A snp molecular marker combination of alfalfa, a chip and application thereof

Abstract

The application discloses a Medicago polymorphic SNP molecular marker combination, a chip and application thereof. The Medicago polymorphic SNP molecular marker combination comprises at least one of 100,637 SNP molecular markers, and physical positions of the 100,637 SNP molecular markers are determined based on sequence alignment of Medicago reference genome No. 4. The Medicago polymorphic SNP molecular marker combination is obtained by integrating whole genome resequencing data and key trait associated site screening, and has the advantages of high polymorphism, high throughput, high accuracy, low cost, multifunction and wide application scene. The Medicago polymorphic SNP molecular marker combination can be widely applied to whole genome association analysis, germplasm resource identification, genetic diversity evaluation, genetic map construction and QTL positioning, molecular marker assisted selection and whole genome selection breeding, important trait gene identification screening and mining and utilization.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an alfalfa SNP molecular marker combination, chip, and its application. Background Technology

[0002] Alfalfa (Mediacago sativa L.) is one of the world's most important legume forage crops, and its genetic improvement is of great significance for the sustainable development of grassland animal husbandry and ecological restoration. However, due to the high heterozygosity and complexity of its autotetraploid (2n=4x=32) genome, traditional breeding methods are inefficient and difficult to achieve synergistic improvement of multiple traits. Currently, alfalfa genotyping mainly relies on low-throughput SSR (simple sequence repeat) or SNP (single nucleotide polymorphism) markers, which have limited coverage and are costly, failing to meet the needs of genomic selection (GS) and molecular design breeding.

[0003] In recent years, genotyping by pinpoint sequencing of liquid-captured target (cGPS) has shown great potential in crop and livestock genetic breeding due to its advantages of high density, high flexibility, and low cost. However, existing technologies are mostly aimed at diploid plants and animals, lacking products suitable for the complex genome of alfalfa. Furthermore, existing SNP marker sets are mostly developed based on limited germplasm resources, lacking representativeness and failing to cover the broad genetic diversity of alfalfa, thus limiting the accuracy of genotype detection. Therefore, there is an urgent need to develop a high-density liquid-phase breeding chip for highly polymorphic alfalfa, which integrates whole-genome resequencing data and key trait-associated loci to screen for highly polymorphic and universal SNP markers. This chip will enable high-throughput, low-cost genotyping of alfalfa germplasm resources, significantly improving the efficiency of genome-wide selection breeding, promoting the transformation of alfalfa breeding from traditional empirical models to precise molecular design models, and providing core technical support for the breeding of high-yield, high-quality, and stress-resistant new varieties. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes a combination of SNP molecular markers for alfalfa.

[0005] The present invention also proposes a primer set and / or probe for detecting the above-mentioned alfalfa SNP molecular marker combination.

[0006] The present invention also proposes a chip.

[0007] The present invention also proposes a reagent kit.

[0008] This invention also proposes a method for screening the above-mentioned combinations of SNP molecular markers in alfalfa.

[0009] This invention also proposes an application of the above-mentioned alfalfa SNP molecular marker combination, primer set and / or probe, chip or kit.

[0010] This invention also proposes a breeding method for alfalfa.

[0011] According to one aspect of the present invention, an asparagus SNP molecular marker combinatorial is proposed, comprising at least one of 100,637 SNP molecular markers, wherein the physical locations of the 100,637 SNP molecular markers are determined based on sequence alignment of alfalfa reference genome α4, and the specific site information is shown in Table 1 below.

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[0144] In some embodiments of the present invention, the molecular marker position information is distinguished by a semicolon (;).

[0145] The reference genome of this invention is selected from the haploid reference genome ZM-4, which converts the complex homotetraploid into a diploid reference genome, thereby achieving genome diploidization. This has the following beneficial effects:

[0146] (1) Simplified genome analysis and genotyping: a) Reduced complexity of genotype interpretation: In tetraploid genomes, each SNP locus may have 4 allele copies (e.g., AAAA, AAAB, AABB, etc.), making genotype interpretation and statistical models more complex. After diploidization, only 2 alleles (e.g., AA, AB, BB) need to be considered for each locus, greatly simplifying genotyping and association analysis. b) Improved efficiency of molecular marker development: Applicable to existing diploid bioinformatics tools (e.g., GATK, PLINK), eliminating the need to develop additional tetraploid-specific algorithm tools.

