An SNP molecular marker closely linked to the branching trait of alfalfa and its application

Abstract

The present invention discloses an SNP molecular marker closely linked to the branching trait of alfalfa and its application. The SNP molecular marker is located at the base position 78614933 on chromosome 7 of the alfalfa reference genome ZM-4 alfalfa genome, and the polymorphism is G / A. The SNP molecular marker closely linked to the branching trait of alfalfa in the present invention has a PIC value > 0.3, a sample data detection rate > 98%, and the marker is a codominant marker, featuring high specificity, high sensitivity, high resolution and high genotyping quality; it can rapidly, highly-throughput and low-cost detect the mutants of the branching number of alfalfa according to the genotype, and then determine the basal branching number and yield of alfalfa, and can be used for molecular marker-assisted breeding for improving the high-yield plant type of alfalfa, 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 branching trait of alfalfa and its application. Background Art

[0002] Alfalfa (Medicago sativa L.) is the most widely cultivated and important economic leguminous forage in the world. It is famous for its high nutritional value, great production potential, and characteristics such as drought resistance and salt tolerance. It is known as the "king of forages" and plays an irreplaceable role in promoting the adjustment of the agricultural industrial structure, the development of high-efficiency and high-quality animal husbandry, and ecological restoration.

[0003] The yield of alfalfa is determined by multiple components, such as plant height, stem diameter, leaf area, number of branches, stem-leaf ratio, etc. The number of alfalfa branches refers to the branches generated from the root neck, main stem or branches, which determines the size of the alfalfa plant cluster. Many studies have shown that the number of basal branches close to the ground of alfalfa is the main factor or even the decisive factor affecting the grass yield, and it has a greater direct effect and contribution to yield formation. At present, alfalfa breeding in China is still in the stage of conventional breeding, and there is less work on the precise evaluation and identification of germplasm resources. The identification efficiency 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 the development and utilization of excellent germplasm resources are seriously insufficient. The identification and evaluation of alfalfa mainly rely on the investigation after the phenotypic traits of germplasm 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 environmental 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] The DNA molecular marker method is a commonly used technical method in current crop variety breeding. DNA molecular markers are genetic markers that directly reflect DNA differences (polymorphisms). Currently, they mainly include SSR (Simple Sequence Repeat) and SNP (Single Nucleotide Polymorphism), etc. SSR markers have the characteristics of simple operation, low cost, good repeatability, and reliable results. Compared with the SSR marker method, the SNP marker technology is more convenient, easy to automate, has a high detection throughput, and is fast; the detection cost per unit data point is low; the data results of different detection laboratories can be compared and verified with each other, and the data has general comparability; it is the most commonly used method for quickly, simply, sensitively, accurately, stably, and low-costly identifying functional genes. There is relatively little research work on molecular markers of functional genes in alfalfa. Currently, there is still a lack of a high-throughput genotype identification and analysis technology system and molecular markers with important breeding value. There are few reports on the selection method based on SNP markers for alfalfa branching traits. 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 purpose, the present invention provides a SNP molecular marker closely linked to the alfalfa branching trait.

[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 detecting or assisting in detecting the number of branches of alfalfa.

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

[0012] According to the first aspect of the present invention, there is provided a SNP molecular marker closely linked to the alfalfa branching trait. The SNP molecular marker is located at the base position 78614933 on chromosome 7 of the alfalfa reference genome ZM-4 alfalfa genome, and the polymorphism is G / A.

[0013] In some embodiments of the present invention, the branching trait includes the number of branches.

[0014] According to the second aspect of the present invention, there is provided a primer set for amplifying the above SNP molecular marker.

[0015] In some embodiments of the present invention, the primer set includes a first specific primer as shown in SEQ ID NO.13 (Table 2) and a second specific primer sequence as shown in SEQ ID NO.14 (Table 2).

[0016] In some embodiments of the present invention, different fluorescent linker sequences are respectively linked to the first specific primer and the second specific primer.

[0017] In some embodiments of the present invention, the fluorescent linker sequences are selected from FAM and HEX.

[0018] 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.15 (Table 2).

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

[0020] According to the fourth aspect of the present invention, a gene chip is provided, and the gene chip includes the above-mentioned primer set.

[0021] 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:

[0022] (1) Detecting the number of branches of alfalfa;

[0023] (2) Identifying and screening alfalfa with different numbers of branches;

[0024] (3) Breeding alfalfa with multi-branch traits;

[0025] (4) Molecular marker-assisted breeding of alfalfa;

[0026] (5) Breeding of alfalfa;

[0027] (6) Preparing products for alfalfa breeding.

[0028] In some embodiments of the present invention, the alfalfa with different numbers of branches includes alfalfa with more than 30 branches and alfalfa with less than 25 branches.

[0029] In some embodiments of the present invention, the multi-branch means that the number of branches is greater than 30.

