A grape seed germination rate related snp marker and application thereof

Abstract

The present application relates to the technical field of molecular markers, in particular to a grape seed germination rate related SNP marker and application thereof. The SNP marker is located at the 482011th (base G or A), 485116th (base C or T), 489064th (base C or T) and 496078th (base A or G) positions of the 4th chromosome gene VIT_04s0008g00560 in the grape reference genome version 23; the dominant haplotype is HAP4. Through the haplotype of the SNP marker site, the high and low of the grape seed germination rate can be accurately and quickly judged at the seedling stage, and the plant does not need to grow fruits. The present application can effectively improve the grape hybrid breeding efficiency, shorten the breeding time, and is suitable for grape genetic breeding work.

Description

Technical Field

[0001] This invention relates to the field of molecular marker technology, and in particular to a SNP marker related to grape seed germination rate and its application. Background Technology

[0002] In grape breeding programs, seeds obtained through controlled hybridization are crucial propagation material. However, low germination rates are a major obstacle for breeders. Therefore, identifying candidate genes that influence grape seed germination rates is of great significance for improving breeding efficiency.

[0003] Single nucleotide polymorphism (SNP) molecular markers are genetic markers formed by a single nucleotide variation in the genome. They are the third generation of DNA molecular marker technology after microsatellite markers, and have advantages such as large number, wide distribution, genetic stability, and ease of automated detection.

[0004] With the advent of the genomic era, clarifying the functions of grape germination-related genes and their significant impact on improving breeding efficiency is crucial. Identifying key candidate genes influencing seed germination traits and developing corresponding molecular markers is a vital task in improving grape breeding efficiency. Utilizing SNP markers for screening grape varieties with high germination rates and promoting the breeding of high-quality new grape varieties has important theoretical guiding significance. Therefore, developing molecular markers related to grape seed germination rate and applying them to grape breeding work is of great importance for improving grape breeding efficiency. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a SNP marker related to grape seed germination rate and its application. By using this SNP marker in grape hybridization breeding, the germination rate of grape seeds can be accurately and quickly determined at the seedling stage based on the genotype of the SNP marker locus, without requiring the plant to develop fruit. This invention can effectively improve the efficiency of grape hybridization breeding, shorten breeding time, and is suitable for grape genetic breeding work.

[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a SNP marker related to grape seed germination rate, wherein the SNP marker is located on chromosome 4 of the grape reference genome, version 23. VIT_04s0008g00560 The bases at positions 482011, 485116, 489064, and 496078 are: position 482011, which is either G or A; position 485116, which is either C or T; position 489064, which is either C or T; and position 496078, which is either A or G.

[0007] Preferably, the grape seed germination rate is highest when the base at position 482011 is A, the base at position 485116 is T, the base at position 489064 is T, and the base at position 496078 is A.

[0008] The present invention also provides a primer pair for detecting the SNP marker described in the above technical solution, wherein the sequence of the upstream primer is shown in SEQ ID No. 1 and the sequence of the downstream primer is shown in SEQ ID No. 2.

[0009] The present invention also provides a kit for detecting the SNP markers described in the above-described technical solutions, comprising the primer pairs described in the above-described technical solutions.

[0010] This invention also provides the application of the SNP markers, primer pairs, or kits described in the above-mentioned technical solutions in determining the germination rate of grape seeds.

[0011] Preferably, the germination rate of grape seeds is determined during the grape seedling stage.

[0012] This invention also provides a method for determining the germination rate of grape seeds, comprising the following steps: 1) RNA was extracted from leaves of different grape varieties and reverse transcribed into cDNA; 2) Using the cDNA described in step 1) as a template, perform full-length TA cloning of the gene using the primer pairs described in the above technical solution to obtain the amplification results; 3) Based on the amplification results described in step 2), perform transformation and plating, single clone screening, plasmid extraction and sequencing, sequence analysis and haplotype determination.

[0013] Preferably, the amplification system in step 2) is: 2.5 µL cDNA, 1.0 µL of upstream primer with a concentration of 7.5 µM, 1.0 µL of downstream primer with a concentration of 7.5 µM, 12.5 µL of 2×q-PCR mixture and 8.0 µL of ddH2O, with a total volume of 25 µL.

[0014] Preferably, the amplification program in step 2) is as follows: 94 °C pre-denaturation for 5 min; 94 °C denaturation for 1 min, 55 °C annealing for 1 min, 72 °C extension for 1.5 min, for 40 cycles.