[0147] (2) Improve the accuracy of genetic mapping and QTL analysis: a) Reduce interference from allele dosage effects. In tetraploids, gene dosage effects may lead to nonlinear relationships between traits and genotypes, increasing noise in QTL (quantitative trait loci) analysis. Diploid models can more clearly resolve dominant / recessive genetic effects and improve the detection capability of trait-marker associations. b) Optimize genome-wide selection (GS) models: Most GS algorithms (such as RR-BLUP and GBLUP) are based on the diploid assumption, and directly using tetraploid data may lead to prediction bias.

[0148] (3) Reduce computing resource consumption: a) Reduce data storage and computing pressure. Tetraploid genomes have a large amount of data due to the diversity of allele combinations (e.g., the size of VCF files may double). Diploidization can significantly reduce storage and computing costs. b) Accelerate bioinformatics workflow: There is no need to adapt to tetraploid-specific software (such as TetraploidSNPMap), and mainstream diploid analysis workflows (such as BWA-GATK) can be used directly.

[0149] In some embodiments of the present invention, the genotype information of the molecular marker is represented in the form of "ref / alt (reference base / variant base)".

[0150] In a second aspect of the invention, a primer set and / or probe for detecting the above-mentioned alfalfa SNP molecular marker combination is provided.

[0151] In some embodiments of the present invention, the probe has a length of 80-120 bp.

[0152] In some embodiments of the present invention, the probe is approximately 100 bp in length.

[0153] In some embodiments of the present invention, the GC content of the probe is between 20% and 80%.

[0154] In some embodiments of the present invention, the number of SNP variant sites on the probe is ≤5.

[0155] In some embodiments of the present invention, the probe has no SSR region and N region.

[0156] In a third aspect of the invention, a chip is provided that includes the aforementioned primer set and / or probes.

[0157] In some embodiments of the present invention, the chip includes a liquid phase chip.

[0158] In a fourth aspect of the invention, a kit is provided comprising the above-described primer set and / or probe.

[0159] In a fifth aspect of the present invention, a method for screening the above-mentioned combinations of alfalfa SNP molecular markers is proposed, the method comprising the following steps:

[0160] A1) After comparing and detecting variants in the whole genome sequencing data of alfalfa samples using Sentieon software, preliminary hard filtering was performed to obtain a file containing SNP variant information of all samples.

[0161] A2) The target SNPs are obtained by mining and screening the file containing SNP variation information of all samples; the mining and screening parameters are: MAF≥0.05, detection rate≥90%;

[0162] A3) Probe design is performed on the target site SNPs obtained in step (2) and the SNPs related to the traits and functions related to yield, quality and stress resistance. SNPs that meet the requirements of the probes are screened to obtain the molecular marker combination of alfalfa SNPs.

[0163] In some embodiments of the present invention, the comparison and variation detection of whole-genome sequencing data of alfalfa samples using Sentieon software specifically includes the following steps:

[0164] B1) Using Sentieon software, the whole genome sequencing data of the alfalfa sample were aligned to alfalfa reference genome 4, and positional sorting and repeat read marking were performed;

[0165] B2) Use Sentieon software to detect variant sites for each sample and obtain the gVCF for each sample;

[0166] B3) Use Sentieon to perform joint-calling and perform joint analysis of gVCF for all samples to obtain the variation results for each individual in the population.

[0167] In some embodiments of the present invention, the hard filtering criteria are as follows: QD < 2.0 || FS > 60.0 || MQ < 40.0 || SOR > 3.0 || MQRankSum < -12.5 || ReadPosRankSum < -8.0.

[0168] In some embodiments of the present invention, the probe has a length of 80-120 bp.

[0169] In some embodiments of the present invention, the probe is approximately 100 bp in length.

[0170] In some embodiments of the present invention, the GC content of the probe is between 20% and 80%.

[0171] In some embodiments of the present invention, the number of SNP variant sites on the probe is ≤5.

[0172] In some embodiments of the present invention, the probe has no SSR region and N region.

