WO2022134553A1 - Molecular marking method for detecting pass rate of hatching goose eggs - Google Patents

Molecular marking method for detecting pass rate of hatching goose eggs Download PDF

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WO2022134553A1
WO2022134553A1 PCT/CN2021/106541 CN2021106541W WO2022134553A1 WO 2022134553 A1 WO2022134553 A1 WO 2022134553A1 CN 2021106541 W CN2021106541 W CN 2021106541W WO 2022134553 A1 WO2022134553 A1 WO 2022134553A1
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goose
reaction
detecting
genome
snp
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Chinese (zh)
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高广亮
王启贵
赵献芝
吴睿
许国洋
张克山
谢友慧
王海威
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重庆市畜牧科学院
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/124—Animal traits, i.e. production traits, including athletic performance or the like
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/172—Haplotypes

Definitions

  • the invention relates to a molecular marker method for detecting the qualification rate of goose eggs, and mainly relates to the field of molecular biology.
  • Single nucleotide polymorphism mainly refers to the polymorphism of the DNA sequence caused by the variation of a single nucleotide in the genome series. It is the most common heritable variation in the genome of animals and plants, and it exists widely in the genome. SNP has low mutation rate, high accuracy and stability, and is widely used as one of the molecular markers of animals and plants. It is widely used in locating disease target genes, individual screening in the process of animal and plant breeding, analysis of species evolution and identification of kinship relationships.
  • Sichuan White Goose is one of the seasonal breeding waterfowl. It has the characteristics of strong stress resistance, good adaptability, roughage resistance, strong disease resistance and roughage resistance. It provides meat, eggs, down, fatty liver, etc. to consumers. . Widely bred throughout China and further used for the improvement and creation of other breeds. Egg qualification rate is an important economic trait and a complex quantitative trait, which is affected by genetics, nutrition, feeding methods, and external environment. Using biological and other methods for research, molecular markers such as duck body size and egg shell color have been discovered and widely used in molecular marker-assisted selection, which has greatly promoted the research progress in the qualification rate of poultry eggs. The reproduction of Sichuan white geese is seasonal.
  • the pass rate of goose breeding eggs is one of the important indicators for evaluating goose fecundity. It refers to the percentage of eggs produced by female geese within 70 weeks of age that meet the requirements of the breed and strain in the total number of eggs produced.
  • the qualified rate of breeding eggs directly affects the hatchability of geese and the quality of goslings, which directly increases the economic benefits of the goose industry and significantly reduces feeding costs.
  • the qualified rate of breeding eggs is affected by many aspects such as heredity, environment and disease. But at present, there is no corresponding detection method to judge the qualification rate of goose breeding eggs. The traditional method consumes a lot of manpower and material resources to detect the qualified rate of breeding eggs, and the cost is relatively high; and it is difficult to select the corresponding female geese for the qualified rate of goose laying eggs.
  • the present invention proposes a molecular marker method for detecting the qualification rate of goose eggs.
  • the molecular marker method is used to select and breed female geese with a higher qualification rate of eggs, which can quickly and accurately detect the qualification rate of goose eggs at the gosling stage. Breeding individuals with target traits greatly improves the efficiency of selection and reduces the cost of feeding.
  • the technical scheme of the present invention includes the following steps: S1: compare the whole genome resequencing data of the goose to be detected to the reference genome sequence, combine the pass rate data of the goose egg to be detected, and perform a genome-wide association analysis, 7 candidate SNP loci to obtain the pass rate of the goose eggs to be tested; S2: 7 SNP loci (SNP289, SNP290, SNP292, SNP294, SNP296, SNP297 and SNP298) and 1 single The distribution frequency of the ploidy module (constructed from 5 SNPs including SNP290, SNP292, SNP294, SNP296 and SNP297) in Sichuan white goose individuals, and the correlation analysis with the egg qualification rate traits, the results show that the above SNP loci and haplotypes Modules were significantly or extremely significantly correlated with hatching egg qualification rate traits.
  • D1 collect blood from the wing veins of the goose to be tested in the early stage of egg laying, and extract the whole genome DNA
  • D2 perform PCR reaction on the DNA in step D1, the system is 5 ⁇ l: 10*buffer 0.5 ⁇ l, Mg 2+ 0.4 ⁇ l, 0.1 ⁇ l of dNTP, 0.2 ⁇ l of Hotstar, 1 ⁇ l of upstream and downstream primer mix, 1.8 ⁇ l of triple distilled water, 1 ⁇ l of DNA sample to be detected (20ng-50ng); PCR amplification program: pre-denaturation at 95°C for 2 min, 45 cycles (95 Denaturation at °C for 30 s, annealing at 56 °C for 30 s, extension at 72 °C for 60 s, extension at 72 °C for 5 min, and storage at 25 °C.
  • D3 SAP enzyme digestion reaction: prepare the total volume of SAP enzyme Mix reaction 2*460 ⁇ l, triple-distilled water 1.53*460 ⁇ l, SAPBuffer 0.17*460 ⁇ l, SAPEnzyme 0.3*460 ⁇ l according to the following sequence, follow the following procedure, in PCR Perform SAP enzyme digestion in the instrument: 37°C for 40 min, 85°C for 5 min, and store at 25°C.
  • D4 single-base extension reaction: prepare the single-base extension reaction Mix in the following order: reaction system 2*460 ⁇ l, triple distilled water 0.619*460 ⁇ l, 10*iplexbuffer 0.2*460 ⁇ l, Terminatormix 0.2*460 ⁇ l, single Base extension probe 0.94*460 ⁇ l, single base elongase 0.041*460 ⁇ l; perform single base extension reaction in PCR machine according to the following procedure: pre-denaturation at 94°C for 30s, 94°C for 5s, 52°C for 5s, 80°C 5s, 72°C for 3min, and stored at 25°C.
  • D5 resin purification: add 16 ⁇ l of triple-distilled water to the 384-well plate of the reaction product, centrifuge at 2000 rpm in a centrifuge for 3 min; add resin, perform resin purification reaction on an inversion shaker for 35 min, and desalt; After the reaction is completed, centrifuge at 2000 rpm for 3 min in a centrifuge; place the desalted sample on the sample target and crystallize naturally.
