CN117604157A - SNP molecular marker closely linked with brassica napus leaf chromogen BnA03.PL1 and application thereof - Google Patents
SNP molecular marker closely linked with brassica napus leaf chromogen BnA03.PL1 and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and discloses an SNP molecular marker closely linked with a brassica napus leaf chromophore BnA03.PL1 and application thereof. The SNP locus of the SNP molecular marker is positioned in leaf color gene BnA03.PL1, is used for the accurate identification of leaf color gene BnA03.PL1 mutant, has high marking type quality, single copy and high polymorphism, has a sample data detection rate of more than 98 percent, can be used for marking auxiliary breeding of the improvement of the leaf color of cabbage type rape, and has wide application universality. The application of the KASP marking primer and the kit for the SNP molecular marking and the identification of the brassica napus leaf color gene BnA03.PL1 is also disclosed, and the method is simple, high in automation degree, high in detection flux, high in speed, accurate in detection result and good in repeatability and stability.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a SNP molecular marker closely linked with a brassica napus leaf chromophore BnA03.PL1, a KASP marker primer, a kit and application thereof.
Background
Cabbage type rape is the third largest vegetable oil source in the world and its yield is about 13% of the global edible oil yield. According to statistics, the oil content of the rape is improved by 1%, the rape planting area of the main production area of the Yangtze river basin corresponding to 2.3-2.5 percent of yield increase of the rape is 90% of the total rape production area in China, and the oil content of the rape in the main production area of the Yangtze river basin is 41-42%.
DNA molecular labeling is the most common method for detecting purity of crop varieties. The DNA molecular marker is a genetic marker directly reflecting DNA difference (polymorphism), and is mainly SSR (Simple Sequence Repeat, simple repeated sequence) and SNP (Single Nucleotide Polymorphism ) at present. The prior SSR has the advantages of large-area use, including simple laboratory operation, low cost, better repeatability, true and reliable results, and the like. Compared with an SSR labeling method, the SNP labeling method has simpler technology, easy automation, high detection flux and high speed; the unit data point detection cost is low; the data results of different detection laboratories can be compared and verified, and the data has universal comparability; is the most common method for identifying functional genes rapidly, simply, sensitively, accurately, stably and with low cost.
The brassica napus purple leaf trait is a morphological variation due to the accumulation of anthocyanins in leaves. Besides being used as an important indication character for hybrid seed production, the purple leaf is rich in anthocyanin and has various biological health care functions of light protection, drought resistance, cold resistance, plant diseases and insect pests reduction, oxidization resistance, tumor resistance, cardiovascular improvement and the like.
Therefore, the rape leaf color character is one of very important characters for breeding, and the development of the functional linkage SNP marker of the leaf color gene BnA03.PL1 based on the KASP technology can be used as a high-efficiency identification marker, so that the condition of identifying functional genes with high flux, low cost and high accuracy in the rape breeding process is supported, and the breeding selection of rape is accelerated.
Disclosure of Invention
The invention aims to solve the technical problems and overcome the defects and shortcomings in the background technology, and provides a SNP molecular marker, a KASP marker primer and a kit which are closely linked with a brassica napus leaf color gene BnA03.PL1 and application thereof, so that the identification of the gene mutant of the leaf color gene BnA03.PL1 can be realized rapidly with high flux and low cost.
In order to solve the technical problems, the technical scheme provided by the invention is as follows:
a SNP molecular marker closely linked with a brassica napus leaf chromogen BnA03.PL1 takes Darmor-bzh genome version of brassica napus as a reference, and the SNP molecular marker has polymorphism at 3879543 th base on a brassica napus A03 chromosome, wherein the polymorphism is A or G.
Use of said SNP molecular markers for identifying the leaf color phenotype of brassica napus; identifying that the 3879543 base on the A03 chromosome of the brassica napus is homozygous A or A/G, wherein the leaf color phenotype of the brassica napus is green; and identifying that the 3879543 base on the A03 chromosome of the brassica napus is homozygous G, and the leaf color phenotype of the brassica napus is purple.
For the above application, preferably, the nucleotide sequence of the KASP marker primer used for identifying the leaf color phenotype of brassica napus is as follows from the 5 'end to the 3' end:
specific primer X: GAGATCCCGATCGTTCAGGTT (SEQ ID NO: 1);
specific primer Y: GAGATCCCGATCGTTCAGGTC (SEQ ID NO: 2);
general primer C: GACATTAGCCAAAGGATTCCACTTG (SEQ ID NO: 3).
