CN117587159A - Chilli SNP molecular marker combination, SNP chip and application thereof - Google Patents

Chilli SNP molecular marker combination, SNP chip and application thereof Download PDF

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CN117587159A
CN117587159A CN202311662361.8A CN202311662361A CN117587159A CN 117587159 A CN117587159 A CN 117587159A CN 202311662361 A CN202311662361 A CN 202311662361A CN 117587159 A CN117587159 A CN 117587159A
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CN117587159B (en
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雷雨婷
刘峰
熊程
李为国
唐顺学
何轮
邹学校
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Abstract

The invention provides a capsicum SNP molecular marker combination, an SNP chip and application thereof, wherein the SNP molecular marker combination consists of 51172 SNP molecular markers with high polymorphism and high detection rate, and the SNP molecular markers consist of 47589 SNP loci, 3194 MNP marker loci and 389 loci related to important agronomic traits/genes such as capsicum disease resistance, capsicum fruit color, capsicum peppery taste, capsicum male sterility and the like which are uniformly distributed on chromosomes. The invention also provides a 50K liquid-phase pepper breeding chip by combining with the molecular marker. The chip has high detection flux, high target site detection rate and accurate and reliable typing result, and can be applied to the analysis of new pepper variety identification, molecular marker assisted selection breeding, gene positioning, whole genome selection, population genetics and population evolution.

