CN110527739B - Major QTL (quantitative trait locus) site of glucosinolate content of brassica napus seeds, SNP (Single nucleotide polymorphism) molecular marker and application thereof - Google Patents
Major QTL (quantitative trait locus) site of glucosinolate content of brassica napus seeds, SNP (Single nucleotide polymorphism) molecular marker and application thereof Download PDFInfo
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Abstract
The invention provides a major QTL site of the glucosinolate content of brassica napus seeds, which is positioned between the 1798327 th base and the 4773555 th base of the A09 chromosome of the brassica napus. Preferably, the contribution rate to the glucosinolate content of the brassica napus seeds is 72.11%. Closely linked to the first SNP molecular marker, which is located at base 1798327 and is either C or T, the mutation results in a polymorphism. Closely linked to a second SNP molecular marker located at base 4773555, being either A or G, the mutation resulting in a polymorphism. Closely linked to the peak SNP molecular marker, located at base 2629811, either C or T, the mutation resulting in a polymorphism. Also provides related SNP molecular markers and application. The major QTL site of the glucosinolate content of the brassica napus seeds has high contribution rate to the glucosinolate content of the brassica napus seeds, plays a key role in regulating and controlling the glucosinolate content of the brassica napus seeds, can be used for site cloning and molecular marker assisted selection, and is suitable for large-scale popularization and application.
Description
Technical Field
The invention relates to the technical field of molecular biology and rape breeding, in particular to the technical field of the glucosinolate content of brassica napus seeds, and specifically relates to an SNP molecular marker closely linked with a major QTL site of the glucosinolate content of the brassica napus seeds and application thereof.
Background
Rape (Brassica napus) is an important oil crop in Brassica plants in cruciferae, is a main oil crop widely planted worldwide and is the only winter oil crop in China. Therefore, the development of rape production is taken as the key point for ensuring the safe supply of the edible oil in China, and the method has important practical significance. The cabbage type rape in China becomes a main cultivated variety due to the characteristics of disease resistance, high yield, wide adaptability, strong adverse-resistant performance and the like. The yield of the rape is increased mainly by three ways of increasing the rape yield per unit area, increasing the oil content of the rape seeds and enlarging the planting area. The main goal of rape breeders is now a high oil content, yield and quality of effort. Because the oil content, yield, quality and other characters of the rape are complex quantitative characters and are greatly influenced by the environment, the traditional breeding method and the traditional breeding technology are difficult to have larger breakthrough on the existing basis, so that the quantitative genetics and the molecular marker technology are combined, and a new opportunity is provided for the development period of the rape genetic breeding.
The SNP-based molecular marker technology is considered as a third-generation molecular marker appearing after RFLP and SSR, and refers to the difference of individual nucleotides or only small deletion, mutation, insertion and the like between different alleles of the same locus, and automatic batch detection can be realized by methods such as a DNA chip technology based on sequencing or PCR and the like, so that the SNP-based molecular marker technology has incomparable superiority and potential in the research of gene positioning.
Glucosinolates (glucosinolates) are a class of nitrogen and sulfur containing secondary metabolites of plants. The cake dregs after rape seed oil extraction contain rich protein, and is a good natural animal feed. However, the glucosinolate is a harmful component in the cabbage type rape seeds, and the rape cake dregs containing the high glucosinolate and the hydrolysate thereof can cause toxicity to livestock when being used for feeding the livestock, so the content of the glucosinolate in the rape seeds determines the value of the rape cake dregs as feed.
In the prior art, Jiangjian Xixia and the like (Shanghai agricultural science, 2019) indicate that reducing the content of glucosinolates in rape seeds is one of important breeding targets for improving the quality of the rape seed oil. In recent years, with the completion of whole genome sequencing, the wide application of molecular marker technology, the continuous development of molecular Marker Assisted Selection (MAS) technology, and the research results of locating molecular markers and QTLs related to important quality traits such as glucosinolates of rape are increasing, but most of them are in the initial locating stage. Therefore, by means of molecular marker and Quantitative Trait Locus (QTL) positioning, the glucosinolate content character of the cabbage type rape seeds is further researched at a molecular level, so that the rapeseed oil quality is improved, and a foundation is laid for disclosing the genetic mechanism and the molecular mechanism of the glucosinolate content in the cabbage type rape.
