SNP molecular markers, related detection primers or probes, and applications of major QTL loci for 1000-seed weight traits in Brassica napus

Abstract

The present invention provides a SNP molecular marker for the main effect QTL site of the 1000-grain weight trait of Brassica napus, which is tightly linked to the main effect QTL site of the 1000-grain weight trait of Brassica napus. The main effect QTL site of the 1000-grain weight trait of Brassica napus is located between bases 20082973 and 20310902 on the A07 chromosome of Brassica napus. Also provided are applications of the SNP molecular marker, primers or probes for detecting the SNP molecular marker, and applications thereof. The SNP molecular marker for the main effect QTL site of the 1000-grain weight trait of Brassica napus of the present invention can detect the size of the 1000-grain weight of Brassica napus, can predict the size of the 1000-grain weight of Brassica napus, can effectively select the size of the 1000-grain weight of Brassica napus, and can also be used for molecular marker-assisted breeding of Brassica napus with large 1000-grain weight, thereby accelerating the process of 1000-grain weight breeding of Brassica napus, and is suitable for large-scale promotion and application.

Description

Technical Field

[0001] The present invention relates to the technical fields of molecular biology and rapeseed genetic breeding, in particular to the technical field of 1000-grain weight traits of Brassica napus, specifically to a SNP molecular marker of a major effect QTL site of the 1000-grain weight trait of Brassica napus, related detection primers or probes, and applications. Background Art

[0002] Grain weight is one of the three key factors contributing to high yield in rapeseed. Creating high-yield varieties that double yield by increasing grain weight is a key approach to developing high-density-tolerant, high-yield, and multi-resistant varieties. Thousand-grain weight in rapeseed is a typical quantitative trait controlled by a combination of major and multiple genes, and is significantly influenced by the environment. Additive effects are the primary component of the genetic effect of the thousand-grain weight gene and form the crucial genetic basis for increased grain weight through gene aggregation. Major breakthroughs are difficult to achieve with traditional breeding techniques. Therefore, combining molecular marker technology with quantitative genetics is crucial to developing novel molecular markers for the genetic breeding of thousand-grain weight in rapeseed.

[0003] Quantitative trait loci (QTL) mapping has been shown to be an effective strategy for dissecting its complex genetic basis and facilitating marker-assisted selection to accelerate rapeseed breeding. Previous studies have reported multiple QTLs for rapeseed seed yield-related traits, which are distributed across all chromosomes of the rapeseed genome. Before the advent of the Brassica napus reference genome, QTL mapping was performed using simple sequence repeat (SSR) and low-density amplified fragment length polymorphism (AFLP) markers. In recent years, with the completion of whole-genome sequencing, the widespread application of molecular marker technology, and the continuous development of marker-assisted selection (MAS) technology, more and more research results have been reported on molecular markers and QTL mapping related to the 1000-grain weight trait of rapeseed.

[0004] Therefore, further research on the 1000-grain weight trait of Brassica napus seeds at the molecular level with the help of molecular markers and quantitative trait loci (QTL) positioning will help improve rapeseed yield and lay the foundation for revealing the genetic structure and molecular mechanism of 1000-grain weight in Brassica napus.

[0005] Therefore, it is hoped to provide a SNP molecular marker for the major effect QTL site of the thousand-grain weight trait of Brassica napus, which can detect the size of the thousand-grain weight of Brassica napus, predict the size of the thousand-grain weight of Brassica napus, effectively select the size of the thousand-grain weight of Brassica napus, and can also be used for molecular marker-assisted breeding of Brassica napus with large thousand-grain weight, thereby accelerating the process of breeding Brassica napus with thousand-grain weight. Summary of the Invention

[0006] In order to overcome the shortcomings of the above-mentioned prior art, one object of the present invention is to provide a SNP molecular marker for the main effect QTL site of the 1000-grain weight trait of Brassica napus, which can detect the size of the 1000-grain weight of Brassica napus, predict the size of the 1000-grain weight of Brassica napus, effectively select the size of the 1000-grain weight of Brassica napus, and can also be used for molecular marker-assisted breeding of Brassica napus with large 1000-grain weight, accelerate the process of breeding of Brassica napus with 1000-grain weight, and is suitable for large-scale promotion and application.