Description

Chilli SNP molecular marker combination, SNP chip and application thereof
Technical Field
The invention belongs to the technical field of plant molecular genetic breeding, and particularly relates to a capsicum SNP molecular marker combination, a SNP chip and application thereof.
Background
Capsicum (Capsicum annuum l.) belongs to the family of solanaceae and is an important condiment and colorant. The pepper cultivation history is long, variety resources are rich and complex, and in recent years, conventional pepper breeding achieves great achievement, but with the upgrading of consumption requirements, requirements on variety of pepper varieties such as taste, color, health, quality and the like are deepened, and the provision of varieties with good quality is the development direction of pepper breeding at present.
Conventional pepper breeding mainly adopts field plant morphology identification, but the method is limited by factors such as low accuracy, long identification time, high input cost, easiness in environmental influence and the like, and as the number of pepper varieties is increased, the phenotype difference among varieties is smaller and smaller, and the morphological identification is more difficult. The new variety is expanded in China, and meanwhile, high-quality germplasm resources are introduced, and particularly, the wild resources and the specific resources are relatively lacking, so that the requirements for breeding high-quality pepper varieties by combining modern molecular breeding technology are increased.
The molecular marker identification methods commonly used at present are RFLP, RAPD and SSR technologies, and have the following disadvantages: RFLP (restriction endonuclease fragment length polymorphism) technology has complex operation, high requirement on DNA quality, higher cost and harmful radioisotope to human body; the RAPD technique (randomly amplifying polymorphic DNA) results are unstable and have poor repeatability; the SSR primer has high development cost and is not suitable for large-scale commercial identification; the requirements of an ideal detection method are not met.
Compared with other detection modes, the liquid-phase breeding chip based on SNP markers has the characteristics of high marker density, strong automation, high detection flux and the like, and can establish a modern breeding system and promote the development of pepper breeding.
Disclosure of Invention
In order to overcome the problems in the prior art, the invention provides a capsicum SNP molecular marker combination, a SNP chip and application thereof, which can be used for capsicum new variety identification, molecular marker assisted selection breeding, gene positioning, whole genome selection, population genetics, population evolution and the like.
In order to solve the technical problems, the technical scheme provided by the invention is as follows:
the first aspect of the present invention provides a combination of capsicum SNP molecular markers, wherein the combination of SNP molecular markers comprises 51172 SNP molecular markers, the 51172 SNP molecular markers are shown in table 1, and the positions and variation information of the SNP loci adopt chromosomes: physical location: the physical position of the SNP molecular marker combination is subjected to positioning analysis based on a camphor tree port capsicum reference genome.
The camphor tree harbor pepper reference genome source is shown in the document Genomes of cultivated and wild Capsicum species provide insights into pepper domestication and population differentiation.
Specific information of 51172 SNP markers is shown in Table 1 below.
Table 1: SNP molecular marker information
Based on the same technical concept, the second aspect of the invention also provides a capsicum SNP chip, which comprises a probe or a primer for detecting the capsicum SNP molecular marker combination.
The invention also provides application of the capsicum SNP molecular marker combination or the capsicum SNP chip in capsicum genetic diversity analysis.
The invention also provides application of the capsicum SNP molecular marker combination or the capsicum SNP chip in capsicum colony structure analysis.
The invention also provides application of the capsicum SNP molecular marker combination or the capsicum SNP chip in capsicum genetic background breeding analysis.
The invention also provides application of the capsicum SNP molecular marker combination or the capsicum SNP chip in the identification of the capsicum relatedness.
The invention also provides application of the capsicum SNP molecular marker combination or the capsicum SNP chip in capsicum whole genome association analysis.
The invention also provides application of the capsicum SNP molecular marker combination or the capsicum SNP chip in capsicum variety identification.
The invention also provides application of the capsicum SNP molecular marker combination or the capsicum SNP chip in molecular marker assisted selective breeding.
Compared with the prior art, the invention has the beneficial effects that:
the invention utilizes the resequencing sequence of pepper germplasm resources such as main cultivars of domestic and foreign peppers, improved varieties in Hunan places and the like to screen SNP loci of the whole genome, which are evenly distributed, with high polymorphism (PIC mean value 0.30) and high detection rate (mean value 99.5); MNP marker loci for plant variety identification; also added are important agronomic trait genes of 26 capsicum: the loci such as the spicy gene, the disease resistance gene and the fruit development gene develop a set of 51172 SNP molecular markers to form the high-density liquid-phase breeding chip of the capsicum. The chip has high detection flux, high target site detection rate and accurate and reliable parting result, and can be applied to the identification of new pepper varieties, molecular marker assisted selection breeding, gene positioning, whole genome selection, population genetics, population evolution and the like.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a flow chart of site selection in example 1 of the present invention;
FIG. 2 is a graph showing PIC value distribution of 51172 SNP sites in example 1 according to the invention;
FIG. 3 is a distribution diagram of 51172 SNP sites on a chromosome in example 1 according to the invention;
fig. 4 is a group structural analysis chart of 139 capsicum materials in example 2 of the present invention;
fig. 5 is a PCA scatter plot of 139 capsicum materials in example 2 of the present invention.
Detailed Description
The present invention will be described more fully hereinafter with reference to the accompanying drawings, in which preferred embodiments are shown, for the purpose of illustrating the invention, but the scope of the invention is not limited to the specific embodiments shown.
Unless defined otherwise, all technical and scientific terms used hereinafter have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the scope of the present invention.
Unless otherwise specifically indicated, the various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or may be prepared by existing methods.
Example 1
The embodiment provides a capsicum SNP molecular marker combination, which consists of 51172 SNP molecular markers, wherein 51172 SNP molecular markers are shown in the table 1 of the summary of the invention, the screening process is shown in fig. 1, and the method specifically comprises the following steps:
1. pepper germplasm resource collection
To ensure the representativeness of the capsicum material and the universality of the liquid chip, 176 parts of capsicum germplasm resource data provided by Hunan agricultural university are collected and resequenced.
And simultaneously collecting the resequencing data of more than 300 parts of pepper cultivars from Asia, america, africa and European gene libraries, such as wild species, breeding varieties with different origins and various fruit shapes, and the like.
2. Full genome resequencing of capsicum
The collected 176 parts of capsicum material is subjected to genome-wide re-sequencing, and the method comprises the following specific steps:
(1) Extracting the DNA of the capsicum material by a magnetic bead method.
(2) And (3) constructing a DNA-seq sequencing library by adopting an MGI standard library construction method, wherein the quality inspection is qualified.
(3) The qualified library was sequenced using a Huada sequencing platform (MGI), the sequencing strategy was PE150, the sequencing depth was 10×, and 30Gb per strain.
176 parts of resequenced data are compared and subjected to mutation detection, and the analysis flow is as follows:
(1) Reads were aligned to correspond to the capsicum reference genome (capsicum camphora harbor) using Sentieon, position ordered and labeled repeatedly Reads.
(2) And detecting a mutation site of each sample to obtain mutation information of each sample.
(3) And carrying out joint analysis on gVCF of all samples to obtain a variation result of each individual in the population.
3. Chilli liquid phase breeding chip development
(1) Full genome site
a. Resequencing variant site acquisition: 176 parts of capsicum germplasm resource data provided by Hunan agricultural university are subjected to resequencing, and about 1.3 hundred million SNP mutation sites are extracted. The resequencing data of more than 300 capsicum seeds collected at home and abroad are used for extracting about 7600 thousands of SNP mutation sites. The number of common SNP mutation sites is 6600 ten thousand.
