CN110527738B - Major QTL (quantitative trait loci) site and SNP (single nucleotide polymorphism) molecular marker for oleic acid content of brassica napus seeds and application of major QTL site and SNP molecular marker - Google Patents
Major QTL (quantitative trait loci) site and SNP (single nucleotide polymorphism) molecular marker for oleic acid content of brassica napus seeds and application of major QTL site and SNP molecular marker Download PDFInfo
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Abstract
The invention provides a main effect QTL site of the oleic acid content of cabbage type rape seeds, which is positioned between the 8024751 th base and the 11299788 th base of the A08 chromosome of cabbage type rape. Preferably, the contribution rate to the oleic acid content of the brassica napus seeds is 71.00%. Closely linked to the first SNP molecular marker, which is located at base 8024751 and is C or G, the mutation results in a polymorphism. Closely linked to a second SNP molecular marker located at base 11299788, being either A or G, the mutation resulting in a polymorphism. Closely linked to the peak SNP molecular marker, located at base 10445600, at either a or G, the mutation resulting in a polymorphism. Also provides related SNP molecular markers and application. The main effect QTL site of the oleic acid content of the cabbage type rape seeds has high contribution rate to the oleic acid content of the cabbage type rape seeds, plays a key role in regulating and controlling the oleic acid content of the cabbage type rape seeds, can be used for map 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 oleic acid content of brassica napus seeds, and specifically relates to an SNP molecular marker closely linked with major QTL (quantitative trait loci) of the oleic acid 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. Cabbage type rape in China becomes a main cultivated variety due to the characteristics of disease resistance, high yield, wide adaptability, strong adverse resistance 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 great breakthrough on the existing basis, so that the combination of quantitative genetics and a molecular marker technology provides a new opportunity 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.
The contents of oleic acid and linolenic acid in rape have important influence on the aspects of eating quality, nutritional quality, storage and the like. In the aspect of edible quality, the rapeseed oil with high oleic acid content has relatively low content of polyunsaturated fatty acid, strong thermal stability and difficult oxidation in the purification and heating process, and the linolenic acid is easy to be oxidized under the conditions of high temperature or strong light to cause the deterioration of grease and is not favorable for storage. In terms of nutritional quality, monounsaturated fatty acids such as oleic acid (C18: 1) and the like in the rapeseed oil are more beneficial to human health, and linolenic acid (C18: 3) contains three unsaturated bonds, is very easy to oxidize to form free radicals, generates lipid peroxidation, and damages the structures and functions of cells and cell membranes.
In the prior art, Jiangxianxia and the like (Shanghai agricultural science, 2019) point out that the influence of the contents of oleic acid and linolenic acid in rapeseed oil on human health and storage is comprehensively considered, the content of oleic acid in the rapeseed oil is improved, and the content of linolenic acid is reduced, which is an important breeding target in quality breeding of the rapeseed 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 molecular markers related to important quality traits such as oleic acid content of rape and QTL localization are increasing, but most of them are in the initial localization stage. Therefore, by means of molecular markers and Quantitative Trait Locus (QTL) positioning, the oleic acid content of the brassica napus seeds is further researched at a molecular level, so that the quality of the rapeseed oil is improved, and a foundation is laid for disclosing the genetic mechanism and the molecular mechanism of the oleic acid content of the brassica napus seeds.
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 oleic acid content in brassica napus seeds, wherein the major QTL locus of oleic acid content in brassica napus seeds is located between the 8024751 st base and the 11299788 th base of the chromosome a08 of brassica napus, has a high contribution rate to the oleic acid content of brassica napus seeds, plays a key role in the regulation and control of the oleic acid content of brassica napus seeds, can be used for sital cloning and molecular marker-assisted selection, and is suitable for large-scale popularization and application.
Preferably, the major QTL site of the oleic acid 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 8024751 th base of the A08 chromosome of Brassica napus, the 8024751 th base is C or G, and the mutation results in polymorphism.
Preferably, the second SNP molecular marker is located at the 11299788 th base of the A08 chromosome of Brassica napus, the 11299788 th base is A or G, and the mutation results in polymorphism.
Preferably, the peak SNP molecular marker is located at the 10445600 th base of the A08 chromosome of Brassica napus, and the 10445600 th base is A or G, 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 oleic acid content of the brassica napus seeds.
