CN117210596A - Melon SNP locus marker combination, SNP locus marker detection probe combination, liquid phase chip and application - Google Patents
Melon SNP locus marker combination, SNP locus marker detection probe combination, liquid phase chip and application Download PDFInfo
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Abstract
The invention belongs to the technical field of plant genotyping detection, and particularly relates to a melon SNP (single nucleotide polymorphism) marker locus combination, a SNP locus detection probe combination, a liquid phase chip and application. The probe can be used for quickly typing the genotype of the melon sample, and is used for genetic diversity analysis, variety identification, genetic linkage map construction, gene positioning, genetic background selection and molecular auxiliary breeding of melon germplasm resources, so that efficient molecular breeding of the melon is realized.
Description
Technical Field
The invention belongs to the technical field of plant genotyping detection, and particularly relates to a melon SNP (single nucleotide polymorphism) marker locus combination, a SNP locus detection probe combination, a liquid phase chip and application.
Background
Melon (cutemis melo l.) is an important commercial crop, and its abundant commodity type is highly favored by consumers. The assembly of the melon high-quality genome and the completion of large-scale melon germplasm genome re-sequencing mark that the molecular research of the melon breaks through the limitation of the number of marks, and simultaneously, precious genetic resources and variation information are provided for genome-assisted melon breeding and melon variety identification management. However, by using the resequencing technology, the application of such high-throughput molecular markers to actual breeding and variety identification management works requires higher computational resources, special technical requirements and higher detection cost, which limits the wider application in melon molecular breeding. Therefore, a set of genotype identification method which is rapid, efficient, mature, stable, low in cost and high in flux is established at present, so that the sample detection efficiency in melon variety identification and breeding is improved, and melon researchers are facilitated to promote melon breeding and high-quality development of molecular biology.
The genomic polymorphism caused by a single nucleotide transition, a transversion, a deletion or an insertion is called a Single Nucleotide Polymorphism (SNP). Advances in second generation sequencing technology have driven high throughput development of SNP detection, and the SNP detection has the advantages of high genome coverage density, strong representativeness of genetic characteristics, stable inheritance, automation, easy operation and the like, and has wider application in genetic and breeding aspects. By analyzing the base differences in genome among samples, the method can be used for identifying the genotype and genetic background of the detected samples and determining the information of the genetic relationship among the samples, the population structure and the like. SNP markers have various applications in genetic analysis, such as: marker assisted population selection, genetic linkage map construction, QTL positioning, population genetic polymorphism analysis, population system evolution relation inference and the like. In addition, SNP markers are also extremely widely used in breeding, such as: SNP markers can help the breeder to identify and distinguish different varieties more accurately; the SNP markers can help the seed industry practitioners to effectively regulate and control the crop characters and develop new good varieties; the SNP marker can select good varieties more quickly and accurately during selective breeding, and defects such as tedious, time-consuming, inaccurate and the like existing in the traditional selection method are avoided; SNP markers can also help the germplasm staff to better protect and utilize rare genetic resources and to evaluate germplasm resources.
Whole genome sequencing can cover a whole genome sequence to a great extent, however, the important application index sequencing price of the whole genome sequencing is often increased along with the increase of the sequencing depth, and the advantages of precision and price cannot be well considered. Simplified genome sequencing is generally a random restriction of DNA sequences by restriction enzymes, and sequencing is relatively inexpensive, but there are limitations due to the randomness of the simplified genome sequencing cleavage sites and the false positives and false negatives of the detection results. The targeted sequencing typing is to perform deep sequencing from a genome at fixed points and obtain a marker locus with relatively fixed physical position and high reliability, the sequencing cost varies according to the size of the marker flux, and the sample detection price is relatively low. Whole genome sequencing is often used as priori data and important data support for targeted genome sequencing because of wide genome coverage, and the combined application of the two can achieve the advantage complementation of a sequencing strategy. The target sequencing parting platform has the advantages of accurate target, high sequencing depth of variation sites, low parting cost, high cost performance and the like, and in recent researches, a liquid chip technology is used as a carrier and based on variation group data of group weight sequencing, and the screening of representative SNP becomes an efficient and reliable method in variety identification and breeding improvement. At present, agricultural crops such as corns, paddy rice, wheat, soybeans, peanuts, cotton and the like (Guo et al, 2019; guo et al, 2021; liu et al, 2022); vegetable or fruit crops such as cabbages, peppers, eggplants, cucumbers, watermelons (Su et al, 2018; du et al, 2019; liu et al, 2019; zhang et al, 2020; shen et al, 2021; zhang et al, 2022) have found widespread use.
