An SNP molecular marker closely linked to the above-ground dry weight trait of alfalfa and its application

Abstract

The present invention discloses an SNP molecular marker closely linked to the above-ground dry weight trait of alfalfa and its application. The SNP molecular marker is located at the base position 6021492 on chromosome 4 of the alfalfa reference genome ZM-4 alfalfa genome, and the polymorphism is C / T. The present invention provides an SNP molecular marker closely linked to the dry weight trait of alfalfa, which is a co-dominant marker and has the characteristics of high specificity, high sensitivity, high resolution and high genotyping quality; the marker is not affected by environmental conditions and can be detected using seeds or different types of plant tissues, with accurate results, good repeatability and stability; different detection laboratories and different data results can be compared and verified with each other, and the detection of mutants of the alfalfa dry weight trait can be achieved quickly, high-throughput and at low cost according to the genotype, and the above-ground dry weight of alfalfa can be determined, with wide application universality.

Description

Technical Field

[0001] The present invention belongs to the field of agricultural molecular biology, and particularly relates to an SNP molecular marker closely linked to the above-ground dry weight trait of alfalfa and its application. Background Art

[0002] Alfalfa (Medicago sativa L.) is one of the most important perennial leguminous forages in the world. It has a large biomass, high nutritional value, and characteristics such as drought resistance and salt tolerance. It is known as the "king of forages" and occupies an important position in the forage industry. It plays an irreplaceable role in the development of high-quality animal husbandry and ecological governance and restoration. At present, the variety breeding of alfalfa mainly relies on traditional breeding methods such as hybridization and selection, resulting in low efficiency of variety development, poor stability, and few excellent alfalfa varieties with outstanding traits, seriously restricting the development of the alfalfa industry. Therefore, it is necessary to deeply analyze the molecular genetic mechanism of important traits of alfalfa, develop molecular genetic markers with the characteristics of high efficiency, accuracy, and low cost for alfalfa genotype identification and related trait prediction, break through the bottleneck problems in alfalfa germplasm resource evaluation and variety cultivation, improve breeding efficiency, and help alfalfa enter molecular design breeding from conventional breeding.

[0003] At present, there is little work on the precise evaluation and identification of alfalfa germplasm resources. The identification efficiency of germplasm resources is low and the accuracy is not high. The excavation and identification of its important traits, especially yield, nutritional quality, and stress resistance traits, are not deep enough, and excellent alfalfa germplasm materials cannot be effectively obtained. The identification and evaluation of alfalfa mainly rely on the investigation after the phenotypic traits of breeding materials are shown during the growth process. This type of evaluation method has the following problems: it usually depends on the visual recognition of morphological characteristics and biological characteristics, the judgment criteria are difficult to accurately quantify, and the subjectivity is strong; it is easily affected by the environment and cultivation conditions, and the accuracy and stability are poor; it takes a long time and the timeliness is poor; it requires a large amount of manpower and material resources, and the cost is high.

[0004] DNA molecular marker method is a commonly used technical method in current crop variety breeding. DNA molecular marker is a genetic marker that directly reflects DNA differences (polymorphisms). Currently, it mainly includes SSR (Simple Sequence Repeat) and SNP (Single Nucleotide Polymorphism), etc. SSR markers have the characteristics of simple operation, low cost, good repeatability, and true and reliable results. Compared with SSR marker method, SNP marker technology is more simple, easy to automate, has a high detection throughput, fast speed; the detection cost per data point is low; the data results of different detection laboratories can be compared and verified with each other, and the data has universal comparability; it is the most commonly used method for quickly, simply, sensitively, accurately, stably, and low-costly identifying functional genes. There is less research work on alfalfa in terms of functional gene markers. Currently, there is still a lack of a high-throughput genotype identification and analysis technology system and molecular markers with important breeding value. The selection method based on SNP marker selection for the above-ground dry weight trait of alfalfa is rarely reported. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. For this reason, the present invention provides a SNP molecular marker closely linked to the above-ground dry weight trait of alfalfa.

[0006] The present invention also provides a primer set for detecting the above SNP molecular marker.

[0007] The present invention also provides a kit.

[0008] The present invention also provides a gene chip.

[0009] The present invention also provides the application of the above SNP molecular marker, primer set, kit and / or gene chip.

[0010] The present invention also provides a method for identifying or assisting in identifying the above-ground dry weight of alfalfa.

[0011] The present invention also provides a breeding method for alfalfa.

[0012] According to the first aspect of the present invention, a SNP molecular marker closely linked to the above-ground dry weight trait of alfalfa is provided. The SNP molecular marker is located at the base position 60214922 of chromosome 4 of the alfalfa reference genome ZM-4alfalfa genome, and the polymorphism is C / T.

