Cauliflower variety identification method and primer group based on nucleic acid mass spectrometry technology

Abstract

The invention discloses a cauliflower variety identification method based on a nucleic acid mass spectrometry technology and a primer group. The primer group comprises amplification primer pairs and extension primers of 30 cauliflower SNP (Single Nucleotide Polymorphism) sites, wherein the nucleotide sequences of the 30 pairs of amplification primer pairs are respectively shown as SEQ ID NO.1-NO.60; the nucleotide sequences of the 30 extension primers are respectively as shown in SEQ ID NO. 61 to SEQ ID NO. 90. The detection primer group is designed according to the nucleic acid sequences of the 30 SNP sites of the cauliflower, and the cauliflower variety is subjected to SNP typing analysis by using a nucleic acid mass spectrometry technology, so that the result of identifying the cauliflower variety is achieved. The detection method provided by the invention can realize detection of 30 SNP loci in a single tube, and has the characteristics of high throughput, high accuracy, high sensitivity and the like.

Description

Technical Field

[0001] The invention belongs to the field of crop breeding and application technology, and in particular relates to a technology for authenticating crop varieties, which is a cauliflower variety identification method and a primer set based on nucleic acid mass spectrometry technology. Background Art

[0002] brocoli( Brassica oleracea L. var. botrytis ) is also known as cauliflower and cauliflower. It is a subspecies of Brassica oleracea in the Cruciferae family. It has good flavor, rich nutritional value, and anti-cancer effects. It is one of the important vegetable crops in the world. Cauliflower occupies an important position in my country's vegetable industry. With the development of the cauliflower industry, some hybrids have appeared in the seed market, resulting in a decline in the quality and yield of cauliflower, affecting the income of growers; in addition, the emergence of counterfeit seeds disrupts the market order and has an adverse impact on variety innovation, the stability of agricultural production and sustainable development. Cauliflower variety identification plays a vital role in the cauliflower industry. It is an important link in ensuring the quality of cauliflower seeds and the authenticity of varieties. It is of great significance in ensuring national food security, protecting the interests of breeders and breeding companies, and promoting high-yield and high-quality cauliflower varieties.

[0003] As one of the genetically stable polymorphic markers, SNP markers have been widely used in cauliflower variety identification. However, the existing cauliflower variety identification technology is mainly based on competitive allele-specific PCR based on the KASP fluorescence in situ scanning platform. Each reaction system can only detect one SNP site in a single sample. When the number of SNP sites and sample size are large, this method is time-consuming and labor-intensive, and cannot meet the needs of biobreeding industrialization service support. Therefore, it is urgent to develop a new detection method that can simultaneously detect multiple SNP sites in the same sample in one reaction system, and accurately and efficiently, specifically and sensitively, and simply and quickly complete the cauliflower variety identification.

[0004] Nucleic acid mass spectrometry SNP detection technology combines multiplex PCR technology, iPLEX single base extension technology and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to perform genotyping detection on SNP sites. According to the different molecular weights of the extension products, the products of different molecular weights are distinguished by time-of-flight mass spectrometry, thereby achieving the purpose of genotyping. Multiple SNP sites can be detected simultaneously in the same reaction tube, which can meet the large-scale rapid screening of SNP sites, and has the characteristics of high sensitivity, high throughput, high accuracy, low cost, low pollution, easy interpretation of results, and high degree of automation. Since multiple amplifications are to be performed in the same reaction system, the designed primer set should be specific and should avoid mutual interference. Therefore, it is necessary to design the optimal primer combination corresponding to the SNP marker for cauliflower variety identification. Summary of the invention

[0005] In view of the fact that the existing cauliflower variety identification and detection means have low detection throughput and cannot meet the needs of the national biological breeding industry, the main purpose of the present invention is to establish a cauliflower variety identification method and primer set that can simultaneously detect multiple SNP sites.

[0006] A primer set of 30 SNP sites for cauliflower variety identification, SEQ ID NO.1-NO.90, characterized in that the nucleotide sequences of the 30 amplification primer pairs in the primer set are as shown in SEQ ID NO.1-NO.60; the nucleotide sequences of the 30 extension primers in the primer set are as shown in SEQ ID NO.61-NO.90.

[0007]

[0008]

[0009]

[0010]

[0011]

[0012] The amplification primers, extension primers and / or primer sets described in the present invention can be used for amplifying nucleic acid sequences of 30 SNP sites; the length of the amplification primers is 29bp-30bp, and the GC content is 46.67-53.33%; the length of the extension primers is 15bp-28bp, and the GC content is 38.46-70.59%.