Preferably, the method of application comprises the steps of:
(1) Extracting total DNA of a brassica napus sample to be detected;
(2) Taking the DNA extracted in the step (1) as a template, respectively carrying out PCR amplification by using the KASP labeled primer, and then carrying out fluorescent signal scanning and genotyping; if only fluorescence of the specific primer X is detected in the sample, the genotype of the sample is homozygous allele X; if only fluorescence of the specific primer Y is detected, the genotype of the sample is homozygous allele Y; if the fluorescence of the specific primer X and the fluorescence of the specific primer Y are detected at the same time, the genotype of the sample is heterozygous;
(3) And carrying out data analysis according to the genotyping result to obtain the leaf color gene BnA03.PL1 of the brassica napus sample to be tested.
Preferably, the method is performed using a Douglas Array Tape platform; the PCR amplification system comprises: 100 mu M universal primer C, 100 mu M specific primer X, 100 mu M specific primer Y, 2 xKASP Master Mix, DNA of a cabbage type rape sample to be tested, ultrapure water.
Preferably, PCR amplification is performed with SOELLEX under the following conditions: 94 ℃ for 15 minutes; 94 ℃ for 20 seconds, 65 ℃ to 57 ℃ for 60 seconds, 10 cycles; 94℃for 20 seconds, 57℃for 60 seconds, 33 cycles.
Based on a general inventive concept, the present invention also provides a KASP marker primer for identifying brassica napus leaf color gene bna03.pl1, wherein the nucleotide sequence of the KASP marker primer is as follows from the 5 'end to the 3' end:
specific primer X: GAGATCCCGATCGTTCAGGTT (SEQ ID NO: 1);
specific primer Y: GAGATCCCGATCGTTCAGGTC (SEQ ID NO: 2);
general primer C: GACATTAGCCAAAGGATTCCACTTG (SEQ ID NO: 3).
Based on a general inventive concept, the invention also provides a kit for identifying brassica napus leaf color gene BnA03.PL1, which comprises the KASP marking primer.
In the above kit, preferably, the concentration ratio of the specific primer X, the specific primer Y and the universal primer C in the PCR reaction system is 10-12:10-12:25-30.
Preferably, the kit further comprises 2×kasp Master Mix and ultrapure water.
Based on a general inventive concept, the invention also provides an application of the KASP labeling primer or the kit in identifying brassica napus leaf color gene BnA03.PL1.
Compared with the prior art, the invention has the beneficial effects that:
(1) The invention screens out a KASP mark primer group and a kit of SNP mark closely linked with cabbage type rape leaf color gene BnA03.PL1, which are used for the accurate identification of leaf color gene BnA03.PL1 mutant type, the marking mark has high quality, single copy and high polymorphism (PIC value in the existing cabbage type rape resource is higher than 0.3), the sample data detection rate is higher than 98%, and the invention can be used for the marking auxiliary breeding of the leaf color breeding improvement of cabbage type rape and has wide application universality.
(2) The invention also provides an application method for identifying the brassica napus leaf color gene BnA03.PL1 by using the KASP marking primer and the kit, and the detection method is simple and has the automation degree as high as 90%; the detection flux is high, the speed is high (122,880 data points can be obtained in 8 hours, which is 10 times of that of the traditional 96-well plate SNP genotyping method); the detection reagent consumption is small (only 0.8 uL/reaction), the reagent consumable cost is low (compared with the traditional 96-well plate SNP genotyping method, the reagent consumable cost is reduced by 70% -90%); the detection result is accurate, the repeatability and the stability are good, the data results of different detection laboratories can be compared and verified, and the data has universal comparability; the invention provides a universal method for rapid, efficient, low-cost and accurate detection.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of genotyping of SNP molecular markers in rape diversity material according to an embodiment of the invention.
Detailed Description
The present invention will be described more fully hereinafter with reference to the accompanying drawings, in which preferred embodiments are shown, for the purpose of illustrating the invention, but the scope of the invention is not limited to the specific embodiments shown.
Unless defined otherwise, all technical and scientific terms used hereinafter have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the scope of the present invention.
Unless otherwise specifically indicated, the various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or may be prepared by existing methods.
Examples:
the invention develops an SNP molecular marker closely linked with a brassica napus leaf color gene BnA03.PL1. The molecular marker is obtained by developing and verifying high-quality loci obtained by carrying out gene sequence extraction and allele comparison on the gene interval of the brassica napus leaf color gene BnA03.PL1, is tightly linked with the leaf color gene BnA03.PL1, and can rapidly select whether a breeding material contains the leaf color gene BnA03.PL1 according to genotypes.
