High-precision grape variety identification DNA bar code method based on whole genome variation map

Abstract

The invention discloses a high-precision grape variety identification DNA bar code method based on a whole genome variation map, and relates to the technical field of grape cultivation, the method comprises the following steps: 1, selecting main representative grape cultivation varieties covering wine brewing, drying and fresh eating from different grape cultivation areas in the world, and obtaining a plurality of individuals as samples; 2, extracting genome DNA from the grape leaves by using a CTAB method, and re-sequencing the extracted DNA by using a high-throughput sequencing technology; 3, comparing the re-sequencing data to a reference genome, screening out SNP and InDel variation sites with variety specificity, annotating the SNP and InDel variation sites, and selecting out proper variation sites as DNA bar codes; and 4, aiming at the screened DNA bar code, extracting upstream and downstream 150bp corresponding sequences of a variation site of the DNA bar code, and designing a primer by using primer3 software.

Description

Technical Field

[0001] The present invention relates to the technical field of grape cultivation, and in particular to a DNA barcode method for high-precision grape variety identification based on a whole genome variation map. Background Art

[0002] With the continuous development of grape cultivation and the improvement of varieties, the unique variations of different varieties have become an important basis for variety differentiation. These variations are not only reflected in morphological characteristics, but also show obvious differences at the genetic level. However, traditional grape variety identification mainly relies on morphological characteristics, cultivation habits and fruit characteristics. Since grapes are woody vines with a long growth period, these methods are not only time-consuming and laborious, but also limited by grape growth and changes in environmental factors, which can easily lead to misjudgment. Therefore, accurate and rapid identification and differentiation of different grape cultivars is of great significance in grape planting management, maintaining variety purity, and coping with environmental stress, and has extremely high application value in preventing variety mixing, improving the market competitiveness of grape products, and protecting intellectual property rights.

[0003] Accurate identification of grape cultivars is of great significance for horticultural production, product quality and scientific research and breeding of grapes. However, traditional identification methods mainly rely on the external morphological characteristics of grapes (such as leaf morphology, berry shape, color, etc.) and growth traits (such as maturity, disease resistance, etc.). These methods have the disadvantages of being greatly affected by the environment, relying on the specific phenological period of grapes, being cumbersome and time-consuming, having low resolution, relying on the subjectivity of the identifier, and being unable to provide DNA-level characteristics. They cannot meet the needs of modern agricultural production and scientific research that require high precision and high efficiency.

[0004] Traditional DNA barcodes are mainly designed based on fragments of conserved sequences and contain limited genetic information. These single fragments have low resolution for closely related species and can easily lead to identification errors. Moreover, these barcodes have poor adaptability and are weak in detecting closely related species, hybrids, and variants. At the same time, these barcodes have weak ability to identify intraspecific variations and are mainly used for interspecific identification. Most importantly, traditional barcodes are generally based on Sanger sequencing technology, which has a limited number of samples that can be processed and has low throughput, making them unsuitable for large-scale, high-throughput research. Summary of the invention

[0005] In view of the shortcomings of the prior art, the technical solution adopted by the present invention to solve the technical problems is: to provide a method for developing a high-precision grape variety identification DNA barcode using a whole genome variation map. Specifically, firstly, the whole genome resequencing data of different grape varieties are compared with the reference genome and the variation sites are extracted to construct a whole genome variation map. Then, high-quality and population-specific variation sites are filtered out and annotated. Suitable variation sites are selected as DNA barcodes, and specific primers are designed for each DNA barcode for identification of grape cultivars.

[0006] Through these DNA barcodes, different grape varieties can be accurately and quickly identified, overcoming the limitations of traditional identification methods and providing an effective tool for grape breeding, planting management and product quality control. The specific scheme of the present invention is as follows:

[0007] The present invention uses whole genome re-testing technology to develop DNA barcodes for nine grape cultivars. These markers are stable nucleic acid sequences that can show significant differences between different varieties, thereby achieving accurate variety differentiation. Specifically, the following steps are included:

[0008] Step 1: Sample collection: 9 major representative cultivars covering winemaking, dried wine making and fresh consumption were selected from different grape-growing areas around the world, including Chardonnay, Cabernet Sauvignon, Merlot, Pinot Noir, Riesling, Sauvignon Blanc, Thopson Seedless, Zinfandel and Merlot, totaling 477 individuals.

[0009] Step 2: Genomic DNA extraction and resequencing: Genomic DNA was extracted from grape leaves using the CTAB method to ensure DNA purity and integrity.

