CN118813858A - A SNP molecular marker tightly linked to the aboveground dry weight trait of alfalfa and its application - Google Patents

Abstract

The invention discloses a SNP molecular marker tightly linked to the aboveground dry weight trait of alfalfa and its application. The SNP molecular marker is located at the base of position 6021492 of chromosome 4 of the alfalfa reference genome ZM‑4alfalfa genome, and the polymorphism is C/T. The invention provides a SNP molecular marker tightly linked to the dry weight trait of alfalfa, which is a co-dominant marker with the characteristics of high specificity, high sensitivity, high resolution and high typing quality; the marker is not affected by environmental conditions, can be detected using seeds or different types of plant tissues, and the result is accurate, repeatable and stable; different testing laboratories and different data results can be compared and verified with each other, and the detection of mutant types of dry weight traits of alfalfa can be realized quickly, with high throughput and low cost according to the genotype, and the aboveground dry weight of alfalfa can be determined, which has wide application universality.

Description

A SNP molecular marker tightly linked to the aboveground dry weight trait of alfalfa and its application

Technical Field

The invention belongs to the field of agricultural molecular biology, and in particular relates to a SNP molecular marker tightly linked to the aboveground dry weight trait of alfalfa and an application thereof.

Background Art

Alfalfa (Medicago sativa L.) is one of the most important perennial legumes in the world. It has a large biomass, high nutritional value, and is drought-resistant and salt-tolerant. 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 selection and breeding of alfalfa varieties mainly rely on traditional breeding methods such as hybridization and selection, resulting in low efficiency and poor stability in variety research and development. There are few excellent alfalfa varieties with outstanding traits, which seriously restricts 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 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 breeding, improve breeding efficiency, and help alfalfa move from conventional breeding to molecular design breeding.

At present, there is little work on the precise evaluation and identification of alfalfa germplasm resources. The efficiency and accuracy of germplasm identification are low, and the exploration and identification of its important traits, especially yield, nutritional quality and stress resistance, are not in-depth enough, and excellent alfalfa germplasm materials cannot be effectively obtained. The identification and evaluation of alfalfa mainly rely on the investigation of breeding materials after they show phenotypes 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, and the judgment criteria are difficult to quantify accurately and are highly subjective; it is easily affected by environmental and cultivation conditions, and has poor accuracy and stability; it takes a long time and has poor timeliness; it requires a lot of manpower and material resources, and the cost is high.

DNA molecular markers are a commonly used technical method for crop variety selection. DNA molecular markers are genetic markers that directly respond to DNA differences (polymorphisms). Currently, they mainly include SSR (Simple Sequence Repeat) and SNP (Single Nucleotide Polymorphism). SSR markers have the characteristics of simple operation, low cost, good repeatability, and reliable results. Compared with SSR markers, SNP marker technology is simpler and easy to automate, with high detection throughput and fast speed; the detection cost per data point is low; the data results of different testing laboratories can be compared and verified with each other, and the data are universally comparable; it is the most commonly used method for identifying functional genes quickly, simply, sensitively, accurately, stably, and at low cost. There is little research on functional gene markers in alfalfa. At present, there is still a lack of high-throughput genotype identification and analysis technology systems and molecular markers with important breeding value. The selection method based on SNP marker selection method for alfalfa aboveground dry weight traits is rarely reported.

Summary of the invention

The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes a SNP molecular marker that is closely linked to the aboveground dry weight trait of alfalfa.

The present invention also provides a primer set for detecting the above SNP molecular markers.

The present invention also provides a kit.

The invention also provides a gene chip.

The present invention also proposes the application of the above-mentioned SNP molecular marker, primer set, kit and/or gene chip.

The invention also provides a method for identifying or assisting in identifying the dry weight of the aboveground part of alfalfa.

The invention also provides a breeding method for alfalfa.

According to the first aspect of the present invention, a SNP molecular marker tightly linked to the aboveground dry weight trait of alfalfa is proposed. The SNP molecular marker is located at the base 60214922 of chromosome 4 of the alfalfa reference genome ZM-4alfalfa genome, and the polymorphism is C/T.

According to a second aspect of the present invention, a primer set for amplifying the above-mentioned SNP molecular marker is proposed.

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).

In some embodiments of the present invention, the first specific primer and the second specific primer are respectively connected with different fluorescent linker sequences.

In some embodiments of the present invention, the fluorescent linker sequence is selected from FAM and HEX.

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

According to a third aspect of the present invention, a kit is provided, the kit comprising the above-mentioned primer set.

According to a fourth aspect of the present invention, a gene chip is provided, wherein the gene chip comprises the above-mentioned primer set.

According to a fifth aspect of the present invention, the use of the above-mentioned SNP molecular marker, primer set, kit or gene chip in any of the following is proposed:

(1) Detecting the aboveground dry weight of alfalfa;

(2) Identify and select alfalfa germplasm with different aboveground dry weights;

(3) Breeding alfalfa with an aboveground dry weight of ≥150 g per plant;

(4) Molecular marker-assisted breeding of alfalfa;

(5) Alfalfa breeding;

(6) Prepare alfalfa breeding products.