Disclosure of Invention
In order to solve the problems in the prior art, an object of the present invention is to provide an SNP molecular marker for a major QTL locus of the glucosinolate content in brassica napus seeds, wherein the major QTL locus of the glucosinolate content in brassica napus seeds is located between the 1798327 st base and the 4773555 th base of the chromosome a09 of brassica napus, has a high contribution rate to the glucosinolate content in brassica napus seeds, plays a key role in the regulation of the glucosinolate content in brassica napus seeds, can be used for site-directed cloning and molecular marker-assisted selection, and is suitable for large-scale popularization and application.
Preferably, the major QTL site of the glucosinolate content of the brassica napus seeds is closely linked with SNP molecular markers, and the SNP molecular markers are a first SNP molecular marker, a second SNP molecular marker and/or a peak SNP molecular marker.
Preferably, the first SNP molecular marker is located at the 1798327 th base of the A09 chromosome of Brassica napus, the 1798327 th base is C or T, and the mutation results in polymorphism.
Preferably, the second SNP molecular marker is located at the 4773555 th base of the A09 chromosome of Brassica napus, the 4773555 th base is A or G, and the mutation results in polymorphism.
Preferably, the peak SNP molecular marker is located at the 2629811 th base of the A09 chromosome of Brassica napus, and the 2629811 th base is C or T, and the mutation causes polymorphism.
The invention also aims to provide the application of the SNP molecular marker of the major QTL site of the glucosinolate content of the brassica napus seeds.
Preferably, the method is used for detecting the glucosinolate content of the Brassica napus seeds, or predicting the glucosinolate content of the Brassica napus seeds, or effectively selecting the glucosinolate content of the Brassica napus seeds.
Preferably, the molecular marker is used for the molecular marker-assisted breeding of the Brassica napus or used for accelerating the process of the breeding of the glucosinolate content of the Brassica napus.
The invention also aims to provide a primer or a probe of the SNP molecular marker of the major QTL site of the glucosinolate content of the brassica napus seeds.
Preferably, the primer or probe is designed by taking a DNA fragment containing 400bp sequences (801 bp in total) before and after the Brassica napus chrA 09-1798327 (C/T) as a template, and the DNA fragment is shown as SEQ ID NO. 1.
Preferably, the primer or the probe is designed by taking a DNA fragment containing 400bp sequences (801 bp in total) before and after the Brassica napus chrA 09-4773555 (A/G) as a template, and the DNA fragment is shown as SEQ ID NO. 2.
Preferably, the primer or probe is designed by taking a DNA fragment containing 400bp sequences (801 bp in total) before and after the Brassica napus chrA 09-2629811 (C/T) as a template, and the DNA fragment is shown as SEQ ID NO. 3.
Preferably, the primer or probe is labeled with a fluorophore comprising FAM, HEX, VIC, ROX.
The invention also aims to provide application of the primer or the probe of the SNP molecular marker of the major QTL site of the glucosinolate content of the brassica napus seeds in detecting and/or predicting the glucosinolate content of the brassica napus seeds or in molecular marker-assisted breeding of the brassica napus seeds.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention provides a major QTL site of the glucosinolate content of cabbage type rape seeds, which is closely linked with an SNP molecular marker, has high contribution rate to the glucosinolate content of the cabbage type rape seeds, plays a key role in regulating and controlling the glucosinolate content of the cabbage type rape seeds, can be used for site-directed cloning and molecular marker-assisted selection, and is suitable for large-scale popularization and application.
(2) The SNP molecular marker of the major QTL site of the glucosinolate content of the cabbage type rape seeds comprises a SNP molecular marker of a 1798327 th base of an A09 chromosome of the cabbage type rape, a SNP molecular marker of a 4773555 th base of an A09 chromosome of the cabbage type rape and a peak SNP molecular marker of a 2629811 th base of an A09 chromosome of the cabbage type rape.