[0013] The 30 cauliflower SNP sites are: Chr1:860653[C / G]; Chr1:1581568[C / A]; Chr1:2022083[A / T]; Chr1:9494597[G / T]; Chr1:2022902[C / T]; Chr1:45449565[T / A]; Chr2:3174530[A / G]; hr2:14297682[G / A]; Chr2:39230790[T / A]; Chr2:65005201[C / T]; Chr3:6005226[T / C]; Chr4:14063327[C / G]; Chr4:52102326[G / A]; Chr5:311091[C / T]; Chr5:4246754[ G / C]; Chr5:42917099[G / C]; Chr5:42919228[T / C]; Chr6:34494441[G / C]; Chr6:4058 2776[G / T]; Chr7:6266029[G / A]; Chr7:7108476[T / G]; Chr7:18611643[C / G]; Chr7:4 9710841[C / T]; Chr8:685258[A / G]; Chr8:7993815[A / T]; Chr8:10333745[C / G]; Chr8 :10995531[G / A]; Chr8:23662826[C / A]; Chr8:51980792[T / C]; Chr9:2367375[C / T].

[0014] The cauliflower variety identification method based on nucleic acid mass spectrometry technology of the present invention uses the genomic DNA of the tested cauliflower variety as a template, amplifies 30 SNP sites, and performs SNP typing analysis on the cauliflower, comprising the following steps: The nucleic acid sequences of 30 SNP sites of cauliflower were obtained; the primer sets SEQ ID NO.1-NO.90 were designed; Extracting genomic DNA from a cauliflower sample, using the obtained genomic DNA fragment as a template and using the primer set as an amplification system to perform an amplification reaction to obtain an amplification product; The above amplified product is subjected to SAP digestion, and an extension reaction is performed using the primer set as an extension system to obtain an extension product; The above extension products were subjected to resin desalting treatment, and the obtained samples were subjected to mass spectrometry detection by time-of-flight mass spectrometer to determine the single nucleotide types at 30 SNP sites in the cauliflower samples.

[0015] The present invention also discloses the application of primer genome in genotyping and identification of cauliflower varieties. Experimental results show that the detection method of the present invention can detect 30 SNP sites in a single tube, and has the characteristics of high throughput, high accuracy, high sensitivity, etc.

[0016] The cauliflower variety identification method and primer set based on nucleic acid mass spectrometry disclosed in the present invention have the following advantages compared with the prior art: The present invention discloses a cauliflower variety identification method and primer set based on nucleic acid mass spectrometry technology. The method can detect 30 SNP sites in a single tube, has high accuracy, high sensitivity and high specificity, can solve the defects of other detection technologies such as low throughput, too few reaction sites in a single tube, high cost and complicated operation, and can be applied to cauliflower variety identification.

[0017] The present invention is described in more detail as follows: A cauliflower variety identification method and primer set based on nucleic acid mass spectrometry technology: The primer set for detecting 30 SNP sites in cauliflower has a nucleic acid sequence as shown in SEQ ID NO.1-NO.90. The nucleotide sequences of the 30 pairs of amplification primers in the primer set are shown in SEQ ID NO.1-NO.60; the nucleotide sequences of the 30 extension primers in the primer set are shown in SEQ ID NO.61-NO.90.

[0018] The primer set comprises multiple detection primer pairs; the primer set comprises 30 pairs of amplification primers, the nucleotide sequences of which are shown in SEQ ID NO.1-NO.60, and 30 extension primers, the nucleotide sequences of which are shown in SEQ ID NO.61-NO.90. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1Gel electrophoresis after PCR amplification using the primers in Table 1; Lane M is a marker, and lanes 1-30 are 30 SNP sites: Chr1:860653[C / G]; Chr1:1581568[C / A]; Chr1:2022083[A / T]; Chr1:9494597[G / T]; Chr1:2022902[C / T]; Chr1:4544 9565[T / A]; Chr2:3174530[A / G]; Chr2:14297682[G / A]; Chr2:39230790[T / A]; Chr2:65005 201[C / T]; Chr3:6005226[T / C]; Chr4:14063327[C / G]; Chr4:52102326[G / A]; Chr5:311091 [C / T]; Chr5:4246754[G / C]; Chr5:42917099[G / C]; Chr5:42919228[T / C]; Chr6:34494441 [G / C]; Chr6:40582776[G / T]; Chr7:6266029[G / A]; Chr7:7108476[T / G]; Chr7:18611643[C / G]; Chr7:49710841[C / T]; Chr8:685258[A / G]; Chr8:7993815[A / T]; Chr8:10333745[C / G] ; Chr8:10995531[G / A]; Chr8:23662826[C / A]; Chr8:51980792[T / C]; Chr9:2367375[C / T]. DETAILED DESCRIPTION