  • D6 mass spectrometry detection and data analysis: the reaction results obtained from D1-D5 are detected on a nucleic acid flight mass spectrometer, the mass spectrometry peaks are detected by the Typer 4.0 software, and the genotype of each sample target site is interpreted according to the mass spectrometry peak map.
  • the detection of 7 SNP molecular marker primer pairs is:
  • the technical scheme utilizes the method of molecular markers to select and breed female geese with high egg qualification rate, and can quickly and accurately select and breed individuals with target traits at the gosling stage, It greatly improves the efficiency of breeding and reduces the cost of feeding.
  • Fig. 1 is the flight mass spectrometry primer chart of 7 SNP molecular markers according to the embodiment of the present invention
  • Fig. 2 is the frequency chart of 7 SNP molecular markers and haplotype modules according to the embodiment of the present invention
  • Fig. 3 is the genome-wide association analysis result of the hatching egg qualification rate trait and the genome-wide SNP site according to the embodiment of the present invention
  • FIG. 5 is the result of the flight mass spectrometry of SNP289 according to the embodiment of the present invention. Note: No call: individuals who failed the test; C: CC genotype; T: TT genotype.
  • FIG. 6 is the result of the flight mass spectrometry of SNP290 according to the embodiment of the present invention. Note: No call: individuals who failed the test; T: TT genotype; C: CC genotype.
  • FIG. 7 is the result of the flight mass spectrometry of SNP292 according to the embodiment of the present invention. Note: No call: individuals who failed the test; T: TT genotype; C: CC genotype.
  • FIG. 8 is the result of the flight mass spectrometry of SNP294 according to the embodiment of the present invention. Note: No call: individuals who failed the test; G: GG genotype; A: AA genotype.
  • FIG. 9 is the result of the flight mass spectrometry of SNP296 according to the embodiment of the present invention. Note: No call: individuals who failed the test; G: GG genotype; A: AA genotype.
  • FIG. 10 is the result of SNP297 in-flight mass spectrometry of the embodiment of the present invention. Note: No call: individuals who failed the test; A: AA genotype; T: TT genotype.
  • FIG. 11 is the result of the flight mass spectrometry of SNP298 according to the embodiment of the present invention. Note: No call: individuals who failed the test; G: GG genotype; T: TT genotype.
  • the embodiment of the present invention includes the following steps:
  • the whole genome resequencing data of Sichuan white goose was compared with the reference genome sequence, combined with the egg qualification rate data of Sichuan white goose, the 7 candidate SNP loci for obtaining the qualification rate of goose eggs were screened by genome-wide association analysis (GWAS). .
  • GWAS genome-wide association analysis
  • 7 SNP loci and 5 SNP loci were constructed to construct the distribution frequency of haplotype modules in Sichuan white goose individuals and do correlation analysis.
  • the above SNPs The loci and haplotype modules were significantly or extremely significantly correlated with the egg yield traits (p ⁇ 0.05 or p ⁇ 0.01).
  • the re-sequencing is the whole genome re-sequencing sequence of Sichuan White Goose, and the reference genome is the reference genome sequence of Sichuan White Goose.
  • the method includes the following steps:
  • D2 Perform PCR reaction on the DNA in step S1, the system is 5 ⁇ l: 10*buffer 0.5 ⁇ l, Mg 2+ 0.4 ⁇ l, dNTP 0.1 ⁇ l, Hotstar 0.2 ⁇ l, upstream and downstream primer mix 1 ⁇ l, triple distilled water 1.8 ⁇ l, DNA to be detected Sample 1 ⁇ l (20ng-50ng).
  • PCR amplification procedure pre-denaturation at 95°C for 2 min, 45 cycles (denaturation at 95°C for 30s, annealing at 56°C for 30s, extension at 72°C for 60s), extension at 72°C for 5 min, and storage at 25°C.
  • SAP enzyme digestion reaction prepare the total volume of SAP enzyme Mix reaction 2*460 ⁇ l, triple-distilled water 1.53*460 ⁇ l, SAPBuffer 0.17*460 ⁇ l, SAPEnzyme 0.3*460 ⁇ l according to the following procedure, follow the following procedure, in the PCR machine Digest with SAP enzyme in: 37 °C for 40 min, 85 °C for 5 min, and store at 25 °C.
  • D4 Single-base extension reaction: prepare a single-base extension reaction Mix in the following order: reaction system 2*460 ⁇ l, triple distilled water 0.619*460 ⁇ l, 10*iplexbuffer 0.2*460 ⁇ l, Terminatormix 0.2*460 ⁇ l, single-base extension Probe 0.94*460 ⁇ l, single base elongase 0.041*460 ⁇ l.
  • the single-base extension reaction was performed in a PCR machine according to the following procedure: pre-denaturation at 94°C for 30s, 94°C for 5s, 52°C for 5s, 80°C for 5s, 72°C for 3 min, and storage at 25°C.
  • D5 Resin purification: add 16 ⁇ l of triple-distilled water to the 384-well plate of the reaction product, centrifuge at 2000 rpm for 3 min in a centrifuge; add resin, perform resin purification reaction on an inversion shaker for 35 min, and desalt; after the reaction is completed Centrifuge at 2000 rpm for 3 min in a centrifuge; place the desalted sample on the sample target and crystallize naturally;
  • D6 Mass spectrometry detection and data analysis: The reaction results obtained from D1-D5 were detected on a nucleic acid flight mass spectrometer, the mass spectrometry peaks were detected by the Typer 4.0 software, and the genotypes of the target sites of each sample were interpreted according to the mass spectrometry peak map.
  • Sichuan white goose female geese in the egg-laying period (30-60 weeks) are used as the research animals (209), the female geese are raised in individual egg-laying cages, and the male and female geese are bred in a ratio of 1:4. Breeding is carried out, and during the period from 35 weeks to 60 weeks, the qualified rate of hatching eggs is counted every day.
