Mustard core SNP (Single Nucleotide Polymorphism) molecular marker set as well as screening method and application thereof

Abstract

The invention belongs to the technical field of molecular biology and plant molecular breeding, and particularly relates to a mustard core SNP molecular marker set and a screening method and application thereof. The invention provides a core SNP marker set (33) which is subjected to whole genome re-sequencing screening and experimental verification and is suitable for mustard germplasm resource identification and genetic analysis for the first time. The marker set is high in polymorphism and good in stability, covers the whole genome and can effectively distinguish different leaf mustard germplasms. By utilizing the core marker set and the matched KASP primer, the genetic typing of the leaf mustard germplasm resources can be quickly and accurately performed with high throughput, and the defects that the traditional morphological identification is time-consuming, labor-consuming and poor in accuracy are overcome. The marker set can be used for analyzing the genetic diversity, the population structure and the phylogenetic relationship of the leaf mustard germplasm resources, and a molecular basis is provided for collection, preservation and evaluation of the germplasm resources and breeding parent matching.

Description

Technical Field

[0001] The present invention belongs to the technical fields of molecular biology and plant molecular breeding, and particularly relates to a core SNP molecular marker set of mustard, a screening method thereof, and an application thereof. Background Art

[0002] Mustard ( Brassica juncea (L.) Czern.) is an allopolyploid (AABB) cash crop of the genus Brassica in the family Brassicaceae, including two major categories: vegetable mustard and oilseed mustard. Based on morphological observation and comparison, vegetable mustard is divided into four major categories: leaf mustard, stem mustard, root mustard, and bolt mustard, with 16 varieties. Mustard is widely cultivated throughout China. Among them, leaf mustard has the widest distribution, stem mustard is mainly distributed in Chongqing, Sichuan, Zhejiang and other places, root mustard is mainly distributed in the southwest and the middle and lower reaches of the Yangtze River, and oilseed mustard has a certain planting area in the northwest of China. The variation of mustard leaves is the most complex. In addition to changes in leaf shape, leaf color, leaf margin, etc., it also includes variations such as elongation of the petiole, widening and thickening of the petiole and midrib, hearting, and heading. Moreover, mustard is an often cross-pollinated plant with insect pollination. Under natural conditions, it is extremely easy to hybridize between different mustard varieties, and there is a small natural hybridization rate between mustard and Chinese cabbage, pakchoi, turnip, and Brassica rapa with the A chromosome group in the genus Brassica of the family Brassicaceae, resulting in rich genetic diversity of mustard germplasm resources. In addition, mustard has different names in different regions. In Sichuan Province, Chongqing City, and western Hunan, it is generally called green vegetable, and in other regions, it is also called spicy vegetable, bitter vegetable, etc. During the process of sorting, protecting, and identifying mustard germplasm resources across the country, there are a large number of phenomena of different plants with the same name or the same plant with different names, which brings certain troubles to the sorting and identification of mustard germplasm resources. In addition, with the continuous increase of mustard cultivated varieties, intellectual property disputes occur from time to time. Therefore, it is urgent to accurately classify and identify mustard germplasm resources or varieties.

[0003] For a long time, the identification and evaluation of mustard germplasm and varieties mainly relied on phenotypic investigation, which was time-consuming and laborious and was easily affected by human, environmental and other factors. With the development of modern biotechnology, DNA molecular markers provide new means and methods for crop genetic diversity research and germplasm identification. With the development of DNA molecular marker technology and the breakthrough of SNP marker genotyping technology, the superiority and application prospects of SNP markers have become very prominent, and they have begun to gradually replace early-generation molecular markers such as SSR and AFLP.

[0004] At present, there is no publicly reported core SNP marker set screened and verified for accurate identification and genetic analysis of germplasm resources in the whole genome range of mustard. Therefore, developing a set of SNP core marker sets covering the genome, with high polymorphism and good stability is of great significance for mustard germplasm resource management, variety right protection and molecular breeding. Summary of the Invention

[0005] The purpose of this invention is to address existing problems by providing a core SNP molecular marker set for mustard, its screening method, and its application.

