Molecular marker of alfalfa plant height SNP, KASP primer set and application thereof

Abstract

The application discloses an alfalfa plant height SNP molecular marker, a KASP primer group and application thereof, and belongs to the technical field of biotechnology and plant molecular breeding technology.The allelic variation base of the SNP molecular marker is G / A, and is located at the 52424701th position of alfalfa chromosome 6.The application further provides a KASP primer group for detecting the SNP molecular marker.The application further provides application of the KASP primer group in preparation of a kit for identifying an alfalfa plant height trait, a kit containing the primer group and an alfalfa plant height SNP genotype detection method for non-breeding purposes.The typing result is completely corresponding to the actual plant height phenotype, and the accuracy rate reaches 100%.The application can be used for early prediction of the alfalfa plant height at the seedling stage, has the advantages of simple operation, low cost, high flux, accurate and reliable result, is suitable for large-scale molecular marker assisted breeding screening, significantly shortens the alfalfa breeding period, and improves the breeding efficiency.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and plant molecular breeding technology, specifically a high SNP molecular marker for alfalfa plants, a KASP primer set and its application. Background Technology

[0002] Alfalfa, a perennial forage grass belonging to the genus *Alfalfa* in the legume family, is known as the "king of forage grasses" due to its rich nutritional content and high protein content. Alfalfa yield is an important indicator of its production and economic performance, while plant height is a key indicator reflecting the growth status of the forage grass and evaluating its yield.

[0003] Traditional breeding methods for alfalfa varieties with ideal plant height involve selecting individual plants by measuring the height of offspring. This method is time-consuming and has low genetic efficiency. Advances in modern molecular biology techniques allow us to use molecular markers closely linked to relevant traits for parental selection or pre-screening of hybrid offspring, thereby improving breeding efficiency. Therefore, establishing an efficient DNA molecular marker and its detection method for early selection in breeding is crucial for reducing breeding workload, accelerating high-yield alfalfa breeding, and yielding significant economic benefits.

[0004] Kompetitive allele-specific PCR (KASP) is a technique based on primer terminal base specific matching and universal fluorescent probes to perform biallelic genotyping of SNPs and InDels at specific loci. Genotyping can be performed using a quantitative real-time PCR instrument or a conventional PCR instrument combined with an enzyme-linked immunosorbent assay (ELISA) reader. It offers advantages such as good stability, high accuracy, low detection cost, and high throughput. Developing and designing KASP molecular markers related to alfalfa plant height, and validating genotyping in natural and hybrid populations, will support the use of KASP markers for alfalfa parental selection and early selection of hybrid offspring, helping to accelerate the alfalfa breeding selection process and improve breeding efficiency. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides an alfalfa plant height SNP molecular marker, a KASP primer set, and their applications. The SNP molecular marker and KASP primer set can provide early auxiliary prediction and identification of alfalfa germplasm plant height phenotype. The operation is simple and convenient, with low cost and high selection efficiency. It can efficiently screen target single plants or a large number of progeny with potential superior qualities, shorten the alfalfa breeding cycle, and improve breeding efficiency.

[0006] Specifically as follows: A molecular marker for high SNP in alfalfa plants, characterized in that the nucleotide sequence of the SNP molecular marker is shown in SEQ ID No. 4; the allelic variant of the SNP molecular marker is G / A, and the allelic variant is located at position 52424701 on chromosome 6 of the alfalfa genome.

[0007] Furthermore, when the allelic variation of the SNP molecular marker site is G, the corresponding alfalfa plant height phenotype is dwarf; when the allelic variation of the SNP molecular marker site is A, the corresponding alfalfa plant height phenotype is tall; when the allelic variation of the SNP molecular marker site is G / A, i.e., the genotype is GA heterozygous, the corresponding alfalfa plant height phenotype is medium.

[0008] Secondly, the present invention provides a KASP primer set for alfalfa plants with high SNP molecular markers, the KASP primer set consisting of forward primer Fc, forward primer Ft and universal reverse primer R, the nucleotide sequence of the forward primer Fc is shown in SEQ ID No. 1, the nucleotide sequence of the forward primer Ft is shown in SEQ ID No. 2, and the nucleotide sequence of the universal reverse primer R is shown in SEQ ID No. 3.

[0009] Furthermore, the 5' end of the forward primer Fc is connected to a FAM fluorescent tag sequence, and the 5' end of the forward primer Ft is connected to a HEX fluorescent tag sequence.