[0007] Another object of the present invention is to provide a SNP molecular marker for the major QTL locus of the thousand-grain weight trait of Brassica napus, which has a clever design, is simple and fast to detect, is low in cost, is not affected by the environment, and is suitable for large-scale promotion and application.

[0008] Another object of the present invention is to provide an application of a SNP molecular marker at the main effect QTL site of the 1000-grain weight trait of Brassica napus, which can be used to detect the size of the 1000-grain weight of Brassica napus, can be used to predict the size of the 1000-grain weight of Brassica napus, can be used to effectively select the size of the 1000-grain weight of Brassica napus, and can also be used for molecular marker-assisted breeding of Brassica napus with large 1000-grain weight, thereby accelerating the process of breeding of Brassica napus with 1000-grain weight, and is suitable for large-scale promotion and application.

[0009] Another object of the present invention is to provide an application of a SNP molecular marker for the major effect QTL site of the thousand-grain weight trait of Brassica napus, which has a clever design, is simple and fast to detect, is low in cost, is not affected by the environment, and is suitable for large-scale promotion and application.

[0010] Another object of the present invention is to provide a primer or probe for detecting SNP molecular markers at the main effect QTL site of the 1000-grain weight trait of Brassica napus. The primer or probe can detect the size of the 1000-grain weight of Brassica napus, predict the size of the 1000-grain weight of Brassica napus, effectively select the size of the 1000-grain weight of Brassica napus, and can also be used for molecular marker-assisted breeding of Brassica napus with large 1000-grain weight, thereby accelerating the process of breeding of Brassica napus with 1000-grain weight, and is suitable for large-scale promotion and application.

[0011] Another object of the present invention is to provide a primer or probe for detecting SNP molecular markers of the main effect QTL site of the thousand-grain weight trait of Brassica napus, which has a clever design, is simple and fast to detect, is low in cost, is not affected by the environment, and is suitable for large-scale promotion and application.

[0012] Another object of the present invention is to provide an application of a primer or probe for detecting SNP molecular markers at the main effect QTL site of the 1000-grain weight trait of Brassica napus. The primer or probe can be used to detect the size of the 1000-grain weight of Brassica napus, can be used to predict the size of the 1000-grain weight of Brassica napus, can be used to effectively select the size of the 1000-grain weight of Brassica napus, and can also be used for molecular marker-assisted breeding of Brassica napus with large 1000-grain weight, thereby accelerating the process of breeding of Brassica napus with 1000-grain weight, and is suitable for large-scale promotion and application.

[0013] Another object of the present invention is to provide an application of a primer or probe for detecting SNP molecular markers at the main effect QTL site of the thousand-grain weight trait of Brassica napus. The primer or probe has an ingenious design, is simple and fast to detect, has low cost, is not affected by the environment, and is suitable for large-scale promotion and application.

[0014] To achieve the above objectives, in the first aspect of the present invention, a SNP molecular marker for the major QTL site of the thousand-grain weight trait of Brassica napus is provided, which is characterized in that the SNP molecular marker is tightly linked to the major QTL site of the thousand-grain weight trait of Brassica napus, and the major QTL site of the thousand-grain weight trait of Brassica napus is located between bases 20082973 and 20310902 on chromosome A07 of Brassica napus.

[0015] Preferably, the SNP molecular marker is located at the 20082973rd base, the 20082973rd base is A or G, and the mutation causes polymorphism.

[0016] Preferably, the SNP molecular marker is located at the 20310902nd base, the 20310902nd base is G or A, and the mutation causes polymorphism.

[0017] In the second aspect of the present invention, the application of the SNP molecular markers of the major effect QTL sites of the above-mentioned 1000-grain weight trait of Brassica napus to detecting the size of the 1000-grain weight of Brassica napus, predicting the size of the 1000-grain weight of Brassica napus, selecting the size of the 1000-grain weight of Brassica napus, or molecular marker-assisted breeding of Brassica napus with large 1000-grain weight is provided.

[0018] In a third aspect of the present invention, primers or probes for detecting SNP molecular markers at the major effect QTL loci of the aforementioned 1000-grain weight trait of Brassica napus are provided.