In some embodiments of the present invention, the alfalfa germplasms with different aboveground 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.

According to a sixth aspect of the present invention, a method for identifying or assisting in identifying the aboveground dry weight of alfalfa is provided, the method comprising the following steps:

S1, extracting genomic DNA from alfalfa materials;

S2. Performing polymorphism detection of the SNP molecular marker on the genomic DNA extracted in step S1, and determining the aboveground dry weight of the alfalfa material according to the genotype.

In some embodiments of the present invention, if the genotype obtained by the SNP molecular marker detection is CC, the dry weight of alfalfa material is ≥150g; if the genotype obtained by the SNP molecular marker detection is TT, the dry weight of alfalfa material is <150g; if the genotype obtained by the SNP molecular marker detection is TC, the dry weight of alfalfa material is ≥150g.

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

In some embodiments of the present invention, in step S2, the SNP molecular markers are detected using KASP (competitive allele-specific PCR) technology.

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

In some embodiments of the present invention, the amplification program for detecting SNP molecular markers using 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.

According to a seventh aspect of the present invention, a method for breeding alfalfa is proposed, comprising the following steps: using the above method, selecting alfalfa germplasm with a single plant aboveground dry weight of ≥150g for subsequent breeding.

According to some embodiments of the present invention, at least the following beneficial effects are achieved: the present invention provides a SNP molecular marker tightly linked to the aboveground dry weight trait of alfalfa, the marker is a co-dominant marker, the PIC value is higher than> 0.3, the sample data detection rate is> 98%, and it has the characteristics of high specificity, high sensitivity, high resolution and high typing quality; the marker is not affected by environmental conditions, and can be detected using seeds or different types of plant tissues, and the results are accurate, repeatable and stable; different testing laboratories and different data results can be compared and verified with each other, and the data have universal comparability, and can realize rapid, high-throughput and low-cost detection of alfalfa dry weight mutants according to genotype, determine the aboveground dry weight of a single plant, and be used for marker-assisted breeding for improving high-yield plant types of alfalfa, and has wide application universality.

The detection method of the invention is combined with the KASP detection technology and can be used for detecting the aboveground dry weight mutant of alfalfa. The detection method is simple and fast, has low detection cost, and is applicable to different detection instruments and equipment.

Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

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

FIG1 is a flow chart of molecular marker development in Example 1 of the present invention;

FIG. 2 is a diagram showing typical typing results of the molecular marker Me900010 in Example 1 of the present invention.

DETAILED DESCRIPTION

The following will clearly and completely describe the concept of the present invention and the technical effects produced in combination with the embodiments, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by technicians in this field without creative work are all 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, unless otherwise specified, can be obtained from commercial channels.

Embodiment of the present invention: A tightly linked SNP molecular marker associated with the aboveground dry weight trait of alfalfa and its application

The design process of the molecular marker is shown in Figure 1. A breeding germplasm resource database is constructed based on the resequencing data of 63 core germplasm resources of alfalfa. According to the analysis of the phenotypic survey data of existing resources and the whole genome resequencing data, the closely associated sites of alfalfa aboveground dry weight are located at multiple sites in the interval shown in Table 1 below. The reference genome version of alfalfa is ZM-4alfalfa genome, which is derived from the assembled genome. The obtained data is extracted based on the sequence information, and the gene sequence is subjected to SNP polymorphism analysis. Finally, multiple SNP sites are obtained and about 150bp before and after its flanking sequence are obtained for marker design and synthesis.

Table 1

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

The steps for screening and validating SNP molecular markers closely linked to the dry weight trait of alfalfa are as follows:

1 Primer design

For the molecular markers screened in Table 1 above, KASP marker primers were designed based on the alfalfa reference genome ZM-4alfalfagenome using the online primer design website BatchPrimer3 (http://probes.pw.usda.gov/batchprimer3/). Each set of markers has three primers, two of which are connected to FAM and HEX fluorescent sequences at the 5' end. After the design is completed, the primer sequence is further analyzed for the whole genome copy number, and finally a high-quality single-copy KASP marker is obtained. The site design information is shown in Table 2 below. The primers were commissioned to be synthesized by Sangon Biotech (Shanghai) Co., Ltd.

By using the molecular markers obtained by the above-mentioned design, high-throughput detection can be carried out on the aboveground dry weight of alfalfa materials. When the detected molecular marker is a genotype containing the base corresponding to Allele X, it indicates that the aboveground 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 base corresponding to Allele X, it indicates that the aboveground 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 aboveground dry weight of the alfalfa material is ≥150g, and has a high dry weight (biomass) phenotype.

Table 2

2. Sample testing

DNA extraction: Genomic DNA was extracted from alfalfa using the simplified CTAB method.

KASP reaction test: The validation and detection of KASP markers were performed using the Douglas Scientific Array Tape system. 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.

