinese Academy of Sciences.

[0140] The research team led by Wu Zinian from the Grassland Institute of the Chinese Academy of Agricultural Sciences provided 50 sources of Leymus chinensis seeds.

[0141] 400 sources of Leymus chinensis were collected in Xiwuzhumuqin.

[0142] 2. DNA Extraction

[0143] DNA from 24 different individuals of Leymus chinensis was extracted using the EDTA method. The fully ground samples were placed in EDTA extraction solution, treated at 60°C for 10 minutes, centrifuged and the supernatant was taken. After adding RNase treatment for 15 minutes, 1 / 3 volume of chloroform was added and vortexed for 1 minute, and the supernatant was taken by centrifugation. An equal volume of isopropanol was added, and the precipitate was taken in a centrifuge tube. After adding 3 volumes of 70% ethanol and vortexed for 1 minute, the precipitate was taken by centrifugation. After the liquid in the precipitate was completely evaporated, distilled water was added, vortexed for 1 minute, and the supernatant was taken by centrifugation.

[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 universal balanced fertilizer for 1 hour to supplement water and fertilizer. Supplementation was done every 3 days. After another 15 days, the survival of different Leymus chinensis individuals was counted.

[0146] 4. Obtaining SNP molecular markers related to drought resistance in Leymus chinensis

[0147] (1) Identifying SNPs associated with drought resistance

[0148] The same source of Leymus chinensis at the three-leaf stage was divided into an experimental group where watering was stopped and a control group where water was supplemented every 3 days. The leaves of the treatment group and the control group were harvested on the 9th day, and RNA was extracted using an RNA extraction kit. The RNA samples were sent to Novogene for RNA-Seq analysis. After differential expression analysis, genes with large up-regulation under drought stress were identified. The Arabidopsis homologous genes of these genes were searched on the Arabidopsis TAIR website, and the homologous genes Lc3Ns024690, Lc3Xm072595, Lc6Ns028159, and Lc1Xm058728 that were clearly marked as affecting drought resistance were used as candidate genes for discovering drought resistance molecular markers ( Figure 2 The relevant function predictions are shown in Table 2.

[0149] Table 2: Function prediction of candidate genes

[0150]

[0151] Using the genome of Leymus_chinensis_Lc6-5 version, and the obtained CDS and pep fasta files, search for the 600bp sequence upstream of the promoter of Lc3Ns024690, Lc3Xm072595, Lc6Ns028159 and Lc1Xm058728 genes. The mixed DNA of the first true leaf of 600 different provenances of Leymus chinensis seedlings was extracted by EDTA method. Each provenance of Leymus chinensis took a true leaf of about 1cm long. According to the Hi-tom technical requirements, sequencing primers were designed. Each pair of primers amplified a 300bp fragment. Therefore, in order to amplify the promoter 0bp to -600bp sequence, two pairs of primers were designed for each gene. The sequence was analyzed by Hi-tom, and SNP sites that may affect the drought resistance of plants were found. In this step, the filtering threshold of Hi-tom was set to 0.1%, and the data volume of each sample was set to 10000.

[0152] (2) Weighted Scoring

[0153] Hi-tom sequencing was performed on the DNA regions where the mutation elements of 61 drought-resistant Leymus chinensis sources were located, and the proportions of elements and mutations in 600 Leymus chinensis sources and 61 drought-resistant Leymus chinensis sources were compared. Hi-tom was used to detect the presence of drought-related elements in all 600 Leymus chinensis and 61 drought-resistant Leymus chinensis used in the experiment. The results are shown in Table 3. Weight scores were given to different molecular marker types, and the value was the proportion of DNA sequences with elements in 61 drought-resistant Leymus chinensis / the proportion of DNA sequences with elements in 600 total Leymus chinensis. The larger the ratio, the more it reflects the weight of the column in the drought resistance assessment. When only drought-related elements are detected, the value is +; when drought-resistant elements and mutation elements are detected at the same time, the value is 0; when only mutation elements are detected, the value is -. The larger the value, the stronger the drought resistance of the plant. The results are shown in Table 4, where positive numbers indicate that the plant is drought-resistant and negative numbers indicate that the plant is not drought-resistant.