[0013] According to the second aspect of the present invention, a primer set for amplifying the above SNP molecular marker is provided.

[0014] In some embodiments of the present invention, the primer set includes a first specific primer as shown in SEQ ID NO.4 (Table 2) and a second specific primer sequence as shown in SEQ ID NO.5 (Table 2).

[0015] In some embodiments of the present invention, different fluorescent linker sequences are respectively linked to the first specific primer and the second specific primer.

[0016] In some embodiments of the present invention, the fluorescent linker sequences are selected from FAM and HEX.

[0017] According to some embodiments of the present invention, the primer set further includes a universal primer, and the sequence of the universal primer is as shown in SEQ ID NO.6 (Table 2).

[0018] According to the third aspect of the present invention, a kit is provided, and the kit includes the above-mentioned primer set.

[0019] According to the fourth aspect of the present invention, a gene chip is provided, and the gene chip includes the above-mentioned primer set.

[0020] According to the fifth aspect of the present invention, the above-mentioned SNP molecular marker, primer set, kit or gene chip is applied in any one of the following:

[0021] (1) Detecting the above-ground dry weight of alfalfa;

[0022] (2) Identifying and screening alfalfa germplasms with different above-ground dry weights;

[0023] (3) Breeding alfalfa with a single-plant above-ground dry weight ≥ 150 g;

[0024] (4) Molecular marker-assisted breeding of alfalfa;

[0025] (5) Breeding of alfalfa;

[0026] (6) Preparing products for alfalfa breeding.

[0027] In some embodiments of the present invention, the alfalfa germplasms with different above-ground dry weights include alfalfa germplasms with a single-plant dry weight ≥ 150 g and / or alfalfa germplasms with a single-plant dry weight < 150 g.

[0028] According to the sixth aspect of the present invention, a method for identifying or assisting in identifying the above-ground dry weight of alfalfa is provided, and the method includes the following steps:

[0029] S1. Extracting genomic DNA from alfalfa materials;

[0030] S2. Perform polymorphism detection of the SNP molecular markers on the genomic DNA extracted in step S1, and determine the above-ground dry weight of the alfalfa material according to the genotype.

[0031] In some embodiments of the present invention, when the genotype detected by the SNP molecular marker is CC, the dry weight per plant of the alfalfa material ≥ 150 g; when the genotype detected by the SNP molecular marker is TT, the dry weight per plant of the alfalfa material < 150 g; when the genotype detected by the SNP molecular marker is TC, the dry weight per plant of the alfalfa material ≥ 150 g.

[0032] In some embodiments of the present invention, in step S1, the genomic DNA is extracted from the alfalfa material using the simplified CTAB method (cetyltrimethylammonium bromide method).

[0033] In some embodiments of the present invention, in step S2, the SNP molecular markers are detected using the KASP (Kompetitive Allele Specific PCR) technology.

[0034] In some embodiments of the present invention, the composition of the KASP reaction mixture for detecting the SNP molecular markers using the KASP technology is as follows:

[0035]

[0036]

[0037] In some embodiments of the present invention, the amplification program for detecting the SNP molecular markers using the KASP technology is: 94 °C for 15 min; 94 °C for 20 s, 65 °C - 57 °C for 60 s, 10 cycles; 94 °C for 20 s, 57 °C for 60 s, 33 cycles.

[0038] According to the seventh aspect of the present invention, an alfalfa breeding method is provided, including the following steps: using the above method, select alfalfa germplasms with an above-ground dry weight per plant ≥ 150 g for subsequent breeding.

[0039] According to some embodiments of the present invention, it has at least the following beneficial effects: The present invention provides an SNP molecular marker tightly linked to the above-ground dry weight trait of alfalfa. The marker is a co-dominant marker with a PIC value higher than 0.3 and a sample data detection rate higher than 98%. It has the characteristics of high specificity, high sensitivity, high resolution, and high genotyping quality. The marker is not affected by environmental conditions and can be detected using seeds or different types of plant tissues with accurate, repeatable, and stable results. Different detection laboratories and different data results can be compared and verified with each other. The data has universal comparability, and the dry weight mutants of alfalfa can be detected quickly, in a high-throughput manner, and at low cost according to the genotype, and the above-ground dry weight of individual plants can be determined, which is used for marker-assisted breeding in improving the high-yield plant type of alfalfa and has wide application universality.

[0040] The detection method of the present invention combines the KASP detection technology and can be used for the detection of above-ground dry weight mutants of alfalfa. The detection method is simple, fast, and has low detection cost, and is applicable to different detection instrument devices.