[0020] The present invention is described below by specific embodiments. Unless otherwise specified, the technical means used in the present invention are methods known to those skilled in the art. In addition, the embodiments should be understood as illustrative rather than limiting the scope of the present invention, and the essence and scope of the present invention are limited only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments, without departing from the essence and scope of the present invention, also belong to the protection scope of the present invention. Unless otherwise specified, the reagents and materials used in the following examples are commercially available. Example

[0021] 1. Primer Design Combined with the SNP sites reported in the literature for cauliflower variety identification, combined with two groups of core site combinations, 30 SNP sites were selected for primer design, which are: Chr1:860653[C / G]; Chr1:1581568[C / A]; Chr1:2022083[A / T]; Chr1:9494597[G / T ]; Chr1:2022902[C / T]; Chr1:45449565[T / A]; Chr2:3174530[A / G]; Chr2:1429768 2[G / A]; Chr2:39230790[T / A]; Chr2:65005201[C / T]; Chr3:6005226[T / C]; Chr4:1 4063327[C / G]; Chr4:52102326[G / A]; Chr5:311091[C / T]; Chr5:4246754[G / C]; Ch r5:42917099[G / C]; Chr5:42919228[T / C]; Chr6:34494441[G / C]; Chr6:40582776[ G / T]; Chr7:6266029[G / A]; Chr7:7108476[T / G]; Chr7:18611643[C / G]; Chr7:4971 0841[C / T]; Chr8:685258[A / G]; Chr8:7993815[A / T]; Chr8:10333745[C / G]; Chr8: 10995531[G / A]; Chr8:23662826[C / A]; Chr8:51980792[T / C]; Chr9:2367375[C / T].

[0022] Amplification primers (SEQ ID NO.1-NO.60) and extension primers (SEQ ID NO.61-NO.90) were designed according to the above 30 SNP sites, and the primer sequences are shown in Table 1.

[0023]

[0024]

[0025]

[0026]

[0027]

[0028] The amplification primers (SEQ ID NO.1-NO.60) in Table 1 were used to perform PCR amplification on the same cauliflower DNA, and the specificity of each amplification primer was verified by gel electrophoresis. Figure 1 As shown, each amplification primer can successfully amplify specific bands and can be used for subsequent experiments.

[0029] 2. Cauliflower variety identification method based on SNP loci 1. DNA Extraction Cauliflower leaves were collected and the genomic DNA of cauliflower leaves was extracted using the Kangwei New Plant Genomic DNA Extraction Kit (Cat. No.: CW0531M) (refer to the attached instruction manual for specific steps). The extracted DNA concentration was measured using NanoDrop 2000 and diluted to 20 ng / µL with deionized water.

[0030] 2. PCR Amplification The amplification primer mixture is obtained by mixing the amplification primers (SEQ ID NO.1-NO.60) shown in Table 1. The concentration of the amplification primers with sequences shown in SEQ ID NO.1-NO.60 in the amplification primer mixture is 0.5 μM.

[0031] The PCR amplification system is shown in Table 2. In the PCR amplification reaction system, the final concentration of each amplification primer having a sequence as shown in SEQ ID NO.1-NO.60 is 0.1 μM.

[0032] Table 2 PCR amplification reaction system Reagents Volume (μL) Deionized water 1.75 10×PCR Buffer 0.625 MgCl2 (25mM) 0.325 dNTP mix (25 mM ) 0.1 HotStarTaq enzyme (5U / μL) 0.2 Amplification primer mix 1 DNA 2 Total volume 5 The PCR amplification reaction conditions are shown in Table 3.

[0033] Table 3 PCR amplification reaction conditions

[0034] 3.SAP purification reaction Prepare the SAP mixture according to Table 4 and add it to the above amplification reaction wells.

[0035] Table 4 SAP digestion reaction system Reagents Volume (μL) Deionized water 1.53 SAP Buffer (10 X) 0.17 SAP Enzyme (1.7U / μL) 0.3 Total Volume 2 The SAP purification reaction conditions are shown in Table 5.