[0010] Thirdly, this invention provides the application of the KASP primer set in the preparation of a kit for identifying alfalfa plant height traits.

[0011] Fourthly, the present invention provides a kit for identifying the high SNP genotype of alfalfa plants, the kit comprising the KASP primer set as described in claim 3 or 4.

[0012] Furthermore, the kit also contains 2×PARMS mastermix.

[0013] Fifthly, this invention provides a method for detecting high SNP genotypes in alfalfa plants for non-breeding purposes, comprising the following steps: S1. Extract genomic DNA from the alfalfa germplasm to be identified, and perform PCR amplification using the KASP primer set described in claim 3 or 4 to obtain PCR amplification products; S2. Detect the fluorescence signal of the PCR amplification product, and determine the SNP genotype of the alfalfa germplasm to be identified based on the fluorescence signal type; If a FAM fluorescence signal is detected, the genotype of the SNP molecular marker site is determined to be GG homozygous. If a HEX fluorescence signal is detected, the genotype of the SNP molecular marker site is determined to be homozygous AA.

[0014] Further, in step S1, the PCR amplification system is as follows: 2 μL template DNA, 5 μL 2×PARMS mastermix, 0.15 μL 10 μM forward primer Fc, 0.15 μL 10 μM forward primer Ft, 0.4 μL 10 μM reverse primer R, and ddH2O to a final volume of 10 μL.

[0015] Further, in step S1, the PCR amplification program is as follows: 94℃ pre-denaturation for 20 min; 94℃ denaturation for 20 s, 65-57℃ extension for 1 min, 10 cycles, with the extension temperature decreasing by 0.8℃ in each cycle; 94℃ denaturation for 20 s, 57℃ extension for 1 min, 32 cycles.

[0016] Furthermore, in step S2, when the genotype of the SNP molecular marker site is determined to be GG homozygous, the alfalfa germplasm to be identified is a dwarf material; when the genotype of the SNP molecular marker site is determined to be AA homozygous, the alfalfa germplasm to be identified is a tall material.

[0017] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention, through association analysis, reveals for the first time a significant correlation between the G / A allelic variation at locus 52424701 on chromosome 6 of alfalfa and the plant height trait. Experiments show that the GG genotype at this SNP locus corresponds to dwarf plants (average plant height 18cm), the AA genotype corresponds to tall plants (average plant height 46.8cm), and the GA heterozygous genotype corresponds to medium plants (average plant height 37.1cm). The differences in plant height among the three genotypes are all highly significant. The discovery of this SNP molecular marker provides a novel target site for marker-assisted breeding of alfalfa plant height.

[0018] 2. Traditional alfalfa height selection methods require cultivating plants to maturity and then manually measuring the height of each plant for individual selection, which takes up to a complete growth cycle. This invention only requires extracting DNA from a small number of leaves during the alfalfa seedling stage, performing PCR amplification using KASP primers, and reading the fluorescence signal with an enzyme-linked immunosorbent assay (ELISA) reader. Genotyping can be completed within a few hours, eliminating the need to wait for plants to mature or for large-scale planting. This significantly shortens the breeding cycle and saves breeding land and labor costs.

[0019] 3. Based on the KASP technology platform, this invention allows PCR amplification and fluorescence signal reading to be performed in 96-well or 384-well plates. Hundreds of samples can be genotyped simultaneously in a single experiment. This enables rapid early screening of a large number of progeny plants in a large-scale breeding population. Materials carrying the dwarf genotype can be eliminated at the seedling stage, and only individuals with the tall genotype or heterozygous individuals can be retained for subsequent breeding processes, significantly improving breeding efficiency.

[0020] 4. This invention validated the KASP typing of 120 alfalfa germplasm accessions and the effectiveness of the typing on 15 alfalfa varieties, demonstrating that the fluorescence signal typing results completely corresponded to the actual plant height phenotype, with a verification accuracy of 100%. The average plant heights corresponding to the three fluorescence signal types (FAM for dwarf plants, FAMHEX for medium plants, and HEX for tall plants) were 18cm, 37.1cm, and 46.8cm, respectively, with highly significant differences between groups. This indicates that the KASP primer set of this invention has excellent resolution and specificity, can clearly distinguish the three genotypes, and the typing results are accurate and reliable.

