CN114752702A - Molecular marker BnCa-2C2 closely linked with rape calcium content trait QTL and application thereof - Google Patents
Molecular marker BnCa-2C2 closely linked with rape calcium content trait QTL and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of molecular biology and genetic breeding, and discloses a molecular marker BnCa-2C2 closely linked with a rape calcium content trait QTL and application thereof. The invention obtains an SNP (single nucleotide polymorphism) marker which is obviously related to the calcium content of rape, is positioned at the 56463993 th base position on the C02 chromosome of a Darmor-bzh v10 genome, and can explain 20.7 percent of phenotypic variation. The KASP detection primer designed according to the molecular marker is used for auxiliary selection, so that the screening efficiency of the high-calcium rape single plant can be improved.
Description
Technical Field
The invention belongs to the technical field of molecular biology and genetic breeding, and particularly relates to a molecular marker BnCa-2C2 closely linked with a rape calcium content trait QTL and application thereof.
Background
The cabbage type rape is formed by natural interspecific hybridization and double evolution of cabbage and Chinese cabbage, and the vegetable attribute of the cabbage type rape has scientific basis on origin and evolution. With the wide popularization of the varieties of the double-low rape, the rape seedlings and the flowering Chinese cabbage are gradually accepted by consumers as delicious vegetables. Researches show that the rape is a good plant transformation carrier of calcium element, and the cultivation of a new rape variety with high-efficiency utilization of the calcium element is an important technical means for improving the mineral nutrient deficiency of human bodies and promoting the development and utilization of a rape full-value chain.
The traditional breeding technology is applied to breeding and improving the crops with mineral nutrients such as calcium, and has the defects of low selection efficiency, long breeding period and the like. With the rapid development of molecular biology and sequencing technology, a breeding means assisted by genotype selection is proved by practice to be an effective way for solving the traditional breeding dilemma, and the development of molecular markers with closely linked characters is an important prerequisite for the application of the technology. The molecular marker closely related to the calcium content accumulation in the rape is selected and detected by utilizing the molecular marker assistance, so that the complicated phenotype identification working scale can be reduced, the accurate introduction or aggregation of characters can be guided, and the breeding efficiency can be greatly improved.
The invention takes rape core germplasm resource groups as materials, utilizes whole genome correlation analysis to identify the QTL sites related to the calcium content in the rape with breeding application potential, develops molecular markers based on QTL site information, and has positive effects on accelerating the breeding of new varieties of calcium-rich rape and improving the production benefit of the rape.
Disclosure of Invention
The invention aims to provide application of a reagent for detecting 56463993 th base on C02 chromosome of a genome of Brassica napus Darmor-bzh v10 in calcium content screening breeding of Brassica napus. The screening and breeding of the high-calcium rape can be realized by detecting the genotype of the 56463993 th basic group on the C02 chromosome of the cabbage type rape Darmor-bzh v10 genome.
In order to achieve the purpose, the invention adopts the following technical measures:
and obtaining a cabbage type rape calcium content trait QTL site qBnCa-2C 2:
(1) 327 parts of brassica napus inbred lines from various countries in the world are collected as rape related groups, single leaves of each line of the related groups are collected, total DNA is extracted by a CTAB method, and genotype analysis is carried out on each sample by using a rape 50K Illumina SNP chip developed by Wuhan double-greenery core-invasive science research institute Limited.
(2) The Illumina BeadStudio genotyping software (http:// www.illumina.com /) was used to calculate the marker heterozygosity rate (heterozygosity rate), deletion rate (missing rate), and minimum allele frequency (minor allele frequency) of the population material at each locus. Carrying out SNP marker filtration by taking the deletion rate of less than or equal to 0.2, the heterozygosity rate of less than or equal to 0.2, the minimum allele frequency of more than 0.05 and the unique matching of the SNP markers in a Brassica napus Darmor genome (Chalhoub et al, 2014) as screening standards, and finally obtaining 21,243 high-quality SNP markers for whole genome association analysis.