[0006] This invention is achieved through the following technical solution: The core molecular marker set of SNPs used for identification and genetic analysis of mustard germplasm resources consists of 33 SNP loci located in the mustard genome at the following positions: The following loci are located on chromosomes ChrA01: 999881, 6161911, 13184011, 15610616, 34854190, 38545092, 38662141; 4076896; and others. The locus at position 20422094 on chromosome 4, the locus at position 26587494 on chromosome 4, the locus at position 8486177 on chromosome 6, the locus at position 9108186 on chromosome 6, the locus at position 11417300 on chromosome 6, the locus at position 23349540 on chromosome 7, the locus at position 28038309 on chromosome 8, the locus at position 30495962 on chromosome 8, and the locus at position 9108186 on chromosome 6. The following loci are listed: 31074770, 8248951, 8942556, 15194056, 50511602, 274261, 20523681 on chromosome 10, 6604305 on chromosome 10, and 51492614 on chromosome 10. The loci are located at the following locations: ChrB03 chromosome at position 11648229, ChrB03 chromosome at position 25135725, ChrB04 chromosome at position 10251914, ChrB04 chromosome at position 11472137, ChrB05 chromosome at position 7461483, ChrB07 chromosome at position 22433919, ChrB08 chromosome at position 5434183, and ChrB08 chromosome at position 5885694.

[0007] Furthermore, the 33 SNP sites cover the A and B genomes of mustard and are evenly distributed on the chromosomes.

[0008] A KASP genotyping primer set for detecting any site in the core molecular marker set of the SNP, the primer set comprising 33 primer sets with nucleotide sequences as shown in SEQ ID NO: 1 to SEQ ID NO: 99, each primer set being used to detect a specific SNP site, each primer set comprising one FAM-labeled allele-specific forward primer, one HEX-labeled allele-specific forward primer and one universal reverse primer.

[0009] A method for screening the core molecular marker set of the SNP includes the following steps: (1) Based on the whole genome resequencing data of multiple mustard germplasm resources, the SNPs obtained were initially screened according to the evenness of their distribution on chromosomes, site polymorphism, minor allele frequency and flanking sequence uniqueness to obtain a set of candidate SNPs. (2) Design KASP genotyping primers for each site in the candidate SNP set; (3) Genotyping experiments were conducted on representative mustard materials using the KASP primers. Based on the genotyping results, SNP sites with clear genotyping and high stability were selected to form the core molecular marker set.

[0010] Furthermore, in step (1), the preliminary screening conditions include: the selected SNP site has no other SNP sites within 100 bp of its upstream and downstream sequences.

[0011] Furthermore, in step (3), the screening based on the typing effect includes: removing markers with unclear clustering or weak signals in the KASP typing diagram, and retaining markers that can clearly distinguish different genotypes.

[0012] Furthermore, the application of the SNP core molecular marker set or the KASP genotyping primer set in the identification of mustard germplasm resources or varieties.

[0013] Furthermore, the application of the SNP core molecular marker set or the KASP genotyping primer set in constructing a DNA fingerprint or molecular identity card for mustard.

[0014] Furthermore, the SNP core molecular marker set or the KASP genotyping primer set are used in the analysis of genetic diversity, population structure, or phylogenetic evolution of mustard.

[0015] A method for identifying mustard germplasm resources or varieties, comprising the following steps: (1) Extract genomic DNA from the mustard sample to be tested; (2) Using the DNA obtained in step (1) as a template, the KASP genotyping primer set is used to perform genotyping on all or part of the sites in the SNP core molecular marker set by competitive allele-specific PCR technology. (3) Compare the genotyping results obtained in step (2) with the reference fingerprint data of known varieties to achieve identification.

[0016] The present invention has the following advantages over the prior art: This invention provides for the first time a set of 33 core SNP markers, screened and experimentally validated through whole-genome resequencing, suitable for the identification and genetic analysis of mustard germplasm resources. This marker set exhibits high polymorphism, good stability, and covers the entire genome, effectively distinguishing different mustard germplasm.