[0021] 5. Compared with traditional genotyping techniques such as the TaqMan probe method, the KASP technology does not require the synthesis of fluorescently labeled probes. Genotyping detection can be completed simply by attaching fluorescent tags to universal primers. This invention does not require high-end equipment. The entire detection process can be completed using a real-time PCR instrument or a regular PCR instrument combined with an enzyme-linked immunosorbent assay (ELISA) reader. The equipment investment cost is low, and it is easy to promote and apply in ordinary breeding laboratories and technology extension units, with good accessibility and practicality.

[0022] 6. This invention can be directly applied to the selection of alfalfa parents and the early selection of hybrid offspring, elevating breeding selection from traditional phenotypic selection to genotypic selection. In the parent selection stage, this invention can be used to screen germplasm materials with tall genotypes as parents; in the hybrid offspring selection stage, genotypic identification can be performed on individual offspring plants during the seedling stage to selectively retain individuals with tall genotypes. The implementation of this invention helps to accelerate the breeding process of new alfalfa varieties with ideal plant types, provides strong technical support for the genetic improvement of alfalfa yield, and has significant economic benefits and application prospects.

[0023] 7. The detection method provided by this invention can complete genotyping at the seedling stage, and is applicable to non-breeding purposes such as alfalfa germplasm resource identification and variety purity testing, providing reliable technical support for molecular marker-assisted selection of alfalfa. Detailed Implementation

[0024] Example 1: Discovery of SNP molecular markers related to alfalfa plant height 1. Experimental materials and planting conditions This example uses 120 alfalfa germplasm resources, planted in Helingeer New District, Hohhot City, Inner Mongolia Autonomous Region, with minimum allele frequencies ranging from 39°58' to 40°41' north latitude and 111°26' to 112°18' east longitude. In 2024, the average annual high temperature in Helingeer County was approximately 15°C, and the average low temperature was approximately 1°C. The extreme maximum temperature occurred on July 23, reaching 38°C; the extreme minimum temperature occurred on January 23, dropping to -34°C. The total annual precipitation was approximately 222.8 mm. Precipitation was mainly concentrated in summer, especially in July and August. The soil in Helingeer County is mainly black calcareous soil and chestnut calcareous soil, with high soil fertility, suitable for crop growth. The soil is generally alkaline, containing 65.3 mg / kg of available potassium, 23.4 mg / kg of available phosphorus, and 39.83 mg / kg of available nitrogen.

[0025] The experimental population was designed using a randomized block design with three replicates. Each block was placed in the same plot of land, with a plot size of 12m². 2 Minimum allele frequency (length 4 m × width 3 m). A 0.5 m protective row was established around the entire experimental plot. The soil quality and fertility of the experimental plot were investigated and analyzed before sowing, requiring meticulous cultivation of the seedbed. Sowing was done in rows with a row spacing of 30 cm, 10 rows per plot, and a sowing depth of 0.5-1.0 cm. The seeding rate was 15 g per plot (10.05 kg / hm²). 2 ).

[0026] 2. Determination of plant height During the initial and full-blown flowering stages, the plant height of each alfalfa sample was measured manually to obtain plant height values ​​for different replicates and at different times for each material, and the average value was calculated. This average value was used for subsequent GWAS analysis.

[0027] 3. Genomic DNA extraction and library construction Approximately 0.02–0.05 g of young plant leaves were selected and flash-frozen in liquid nitrogen. Genomic DNA was extracted using the CTAB method: samples were thoroughly ground using a tissue homogenizer, lysed at 65°C for 30 min, and impurities were removed by chloroform extraction. High-quality genomic DNA was obtained through magnetic bead purification. DNA concentration was detected using a Qubit 3.0 quantitative PCR instrument (Invitrogen, USA) and the dsDNAHS Assay Kit (YEASEN, Shanghai, China), and DNA integrity was assessed by agarose gel electrophoresis. 500 ng of qualified DNA was collected and fragmented, end-repaired, A-tailed, ligated, and amplified using the Hieff NGS DNA Library Prep Kit (YEASEN, Shanghai, China) to construct the genomic library. The libraries were quantitatively analyzed using the VAHTS Library Quantification Kit (Vazyme, Nanjing, China) on a LightCycler 480 qPCR instrument (Roche, Switzerland). Qualified libraries were transformed and circularized using the MGI Easy Universal Library Conversion Kit and Library Circulation Kit (MGI Tech, Shenzhen, China). Finally, paired-end sequencing was performed on the DNBSEQ-T7RS high-throughput sequencing platform (MGI Tech, Shenzhen, China). Approximately 40GB of raw data was obtained from each material.