[0010] Step 3: Screening for specific variant sites: Use high-throughput sequencing technology to resequence the whole genomes of multiple grape varieties, and use bioinformatics analysis to compare the resequencing data to the reference genome of Cabernet Sauvignon grapes to screen out variety-specific SNP (single nucleotide polymorphism) and InDel (insertion / deletion) variant sites. These variant sites are then annotated. Appropriate variant sites will be used as DNA barcodes and targeted primers will be designed.

[0011] Step 4: Primer design: Extract the corresponding sequence of 150bp upstream and downstream of the mutation site, use primer3 to design primers for the template, and screen out primer sequences with similar Tm for final variety identification. The primer design principles are as follows:

[0012] The primer length is generally 18-27 bp, the annealing temperature is 55-60°C, the GC content is controlled between 40% and 60%, and the 3' end of the primer should avoid forming a hairpin structure or dimer. The DNA barcode of the present invention can accurately identify multiple grape varieties.

[0013] The beneficial effects of the present invention are as follows:

[0014] 1. The variant site identification and DNA barcode development method proposed in the present invention integrates the methods of resequencing data comparison, variant site detection, DNA barcode development and primer design, and can design detection primers for a large number of identified variant sites in an individualized, accurate and efficient manner. Under the premise of ensuring accuracy, the efficiency and accuracy of identification are greatly improved, which is of great significance in practical applications.

[0015] 2. This method is accurate, efficient and high-throughput, and can be widely used in grape production, breeding, intellectual property protection and other fields. It fills the shortcomings and gaps of existing traditional DNA barcodes and reliance on morphological characteristics in grape variety identification. Compared with traditional morphological identification methods, the method of the present invention is easy to operate, has a short detection time and higher throughput. DNA barcode markers can effectively distinguish grape varieties with similar appearance or close relationship, and can be widely used in vineyard management, variety authenticity verification and new variety development. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a flow chart of the present invention. DETAILED DESCRIPTION

[0017] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. The embodiments of the present invention are provided for the purpose of illustration and description, and are not intended to be exhaustive or to limit the present invention to the disclosed forms. Many modifications and variations will be apparent to those of ordinary skill in the art. The embodiments are selected and described in order to better illustrate the principles and practical applications of the present invention, and to enable those of ordinary skill in the art to understand the present invention and thereby design various embodiments with various modifications suitable for specific uses.

[0018] Example:

[0019] A high-precision DNA barcoding method for grape variety identification based on a whole-genome variation map, including:

[0020] Step 1: Select major representative grape cultivars covering winemaking, dried grapes, and fresh grapes from different grape-growing areas around the world, and obtain multiple individuals as samples;

[0021] Step 2: Use the CTAB method to extract genomic DNA from grape leaves, and use high-throughput sequencing technology to resequence the extracted DNA;

[0022] Step 3: Align the resequencing data to the reference genome, screen out species-specific SNP and InDel variant sites, annotate them, and select appropriate variant sites as DNA barcodes. During the screening, MAF is set to be no less than 0.1, MinDP is set to be no less than 40, and Max-missing is set to be no more than 1;

[0023] Step 4: For the screened DNA barcodes, extract the corresponding sequences 150bp upstream and downstream of the variant sites, and use primer3 software to design primers with a primer length of 18-27bp, an annealing temperature of 55-60°C, a GC content controlled between 40%-60%, and the 3' end of the primer should avoid forming a hairpin structure or dimer.

[0024] In step 1, the selected grape cultivars include Chardonnay, Cabernet Sauvignon, Merlot, Pinot Noir, Riesling, Sauvignon Blanc, Seedless White, Zinfandel, and Merlot.

[0025] In step 2, the quality of the extracted DNA was tested by agarose gel electrophoresis and NanoDrop photometer, and resequencing was performed using Illumina sequencing technology to generate whole-genome sequencing data with 30x coverage. The data was cleaned and filtered using quality control software to remove low-quality sequences and adapter sequences.

[0026] In step 3, the reference genome is the reference genome of Cabernet Sauvignon grapes, the alignment software uses BWA or Bowtie2, the alignment results are evaluated using SAMtools software, and variation detection is performed using GATK software.

[0027] Step 4 also includes screening and optimization of primers, selecting non-synonymous mutations or frameshift mutation sites that can cause changes in protein function for primer design, and screening out primer pairs with similar annealing temperatures based on the return temperature Tm value.