[0173] In a sixth aspect of the invention, the application of the above-described alfalfa SNP molecular marker assemblies, primer sets and / or probes, chips and kits in any of the following is proposed:

[0174] C1) Identification of alfalfa germplasm resources;

[0175] C2) Assessment of genetic diversity in alfalfa;

[0176] C3) Construction of genetic map and QTL mapping of alfalfa;

[0177] C4) Genome-wide association analysis and genome-wide prediction of alfalfa;

[0178] C5) Marker-assisted selection breeding of alfalfa;

[0179] C6) Targeted improvement of alfalfa;

[0180] C7) Alfalfa multi-gene aggregation breeding, whole-genome selection breeding, or intelligent design breeding;

[0181] C8) Alfalfa genotyping detection;

[0182] C9) Cluster analysis and phylogenetic identification of alfalfa;

[0183] C10) Discovery and identification of important trait genes in alfalfa;

[0184] C11) Analysis of alfalfa population structure.

[0185] In some embodiments of the present invention, the application can be implemented through the following methods:

[0186] S1. Genotyping of the sample to be tested is performed using at least one of the following: alfalfa SNP molecular marker combination, primer set and / or probe, chip and kit, to obtain genotyping results;

[0187] S2. Analyze the genotyping results obtained in step S1.

[0188] In a seventh aspect of the present invention, a method for breeding alfalfa is proposed, comprising the following steps: detecting the DNA of alfalfa to be tested using at least one of the above-mentioned alfalfa SNP molecular marker combination, primer set and / or probe, chip and kit, and selecting alfalfa for subsequent breeding.

[0189] In some embodiments of the present invention, the detection is performed based on liquid-phase probe capture sequencing genotyping technology.

[0190] The present invention has at least the following beneficial effects:

[0191] The alfalfa SNP molecular marker combinatorial scheme of this invention is obtained by integrating whole-genome resequencing data and screening of key trait-related loci. It has the advantages of high polymorphism, high throughput, high accuracy, low cost, multifunctionality, and wide application scenarios. At the same time, the alfalfa SNP molecular marker combinatorial scheme also covers 2,358 functional loci related to yield, quality, and stress resistance. It can be widely used in alfalfa whole-genome association analysis, germplasm resource identification, genetic diversity assessment, genetic map construction and QTL mapping, molecular marker-assisted selection and whole-genome selection breeding, identification, screening, and utilization of important trait genes.

[0192] The alfalfa whole-genome SNP liquid-phase breeding chip prepared by combining SNP molecular markers according to the present invention has the advantages of high throughput, high accuracy, low cost, multifunctionality, and wide application scenarios. It can be widely used in alfalfa whole-genome association analysis, germplasm resource identification, genetic diversity assessment, genetic map construction and QTL mapping, molecular marker-assisted selection, and whole-genome selection breeding. It effectively reduces the cost of alfalfa genetic diversity analysis, QTL mapping, GWAS analysis and other applications in scientific research. It can realize high-throughput and low-cost genotyping of alfalfa germplasm resources, significantly improve the efficiency of whole-genome selection breeding, promote the transformation of alfalfa breeding from traditional experience model to precise molecular design model, provide core technical support for the breeding of high-yield, high-quality and stress-resistant new varieties, and accelerate the process of basic research and breeding of alfalfa. Attached Figure Description

[0193] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0194] Figure 1 This is a chromosome density distribution map of alfalfa 100K cGPS liquid-phase chip sites in an embodiment of the present invention;

[0195] Figure 2 This is a schematic diagram of the cGPS liquid phase chip process detection in an embodiment of the present invention;

[0196] Figure 3 This is a graph showing the results of genome-wide association analysis in an embodiment of the present invention;

[0197] Figure 4 This is a diagram showing the detection results of the alfalfa 100K cGPS liquid phase chip in this embodiment of the invention, which distinguishes alfalfa varieties from different countries.

[0198] Figure 5 The image shows the detection results of 97 domestic alfalfa varieties distinguished by the 100K cGPS liquid phase chip in this embodiment of the invention.

