CN117363780A - White gourd 10K SNP liquid-phase breeding chip and application thereof - Google Patents
White gourd 10K SNP liquid-phase breeding chip and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and discloses a wax gourd 10K SNP liquid-phase breeding chip and application thereof. The 10K liquid-phase white gourd breeding chip has 10722 SNP loci, has good locus representativeness, strong specificity and high polymorphism, is a set of accurate and efficient molecular breeding chip, and can be widely applied to detection of white gourd materials of different varieties. The detection flux is high, the data quantity is large in one-time output, and detection of nearly thousands of materials can be covered simultaneously; meanwhile, the method is suitable for the mainstream platform of secondary sequencing such as illuminea and MGI, and has the wide adaptability of the platform. The chip can be applied to molecular marker fingerprint analysis of white gourd varieties, identification of offspring genotypes of hybridization groups, identification of variety authenticity, genetic background analysis and screening of breeding materials, association analysis of whole genome, genetic diversity analysis of germplasm resources and genetic relationship identification.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a wax gourd 10K SNP liquid-phase breeding chip and application thereof.
Background
White gourd (Benincasa hispida cognin.) is an important vegetable crop of Cucurbitaceae, and has a long cultivation history, and is now one of the main vegetable varieties in China. However, at present, white gourd varieties still occupy a considerable market proportion, and have the defects of unstable quality, yield and resistance, seed degeneration and the like. With the development of biotechnology, breeders also put more strict requirements on the yield, quality and resistance of white gourd, and the existing germplasm resources are difficult to meet the breeding requirements. Therefore, the cultivation of excellent new varieties of white gourd is quickened, and the sustainable development of the white gourd industry is promoted.
At present, genetic background among white gourd materials is narrow, germplasm resources are relatively few, and research based on molecular level is obviously lagged behind other vegetable crops. Vegetables such as cucumbers and tomatoes successfully solve the serious scientific problems of bitter taste of the cucumbers, flavor of the tomatoes and the like on the basis of whole genome sequencing, provide a brand-new breeding concept and strategy for breeding, and are successfully applied to breeding practice. The research of the white gourd also has a great need to break through the natural mode of traditional breeding, combine the technologies of genomics, high-throughput genotyping, gene editing and the like which are developed at high speed nowadays, improve the breeding strategy, promote the traditional breeding of the white gourd to change the direction of accurate and efficient molecular breeding, realize the accurate control of the target gene of the white gourd, effectively select the genetic background and accurately analyze and identify the breeding variety.
At present, molecular research of white gourd mainly adopts molecular marker technologies such as SSR (simple sequence repeat) markers, ISSR markers, AFLP and the like, and the SSR markers have low flux and high development cost and are not suitable for large-scale commercial identification; ISSR markers are mostly dominant markers, and cannot distinguish dominant homozygous and heterozygous genotypes; AFLP markers have high requirements on reaction conditions, are unevenly distributed on chromosomes, and have unstable results and poor repeatability. The SNP liquid phase chip has the advantages of convenience in detection, low cost, high flux, high sensitivity and the like. At present, no related report of the white gourd SNP liquid phase chip is found in China. Therefore, the development of the first high-density wax gourd SNP liquid phase chip can provide important technical support for molecular breeding of wax gourd.
The invention develops a set of 10K SNP liquid-phase breeding chip for white gourd, which can carry out fingerprint analysis on white gourd variety resource molecular markers, identification on filial generation genotypes of hybrid groups, identification on variety authenticity, analysis and screening on genetic background of breeding materials, association analysis on whole genome, genetic diversity analysis on germplasm resources and genetic relationship identification.
Disclosure of Invention
The invention aims to solve the technical problems and overcome the defects and shortcomings in the background technology, and provides a white gourd 10K SNP liquid-phase breeding chip and application thereof, wherein the chip sites are uniformly distributed on chromosomes, have good site representativeness, strong specificity and high polymorphism, and are a set of accurate and efficient molecular breeding chips. Can be widely used for detecting and applying different types of white gourd materials such as wild species, local species, cultivated species and the like. Provides an indispensable important tool for molecular breeding research such as high-throughput genotyping, gene positioning, fingerprint imprinting, whole genome selection and the like of white gourd.
In order to solve the technical problems, the technical scheme provided by the invention is as follows:
a white gourd 10K SNP liquid-phase breeding chip, the genotyping object of the chip comprises 10722 SNP loci on the white gourd whole genome, the positional information of the SNP loci is shown in an instruction table 1, and the reference genome of the SNP loci is white gourd genome "pf3_genome.v1.1".
Based on a general inventive concept, the invention also provides application of the white gourd 10K SNP liquid-phase breeding chip in white gourd germplasm resource genetic diversity analysis, population structure analysis, genetic and evolutionary analysis, genetic relationship identification, whole genome association analysis or genome selection.
The above application, preferably, the method of application includes the following steps: and genotyping the wax gourd sample by utilizing the wax gourd 10K SNP liquid-phase breeding chip.