[0028] 4. Mutation detection and SNP screening The raw sequencing data was cleaned using Fastp (version 0.20.0) software to remove the adapter and filter out low-quality base sequences. Then, BWA-MEM2 (version 2.2.1) was used to align the cleaned sequencing data to the "alfalfa haplotype reference genome". SAMtools (version 1.9) was used to filter multiple alignments and low-quality sequences, resulting in a filtered BAM file which was then sorted. PCR repetitive sequences were marked using the Mark Duplicates function in the Picard toolkit (version 2.17.3). Finally, the processed BAM file was used for variant detection using GATK Haplotype Caller (version 4.3.0).

[0029] In the SNP filtering strategy, the following parameters were set as screening criteria: Quality ByD (QD) less than 2.0, Fisher Strand (FS) greater than 60.0, Mapping Quality Rank Sum Test (MQ Rank Sum Test) less than -12.5, Read Pos Rank Sum (Read Pos Rank Sum) less than -8.0, Strand Odds Ratio (SOR) greater than 3.0, and Mapping Quality (MQ) less than 40.0. Subsequently, the screening conditions for a subset of alfalfa SNP markers were defined, including: (1) using PLINK2 (version 2.0) to remove SNP markers with a deletion rate greater than 20% and a Minor Allele Frequency (MAF) less than 0.05, thereby creating a basic SNP set containing only biallelic SNP markers; Similarly, VCFtools was used to filter Indel markers, retaining only biallelic markers with a deletion rate less than 20% and a MAF greater than 0.05.

[0030] 5. GWAS Association Analysis GWAS analysis was performed using EMMAX with 8,017,931 high-quality SNP markers. Model reliability was assessed using QQ plots. Principal component analysis was performed on the SNP data, with the first five principal component variables used as covariates to correct for population structure. Manhattan plot visualization was performed using the R package CMplot (Yin et al., 2021). Linkage disequilibrium (LD) analysis of associated SNPs was calculated using LDBlockShow (Dong et al., 2021), and the data was visualized.

[0031] 6. Identification of significantly associated SNP sites A locus (chr6.3_52424701) was found near chromosome 6 at 52.42 Mb, which was most significantly associated with the GWAS of plant height on April 25th. The allelic variation was G / A, with a LOD value of 9.21 and an explainable phenotypic contribution of 61.55%. This locus corresponds to position 52424701 on chromosome 6 of the alfalfa genome. The nucleotide sequence of the alfalfa plant height SNP molecular marker is shown in SEQ ID No. 4. When the allelic variation of this SNP molecular marker locus is G, the genotype is GG or GA; when the allelic variation is A, the genotype is AA. With genotype GG, the alfalfa is a dwarf plant with an average plant height of 18 cm; with genotype AA, the alfalfa is a tall plant with an average plant height of 46.8 cm; and with genotype GA, the alfalfa is a medium plant with an average plant height of 37.1 cm.

[0032] Example 2 KASP Primer Set A KASP primer set for detecting high SNP molecular markers in alfalfa plants is provided. The KASP primer set consists of forward primer Fc, forward primer Ft, and universal reverse primer R, and their sequences are shown in Table 1 below.

[0033] Table 1. Molecular detection primer sequence

[0034] Example 3: High specificity of KASP primer set and alfalfa plant 1. At maturity, 120 alfalfa germplasm samples were selected to determine plant height. Three healthy, lodging-free plants were selected from each germplasm sample. After straightening the plants, the height from the ground surface to the top leaf tip was measured with a ruler. The average value was taken as the germplasm plant height. After measurement, leaves were collected simultaneously to extract DNA for KASP molecular marker typing. 2. Construct the PCR amplification reaction system (Table 2), and perform PCR amplification according to the PCR amplification procedure in Table 3; Table 2 PCR amplification reaction system

[0035] Table 3 PCR amplification program

[0036] After PCR amplification, the fluorescence signal was read using a TECAN Infinite M1000 microplate reader, and the genotype results were output. The results are shown in Table 4-5.

[0037] Table 4. Phenotypic results of plant height and fluorescence signal in different alfalfa germplasms

[0038] Table 5. KASP Classification Statistical Results

[0039] Using Tables 4 and 5, the KASP primer set of this invention was used to genotype 120 alfalfa germplasm samples. Combined with the actual plant height type of the alfalfa germplasm samples, a high degree of fit was found with the actual plant height phenotype. Furthermore, there were significant differences in the fluorescence signals of the PCR amplification products from the KASP primer set. This indicates that the KASP primer set of this invention is specific for alfalfa plant height phenotype.