(3) And (3) introducing the obtained genotype data of the association analysis population into STRUCTURE v.2.3.4 for population STRUCTURE analysis, and dividing 327 brassica napus germplasm resources into 3 subgroups. The SPAGeDi software was used to calculate the genetic relationship between 327 Brassica napus germplasm resources (Hardy and Vekemans, 2002).
(4) 327 parts of materials are cultured by a rape water culture system containing 5mM of calcium element, and vegetable seedling samples are collected for calcium content determination after the materials are cultured to five-leaf stage. Setting 3 biological repetitions, uniformly crushing 5 plants of materials from each sample, and measuring the content of calcium element in the sample by a flame atomic absorption method.
(5) By combining genotype data, population structure and rape seedling-stage calcium content data and performing association analysis by using TASSEL 5.0 software (Bradbur y et al, 2007), an SNP marker Bn-C02_41808877 which is significantly associated with the rape calcium content is detected on a C02 chromosome, the maximum can explain 20.7% of phenotypic variation, the SNP variation site (variation from C to A) is located at the 56463993 th base of a cabbage type rape Darmor-bzh v10(Rousseau-Gueutin et al, 2020) genome C02 chromosome, and the calcium efficient enrichment main effect QTL site which is closely linked with the SNP site is named as BqBnCa-2C 2.
The application of the reagent for detecting 56463993 th base on C02 chromosome of cabbage type rape Darmor-bzh v10 genome in rape calcium enrichment screening breeding belongs to the protection range of the invention.
The application of the reagent for detecting the rape sequence containing 56463993 th base on the C02 chromosome of the cabbage type rape Darmor-bzh v10 genome in the calcium enrichment screening breeding of the rape also belongs to the protection range of the invention.
In the above application, preferably, the sequence of the rape is shown in SEQ ID NO. 1.
The application of the primer designed aiming at the 56463993 th base on the C02 chromosome of the cabbage type rape Darmor-bzh v10 genome in the calcium enrichment screening breeding of the rape also belongs to the protection range of the invention.
In the above applications, the applicant developed a KASP marker BnCa-2C2 of the antisense strand based on the SNP site, and the primers designed based on the marker were:
low-calcium-content allele-specific primer BnCa-2C 2-F1: GAGTAAACCATTGCTCTTATGTTCAG
High calcium content allele specific primer BnCa-2C 2-F2: GAGTAAACCATTGCTCTTATGTTCAT
Reverse primer BnCa-2C 2-R: CGTTTATTTGACGAATAAAATAAAGG is added.
The primers need to be based on the principle of KASP label development, and a KASP-labeled universal linker needs to be added before use.
Compared with the prior art, the invention has the following advantages:
(1) the invention obtains the main effect QTL locus qBnCa-2C2 which is obviously related to the calcium content character of rape seedlings for the first time, can explain 20.7 percent of phenotypic variation at most, and can be effectively applied to the genetic improvement of the calcium enrichment character of rape.
(2) The first research discovers a molecular marker BnCa-2C2 which is obviously related to the enrichment of calcium in rape, and provides a reliable molecular marker source for the pre-selection of the high-efficiency enrichment character of calcium in rape.
(3) The excellent allelic variation of qBnCa-2C2 in rape varieties or strains can be quickly selected by utilizing the molecular marker BnCa-2C2 in the growth period of rape seedlings, so that the workload of breeding and screening can be greatly reduced, the breeding period is shortened, and the breeding process of high-calcium rape is accelerated.
Detailed Description
The technical scheme of the invention is the conventional technology in the field if not particularly described; the reagents or materials, if not specifically mentioned, are commercially available. In the present invention, the Brassica napus genomes are all referenced to Darmor-bzh v10(Rousseau-Gueutin et al, 2020), unless otherwise specified. The cabbage type rape reference of the invention is as follows: the wheel-Genome sequencing of a world Wide Collection of ordered accesses of the Ge scientific Basis of Ecotype dictionary.
Example 1:
obtaining a rape calcium content character major QTL site qBnCa-2C 2:
(1) 327 parts of cabbage type rape inbred lines from various countries in the world are collected as a rape related group, single leaves of various strains of the related group are collected, total DNA is extracted by a CTAB method, and genotype analysis is carried out on each sample by using a rape 50K Illumina SNP chip developed by Wuhan Shuanglv resource core-invasive science and technology research institute Limited.
