CN117385093A - SNP molecular marker closely linked with brassica napus anti-cracking gene BnMAN7A07 and application thereof - Google Patents
SNP molecular marker closely linked with brassica napus anti-cracking gene BnMAN7A07 and application thereof Download PDFInfo
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- 240000002791 Brassica napus Species 0.000 title claims abstract description 46
- 235000011293 Brassica napus Nutrition 0.000 title claims abstract description 46
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 39
- 238000005336 cracking Methods 0.000 title claims abstract description 35
- 239000003147 molecular marker Substances 0.000 title claims abstract description 14
- 238000000034 method Methods 0.000 claims abstract description 14
- 239000003550 marker Substances 0.000 claims description 23
- 238000003205 genotyping method Methods 0.000 claims description 15
- 238000012408 PCR amplification Methods 0.000 claims description 13
- 240000007124 Brassica oleracea Species 0.000 claims description 9
- 108700028369 Alleles Proteins 0.000 claims description 8
- 210000000349 chromosome Anatomy 0.000 claims description 8
- 235000003899 Brassica oleracea var acephala Nutrition 0.000 claims description 7
- 235000011301 Brassica oleracea var capitata Nutrition 0.000 claims description 7
- 235000001169 Brassica oleracea var oleracea Nutrition 0.000 claims description 7
- 238000006243 chemical reaction Methods 0.000 claims description 7
- 238000007405 data analysis Methods 0.000 claims description 5
- 239000002773 nucleotide Substances 0.000 claims description 4
- 125000003729 nucleotide group Chemical group 0.000 claims description 4
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 4
- 239000012498 ultrapure water Substances 0.000 claims description 4
- 230000023753 dehiscence Effects 0.000 claims description 3
- 235000011303 Brassica alboglabra Nutrition 0.000 claims description 2
- 235000011302 Brassica oleracea Nutrition 0.000 claims description 2
- 238000001514 detection method Methods 0.000 abstract description 23
- 238000009395 breeding Methods 0.000 abstract description 12
- 230000001488 breeding effect Effects 0.000 abstract description 12
- 230000004907 flux Effects 0.000 abstract description 7
- 230000035772 mutation Effects 0.000 abstract description 2
- 239000000523 sample Substances 0.000 description 14
- 239000000463 material Substances 0.000 description 7
- 239000003153 chemical reaction reagent Substances 0.000 description 6
- 238000013461 design Methods 0.000 description 4
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 3
- 238000012795 verification Methods 0.000 description 3
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 2
- 235000006008 Brassica napus var napus Nutrition 0.000 description 2
- 240000000385 Brassica napus var. napus Species 0.000 description 2
- 238000007844 allele-specific PCR Methods 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
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- 235000014698 Brassica juncea var multisecta Nutrition 0.000 description 1
- 235000010149 Brassica rapa subsp chinensis Nutrition 0.000 description 1
- 235000006618 Brassica rapa subsp oleifera Nutrition 0.000 description 1
- 235000000536 Brassica rapa subsp pekinensis Nutrition 0.000 description 1
- 241000499436 Brassica rapa subsp. pekinensis Species 0.000 description 1
- 235000004977 Brassica sinapistrum Nutrition 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 238000012271 agricultural production Methods 0.000 description 1
- 230000009418 agronomic effect Effects 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
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- 239000000828 canola oil Substances 0.000 description 1
- 235000019519 canola oil Nutrition 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
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- 230000007226 seed germination Effects 0.000 description 1
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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 anti-cracking gene BnMAN7A07 and application thereof. The SNP locus of the SNP molecular marker is positioned in an anti-cracking horn gene BnMAN7A07, is used for accurately identifying mutation type of the anti-cracking horn gene BnMAN7A07, 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 breeding improvement of anti-cracking horn of brassica napus, 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 anti-cracking gene BnMAN7A07 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 anti-cracking gene BnMAN7A07, 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.
The manifestation of the canola oil crack trait is seed cracking, which typically occurs during the mature period. To a certain extent, the corner cracking character is a serious problem in the rape production process, as seed cracks can damage seed quality, so that seed germination rate and germination capacity are reduced, and yield and quality are affected. Therefore, the rape anti-cracking character is an important research direction for the development of grain safety and agricultural sustainable performance.
Therefore, the anti-cracking character of rape is one of important characters for breeding, and the development of the functional linkage SNP marker of the anti-cracking gene BnMAN7A07 based on the KASP technology can be used as an efficient identification marker to support the condition of identifying functional genes with high flux, low cost and high accuracy in the rape breeding process, so that 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 art, and provides a SNP molecular marker, a KASP marker primer and a kit which are closely linked with a brassica napus anti-cracking gene BnMAN7A07 and application thereof, so that the identification of the gene mutant of the anti-cracking gene BnMAN7A07 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 anti-cracking gene BnMAN7A07 takes Darmor-bzh genome version of brassica napus as a reference, wherein the SNP molecular marker has polymorphism at 11348253 th base on a brassica napus A07 chromosome, and the polymorphism is A or C.
The above-mentioned use, preferably, for identifying a dehiscence phenotype of brassica napus; identifying that the 11348253 base on the A07 chromosome of the brassica napus is homozygous A, wherein the anti-dehiscence phenotype of the brassica napus is non-dehiscence; identifying the anticracking angle of the brassica napus as the phenotype of the anticracking angle when the 11348253 base on the A07 chromosome of the brassica napus is homozygote C or A/C.