  • the above 209 geese were subjected to whole-genome resequencing (the data volume of each individual was greater than 11G, and the genome coverage was greater than 10), and the whole-genome data was compared to the goose genome using BWA software, and the GATK method was used for SNP extraction and filtering. After merging, 9,279,339 SNPs and 209 individuals passed the quality control.
  • GWAS genome-wide association analysis
  • MLM Mixed linear model
  • GWAS results screened 7 SNP molecular markers related to egg qualification rate traits, as shown in Figure 2.
  • Haplotypes were constructed for the above SNP loci using Plink software.
  • the SNP loci and haplotypes above were genotyped in the goose population by the method of flight mass spectrometry.
  • the genotype and frequency of the above seven SNPs in Sichuan white goose population were detected by time-of-flight mass spectrometry.
  • five SNP loci SNP290, SNP292, SNP294, SNP296 and SNP297 were constructed into a haplotype module, as shown in Figure 3.
  • SNP SNP-specific nuclease
  • RLFP restriction endonuclease
  • KASP direct sequencing and flight mass spectrometry
  • the Sichuan white goose samples in the following examples are all from the Anfu Waterfowl Experimental Base, Rongchang District, Chongqing City, and all individuals are hatched in the same batch and raised in the same environment. After entering the breeding period, single-cage breeding was adopted, and samples of Sichuan white geese were obtained based on the record of the pass rate of breeding eggs of Sichuan white geese from 35 weeks to 60 weeks.
  • the above 209 geese were subjected to whole-genome resequencing (the data volume of each individual was greater than 11G, and the genome coverage was greater than 10), and the whole-genome data was compared to the goose genome using BWA software, and the GATK method was used for SNP extraction and filtering. After merging, 9,279,339 SNPs and 209 individuals passed the quality control.
  • the present invention performs genome-wide association analysis (GWAS) on the above-mentioned 209 geese's egg qualification rate traits and genome-wide SNP sites, and uses GEMMA software to complete the analysis based on a mixed linear model (Mixed linear model, MLM) method.
  • the GWAS results screened 7 SNP molecular markers related to egg qualification rate traits, as shown in Figure 2.
  • the whole genome DNA of the above 209 geese was selected as the experimental material, and time-of-flight mass spectrometry was performed for the above SNP loci.
  • Primer design According to the SNP site, primer design is carried out by the AssayDesign3.1 software of Agena Company, as shown in Figure 1.
  • Synthesis of primers and quality inspection Synthesize the primers synthesized by the company, and conduct quality inspection by matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF) to check whether the actual molecular weight is consistent with the theoretical molecular weight, and whether the purity of the primers meets the experimental requirements. .
  • the specific operation is: draw 2ul of each extension primer synthesized into Mix, draw 2ul from the extension primer Mix and add it to 40 ⁇ l ddH2O, carry out mass spectrometry detection, the molecular weight of the obtained peak map should be consistent with the theoretical value, no impurity peaks.
  • PCR amplification procedure pre-denaturation at 95°C for 2 min, 45 cycles (denaturation at 95°C for 30s, annealing at 56°C for 30s, extension at 72°C for 60s), extension at 72°C for 5 min, and storage at 25°C.
  • the PCR product was digested with SAP enzyme: the total volume of the SAP enzyme Mix reaction was prepared in the following order: 2*460 ⁇ l, three-distilled water 1.53*460 ⁇ l, SAPBuffer 0.17*460 ⁇ l, SAPEnzyme 0.3*460 ⁇ l, according to the following procedures, in PCR Perform SAP enzyme digestion in the instrument: 37°C for 40 min, 85°C for 5 min, and store at 25°C.
  • the product is then subjected to a single-base extension reaction: prepare a single-base extension reaction Mix in the following order: reaction system 2*460 ⁇ l, triple distilled water 0.619*460 ⁇ l, 10*iplexbuffer0.2*460 ⁇ l, Terminatormix0.2*460 ⁇ l, single Base extension probe 0.94*460 ⁇ l, single base extendase 0.041*460 ⁇ l.
  • the single-base extension reaction was performed in a PCR machine according to the following procedure: pre-denaturation at 94°C for 30s, 94°C for 5s, 52°C for 5s, 80°C for 5s, 72°C for 3 min, and storage at 25°C.
  • the reaction product was subjected to resin purification, put into a 384-well plate, added 16 ⁇ l of triple-distilled water, and centrifuged at 2000 rpm in a centrifuge for 3 min; added resin, performed a resin purification reaction on an inversion shaker for 35 min, and desalted; after the reaction was completed Centrifuge at 2000 rpm for 3 min in a centrifuge; spot the desalted sample on the sample target and crystallize naturally.
  • Mass spectrometry detection and data analysis The reaction results obtained above were detected on a nucleic acid flight mass spectrometer, the mass spectrometry peaks were detected by the Typer 4.0 software, and the genotype of each sample target site was interpreted according to the mass spectrometry peak map.
  • the qualified rate of eggs of individuals with CC genotype of SNP289 was significantly higher than that of individuals of other genotypes (p>0.05); the qualified rate of eggs of individuals of TT genotype of SNP290 was the highest, and the qualified rate of individuals of CC genotype was the lowest; Individuals with CC genotype had the highest egg qualified rate; SNP294 had the highest egg qualified rate with GG genotype; SNP296 had AA genotype significantly higher than other genotypes; SNP298 had GT genotype with significantly higher egg qualified rate than TT genotype.
  • Haplotype 1 (TTCTAGAAAT) individuals had the highest egg qualification rate, and haplotype 6 (CCTTAAGGAA) individuals had the lowest egg qualification rate.

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Abstract

The present disclosure relates to the technical field of SNP molecular marking, and provides a molecular marking method for detecting the pass rate of hatching goose eggs, comprising: obtaining whole genome SNP loci by comparing the whole genome re-sequencing data with a reference goose genome sequence, and acquiring 7 candidate SNP molecular markers of hatching goose egg pass rate traits by means of genome-wide association analysis; and detecting the polymorphism of the 7 SNP molecular markers by means of time-of-flight mass spectrometry, and counting the genotype frequency thereof. One haplotype block is also extremely significantly correlated with the egg-producing pass rate. The polymorphic SNP molecular markers and the primer pair comprise at least one of the 7 SNP molecular markers. The screening method allows for the polymorphic SNP molecular markers common to Sichuan white geese to be obtained, and provides 7 valid SNP molecular markers, enriching the library of Sichuan white geese molecular markers. Use of the method allows for identification of individual geese having a high or low hatching egg pass rate, and the time and cost of conventional goose breeding are reduced.