Table 3 PCR amplification system for KASP marker genotyping

PCR amplification reaction conditions: SOELLEX was used for PCR amplification, and the amplification conditions were as follows: 94°C for 15 min; 94°C for 20 s, 65°C-57°C (annealing temperature decreased by 0.8°C each cycle) for 60 s, 10 cycles; 94°C for 20 s, 57°C for 60 s, 33 cycles.

Signal scanning and genotyping: After the PCR reaction is completed, ARAYA is used to scan the fluorescence signal of the reaction system; then INTELLICS is used for genotyping and data analysis.

3 Marker typing data

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

A typical KASP marker genotyping diagram is shown in FIG2 . As can be seen from the figure, in the KASP marker genotyping test, the genotypes of the samples are divided into three clusters, namely, cluster X, cluster Y, and heterozygous genotype cluster, wherein cluster X indicates that the sample contains a homozygous X allele genotype at the KASP marker site (marked in red in the genotyping diagram, located in the upper left corner of the diagram), cluster Y indicates that the sample contains a homozygous Y allele genotype at the KASP marker site (marked in blue in the genotyping diagram, located in the lower right corner of the diagram), and heterozygous genotype cluster indicates that the sample contains a heterozygous X and Y allele genotype at the KASP marker site (marked in purple in the genotyping diagram).

The quality verification results showed that only the two homozygous and heterozygous clusters of KASP marker Me900010 were well-typed and compact, with a single copy of the locus, and the detection rate was higher than 98% and the consistency with the phenotype was high. The genotyping quality of KASP markers can fully meet the accurate detection of aboveground dry weight of alfalfa. Therefore, Me900010 was selected for subsequent experimental verification.

4 Specificity and practicality of detection

In order to detect the specificity and practicality of the marker Me900010 in the present invention, the strains of alfalfa from different regions were collected for field planting, and the genotype and actual phenotype detection and verification were performed according to the above-mentioned detection method. In order to ensure the accuracy of the aboveground dry weight phenotype measurement, every 10 strains of alfalfa from each strain were selected as a measurement sample, and the average value of 3 repetitions was used as the phenotypic measurement value, and the aboveground dry weight (g) was classified, and the aboveground dry weight of a single plant ≥150g was a high dry weight germplasm material; the aboveground dry weight of a single plant <150g was a low dry weight germplasm material, and the phenotype was determined in this way.

Table 4

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 aboveground dry weight (biomass) of alfalfa and can quickly and accurately identify the aboveground dry weight level of the tested alfalfa germplasm material.

The reagents and consumables of the Douglas Arraytape genotyping platform used in the present invention were purchased from LGC, UK. Advantages of KASP marker detection based on the Douglas Array Tape platform: The automation of KASP markers reaches 90%, which greatly reduces the manpower 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 0.8uL/reaction), and the cost of reagents and consumables is reduced by 70%-90% compared with the traditional 96-well plate SNP genotyping method.

The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims (10)

1. A SNP molecular marker tightly linked to the aboveground dry weight trait of alfalfa, characterized in that the SNP molecular marker is located at the base 60214922 of chromosome 4 of the alfalfa reference genome ZM-4alfalfa genome, and the polymorphism is C/T. 2. A primer set for amplifying the SNP molecular marker as described in claim 1. 3. The primer set according to claim 2, characterized in that the primer set comprises a first specific primer sequence as shown in SEQ ID NO.4 and a second specific primer sequence as shown in SEQ ID NO.5. 4. The primer set according to claim 3, characterized in that the first specific primer and the second specific primer are respectively connected with different fluorescent linker sequences; preferably, the fluorescent linker sequence is selected from FAM and HEX. 5 . The primer set according to claim 2 , characterized in that the primer set further comprises a universal primer, and the nucleotide sequence of the universal primer is shown in SEQ ID NO.6. 6. A kit, characterized in that the kit comprises the primer set according to any one of claims 2 to 5. 7. A gene chip, characterized in that the gene chip comprises the primer set according to any one of claims 2 to 5. 8. Use of the SNP molecular marker according to claim 1, the primer set according to any one of claims 2 to 5, the kit according to claim 6 or the gene chip according to claim 7 in any of the following: (1) Detecting the dry weight of alfalfa; (2) Identify and select alfalfa with different aboveground dry weights; (3) Breeding alfalfa with an aboveground dry weight of ≥150 g per plant; (4) Molecular marker-assisted breeding of alfalfa; (5) Alfalfa breeding; (6) Prepare alfalfa breeding products. 9. A method for identifying or assisting in identifying the aboveground dry weight of alfalfa, characterized in that the method comprises the following steps: S1, extracting genomic DNA from alfalfa 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 aboveground dry weight of the alfalfa material according to the genotype. 10. A method for breeding alfalfa, characterized in that it comprises the following steps: using the method according to claim 9, selecting alfalfa materials with a single plant aboveground dry weight of ≥150g for subsequent breeding.