[0041] Additional aspects and advantages of the present invention will be given in part in the following description, become apparent in part from the following description, or be understood through the practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The following further describes the present invention in conjunction with the drawings and embodiments, where:

[0043] Figure 1 is the flow chart of molecular marker development in Example 1 of the present invention;

[0044] Figure 2 is the typical genotyping result diagram of the molecular marker Me900010 in Example 1 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0045] The following will clearly and completely describe the concept and technical effects generated by the present invention in conjunction with the embodiments 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 embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention. The test methods used in the embodiments are all conventional methods unless otherwise specified; the materials, reagents, etc. used are all commercially available unless otherwise specified.

[0046] Example of the present invention: A tightly linked SNP molecular marker related to the above-ground dry weight trait of alfalfa and its application

[0047] The design process of this molecular marker is as Figure 1As shown, a breeding germplasm resource database was constructed based on the resequencing data of 63 core germplasm resources of alfalfa. According to the analysis of the phenotypic survey data and whole-genome resequencing data of the existing resources, the tightly linked loci of the above-ground dry weight of alfalfa were located at multiple loci in the interval shown in Table 1 below. The reference genome version of alfalfa is ZM-4 alfalfa genome, and the source is the assembled genome. The obtained data was extracted based on sequence information, and SNP polymorphism analysis was performed on the gene sequences. Finally, multiple SNP loci were obtained, and the flanking sequences about 150bp before and after were obtained for marker design and synthesis.

[0048] Table 1

[0049] Phenotype Target_Type Chr Position DryWeight.GLM SNP chr1 51340716 DryWeight.GLM SNP chr4 60214922 DryWeight.GLM SNP chr5 43772478 DryWeight.GLM SNP chr3 23749587

[0050] The screening and verification steps of SNP molecular markers tightly linked to the dry weight trait of alfalfa are as follows:

[0051] 1 Primer design

[0052] For the molecular markers screened in Table 1 above, based on the alfalfa reference genome ZM-4 alfalfa genome, the online primer design website BatchPrimer3 (http: / / probes.pw.usda.gov / batchprimer3 / ) was used to design KASP marker primers for them. Each group of markers has three primers, and FAM and HEX fluorescent sequences are respectively connected to the 5' ends of two of the specific primers. After the design is completed, further analysis of the copy number of the primer sequences in the whole genome is carried out, and finally high-quality single-copy KASP markers are obtained. The locus design information is shown in Table 2 below. The primers were entrusted to Sangon Biotech (Shanghai) Co., Ltd. for synthesis.

[0053] Using the molecular markers designed above, high-throughput detection of the above-ground dry weight of alfalfa materials can be carried out. When the detected molecular marker is a genotype containing the corresponding base of Allele X, it indicates that the above-ground dry weight of the alfalfa material is ≥150g and has a high dry weight (biomass) phenotype; when the detected genotype is a genotype that does not contain the corresponding base of Allele X, it indicates that the above-ground dry weight of the alfalfa material is <150g and has a low dry weight (biomass) phenotype. When the detected genotype is a heterozygous genotype, it also indicates that the above-ground dry weight of the alfalfa material is ≥150g and has a high dry weight (biomass) phenotype.

[0054] Table 2

[0055]

[0056]

[0057] 2 Sample Detection

[0058] DNA Extraction: Genomic DNA was extracted from alfalfa using the simplified CTAB method.

[0059] KASP Reaction Test: The verification and detection of KASP markers were carried out using the Array Tape system of Douglas Scientific. The Array Tape genotyping platform includes NEXAR for PCR amplification system assembly, SOELLEX for PCR amplification, ARAYA for fluorescence signal scanning, and INTELLICS for data analysis. The amplification system is shown in Table 3.

[0060] Table 3 PCR Amplification System for KASP Marker Genotyping

[0061]

[0062] The PCR amplification reaction conditions were as follows: PCR amplification was carried out using SOELLEX, and the amplification conditions were as follows: 94°C for 15 min; 94°C for 20 s, 65°C - 57°C (the annealing temperature decreases by 0.8°C per cycle) for 60 s, 10 cycles; 94°C for 20 s, 57°C for 60 s, 33 cycles.

[0063] Signal Scanning and Genotyping: After the PCR reaction was completed, the fluorescence signal of the reaction system was scanned using ARAYA; then genotype typing and data analysis were carried out using INTELLICS.

[0064] 3 Marker Genotyping Data

[0065] According to the above detection method, 63 alfalfa materials were used to verify the KASP reaction for the markers Me900009, Me900010, Me900011, and Me900019 in Table 2 above.