Description

一种检测鹅种蛋合格率的分子标记方法A molecular marker method for detecting the qualification rate of goose eggs 技术领域technical field
本发明涉及一种检测鹅种蛋合格率的分子标记方法，主要涉及分子生物学领域。The invention relates to a molecular marker method for detecting the qualification rate of goose eggs, and mainly relates to the field of molecular biology.
背景技术Background technique
单核苷酸多态性(single nucleotide polymorphism，SNP)，主要指在基因组系列上有单个核苷酸的变异所引起的DNA序列的多态性。是动植物基因组可遗传变异中最常见的一种变异，广泛存在基因组中。SNP具有突变率低、准确性和稳定性高，被广泛应用于动植物的分子标记之一。在定位疾病靶基因、动植物育种过程中个体筛选、物种进化过程的分析和亲缘关系鉴定等方面广泛的应用。Single nucleotide polymorphism (single nucleotide polymorphism, SNP), mainly refers to the polymorphism of the DNA sequence caused by the variation of a single nucleotide in the genome series. It is the most common heritable variation in the genome of animals and plants, and it exists widely in the genome. SNP has low mutation rate, high accuracy and stability, and is widely used as one of the molecular markers of animals and plants. It is widely used in locating disease target genes, individual screening in the process of animal and plant breeding, analysis of species evolution and identification of kinship relationships.
我国是养鹅最多的国家，但由于各种原因，养鹅业的发展速度较慢，尤其是规模化养鹅的效益较差，甚至出现亏损的现象，究其原因，繁殖性能低下是制约养鹅生产发展的重要因素之一。大多数鹅的繁殖具有季节性、就巢性等特点，这就导致母鹅产蛋量很低，增加了种鹅的繁殖成本，提高了商品鹅的生产成本，严重制约了产业的发展。鹅繁殖性状受环境因素影响较大，很难由表型选择得到正确的基因型，这就限制了传统数量遗传学在繁殖性状改良中的应用，使得传统选育进展甚微。分子遗传标记为解决这一棘手问题提供了良好的契机，是有效的展开高产新品种鹅分子育种的关键。my country is the country with the largest number of geese, but due to various reasons, the development of the goose industry is slow, especially the large-scale goose breeding is less efficient, and even suffers losses. One of the important factors in the development of goose production. The breeding of most geese has the characteristics of seasonality and nesting, which leads to low egg production of female geese, increases the breeding cost of breeding geese, increases the production cost of commercial geese, and seriously restricts the development of the industry. The reproductive traits of goose are greatly affected by environmental factors, and it is difficult to obtain the correct genotype by phenotypic selection, which limits the application of traditional quantitative genetics in the improvement of reproductive traits, and makes little progress in traditional breeding. Molecular genetic markers provide a good opportunity to solve this thorny problem and are the key to effective molecular breeding of new high-yield breeds of goose.
四川白鹅是季节性繁殖水禽之一，具有抗逆性强、适应性好、耐粗饲、抗病性强和耐粗饲等特点，为广大消费者提供肉、蛋、羽绒、肥肝等。在中国各地被广泛饲养并被进一步用于其他品种的改良和创制。种蛋合格率性状是重要的经济性状，也是复杂的数量性状，且均受到遗传、营养、饲养方式、外界环境等影响。利用生物学等方法进行研究，鸭体尺、鸡蛋壳颜色等分子标记被发现及广泛应用到分子标记辅助选择，极大地推进了禽类种蛋合格率等方面的研究进展。四川白鹅繁殖具有季节性，每年九月份至次年五月份产蛋，其余时间 休产。这种独特的习性导致母鹅产蛋量较低，增加了种鹅的繁殖成本，提高了商品鹅的生产成本，严重影响经济效益，制约了养鹅业的发展。而且由于种蛋合格率性状的选择是一个长时间人工选育的过程，收到选择性状、检测指标方法、成本、营养水平、性状检测技术手段以及环境等原因的作用，大大提高了对种蛋合格率性状选育的难度，同时也进一步限制了鹅产业进一步利用的可能。Sichuan White Goose is one of the seasonal breeding waterfowl. It has the characteristics of strong stress resistance, good adaptability, roughage resistance, strong disease resistance and roughage resistance. It provides meat, eggs, down, fatty liver, etc. to consumers. . Widely bred throughout China and further used for the improvement and creation of other breeds. Egg qualification rate is an important economic trait and a complex quantitative trait, which is affected by genetics, nutrition, feeding methods, and external environment. Using biological and other methods for research, molecular markers such as duck body size and egg shell color have been discovered and widely used in molecular marker-assisted selection, which has greatly promoted the research progress in the qualification rate of poultry eggs. The reproduction of Sichuan white geese is seasonal. Eggs are laid from September to May of the following year, and rest during the rest of the year. This unique habit leads to lower egg production of female geese, increases the breeding cost of breeding geese, increases the production cost of commercial geese, seriously affects economic benefits, and restricts the development of the goose breeding industry. Moreover, because the selection of the qualification rate of breeding eggs is a long-term artificial breeding process, the selection of characteristics, detection index methods, costs, nutritional levels, technical means of trait detection, and the environment have greatly improved the qualification rate of breeding eggs. The difficulty of trait breeding also further limits the possibility of further utilization in the goose industry.