[0199] Figure 6 This is a diagram of the whole-genome prediction detection results in an embodiment of the present invention;

[0200] Figure 7 This is a diagram showing the whole-genome prediction detection results in an embodiment of the present invention. Detailed Implementation

[0201] The following will describe the concept and technical effects of the present invention clearly and completely with reference to the embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0202] Example 1

[0203] This embodiment provides an alfalfa SNP molecular marker combinatorial. The alfalfa SNP molecular marker combinatorial includes 100,637 SNP loci. The physical locations of these 100,637 SNP loci were determined by sequence alignment based on the ZM-4 reference genome (Pan-genomic analysis highlights genes associated with agronomic traits and enhances genomics-assisted breeding in alfalfa, https: / / www.nature.com / articles / s41588-025-02164-8). Specific information on the 100,637 SNP loci is shown in Table 1 of the specification. The screening process is as follows:

[0204] 1. Collection of alfalfa germplasm resources

[0205] To obtain genetically rich whole-genome loci, 176 alfalfa varieties from different countries and regions, including China, Turkey, and Europe, were collected.

[0206] 2. Alfalfa whole genome resequencing

[0207] Whole-genome resequencing was performed on 176 alfalfa samples collected. The specific steps included: (1) DNA extraction using magnetic beads; (2) using the MGI library standard method to construct DNA-seq sequencing libraries that passed quality control; and (3) sequencing of the libraries using the BGI sequencing platform (MGI) with the PE150 sequencing strategy after passing quality control. The resequencing data of the 176 alfalfa samples were compared and variants were detected using Sentieon. The analysis process is as follows:

[0208] 1) Sentieon was used to align reads to their corresponding alfalfa reference genome (database, literature, or link), orient them by position, and mark duplicate reads. 2) Sentieon was used to detect variant sites in each sample, obtaining the gVCF for each sample. 3) Sentieon was used for joint-calling, performing joint analysis of the gVCF of all samples to obtain the variant results for each individual in the population. To ensure SNP accuracy, preliminary hard filtering was applied to the SNP sites obtained after joint analysis (SNP hard filtering criteria: "QD < 2.0 || FS > 60.0 || MQ < 40.0 || SOR > 3.0 || MQRankSum < -12.5 || ReadPosRankSum < -8.0").

[0209] After filtering, a .vcf file containing SNP variation information for all samples was obtained, totaling 2,043,025 SNP sites.

[0210] 3. Site screening

[0211] The MAF value, detection rate, and heterozygosity rate of the loci were calculated and statistically analyzed from the .vcf file containing SNP variation information of all samples. The target locus set was screened according to MAF≥0.05 and SNP locus detection rate≥90%. Probes were designed for the screened target locus set. The probe design principles were: a. Design probes within 100 bp to the left and right of all target loci, with a probe length of generally 100 bp; b. Calculate the GC content of the target region; c. Calculate the number of homologous regions of the target probe. The probe screening criteria were as follows: a. GC content of the probe was between 20% and 80%; b. Number of homologous regions < 5; c. Number of SNP variant sites on the probe ≤ 5; d. No SSR or N regions on the probe; e. After screening the probe sequences that finally met the requirements, based on the principle of uniform distribution on the reference genome, 90K SNP sites were selected as whole-genome sites. Simultaneously, based on previous research, 2,358 functional loci related to yield, quality, and stress resistance were added. Based on the principle of uniform distribution on chromosomes, gaps were filled, finally constructing an alfalfa 100K liquid-phase microarray with 100,637 sites and an average spacing of 8.1 Kb (liquid-phase microarray site chromosome density distribution diagram is shown below). Figure 1 As shown in the figure), the site information is shown in Table 1 of the instruction manual.

[0212] Example 2

[0213] This embodiment provides an alfalfa 100K liquid phase chip.

[0214] The 100,637 SNP loci screened in Example 1 were used to develop an alfalfa 100K SNP liquid-phase breeding chip based on the probe design principles in Example 1 and Huazhi's independently developed liquid-phase probe precise localization sequencing and genotyping technology (cGPS). cGPS is based on an optimized thermodynamic stability algorithm model, which designs specific probes for target region sequences. Then, the synthesized specific probes are used to capture and enrich multiple different target sequences located at different genomic positions using liquid-phase hybridization. Finally, sequencing libraries are constructed and high-throughput sequencing is performed on the captured and enriched target genomic sequences to obtain the genotypes of all SNP / InDel loci within the target region. A schematic diagram of the detection process is shown below. Figure 2 As shown.