Therefore, it is necessary to develop a rapid genotyping detection method based on SNP sites, which can be used in melon samples.
Disclosure of Invention
Aiming at the problems, the invention aims to provide a probe for detecting SNP loci of muskmelon, which is used for detecting and screening 2000 SNP locus combinations of the muskmelon, can realize rapid genotyping of a muskmelon sample, and is used for genetic diversity analysis, variety identification, genetic linkage map construction, gene positioning, genetic background selection and molecular assisted breeding of muskmelon germplasm resources so as to realize efficient molecular breeding of the muskmelon.
In order to achieve the above purpose, the present invention may adopt the following technical scheme:
the invention provides a melon SNP marker locus combination, which is characterized by comprising 2000 SNP marker loci, wherein each SNP marker locus comprises 1 base variation locus; the SNP marker locus positions and mutation loci are shown in Table 1.
The invention also provides a probe combination for detecting the melon SNP marker locus combination, which is characterized in that each SNP marker locus is correspondingly provided with a group of probes for detecting the corresponding SNP marker locus to form the probe combination.
In a further aspect, the invention provides a reagent for identifying melon genotyping, comprising the probe combination for detecting melon SNP marker loci.
In a further aspect, the invention provides a liquid-phase chip comprising the probe combination for detecting melon SNP marker loci or the reagent for identifying melon genotyping.
In a further aspect, the invention provides an application of the melon SNP marker locus combination, the probe combination for detecting the melon SNP marker locus, the reagent for identifying melon genotyping or the liquid chip in identifying melon genotyping.
In still another aspect, the invention provides an application of the melon SNP marker locus combination, the probe combination for detecting the melon SNP marker locus, the reagent for identifying melon genotyping or the liquid chip in genetic diversity analysis, variety identification, genetic linkage map construction, QTL or gene positioning, genetic background selection or molecular assisted breeding of melons.
The beneficial effects of the invention at least comprise: the probe for detecting melon SNP loci is used for detecting and screening 2000 SNP locus combinations of melons, and genetic diversity analysis and variety identification can be performed on melon germplasm resources based on the probe; the mutation spectrum features of resequencing level can be achieved by carrying out linkage spectrum construction, gene positioning, genetic background selection and molecular auxiliary breeding on melon genetic groups, the mutation spectrum features have extremely high accuracy and reliability, and meanwhile, the mutation spectrum features have lower price advantage compared with sample typing by resequencing.
Drawings
FIG. 1 shows distribution of 2K SNP sites in melon genome-wide chromosomes;
FIG. 2A is a graph showing MAF frequency distribution analysis results of 2000 SNP loci;
FIG. 2B is a phylogenetic tree constructed based on 2000 SNP locus generations and a phylogenetic tree constructed of all SNP loci of the whole genome;
FIG. 2C is a genetic relationship matrix diagram based on genetic similarity between 2000 SNP loci and two individuals of all SNP loci of the whole genome;
FIG. 3A is a representative germplasm fingerprint of a core SNP construct based on melon liquid phase chip 2K SNP site screening;
FIG. 3B shows the difference distribution of core SNP individuals based on melon liquid phase chip 2K SNP locus screening;
FIG. 4A is a genetic background map of a backcross population constructed using melon liquidoid chips;
FIG. 4B is a graph showing the statistics of background revertants for the construction of backcross populations using melon liquid phase chips.
Detailed Description
The examples are presented for better illustration of the invention, but the invention is not limited to the examples. Those skilled in the art will appreciate that various modifications and adaptations of the embodiments described above are possible in light of the above teachings and are intended to be within the scope of the invention.