(3) The invention specifically provides three SNP molecular markers of the glucosinolate content of cabbage type rape seeds, wherein the peak value SNP marker is as follows: chrA 09-2629811 (C/T), corresponding to the seed thioglycoside phenotype grouping: when the SNP at the position chrA 09-2629811 is C, the average seed glucosinolate content of the material is 39.26 mu mol/g; at T, the average seed glucosinolate content of the material is 102.81 mu mol/g; the contribution rate of this peak SNP was 72.11%;
one boundary SNP marker for seed glucosinolate content is: chrA 09-1798327 (C/T), corresponding to the seed thioglycoside phenotype grouping: when the SNP at the position chrA 09-1798327 is C, the average seed glucosinolate content of the material is 40.98 mu mol/g; at T, the average seed glucosinolate content of the material is 84.89 mu mol/g; the contribution rate of the border SNP was 28.04%;
another border SNP marker for seed thioglycoside content is: chrA09_4773555(A/G), corresponding to the seed thioglycoside phenotype grouping: when the SNP at the position chrA 09-4773555 is A, the average seed glucosinolate content of the material is 93.01 mu mol/g; g, the average seed glucosinolate content of the material is 42.55 mu mol/G; the contribution rate of this border SNP was 42.22%.
Drawings
FIG. 1 is a diagram showing the results of the distribution of the glucosinolate content of Brassica napus seeds according to the present invention.
FIG. 2 is a schematic diagram of the major QTL site location of the glucosinolate content of Brassica napus seeds of the present invention.
FIG. 3 is a schematic diagram of allelic analysis using peak SNP molecular markers of major QTL sites of glucosinolate content of Brassica napus seeds in accordance with the present invention.
Detailed Description
The present invention will be described in further detail with reference to specific examples below, which are not intended to limit the scope of the present invention, so that those skilled in the art can better understand the present invention and practice it.
The experimental procedures used in the following examples are conventional unless otherwise specified. The materials, reagents and the like used are commercially available unless otherwise specified.
Example 1 determination of phenotype of the glucosinolate content of Brassica napus seeds
1. Determination of the seed thioglycoside content phenotype of a related population
(1) 324 parts of cabbage type rape advanced generation strains from all over the world form a natural population, and the investigation of the content of the seed glucosinolates at 2 o' clock in 3 years is completed in a yang logical test base in Wuhan city and a test base in a farm academy in Yangzhou city.
(2) Three repeats of direct seeding and final singling are randomly designed, wherein the row spacing is 33cm, the plant spacing is 15cm, and each cell is 4 rows. And (4) planting protective rows around the test material field.
(3) The content of the seed glucosinolate is as follows: mature seeds of 10 plants of the material are taken from each cell, and the content of the glucosinolate is measured by a near infrared measuring instrument.
(4) The multi-year multi-point phenotype data was integrated by the BLUP method (http:// www.extension.org/pages/61006) to obtain seed thioglycoside breeding values as seed thioglycoside phenotype.
The tabular values for all environments were averaged for 324 parts of material and the results are summarized as follows:
TABLE 1324 seed thioglycoside content profile values for all environments of material (. mu. mol/g)
The result of the distribution of the content of the glucosinolates in the seeds of the related groups shows that the content of the glucosinolates in the seeds shows that the distribution shows continuous distribution and two peak values, which proves that the content of the glucosinolates in the seeds can have two major gene loci, as shown in figure 1.