[0019] Preferably, the primers or probes are designed based on a DNA fragment of 400 bp sequence before and after bases 20082973 of the A07 chromosome of Brassica napus as a template, and the DNA fragment is shown in SEQ ID NO: 1.

[0020] Preferably, the primers or probes are designed based on a DNA fragment of 400 bp sequence before and after base 20310902 of the A07 chromosome of Brassica napus as a template, and the DNA fragment is shown in SEQ ID NO: 2.

[0021] In the fourth aspect of the present invention, primers or probes of SNP molecular markers at the major effect QTL sites of the above-mentioned 1000-grain weight trait of Brassica napus are provided for use in detecting the size of the 1000-grain weight of Brassica napus, predicting the size of the 1000-grain weight of Brassica napus, selecting the size of the 1000-grain weight of Brassica napus, or in molecular marker-assisted breeding of Brassica napus with large 1000-grain weight.

[0022] The beneficial effects of the present invention are mainly:

[0023] 1. The SNP molecular marker of the main effect QTL site of the 1000-grain weight trait of Brassica napus of the present invention is closely linked to the main effect QTL site of the 1000-grain weight trait of Brassica napus. The main effect QTL site of the 1000-grain weight trait of Brassica napus is located between bases 20082973 and 20310902 on chromosome A07 of Brassica napus. The SNP molecular marker can detect the size of the 1000-grain weight of Brassica napus, predict the size of the 1000-grain weight of Brassica napus, effectively select the size of the 1000-grain weight of Brassica napus, and can also be used for molecular marker-assisted breeding of Brassica napus with high 1000-grain weight, thereby accelerating the process of breeding of Brassica napus with 1000-grain weight, and is suitable for large-scale promotion and application.

[0024] 2. The SNP molecular marker of the main effect QTL locus of the 1000-grain weight trait of Brassica napus of the present invention is closely linked to the main effect QTL locus of the 1000-grain weight trait of Brassica napus. The main effect QTL locus of the 1000-grain weight trait of Brassica napus is located between bases 20082973 and 20310902 on the A07 chromosome of Brassica napus. The SNP molecular marker is cleverly designed, simple and fast to detect, low in cost, not affected by the environment, and suitable for large-scale promotion and application.

[0025] 3. The application of the SNP molecular marker of the main effect QTL site of the 1000-grain weight trait of Brassica napus in the present invention can be used to detect the size of the 1000-grain weight of Brassica napus, can be used to predict the size of the 1000-grain weight of Brassica napus, can be used to effectively select the size of the 1000-grain weight of Brassica napus, and can also be used for molecular marker-assisted breeding of Brassica napus with large 1000-grain weight, thereby accelerating the process of Brassica napus 1000-grain weight breeding, and is suitable for large-scale promotion and application.

[0026] 4. The application of the SNP molecular marker of the major QTL locus of the 1000-grain weight trait of Brassica napus in the present invention is ingeniously designed, simple and fast to detect, low in cost, unaffected by the environment, and suitable for large-scale promotion and application.

[0027] 5. The primers or probes for detecting SNP molecular markers at the major effect QTL loci of the 1000-grain weight trait of Brassica napus of the present invention can detect the size of the 1000-grain weight of Brassica napus, can predict the size of the 1000-grain weight of Brassica napus, can effectively select the size of the 1000-grain weight of Brassica napus, and can also be used for molecular marker-assisted breeding of Brassica napus with large 1000-grain weight, accelerate the process of breeding of Brassica napus with 1000-grain weight, and are suitable for large-scale promotion and application.

[0028] 6. The primers or probes for detecting SNP molecular markers at the major QTL loci for the thousand-grain weight trait of Brassica napus of the present invention are cleverly designed, simple and fast to detect, low in cost, unaffected by the environment, and suitable for large-scale promotion and application.

[0029] 7. The use of primers or probes for detecting SNP molecular markers at the major effect QTL loci of the 1000-grain weight trait of Brassica napus according to the present invention can be used to detect the size of the 1000-grain weight of Brassica napus, can be used to predict the size of the 1000-grain weight of Brassica napus, can be used to effectively select the size of the 1000-grain weight of Brassica napus, and can also be used for molecular marker-assisted breeding of Brassica napus with large 1000-grain weight, thereby accelerating the process of breeding of Brassica napus with 1000-grain weight, and is suitable for large-scale promotion and application.