The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure. Unless the context clearly differs, singular forms of expression include plural forms of expression. As used herein, it is understood that terms such as "comprising," "having," "including," and the like are intended to indicate the presence of a feature, number, operation, component, part, element, material, or combination. The terms of the present invention are disclosed in the specification and are not intended to exclude the possibility that one or more other features, numbers, operations, components, elements, materials or combinations thereof may be present or added. As used herein, "/" may be interpreted as "and" or "as appropriate.
The embodiment of the invention provides a melon SNP marker locus combination, which is characterized by comprising 2000 SNP marker loci, wherein each SNP marker locus comprises 1 base variation locus; the SNP marker locus positions and mutation loci are shown in Table 1.
TABLE 1 2000 SNP marker loci
The other embodiment of the invention provides a probe combination for detecting the melon SNP marker locus combination, which is characterized in that each SNP marker locus is correspondingly provided with a group of probes for detecting the corresponding SNP marker locus to form the probe combination.
It should be noted that, the 2000 SNP site combinations in table 1 described above can greatly reflect the genetic characteristics of a representative germplasm population of melon; the density of the liquid phase chip and the uniform distribution degree on the chromosome are highest in the current melon DNA chip report, can be applied to different application scenes in a substantive way, and have great application value in melon breeding improvement and variety identification.
In addition, the 2000 SNP loci were screened as follows: 823 representative germplasm was screened by re-sequencing data of 1175 melon (NCBI BioProject accession: PRJNA 565104) based on PCA analysis of phenotype and genotype; 8032592 SNP loci are identified by GATK pipeline by taking 'DHL94v4.0' as a reference genome, and firstly, non-two-level loci, rare mutation loci (minimum allele count 40), high deletion rate and high heterozygosity rate loci (more than 0.1), low sequencing coverage loci (less than 4) and low alignment scoring loci (less than 20) are filtered out by adopting a conventional principle to ensure the reliability of mutation loci. Then aiming at the development of melon whole genome liquid phase chip, the following screening criteria are adopted: 1) At least more than 30 markers among any two melon resources have genotype differences so as to ensure genetic diversity among individuals; 2) The GC content of 55bp flanking sequences at the upstream and downstream of the SNP locus is between 30% and 70% to ensure the combination of probes; 3) The number of other variants of the upstream 55bp flanking sequence and the downstream 55bp flanking sequence of the SNP locus cannot exceed 1 so as to ensure typing accuracy; 4) The similarity between the upstream and downstream 55bp flanking sequences of the SNP locus and other areas of the genome cannot exceed 10 percent so as to ensure the uniqueness of probe hybridization; 5) A linkage disequilibrium coefficient (VIF) between any two SNPs in each 100Kb window of less than 2 to ensure non-redundancy of the marker; 6) Bin was drawn for the remaining SNPs using 170kb as a sliding window, and the final 2000 SNPs were selected based on their criteria for even distribution on the chromosome.
In some embodiments, the probes corresponding to each SNP marker site are as set forth in Table 2. The probe numbers in table 2 correspond to the mutation sites and positions corresponding to the SNP numbers in table 1, for example, probe No. 1 is a probe of SNP marker site No. 1.
TABLE 2 Probe corresponding to SNP marker loci
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In yet another embodiment, the invention provides a reagent for identifying melon genotyping, comprising the probe combination for detecting melon SNP marker loci.
In still another embodiment, the invention provides a liquid chip comprising the probe combination for detecting melon SNP marker loci or the reagent for identifying melon genotyping.
It should be noted that, the above-mentioned 2000 SNP locus combinations in the liquid phase chip can greatly reflect the genetic characteristics of representative germplasm groups of melon; the density of the liquid phase chip and the uniform distribution degree on the chromosome are highest in the current melon DNA chip report, can be applied to different application scenes in a substantive way, and have great application value in melon breeding improvement and variety identification.