[0036] Table 5 SAP digestion reaction conditions Temperature (℃) time Number of cycles 37 40s 1 85 5s 1 4 ∞ 1 4. Single base extension reaction A single base extension reaction was performed. The extension primer mixture was obtained by mixing the extension primers (SEQ ID NO.61-NO.90) shown in Table 1. The concentrations of the extension primers with sequences shown in SEQ ID NO.61-NO.90 in the extension primer mixture were shown in Table 6.

[0037]

[0038] The single base extension reaction system is shown in Table 7.

[0039] Table 7 Single base extension reaction system Reagents Volume (μL) Deionized water 0.619 iPLEX Buffer plus 0.2 iPLEX terminator 0.2 iPLEX enzyme 0.041 Extension Primer Mix 0.94 Total volume 2 The single base extension reaction conditions are shown in Table 8.

[0040]

[0041] The reagents used in the above-mentioned PCR amplification reaction, SAP digestion reaction and single base extension reaction are all Agena chip supporting reagents, and the Agena chip supporting reagents are shown in Table 9.

[0042]

[0043] 5. Nucleic acid mass spectrometry detection 18 µL of deionized water was added to the reaction product, mixed well, and then 6 mg of resin was added for desalting. The mixture was rotated for 15 minutes and centrifuged at 2000 rpm for 5 minutes. The desalted sample was tested by mass spectrometer.

[0044] Import the edited site assay information into the Typer software, select the reaction well position of the test sample to build the plate, import the corresponding sample name, connect the test chip, place the 384-well plate and the test chip in the corresponding position of the mass spectrometer, and start the time-of-flight mass spectrometry detection.

[0045] 6. Experimental Results The extension products were subjected to mass spectrometry analysis, the flight time of the extension products in the vacuum tube was detected, the flight mass spectrum peak of the extension products was obtained, the molecular weight of the extension products and the preset molecular weight were calculated (Table 10), the single nucleotide type represented by the peak of the extension products was determined, and the sample genotype test results were obtained. The test results of the 6 samples are shown in Table 11.

[0046]

[0047]

[0048]

[0049]

[0050]

[0051] It can be seen from the test results that the cauliflower variety identification method based on nucleic acid mass spectrometry technology and the primer set of the present invention can successfully detect the genotypes of the 30 SNP sites in the above 6 samples.

[0052] The above embodiments are only preferred implementation modes of the present invention and are only descriptions of the preferred implementation modes of the present invention, but are not intended to limit the scope of the present invention. Without departing from the design spirit and principles of the present invention, various changes, modifications, substitutions, combinations, simplifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field are included in the protection scope of the present invention.

Claims

1. A primer genome for cauliflower variety identification based on nucleic acid mass spectrometry technology, characterized in that: The primer set includes amplification primers; the nucleotide sequences of the amplification primers are shown in SEQ ID NO.1-NO.

60.

2. A primer genome for cauliflower variety identification based on nucleic acid mass spectrometry technology, characterized in that: The primer set includes an extension primer; the nucleotide sequence of the extension primer is shown as SEQ ID NO.61-SEQ ID NO.

90.

3. A primer genome for cauliflower variety identification based on nucleic acid mass spectrometry technology, characterized in that: The primer set comprises 30 pairs of amplification primers SEQ ID NO.1-NO.60 described in claim 1 and 30 extension primers SEQ ID NO.61-NO.90 described in claim 2: 。 4. Use of the amplification primer according to claim 1, the extension primer according to claim 2 and / or the primer set according to claim 3 for genotyping cauliflower varieties.

5. Use of the amplification primer according to claim 1, the extension primer according to claim 2 and / or the primer set according to claim 3 in identifying cauliflower varieties.

6. A method for identifying cauliflower varieties based on nucleic acid mass spectrometry technology, characterized in that: The method comprises: Obtaining nucleic acid sequences of 30 SNP sites of cauliflower; designing the primer genome described in claims 1-3; Extracting genomic DNA from a cauliflower sample, using the obtained genomic DNA fragment as a template, and performing an amplification reaction using the primer set described in claim 3 as an amplification system to obtain an amplified product; The amplified product is subjected to SAP digestion, and an extension reaction is performed using the primer set described in claim 3 as an extension system to obtain an extension product; The above extension products were subjected to resin desalting treatment, and the obtained samples were detected by time-of-flight mass spectrometer to determine the single nucleotide types at 30 SNP sites in the cauliflower samples.

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