[0040] 3. Validity verification Fifteen varieties were selected, and the plant height of each variety was measured with a ruler. Three plants of each variety were measured, and the average height was taken as the actual height of that variety. The above steps and methods were then followed, and the results are shown in Table 6.

[0041] Table 6. Results of plant height phenotype and fluorescence signal type in alfalfa germplasm.

[0042] As shown in Table 6, the plant height of different alfalfa germplasm phenotypes corresponds to the fluorescence signal type detected by the KASP primer set of this invention, and the verification accuracy is 100%.

[0043] In summary, the KASP primer set of this invention can be used as a primer set for detecting SNP molecular markers of alfalfa plant height, and can assist in the prediction and identification of alfalfa germplasm plant height phenotype.

Claims

1. A molecular marker for high SNP levels in alfalfa plants, characterized in that, The nucleotide sequence of the SNP molecular marker is shown in SEQ ID No. 4; the allelic variant of the SNP molecular marker is G / A, and the allelic variant is located at position 52424701 on chromosome 6 of the alfalfa genome.

2. The alfalfa plant high SNP molecular marker according to claim 1, characterized in that, When the allelic variant base at the SNP molecular marker site is G, the genotype is GG or GA; when the allelic variant base is A, the genotype is AA; when the allelic variant base at the SNP molecular marker site is G / A, the genotype is GA.

3. A KASP primer set for detecting high SNP molecular markers in alfalfa plants as described in claim 1 or 2, characterized in that, The KASP primer set consists of forward primer Fc, forward primer Ft, and universal reverse primer R. The nucleotide sequence of the forward primer Fc is shown in SEQ ID No. 1, the nucleotide sequence of the forward primer Ft is shown in SEQ ID No. 2, and the nucleotide sequence of the universal reverse primer R is shown in SEQ ID No.

3.

4. The KASP primer set according to claim 3, characterized in that, The 5' end of the forward primer Fc is connected to a FAM fluorescent tag sequence, and the 5' end of the forward primer Ft is connected to a HEX fluorescent tag sequence.

5. The use of the KASP primer set according to claim 3 or 4 in the preparation of a kit for identifying alfalfa plant height trait.

6. A kit for identifying high SNP genotypes in alfalfa plants, characterized in that, The kit contains the KASP primer set as described in claim 3 or 4.

7. A method for detecting alfalfa plant height SNP genotypes for non-breeding purposes, the method being used for identifying plant height traits or detecting varietal purity in alfalfa germplasm resources, characterized in that... Includes the following steps: S1. Extract genomic DNA from the alfalfa germplasm to be identified and perform PCR amplification using the KASP primer set described in claim 3 or 4 to obtain PCR amplification products. S2. Detect the fluorescence signal of the PCR amplification product, and determine the SNP genotype of the alfalfa germplasm to be identified based on the fluorescence signal type. If a FAM fluorescence signal is detected, the genotype of the SNP molecular marker site is determined to be GG homozygous. If a HEX fluorescence signal is detected, the genotype of the SNP molecular marker site is determined to be AA homozygous. If both FAM and HEX fluorescence signals are detected simultaneously, the genotype is determined to be GA heterozygous.

8. The method according to claim 7, characterized in that, In step S1, the PCR amplification system is as follows: 2 μL template DNA, 5 μL 2×PARMS mastermix, 0.15 μL 10 μM forward primer Fc, 0.15 μL 10 μM forward primer Ft, 0.4 μL 10 μM reverse primer R, and ddH2O to a final volume of 10 μL.

9. The method according to claim 7, characterized in that, In step S1, the PCR amplification program is as follows: 94℃ pre-denaturation for 20 min; 94℃ denaturation for 20 s, 65-57℃ extension for 1 min, 10 cycles, with the extension temperature decreasing by 0.8℃ in each cycle; 94℃ denaturation for 20 s, 57℃ extension for 1 min, 32 cycles.

10. The method according to any one of claims 7-9, characterized in that, In step S2, when the genotype of the SNP molecular marker locus is determined to be GG homozygous, the alfalfa germplasm to be identified is a dwarf material; when the genotype of the SNP molecular marker locus is determined to be AA homozygous, the alfalfa germplasm to be identified is a tall material; and when the genotype of the SNP molecular marker locus is determined to be GA heterozygous, the alfalfa germplasm to be identified is a medium-sized material.

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