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

[0155]

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

[0157]

[0158]

[0159] (3) DNA extraction and PCR amplification

[0160] Molecular marker-specific primers were used to amplify the Lc3Ns024690, Lc3Xm072595, Lc6Ns028159 and Lc1Xm058728 gene fragments of 24 different Leymus chinensis individuals. The system was to add 100ng of DNA to the 15ul system, 1ul of 10uM / L upstream and downstream primers, anneal at 53℃ for 30s, extend at 72℃ for 55s, and 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.

[0161] (4) Regression detection

[0162] After comparison, it was found that among the 24 different Leymus chinensis individuals, the Leymus chinensis provenances with high molecular marker scores basically survived after drought stress, and the two showed a positive correlation (as shown in Table 5), which shows that the molecular marker method can well predict the drought resistance of different Leymus chinensis individuals.

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

[0164]

[0165]

[0166] Example 2 Correspondence between SNP molecular markers and drought tolerance

[0167] 1. Experimental system for high-throughput evaluation of drought resistance of different Leymus chinensis provenances

[0168] (1) Seed germination

[0169] 0.6 g of seeds were sown on the surface of peat soil. At 21:00 every day, the seeds were placed in a 16°C dark incubator with the lid of the culture dish closed. At 9:00 the next day, the seeds were placed in a 22°C 16h light / 8h dark incubator with the lid of the culture dish opened. The humidity in the incubator was 55% and the light intensity was 100 μmol / (m 2 ·s). When the surface of the peat soil is dry, add tap water. When the seeds germinate, place them in the incubation room all day.

[0170] (2) Seedling planting and cultivation

[0171] When the seedlings grow to about 4 cm long, add tap water to the culture soil, and carefully pry the culture soil to separate the roots of the seedlings from the culture soil. After selecting the seedlings with consistent growth and undamaged roots, plant them in the plug tray, plant one plant in one planting slot, plant 5 different varieties of Leymus chinensis in one plug tray, and plant the same variety of Leymus chinensis in every 4 adjacent planting slots to form a small unit arranged in a field shape. The small units in the field shape are planted in the plug tray in turn, and each variety contains 6 or 7 small units arranged in a field shape in one plug tray, for a total of 20 Leymus chinensis from different sources. After planting, place it in the culture room for cultivation. Every 3 days, immerse the plug tray in the culture solution containing 4g / 5L universal balanced fertilizer for 1 hour to supplement water and fertilizer. At least 12 small units arranged in a field shape are planted for each source, and they are distributed in at least two different plug trays.

[0172] (3) Relative water content detection

[0173] On the 0th, 9th and 15th days of drought, the leaf pulvinus of 5 plants of different drought treatment varieties were harvested, and the fresh weight FW was measured with a 1 / 1000 balance. Then the samples were placed in a 5 ml pipette tip, sealed with aluminum foil, and immersed in distilled water for 12 h. Then, they were rotated in a centrifuge at 2000 rpm for 1 min to dry the water attached to the leaves. Then, the saturated weight TW was measured. After that, the leaves were placed in a 60 °C oven for 12 h, and the dry weight DW was measured.

[0174] According to the relative water content calculation formula, RWC = (FW-DW / TW-DW) * 100, the relative water content decrease is calculated. The sheepgrass provenance with a small decrease is considered to have strong drought resistance. The decrease is calculated as the slope of its water 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 culture solution containing 4g / 5L universal balanced fertilizer for 1 hour to supplement water and fertilizer. Supplementation was done every 3 days. After the 15th day of water supplementation, the number of plants with green leaves re-extracted from each variety in the drought treatment group was counted, and this number was recorded as the number of plants that survived. The Leymus chinensis provenances with high survival rates were considered to have strong drought resistance, and the drought resistance of the Leymus chinensis was ranked.