[0017] Using the core marker set and matching KASP primers of this invention, genotyping of mustard germplasm resources can be performed quickly, accurately, and with high throughput, overcoming the shortcomings of traditional morphological identification, which is time-consuming, labor-intensive, and inaccurate.

[0018] The marker set of this invention can be used to construct DNA fingerprint maps of mustard varieties, solve the problem of "different names for the same thing" and "different names for the same thing", and provide a reliable technical means for variety rights protection, seed market supervision and authenticity identification.

[0019] The marker set of this invention can be used to analyze the genetic diversity, population structure and phylogenetic relationships of mustard germplasm resources, providing a molecular basis for the collection, preservation, evaluation and selection of breeding parents of germplasm resources. Attached Figure Description

[0020] Figure 1 This is a marker for poor fractal characteristics; Figure 2 For well-defined markers; Figure 3 Fingerprint profiles of 207 mustard samples; Figure 4 The results are based on the evolutionary and population structure analysis of 169 materials. Detailed Implementation

[0021] To further explain the present invention, the following specific embodiments are described.

[0022] Example 1: Screening of SNP core molecular marker set 1.1 Sources of Materials and Data 169 representative mustard germplasm resources were collected and whole-genome resequencing was performed to obtain genome-wide SNP data. The reference genome used was the Braju_tum_V3.0 version of the mustard T84-66 reference genome.

[0023] 1.2 Screening of Core SNP Tags (1) 1812 core molecular markers were initially screened, and the screening criteria were as follows: a) Site detection rate greater than 0.95; b) Site polymorphism greater than 0.4; c) MAF is greater than 0.15; d) Heterozygosity is less than 0.1; e) The SNP density of each chromosome is greater than 50 kb / SNP.

[0024] (2) The number of core molecular markers was reduced to 118 for subsequent KASP validation, and 100bp sequences upstream and downstream of the SNPs were provided for KASP primer design. The screening criteria are as follows: a) Increase the screening criteria in (1) and continue filtering; Filtering criteria: ① Site detection rate > 99%; ②MAF is greater than 0.15; ③ Heterozygosity rate is less than 0.03; ④ Remove redundant markers; ⑤ Genetic linkage and correlation: Select mutually independent SNP sites and avoid selecting sites that are too densely packed in the same gene or genomic region (there should be no other SNPs within 100 bp before and after the selected SNP site). ⑥PIC >= 0.2; ⑦ Site polymorphism greater than 0.4; b) The selected 100 SNPs must be able to distinguish all population materials in the resequencing; c) The selected SNPs must not have other SNP sites within 100 bp above and below them. d) The selected SNPs should be distributed as evenly as possible across each chromosome and the upper, middle and lower parts of the chromosome.

[0025] (3) KASP primer design and synthesis: For the 118 candidate SNP sites mentioned above, 100 bp of genomic sequence was extracted upstream and downstream of each site. KASP genotyping primers were designed, and markers with excessive GC content in the primers were removed. The remaining markers were verified by KASP. The primers included two allele-specific forward primers (with universal adapter sequences carrying FAM and HEX fluorescent tags, respectively) and one universal reverse primer.

[0026] 1.3 KASP Experimental Validation and Core Label Determination: (1) DNA preparation: Genomic DNA was extracted from 207 mustard materials and the concentration was uniformly adjusted to 20-30 ng / μL. The DNA samples were prepared to a uniform and suitable concentration (20-30 ng / μL) and added to 96-well PCR plates. Two negative controls (NTC) were added to each PCR plate. (2) Preparation of KASP genotyping mixture Refer to Table 1 below to prepare the required mixtures for the reaction.

