A primer set for identifying cabbage vegetable varieties, a kit and application thereof

Abstract

The present disclosure provides a primer set for identifying cabbage vegetable varieties and a kit and application thereof, which belongs to the field of gene detection. The primer set includes: the first primer pair to the 147th primer pair, each of which includes a forward primer and a reverse primer, and the forward primer of the first primer pair, the reverse primer of the first primer pair to the forward primer of the 147th primer pair, and the reverse primer of the 147th primer pair are shown in sequence as SEQ ID NO: 1 to SEQ ID NO: 294 in the sequence table. The primer set provided by the present disclosure is used to identify cabbage vegetable varieties and evaluate the genetic diversity of different varieties of cabbage vegetables, and the results are accurate.

Description

Technical Field

[0001] The present disclosure relates to the field of gene detection, and in particular to a primer set for identifying cabbage vegetable varieties, a kit and an application thereof. Background Art

[0002] Cabbage vegetables mainly include: broccoli, cauliflower, cabbage, kale, Brussels sprouts, Brussels sprouts and kale, etc. They are edible with flower heads, leaves, leaf heads, shortened corms, flower stalks, side shoots or flower buds.

[0003] In recent years, the planting area of ​​broccoli in my country has been increasing. Therefore, achieving localization of broccoli varieties and replacing imported varieties is the primary goal of broccoli breeding. However, traditional molecular marker detection technologies such as RAPD, SSR and KASP are time-consuming, high-priced and low-throughput, and are not suitable for molecular marker-assisted breeding across the entire genome; gene chips are expensive and the core technology is owned by foreign countries, which cannot be applied on a large scale and cannot meet personalized needs. At the same time, there are technical barriers. Given the current international situation, it is not conducive to the safety of domestic breeding. Therefore, the development of a low-cost, high-throughput molecular marker detection system covering the entire genome of broccoli is of great significance for the molecular breeding of broccoli. It can also be applied to the molecular breeding of a variety of important cabbage vegetables belonging to the same cabbage variant C genome, such as cabbage, cauliflower, kale, Brussels sprouts and Brussels sprouts, to achieve the process and informationization of molecular design breeding of cabbage vegetables. It can not only be used to evaluate the genetic diversity of different varieties of cabbage vegetables, but also can be used in backcross breeding, gene positioning, variety authenticity analysis and other fields, greatly improving the breeding efficiency of cabbage and other cabbage vegetables in my country, shortening the breeding cycle, and promoting the development of vegetable seed industry.

[0004] Public Content

[0005] In order to solve the problems of the prior art, the present disclosure provides a primer set and a kit and application thereof for identifying cabbage vegetable varieties. The technical solution is as follows:

[0006] On the one hand, the present disclosure provides a primer set for identifying cabbage vegetable varieties, the primer set comprising: the 1st primer pair to the 147th primer pair, each of the primer pairs comprising a forward primer and a reverse primer, the forward primer of the 1st primer pair, the reverse primer of the 1st primer pair to the forward primer of the 147th primer pair, and the reverse primer of the 147th primer pair are respectively as shown in SEQ ID NO: 1 to SEQ ID NO: 294 in the sequence listing.

[0007] In another aspect, the present disclosure provides a kit for identifying cabbage vegetable varieties, the kit comprising the above primer set.

[0008] In yet another aspect, the present disclosure provides an application of a primer set for identifying cabbage vegetable varieties, the application comprising: using the primer set for identifying cabbage vegetable varieties.

[0009] Specifically, the application includes: using the above primer set to identify broccoli varieties.

[0010] Specifically, the application includes: using the above primer set to evaluate the genetic diversity of different varieties of cabbage vegetables.

[0011] Furthermore, the application also includes:

[0012] Extracting genomic DNA from the sample to be tested;

[0013] Using the primer set to perform a first round of PCR amplification on the genomic DNA of the sample to be tested to obtain a first round of amplification products;

[0014] Using the first round amplification product as a template and the primer set to perform a second round of PCR amplification to obtain a second round of amplification product;

[0015] mixing the second round amplification products to obtain a mixed amplification product;

[0016] Purifying the mixed amplification product and performing sequencing analysis to obtain raw data;

[0017] The raw data were analyzed, and the pairwise genetic distance matrix was calculated using MEGA software to construct a phylogenetic tree;

[0018] The phylogenetic tree is visualized using iTOL to obtain the differentiation order branch tree and evolutionary relationship between the samples to be tested, which is used to evaluate the genetic diversity of different varieties of the cabbage vegetables and identify the cabbage vegetable varieties.

[0019] Furthermore, each 10 μL of the reaction system for the first round of PCR amplification includes: 1.0 μL of genomic DNA, 2.5 μL of 4×VAHTS Multi-PCR Mix, 1.0 μL of primer set and 5.5 μL of water.