Compared with the prior art, the invention has the beneficial effects that:
(1) The 10K liquid-phase white gourd breeding chip has the characteristics of good site representativeness, strong specificity, high polymorphism and the like, is a set of accurate and efficient molecular breeding chip, and can be widely applied to detection of white gourd materials of different varieties.
(2) Based on a high-throughput sequencing technology, the detection method has high detection throughput and large data output at one time, and can cover detection of nearly thousands of materials at the same time; meanwhile, the method is suitable for the mainstream platform of secondary sequencing such as illuminea and MGI, and has the wide adaptability of the platform.
(3) The chip can carry out molecular marker fingerprint analysis on white gourd variety resources, identification on genotype of filial generation, identification on variety authenticity, analysis and screening on genetic background of breeding materials, association analysis on whole genome, genetic diversity analysis on germplasm resources, genetic relationship identification, wide adaptability, strong specificity, high polymorphism and simple and rapid operation.
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 cGPS sequencing library construction in example 1;
FIG. 2 is a flowchart of the label development in example 1;
FIG. 3 is a graph showing PIC value distribution of 10K locus of wax gourd in example 1;
FIG. 4 is a diagram showing the distribution of 10K locus of wax gourd on chromosome of wax gourd in example 1;
FIG. 5 is a annotation analysis of SNP sites in example 2;
FIG. 6 is a population structure analysis PCA scattergram of the white gourd seed material of example 2;
FIG. 7 is a phylogenetic tree diagram of the population structure analysis of the white gourd breeding material of example 2.
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, the technical 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 invention relates to a wax gourd 10K liquid phase breeding chip.
The invention is based on white gourd materials of different types (wild germplasm, local varieties and cultivars), and obtains more than 1 hundred million SNP loci by carrying out re-sequencing analysis in combination with materials provided by vegetable research institute of Hunan province. And then, according to indexes of the SNP loci, 10444 SNP loci with strong representativeness, good polymorphism and even chromosome distribution and 278 functional gene loci related to important economic traits such as stress resistance, fruit traits, production development and the like of the white gourd are selected as 10K liquid-phase SNP chips of the white gourd. The chip is specifically obtained by the following steps:
1. white gourd germplasm resource collection
In order to ensure the representativeness of the white gourd materials and the universality of liquid chip detection, 93 parts of white gourd diversity materials are collected from vegetable institute of Hunan province, and the resequencing data of 146 parts of materials of different regions and different varieties (wild germplasm, local variety and cultivar) are collected on a collecting network.
2. Wax gourd whole genome resequencing
Carrying out whole genome re-sequencing on 93 parts of collected white gourd materials, wherein the method comprises the following specific steps of: (1) DNA extraction by the magnetic bead method. (2) And (3) constructing a quality inspection qualified DNA-seq sequencing library by adopting an MGI library standard method. (3) After the quality of the library is checked, the library is sequenced by adopting a Huada sequencing platform (MGI), the sequencing strategy is PE150, the sequencing depth is 10×, and each strain is 9.2Gb.
The analysis procedure was as follows, with Sentieon for comparison and mutation detection of 239 parts of resequenced data: (1) Reads were aligned to correspond to the wax gourd reference genome (pf3_genome. V1.1), position ordered and labeled using Sentieon repeat reads.
(2) Mutation site detection was performed on each sample using Sentieon to obtain gccf for each sample.
(3) Joint-rolling is performed by using Sentieon, and gVCF of all samples is subjected to joint analysis, so that a mutation result of each individual in the population is obtained. To ensure SNP accuracy, the SNP sites obtained after the association analysis are subjected to preliminary hard filtration (SNP hard filtration standard: QD < 2.0|FS > 60.0|MQ < 40.0|SOR > 3.0|MQRankSum < -12.5|ReadPosRankSum < -8.0 ").
3. White gourd liquid phase breeding chip development
(1) Whole genome site selection:
a. candidate site selection: calculating quality indexes of resequencing sites, screening the SNP polymorphic sites with the heterozygosity rate less than 0.3, the site deletion frequency less than 0.1, the minimum allele frequency more than or equal to 0.1 and the sequencing depth more than or equal to 5x as candidate sites to obtain 920418 sites in total.
b. 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 263405 SNP loci which can be used for chip development.
c. Site density screening: according to the principle of uniform distribution of sites, SNP sites uniformly distributed on a white gourd chromosome are screened, wherein the average spacing of the sites is 88Kb, and 10444 SNP sites are included.
4. Determination of important functional sites
For reported functional genes related to important economic traits such as stress resistance and fruit traits of white gourd, screening SNP loci on the genes as candidate loci, wherein the candidate loci comprise 278 SNP loci, and the functional genes comprise: 130 markers of stress-resistance related sites; the number of markers of disease-resistant related sites is 5; the number of markers for the growth-related sites is 45; the number of markers for seed-related sites was 6; the number of markers at the fruit shape-related sites was 92.