2. Acquisition of related population high quality SNP data set
The method comprises the following steps of (1) extracting total DNA of leaves by adopting a CTAB method, and extracting the total DNA of the leaves of each material of a related population, wherein the specific method comprises the following steps:
rinsing the young and tender leaves in 10% ethanol; then shearing 0.1-0.2g of blades, putting the blades into a bowl mill, quickly milling the blades into powder by using liquid nitrogen, and putting the powder into a 2mL centrifuge tube; adding 700 mu L of preheated DNA extracting solution; mixing, placing in 65 deg.C water bath for 1h, and mixing for 1 time every 10-15 min; adding 700 μ L of mixed solution (phenol: chloroform: isoamyl alcohol 25: 24: 1), and mixing by gentle inversion for 10 min; centrifuging at 10000 Xg for 15min at room temperature; absorbing the supernatant into a new 2mL centrifuge tube; adding equal volume of mixed solution (chloroform: isoamyl alcohol is 24: 1), mixing, standing for 5min, centrifuging for 15min at 10000 Xg, and sucking supernatant into a new centrifuge tube with a gun; adding 2 times volume of anhydrous ethanol, mixing, standing at-20 deg.C for 1 hr at 10000 Xg, centrifuging for 10min, and removing supernatant; adding 500 μ L of precooled 75% ethanol, washing the precipitate, and removing the supernatant; washing and precipitating for 2 times continuously, and then airing; adding 100 μ L RNase A solution containing 2% RNase A, standing at 37 deg.C for 1h, and standing at 4 deg.C overnight; re-extracting DNA solution with equal volume of mixed solution (chloroform: isoamyl alcohol: 24: 1), reversing, mixing, standing for 10min, 10000 Xg, centrifuging for 15 or 20min, removing RNase A, sucking supernatant (about 60 μ L), and centrifuging again for 1 min; detecting the concentration, quality and integrity of the DNA by agarose gel electrophoresis (0.8%) and an ultraviolet spectrophotometer; the ratio of the absorbance 260/280 was determined to be between 1.8 and 2.0 for all DNA samples. The DNA samples were then transported on dry ice to sequencing Inc. (Huada science and technology, Inc.), each material having a sequencing depth of about 7X.
After obtaining high quality DNA as described above, the sequencing company (Huada science and technology Co., Ltd.) performed 7 Xcoverage depth sequencing and returned data, and performed sequencing quality evaluation using FastQC software, and then adapter and low quality reads filtration on the sequencing sequence. Obtaining clear data of double-end sequencing of each material, then using bwa software to carry out mapping and GATK software to carry out mutation detection, after obtaining a total SNP data set of a related group, carrying out SNP data set quality filtration according to the minimum allele frequency of more than or equal to 0.05, the deletion rate of less than or equal to 0.1 and the heterozygosity rate of less than or equal to 0.1, and finally obtaining a high-quality group SNP data set for subsequent analysis.
3. Genome-wide association analysis
Format conversion is carried out on the VCF file of the high-quality SNP data set generated in the last step by using plink software, then EMMAX software is used for carrying out whole-gene association analysis on the obtained seed glucosinolate content phenotype and the SNP data set to obtain the P value of each SNP locus, and when the P value is less than 7.2 multiplied by 10 -7 The SNP is the obvious SNP, the SNP with the minimum P value is the peak SNP, the materials are grouped by different allele types of the peak SNP in a group, variance analysis is carried out, and the percentage of the ratio of the variance between the groups to the total variance is the contribution rate of the peak SNP.
Through analysis, the interval of the major QTL site of the glucosinolate content of the brassica napus seeds is limited between the 1798327 th base and the 4773555 th base of the A09 chromosome of the brassica napus, the corresponding SNPs are chrA 09-1798327 (C/T), chrA 09-4773555 (A/G), and the peak SNP is: and chrA 09-2629811 (C/T), wherein the contribution rate of the QTL to the glucosinolate content of the brassica napus seeds is 72.11% (the materials are grouped according to different allele types of peak SNP, the analysis of single-factor variance is carried out, and the percentage of the variance between groups divided by the total variance is the contribution rate).
The peak SNP of the seed glucosinolate content is as follows: chrA 09-2629811 (C/T), corresponding to the seed thioglycoside phenotype grouping: when the SNP at the position chrA 09-2629811 is C, the average seed glucosinolate content of the material is 39.26 mu mol/g; at T, the average seed glucosinolate content of the material is 102.81 mu mol/g; the contribution rate of this peak SNP was 72.11%.