[0020] Furthermore, each 10 μL of the reaction system of the second round of PCR amplification includes: 1.0 μL of the diluted first round amplification product, 2.5 μL of 4×VAHTS Multi-PCRMix, 1.5 μL of primer set, 0.5 μL of barcode and 4.5 μL of water.

[0021] Specifically, the application includes: using the primer set in cabbage backcross breeding and background selection, molecular marker-assisted breeding, and whole genome breeding.

[0022] Specifically, the application includes: using the above primer set in cabbage germplasm resource genotyping, species evolution analysis, seed purity detection, and variety authenticity analysis.

[0023] The beneficial effects of the technical solution provided by the embodiments of the present disclosure are as follows: the embodiments of the present disclosure provide a primer set for identifying cabbage vegetable varieties, a kit and an application thereof, the primer set is used to identify cabbage vegetable varieties and evaluate the genetic diversity of different varieties of cabbage vegetables, and the results are accurate. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present disclosure, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present disclosure. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0025] Figure 1 This is a schematic diagram of the distribution of 147 SNP sites on chromosomes provided in Example 1 of the present disclosure;

[0026] Figure 2 is a schematic diagram of the phylogenetic tree of 114 Brassica vegetables provided in Example 3 of the present disclosure;

[0027] Figure 3 It is a heat map of similarity analysis results in the backcross population provided in Example 4 of the present disclosure. DETAILED DESCRIPTION

[0028] In order to make the objectives, technical solutions and advantages of the present disclosure more clear, the embodiments of the present disclosure will be further described in detail below with reference to the accompanying drawings.

[0029] The plant materials used in the embodiments of the present invention are all from the laboratory of Horticulture Research Institute of Shanghai Academy of Agricultural Sciences.

[0030] Embodiment 1

[0031] The present embodiment provides a primer set for identifying cabbage vegetable varieties, the primer set comprising: the 1st primer pair to the 147th primer pair, each primer pair comprising a forward primer and a reverse primer, the forward primer of the 1st primer pair, the reverse primer of the 1st primer pair to the forward primer of the 147th primer pair, and the reverse primer of the 147th primer pair are respectively as shown in SEQ ID NO: 1 to SEQ ID NO: 294 in the sequence listing.

[0032] The first primer pair to the 147th primer pair correspond to 147 SNP markers, respectively. Figure 1As shown, the distribution of the 147 SNP markers on chromosomes is as follows: 12 SNPs on chromosome C01; 16 SNPs on chromosome C02; 19 SNPs on chromosome C03; 14 SNPs on chromosome C04; 13 SNPs on chromosome C05; 13 SNPs on chromosome C06; 13 SNPs on chromosome C07; 18 SNPs on chromosome C08; 29 SNPs on chromosome C09; about 16 SNPs per chromosome on average. The primer pairs in the primer set provided in this embodiment are mixed together at equimolar concentrations and can be amplified in the same PCR reaction.

[0033] The primer set provided in this example is shown in Table 1.

[0034] Table 1 shows the information of 147 SNP sites and the corresponding primer pair sequences

[0035]

[0036]

[0037]

[0038]

[0039]

[0040] In Table 1, base 1 and base 2 are two single nucleotide polymorphisms (SNPs) to be detected.

[0041] Embodiment 2

[0042] This embodiment provides a kit for identifying cabbage vegetable varieties, and the kit includes the primer set provided in Example 1.

[0043] Embodiment 3

[0044] This embodiment provides an application of a primer set for identifying cabbage vegetable varieties, and the application includes: using the primer set provided in Example 1 to identify cabbage vegetable varieties.

[0045] Specifically, the application includes: using the primer set provided in Example 1 to identify broccoli varieties.

[0046] Specifically, the application includes: using the above primer set to evaluate the genetic diversity of different varieties of cabbage vegetables.

[0047] Furthermore, the application also includes:

[0048] Extracting genomic DNA from the sample to be tested, preferably from young and tender tissue of the sample to be tested;

[0049] The first round of PCR amplification is performed on the genomic DNA of the sample to be tested using the primer set to obtain the first round of amplification products;

[0050] A second round of PCR amplification is performed using the first round amplification product as a template and a primer set to obtain a second round of amplification product;

[0051] mixing the second round amplification products to obtain a mixed amplification product;

[0052] The mixed amplification products are purified and sequenced to obtain raw data;

[0053] The original data were analyzed, and the pairwise genetic distance matrix was calculated using MEGA software to construct a phylogenetic tree;

[0054] The phylogenetic tree was visualized using iTOL to obtain the differentiation order branch tree and evolutionary relationship between at least two samples to be tested, which was used to evaluate the genetic diversity of different varieties of cabbage vegetables and identify cabbage vegetable varieties.