Preferably, the method is used for detecting the content of the oleic acid in the brassica napus seeds, or predicting the content of the oleic acid in the brassica napus seeds, or effectively selecting the content of the oleic acid in 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 oleic acid content of the Brassica napus.
The invention also aims to provide a primer or a probe of the SNP molecular marker of the main effect QTL site of the oleic acid content of the brassica napus seeds.
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 08-8024751 (C/G) as a template, and the DNA fragment is shown as SEQ ID NO. 1.
Preferably, the primer or probe is designed by taking a DNA fragment containing 400bp sequences (801 bp in total) of the brassica napus chrA 08-11299788 (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) of the brassica napus chrA 08-10445600 (A/G) 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 main QTL site of the oleic acid content of the brassica napus seeds in detecting and/or predicting the oleic acid 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 main effect QTL site of the oleic acid content of a cabbage type rape seed, which is closely linked with an SNP molecular marker, has high contribution rate to the oleic acid content of the cabbage type rape seed, plays a key role in regulating and controlling the oleic acid content of the cabbage type rape seed, can be used for map-based cloning and molecular marker-assisted selection, and is suitable for large-scale popularization and application.
(2) The SNP molecular markers of the major QTL site of the oleic acid content of the cabbage type rape seeds comprise a 8024751 th base SNP molecular marker located on an A08 chromosome of the cabbage type rape, a 11299788 th base SNP molecular marker located on an A08 chromosome of the cabbage type rape and a peak value SNP molecular marker located on a 10445600 th base of an A08 chromosome of the cabbage type rape.
(3) The invention specifically provides three SNP molecular markers of the oleic acid content of cabbage type rape seeds, wherein the peak value SNP marker is as follows: chrA08_10445600(A/G), corresponding to the seed oleic acid content phenotype grouping: when the SNP at position chrA 08-10445600 is A, the average seed oleic acid content of the material is 19.53%; at G, the average seed oleic acid content of the material was 52.85%; the contribution rate of the peak SNP is 71.00%;
one border SNP marker for seed oleic acid content was: chrA08_8024751(C/G), corresponding to the seed oleic acid content phenotype grouping: when the SNP at position chrA 08-8024751 is C, the average seed oleic acid content of the material is 50.37%; g, the average seed oleic acid content of the material is 25.23%; the contribution rate of the border SNP was 41.14%;
another boundary SNP marker for seed oleic acid content is: chrA08_11299788(A/G), corresponding to the seed oleic acid content phenotype grouping: when the SNP at the position chrA 08-11299788 is A, the average seed oleic acid content of the material is 50.08%; at G, the average seed oleic acid content of the material is 25.23%; the contribution rate of this border SNP was 39.52%.
Drawings
FIG. 1 is a graph showing the distribution results of the oleic acid content of Brassica napus seeds according to the present invention, i.e., the phenotypic frequency distribution of the oleic acid content of seeds of related groups.
FIG. 2 is a schematic diagram of the major QTL site location of the oleic acid content of Brassica napus seeds of the present invention, i.e. Manhattan plot for the correlation analysis of the oleic acid content of seeds of the correlated population.
FIG. 3 is a schematic diagram of allelic analysis by using the peak SNP molecular marker of the main effect QTL site of the oleic acid content of Brassica napus seeds in the invention, namely an allelic analysis diagram of the peak SNP (chrA 08-10445600) molecular marker of the main effect QTL site.
Detailed Description
The present invention will be described in further detail with reference to specific examples below so that those skilled in the art can better understand the present invention and practice the present invention, but the examples are not intended to limit the present invention.
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 the phenotype of the oleic acid content of Brassica napus seeds
1. Determination of seed oleic acid content phenotype of related populations
(1) 324 parts of cabbage type rape advanced generation strains from all over the world form a natural population, and the investigation of the oleic acid content of seeds 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 oleic acid content of the seeds is as follows: mature seeds of 10 plants of the material are taken from each cell, and the oleic acid content is measured by a near infrared tester.
(4) The multi-year multi-point phenotype data was integrated by the BLUP method (http:// www.extension.org/pages/61006) to obtain seed oleic acid content breeding values as seed oleic acid content phenotypes.
The tabular values for all environments were averaged for 324 parts of material and the results are summarized as follows:
TABLE 1324 parts of materials table values for seed oleic acid content for all environments (%)
The distribution result of the oleic acid content of the seeds of the related groups shows that the oleic acid content of the seeds presents continuous distribution and presents two peak values, and the oleic acid content of the seeds is proved to have two major gene loci, as shown in figure 1.