[0015] Preferably, step 3) involves transformation and plating using DH5α chemocompetent cells, which are placed on ice for 30 minutes, heat-shocked at 42°C for 45 seconds, and then quickly returned to ice for 2-3 minutes; 600 μL of LB medium is added, and the cells are shaken at 37°C for 1 hour to improve recovery efficiency; the cells are then plated on LB agar plates containing ampicillin and incubated at 37°C for 12-16 hours. Step 3) The method of single-clone screening is to select no less than 10 single clones for each sample to cover allele diversity; use colony PCR for initial screening, using universal primers for vectors, and confirm that the inserted fragment size is correct; Step 3) The plasmid extraction and sequencing method is as follows: plasmid is extracted using a miniprep kit at a concentration ≥50 ng / μL, with an A260 / A280 ratio between 1.8 and 2.0; M13 universal primers are used for sequencing to ensure full sequence coverage. Step 3) The method for sequence analysis and haplotype determination is to align the sequencing results to the reference sequence, identify SNP sites, and clarify the haplotype relationship between different varieties.

[0016] The beneficial effects of this invention are: Based on the fact that a higher germination rate of offspring seeds during artificial cross-pollination of grapes helps improve breeding efficiency, this invention provides a SNP marker related to grape germination rate: this SNP marker is located at... VIT_04s0008g00560Gene positions 480863_480916_480949_481191_481363_481394_481485_481512_481524_482011_482237_482272_482603_483059_483079_483193_483200_483336_483417_483730_483880_484006_484062_484416_484429_484597_484750_484811_485116_485195_485370_485431_485595_485941_486076_486288_486584_486732_486787_486935_486940_487170_487328_487770_487827_487908_488108_488590_488636_488753_488924_488938_488939_488991_489049_489064_489083_489109_489159_489519_489584_489866_489967_490208_490887_490895_490973_491144_491263_492303_492658_492795_493163_493174_493469_493618_493841_493898_493916_494762_494978_495190_495455_495668_495680_495880_495923_496038_496078_496243_496527_496528_496557_496656_496786_496817_496901_496922_496929_497027_497039_497042_497060, and its advantageous haplotype HAP4 sequence is (SEQ ID No. 3): G_T_C_G_G_T_G_G_G_A_T_C_A_C_A_T_T_G_T_T_T_C_T_C_A_G_A_T_T_G_G_A_T_T_A_A_G_C_C_A_A_T_A_A_G_G_A_T_C_T_G_C_G_T_C_T_G_G_T_T_C_A_T_C_A_C_C_G_T_G_G_T_A_C_C_G_T_C_A_A_A_A_T_G_C_A_G_T_A_C_C_G_A_C_T_A_A_A_G_T_G_T_G.

[0017] By identifying different haplotypes of this SNP marker locus, the germination rate of grape seeds can be accurately and quickly determined during the seedling stage, without requiring the plant to develop fruit. This invention can effectively improve the efficiency of grape hybridization breeding, shorten breeding time, and is applicable to grape genetic breeding work. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0019] Figure 1 Manhattan plot and QQ plot of grape seed germination rate based on genome-wide association study (GWAS), where Figure 1 The left side is a Manhattan plot, which is a graph showing the genetic marker effect value, i.e., the genome-wide P value after the F test, ordered by the physical location on the chromosome. The horizontal axis is the genome coordinate, and the vertical axis is -log10P. The smaller the P value, the stronger the association. The black dashed line is the threshold line for screening significant sites. SNP sites above the threshold line are candidate sites associated with the phenotype. The right side is a Quantile-quantile plot, which shows the distribution of the actual P value (vertical axis) and the expected P value of the null hypothesis without association (horizontal axis). It is used to detect the influence of population stratification and individual kinship on association analysis. The closer the blue line is to the red line, the less the results are affected by population stratification.

[0020] Figure 2 Comparison of different haplotype phenotypes of key candidate genes affecting grape seed germination rate; Figure 3 for VIT_04s0008g00560 Comparison of relative gene expression levels in different haplotypes (high seed germination rate, HAP4, steel blue; low seed germination rate, HAP2, orange). Detailed Implementation

[0021] This invention provides a SNP marker related to grape seed germination rate, wherein the SNP marker is located on chromosome 4 of the grape reference genome, version 23. VIT_04s0008g00560 The bases at positions 482011, 485116, 489064, and 496078 are specified, with the base at position 482011 being either G or A, the base at position 485116 being either C or T, the base at position 489064 being either C or T, and the base at position 496078 being either A or G. In this invention, the grape seed germination rate is highest when the bases at positions 482011, 485116, 489064, and 496078 are all A.