[0177] 2. Drought resistance score

[0178] DNA from different sources of Leymus chinensis was extracted by EDTA method, and 2ml long Leymus chinensis leaf samples were extracted with 300ul extraction solution. After 100ul chloroform was used to remove proteins, the samples were rinsed with 70% alcohol. Molecular marker-specific primers were used to amplify the gene fragments of Leymus chinensis Lc3Ns024690, Lc3Xm072595, Lc6Ns028159 and Lc1Xm058728. The system was to add 100ng of DNA to the 15ul system, 1ul of 10uM / L upstream and downstream primers, anneal at 53℃ for 30s, extend at 72℃ for 55s, and 34 cycles. The amplification of the bands was recorded, and the drought resistance of Leymus chinensis was scored.

[0179] 3. Regression Detection

[0180] Compare the relationship between molecular marker scores and drought tolerance of Leymus chinensis. After comparison, it was found that Leymus chinensis provenances with high molecular marker scores had strong drought resistance and high survival rate after drought stress. The two showed a positive correlation, and the determination coefficient of the regression equation was 0.4727 ( Figure 3 As shown in Table 6), it shows that the molecular marker method can well predict the drought resistance of Leymus chinensis from different sources. The survival rate of Leymus chinensis with a survival rate of 0% often has a water decline slope greater than -40% (as shown in Table 6). Therefore, the survival rate of Leymus chinensis after drought directly reflects the drought resistance level of Leymus chinensis. The water decline slope can assist in judging the drought resistance of Leymus chinensis. The most drought-resistant Leymus chinensis genotype combination CC-GGCC-TT-AATTGG has a high survival rate after drought and a small water decline slope during drought stress compared to other genotype combinations (Table 6).

[0181] Table 6: Test results of drought resistance of 20 different Leymus chinensis provenances

[0182]

[0183] Example 3: Detect the drought resistance of eight Leymus chinensis

[0184] 1. Detect the drought resistance of eight Leymus chinensis in the same way as in Example 2.

[0185] In the step of "seedling planting and cultivation", at 21:00 every day, put it into an incubator at 16 °C in the dark, cover the culture dish lid, and place it in a culture room at 22 °C with 16 h light / 8 h dark at 9:00 the next day, open the culture dish lid, the humidity of the culture room is 55%, and the light intensity is 100 μmol / (m 2 ·s). Compared with the constant temperature treatment, the seedlings obtained by the variable temperature treatment are stronger, there are fewer molds during the seed germination process, and the germination rate is higher.

[0186] In the step of "seedling planting and cultivation", different varieties of Leymus chinensis are planted in one seedling tray. Every 4 adjacent planting grooves plant the same variety of Leymus chinensis to form a small unit arranged in a "field" shape. The "field"-shaped small units are sequentially planted in the seedling tray, and each variety contains several "field"-shaped arranged small units in one seedling tray. The "field"-shaped arranged small units can reduce the drought resistance differences of different Leymus chinensis caused by the different positions of the plants in the seedling tray and reduce the contingency.

[0187] In the step of "seedling planting and cultivation", the seedling tray is a seedling tray with 128 planting grooves, 8 in a row, a total of 16 rows. The upper mouth width of each planting groove is 3 cm, the lower mouth width is 1.5 cm, and the depth is 4 cm; the new seedling tray is a seedling tray with 32 planting grooves, 4 in a row, a total of 8 rows; place the seedling tray with 128 planting grooves on the empty seedling tray with 32 planting grooves, and place a tray under the two seedling trays to make the culture soil fixing the plants suspended to ensure that the experimental results are not affected by waterlogging.

[0188] In the step of "relative water content detection", on the 0th, 9th, and 15th days of drought, harvest the parts above the leaf sheaths of the plants treated with different drought days respectively, and calculate the relative water content of the leaves. Among them, the 0th day is the initial state without treatment, and its sufficient water content reflects the health state of the Leymus chinensis used in the experiment, that is, the Leymus chinensis without drought treatment does not wilt due to other factors such as diseases; the 9th day is the time node when more than half of the Leymus chinensis show visible wilting to the naked eye. This node is used to distinguish the "drought resistance" of different Leymus chinensis, that is, the ability to maintain water in drought; the 15th day is the time node when all the Leymus chinensis wilt and the above-ground part shows an air-dried state. This node is used to judge the "drought tolerance" of the plant, that is, the ability to tolerate dehydration and drought.