鹅种蛋合格率是评定鹅繁殖力的重要指标之一，是指母鹅在70周龄以内所产的符合本品种、品系要求的种蛋数占总产蛋数的百分比。种蛋合格率直接影响鹅的孵化率和雏鹅的质量，直接增加鹅产业的经济效益，显著降低饲养成本。种蛋合格率受到遗传、环境和疾病等多方面影响。但目前尚没有相应的检测手段判断鹅种蛋合格率的高低。传统的方法耗费大量的人力、物力检测种蛋合格率，成本较大；且针对鹅产蛋合格率对应到相应母鹅进行选育难度较大。The pass rate of goose breeding eggs is one of the important indicators for evaluating goose fecundity. It refers to the percentage of eggs produced by female geese within 70 weeks of age that meet the requirements of the breed and strain in the total number of eggs produced. The qualified rate of breeding eggs directly affects the hatchability of geese and the quality of goslings, which directly increases the economic benefits of the goose industry and significantly reduces feeding costs. The qualified rate of breeding eggs is affected by many aspects such as heredity, environment and disease. But at present, there is no corresponding detection method to judge the qualification rate of goose breeding eggs. The traditional method consumes a lot of manpower and material resources to detect the qualified rate of breeding eggs, and the cost is relatively high; and it is difficult to select the corresponding female geese for the qualified rate of goose laying eggs.
发明内容SUMMARY OF THE INVENTION
针对以上现有技术的不足，本发明提出一种检测鹅种蛋合格率的分子标记方法，利用分子标记的方法对种蛋合格率较高的母鹅进行选育，能够快速且精准的在雏鹅阶段对具有目标性状的个体进行选育，大大提高了选育的效率，并减少了饲养的成本。In view of the above deficiencies of the prior art, the present invention proposes a molecular marker method for detecting the qualification rate of goose eggs. The molecular marker method is used to select and breed female geese with a higher qualification rate of eggs, which can quickly and accurately detect the qualification rate of goose eggs at the gosling stage. Breeding individuals with target traits greatly improves the efficiency of selection and reduces the cost of feeding.
为达到上述目的，本发明的技术方案是：包括如下步骤：S1：以待检测鹅的全基因组重测序数据比对到参考基因组序列，结合待检测鹅种蛋合格率数据，通过全基因组关联分析，筛选获得待测鹅蛋合格率的候选的7个SNP位点；S2：经过飞行时间质谱的技术检验7个SNP位点(SNP289，SNP290，SNP292，SNP294，SNP296，SNP297和SNP298)和1个单倍型模块(由SNP290，SNP292，SNP294，SNP296和SNP297等5个SNP构建)在四川白鹅个体中的分布频率，并与种蛋合格率性状做关联分析，结果表明上述SNP位点和单倍型模块与种蛋合格率性状显著或极显著相关。In order to achieve the above object, the technical scheme of the present invention includes the following steps: S1: compare the whole genome resequencing data of the goose to be detected to the reference genome sequence, combine the pass rate data of the goose egg to be detected, and perform a genome-wide association analysis, 7 candidate SNP loci to obtain the pass rate of the goose eggs to be tested; S2: 7 SNP loci (SNP289, SNP290, SNP292, SNP294, SNP296, SNP297 and SNP298) and 1 single The distribution frequency of the ploidy module (constructed from 5 SNPs including SNP290, SNP292, SNP294, SNP296 and SNP297) in Sichuan white goose individuals, and the correlation analysis with the egg qualification rate traits, the results show that the above SNP loci and haplotypes Modules were significantly or extremely significantly correlated with hatching egg qualification rate traits.
优选地，D1：对产蛋前期的待检测鹅母鹅的翅静脉采血，并提取全基因组DNA；D2：将步骤D1中的DNA进行PCR反应，体系5μl：10*buffer 0.5μl，Mg 2+0.4μl，dNTP 0.1μl，Hotstar 0.2μl，上下游引物混合物1μl，三蒸水1.8μl，待检测DNA样品1μl(20ng-50ng)；PCR扩增程序：预变性95℃2min，45个循环(95℃变性30s，56℃退火30s，72℃延伸60s，)72℃延伸5min，25℃保存。 Preferably, D1: collect blood from the wing veins of the goose to be tested in the early stage of egg laying, and extract the whole genome DNA; D2: perform PCR reaction on the DNA in step D1, the system is 5 μl: 10*buffer 0.5 μl, Mg 2+ 0.4 μl, 0.1 μl of dNTP, 0.2 μl of Hotstar, 1 μl of upstream and downstream primer mix, 1.8 μl of triple distilled water, 1 μl of DNA sample to be detected (20ng-50ng); PCR amplification program: pre-denaturation at 95°C for 2 min, 45 cycles (95 Denaturation at ℃ for 30 s, annealing at 56 ℃ for 30 s, extension at 72 ℃ for 60 s, extension at 72 ℃ for 5 min, and storage at 25 ℃.
优选地，D3：SAP酶消化反应：按照下述顺序配制SAP酶Mix反应物总体积2*460μl，三蒸水1.53*460μl，SAPBuffer 0.17*460μl，SAPEnzyme 0.3*460μl，按照下述程序，在PCR仪中进行SAP酶消化：37℃40min，85℃5min，25℃保存。Preferably, D3: SAP enzyme digestion reaction: prepare the total volume of SAP enzyme Mix reaction 2*460μl, triple-distilled water 1.53*460μl, SAPBuffer 0.17*460μl, SAPEnzyme 0.3*460μl according to the following sequence, follow the following procedure, in PCR Perform SAP enzyme digestion in the instrument: 37°C for 40 min, 85°C for 5 min, and store at 25°C.
优选地，D4：单碱基延伸反应：按照下述顺序配制单碱基延伸反应Mix：反应物体系2*460μl，三蒸水0.619*460μl，10*iplexbuffer 0.2*460μl，Terminatormix 0.2*460μl，单碱基延伸探针0.94*460μl，单碱基延伸酶0.041*460μl；按照下述程序，在PCR仪中进行单碱基延伸反应：预变性94℃30s，94℃5s，52℃5s，80℃5s，72℃3min，25℃保存。Preferably, D4: single-base extension reaction: prepare the single-base extension reaction Mix in the following order: reaction system 2*460μl, triple distilled water 0.619*460μl, 10*iplexbuffer 0.2*460μl, Terminatormix 0.2*460μl, single Base extension probe 0.94*460μl, single base elongase 0.041*460μl; perform single base extension reaction in PCR machine according to the following procedure: pre-denaturation at 94°C for 30s, 94°C for 5s, 52°C for 5s, 80°C 5s, 72°C for 3min, and stored at 25°C.