[0022] The present invention also provides a primer pair for detecting the SNP marker described in the above technical solution, wherein the sequence of the upstream primer is shown in SEQ ID No. 1 and the sequence of the downstream primer is shown in SEQ ID No. 2.

[0023] SEQ ID No. 1: 5'-CAAAGGTCAATGGGAACCCC-3'; SEQ ID No. 2: 5'-CCCCTTCTGTACAGCCCTTG-3'.

[0024] The present invention also provides a kit for detecting the SNP markers described in the above-described technical solutions, comprising the primer pairs described in the above-described technical solutions.

[0025] This invention also provides the application of the SNP markers, primer pairs, or kits described in the above-mentioned technical solutions in determining the germination rate of grape seeds. In this invention, the germination rate of grape seeds is determined during the grape seedling stage.

[0026] This invention also provides a method for determining the germination rate of grape seeds, comprising the following steps: 1) RNA was extracted from leaves of different grape varieties and reverse transcribed into cDNA; 2) Using the cDNA described in step 1) as a template, amplification is performed using the primer pair described in the above technical solution to obtain the amplification result; 3) Based on the amplification results described in step 2), perform transformation and plating, single clone screening, plasmid extraction and sequencing, and sequence analysis and haplotype determination to determine the germination rate of grape seeds.

[0027] In this invention, the preferred amplification system consists of: 2.5 µL cDNA, 1.0 µL of 7.5 µM upstream primer, 1.0 µL of 7.5 µM downstream primer, 12.5 µL of 2×q-PCR mixture, and 8.0 µL of ddH2O, for a total volume of 25 µL. The preferred amplification program is: 94 °C pre-denaturation for 5 min; 94 °C denaturation for 1 min, 55 °C annealing for 1 min, and 72 °C extension for 1.5 min, for 40 cycles. In this invention, the transformation and plating method preferably uses DH5α chemocompetent cells, which are placed on ice for 30 minutes, heat-shocked at 42°C for 45 seconds, and quickly returned to ice for 2-3 minutes; 600 μL of LB medium is added, and the cells are shaken at 37°C for 1 hour to improve recovery efficiency; the cells are then plated on LB plates containing ampicillin and incubated at 37°C for 12-16 hours; the single-clone screening method preferably involves picking no less than 10 single clones from each sample to cover allele diversity; colony PCR is used for initial screening, using universal vector primers to confirm the correct size of the inserted fragment; the plasmid extraction and sequencing method preferably uses a miniprep kit to extract plasmids at a concentration ≥50 ng / μL, with an A260 / A280 ratio between 1.8 and 2.0; universal M13 primers are used for sequencing to ensure full sequence coverage; the sequence analysis and haplotype determination method preferably involves aligning the sequencing results to a reference sequence, identifying SNP sites, and clarifying the haplotype relationship between different varieties.

[0028] To further illustrate the present invention, the following detailed description is provided in conjunction with embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0029] Example 1 This embodiment provides a specific method for mining SNP markers related to grape seed germination rate based on GWAS analysis.

[0030] 1. Preparation of test materials The test materials consisted of 269 natural grape populations obtained from the experimental nursery of the Grape and Fruit Research Institute of Xinjiang Uygur Autonomous Region (see Table 1).

[0031] Table 1. Seed germination rates of 269 natural grape populations Note: NA indicates missing data.

[0032] Table 2 Key candidate genes affecting grape seed germination rate VIT_04s0008g00560 Different haplotype distributions of (calcineurin-binding protein 1) 2. Determination of seed germination rate of grape natural population materials.

[0033] Mature grape berries collected from the 269 natural populations listed in the table above were manually seeded, washed with tap water to remove shriveled seeds, and then air-dried for later use. For grape seeds that had undergone low-temperature stratification, 30 plump seeds from each variety were selected, with three replicates. The seeds were soaked in a 0.5% (v / v) sodium hypochlorite (NaClO) solution for 10 minutes, followed by thorough rinsing with sterile deionized water. The seeds were evenly placed in 90 mm diameter plastic petri dishes lined with two layers of filter paper. All petri dishes were incubated at 28°C for 20 days, with alternating light and dark cycles for 12 hours. Germination was defined as the radicle penetrating the seed coat and sprouting ≥2 mm. Germination counts were recorded every 24 hours for 20 consecutive days. Germination parameters were evaluated according to the rules of the International Seed Testing Association (ISTA), and the evaluation method is as follows: Germination rate (GR) is expressed as (G%): G% = n / N × 100, where n is the number of germinated seeds at the end of the experiment and N is the total number of seeds sown.