[0189] In the step "Relative moisture content detection", the method for measuring the water saturated weight TW is: carefully stuff the sheep grass into a 5 ml pipette tip, try not to damage the sheep grass, seal the tail of the pipette tip with aluminum foil, and then soak it in water for 12 hours. After the plant absorbs enough water, put the pipette tip with the thin end facing down into a centrifuge and rotate it at 1000 rpm for 2 minutes to completely remove the moisture on the surface of the sheep grass leaves. Then weigh the sheep grass, which is the water saturated weight TW.

[0190] 2. Drought resistance score

[0191] DNA from eight different sources of Leymus chinensis was extracted using the EDTA method, and molecular marker-specific primers were used to amplify gene fragments of Lc3Ns024690, Lc3Xm072595, Lc6Ns028159, and Lc1Xm058728 of Leymus chinensis. The system is to add 100ng of DNA to the 15ul system, 1ul of 10uM / L upstream and downstream primers, anneal at 53℃ for 30s, extend at 72℃ for 55s, and 34 cycles. The amplification results of the bands were recorded, and the drought resistance of Leymus chinensis was scored. Molecular marker gel images of drought-resistant gene elements of some Leymus chinensis sources are shown below. Figure 4 As shown, wherein: M: Marker, 1: Detection of the presence of SNP: G in Lc1Xm058728, 2: Detection of the presence of SNP: C in Lc1Xm058728, 3: Detection of the presence of SNP: GGC in Lc6Ns028159, 4: Detection of the presence of SNP: ATG in Lc6Ns028159, 5: Detection of the presence of SNP: CA in Lc3Ns024690, 6: Detection of the presence of SNP: GC in Lc3Ns024690, 7: Detection of the presence of SNP: G in Lc3Xm072595, 8: Detection of the presence of SNP: T in Lc3Xm072595.

[0192] 3. Regression Detection

[0193] Compare the relationship between molecular marker scores and drought resistance of Leymus chinensis. After comparison, it was found that Leymus chinensis 16 and 3, the two seed sources with the highest molecular marker scores, had the highest survival rate after drought stress, and Leymus chinensis 4 and 24, the two seed sources with the lowest marker scores, had the lowest survival rate after drought stress (as shown in Table 7), which shows that the molecular marker can well predict the drought resistance of Leymus chinensis.

[0194] Table 7: Test results of drought resistance of eight different Leymus chinensis provenances

[0195]

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

Claims

1. SNP molecular markers related to drought resistance of Leymus chinensis, characterized in that: 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 a 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 a GC / CA polymorphism; The molecular marker SNP3 is located at the 277th base of the nucleotide sequence shown in SEQ ID NO: 3 and has a T / G polymorphism; The molecular marker SNP4 is located at the 394th, 395th and 396th bases of the nucleotide sequence shown in SEQ ID NO: 4, and has an ATG / GGC polymorphism.

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

3. A molecular marker combination related to drought resistance of Leymus chinensis, characterized in that: The molecular marker combination is composed of two or more combinations of SNP1, SNP2, SNP3 and SNP4.

4. The molecular marker combination according to claim 3, characterized in that: The genotype of the SNP1 marker is CC and GG, the genotype of the SNP2 marker is GGCC and CCAA, the genotype of the SNP3 marker is TT and GG, and the genotype of the SNP4 marker is AATTGG / GGGGCC; When the molecular marker combination is C, GC, T, and ATG homozygous genotypes according to SNP1 to SNP4, that is, CC-GGCC-TT-AATTGG genotype combination, the corresponding Leymus chinensis has the strongest drought resistance; The haplotype molecular markers are GG, CCAA, GG, GGGGCC homozygous genotypes according to SNP1 to SNP4, that is, when the genotype combination is GG-CCAA-GG-GGGGCC, the corresponding Leymus chinensis has the weakest drought resistance.