优选地，D5：树脂纯化：将反应产物的384孔板中加入16μl三蒸水，在离心机中离心2000转离心3min；加入树脂，在反转摇匀仪上做树脂纯化反应35min，脱盐；反应完成后在离心机中离心2000转离心3min；将脱盐处理后的样品点在样品靶上，自然结晶。Preferably, D5: resin purification: add 16 μl of triple-distilled water to the 384-well plate of the reaction product, centrifuge at 2000 rpm in a centrifuge for 3 min; add resin, perform resin purification reaction on an inversion shaker for 35 min, and desalt; After the reaction is completed, centrifuge at 2000 rpm for 3 min in a centrifuge; place the desalted sample on the sample target and crystallize naturally.
优选地，D6：质谱检测及数据分析：将D1-D5所得的反应结果在核酸飞行质谱仪进行检测，利用Typer4.0软件检测质谱峰，并根据质谱峰图判读各样本目标位点基因型。Preferably, D6: mass spectrometry detection and data analysis: the reaction results obtained from D1-D5 are detected on a nucleic acid flight mass spectrometer, the mass spectrometry peaks are detected by the Typer 4.0 software, and the genotype of each sample target site is interpreted according to the mass spectrometry peak map.
优选地，检测7个SNP分子标记引物对为：Preferably, the detection of 7 SNP molecular marker primer pairs is:
Figure PCTCN2021106541-appb-000001
Figure PCTCN2021106541-appb-000001
本发明的技术原理及有益效果如下：本技术方案利用分子标记的方法对种蛋合格率较高的母鹅进行选育，能够快速且精准的在雏鹅阶段对具有目标性状的个体进行选育，大大提高了选育的效率，并减少了饲养的成本。The technical principle and beneficial effects of the present invention are as follows: the technical scheme utilizes the method of molecular markers to select and breed female geese with high egg qualification rate, and can quickly and accurately select and breed individuals with target traits at the gosling stage, It greatly improves the efficiency of breeding and reduces the cost of feeding.
附图说明Description of drawings
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅 仅是本发明的其中11幅，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。In order to illustrate the embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings used in the description of the embodiments. Obviously, the drawings in the following description are only for the present invention. For 11 of them, for those of ordinary skill in the art, other drawings can also be obtained from these drawings without any creative effort.
图1为本发明实施例的7个SNP分子标记的飞行质谱引物图表；Fig. 1 is the flight mass spectrometry primer chart of 7 SNP molecular markers according to the embodiment of the present invention;
图2为本发明实施例的7个SNP分子标记的及单倍型模块的频率图表；Fig. 2 is the frequency chart of 7 SNP molecular markers and haplotype modules according to the embodiment of the present invention;
图3为本发明实施例的种蛋合格率性状与全基因组SNP位点的全基因组关联分析结果；Fig. 3 is the genome-wide association analysis result of the hatching egg qualification rate trait and the genome-wide SNP site according to the embodiment of the present invention;
图4为本发明实施例的位于染色体1的紧密连锁的单倍型图；4 is a closely linked haplotype map located on chromosome 1 according to an embodiment of the present invention;
图5为本发明实施例的SNP289飞行质谱的结果。注：No call：检测失败个体；C：CC基因型；T：TT基因型。FIG. 5 is the result of the flight mass spectrometry of SNP289 according to the embodiment of the present invention. Note: No call: individuals who failed the test; C: CC genotype; T: TT genotype.
图6为本发明实施例的SNP290飞行质谱的结果。注：No call：检测失败个体；T：TT基因型；C：CC基因型。FIG. 6 is the result of the flight mass spectrometry of SNP290 according to the embodiment of the present invention. Note: No call: individuals who failed the test; T: TT genotype; C: CC genotype.
图7为本发明实施例的SNP292飞行质谱的结果。注：No call：检测失败个体；T：TT基因型；C：CC基因型。FIG. 7 is the result of the flight mass spectrometry of SNP292 according to the embodiment of the present invention. Note: No call: individuals who failed the test; T: TT genotype; C: CC genotype.
图8为本发明实施例的SNP294飞行质谱的结果。注：No call：检测失败个体；G：GG基因型；A：AA基因型。FIG. 8 is the result of the flight mass spectrometry of SNP294 according to the embodiment of the present invention. Note: No call: individuals who failed the test; G: GG genotype; A: AA genotype.
图9为本发明实施例的SNP296飞行质谱的结果。注：No call：检测失败个体；G：GG基因型；A：AA基因型。FIG. 9 is the result of the flight mass spectrometry of SNP296 according to the embodiment of the present invention. Note: No call: individuals who failed the test; G: GG genotype; A: AA genotype.
图10为本发明实施例的SNP297飞行质谱的结果。注：No call：检测失败个体；A：AA基因型；T：TT基因型。FIG. 10 is the result of SNP297 in-flight mass spectrometry of the embodiment of the present invention. Note: No call: individuals who failed the test; A: AA genotype; T: TT genotype.
图11为本发明实施例的SNP298飞行质谱的结果。注：No call：检测失败个体；G：GG基因型；T：TT基因型。FIG. 11 is the result of the flight mass spectrometry of SNP298 according to the embodiment of the present invention. Note: No call: individuals who failed the test; G: GG genotype; T: TT genotype.
具体实施方式Detailed ways
下面将结合附图，对本发明中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明的较佳实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only preferred embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.
实施例Example
如图1所示，本发明实施例包括以下步骤：As shown in Figure 1, the embodiment of the present invention includes the following steps:
四川白鹅的全基因组重测序数据比对到参考基因组序列，结合四川白鹅种蛋合格率数据，通过全基因组关联分析(GWAS)的方法，筛选获得鹅种蛋合格率的候选的7个SNP位点。经过飞行时间质谱的技术检验7个SNP位点和5个SNP位点(SNP290，SNP292，SNP294，SNP296和SNP297)构建单倍型模块在四川白鹅个体中的分布频率并且做关联分析，上述SNP位点和单倍型模块与种蛋合格率性状显著或极显著相关(p<0.05或p<0.01)。The whole genome resequencing data of Sichuan white goose was compared with the reference genome sequence, combined with the egg qualification rate data of Sichuan white goose, the 7 candidate SNP loci for obtaining the qualification rate of goose eggs were screened by genome-wide association analysis (GWAS). . After the technology of time-of-flight mass spectrometry, 7 SNP loci and 5 SNP loci (SNP290, SNP292, SNP294, SNP296 and SNP297) were constructed to construct the distribution frequency of haplotype modules in Sichuan white goose individuals and do correlation analysis. The above SNPs The loci and haplotype modules were significantly or extremely significantly correlated with the egg yield traits (p<0.05 or p<0.01).