[0215] Example 3

[0216] This embodiment provides a method for genotyping alfalfa samples using the alfalfa 100K liquid phase chip from Embodiment 2. The steps are as follows:

[0217] 1. Extraction and detection of alfalfa gDNA

[0218] Fresh alfalfa leaves were collected, and gDNA was extracted from the tissue using the magnetic bead method. The concentration was accurately quantified using Qubit, and the integrity of the gDNA was analyzed by 1% agarose gel electrophoresis. Samples that passed quality control proceeded to the next step of the experiment.

[0219] 2. cGPS Experiment Procedure

[0220] (1) Take 200ng of qualified gDNA, digest it into 100-500 bp fragments using enzyme digestion reagent, and then add Taq enzyme for end repair.

[0221] (2) Use T4 ligase to ligate the adapter fragments to both ends of the DNA, and use fragment sorting magnetic beads to purify the ligation product and remove small fragments;

[0222] (3) Use primers with tagged sequences to perform PCR amplification on the purified ligation product. The amplification product is purified using magnetic beads to remove impurity fragments from the PCR process and complete the library construction.

[0223] (4) The library concentration was detected using Qubit, and the library quality was detected using Agilent 2100 / 2200 Bioanalyzer. The library fragment size was between 250-500 bp.

[0224] (5) Mix 10 qualified libraries in equal amounts to form a hybridization library. Use a vacuum concentrator to concentrate the mixed library. Then add the genome blocking reagent, RNase inhibitor, and probe to the concentrated library. After mixing, place the sample on a PCR instrument for hybridization reaction and incubate overnight at 55°C (16-24 h).

[0225] (6) Use streptavidin magnetic beads to capture the hybridization product. Add the capture magnetic beads to the hybridization product to adsorb the probe and target region binding. Bind at room temperature for 30-60 min. Then wash the magnetic beads with washing solution to remove non-specific binding fragments. Finally, perform PCR amplification on the captured target region. Purify the amplification product with fragment sorting magnetic beads to remove excess non-target products.

[0226] (7) Library quality control, same as step (4). After the library quality control is qualified, PE150 sequencing is performed using the BGI sequencing platform.

[0227] (8) The raw data after high-throughput sequencing underwent quality control filtering and other processing. FastP software was used to remove reads containing adapter contamination and low-quality reads. BWA software was used to align the data with the target genome, and GATK software was then used to analyze the mutation sites in the sequencing results to obtain the genotyping results of the target loci. A flowchart is shown below. Figure 2 As shown.

[0228] Example 4: Evaluation of the genotyping effect of alfalfa 100K liquid phase chip

[0229] To verify the genotyping effect of the 100K liquid phase chip, the genotyping detection of 24 different alfalfa materials (including 4 duplicate samples) was performed using the alfalfa 100K liquid phase chip obtained in Example 2 (see Example 3 for specific operation method).

[0230] Table 2 Detection rate of alfalfa samples

[0231]

[0232] Table 3. Average Genotypic Consistency Rate of Technical Replicated Samples

[0233]

[0234] The results are shown in Tables 2 and 3. Table 2 shows that, after sequencing and data analysis, the detection rate of loci in the 24 samples was between 97.95% and 99.54%, with an average detection rate of 99.07%. Table 3 shows that the genotypic consistency rate of the four technical replicates was between 97.33% and 97.95%, with an average consistency rate of 97.61%. The genotyping effect evaluation results of alfalfa 100K liquid phase chip indicate that the alfalfa 100K liquid phase chip has a high genotyping detection rate, a high average genotyping consistency rate, and accurate and reliable genotyping results.

[0235] Example 5: Population Structure Analysis and Kinship Identification

[0236] Genotyping of alfalfa varieties from different countries was performed using the 100K liquid phase chip prepared in Example 1, including 31 Chinese varieties, 93 European varieties, and 52 Turkish varieties. Genetic distance matrices were calculated using the IBS method in Plink software, and cluster analysis and phylogenetic tree construction were conducted. The results are as follows: Figure 3 As shown in the figure, the alfalfa 100K liquid phase chip prepared by the present invention can be effectively used to distinguish alfalfa varieties from different countries, and the classification effect is consistent with the actual classification results.