One of the border SNPs for seed glucosinolate content is: chrA 09-1798327 (C/T), corresponding to the seed thioglycoside phenotype grouping: when the SNP at the position chrA 09-1798327 is C, the average seed glucosinolate content of the material is 40.98 mu mol/g; at T, the average seed glucosinolate content of the material is 84.89 mu mol/g; the contribution rate of this border SNP was 28.04%.
Another boundary SNP for seed glucosinolate content is: chrA09_4773555(A/G), corresponding to the seed thioglycoside phenotype grouping: when the SNP at the position chrA 09-4773555 is A, the average seed glucosinolate content of the material is 93.01 mu mol/g; g, the average seed glucosinolate content of the material is 42.55 mu mol/G; the contribution rate of this border SNP was 42.22%.
The whole genome sequence of Brassica napus is published, wherein 400bp sequences (801 bp in total) before and after the sequence containing chrA 09-1798327 (C/T) are shown as SEQ ID NO:1, 400bp sequences (801 bp in total) before and after the sequence containing chrA 09-4773555 (A/G) are shown as SEQ ID NO:2, and 400bp sequences (801 bp in total) before and after the sequence containing chrA 09-2629811 (C/T) are shown as SEQ ID NO: 3. The skilled in the art can design a specific primer or probe for detecting the SNP site according to a known sequence by using a conventional method, and the primer or probe can also be marked with a fluorescent group such as FAM, HEX, VIC, ROX and the like and a quenching group such as BHQ1 or TAMRA by using the conventional technology in the art, so that the genotype of the SNP site can be detected by using the conventional method in the art such as sequencing or PCR and the like, and therefore, the content of the glucosinolate in the Brassica napus seeds can be detected, the content of the glucosinolate in the Brassica napus seeds can be predicted, and further, the content of the glucosinolate in the Brassica napus seeds can be effectively selected, so that the specific primer or probe is used for molecular marker assisted breeding of the Brassica napus with the glucosinolate content, and the breeding process of the glucosinolate content of the Brassica napus can be accelerated.
Therefore, the invention detects a major QTL locus of the glucosinolate content of the cabbage type rape seeds on the chromosome A09 of the cabbage type rape through the phenotypic analysis and the whole genome re-sequencing of the glucosinolate content of the seeds and then the whole genome correlation analysis, and the contribution rate of the major QTL locus to the glucosinolate content of the cabbage type rape seeds is 72.11 percent. The major QTL site of the glucosinolate content of the cabbage type rape seeds is positioned between the 1798327 th base and the 4773555 th base of an A09 chromosome of the cabbage type rape, the obvious SNP of the boundary is chrA 09-1798327 (C/T), chrA 09-4773555 (A/G), the peak SNP is chrA 09-2629811 (C/T), and according to SNP molecular markers which are closely linked with the major QTL site, the method can be used for detecting the content of the glucosinolate content of the cabbage type rape seeds, predicting the content of the glucosinolate content of the cabbage type rape seeds, effectively selecting the content of the glucosinolate content of the cabbage type rape seeds, assisting breeding by using the molecular markers of the cabbage type rape seeds with the glucosinolate content, and accelerating the breeding process of the glucosinolate content of the cabbage type rape seeds.