检测7个SNP分子标记引物如图1所示：飞行质谱引物The detection of 7 SNP molecular marker primers is shown in Figure 1: Flight mass spectrometry primers
所述重测序为四川白鹅全基因组重测序序列，所述参考基因组为四川白鹅参考基因组序列。所述的方法包括以下步骤：The re-sequencing is the whole genome re-sequencing sequence of Sichuan White Goose, and the reference genome is the reference genome sequence of Sichuan White Goose. The method includes the following steps:
D1：对产蛋前期的四川白鹅母鹅的翅静脉采血，并提取全基因组DNA；D1: Blood was collected from the wing veins of the Sichuan white goose female goose in the early stage of laying, and the whole genome DNA was extracted;
D2：将步骤S1中的DNA进行PCR反应，体系5μl：10*buffer 0.5μl，Mg 2+0.4μl，dNTP 0.1μl，Hotstar 0.2μl，上下游引物混合物1μl，三蒸水1.8μl，待检测DNA样品1μl(20ng-50ng)。PCR扩增程序：预变性95℃2min，45个循环(95℃变性30s，56℃退火30s，72℃延伸60s)72℃延伸5min，25℃保存。 D2: Perform PCR reaction on the DNA in step S1, the system is 5 μl: 10*buffer 0.5 μl, Mg 2+ 0.4 μl, dNTP 0.1 μl, Hotstar 0.2 μl, upstream and downstream primer mix 1 μl, triple distilled water 1.8 μl, DNA to be detected Sample 1 μl (20ng-50ng). PCR amplification procedure: pre-denaturation at 95°C for 2 min, 45 cycles (denaturation at 95°C for 30s, annealing at 56°C for 30s, extension at 72°C for 60s), extension at 72°C for 5 min, and storage at 25°C.
D3：SAP酶消化反应：按照下述顺序配制SAP酶Mix反应物总体积2*460μl，三蒸水1.53*460μl，SAPBuffer0.17*460μl，SAPEnzyme0.3*460μl，按照下述程序，在PCR仪中进行SAP酶消化：37℃40min，85℃5min，25℃保存。D3: SAP enzyme digestion reaction: prepare the total volume of SAP enzyme Mix reaction 2*460μl, triple-distilled water 1.53*460μl, SAPBuffer 0.17*460μl, SAPEnzyme 0.3*460μl according to the following procedure, follow the following procedure, in the PCR machine Digest with SAP enzyme in: 37 °C for 40 min, 85 °C for 5 min, and store at 25 °C.
D4：单碱基延伸反应：按照下述顺序配制单碱基延伸反应Mix：反应物体系2*460μl，三蒸水0.619*460μl，10*iplexbuffer 0.2*460μl，Terminatormix 0.2*460μl，单碱基延伸探针0.94*460μl，单碱基延伸酶0.041*460μl。按照下述程序，在PCR仪中进行单碱基延伸反应：预变性94℃30s，94℃5s，52℃5s，80℃5s，72℃3min，25℃保存。D4: Single-base extension reaction: prepare a single-base extension reaction Mix in the following order: reaction system 2*460μl, triple distilled water 0.619*460μl, 10*iplexbuffer 0.2*460μl, Terminatormix 0.2*460μl, single-base extension Probe 0.94*460μl, single base elongase 0.041*460μl. The single-base extension reaction was performed in a PCR machine according to the following procedure: pre-denaturation at 94°C for 30s, 94°C for 5s, 52°C for 5s, 80°C for 5s, 72°C for 3 min, and storage at 25°C.
D5：树脂纯化:将反应产物的384孔板中加入16μl三蒸水，在离心机中离心2000转离心3min；加入树脂，在反转摇匀仪上做树脂纯化反应35min，脱盐；反应完成后在离心机中离心2000转离心3min；将脱盐处理后的样品点在样品靶上，自然结晶；D5: Resin purification: add 16 μl of triple-distilled water to the 384-well plate of the reaction product, centrifuge at 2000 rpm for 3 min in a centrifuge; add resin, perform resin purification reaction on an inversion shaker for 35 min, and desalt; after the reaction is completed Centrifuge at 2000 rpm for 3 min in a centrifuge; place the desalted sample on the sample target and crystallize naturally;
D6：质谱检测及数据分析：将D1-D5所得的反应结果在核酸飞行质谱仪进行检测，利用Typer4.0软件检测质谱峰，并根据质谱峰图判读各样本目标位点基因型。D6: Mass spectrometry detection and data analysis: The reaction results obtained from D1-D5 were detected on a nucleic acid flight mass spectrometer, the mass spectrometry peaks were detected by the Typer 4.0 software, and the genotypes of the target sites of each sample were interpreted according to the mass spectrometry peak map.
本项目以处于产蛋期(30-60周)四川白鹅母鹅为研究动物(209只)，将母鹅在个体产蛋笼中进行饲养，并以1：4比例进行公鹅和母鹅进行配种，35周至60周期间，每天统计种蛋合格率。In this project, Sichuan white goose female geese in the egg-laying period (30-60 weeks) are used as the research animals (209), the female geese are raised in individual egg-laying cages, and the male and female geese are bred in a ratio of 1:4. Breeding is carried out, and during the period from 35 weeks to 60 weeks, the qualified rate of hatching eggs is counted every day.