b. Candidate site selection: calculating the quality index of the loci, screening the SNP polymorphism loci with the heterozygosity rate less than 0.2, the locus deletion frequency less than 0.1, the minimum allele frequency more than 0.1, the locus polymorphism more than 0.15 and the sequencing depth more than 5x as candidate loci, and obtaining about 200 ten thousand SNP loci in total.
c. Site probe design and screening: extracting 50bp sequences at the upstream and downstream of the SNP locus, analyzing the specificity, the GC content and the like of the SNP locus, judging whether a probe is properly designed, and screening to obtain about 86 ten thousand SNP loci which can be used for chip development.
d. Site density screening: according to the principle of uniform distribution of loci, SNP loci which are uniformly distributed on capsicum chromosomes are screened, wherein the total of 47589 SNP loci are contained, and the average interval is 60Kb.
(2) Pepper MNP site
According to the sequence of a pepper MNP marking primer published in plant variety identification-MNP marking method (GB/T38551-2020), comparing to a pepper reference genome, obtaining 831 target intervals altogether, extracting high-quality SNP loci in the intervals as candidate loci, and totally comprising 3194 SNP loci.
(3) Determination of important Gene/functional loci
Genes related to important agronomic traits such as disease resistance, pepper fruit color, pepper pungency, pepper male sterility and the like of the peppers reported in the excavated literature are screened, high-quality SNP loci in a gene region and a 1K region at the upper and lower streams of the gene region are screened, the genes comprise 389 SNP loci altogether, and the selected gene types and marker numbers are shown in the following table 2:
table 2: gene type and number of markers
4. Development of 50K SNP liquid-phase chip for capsicum
The SNP locus is obtained by collecting and combining pepper germplasm resources provided by Hunan agricultural university based on different types of pepper materials (international main cultivar, hunan local variety, breeding varieties with different origins and various fruit shapes) and carrying out resequencing analysis. And according to the quality indexes of the SNP loci, 47589 SNP loci, 3194 MNP marker loci and 389 loci related to important agronomic characters/genes such as disease resistance, pepper fruit color, pepper pungency and pepper male sterility are selected out, wherein the loci are strong in representativeness, good in polymorphism and uniformly distributed on a chromosome, and the loci are combined to form a 50K liquid phase chip of the pepper.
Polymorphism analysis of SNP loci of 50K chips of peppers: the polymorphism of the 50K SNP locus of the screened capsicum is counted, the average Polymorphism Information Content (PIC) is 0.30, and the PIC value distribution diagram is shown in figure 2.
Chromosome distribution of 50K chip SNP loci of capsicum: the distribution of the screened 50K chips on 12 chromosomes is counted, the 50K SNP loci are uniformly distributed on 12 chromosomes of the capsicum, the average interval of the SNP loci on the chromosomes is 60Kb, and the locus distribution diagram is shown in figure 3.
The 51172 SNP loci selected are precisely positioned and sequenced by using a liquid-phase probe accurately developed by the intelligent autonomous research (Genotyping by Pinpoint Sequencing of liquid captured targets,cGPS) was developed into a 50K liquid chip of capsicum. A liquid phase chip technology, wherein,the method is characterized in that a target interval (3K-150K target interval) sequence is subjected to specific probe design based on an optimized thermodynamic stability algorithm model, a plurality of different target sequences positioned at different genome positions are subjected to liquid phase hybridization capture enrichment by utilizing a synthesized specific probe, and then library construction and second generation sequencing are performed on the capture enriched target interval, so that the genotype of a target SNP locus is obtained.
The technique involves the steps of:
(1) Sample DNA extraction and quality control: sample DNA extraction was performed using the magnetic bead method. Detecting the concentration of the DNA sample by using a Qubit fluorescent quantitative instrument; the integrity of the DNA samples was checked by 1% agarose gel electrophoresis and samples of acceptable quality control were used for library preparation.
(2) Library construction and quality control: a. and (3) enzyme cutting is carried out on the DNA sample by utilizing the fragmenting enzyme, the enzyme cutting end is repaired, an A base is added at the 3' end, and agarose gel electrophoresis is used for detecting the size of the fragment. b. The sequencing linker was ligated to the DNA fragment using T4 ligase and the ligation product was purified using magnetic beads. The concentration of the purified product is detected by a Qubit fluorescent quantitative instrument, and the size of the fragment is detected by agarose gel electrophoresis. c. And (3) carrying out PCR amplification on the purified connection product, and carrying out fragment screening on the amplified product by using magnetic beads. The concentration of the products after fragment screening is detected by a Qubit fluorescent quantitative instrument, and the size of the fragments is detected by agarose gel electrophoresis. d. 200ng of the library thus constructed was taken, and after adding the probe and the hybridization reagent, the hybridization reaction was completed by incubating at 50℃for 16 to 24 hours. Target segment capture is carried out by using streptavidin magnetic beads, a cleaning solution is used for cleaning a capture product, non-specific binding fragments are removed, and a round of PCR amplification is carried out. And detecting the concentration of the library by using a Qubit fluorescent quantitative instrument, detecting the size of the fragments by agarose gel electrophoresis, and completing the construction of the sequencing library after the concentration and the fragment size are determined to be qualified. The prepared library is subjected to high-throughput sequencing by using a Huada sequencer, and the sequencing strategy is PE150.
(3) Bioinformation analysis: (1) filtering the original data: and filtering the original sequencing sequence (Raw Reads) obtained by sequencing to obtain high-quality Clean Reads, and performing quality control on the off-line data by using FASTP software. (2) Pollution detection: sequences were aligned to the NCBI NT database using BLAST software for contamination assessment. (3) Reference genome alignment: the sequencing Reads were aligned to the reference genome using BWA software and position-sequenced to obtain sample-sequenced bam files. (4) Mutation detection: a. and detecting mutation sites of each sample by using GATK software to obtain mutation result files of each sample and each group. (5) Genotyping the target site: judging according to the number proportion of supported Reads of different Alles of the locus, judging the locus as a homozygous genotype when the supported proportion of the mutant Reads is more than or equal to 0.8 or less than or equal to 0.2, and judging as a heterozygous genotype when the supported proportion of the mutant Reads is between 0.2 and 0.8. Finally, a variation result file after 28 conversion is obtained, and the genotyping result of each target SNP in a specific individual can be obtained with high flux, so that high-flux SNP genotyping is realized.
Example 2
The application of the capsicum SNP molecular marker combination in polymorphism and colony structure analysis of capsicum breeding materials.
And 139 parts of pepper materials are detected by using a developed 50K liquid-phase pepper breeding chip, the genotype of the extracted target site is typed, the average detection rate of samples is 99.63%, the repeated consistency rate of the materials in the same batch is 99.99%, and the scheme has the advantages of high detection rate of the target site of the liquid-phase pepper chip and accurate and reliable typing result.
And calculating a genetic distance matrix by using Plink software, carrying out cluster analysis on pepper breeding materials, and constructing a phylogenetic tree graph to judge the genetic relationship, evolutionary relationship and composition structure of different materials. The 139 pepper materials from different groups are subjected to clustering analysis by using the method, the pepper breeding materials are divided into 3 subgroups, the effect is consistent with that of actual grouping, and the analysis result is shown in figure 4, so that the 51172 SNP loci screened out are high in representativeness.
And analyzing PCA (principal component analysis) composition components of the detection material by using PLink software, and constructing a PCA scatter diagram. Each point in the scatter plot represents a sample, and the further the two samples are from each other in the plot, the greater the difference between the genetic backgrounds of the two samples is, and the individuals with similar genetic backgrounds are grouped into one category in the plot. The pepper breeding material was found to divide into 3 distinct populations, and the results are shown in figure 5.
The foregoing is a further detailed description of the invention in connection with the preferred embodiments, and it is not intended that the invention be limited to the specific embodiments described. It will be apparent to those skilled in the art that several simple deductions or substitutions may be made without departing from the spirit of the invention, and these should be considered to be within the scope of the invention.