[0055] Young and tender leaves of broccoli were selected as samples to be tested. In this embodiment, 114 different types of cabbage vegetable varieties were selected, including: 12 kale, 30 broccoli, 24 cauliflower and 48 cabbage.

[0056] The genomic DNA from young leaves of broccoli was extracted using the modified CTAB (cetyltrimethylammonium bromide) method. The specific extraction method is as follows: take a thumb-sized young leaf and place it in a 2mL centrifuge tube and mark the number; add 2 steel balls to the centrifuge tube, then add 100μL CTAB, and use a grinder to fully grind the sample to be tested. After grinding, centrifuge lightly, open the lid and pour out the steel balls, then add 500μL CTAB to the centrifuge tube, and mix it upside down; put it in a 65℃ water bath for 40min, and mix it upside down every 10min; add an equal volume of 24:1 (chloroform: isoamyl alcohol) to the centrifuge tube, and shake it thoroughly; centrifuge it in a centrifuge at 12,000r / min at 4℃ for 15min, then take it out and aspirate the supernatant. Take another new 1.5mL centrifuge tube, add 400μL of isopropanol to the new centrifuge tube, then add 400μL of supernatant, mix thoroughly by inversion, and store at -20℃ for 10min; centrifuge at 12,000r / min at 4℃ for 5min, take out, discard the supernatant, retain the precipitate, add 800μL of 75% ethanol to each tube and wash twice; after drying in a fume hood, add 50μL of 0.1% RNase to dissolve the genomic DNA, and place in a water bath at 37℃ for 30min to fully dissolve the genomic DNA, cool and store at -20℃ for later use.

[0057] The quality of genomic DNA was tested by agarose gel electrophoresis and ultraviolet spectrophotometer, respectively. Specifically, agarose gel electrophoresis should show that the main band of genomic DNA is complete and clear, indicating that there is no degradation and RNA contamination; the OD260 / 280 ratio detected by ultraviolet spectrophotometer should be between 1.8 and 2.2, indicating that there is no protein and visible impurity contamination. The quality assessment test results of genomic DNA provided in this example all meet the relevant quality requirements, and subsequent experiments can be carried out.

[0058] The genomic DNA of the samples to be tested (114 different varieties of cabbage vegetables) was used as a template to amplify the target site using VAHTS Multi-PCR Mix to obtain a large number of first-round amplification products (fragments).

[0059] Specifically, each 10 μL of the first round PCR amplification reaction system includes: 1.0 μL of genomic DNA of the sample to be tested, 2.5 μL of 4×VAHTS Multi-PCR Mix, 1.0 μL of primer set (forward primer and reverse primer are both 0.2 μM) and 5.5 μL of water.

[0060] The reaction procedure for the first round of PCR amplification is:

[0061]

[0062] Specifically, each 10 μL second-round PCR amplification reaction system includes: 1.0 μL diluted first-round amplification product (in this embodiment, the dilution concentration is 0.2 μM for each forward primer and each reverse primer), 2.5 μL 4×VAHTS Multi-PCR Mix, 1.5 μL primer set, 0.5 μL barcode and 4.5 μL water.

[0063] The reaction procedure for the second round of PCR amplification is:

[0064]

[0065] After two rounds of PCR amplification, the second round of amplification products are mixed to obtain a mixed amplification product; the mixed amplification product is purified using AMPure XP Beads to obtain a purified mixed amplification product for library construction. After the library is constructed, it needs to undergo quality control, that is, the library is quality controlled and quantified by agarose gel electrophoresis, real-time fluorescence quantitative PCR and Agilent 2100 respectively. After the library is qualified, the second-generation sequencing platform Illumina HiSeq is used to perform Target-seq sequencing on the sequencing library to obtain the original data. AMPure XP Beads will be used for purification. After the library is constructed, it needs to undergo quality control, that is, gel electrophoresis, real-time fluorescence quantitative PCR and Agilent 2100 are used to perform quality control and quantification. After the library is qualified, the second-generation sequencing platform Illumina HiSeq is used to perform Target-seq sequencing on the sequencing library to obtain the original data.

[0066] The raw data obtained by sequencing was subjected to data quality control to obtain high-quality clean data (valid data), and then the clean data was decomposed into each target site using BWA software to obtain the alignment results in SAM format, and then the SAM format file was converted into BAM format using the software samtools, and then the reads in the BAM file were sorted using the SortSam in the Picard tool to obtain the final BAM file. The genotype of each target site can be determined using GATK. This result achieved the detection of 114×147=79800 markers in one experiment, of which 84.62% of the genotypes had a coverage depth of more than 30×, totaling 67,527, and the average coverage depth of each site was 171.96×. The genotyping results showed that only 5 sites (3.43%) did not detect the target sequence, and the proportion of valid sites reached 96.57%. As can be seen from the above, the primer set provided in this embodiment embodies the efficiency and accuracy of SNP-Panel.