Compared with the prior art that leaf color phenotype of cabbage type rape can be determined by phenotypic observation after field planting, SNP markers developed by the invention can be directly detected and typed, so that the selection efficiency of breeding is greatly improved; the SNP marker detection technology based on the KASP (Kompetitive Allele Specific PCR, competitive allele-specific PCR) detection technology of the Douglas Array Tape platform has the advantages of simple detection method, high automation degree, high flux, high speed, less reagent consumption and low detection cost, and can be widely popularized and used and selected for breeding. The specific research and development process is as follows:
1. construction of cabbage type rape core germplasm resource database
The disclosed rape germplasm resources are used for selecting 49 parts of rape core germplasm resources according to the classification of spring rape, semi-winter rape and winter rape to construct a cabbage type rape germplasm resource database.
2. Information mining of cloned leaf color gene BnA03.PL1
Through extensive literature searches, it was determined that the leaf color gene BnA03.PL1 was designated on the Brassica napus A03 chromosome with the gene symbol BnaA03g59800D and the reference genome version of Brassica napus was Darmor-bzh.
3. Extraction and sequence acquisition of SNP locus of brassica napus
The obtained 49 pieces of core germplasm resource data are extracted based on the sequence of a gene BnaA03g59800D, SNP polymorphism analysis is carried out on the gene sequence, the interval of the gene is relatively conserved, finally 1 high-quality SNP locus (3879543 base on a cabbage type rape A03 chromosome) is obtained, and the SNP locus is extracted and the flanking sequences are obtained about 100bp before and after.
4. Mark design and synthesis
And (3) carrying out KASP primer mark design on the SNP locus obtained by the method based on the reference genome Darmor-bzh of the brassica napus by utilizing an online primer design website BatchPrimer3 (http:// probes. Pw. Usda. Gov/bat Primer3 /). Three primers are provided, and FAM and HEX fluorescent sequences are respectively connected to the 5' ends of two specific primers. After the design is completed, the primer sequence is further subjected to genome-wide copy number analysis, and finally a high-quality single-copy KASP marker locus is obtained, wherein the information is shown in the following table 1:
table 1: site information and primer sequences of KASP marker of leaf color gene BnA03.PL1 (sequence is shown as SEQ ID NO:1-3 in sequence from left to right)
5. Detection and verification of markers
In order to verify the quality of the marker phenotype and the consistency of the genotype of the marker and the sample phenotype, the varieties with the top popularization area in the market in the last 3 years of 2021-2023 are collected, and the samples of the varieties are used for verification, and the method comprises the following steps:
(1) Extracting total DNA of a brassica napus sample to be detected.
(2) Taking the DNA extracted in the step (1) as a template, respectively carrying out PCR amplification by using the KASP labeled primer, and carrying out fluorescent signal scanning of a reaction system by using ARAYA after the PCR reaction is completed; genotyping and data analysis were then performed with INTELLICS; in the KASP marker genotyping detection, the genotypes of the samples are divided into 3 clusters, namely an X cluster, a Y cluster and a heterozygous genotype cluster; wherein the X cluster indicates that the sample contains a homozygous X allele at this KASP marker locus (labeled red in the top left of the figure), the Y cluster indicates that the sample contains a homozygous Y allele at this KASP marker locus (labeled blue in the bottom right of the figure), and the heterozygous genotype cluster indicates that the sample contains both X and Y heterozygous alleles at this KASP marker locus (labeled purple in the genotyping figure);
the verification and detection of the KASP markers was performed with the Array Tape system of Douglas Scientific. The Array Tape genotyping platform included NEXAR for PCR amplification system assembly, SOELLEX for PCR amplification, ARAYA for fluorescent signal scanning, and INTELLICS for data analysis.
PCR amplification system: automatic assembly of the PCR amplification system was performed using NEXAR, and the PCR amplification system is shown in Table 2 below.
Table 2: PCR amplification system for KASP marker genotyping
And (3) PCR amplification: PCR was performed using SOELLEX under the following conditions: 94 ℃ for 15 minutes; 94 ℃ for 20 seconds, 65 ℃ -57 ℃ (0.8 ℃ for 60 seconds for each cycle of annealing temperature reduction), 10 cycles; 94℃for 20 seconds, 57℃for 60 seconds, 33 cycles.