(2) The Illumina BeadStudio genotyping software (http:// www.illumina.com /) was used to calculate the marker heterozygosity rate (heterozygosity rate), deletion rate (missing rate), and minimum allele frequency (minor allele frequency) of the population material at each locus. Carrying out SNP marker filtration by taking the deletion rate of less than or equal to 0.2, the heterozygosity rate of less than or equal to 0.2, the minimum allele frequency of more than 0.05 and the unique matching of the SNP markers in a Brassica napus Darmor genome (Chalhoub et al, 2014) as screening standards, and finally obtaining 21,243 high-quality SNP markers for whole genome association analysis.
(3) And (3) introducing the obtained genotype data of the association analysis population into STRUCTURE v.2.3.4 for population STRUCTURE analysis, and dividing 327 brassica napus germplasm resources into 3 subgroups. The SPAGeDi software was used to calculate the genetic relationship between 327 Brassica napus germplasm resources (Hardy and Vekemans, 2002).
(4) Planting 327 parts of materials in a water culture greenhouse by utilizing a rape water culture system, wherein the calcium content in a culture solution is 5 mM; after the cultivation period of five leaves, vegetable seedling samples are collected for calcium content determination. Setting 3 biological repetitions, uniformly crushing 5 plants of materials from each sample, and measuring the content of calcium element in the sample by a flame atomic absorption method.
(5) By combining genotype data, population structure and rape seedling-stage calcium content data and performing association analysis by using TASSEL 5.0 software (Bradbur y et al, 2007), an SNP marker Bn-C02_41808877 which is significantly associated with the rape calcium content is detected on a C02 chromosome, 20.7% of phenotypic variation can be maximally explained, the SNP variation site (variation from C to A) is located at the 56463993 th base of a cabbage type rape Darmor-bzh v10(Rousseau-Gueutin et al, 2020) genome C02 chromosome, and the closely linked calcium content trait QTL site of the SNP site is named as qBnCa-2C 2.
Example 2:
the method comprises the following steps of obtaining a molecular marker primer closely linked with a rape calcium content trait QTL locus qBnCa-2C 2:
(1) extracting sequences of 100bp respectively upstream and downstream of 56463993 th base of C02 chromosome of Brassica napus, and developing a KASP molecular marker BnCa-2C2 aiming at an antisense strand thereof according to the design principle of KASP (competitive Alley-Specific PCR) molecular marker primers, wherein the marker comprises two competitive forward primers BnC a-2C2-F1 and BnCa-2C2-F2, complementary sequence bases C and A respectively corresponding to the SNP variation sites, and a reverse universal primer BnCa-2C2-R, and the primer sequences are as follows:
BnCa-2C2-F1：gagtaaaccattgctcttatgttcag
BnCa-2C2-F2：gagtaaaccattgctcttatgttcat
BnCa-2C2-R：cgtttatttgacgaataaaataaagg
The primers need to be based on the principle of KASP label development, and a KASP labeled universal linker needs to be added before use.
Wherein the sequence of the joint added before BnCa-2C2-F1 is gaaggtcggagtcaacggatt, and the sequence of the joint added before BnCa-2C2-F2 is gaaggtgaccaagttcatgct.
The sequence amplified in brassica napus tant is genotype a (i.e. genotype AA), the sequence is shown below: cgtttatttgacgaataaaataaaggatgttaattatctttagagtaaaatgatgaacataagagcaatggtttactc (shown in SEQ ID NO. 1).
The sequence amplified in brassica napus Galant is genotype B (i.e. genotype CC), and the sequence is as follows: cgtttatttgacgaataaaataaaggatgttaattatctttagagtaaaatgctgaacataagagcaatggtttactc (shown in SEQ ID NO. 2).