[0067] Based on the genotyping results of these 114 cabbage vegetables, the pairwise genetic distance matrix was calculated, and the neighbor-joining method in MEGA7 software (version 7.0) was used to construct the evolutionary tree. Finally, the evolutionary tree was visualized using iTOL software (version 6.9) to show the closeness of the relationship between the samples to be tested. The results are shown in Figure 2. Figure 2 As shown. Figure 2 It can be seen that 114 different cabbage vegetables can be clearly distinguished into 4 types by the primer set provided by the embodiment of the present invention, namely cabbage, kale, cauliflower and broccoli. The same type of cabbage vegetables can be grouped together, indicating that they are more closely related. This classification result is completely consistent with the morphological classification of the sample to be tested, which is in line with expectations. The results of the phylogenetic tree not only identify the variety types of these 114 cabbage vegetables, but also reveal the genetic relationship of these 114 cabbage vegetables, which will effectively guide the next step of cabbage vegetable hybridization research.

[0068] Embodiment 4

[0069] Specifically, the applications include: using the primer set in backcross breeding of cabbage vegetable varieties and background selection, molecular marker-assisted breeding, and whole genome breeding.

[0070] Specifically, the applications include: using the above primer set in gene typing of cabbage vegetable variety germplasm resources, species evolution analysis, seed purity detection, and variety authenticity analysis.

[0071] In order to verify the application of the primer set provided in this embodiment in backcross breeding, a total of 48 strains of the backcross population BC1 composed of the TZFD maintenance line and the sterile line were used as samples to be tested, named BC1-1 to BC1-48 in sequence, and multiple PCR amplification, targeted sequencing and data analysis were performed on its 147 SNP markers, and the specific experimental process was the same as in Example 3. After obtaining the genotype data of these 48 copies of BC1, the genetic similarity between different individual plants was calculated, that is, the number of variable sites between the individual genotype and the parental genotype was compared, and the individuals with the least number of variable sites and high genetic similarity were selected for further breeding, until a stable individual containing the target trait gene and the background genotype was basically restored to the same as the backcross parent was obtained. It was found through experiments that the background recovery rate of the backcross population was between 57.88% and 86.62%, and was normally distributed, with an average value of 71.11%, which was in line with the standard value (75%) defined by the law of genetics. In this backcross population, the average background recovery rate of 9 strains was higher than the theoretical value, namely: 79.45% BC1-21; 77.31% BC1-22; 80.23% BC1-23; 76.78% BC1-25; 79.74% BC1-26; 77.39% BC1-31; 82.56% BC1-34; 76.94% BC1-35; 77.43% BC1-38. Genetic similarity was converted into a heat map for visualization and auxiliary selection breeding, as shown in the following figure. Figure 3 As shown by Figure 3 It can be seen that in this backcross group, there are two strains (BC1-23, BC1-34) with a background recovery rate of more than 80%, and their color is also the darkest. In actual backcross breeding work, these two strains are selected to continue backcrossing in order to maintain the excellent traits of the recurrent parents as much as possible and speed up the breeding process.

[0072] The above description is only an optional embodiment of the present disclosure and is not intended to limit the present disclosure. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present disclosure shall be included in the protection scope of the present disclosure.

Claims

1. A primer set for identifying cabbage vegetable varieties, characterized in that: The primer set includes: the 1st primer pair to the 147th primer pair, each of the primer pairs includes a forward primer and a reverse primer, the forward primer of the 1st primer pair, the reverse primer of the 1st primer pair to the forward primer of the 147th primer pair, and the reverse primer of the 147th primer pair are respectively as shown in SEQ ID NO: 1 to SEQ ID NO: 294 in the sequence listing.

2. A kit for identifying cabbage varieties, characterized in that: The kit comprises the primer set as claimed in claim 1.

3. An application of a primer set for identifying cabbage vegetable varieties, characterized in that: The application includes: using the primer set as claimed in claim 1 to identify cabbage vegetable varieties.

4. The use according to claim 3, characterized in that: The application includes: using the primer set to identify broccoli varieties.

5. The use according to claim 3, characterized in that: The application includes: using the primer set to evaluate the genetic diversity of different varieties of cabbage vegetables.

6. The use according to claim 3, characterized in that: The application includes: using the primer set in backcross breeding and background selection, molecular marker-assisted breeding, and whole genome breeding of cabbage vegetables.

7. The use according to claim 3, characterized in that: The application includes: using the primer set in gene typing of germplasm resources of cabbage vegetables, species evolution analysis, seed purity detection, and variety authenticity analysis.

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