[0034] 3. GWAS analysis was performed on 269 natural grape populations using GEMMA software. The results are as follows: Figure 1 .in Figure 1 The left side is a Manhattan plot, which shows the genetic marker effect value, i.e., the genome-wide P-value after the F-test, ordered by the physical location on the chromosome. The horizontal axis is the genome coordinate, and the vertical axis is -log10P. The smaller the P-value, the stronger the association. The black dashed line is the threshold line for screening significant loci; SNPs above the threshold line are candidate loci associated with the phenotype. As can be seen in the figure, the gene related to grape seed germination rate is located on chromosome 4, and there is one significant SNP 4_520551 in this region.

[0035] 4. Obtaining the target SNP Analysis of the 25,000 bp region upstream and downstream of this significant SNP site revealed that this region contains one gene. VIT_04s0008g00560This gene encodes calcineurin-binding protein 1, which may be associated with grape seed germination rate. Haplotype analysis was performed on the identified significant association sites using LDBlockShow (version 1.40) software. Therefore, the haplotype markers associated with grape seed germination rate are located in the grape reference genome (ftp: / / ftp.ensemblgenomes.org / pub / release-23 / plants / fasta / vitis_vinife-). The gene on chromosome 4 of (ra / dna / Vitis_vinifera.IGGP2x.23.dna.toplevel.fa.gz) VIT_04s0008g00560 Positions 482011 (G or A), 485116 (C or T), 489064 (C or T), and 496078 (A or G). Gene. VIT_04s0008g00560 It mainly includes 6 haplotypes ( Figure 2 By comparing the germination rates of different haplotypes, it was found that the dominant haplotype was HAP4, which had the highest germination rate.

[0036] 5. Haplotype analysis and verification of different grape varieties RNA was extracted from leaves of different grape varieties and reverse transcribed into cDNA. Full-length TA cloning was performed using the cDNA as a template, resulting in amplification. Transformation and plating were then performed, followed by single-clone screening, plasmid extraction and sequencing, and sequence analysis and haplotype determination. The amplification system consisted of: 2.5 µL cDNA, 1.0 µL of 7.5 µM upstream primer (SEQ ID No. 1), 1.0 µL of 7.5 µM downstream primer (SEQ ID No. 2), 12.5 µL of 2×q-PCR mixture, and 8.0 µL of ddH2O, for a total volume of 25 µL. The amplification program was: pre-denaturation at 94℃ for 5 min; denaturation at 94℃ for 1 min, annealing at 55℃ for 1 min, extension at 72℃ for 1.5 min, for 40 cycles. Transformation and plating were performed using DH5α chemicompetent cells, incubated on ice for 30 min, heat-shocked at 42℃ for 45 seconds, and then quickly returned to ice for 2-3 minutes. Add 600 μL of LB medium and incubate at 37°C for 1 hour to improve recovery efficiency. Spread the culture on LB plates containing ampicillin and incubate at 37°C for 12-16 hours. For single-clone screening, select at least 10 single clones from each sample to cover allelic diversity. Use colony PCR for initial screening, using universal vector primers (e.g., M13F / R), and confirm the correct insert size. For plasmid extraction and sequencing, use a miniprep kit to extract plasmids at a concentration ≥50 ng / μL, with an A260 / A280 ratio between 1.8 and 2.0. M13 universal primers are recommended for sequencing to ensure full sequence coverage. For sequence analysis and haplotype determination, align the sequencing results to a reference sequence (e.g., NCBI accession number), identify SNP sites, and clarify the haplotype relationships between different varieties (Table 3). Validation showed (Table 3) that the haplotype analysis and resequencing results for different varieties were consistent.

[0037] Table 3. Haplotype analysis and verification of different grape varieties. 6. Expression analysis of key candidate genes Total RNA was extracted from seed tissues of 10 different grape varieties (HAP4: "Black Grape", "False Yellow Grape", "Wine White", "Summer Solstice Red", "White Seigne"; HAP2: "Red Lotus Seed", "Queen of the Vineyard", "Calas Rose", "Lilac", and "Extreme High"). Total RNA was extracted using the CTAB method, and trace amounts of genomic DNA were removed according to the instructions for TaKaRa product DNase I (RNase-free). RNA purity was assessed using a NanoDrop2000 micro-volume spectrophotometer, and 3 μL samples were then subjected to agarose gel electrophoresis to verify RNA integrity. cDNA was synthesized using the RNA samples as templates according to the instructions for the Thermo Product RNA Reverse Transcription Kit.

