CN117701756A - SNP molecular marker closely linked with flowering phase gene BnA03.FLC of brassica napus and application thereof - Google Patents
SNP molecular marker closely linked with flowering phase gene BnA03.FLC of brassica napus 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 flowering phase gene BnA03.FLC and application thereof. The SNP locus of the SNP molecular marker is positioned on an A03 chromosome of a flowering phase gene BnA03.FLC, is used for accurately identifying a flowering phase gene BnA03.FLC mutant type, 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 for improving flowering phase breeding 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 flowering phase gene BnA03.FLC 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 flowering phase gene BnA03.FLC, a KASP marker primer, a kit and application thereof.
Background
Cabbage type rape is derived from natural hybridization between cabbage and Chinese cabbage, and is one of crop varieties with the closest relationship with arabidopsis thaliana. Rape is used as important multipurpose plant such as oil, feed, appreciation, honey, etc., flowering is one of the most important characters, and plays a core role in the production, breeding and hetero-optimal utilization process. The flowering period of the rape is one of important agronomic characters in agricultural production, and proper flowering time and flowering period can keep good consistency of the maturity period of the rape, so that the mechanized harvesting efficiency is increased in the mechanized harvesting process.
Flowering time is controlled by a complex genetic network that integrates many endogenous signals and environmental stimuli, such as vernalization, temperature, photoperiod, gibberellin, etc. The genetic control of the flowering character of the rape is a complex process and is influenced by multiple interactions of environmental factors and self genetic factors.
In order to reveal the molecular biological mechanism of rape flowering control, the important agricultural value and ornamental value are mined, developed and utilized, and the functional linkage SNP marker of the flowering phase gene BnA03.FLC 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 method has very important significance in the technical field.
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 closely linked with a brassica napus flowering phase gene BnA03.FLC, a KASP marker primer, a kit and application thereof, which can rapidly realize identification of a gene mutant of the flowering phase gene BnA03.FLC 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 flowering phase gene BnA03.FLC of the brassica napus is used as a reference, and the 6240320 th base of the brassica napus A03 chromosome of the SNP molecular marker has polymorphism G or A by taking the Darmor-bzh genome version of the brassica napus as a reference.
An application of the SNP molecular marker, wherein the application is to identify the flowering phase phenotype of brassica napus; identifying that the flowering phase phenotype of the brassica napus is early flowering when the polymorphism is G; and when the polymorphism is identified as A, the flowering phase phenotype of the brassica napus is late flowers.
For the above application, preferably, the nucleotide sequence of the KASP marker primer used for identifying the flowering phase phenotype of brassica napus is as follows from the 5 'end to the 3' end:
specific primer X: TTGTCTCCGCCTCCGACAAA (SEQ ID NO: 1);
specific primer Y: TTGTCTCCGCCTCCGACAAG (SEQ ID NO: 2);
general primer C: GTAAAGAGAAGAAAGCTTAAAGGGCAA (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 flowering phase gene BnA03.FLC 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 a brassica napus flowering phase gene bna03.Flc, wherein the nucleotide sequence of the KASP marker primer is as follows from the 5 'end to the 3' end:
specific primer X: TTGTCTCCGCCTCCGACAAA (SEQ ID NO: 1);
specific primer Y: TTGTCTCCGCCTCCGACAAG (SEQ ID NO: 2);
general primer C: GTAAAGAGAAGAAAGCTTAAAGGGCAA (SEQ ID NO: 3).
Based on a general inventive concept, the invention also provides a kit for identifying the flowering phase gene BnA03.FLC of brassica napus, 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 marking primer or the kit in identifying the flowering phase gene BnA03.FLC of brassica napus.
Compared with the prior art, the invention has the beneficial effects that:
(1) The invention screens out a SNP molecular marker closely linked with the flowering phase gene BnA03.FLC of the brassica napus, and a KASP marker primer group and a kit thereof, which are used for the accurate identification of the mutation type of the flowering phase gene BnA03.FLC, the marking type has high quality, single copy and high polymorphism (PIC value in the existing brassica napus 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 flowering phase breeding improvement of the brassica napus and has wide application universality.