[0030] 8. The use of primers or probes for detecting SNP molecular markers at the major QTL loci for the 1000-grain weight trait of Brassica napus in the present invention is ingeniously designed, simple and fast to detect, low in cost, unaffected by the environment, and suitable for large-scale promotion and application.

[0031] These and other objects, features and advantages of the present invention are fully reflected in the following detailed description, drawings and claims, and can be achieved by the means, products and combinations particularly pointed out in the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is the phenotypic identification of the 1000-grain weight trait of the parents and the RIL population under multi-year and multi-site environments, where A is the phenotypic difference of the parents under 7 environments; B is the phenotypic distribution of 158 recombinant inbred lines under 7 environments, where CS: Changshun; TT: Tangtou; JS: Jinsha; BD: Badong.

[0033] Figure 2 It is the constructed high-density genetic map and the colinearity between the high-density genetic map and the physical map of the Brassica napus genome, where A is the genetic linkage map and B is the colinearity between the genetic linkage map and the physical map of the genome.

[0034] Figure 3It is the QTL identification of thousand-grain weight traits under single environment and multiple environments, among which A is the QTL of 19 linkage groups identified in 7 single environments, the vertical axis represents the LOD value, and the horizontal axis represents the 19 linkage groups, among which CS: Changshun; TT: Tangtou; JS: Jinsha; BD: Badong; B is the QTL identification under the LGA07 linkage group under multiple environments, the vertical axis represents the LOD value, and the horizontal axis represents the LGA07 linkage group; C is the genetic position of the QTL on the LGA07 linkage group, among which ME represents multiple environments and SE represents single environment.

[0035] Figure 4 This is the effect analysis of the critical SNP of the main effect QTL site of the 1000-grain weight trait, where AA and BB represent the genotypes of the critical SNP of the parents with large and small 1000-grain weight, respectively; CS represents Changshun. DETAILED DESCRIPTION

[0036] After in-depth research, the inventors have revealed for the first time a SNP molecular marker for the major QTL locus of the 1000-grain weight trait of Brassica napus, which can be used to effectively improve the yield of Brassica napus.

[0037] The SNP molecular marker of the main effect QTL site of the thousand-grain weight trait of Brassica napus of the present invention is closely linked to the main effect QTL site of the thousand-grain weight trait of Brassica napus. The main effect QTL site of the thousand-grain weight trait of Brassica napus is located between the 20082973rd base and the 20310902nd base of the A07 chromosome of Brassica napus.

[0038] The SNP molecular marker can be any suitable SNP molecular marker. Preferably, the SNP molecular marker is located at the 20082973rd base, the 20082973rd base is A or G, and the mutation causes polymorphism.

[0039] The SNP molecular marker can be any suitable SNP molecular marker. Preferably, the SNP molecular marker is located at the 20310902nd base, the 20310902nd base is G or A, and the mutation causes polymorphism.

[0040] Also provided is the application of the SNP molecular markers at the major effect QTL sites of the above-mentioned Brassica napus thousand-grain weight trait in detecting the size of the Brassica napus thousand-grain weight, predicting the size of the Brassica napus thousand-grain weight, selecting the size of the Brassica napus thousand-grain weight, or in molecular marker-assisted breeding of Brassica napus with large thousand-grain weight.

[0041] Also provided are primers or probes for detecting SNP molecular markers at the major effect QTL loci of the aforementioned 1000-grain weight trait of Brassica napus.

[0042] The primers or probes can be designed based on any suitable DNA fragment as a template. Preferably, the primers or probes are designed based on a DNA fragment of 400 bp sequence before and after bases 20082973 of the A07 chromosome of Brassica napus as a template, and the DNA fragment is shown in SEQ ID NO: 1.

[0043] The primers or probes can be designed based on any suitable DNA fragment as a template. Preferably, the primers or probes are designed based on a DNA fragment of 400 bp sequence before and after base 20310902 of the A07 chromosome of Brassica napus as a template, and the DNA fragment is shown in SEQ ID NO: 2.