The SNP molecular marker disclosed by the invention is used for carrying out molecular marker-assisted selection, the identification method is simple, the selection efficiency is high, and the glucosinolate content of the brassica napus seeds can be predicted. The selection target is clear and is not influenced by the environment. The individual cabbage type rape with early glucosinolate content can be identified in the early growth stage of cabbage type rape, and other individual plants are eliminated. In conclusion, the major QTL site of the glucosinolate content of the brassica napus seeds has high contribution rate to the glucosinolate content of the brassica napus seeds, plays a key role in regulating and controlling the glucosinolate content of the brassica napus seeds, can be used for map-based cloning and molecular marker-assisted selection, and is suitable for large-scale popularization and application.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
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aaccttggaa agaacaagac tagtgaagat gcagctgctg attatgtcca aggagttcat 780
aatttatccc agtatgctga t 801
<210> 4
<211> 20
<212> DNA
<213> Artificial sequence ()
<220>
<221> primer_bind
<222> (1)..(20)
<223> chrA09_1798327(C/T) upstream primer sequence
<400> 4
<210> 5
<211> 20
<212> DNA
<213> Artificial sequence ()
<220>
<221> primer_bind
<222> (1)..(20)
<223> chrA09_1798327(C/T) downstream primer sequence
<400> 5
<210> 6
<211> 20
<212> DNA
<213> Artificial sequence ()
<220>
<221> primer_bind
<222> (1)..(20)
<223> chrA09_4773555(A/G) upstream primer sequence
<400> 6
<210> 7
<211> 20
<212> DNA
<213> Artificial sequence ()
<220>
<221> primer_bind
<222> (1)..(20)
<223> chrA09_4773555(A/G) downstream primer sequence
<400> 7
<210> 8
<211> 20
<212> DNA
<213> Artificial sequence ()
<220>
<221> primer_bind
<222> (1)..(20)
<223> chrA09_2629811(C/T) upstream primer sequence
<400> 8
<210> 9
<211> 20
<212> DNA
<213> Artificial sequence ()
<220>
<221> primer_bind
<222> (1)..(20)
<223> chrA09_2629811(C/T) downstream primer sequence
<400> 9
Claims (3)
1. The application of a primer or a probe of an SNP molecular marker of a major QTL site of the glucosinolate content of cabbage type rape seeds in detecting and/or predicting the glucosinolate content of the cabbage type rape seeds or in molecular marker-assisted breeding of the cabbage type rape seeds;
the SNP molecular marker is positioned at the 401 st base of the nucleotide sequence shown as SEQ ID NO. 1, and the 401 st base is C or T; when this site is T, it has a higher thioglycoside content than when it is C.
2. The application of a primer or a probe of an SNP molecular marker of a major QTL site of the glucosinolate content of cabbage type rape seeds in detecting and/or predicting the glucosinolate content of the cabbage type rape seeds or in molecular marker-assisted breeding of the cabbage type rape seeds;
the SNP molecular marker is located at the 401 st base of the nucleotide sequence shown as SEQ ID NO. 2, and the 401 st base is A or G; when this site is A, it has a higher thioglycoside content than when it is G.
3. The application of a primer or a probe of an SNP molecular marker of a major QTL site of the glucosinolate content of cabbage type rape seeds in detecting and/or predicting the glucosinolate content of the cabbage type rape seeds or in molecular marker-assisted breeding of the cabbage type rape seeds;
the SNP molecular marker is positioned at the 401 st base of the nucleotide sequence shown as SEQ ID NO. 3, and the 401 st base is C or T; when this site is T, it has a higher thioglycoside content than when it is C.
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| CN114836561B (en) * | 2022-03-28 | 2023-09-01 | 浙江省农业科学院 | SNP (Single nucleotide polymorphism) marker related to glucoraphanin content in broccoli, primer and application |
| CN116042889B (en) * | 2022-09-29 | 2023-08-29 | 安徽科技学院 | Molecular marker linked with main effect QTL locus of flowering phase of brassica napus and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| EP2016821A1 (en) * | 2007-06-13 | 2009-01-21 | Syngeta Participations AG | New hybrid system for Brassica napus |
| CN103937790B (en) * | 2014-05-12 | 2016-04-20 | 中国农业科学院油料作物研究所 | A kind of molecule marker of being closely related with Semen Brassicae campestris sulphur resources and application |
| CN105505925B (en) * | 2016-01-10 | 2018-05-22 | 华中农业大学 | A kind of molecular labeling with Oil Content In B. Napus L QTL close linkages |
| CN107723379B (en) * | 2017-11-30 | 2021-03-02 | 华中农业大学 | Molecular marker for controlling flowering phase of cabbage type rape and application |
| CN108754011B (en) * | 2018-06-21 | 2021-05-14 | 贵州省油菜研究所 | Major QTL (quantitative trait locus) site for thousand grain weight trait of brassica napus, SNP (Single nucleotide polymorphism) molecular marker and application |
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