(2) The invention also provides an application method for identifying the flowering phase gene BnA03.FLC of the brassica napus 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 method for detecting universality rapidly, efficiently and accurately with low cost.
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 in the 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 flowering phase gene BnA03.FLC. The molecular marker is obtained by carrying out gene sequence extraction and allele comparison on a gene interval of a brassica napus flowering phase gene BnA03.FLC to obtain a high-quality site through development verification, and is closely linked with the flowering phase gene BnA03.FLC, so that whether a breeding material contains the flowering phase gene BnA03.FLC can be rapidly selected according to genotypes.
Compared with the prior art that the phenotype data can be obtained only by carrying out the height measurement after the plant height phenotype selection of the brassica napus is planted and flowering in the field, the SNP marker developed by the invention can be directly detected and typed, and 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 120 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 flowering phase gene BnA03.FLC
Through a large number of literature searches, it is determined that the flowering phase gene BnA03.FLC is located on the chromosome of the brassica napus A03, the gene symbol is BnaA03g13630D, and the reference genome version of the brassica napus is Darmor-bzh.
3. Extraction and sequence acquisition of SNP locus of brassica napus
Extracting 120 parts of core germplasm resource data based on the sequence of a gene BnaA03g13630D, carrying out SNP polymorphism analysis on the gene sequence, finally obtaining 3 SNP loci (6240320 th, 6240795 th and 6241544 th bases on a cabbage type rape A03 chromosome), extracting the SNP loci and obtaining about 100bp before and after flanking sequences.
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 of KASP (KASP-related fragment thereof) mark of flowering phase gene BnA03.FLC and primer sequence
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), the 3 KASP markers are better and compact in typing, the detection rate is higher than 98%, but the detection typing diagram of the KASP markers BN900116 is polymorphic, the mutation type of the genes cannot be distinguished, and the detection typing diagram cannot be used for genotype detection; the markers BN900115 and BN900117 are good in typing, and the accurate detection of the brassica napus plant height genes is realized.
From the phenotype data of the material and the corresponding result of the genotype data (see table 3), BN900115 marks that the phenotype of the material is early when the genotype is G, and late when the genotype is a, the phenotype of the material is highly consistent with the corresponding relation of the genotype, so that whether the material contains the bna03.Flc gene can be accurately identified through the genotype; the BN900117 marker genotype and phenotype have no obvious correspondence, and cannot be applied to gene detection of BnA03.FLC.
Table 3 phenotype data and genotype data statistics for cultivars
Claims (10)
1. A SNP molecular marker closely linked with a flowering phase gene BnA03.FLC of brassica napus is characterized in that a Darmor-bzh genome version of the brassica napus is taken as a reference, and a 6240320 th base on a chromosome A03 of the brassica napus has a polymorphism, wherein the polymorphism is G or A.
2. The use of the SNP molecular marker of claim 1, wherein the use is to identify the flowering phenotype of brassica napus; identifying that the flowering phase phenotype of the brassica napus is early flowering when the polymorphism is G; and when the polymorphism is identified as A, the flowering phase phenotype of the brassica napus is late flowers.
3. The use according to claim 2, wherein the nucleotide sequence of the KASP marker primer used for identifying the flowering phase phenotype of brassica napus is set forth from 5 'to 3' as follows:
specific primer X: TTGTCTCCGCCTCCGACAAA;
specific primer Y: TTGTCTCCGCCTCCGACAAG;
general primer C: GTAAAGAGAAGAAAGCTTAAAGGGCAA.
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 flowering phase gene BnA03.FLC 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 a flowering phase gene bna03.Flc of brassica napus, wherein the nucleotide sequence of the KASP marker primer is shown from the 5 'end to the 3' end as follows:
specific primer X: TTGTCTCCGCCTCCGACAAA;
specific primer Y: TTGTCTCCGCCTCCGACAAG;
general primer C: GTAAAGAGAAGAAAGCTTAAAGGGCAA.
7. A kit for identifying the flowering phase gene bna03.flc of brassica napus, 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 the flowering phase gene bna03.flc of brassica napus.
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2023
- 2023-12-21 CN CN202311779438.XA patent/CN117701756A/en active Pending
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