[0028] The specific steps for primer screening and optimization are as follows:

[0029] Step A1: Use SnpEff software to analyze the screened specific SNP and InDel sites to determine the sites that are non-synonymous mutations, i.e., mutations that cause changes in the amino acid sequence, and frameshift mutations, i.e., insertions or deletions that cause reading frame shifts and affect protein structure.

[0030] For these key mutation sites, Primer3 software was used to design primers, and the primer length was set in the range of 18 to 27 bases to ensure that the primers had both specificity and amplification efficiency; the GC content of the primers was regulated in the range of 40% to 60% to prevent high GC content from inducing primer secondary structure; the annealing temperature Tm was controlled between 55°C and 60°C to avoid adverse effects of inappropriate annealing temperature on the PCR reaction, generating a series of candidate primer pairs.

[0031] Step A2: Sort the primer pairs according to their annealing temperature Tm values, screen out primer pair combinations with Tm values ​​within ±2°C by comparing the differences in Tm values ​​of adjacent primer pairs, and calculate the annealing temperature Tm values ​​of all designed candidate primer pairs one by one. These primer pairs with similar Tm values ​​can achieve effective annealing under similar temperature conditions during subsequent PCR amplification, which helps maintain the stability and consistency of the amplification reaction and reduces the probability of nonspecific amplification or low amplification efficiency caused by annealing temperature fluctuations.

[0032] Step A3: The selected primer pairs with similar Tm values ​​are compared with the whole genome sequence of grapes, and the sequence comparison algorithm is used to check whether the binding site of the primers in the genome is unique, to ensure that the primers will not mismatch or bind to non-target regions. If the primers are found to have the risk of binding to non-target highly homologous regions, they will be excluded. After strict specificity verification, the primer pairs with strong specificity, high amplification efficiency and similar Tm values ​​are determined from the remaining primer pairs as the final primer combination for grape variety identification.

[0033] The development of DNA barcodes and primer design based on the whole genome variation map of Chardonnay varieties were carried out as an example. Samples were obtained from healthy fresh leaves of Chardonnay grape plants, and genomic DNA was extracted using the conventional CTAB method or a commercially available DNA extraction kit. The quality of the extracted DNA was detected by agarose gel electrophoresis and NanoDrop photometer to ensure that the sample had sufficient concentration and purity for subsequent resequencing analysis. The extracted high-quality Chardonnay genomic DNA was sent to a high-throughput sequencing platform for resequencing, and Illumina sequencing technology was used to generate 30x coverage whole genome sequencing data. The sequencing data was cleaned and filtered by quality control software (FastQC) to remove low-quality sequences and adapter sequences.

[0034] The cleaned Chardonnay genome sequence was aligned with the reference genome of Cabernet Sauvignon (version: Vitis vinifera 12X.v2 or the latest version). The alignment software used was BWA (Burrows-Wheeler Aligner) or Bowtie2 to generate an alignment file (BAM format). The SAMtools software was used to evaluate the alignment results to ensure the coverage and accuracy of the alignment and filter out sequences with multiple alignments or low-quality alignments.

[0035] GATK (Genome Analysis Toolkit) was used to detect SNP and InDel sites in the data comparison and generate a variant site file (VCF format). The following filtering parameters were used to filter the initially detected SNPs and InDels:

[0036] MAF (Minor Allele Frequency): set to 0.05 to ensure that the selected loci have a higher frequency in the Chardonnay population.

[0037] MinDP (Minimum Depth): Set to 40 to ensure that the sequencing coverage of the variant sites is high enough to exclude false positives caused by sequencing errors.

[0038] Max-missing (maximum missing rate): set to 1, requiring all samples to have valid genotype data at the target site.

[0039] The SNP and Indel mutation sites and primer sequences of the nine grape cultivars are shown in Appendix 1 and Appendix 2;

[0040] Appendix 1. SNP variant sites and primers of 9 asexually propagated cultivars

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[0045] Appendix 2. Indel mutation sites and primers of 9 asexually propagated cultivars

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[0052] The screened SNP and InDel sites are compared with populations of other grape varieties (such as Cabernet Sauvignon, Pinot Noir, etc.) to select variant sites unique to the Chardonnay variety. These specific sites must be fixed in the Chardonnay population and not present in other varieties.