[0038] Based on the design principles of quantitative polymerase chain reaction (q-PCR) primers, q-PCR primers for candidate genes were designed using Beacon Designer 8 software; for VIT_04s0008g00560 Gene: Forward primer sequence (SEQ ID No. 9): 5'-CACCAAGGAAGCCCAGGAAT-3'; Reverse primer sequence (SEQ ID No. 10): 5'-GATTGCCAGCAGTGCATCAGA-3'; GAPDH was used as an internal control gene, and the procedure was performed according to the instructions of the SYBR® Green Fast q-PCR Mixed Kit. The amplification system contained 2.5 μL cDNA; 1.0 μL each of upstream and downstream primers (7.5 μM); 12.5 μL 2×q-PCR mixture; and 8.0 μL ddH2O, for a total volume of 25 μL. The reaction program consisted of pre-denaturation at 95°C for 1 min, denaturation at 95°C for 10 sec, annealing at 60°C for 20 sec, and extension at 72°C for 30 sec (40 cycles). Three replicate tests were performed, and 2... -ΔΔCt The method calculated the expression of candidate genes in different haplotype grape varieties.

[0039] The results are as follows Figure 3 As shown, the candidate gene was relatively more expressed in haplotype HAP4, which has a high seed germination rate advantage, than in haplotype HAP2, which has a low seed germination rate advantage.

[0040] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A SNP marker related to grape seed germination rate, characterized in that, The SNP marker is located on chromosome 4 of the grape reference genome, version 23. VIT_04s0008g00560 The bases at positions 482011, 485116, 489064, and 496078 are: position 482011, which is either G or A; position 485116, which is either C or T; position 489064, which is either C or T; and position 496078, which is either A or G.

2. The SNP marker according to claim 1, characterized in that, The germination rate of grape seeds is highest when the base at position 482011 is A, the base at position 485116 is T, the base at position 489064 is T, and the base at position 496078 is A.

3. A primer pair for detecting the SNP marker of claim 1 or 2, characterized in that, The sequence of the upstream primer of the primer pair is shown in SEQ ID No. 1, and the sequence of the downstream primer is shown in SEQ ID No.

2.

4. A kit for detecting the SNP marker of claim 1 or 2, characterized in that, It includes the primer pair as described in claim 3.

5. The application of the SNP marker of claim 1 or 2, the primer pair of claim 3, or the kit of claim 4 in determining the germination rate of grape seeds.

6. The application according to claim 5, characterized in that, To determine the germination rate of grape seeds during the grape seedling stage.

7. A method for determining the germination rate of grape seeds, characterized in that, Includes the following steps: 1) RNA was extracted from leaves of different grape varieties and reverse transcribed into cDNA; 2) Using the cDNA described in step 1) as a template, perform full-length TA cloning of the gene using the primer pair described in claim 3 to obtain the amplification result; 3) Based on the amplification results described in step 2), perform transformation and plating, single clone screening, plasmid extraction and sequencing, sequence analysis and haplotype determination.

8. The method according to claim 7, characterized in that, Step 2) The amplification system is as follows: 2.5 µL cDNA, 1.0 µL upstream primer with a concentration of 7.5 µM, 1.0 µL downstream primer with a concentration of 7.5 µM, 12.5 µL 2×q-PCR mixture and 8.0 µL ddH2O, with a total volume of 25 µL.

9. The method according to claim 7, characterized in that, The amplification program in step 2) is as follows: 94 °C pre-denaturation for 5 min; 94 °C denaturation for 1 min, 55 °C annealing for 1 min, 72 °C extension for 1.5 min, for 40 cycles.

10. The method according to claim 7, characterized in that, Step 3) The transformation and plating method is as follows: use DH5α chemocompetent cells, place them on ice for 30 minutes, heat shock them at 42°C for 45 seconds, and quickly return them to ice for 2-3 minutes; add 600 μL of LB medium, shake them at 37°C for 1 hour to improve recovery efficiency; plating them on LB plates containing ampicillin, and incubating them at 37°C for 12-16 hours. Step 3) The method of single-clone screening is to select no less than 10 single clones for each sample to cover allele diversity; use colony PCR for initial screening, using universal primers for vectors, and confirm that the inserted fragment size is correct; Step 3) The plasmid extraction and sequencing method is as follows: plasmid is extracted using a miniprep kit at a concentration ≥50 ng / μL, with an A260 / A280 ratio between 1.8 and 2.0; M13 universal primers are used for sequencing to ensure full sequence coverage. Step 3) The method for sequence analysis and haplotype determination is to align the sequencing results to the reference sequence, identify SNP sites, and clarify the haplotype relationship between different varieties.

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