The invention also provides an application of the muskmelon SNP marker locus combination, the probe combination for detecting the muskmelon SNP marker locus, the reagent for identifying the muskmelon genotyping or the liquid chip in identifying the muskmelon genotyping.
In still another aspect, the invention provides an application of the melon SNP marker locus combination, the probe combination for detecting the melon SNP marker locus, the reagent for identifying melon genotyping or the liquid chip in genetic diversity analysis, variety identification, genetic linkage map construction, QTL or gene positioning, genetic background selection or molecular assisted breeding of melons.
For a better understanding of the present invention, the content of the present invention is further elucidated below in connection with the specific examples, but the content of the present invention is not limited to the examples below.
In the implementation of the invention, the melon whole genome liquid phase chip kit is manufactured by utilizing a liquid phase probe hybridization-based targeted gene capturing technology (GenoBaits) of Shi family Boruidi company.
EXAMPLE 1 development of melon Whole genome liquid phase chip
823 representative germplasm was screened by re-sequencing data of 1175 melon (NCBI BioProject accession: PRJNA 565104) based on PCA analysis of phenotype and genotype. 8032592 SNP loci are identified by GATK pipeline by taking 'DHL 94v 4.0' as a reference genome, and firstly, non-two-level loci, rare mutation loci (minimum allele count 40), high deletion rate and high heterozygosity rate loci (more than 0.1), low sequencing coverage loci (less than 4) and low alignment scoring loci (less than 20) are filtered out by adopting a conventional principle to ensure the reliability of mutation loci.
Then aiming at the development of melon whole genome liquid phase chip, the following screening criteria are adopted: 1) At least more than 30 markers among any two melon resources have genotype differences so as to ensure genetic diversity among individuals; 2) The GC content of 55bp flanking sequences at the upstream and downstream of the SNP locus is between 30% and 70% to ensure the combination of probes; 3) The number of other variants of the upstream 55bp flanking sequence and the downstream 55bp flanking sequence of the SNP locus cannot exceed 1 so as to ensure typing accuracy; 4) The similarity between the upstream and downstream 55bp flanking sequences of the SNP locus and other areas of the genome cannot exceed 10 percent so as to ensure the uniqueness of probe hybridization; 5) A linkage disequilibrium coefficient (VIF) between any two SNPs in each 100Kb window of less than 2 to ensure non-redundancy of the marker; 6) Drawing Bin for the rest SNP by taking 170kb as a sliding window; based on the standard of uniform distribution of the SNP in the chromosome, the final 2000 SNP are selected, and a Melon whole genome SNP liquid phase chip kit 'Melon 2K' is manufactured by a liquid phase probe hybridization-based targeted gene capturing technology (GenoBaits) of Shi family Boruidi company, wherein the distribution of 2000 SNP loci in the chromosome is shown in figure 1.
EXAMPLE 2 melon Whole genome liquid phase chip for population genetic analysis
In the embodiment of the invention, based on 2000 very representative SNP loci and all SNP loci of the whole genome, the Minimum Allele Frequency (MAF) and the phylogenetic tree of 823 representative melon germplasm are compared and analyzed, so that the representativeness of 2000 representative SNP loci is evaluated. From the analysis result of MAF frequency distribution, 2000 SNP loci and all SNP loci of the whole genome have a very consistent distribution trend on MAF frequency distribution in the whole population and different subgroups, which can correctly reflect genetic diversity in the whole population and subgroups (FIG. 2A, wherein the abscissa of each group is the whole population of individuals, cultivated thin skin, cultivated thick skin, wild thin skin and wild thick skin in sequence from left to right).
In addition, the evolutionary tree constructed by using 2000 SNP locus generations and the evolutionary tree constructed by using all SNP loci in the whole genome have nearly identical topological structures and grouping results, and can reflect four classical melon groups: cultivated veneer, wild veneer and wild veneer (fig. 2B).
In addition, based on 2000 representative SNP sites and all SNP sites of the whole genome, the embodiment of the invention further calculates the genetic similarity between two individuals and constructs the genetic relationship matrix of two sets of data, the pearson correlation coefficient of the two sets of data can reach 0.996 in 823 germplasm, and the average reaches more than 0.99 inside different subgroups and among different subgroups (fig. 2C).