[0237] In addition, cluster analysis and phylogenetic tree construction were performed on 97 different alfalfa samples collected domestically (a population resulting from a cross between the domestic variety Zhongmu 1 and the foreign variety Felicia) using the alfalfa 100K liquid phase chip prepared in Example 1. The results are as follows: Figure 4 As shown in the figure, group 3 is the source of parent 1 of *Clerodendrum cyrtonema*, group 2 is the source of parent 1 of *Felicia*, and group 1 is an intermediate type between the two. The classification effect is consistent with the actual classification results.

[0238] The results show that the alfalfa 100K liquid phase chip prepared in Example 1 of the present invention can be effectively used for alfalfa population structure analysis and kinship identification.

[0239] Example 6 Genome-wide association analysis

[0240] Using 176 alfalfa germplasm resources as an associated population, plant height data were recorded. These resources were planted at the Langfang experimental base. At the alfalfa budding stage, the distance from the ground to the highest point of the plant was measured in centimeters. Five plants were measured per replicate for each material, with three replicates. Genotyping of these 176 materials was performed using a 100K liquid chromatography-mass spectrometry chip prepared in Example 1. Genome-wide association analysis was conducted using a mixed linear model (MLM). Results are as follows: Figure 5 As shown in the figure, 10 SNP loci significantly associated with plant height were located in the alfalfa genome, and these loci are distributed on different chromosomes.

[0241] Example 7: Whole Genome Prediction

[0242] Genotyping of 176 alfalfa germplasm resources was performed using the 100K liquid phase chip prepared in Example 1. The rrBLUP model was used for genome-wide prediction of alfalfa plant height, with a training population of 60%. First, the prediction accuracy was analyzed with different numbers of markers. It was found that the prediction accuracy continuously improved with increasing marker number, reaching a maximum of approximately 0.38 when the number of markers was 4K. However, the prediction accuracy no longer increased with further increases in the number of markers. Figure 6 ).

[0243] Subsequently, GWAS analysis was used to predict the SNPs significantly associated with plant height. These SNPs were then ranked from smallest to largest significance (P-value). It was found that TagSNPs had significantly higher predictive accuracy than randomly selected SNPs. Furthermore, using 2K TagSNPs could improve the predictive accuracy to approximately 0.7. Figure 7 ).

[0244] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. The application of alfalfa SNP molecular marker combinatorial analysis in alfalfa genome-wide association analysis, genome-wide prediction, population structure analysis, and phylogenetic identification, characterized in that, The alfalfa SNP molecular marker assemblage consists of 100,637 SNP molecular markers. The physical locations of these 100,637 SNP molecular markers were determined based on sequence alignment of alfalfa reference genome α4. The specific site information is shown in Table 1 of the specification.

2. The use of the alfalfa SNP molecular marker combination as described in claim 1 in any of the following: 1) Identification of alfalfa germplasm resources; 2) Assessment of genetic diversity in alfalfa; 3) Alfalfa genotyping detection; 4) Cluster analysis of alfalfa.

3. Primer sets and / or probes for detecting the alfalfa SNP molecular marker combination as described in claim 1.

4. The primer set and / or probe according to claim 3, characterized in that, The probe has a length of 80-120 bp.

5. The primer set and / or probe according to claim 3, characterized in that, The GC content of the probe is between 20% and 80%.

6. The primer set and / or probe according to claim 3, characterized in that, The probe has ≤5 SNP mutation sites.

7. A chip, characterized in that, The chip comprises a primer set and / or probe as described in any one of claims 3-6; the chip is a liquid-phase chip.

8. A reagent kit, characterized in that, The kit contains the primer set and / or probe as described in any one of claims 3-6.

9. The use of any one of the primer set and / or probe according to any one of claims 3-6, the chip according to claim 7, and the kit according to claim 8 in any of the following: C1) Identification of alfalfa germplasm resources; C2) Assessment of genetic diversity in alfalfa; C3) Genome-wide association analysis and genome-wide prediction of alfalfa; C4) Alfalfa genotyping; C5) Cluster analysis and phylogenetic identification of alfalfa; C6) Analysis of alfalfa population structure.

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