[0066] A typical KASP marker genotyping map is as Figure 2 shown. It can be seen from the figure that in the KASP marker genotyping detection, the genotypes of the samples are divided into 3 clusters, namely the X cluster, the Y cluster, and the heterozygous genotype cluster. Among them, the X cluster indicates that the sample contains a homozygous X allele genotype at this KASP marker locus (marked as red in the genotyping map, located in the upper left corner of the graph), the Y cluster indicates that the sample contains a homozygous Y allele genotype at this KASP marker locus (marked as blue in the genotyping map, located in the lower right corner of the graph), and the heterozygous genotype cluster indicates that the sample contains X and Y heterozygous allele genotypes at this KASP marker locus (marked as purple in the genotyping map).

[0067] The quality verification results showed that for the KASP markers, only the two homozygous and heterozygous cluster genotyping of Me900010 was good and compact. The locus was single-copy, the detection rate was higher than 98%, and the correspondence with the phenotype was highly consistent. The genotype typing quality of the KASP markers could fully meet the accurate detection of the above-ground dry weight of alfalfa. Therefore, Me900010 was selected for subsequent experimental verification.

[0068] 4 Specificity and practicality detection

[0069] To detect the specificity and practicality of the marker Me900010 in the present invention, alfalfa strains from different regions were collected for field planting. According to the above detection method, genotype and actual phenotype detection and verification were carried out. To ensure the accuracy of the above-ground dry weight phenotype measurement, every 10 plants of each alfalfa strain were selected as a measurement sample, and the average value of 3 repetitions was used as the phenotype measurement value. Classification was carried out according to the above-ground dry weight quality (g). Germplasm materials with a single-plant above-ground dry weight ≥ 150 g were high-dry weight germplasm materials; those with a single-plant above-ground dry weight < 150 g were low-dry weight germplasm materials, and the phenotype was determined accordingly.

[0070] Table 4

[0071]

[0072]

[0073] The results are shown in Table 4. It can be seen from the table that the genotype and phenotype identification results are basically consistent. The marker Me900010 of the present invention has high specificity in detecting the above-ground dry weight (biomass) of alfalfa and can quickly and accurately identify the above-ground dry weight level of the tested alfalfa germplasm materials.

[0074] The reagents and consumables supporting the Douglas Arraytape genotyping platform used in the present invention were all purchased from LGC Company, UK. Advantages of KASP marker detection based on the Douglas Array Tape platform: The automation degree of KASP markers reaches 90%, greatly reducing the labor and human errors in the laboratory; The detection throughput is high, and 122,880 data points can be obtained in 8 hours, which is 10 times that of the traditional 96-well plate SNP genotyping method; The detection reaction volume is small (only 0.8 uL / reaction), and compared with the traditional 96-well plate SNP genotyping method, the reagent and consumable cost is reduced by 70%-90%.

[0075] The embodiments of the present invention have been described in detail above in conjunction with the accompanying drawings. However, the present invention is not limited to the above embodiments. Various changes can be made without departing from the spirit of the present invention within the knowledge scope of those of ordinary skill in the art. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. Use of a reagent for detecting a SNP molecular marker tightly linked to the aboveground dry weight trait of alfalfa in any of the following: (1) Identification and selection of alfalfa with different aboveground dry weights; (2) Breeding alfalfa with an aboveground dry weight of ≥150 g per plant; The SNP molecular marker is located at the base 60214922 of chromosome 4 of the alfalfa reference genome ZM-4 alfalfa genome, and the polymorphism is C / T.

2. A primer set for amplifying the SNP molecular marker as claimed in claim 1, characterized in that: The primer set includes a first specific primer as shown in SEQ ID NO.4, a second specific primer as shown in SEQ ID NO.5 and a universal primer as shown in SEQ ID NO.

6.

3. The primer set according to claim 2, characterized in that The first specific primer and the second specific primer are respectively connected with different fluorescent linkers.

4. The primer set according to claim 3, characterized in that: The fluorescent linker is selected from FAM and HEX.

5. A kit, characterized in that: The kit comprises the primer set according to any one of claims 2 to 4.

6. Use of the primer set according to any one of claims 2 to 4 or the kit according to claim 5 in any one of the following: (1) Identification and selection of alfalfa with different aboveground dry weights; (2) Breed alfalfa with an aboveground dry weight of ≥150g per plant.

7. A method for identifying the aboveground dry weight of alfalfa, characterized in that: The method comprises the following steps: S1. Extract genomic DNA from alfalfa germplasm materials; S2. Performing polymorphism detection of the SNP molecular markers as claimed in claim 1 on the genomic DNA extracted in step S1, and determining the aboveground dry weight of the alfalfa germplasm material according to the genotype.

8. A method for selecting alfalfa germplasm with an aboveground dry weight of ≥150g per plant for breeding, characterized in that: The method comprises the following steps: using the method as claimed in claim 7, selecting alfalfa germplasm material with a single plant aboveground dry weight of ≥150g for subsequent breeding.

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