[0044] Also provided are primers or probes for SNP molecular markers at the major effect QTL loci of the above-mentioned Brassica napus thousand-grain weight trait for use in detecting the size of the Brassica napus thousand-grain weight, predicting the size of the Brassica napus thousand-grain weight, selecting the size of the Brassica napus thousand-grain weight, or in molecular marker-assisted breeding of Brassica napus with high thousand-grain weight.

[0045] The present invention will be further described below with reference to specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental procedures in the following examples, for which specific conditions are not specified, were generally performed under conventional conditions, such as those described in J. Sambrook et al., Molecular Cloning Laboratory Manual, 3rd edition, Science Press, 2002, or according to the conditions recommended by the manufacturer.

[0046] Example 1: Phenotypic determination of 1000-grain weight of Brassica napus parents and RIL populations

[0047] (1) Brassica napus Darmor was used for distant hybridization with white mustard. A large-grain line GRG2462 was selected from the F2:3. A small-grain line GRD328 was also selected for hybridization with GRG2462. A total of 158 recombinant inbred lines were obtained by continuous self-pollination for 11 generations (B. napus Darmor was obtained from the Wuhan Oil Crop Germplasm Resource Bank, and white mustard and small-grain line GRD328 were purchased from Guizhou Hemufu Seed Co., Ltd.). The 1000-grain weight trait was investigated in multiple locations in Guizhou Province over many years (Changshun in 2014-2015, Changshun in 2016-2017, Changshun in 2017-2018, Tangtou in 2017-2018, Jinsha in 2017-2018, Badong in 2017-2018, and Changshun in 2018-2019).

[0048] (2) Direct seeding was used, with a row spacing of 33 cm and a plant spacing of 15 cm. Each plot had 4 rows and three replicates were randomly designed. Protective rows were planted around the test material plots.

[0049] (3) Thousand-grain weight: The weight of 1000 mature seeds from 10 plants in each plot.

[0050] The results of the distribution of thousand-grain weight of RIL populations under multiple years and multiple locations showed that the distribution of thousand-grain weight trait was normal, proving that thousand-grain weight trait was a quantitative trait and there was a major effect gene locus. Figure 1 shown.

[0051] Example 2: Acquisition of a high-quality SNP dataset from a RIL population

[0052] The CTAB method was used to extract total DNA from leaves of each material in the RIL population. The specific method was as follows:

[0053] The young leaves were rinsed in 10% ethanol; 0.1-0.2 g of leaves were cut and placed in a mortar, quickly ground into powder using liquid nitrogen, and placed in a 2 mL centrifuge tube; 700 μL of preheated DNA extraction solution was added; after mixing, the tube was placed in a 65°C water bath for 1 hour, mixing once every 10-15 minutes; 700 μL of a mixture (phenol: chloroform: isoamyl alcohol = 25:24:1) was added and gently inverted to mix for 10 minutes; centrifuged at 10,000 × g for 15 minutes at room temperature; the supernatant was transferred to a new 2 mL centrifuge tube; an equal volume of a mixture (chloroform: isoamyl alcohol = 24:1) was added and inverted to mix, allowed to stand for 5 minutes, and centrifuged at 10,000 × g for 15 minutes. The supernatant was pipetted into a new centrifuge tube; 2 volumes of anhydrous ethanol were added, mixed, and allowed to stand at -20°C for 1 hour, 10 The pellet was centrifuged at 10,000 × g for 10 minutes, and the supernatant discarded. The pellet was then washed with 500 μL of pre-cooled 75% ethanol, and the supernatant discarded. After two consecutive washes, the pellet was air-dried. 100 μL of a 2% RNase A solution was added, and the pellet was incubated at 37°C for 1 hour before incubating at 4°C overnight. The DNA solution was re-extracted with an equal volume of a mixture (chloroform:isoamyl alcohol = 24:1), mixed by inversion, and incubated for 10 minutes. The pellet was centrifuged at 10,000 × g for 15 or 20 minutes to remove the RNase A. The supernatant (approximately 60 μL) was aspirated and centrifuged again for 1 minute. DNA concentration, quality, and integrity were assessed by agarose gel electrophoresis (0.8%) and UV spectrophotometry. The absorbance 260 / 280 ratio for all DNA samples was determined to be between 1.8 and 2.0. The DNA samples were then shipped on dry ice to a sequencing company (BGI Genomics Co., Ltd.), and each sample was sequenced to a depth of approximately 7×.