[0027] Table 1

[0028] (3) Add the KASP genotyping mixture to the array tape membrane containing the DNA template. The IntelliQube SNP gene detection platform was used to develop a program. The 384-well array tape, DNA sample plate, and KASP genotyping mixture were placed into the machine in sequence. The machine was operated to execute the program, which automatically dispensed the DNA sample diluent and KASP genotyping mixture into the 384-well array tape and sealed them.

[0029] (4) Perform PCR cycling reaction PCR reactions can be performed in IntelliQube in SNP genotyping inline mode (single membrane) or in Hydrocycler in SNP genotyping outline mode (multiple membranes) for water bath PCR. The program settings are shown in Table 2 below: Table 2

[0030] (5) Fluorescence data reading and analysis After the PCR reaction was completed, fluorescence data were read and analyzed using an IntelliQube machine (Table 3).

[0031] Table 3

[0032] (6) Loop If the fluorescence signal is low and the clustering is scattered, fluorescence readings can be performed after additional cycles. The conditions for adding cycles are shown in Table 4 below: Table 4

[0033] Based on the KASP validation results, markers with poor classification were removed. Figure 1 ), retain the well-defined markers ( Figure 2Based on their distribution on chromosomes, a set of 33 SNP markers was finally obtained. The nucleotide sequence information of the KASP primer set used to detect these 33 core SNP sites is shown in SEQ ID NO: 1 to SEQ ID NO: 99. The ID, chromosomal location, allele information, and corresponding KASP primer sequences of the 33 core SNP markers are detailed in Table 5.

[0034] Table 5

[0035] Example 2: Application of core SNP marker set in mustard germplasm resource analysis 2.1 DNA fingerprinting construction Genotyping was performed on 207 mustard accessions using the 33 core SNP markers and their KASP primers identified in Example 1. The genotyping data were analyzed using SNPT software to select the optimal marker combination for constructing the DNA fingerprint. Genotype heatmaps were generated using RStudio software, yielding the DNA fingerprints of the 207 mustard accessions (e.g., [image of fingerprints]). Figure 3 (As shown in the image). This map visually illustrates the genotypic differences among the materials and can be used for rapid variety identification, purity analysis, and genetic similarity assessment.

[0036] 2.2 Population genetic structure and evolutionary analysis We selected 33 SNP genotyping data from 169 mustard materials (including different types such as leaf and stem varieties) for population analysis.

[0037] (1) Phylogenetic tree construction: Based on the genetic distance matrix, a phylogenetic tree was constructed using the neighbor-joining (NJ) method and visualized using MEGA-X software. The results showed that most stem mustard varieties clustered into one phylogenetic line, indicating that they had similar genetic backgrounds; at the same time, a few stem mustard varieties were found to cross-cluster with leaf mustard varieties, reflecting gene flow between different varieties (e.g., ... Figure 4 (as shown in part).

[0038] (2) Population structure analysis: Structure 2.3.4 software was used for analysis. The subpopulation number (K) was set to a range of 2-10, and simulation calculations were performed (10,000 burn-in periods, 10,000 MCMC iterations, and 5 repetitions for each K value) to determine the optimal subpopulation number. The analysis results revealed the subpopulation division of mustard germplasm resources, providing a molecular basis for resource classification and parent selection in breeding (e.g., Figure 4 (as shown in part).

[0039] Example 3: Application of Core SNP Marker Sets in Variety Identification (1) Sample preparation: Take the mustard seed or leaf sample to be identified and extract genomic DNA.

[0040] (2) Target site typing: Select all or some of the most discriminative sites of the 33 core SNP markers in Example 1, and use the corresponding KASP primer set (SEQ ID NO: 1-99) to perform competitive allele-specific PCR (KASP) typing. The operation steps are the same as in Example 1.

[0041] (3) Comparison of identification results: The SNP genotyping profile (i.e., DNA fingerprint) obtained from the sample to be tested is compared with the established standard DNA fingerprint database of known varieties. If the fingerprints match completely or are highly similar, the sample can be identified as the variety; if there is no match, it suggests that it may be a new variety or a variant material. This method is applicable to variety authenticity identification, seed purity testing, and market supervision.