KASP label detection advantage based on Douglas Array Tape platform: the automation degree of KASP marking based on Douglas Array Tape platform reaches 90%, and manpower and human error in laboratory are greatly reduced. The detection flux is high, 122,880 data points can be obtained in 8 hours, and the detection flux is 10 times that of the traditional 96-well plate SNP genotyping method. The detection reaction volume is low (only 0.8 uL/reaction), and compared with the traditional 96-well plate SNP genotyping method, the reagent consumable cost is reduced by 70-90%.
(3) And (3) carrying out data analysis according to the KASP marking typing result and the phenotype result to select the optimal site for subsequent application.
According to the detection result (see figure 1), KASP mark has better and compact typing, the detection rate is higher than 98%, and the accurate detection of the brassica napus leaf chromophore BnA03.PL1 can be realized.
From the phenotype data of the material and the corresponding results of the genotype data (see table 3), BN900141 marks that the phenotype of the material is green when the genotype is a or a/G, and purple when the genotype is G, the phenotype of the material is highly consistent with the genotype correspondence, and whether the material contains the leaf color gene bna03.pl1 can be accurately identified through the genotype, so BN900141 can be selected for use as a subsequent auxiliary mark.
Table 3 phenotype data and genotype data statistics for cultivars
Claims (10)
1. A SNP molecular marker closely linked with a brassica napus leaf chromogen BnA03.PL1 is characterized in that a Darmor-bzh genome version of brassica napus is taken as a reference, and the SNP molecular marker has a polymorphism at 3879543 th base on a brassica napus A03 chromosome, wherein the polymorphism is A or G.
2. The use of the SNP molecular marker of claim 1, wherein the use is to identify the leaf color phenotype of brassica napus; identifying that the 3879543 base on the A03 chromosome of the brassica napus is homozygous A or A/G, wherein the leaf color phenotype of the brassica napus is green; and identifying that the 3879543 base on the A03 chromosome of the brassica napus is homozygous G, and the leaf color phenotype of the brassica napus is purple.
3. The use according to claim 2, wherein the nucleotide sequence of the KASP marker primer used for identifying brassica napus leaf color phenotype is as follows from 5 'end to 3' end:
specific primer X: GAGATCCCGATCGTTCAGGTT;
specific primer Y: GAGATCCCGATCGTTCAGGTC;
general primer C: GACATTAGCCAAAGGATTCCACTTG.
4. The application according to claim 3, characterized in that the method of application comprises the steps of:
(1) Extracting total DNA of a brassica napus sample to be detected;
(2) Taking the DNA extracted in the step (1) as a template, respectively carrying out PCR amplification by using the KASP labeled primer, and then carrying out fluorescent signal scanning and genotyping; if only fluorescence of the specific primer X is detected in the sample, the genotype of the sample is homozygous allele X; if only fluorescence of the specific primer Y is detected, the genotype of the sample is homozygous allele Y; if the fluorescence of the specific primer X and the fluorescence of the specific primer Y are detected at the same time, the genotype of the sample is heterozygous;
(3) And carrying out data analysis according to the genotyping result to obtain the leaf color gene BnA03.PL1 of the brassica napus sample to be tested.
5. The use of claim 4, wherein the method is performed using a Douglas Array Tape platform; the PCR amplification system comprises: 100 mu M universal primer C, 100 mu M specific primer X, 100 mu M specific primer Y, 2 xKASP Master Mix, DNA of a cabbage type rape sample to be tested, ultrapure water.
6. A KASP marker primer for identifying brassica napus leaf chromogen bna03.pl1, wherein the nucleotide sequence of the KASP marker primer is shown from the 5 'end to the 3' end as follows:
specific primer X: GAGATCCCGATCGTTCAGGTT;
specific primer Y: GAGATCCCGATCGTTCAGGTC;
general primer C: GACATTAGCCAAAGGATTCCACTTG.
7. A kit for identifying brassica napus leaf chromogen bna03.pl1 comprising a KASP marker primer according to claim 6.
8. The kit according to claim 7, wherein the concentration ratio of the specific primer X, the specific primer Y and the universal primer C in the PCR reaction system is 10-12:10-12:25-30.
9. The kit of claim 7, further comprising 2 x KASP Master Mix and ultrapure water.
10. Use of a KASP marker primer according to claim 6 or a kit according to any one of claims 7 to 9 for identifying brassica napus leaf colour gene bna03.pl1.
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| CN202311776862.9A Pending CN117604157A (en) | 2023-12-21 | 2023-12-21 | SNP molecular marker closely linked with brassica napus leaf chromogen BnA03.PL1 and application thereof |
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| CN (1) | CN117604157A (en) |
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2023
- 2023-12-21 CN CN202311776862.9A patent/CN117604157A/en active Pending
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