[0053] Use SnpEff or ANNOVAR software to annotate the specific SNP and InDel sites screened out, and clarify the gene segment where each variant site is located and its possible functional impact. According to the SnpEff annotation results, focus on variant sites that may cause changes in protein function, such as reading frame shifts caused by insertions or deletions, thereby affecting protein structure. Select appropriate variant sites as DNA barcodes and design primers for each DNA barcode.

[0054] Primer3 software was used to design primers for the specific SNP and InDel sites screened. The design principles include:

[0055] Design primers with a length of 18 to 27 bases to ensure appropriate specificity and amplification efficiency; control the Tm value of the primers between 55°C and 60°C to avoid too low or too high annealing temperature affecting the PCR reaction; keep the GC content of the primers between 40% and 60% to avoid the formation of primer secondary structure caused by high GC content.

[0056] Ensure that primer design targets locations around specific variant sites and avoid designing into regions with high homology in the genome, thereby improving primer specificity and amplification efficiency.

[0057] Prioritize non-synonymous mutations or frameshift mutations that can lead to changes in protein function for primer design, and further screen the most suitable primer pairs for amplification based on the design results. Ensure that the selected primer pairs have similar annealing temperatures (Tm) to optimize PCR amplification conditions and reduce the risk of nonspecific amplification or inefficient amplification.

[0058] Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field and related fields without creative work should fall within the scope of protection of the present invention. The structures, devices and operating methods not specifically described and explained in the present invention are implemented according to the conventional means in the field unless otherwise specified and limited.

Claims

1. A high-precision DNA barcoding method for grape variety identification based on a whole genome variation map, characterized in that: include: Step 1: Select major representative grape cultivars covering winemaking, dried grapes, and fresh grapes from different grape-growing areas around the world, and obtain multiple individuals as samples; Step 2: Use the CTAB method to extract genomic DNA from grape leaves, and use high-throughput sequencing technology to resequence the extracted DNA; Step 3: Align the resequencing data to the reference genome, screen out species-specific SNP and InDel variant sites, annotate them, and select appropriate variant sites as DNA barcodes; Step 4: For the screened DNA barcodes, extract the corresponding sequences 150bp upstream and downstream of the mutation site, and use primer3 software to design primers.

2. The high-precision grape variety identification DNA barcoding method based on the whole genome variation map according to claim 1, characterized in that: In the step 1, the selected grape cultivars include Chardonnay, Cabernet Sauvignon, Merlot, Pinot Noir, Riesling, Sauvignon Blanc, Seedless White, Zinfandel, and Merlot.

3. The high-precision grape variety identification DNA barcoding method based on whole genome variation map according to claim 1, characterized in that: In the step 2, the quality of the extracted DNA is detected by agarose gel electrophoresis and NanoDrop photometer, and the resequencing adopts Illumina sequencing technology to generate 30x coverage whole genome sequencing data, and the data is cleaned and filtered by quality control software.

4. The high-precision grape variety identification DNA barcoding method based on whole genome variation map according to claim 1, characterized in that: In the step 3, the reference genome is the reference genome of Cabernet Sauvignon grapes, the alignment software uses BWA or Bowtie2, the alignment results are evaluated using SAMtools software, and variation detection is performed using GATK software.

5. The high-precision grape variety identification DNA barcoding method based on whole genome variation map according to claim 1, characterized in that: The step 4 also includes screening and optimizing primers, selecting non-synonymous mutations or frameshift mutation sites that can cause changes in protein function for primer design, and screening primer pairs with similar annealing temperatures based on the return temperature Tm value.

6. The high-precision grape variety identification DNA barcoding method based on whole genome variation map according to claim 5, characterized in that: The specific steps of screening and optimizing the primers are as follows: Step A1: Use SnpEff software to analyze the screened specific SNP and InDel sites, determine the sites that belong to non-synonymous mutations and frameshift mutations, enable Primer3 software to carry out primer design, set the primer length in the range of 18 to 27 bases, adjust the GC content of the primer in the range of 40% to 60%, and control the annealing temperature Tm between 55°C and 60°C to generate a series of candidate primer pairs. Step A2: Sort the primer pairs according to their annealing temperature Tm values, screen out primer pair combinations with Tm value differences within ±2°C by comparing the differences in Tm values ​​of adjacent primer pairs, and calculate the annealing temperature Tm values ​​of all designed candidate primer pairs one by one. Step A3: The screened primer pairs with similar Tm values ​​are compared and analyzed with the grape genome sequence. The sequence alignment algorithm is used to check whether the primer binding site in the genome is unique. If it is found that the primer has the risk of binding to non-target highly homologous regions, it is excluded.

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