[0054] After obtaining high-quality DNA as described above, the sequencing company (BGI Genomics) performed 7× coverage depth sequencing and returned data. FastQC software was used to assess sequencing quality, followed by adapter and low-quality read filtering. Clean data from paired-end sequencing of each material was obtained, followed by mapping using bwa software and variant detection using GATK software. After obtaining the total SNP dataset for the RIL population, the SNP dataset was quality-filtered based on a minimum allele frequency ≥ 0.05, a deletion rate ≤ 0.1, and a heterozygosity rate ≤ 0.15, ultimately resulting in a high-quality population SNP dataset for subsequent analysis.

[0055] Example 3: Construction of a high-density genetic map

[0056] A high-quality population SNP dataset was obtained based on Example 2 above, and a high-density SNP genetic linkage map of the RIL population was constructed using MadMapper (http: / / cgpdb.ucdavis.edu / XLinkage / MadMapper / ) and HighMap software (http: / / highmap.biomarker.com.cn / ). To increase computational speed, the RECBIT program in MadMapper software was first used to remove redundant SNPs (two or more SNPs co-segregating within the population), SNPs with a data missing ratio ≥25% in 158 strains, and SNPs with an allelic segregation ratio within the population <0.33. The remaining SNPs were then assigned to linkage groups based on their recombination rates. The Kosambi mapping function in HighMap software was then used to convert the recombination values between SNPs on each linkage group into genetic distances in cm. Finally, HighMap was used to perform a visual inspection of each constructed linkage group. Furthermore, HighMap was used to perform a significance test (P < 0.05) for SNP segregation deviation on each linkage group. After the mapping of the genetic linkage groups of the RIL population was completed, the 19 linkage groups of Brassica napus were named LGA01-LGA10 and LGC01-LGC09 according to their diploid ancestral sources using the international unified naming standards. Figure 2 shown.

[0057] Example 4: Identification of QTLs for 1000-grain weight in single and multiple environments

[0058] Under the same environment, the average of the observed values of all block groups of each strain was used as the phenotypic value for QTL analysis. The Composite Interval Mapping (CIM) method in Windows QTL Cartographer 2.5 software (https: / / brcwebportal.cos.ncsu.edu / qtlcart / WQTLCart.htm) was used to perform a genome-wide QTL scan of the thousand-grain weight trait in the RIL population. When setting the parameters, the backward regression method was selected to screen the cofactor (Cofactor), the number of background markers was set to 5, the scanning step was 1cM, and the window size was 10cM. Under the probability condition of P=0.05, 1000 permutation tests were used to determine the threshold of the LOD value in each environment. QTLs with LOD values greater than or equal to the threshold are called significant QTLs. Through analysis, the main effect QTL sites of the thousand-grain weight trait in a single environment are all on chromosome A07, see Figure 3 As shown in A.

[0059] The ICIM-ADD mapping method in IciMapping V4.1 software (https: / / isbreeding.caas.cn / rj / qtllcmapping / 294445.htm) was used to identify the interaction effect between QTL and environment in multiple environments. The LOD threshold and significance analysis were consistent with those in single environment. The analysis found that the QTL for 1000-grain weight in multiple environments was also on chromosome A07. Figure 3 As shown in B. Combining the QTL results under a single environment, the main QTL locus for 1000-grain weight is located between bases 20082973 and 20310902 on chromosome A07 of Brassica napus.

[0060] Example 5: Effect of the major QTL locus for 1000-grain weight in RIL populations

[0061] Based on the main effect QTL loci detected in a single environment, the haplotype effect of the critical SNP of the main effect QTL loci in the RIL population was analyzed. Through the analysis, the critical SNP of the main effect QTL loci of the 1000-grain weight trait showed significant differences in 158 RIL population lines. Figure 4 shown.