[0030] According to the sixth aspect of the present invention, a method for detecting or assisting in detecting the number of branches of alfalfa is provided, and the method includes the following steps:

[0031] S1. Extract genomic DNA from alfalfa germplasm materials;

[0032] S2. Perform polymorphism detection of the SNP molecular markers on the genomic DNA extracted in step S1, and judge the number of branches of the alfalfa germplasm materials according to the genotypes.

[0033] In some embodiments of the present invention, when the genotype detected by the SNP molecular marker is GG, the number of branches of the alfalfa materials is greater than 30, showing a multi-branched trait; when the genotype detected by the SNP molecular marker is AA, the number of branches of the alfalfa materials is less than 25, showing a few-branched trait; when the genotype detected by the SNP molecular marker is GA, the number of branches of the alfalfa materials is less than 25, showing a few-branched trait.

[0034] In some embodiments of the present invention, in step S1, the genomic DNA of alfalfa materials is extracted by the simplified CTAB method (cetyltrimethylammonium bromide method).

[0035] In some embodiments of the present invention, in step S2, the KASP (Kompetitive Allele Specific PCR) technology is used to detect the SNP molecular markers.

[0036] In some embodiments of the present invention, the composition of the KASP reaction mixture for detecting the SNP molecular markers by the KASP technology is as follows:

[0037]

[0038]

[0039] In some embodiments of the present invention, the amplification program for detecting the SNP molecular markers by 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.

[0040] According to the seventh aspect of the present invention, an alfalfa breeding method is proposed, including the following steps: using the above method, select alfalfa germplasm materials with more than 30 branches for subsequent breeding.

[0041] According to some embodiments of the present invention, it has at least the following beneficial effects: The present invention provides an SNP molecular marker closely linked to the branching trait of alfalfa, with a PIC value > 0.3, a sample data detection rate > 98%, and the marker being a co-dominant marker, featuring high specificity, high sensitivity, high resolution, and high genotyping quality; the marker is not affected by environmental conditions, 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 data has universal comparability, and the mutant germplasm of alfalfa branching number can be detected quickly, high-throughput, and at low cost according to the genotype, the branching number level can be determined, and it can be used for marker-assisted breeding for improving the high-yield plant type of alfalfa, with wide application universality.

[0042] The detection method of the present invention combines the KASP detection technology and is used for the detection of mutant materials of alfalfa branching traits. The detection method is simple, fast, and has a low detection cost, and is applicable to different detection instrument devices.

[0043] The additional aspects and advantages of the present invention will be partly given in the following description, partly will become obvious from the following description, or will be understood through the practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The following further describes the present invention in conjunction with the drawings and embodiments, where:

[0045] Figure 1 is the flow chart of molecular marker development in Example 1 of the present invention;

[0046] Figure 2 is the typical genotyping result diagram of the molecular marker Me900026 in Example 1 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0047] The following will clearly and completely describe the concept of the present invention and the technical effects produced in conjunction with the embodiments to fully understand the purpose, features, and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative efforts shall fall within 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.

[0048] Example of the present invention: An SNP molecular marker closely linked to the branching trait of alfalfa and its application

[0049] The design process of this molecular marker is as Figure 1As shown in the figure, a breeding germplasm resource database was constructed based on the re-sequencing data of 31 core germplasm resources of alfalfa. According to the analysis of the phenotypic survey data and whole-genome re-sequencing data of the existing resources, the closely linked loci of the alfalfa branch number were located at multiple loci in the intervals 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 used for sequence information extraction, 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.

[0050] Table 1

[0051] Phenotype Target_Type Chr Position Tiller.GLM SNP chr2 20411939 Tiller.GLM SNP chr2 20412421 Tiller.GLM SNP chr2 20412600 Tiller.GLM SNP chr7 6358911 Tiller.GLM SNP chr7 78614933

[0052] The screening and verification steps of SNP molecular markers closely linked to the alfalfa branch number are as follows:

[0053] 1 Primer design

[0054] For the molecular markers screened in Table 1 above, based on the reference genome ZM-4 alfalfa genome of alfalfa, 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 whole-genome copy number analysis is performed on the primer sequences, 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.

[0055] Using the molecular markers designed above, high-throughput detection can be carried out on the branch number of alfalfa materials. When the genotype of the detected molecular marker corresponds to the base of the homozygous Favorable Allele, it indicates that the branch number of the alfalfa material is greater than 30 and has a multi-branch phenotype; when the detected genotype does not contain the base corresponding to the Favorable Allele, it means that the branch number of the alfalfa material is less than 25 and has a few-branch phenotype. When the detected genotype is a heterozygous genotype, it also indicates that the branch number of the alfalfa material is less than 25 and has a few-branch phenotype.

[0056] Table 2

[0057]

[0058]

[0059] 2 Sample Detection

[0060] DNA Extraction: Genomic DNA was extracted from alfalfa using the simplified CTAB method.