[0042] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A set of SNP core molecular markers for identification and genetic analysis of mustard germplasm resources, characterized in that, The marker set consists of 33 SNP sites located in the mustard genome at the following locations: The following loci are located on chromosomes ChrA01: 999881, 6161911, 13184011, 15610616, 34854190, 38545092, 38662141; 4076896; and others. The locus at position 20422094 on chromosome 4, the locus at position 26587494 on chromosome 4, the locus at position 8486177 on chromosome 6, the locus at position 9108186 on chromosome 6, the locus at position 11417300 on chromosome 6, the locus at position 23349540 on chromosome 7, the locus at position 28038309 on chromosome 8, the locus at position 30495962 on chromosome 8, and the locus at position 9108186 on chromosome 6. The following loci are listed: 31074770, 8248951, 8942556, 15194056, 50511602, 274261, 20523681 on chromosome 10, 6604305 on chromosome 10, and 51492614 on chromosome 10. The loci are located at the following locations: ChrB03 chromosome at position 11648229, ChrB03 chromosome at position 25135725, ChrB04 chromosome at position 10251914, ChrB04 chromosome at position 11472137, ChrB05 chromosome at position 7461483, ChrB07 chromosome at position 22433919, ChrB08 chromosome at position 5434183, and ChrB08 chromosome at position 5885694.

2. The SNP core molecular marker set according to claim 1, characterized in that, The 33 SNP sites cover the A and B genomes of mustard and are evenly distributed on the chromosomes.

3. A primer set for detecting KASP genotyping at any locus in the SNP core molecular marker set of claim 1 or 2, characterized in that, The primer set contains 33 primer sets with nucleotide sequences as shown in SEQ ID NO: 1 to SEQ ID NO:

99. Each primer set is used to detect a specific SNP site. Each set contains one FAM-labeled allele-specific forward primer, one HEX-labeled allele-specific forward primer, and one universal reverse primer.

4. A method for screening the SNP core molecular marker set as described in claim 1 or 2, characterized in that, Includes the following steps: (1) Based on the whole genome resequencing data of multiple mustard germplasm resources, the SNPs obtained were initially screened according to the evenness of their distribution on chromosomes, site polymorphism, minor allele frequency and flanking sequence uniqueness to obtain a set of candidate SNPs. (2) Design KASP genotyping primers for each site in the candidate SNP set; (3) Genotyping experiments were conducted on representative mustard materials using the KASP primers. Based on the genotyping results, SNP sites with clear genotyping and high stability were selected to form the core molecular marker set.

5. The method according to claim 4, characterized in that, In step (1), the preliminary screening conditions include: the selected SNP site has no other SNP sites within 100 bp of its upstream and downstream sequences.

6. The method according to claim 4, characterized in that, In step (3), the screening based on the typing effect includes: removing markers with unclear clustering or weak signals in the KASP typing diagram, and retaining markers that can clearly distinguish different genotypes.

7. The application of the SNP core molecular marker set as described in claim 1 or 2, or the KASP genotyping primer set as described in claim 3, in the identification of mustard germplasm resources or varieties.

8. The application of the SNP core molecular marker set as described in claim 1 or 2, or the KASP genotyping primer set as described in claim 3, in constructing a DNA fingerprint or molecular identity card for mustard.

9. The application of the SNP core molecular marker set as described in claim 1 or 2, or the KASP genotyping primer set as described in claim 3, in genetic diversity analysis, population structure analysis, or phylogenetic analysis of mustard.

10. A method for identifying mustard germplasm resources or varieties, characterized in that, Includes the following steps: (1) Extract genomic DNA from the mustard sample to be tested; (2) Using the DNA obtained in step (1) as a template, the KASP genotyping primer set described in claim 3 is used to perform genotyping on all or part of the sites in the SNP core molecular marker set described in claim 1 or 2 by competitive allele-specific PCR technology. (3) Compare the genotyping results obtained in step (2) with the reference fingerprint data of known varieties to achieve identification.

Images