Claims (9)

1. The capsicum SNP molecular marker combination is characterized by comprising 51172 SNP molecular markers, wherein 51172 SNP molecular markers are shown in table 1, and the positions and variation information of the SNP loci adopt chromosomes: physical location: the physical position of the SNP molecular marker combination is subjected to positioning analysis based on a camphor tree port capsicum reference genome.
2. A capsicum SNP chip, comprising a probe or primer for detecting the capsicum SNP molecular marker combination of claim 1.
3. Use of the capsicum SNP molecular marker combination as defined in claim 1 or the capsicum SNP chip as defined in claim 2 in capsicum genetic diversity analysis.
4. Use of the capsicum SNP molecular marker combination as defined in claim 1 or the capsicum SNP chip as defined in claim 2 in capsicum population structural analysis.
5. The use of the capsicum SNP molecular marker combination as defined in claim 1 or the capsicum SNP chip as defined in claim 2 in capsicum genetic background breeding analysis.
6. Use of the capsicum SNP molecular marker combination as defined in claim 1 or the capsicum SNP chip as defined in claim 2 in capsicum genetic relationship identification.
7. Use of the capsicum SNP molecular marker combination as defined in claim 1 or the capsicum SNP chip as defined in claim 2 in whole genome association analysis of capsicum.
8. Use of the capsicum SNP molecular marker combination as defined in claim 1 or the capsicum SNP chip as defined in claim 2 in capsicum variety identification.
9. The use of the capsicum SNP molecular marker combination as defined in claim 1 or the capsicum SNP chip as defined in claim 2 in molecular marker assisted selection breeding.
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