[0061] KASP Reaction Test: The verification and detection of KASP markers were performed 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, and the NEXAR was used for the automatic assembly of the PCR amplification system.

[0062] Table 3 PCR Amplification System for KASP Marker Genotyping

[0063]

[0064] 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 decreased by 0.8°C per cycle) for 60 s, for 10 cycles; 94°C for 20 s, 57°C for 60 s, for 33 cycles.

[0065] Signal Scanning and Genotyping: After the PCR reaction was completed, the fluorescence signal of the reaction system was scanned using ARAYA; then, genotyping and data analysis were performed using INTELLICS.

[0066] 3 Marker Genotyping Data

[0067] According to the above detection method, 31 alfalfa materials (including replicates) were used to verify the KASP reactions of the markers Me900014, Me900023, Me900024, Me900018, and Me900026 in Table 2 above.

[0068] 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 the homozygous X allele at this KASP marker locus (marked as red in the genotyping map, located in the upper left corner of the figure), the Y cluster indicates that the sample contains the homozygous Y allele genotype at this KASP marker locus (marked as blue in the genotyping map, located in the lower right corner of the figure), and the heterozygous genotype cluster indicates that the sample contains the X and Y heterozygous alleles at this KASP marker locus (marked as purple in the genotyping map).

[0069] The quality verification results show that for the KASP markers, only the two homozygous and heterozygous cluster genotyping of Me900026 is good and compact. The locus is single-copy, the detection rate is higher than 98%, and the correspondence with the phenotype is highly consistent. The genotype typing quality of the KASP markers can fully meet the accurate detection of the number of branches in alfalfa. Therefore, Me900026 was selected for subsequent experimental verification.

[0070] 4 Specificity and Practicality Detection

[0071] To detect the specificity and practicality of the marker Me900026 in the present invention, alfalfa strains from different regions were collected for field planting, and genotype and actual phenotype detections were carried out according to the above detection methods. The experiment was repeated 3 times. When the number of branches was greater than 30, it was judged as the phenotype with many branches, and when the number of branches was less than 25, it was judged as the phenotype with few branches.

[0072] Table 4

[0073]

[0074]

[0075] 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 Me900026 of the present invention has high specificity in detecting the number of branches in alfalfa and can quickly and accurately identify the high or low number of branches of the tested alfalfa germplasm materials.

[0076] 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 μL / reaction), and compared with the traditional 96-well plate SNP genotyping method, the cost of reagents and consumables is reduced by 70%-90%.

[0077] 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 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 alfalfa branching trait in any of the following: (1) Detect the number of alfalfa branches; (2) Identification and screening of alfalfa with different branch numbers; (3) Breeding alfalfa with more than 30 branches; The SNP molecular marker is located at the base 78614933 of chromosome 7 of the alfalfa reference genome ZM-4 alfalfa genome, and the polymorphism is G / A; If the genotype obtained by the SNP molecular marker detection is GG, the number of branches of the alfalfa is greater than 30, and it has a many-branching trait; if the genotype obtained by the SNP molecular marker detection is AA, the number of branches of the alfalfa is less than 25, and it has a few-branching trait; if the genotype obtained by the SNP molecular marker detection is GA, the number of branches of the alfalfa is less than 25, and it has a few-branching trait.

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.13, a second specific primer as shown in SEQ ID NO.14 and a universal primer as shown in SEQ ID NO.

15.

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) Detect the number of alfalfa branches; (2) Identification and screening of alfalfa with different branch numbers; (3) Breeding alfalfa with more than 30 branches; If the genotype obtained by the SNP molecular marker detection is GG, the number of branches of the alfalfa is greater than 30, and it has a many-branching trait; if the genotype obtained by the SNP molecular marker detection is AA, the number of branches of the alfalfa is less than 25, and it has a few-branching trait; if the genotype obtained by the SNP molecular marker detection is GA, the number of branches of the alfalfa is less than 25, and it has a few-branching trait.

7. A method for detecting the number of branches 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 marker as claimed in claim 1 on the genomic DNA extracted in step S1, and determining the number of basal branches of the alfalfa germplasm material according to the genotype; If the genotype obtained by the SNP molecular marker detection is GG, the number of basal branches of the alfalfa germplasm material is greater than 30, and it has a multi-branching trait; if the genotype obtained by the SNP molecular marker detection is AA, the number of basal branches of the alfalfa germplasm material is less than 25, and it has a few-branching trait; if the genotype obtained by the SNP molecular marker detection is GA, the number of basal branches of the alfalfa germplasm material is less than 25, and it has a few-branching trait.

8. A method for breeding alfalfa, characterized in that: The method comprises the following steps: using the method as claimed in claim 7, selecting alfalfa germplasm material with a base branch number greater than 30 for subsequent breeding; the alfalfa breeding method is to select alfalfa germplasm material with a base branch number greater than 30.

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