[0062] According to the reference genome sequence of Brassica napus (https: / / www.genoscope.cns.fr / brassicanapus / data / ), the 400 bp sequence before and after chrA07_20082973 (A / G) (801 bp in total) is shown in SEQ ID NO: 1, and the 400 bp sequence before and after chrA07_20310902 (G / A) (801 bp in total) is shown in SEQ ID NO: 2.

[0063] Those skilled in the art can use conventional methods to design specific primers or probes for detecting SNP sites based on the above-mentioned known sequences. The primers or probes can also be labeled with fluorescent groups such as FAM, HEX, VIC, ROX, etc., and quenching groups such as BHQ1 or TAMRA through conventional techniques in the art, so that the genotype of the above-mentioned SNP sites can be detected by conventional methods in the art such as sequencing or PCR. In this way, the size of the thousand-grain weight of Brassica napus can be detected, the size of the thousand-grain weight of Brassica napus can be predicted, and then the size of the thousand-grain weight of Brassica napus can be effectively selected, which can be used for molecular marker-assisted breeding of Brassica napus with thousand-grain weight, thereby accelerating the process of breeding of Brassica napus with thousand-grain weight.

[0064] Therefore, the present invention detects the major QTL loci for the thousand-grain weight trait of Brassica napus on chromosome A07 of Brassica napus through phenotypic analysis and whole-genome resequencing of the 1000-grain weight trait. The major QTL loci for the thousand-grain weight trait of Brassica napus are located between bases 20,082,973 and 20,310,902 of chromosome A07 of Brassica napus and between bases 11,378,925 and 11,559,658 of chromosome A07 of Brassica napus, respectively. The major QTL loci for the thousand-grain weight of Brassica napus seeds of the present invention play a key role in regulating the thousand-grain weight of Brassica napus and can be used for map-based cloning and molecular marker-assisted selection. Based on the SNP molecular markers that are closely linked to the above-mentioned major effect QTL loci, they can be used to detect the size of the thousand-grain weight of Brassica napus, predict the size of the thousand-grain weight of Brassica napus, and then effectively select the size of the thousand-grain weight of Brassica napus. They can be used for molecular marker-assisted breeding of Brassica napus with thousand-grain weight, thereby accelerating the process of breeding of Brassica napus with thousand-grain weight.

[0065] Molecular marker-assisted selection (MAS) using the SNP molecular markers disclosed in this invention offers a simple identification method, high selection efficiency, and the ability to predict the 1000-grain weight of Brassica napus. This allows for a clear selection target, unaffected by environmental factors. This allows for the identification of single Brassica napus plants with high 1000-grain weights during the early growth stages of the plant, eliminating the remaining plants.

[0066] In summary, the SNP molecular markers at the major effect QTL sites of the 1000-grain weight trait of Brassica napus of the present invention can detect the size of the 1000-grain weight of Brassica napus, can predict the size of the 1000-grain weight of Brassica napus, can effectively select the size of the 1000-grain weight of Brassica napus, and can also be used for molecular marker-assisted breeding of Brassica napus with large 1000-grain weight, thereby accelerating the process of breeding of Brassica napus with 1000-grain weight, and are suitable for large-scale promotion and application.