Preferably, the nucleotide sequence of the KASP marker primer used for identifying the brassica oleracea anti-angle phenotype is as follows from the 5 'end to the 3' end:
specific primer X: TTGTTCTGGCAACTTTTGGGA (SEQ ID NO: 1);
specific primer Y: TGTTCTGGCAACTTTTGGGC (SEQ ID NO: 2);
general primer C: CAGTCGAGGAGCTTTGGCTA (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 anti-cracking horn gene BnMAN7A07 of the brassica napus sample to be detected.
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 one general inventive concept, the invention also provides a KASP (KASP-labeled primer) for identifying the brassica napus anti-cracking gene BnMAN7A07, wherein the nucleotide sequence of the KASP-labeled primer is shown from the 5 'end to the 3' end as follows:
specific primer X: TTGTTCTGGCAACTTTTGGGA (SEQ ID NO: 1);
specific primer Y: TGTTCTGGCAACTTTTGGGC (SEQ ID NO: 2);
general primer C: CAGTCGAGGAGCTTTGGCTA (SEQ ID NO: 3).
Based on a general inventive concept, the invention also provides a kit for identifying the brassica napus anti-cracking gene BnMAN7A07, 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 brassica napus anti-cracking gene BnMAN7A07.
Compared with the prior art, the invention has the beneficial effects that:
(1) The SNP molecular marker closely linked with the brassica napus anti-cracking gene BnMAN7A07, the KASP marker primer group and the kit thereof are screened out and used for accurately identifying the mutation type of the anti-cracking gene BnMAN7A07, 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 percent, and the method can be used for marking auxiliary breeding for improving the anti-cracking breeding of the brassica napus and has wide application universality.
(2) The invention also provides an application method for identifying the brassica napus anti-cracking gene BnMAN7A07 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 winter universality rapidly, efficiently, with low cost and accurately.
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 in SNP molecular marker canola diverse materials in 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 anti-cracking gene BnMAN7A07. The molecular marker is obtained by developing and verifying high-quality loci obtained by carrying out gene sequence extraction and allele comparison on a gene interval of the brassica napus anti-cracking gene BnMAN7A07, is tightly linked with the anti-cracking gene BnMAN7A07, and can rapidly select whether a breeding material contains the anti-cracking gene BnMAN7A07 according to genotypes.
Compared with the prior art that the data can be obtained only by carrying out phenotype investigation in the period from the time of field seed collection to the time of the crack angle level of the brassica napus in breeding, 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 55 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 anti-cracking corner gene BnMAN7A07
Through a large number of literature searches, it is determined that the anti-dehiscence gene BnMAN7A07 is located on the chromosome of the brassica napus A07, the gene symbol is BnaA07g12590D, and the reference genome version of the brassica napus is Darmor-bzh.
3. Extraction and sequence acquisition of SNP locus of brassica napus
Extracting the obtained 55 core germplasm resources based on the sequence of the gene BnaA07g12590D, carrying out SNP polymorphism analysis on the gene sequence to finally obtain 2 SNP loci (11348253 th and 11348307 th bases on the cabbage type rape A07 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: KASP marked site information and primer sequence of anti-crack corner gene BnMAN7A07 (sequence is shown as SEQ ID NO:1-3 from left to right in 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 marker detection result (see figure 1), the marker BN900154 is better and compact in typing, the detection rate is higher than 98%, and the genotype of the brassica napus anti-cracking gene BnMAN7A07 can be accurately detected. And BN900155 marked typing is obviously lacking and cannot be used for genotyping detection.
From the phenotype data of the material and the corresponding result of the genotype data (see table 3), BN900154 marks that the phenotype of the material is a non-cracking angle when the genotype is a, and the phenotype of the material is a low resistance or a resistance when the genotype is C or a/C, and the phenotype is highly consistent with the genotype corresponding relationship, so that whether the material contains the cracking angle gene bncan 7a07 can be accurately identified through the genotype, and therefore, the material can be selected to be applied as a subsequent auxiliary mark.
Table 3 phenotype data and genotype data statistics for cultivars
Claims (10)
1. The SNP molecular marker closely linked with the brassica napus anti-cracking gene BnMAN7A07 is characterized in that the 11348253 th base on the brassica napus A07 chromosome of the SNP molecular marker has polymorphism A or C by taking the Darmor-bzh genome version of the brassica napus as a reference.
2. The use according to claim 1, wherein the use is for identifying an anti-dehiscence phenotype of brassica napus; identifying that the 11348253 base on the A07 chromosome of the brassica napus is homozygous A, wherein the anti-dehiscence phenotype of the brassica napus is non-dehiscence; identifying the anticracking angle of the brassica napus as the phenotype of the anticracking angle when the 11348253 base on the A07 chromosome of the brassica napus is homozygote C or A/C.
3. The use according to claim 2, wherein the nucleotide sequence of the KASP marker primer used to identify the brassica oleracea anti-angle phenotype is as follows from 5 'to 3':
specific primer X: TTGTTCTGGCAACTTTTGGGA;
specific primer Y: TGTTCTGGCAACTTTTGGGC;
general primer C: CAGTCGAGGAGCTTTGGCTA.
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 anti-cracking horn gene BnMAN7A07 of the brassica napus sample to be detected.
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 brassica napus anti-cracking gene BnMAN7a07, wherein the nucleotide sequence of the KASP marker primer is shown from the 5 'end to the 3' end as follows:
specific primer X: TTGTTCTGGCAACTTTTGGGA;
specific primer Y: TGTTCTGGCAACTTTTGGGC;
general primer C: CAGTCGAGGAGCTTTGGCTA.
7. A kit for identifying the brassica napus anti-dehiscence gene bnan 7a07, comprising the KASP marker primer of 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 8 for identifying the brassica napus anti-dehiscence gene BnMAN7a07.
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