5. Development of wax gourd 10K SNP liquid phase chip
The 10722 candidate SNP loci are developed into a white gourd 10K liquid phase chip by using a targeted sequencing genotyping technology (Genobaits technology). The liquid phase chip technology is to design a characteristic probe for a target interval (3K-150K target interval) sequence based on an optimized thermodynamic stability algorithm model, perform liquid phase hybridization capture enrichment on a plurality of different target sequences positioned at different genome positions by utilizing a synthesized specific probe, and then perform library construction and second generation sequencing on the capture enrichment target interval, so as to obtain the genotype of a marker locus in the target interval.
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) GPS 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 performed by using magnetic beads, the capture products are washed by using a washing liquid, non-specific binding fragments are removed, and a round of PCR amplification is performed. 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 cGPS sequencing library after the concentration and the fragment size are determined to be qualified, wherein a library building flow is shown in a figure. The prepared library is subjected to high-throughput sequencing by using a Huada sequencer, and the sequencing strategy is PE150. The library building flow is shown in figure 1.
(3) Bioinformation analysis: (1) raw data filtration (sequencing data quality control): 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, namely, based on fastq files of Clean Reads after sample filtration, 10,000 sequences are randomly selected from the fastq files of each sample by using seqtk software, and the sequences are aligned to an NCBI NT database by using blastn for pollution evaluation. (3) And (3) reference genome alignment, namely aligning sequencing Reads to a reference genome by using BWA software, and performing position sequencing to obtain a sample sequenced bam file. (4) Mutation detection a, detecting mutation sites of each sample by using a biplotypeCaller in gatk software to obtain gVCF files of each sample. b. And (3) using CombinGCFs analysis in gatk software to obtain a mutation result file of the detection material. c. Genotyping the gccf files of the population using GenotypeGVCFs in gatk software, resulting in the original vcf variation result file. (5) Genotyping the target site, namely judging according to the number proportion of supported Reads of different Alles of the site, judging the site as a homozygous genotype when the support 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 support proportion of the mutant Reads is between 0.2 and 0.8. And processing the original vcf variation result file by using an internally written perl script to obtain a 28-converted vcf variation result file.
Finally, the genotyping result of each target SNP in a specific individual can be obtained with high flux, and high flux SNP genotyping is realized; information of 10722 SNP loci is shown in Table 1. The label development flow is shown in fig. 2.
Table 1: tag sequence information
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A high-quality 10K site is selected based on a plurality of stable white gourd varieties (Guangxi black skin, yuanbao white gourd, xiaojia green jade and the like) provided by vegetables in Hunan province to form a white gourd 10K liquid-phase breeding chip, so that most of white gourd varieties in Hunan are covered, and the chip has a wide chip application scene.
The polymorphism of the 10K SNP locus of the screened white gourd is counted, the average polymorphism information content (PIC, polymorphic Information Content, polymorphism information value) is 0.29, the amplitude is 0.163-0.375, the white gourd belongs to high polymorphism (PIC value is 0 at the lowest and is 0.375 at the highest), and the PIC value distribution diagram is shown in figure 3.
The distribution of the screened 10K chips on 12 chromosomes is counted, the 10K SNP loci are uniformly distributed on 12 chromosomes of white gourd, the average interval value of the SNP loci on the chromosomes is 85Kb, and the locus distribution diagram is shown in figure 4.
The efficacy of SNPs was annotated with the snpfff tool for the selected 10K chip sites, see fig. 5.
Example 2:
the invention relates to application of a white gourd SNP liquid phase chip in polymorphism and population structure analysis of white gourd breeding materials.
And (3) carrying out sample detection on 96 parts of white gourd materials by using the white gourd 10K liquid phase breeding chip designed in the embodiment 1, extracting genotype and typing, and analyzing PCA components of the detection materials by using Plink software to construct 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 white gourd breeding materials are found to be divided into 3 obvious groups through statistics, and the white gourd breeding materials are shown in figure 6.
And calculating a genetic distance matrix by using the Plink software, performing cluster analysis, and constructing a phylogenetic tree graph to judge the genetic relationship, evolutionary relationship and composition structure of different materials. The wax gourd 10K liquid phase breeding chip provided by the invention is used for carrying out group analysis on 96 wax gourd materials, and the effect is consistent with the actual grouping. The screened SNP loci are shown to be highly representative, as shown in FIG. 7.
Claims (3)
1. A white gourd 10K SNP liquid-phase breeding chip is characterized in that a genotyping object of the chip comprises 10722 SNP loci on a white gourd whole genome, the position information of the SNP loci is shown in an instruction table 1, and a reference genome of the SNP loci is white gourd genome "pf3_genome. V1.1".
2. Use of the wax gourd 10K SNP liquid phase breeding chip as set forth in claim 1 in wax gourd germplasm resource genetic diversity analysis, population structure analysis, genetic and evolutionary analysis, genetic relationship identification, whole genome association analysis or genome selection.
3. The application according to claim 2, characterized in that the method of application comprises the steps of: and genotyping the wax gourd sample by utilizing the wax gourd 10K SNP liquid-phase breeding chip.
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