(2) Carrying out genotype typing on the marker in a rape related group by adopting a competitive allele specific PCR technology, wherein an amplification use kit is a five-primer amplification hindered mutation system (PAMS), and a 10 mu L reaction system is designed according to the instruction of a PAMS pro SNP genotyping PCR mix kit: 2 XPAMS master mix 5 uL, Allle X primer (10 uM) 0.15 uL, Allle Y primer (10 uM) 0.15 uL, Common R primer (10 uM) 0.4 uL and rape genomic DNA 10-100 ng. The amplification procedure was: 15min at 94 ℃; circulating for 10 times at 94 deg.C for 20s and 65-57 deg.C (Touch-down) for 1 min; circulating for 30 times at 94 deg.C for 20s and 57 deg.C for 1 min; collecting 1 time of fluorescence signals and outputting genotype results. And performing association analysis by using Tassel software to confirm that the BnCa-2C2 is obviously associated with the calcium content traits of the rape.
Example 3:
the application of the primer designed based on the 56463993 th base of rape C02 chromosome in rape calcium content character screening breeding comprises the following steps:
(1) 25 parts of materials with higher and lower calcium contents in 327 parts of materials are selected respectively, a rape water culture nutrition system containing 5mM calcium is applied to culture to a five-leaf stage, and the content of calcium element in the sample is determined by utilizing a flame atomic absorption method.
(2) The distribution of the two genotypes of the molecular marker BnCa-2C2 in the materials with higher and lower calcium content was examined. The result shows that 18 parts of genotype of the molecular marker BnCa-2C2 in 25 parts of material with higher calcium content are A and 7 parts of genotype are B; while 7 parts of 25 parts of the material with the lower calcium content was a and 18 parts was B (table 1).
(3) The T test result shows that the A and B genotypes detected by the molecular marker BnCa-2C2 have very significant difference (P is less than 0.01) in the calcium content character of the rape seedlings.
The above results are enough to show that the molecular marker BnCa-2C2 prepared by the method is highly correlated with the calcium content of rape seedlings, and therefore, the molecular marker can be used for the molecular marker-assisted selection of the rape calcium content character.
Table 1: genotype of molecular marker BnCa-2C2 in rape seedling calcium acid content extreme material
Sequence listing
<110> institute of oil crop of academy of agricultural sciences of China
<120> molecular marker BnCa-2C2 closely linked with rape calcium content trait QTL and application thereof
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 78
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
cgtttatttg acgaataaaa taaaggatgt taattatctt tagagtaaaa tgatgaacat 60
aagagcaatg gtttactc 78
<210> 2
<211> 78
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
cgtttatttg acgaataaaa taaaggatgt taattatctt tagagtaaaa tgctgaacat 60
aagagcaatg gtttactc 78
<210> 3
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
gagtaaacca ttgctcttat gttcag 26
<210> 4
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
gagtaaacca ttgctcttat gttcat 26
<210> 5
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
cgtttatttg acgaataaaa taaagg 26
<210> 6
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
gaaggtcgga gtcaacggat t 21
<210> 7
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
gaaggtgacc aagttcatgc t 21
Claims (4)
1. The application of the reagent for detecting 56463993 th base on C02 chromosome of Brassica napus Darmor-bzh v10 genome in calcium content screening and breeding of Brassica napus.
2. The use of claim 1, wherein the agent is a primer.
3. The use of claim 2, wherein the primer is BnCa-2C 2-F1: GAGTAAACCATTGCTCTTATGTTCAG, BnCa-2C 2-F2: GAGTAAACCATTGCTCTTATGTTCAT, and BnCa-2C 2-R: CGTTTATTTGACGAATAAAATAAAGG are provided.
4. The application of the reagent for detecting the rape sequence containing 56463993 th base on the C02 chromosome of the cabbage type rape Darmor-bzh v10 genome in screening and breeding of the calcium element enrichment capacity of rape is shown in SEQ ID NO. 1.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115992292A (en) * | 2023-03-21 | 2023-04-21 | 湖南农业大学 | SNP molecular marker combination for brassica napus and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115992292A (en) * | 2023-03-21 | 2023-04-21 | 湖南农业大学 | SNP molecular marker combination for brassica napus and application thereof |
| CN115992292B (en) * | 2023-03-21 | 2023-06-27 | 湖南农业大学 | SNP molecular marker combination for brassica napus and application thereof |
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