5. A primer combination for amplifying the molecular marker combination according to claim 3 or 4, characterized in that: The primer combination consists of primers for amplifying SNP1 to SNP4 respectively, wherein: The primers for amplifying the molecular marker SNP1 include the nucleotide sequences shown in SEQ ID NOs: 5-6; The primers for amplifying the molecular marker SNP2 include the nucleotide sequences shown in SEQ ID NOs: 8-9; The primers for amplifying the molecular marker SNP3 include the nucleotide sequences shown in SEQ ID NOs: 11-12; The primers for amplifying the molecular marker SNP4 include the nucleotide sequences shown in SEQ ID NOs: 14-15; Preferably, the primer combination is: The nucleotide sequences of primers for amplifying the molecular marker SNP1 are shown in SEQ ID NOs: 5-7; The nucleotide sequences of the primers for amplifying the molecular marker SNP2 are shown in SEQ ID NOs: 8-10; The nucleotide sequences of primers for amplifying the molecular marker SNP3 are shown in SEQ ID NOs: 11-13; The nucleotide sequences of primers for amplifying the molecular marker SNP4 are shown in SEQ ID NOs: 14-16.

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

7. A method for obtaining the SNP molecular marker according to claim 1 or 2, or the molecular marker combination according to claim 3 or 4, characterized in that: The method comprises the following steps: Genes upregulated by drought stress were identified based on RNA-Seq; Hi-tom was used to search for SNP sites in the promoter region of genes that were upregulated by drought stress in the total population of Leymus chinensis. Predict the loss of function of components caused by SNP mutations; Hi-tom was used to analyze the provenance of drought-resistant Leymus chinensis and compare the proportion of different SNPs in the drought-resistant population and the total population; Different SNP types were weighted and scored, the association between SNP types and drought resistance traits was analyzed, and SNP molecular markers related to drought resistance traits of Leymus chinensis were obtained.

8. Use of the SNP molecular marker according to claim 1 or 2, the molecular marker combination according to claim 3 or 4, the primer combination according to claim 5, the detection reagent or kit according to claim 6, or the SNP marker or molecular marker combination obtained by the method according to claim 7 in any of the following aspects (1) to (6): (1) Identify or assist in identifying the drought resistance of Leymus chinensis; (2) Screening or assisting in the screening of new varieties of Leymus chinensis with strong drought resistance; (3) Molecular marker-assisted breeding of Leymus chinensis; (4) preparing products for identifying or assisting in identifying the drought resistance of Leymus chinensis; (5) preparing products for screening or assisting in screening new varieties of Leymus chinensis with strong drought resistance; (6) Prepare products for molecular marker-assisted breeding of Leymus chinensis.

9. A method for identifying or screening the drought resistance of Leymus chinensis, characterized in that: The method comprises the steps of detecting the polymorphism or genotype of the SNP molecular marker of Leymus chinensis as claimed in claim 1 or 2, or detecting the genotype of the molecular marker combination as claimed in claim 3 or 4.

10. A method for genetic improvement of Leymus chinensis, characterized in that: The method comprises the steps of successive breeding of homozygous individuals of CC genotype of SNP1 molecular marker, and eliminating individuals of all other genotypes at the site; or successive breeding of homozygous individuals of GGCC genotype of SNP2 molecular marker, and eliminating individuals of all other genotypes at the site; or successive breeding of homozygous individuals of TT genotype of SNP3 molecular marker, and eliminating individuals of all other genotypes at the site; or successive breeding of homozygous individuals of AATTGG genotype of SNP4 molecular marker, and eliminating individuals of all other genotypes at the site; preferably, the method comprises the step of successive breeding of individuals of CC-GGCC-TT-AATTGG genotype combination of the above molecular marker combination, and eliminating individuals of all other genotype combinations of the marker combination.

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