2. Acquisition of related population high quality SNP dataset
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 room temperature at 10000 Xg for 15 min; sucking the supernatant into a new 2mL centrifuge tube; adding equal volume of mixed solution (chloroform: isoamyl alcohol: 24: 1), mixing, standing for 5min, centrifuging for 15min at 10000 Xg, and sucking supernatant with a gun into a new centrifuge tube; 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 mu L of precooled 75% ethanol, washing the precipitate, and removing supernatant; washing the precipitate for 2 times, and air drying; 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 Gene science and technology Co., Ltd.) performed 7 Xcoverage depth sequencing and returned data, and the sequencing quality was evaluated by using FastQC software, and then adapter and low quality reads were performed 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, and after obtaining a total SNP data set of an associated group, carrying out SNP data set quality filtering 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. Whole genome 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 oleic acid content phenotype and the SNP data set, and the P value of each SNP site is obtained, and when the P value is smaller than 7.2 multiplied by 10 -7 The SNP of (single nucleotide polymorphism) is the obvious SNP, the SNP with the minimum P value is the peak SNP, materials are grouped in 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 main effect QTL site of the oleic acid content of the cabbage type rape seeds is limited between the 8024751 th base and the 11299788 th base of the A08 chromosome of the cabbage type rape, the corresponding SNPs are chrA08_8024751(C/G), chrA08_11299788(A/G), and the peak SNP is: and chrA 08-10445600 (A/G), wherein the contribution rate of the QTL to the oleic acid content of the brassica napus seeds is 71.00 percent (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 oleic acid content of the seeds is as follows: chrA08_10445600(A/G), corresponding to the seed oleic acid content phenotype grouping: when the SNP at position chrA 08-10445600 is A, the average seed oleic acid content of the material is 19.53%; at G, the average seed oleic acid content of the material was 52.85%; the contribution rate of the peak SNP is 71.00%;
one of the border SNPs for seed oleic acid content is: chrA08_8024751(C/G), corresponding to the seed oleic acid content phenotype grouping: when the SNP at the position chrA 08-8024751 is C, the average seed oleic acid content of the material is 50.37%; at G, the average seed oleic acid content of the material is 25.23%; the contribution rate of the border SNP was 41.14%;
another boundary SNP for seed oleic acid content is: chrA08_11299788(A/G), corresponding to the seed oleic acid content phenotype grouping: when the SNP at the position chrA 08-11299788 is A, the average seed oleic acid content of the material is 50.08%; g, the average seed oleic acid content of the material is 25.23%; the contribution rate of this border SNP was 39.52%.
The whole genome sequence of Brassica napus is published, wherein 400bp sequences (801 bp in total) before and after the sequence containing chrA 08-8024751 (C/G) are shown as SEQ ID NO:1, 400bp sequences (801 bp in total) before and after the sequence containing chrA 08-11299788 (A/G) are shown as SEQ ID NO:2, and 400bp sequences (801 bp in total) before and after the sequence containing chrA 08-10445600 (A/G) are shown as SEQ ID NO: 3. The skilled person can design a specific primer or probe for detecting the SNP site according to the known sequence by using a conventional method, and the primer or probe can be further labeled 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 method 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, thereby detecting the content of oleic acid in the Brassica napus seeds, predicting the content of oleic acid in the Brassica napus seeds, further effectively selecting the content of oleic acid in the Brassica napus seeds, using the molecular marker for assisted breeding of the Brassica napus seeds with oleic acid content, and accelerating the breeding process of the content of oleic acid in the Brassica napus seeds.
Therefore, the method detects a major QTL site of the oleic acid content of the brassica napus seeds on the chromosome A08 of the brassica napus through phenotypic analysis and whole genome re-sequencing of the oleic acid content of the seeds and then whole genome correlation analysis, and the contribution rate of the major QTL site to the oleic acid content of the brassica napus seeds is 71.00 percent. The main effect QTL site of the oleic acid content of the cabbage type rape seeds is positioned between the 8024751 th base and the 11299788 th base of an A08 chromosome of the cabbage type rape, the obvious SNPs at the boundary are chrA 08-8024751 (C/G), chrA 08-11299788 (A/G), and the peak SNP is chrA 08-10445600 (A/G).