[0067] It can be seen that the objectives of the present invention have been fully and effectively achieved. The functional and structural principles of the present invention have been demonstrated and illustrated in the embodiments. The embodiments may be modified as desired without departing from the principles described. Therefore, the present invention includes all variations within the spirit and scope of the claims. Sequence Listing <110> Guizhou Rapeseed Research Institute <120> SNP molecular markers, related detection primers or probes, and applications of major QTL loci for thousand-grain weight traits in Brassica napus <160> 2 <210> 1 <211> 801 <212> DNA <213> Brassica napus (L.) <220> <221> misc_feature <222> (1)...(801) <223> The genomic sequence containing the 400 bp sequence before and after chrA07_20082973 (A / G) <400> 1 aaacaatcta cgaataatta taaacaaaca aaacctgcca ttccgatgat tcaacttcct 60 ccactagaca cctagtcaaa taataccctt caccagacca ctcactagct gcatccgtgg 120 gagcattggt aagaatgtaa aagtatccat tgcgatgttc aagaaaacac tgcacaccat 180 ggactctctc acgagccctt cgcaaaccag ccagtggctt ctccgcattt actatataaa 240 cctcggacga agtcctcgag ttcgaattga tagtacaaa ctttccatct ttagtactcg 300 taatatccac acagaaacta gatcacttt cagagaacac cacagtatca ccacccccgt 360 ctgattcaac atttgtaca acaccctgt gaggtctttg atgttcatca acacagtgt 420 atacaagct gactccatct aacgcccacg ccaagctcac aactccatca acttccagcc 480 tcggaaccaa acaccactt ctaagatcct taatctggag aaggaacgc tcgttgccct 540 taggatcaac agtgtaggcg aggtatt gatcaggtga gacgacaa acaccacgt 600 gacataacc tatagagag agagtaagtc storm tgttacgatc cgcaccgcag 660 ttaacagtaa caaaatctc tacatatatc atatatctat acttttttat aactgttaga 720 accgcaccgc agttacgg cttgctccgc accgctcaat ccgttgttat cagagcctta 780 gtgatta aggregate t 801 <210> 2 <211> 801 <212> DNA <213> Brassica napus (Brassica napus, L.) <220> <221> misc_feature <222> (1)...(801) <223> The genomic sequence containing the 400 bp sequence before and after chrA07_20310902 (G / A) <400> 2 gcggagggta tattccaagt ggagatgtgg actattgtct tactctgagt ttgccaataa 60 aaaaatatag caagtaatta acttgatttt cataattcct ctaatatatg gtagcatgta 120 aaatgctttt ttctacattt accatttatg ttgtgtcttt tcaaatccga acaaaagata 180 attgttaaaa acaatctaaa ctaaaaaaat aatttaaatt ttagacctga actttttaac 240 attagcccta aaacatacat tgactaagtc aatgttagtt aaccattaat tttggttcaa 300 aactatatca cttttttata acacagattt cattagactg aataaaatta ccgcatgtct 360 aattaaccca taattaggtc caattcaaca gaattcgaaa gaaacaaaga ttgttttgcc 420 aatgaatcgg cttcatcatt gagcaattga gcaacagagg tgctttgtat cagatgtaat 480 ggtatcaaat ctgcaggaca ttgttttgat taattataca aaattaagtc gtttagtttt 540 ttaagaaatt attttataca atttgccact aaagaaaaca ttgcattcca tgtatgataa 600 agtcttgagg aatgagttcc attccttgtt gtgactaatg aaaaatat atttttaaaa 660 atatatttaa aacgacgtaa ttctaagtaa ttattccaaa cgacattttt aagctataat 720 tgcatccaaa attaacagtt gattaatatt gggttagtcg aaataggttt tagtgtaaca 780 gttaaaagtt cggttcgaa a 801

Claims

1. A SNP molecular marker for a major QTL locus of 1000-grain weight in Brassica napus, characterized in that: The SNP molecular marker is closely linked to the major effect QTL site of the thousand-grain weight trait of the Brassica napus. The SNP molecular marker is the nucleotide sequence shown in SEQ ID NO:1, and the nucleotide sequence shown in SEQ ID NO:1 has an A / G base mutation at the 401st position from the 5' end; or, the SNP molecular marker is the nucleotide sequence shown in SEQ ID NO:2, and the nucleotide sequence shown in SEQ ID NO:2 has a G / A base mutation at the 401st position from the 5' end.

2. The use of the SNP molecular marker at the main effect QTL site of the 1000-grain weight trait of Brassica napus according to claim 1 in detecting the size of the 1000-grain weight of Brassica napus, predicting the size of the 1000-grain weight of Brassica napus, selecting the size of the 1000-grain weight of Brassica napus, or in molecular marker-assisted breeding of Brassica napus with large 1000-grain weight.

3. A primer or probe for detecting the SNP molecular marker of the main effect QTL site of the 1000-grain weight trait of Brassica napus according to claim 1, and its use in detecting the size of the 1000-grain weight of Brassica napus, predicting the size of the 1000-grain weight of Brassica napus, selecting the size of the 1000-grain weight of Brassica napus, or molecular marker-assisted breeding of Brassica napus with large 1000-grain weight.

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