The above results fully illustrate the representative of 2000 SNP sites in the liquid phase chip of the invention.
EXAMPLE 3 construction of representative germplasm finger print of melon
In the embodiment of the invention, in order to scientifically and efficiently identify and manage melon germplasm resources, core SNP identification is carried out on 2000 polymorphic SNP loci in 823 representative germplasm of muskmelon by perl language script, 21 core SNP genotype loci capable of effectively distinguishing all representative varieties are screened altogether (see table 3), and 21 core SNP genotypes can be endowed with 823 unique genotype combinations of the muskmelon germplasm by typing results (figure 3A), so that the method can be used for distinguishing different varieties and constructing DNA fingerprint.
TABLE 3 21 core SNP genotyping sites and corresponding probes
In addition, by counting the number of core SNP genotypes that differ between each two individuals, it was found that the inter-individual difference core marker data conforms to a normal distribution on a mathematical model, with the number of minimum inter-individual difference genotypes being 1, the number of maximum difference genotypes being 20, and 95.65% of the number of inter-individual difference genotypes being between 5 and 14 (fig. 3B).
Example 4 melon genetic background selection and Single fragment introgression line background recovery detection
In the embodiment of the invention, in order to verify the functionality of the melon whole genome liquid-phase chip in marker-assisted breeding, a single-segment introgression line is constructed by taking a cultivation melon 13C material as a receptor parent and a wild melon P132 as a donor parent. By backcrossing the two parents with the recipient parent "13C", 223 BC were obtained 1 F 1 And (5) generating a population. Based on liquid phase chip pair BC 1 F 1 The individuals were genotyped and visualized to screen 55 target recombinant individuals that could cover the donor parent "P132" genome (FIG. 4A). Among these recombinant individuals, the highest recovery rate of the receptor parent can reach 86.54%, the lowest recovery rate reaches 62.18%, and the average recovery rate reaches 75.79% (fig. 4B). In the construction of the subsequent high-generation backcross population, the 55 recombinant single plants are selected, so that the construction of the introgression line can be assisted and accelerated.
The results show that the melon whole genome liquid phase chip constructed in the invention is reliable to the genetic background selection result of melon groups, and also proves the application value of the melon whole genome liquid phase chip in molecular breeding.
Finally, it is noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered by the scope of the claims of the present invention.
Claims (7)
1. A melon SNP marker locus combination comprising 2000 SNP marker loci, each SNP marker locus comprising a 1 base variation locus; SNP marker site positions and mutation sites are shown in the following table;
2. a probe combination for detecting the melon SNP marker locus combination according to claim 1, wherein each SNP marker locus is provided with a group of probes for detecting the corresponding SNP marker locus, and the probe combination is formed.
3. The probe combination for detecting melon SNP marker locus combinations according to claim 2, wherein the probe corresponding to each SNP marker locus is as set forth in the following table;
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4. a reagent for identifying melon genotyping, comprising the probe combination for detecting melon SNP marker loci according to claim 2 or 3.
5. A liquid chip comprising the probe combination for detecting melon SNP marker loci according to claim 2 or 3 or the reagent for identifying melon genotyping according to claim 4.
6. Use of the melon SNP marker locus combination according to claim 1 or the probe combination for detecting melon SNP marker loci according to claim 2 or 3 or the reagent for identifying melon genotyping according to claim 4 or the liquid chip according to claim 5 for identifying melon genotyping.
7. Use of the melon SNP marker locus combination according to claim 1 or the probe combination for detecting melon SNP marker loci according to claim 2 or 3 or the reagent for identifying melon genotyping according to claim 4 or the liquid phase chip according to claim 5 in genetic diversity analysis, variety identification, genetic linkage map construction, QTL or gene localization, genetic background selection or molecular assisted breeding of melon.
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| CN109652412A (en) * | 2019-01-22 | 2019-04-19 | 西北农林科技大学 | The method and application of a kind of SNP marker, detection muskmelon flower property type |
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