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 oleic acid 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 oleic acid 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 oleic acid content of the brassica napus seeds has high contribution rate to the oleic acid content of the brassica napus seeds, plays a key role in regulating and controlling the oleic acid content of the brassica napus seeds, can be used for map 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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<222> (1)..(20)
<223> chrA08_11299788(A/G) downstream primer
<400> 7
<210> 8
<211> 20
<212> DNA
<213> Artificial sequence ()
<220>
<221> primer_bind
<222> (1)..(20)
<223> chrA08_10445600(A/G) upstream primer
<400> 8
<210> 9
<211> 20
<212> DNA
<213> Artificial sequence ()
<220>
<221> primer_bind
<222> (1)..(20)
<223> chrA08_10445600(A/G) downstream primer
<400> 9
Claims (3)
1. The application of a primer or a probe of an SNP molecular marker of a main effect QTL site of the oleic acid content of a cabbage type rape seed in detecting and/or predicting the oleic acid content of the cabbage type rape seed or in molecular marker assisted breeding of the cabbage type rape seed;
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 G; when this site is C, it has a higher oleic acid content than when it is G.
2. The application of a primer or a probe of an SNP molecular marker of a main effect QTL site of the oleic acid content of a cabbage type rape seed in detecting and/or predicting the oleic acid content of the cabbage type rape seed or in molecular marker assisted breeding of the cabbage type rape seed;
the SNP molecular marker is positioned 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 oleic acid content than when it is G.
3. The application of a primer or a probe of an SNP molecular marker of a main effect QTL site of the oleic acid content of a cabbage type rape seed in detecting and/or predicting the oleic acid content of the cabbage type rape seed or in molecular marker assisted breeding of the cabbage type rape seed;
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 A or G; when this site is G, it has a higher oleic acid content than when it is A.
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| CN111118204B (en) * | 2020-02-25 | 2023-04-14 | 贵州省油菜研究所 | A08 chromosome major QTL (quantitative trait locus) site with oleic acid content character of brassica napus seeds, SNP molecular marker and application |
| CN111100951B (en) * | 2020-02-25 | 2023-05-12 | 贵州省油菜研究所 | A09 chromosome major QTL locus of cabbage type rape oil content character, SNP molecular marker and application |
| CN111518943B (en) * | 2020-05-26 | 2023-07-28 | 中国林业科学研究院亚热带林业研究所 | SNP molecular marker related to eicosenoic acid content in camellia seed oil and application thereof |
| CN113736903B (en) * | 2021-09-14 | 2023-08-18 | 中国农业科学院油料作物研究所 | Molecular marker closely linked with rape selenium efficient property main effect QTL locus qSe.C03 and application |
| CN113652499B (en) * | 2021-09-16 | 2023-06-09 | 中国农业科学院油料作物研究所 | Molecular marker closely linked with rape selenium efficient property main effect QTL locus qSe.C07 and application |
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| CN107828908A (en) * | 2017-10-27 | 2018-03-23 | 江苏省农业科学院 | The molecule labelling method of cabbage type rape seed high oleic acid content and application |
| CN108239674A (en) * | 2017-11-19 | 2018-07-03 | 华中农业大学 | Cabbage type rape high oleic acid QTL and the molecular labeling with its close linkage |
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| MX2007008435A (en) * | 2005-01-12 | 2007-09-06 | Agrinomics Llc | Generation of plants with altered oil content. |
| CN102766627B (en) * | 2012-08-08 | 2013-06-05 | 中国农业科学院油料作物研究所 | Molecular marker closely linked with oil content character of rapes and application |
| EP3288370A1 (en) * | 2015-04-28 | 2018-03-07 | Bayer CropScience NV | Brassica plants with modified seed oil composition |
| 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 |
| 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 |
| CN108728575B (en) * | 2018-06-21 | 2021-07-02 | 贵州省油菜研究所 | Major QTL site of brassica napus silique length character, SNP molecular marker and application |
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| CN107828908A (en) * | 2017-10-27 | 2018-03-23 | 江苏省农业科学院 | The molecule labelling method of cabbage type rape seed high oleic acid content and application |
| CN108239674A (en) * | 2017-11-19 | 2018-07-03 | 华中农业大学 | Cabbage type rape high oleic acid QTL and the molecular labeling with its close linkage |
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