将上述209只鹅进行全基因组重测序(每个个体数据量大于11G，基因组覆盖率大于10)，利用BWA软件将全基因组数据比对到鹅基因组上，并利用GATK的方法进行SNP提取、过滤合并后，获得有9,279,339个SNP和209个个体通过质控。The above 209 geese were subjected to whole-genome resequencing (the data volume of each individual was greater than 11G, and the genome coverage was greater than 10), and the whole-genome data was compared to the goose genome using BWA software, and the GATK method was used for SNP extraction and filtering. After merging, 9,279,339 SNPs and 209 individuals passed the quality control.
本发明对上述209只鹅的种蛋合格率性状与全进行SNP位点进行全基因组关联分析(GWAS)，并使用GEMMA软件基于混合线性模型(Mixedlinearmodel,MLM)的方法完成该分析，混合线性模型如下：利用GENNA软件利用线性公式进行全基因组关联分析(GWAS)，y＝Wα+xβ+ε,其中α为协变量矩阵(W)相对应的系数向量，β为SNP的效应大小，ε表示随机残差。GWAS结果分别筛选到7个种蛋合格率性状相关SNP分子标记，如图2。In the present invention, genome-wide association analysis (GWAS) is performed on the above-mentioned 209 geese's egg qualification rate traits and SNP sites, and GEMMA software is used to complete the analysis based on the mixed linear model (Mixed linear model, MLM). The mixed linear model is as follows : Genome-wide association analysis (GWAS) using linear formula with GENNA software, y=Wα+xβ+ε, where α is the coefficient vector corresponding to the covariate matrix (W), β is the effect size of the SNP, ε is the random residual Difference. The GWAS results screened 7 SNP molecular markers related to egg qualification rate traits, as shown in Figure 2.
利用Plink软件针对上述SNP位点进行单倍型构建。利用飞行质谱的方法检验上述SNP位点和单位型在鹅群体中进行基因分型。利用飞行时间质谱技术对上述7个SNP在四川白鹅群体中基因型和频率的检测。值得注意的是，5个SNP位点(SNP290，SNP292，SNP294，SNP296和SNP297)构建到一个单倍型模块，如图3。Haplotypes were constructed for the above SNP loci using Plink software. The SNP loci and haplotypes above were genotyped in the goose population by the method of flight mass spectrometry. The genotype and frequency of the above seven SNPs in Sichuan white goose population were detected by time-of-flight mass spectrometry. Notably, five SNP loci (SNP290, SNP292, SNP294, SNP296 and SNP297) were constructed into a haplotype module, as shown in Figure 3.
检测SNP的方式非常多，限制性内切酶、RLFP、KASP、直接测序以及飞行质谱等方法，本发明主要提供7个种蛋合格率的分子标记，具体的检测方式可以根据实际情况而掌握。There are many ways to detect SNP, such as restriction endonuclease, RLFP, KASP, direct sequencing and flight mass spectrometry.
除非特别说明，本发明所用试剂和材料均为市购。Unless otherwise specified, the reagents and materials used in the present invention are commercially available.
以下实施例中的四川白鹅样品均来自重庆市荣昌区安富水禽实验基地，且所有个体均为同一批次孵化，在相同环境下饲养。进入育成期后，采用单笼养殖，基于记录四川白鹅35周-60周种蛋合格率得到四川白鹅样本。The Sichuan white goose samples in the following examples are all from the Anfu Waterfowl Experimental Base, Rongchang District, Chongqing City, and all individuals are hatched in the same batch and raised in the same environment. After entering the breeding period, single-cage breeding was adopted, and samples of Sichuan white geese were obtained based on the record of the pass rate of breeding eggs of Sichuan white geese from 35 weeks to 60 weeks.
实施例1：Example 1:
(1)种蛋合格率全基因组关联分析(1) Genome-wide association analysis of egg qualification rate
全基因组关联分析：Genome-wide association analysis:
四川白鹅20周龄时利用含有肝素钠的真空采血管采集翅静脉2mL血液，利用血液基因组提取试剂盒(北京天根公司，DP332)提取血液基因组DNA，利 用电泳实验和NanoDrop2000分光光度计测量DNA原液浓度和完整性，并将其溶解在TE溶液中在-20℃保存备用。利用干冰运输的方式将基因组DNA送往北京诺禾致源科技股份有限公司进行全基因组重测序。At the age of 20 weeks, 2 mL of blood from the fin vein was collected from a vacuum blood collection tube containing sodium heparin, and the blood genomic DNA was extracted using a blood genome extraction kit (Beijing Tiangen Company, DP332), and the DNA was measured by electrophoresis experiment and NanoDrop2000 spectrophotometer. The concentration and integrity of the stock solution were dissolved in TE solution and stored at -20°C for later use. The genomic DNA was sent to Beijing Nuohezhiyuan Technology Co., Ltd. for whole-genome resequencing by means of dry ice transportation.
四川白鹅20周龄时利用含有肝素钠的真空采血管采集翅静脉2mL血液，利用血液基因组提取试剂盒(北京天根公司，DP332)提取血液基因组DNA，利用电泳实验和NanoDrop2000分光光度计测量DNA原液浓度和完整性，并将其溶解在TE溶液中在-20℃保存备用。利用干冰运输的方式将基因组DNA送往北京诺禾致源科技股份有限公司进行全基因组重测序。At the age of 20 weeks, 2 mL of blood from the fin vein was collected from a vacuum blood collection tube containing sodium heparin, and the blood genomic DNA was extracted using a blood genome extraction kit (Beijing Tiangen Company, DP332), and the DNA was measured by electrophoresis experiment and NanoDrop2000 spectrophotometer. The concentration and integrity of the stock solution were dissolved in TE solution and stored at -20°C for later use. The genomic DNA was sent to Beijing Nuohezhiyuan Technology Co., Ltd. for whole-genome resequencing by means of dry ice transportation.
将上述209只鹅进行全基因组重测序(每个个体数据量大于11G，基因组覆盖率大于10)，利用BWA软件将全基因组数据比对到鹅基因组上，并利用GATK的方法进行SNP提取、过滤合并后，获得有9,279,339个SNP和209个个体通过质控。The above 209 geese were subjected to whole-genome resequencing (the data volume of each individual was greater than 11G, and the genome coverage was greater than 10), and the whole-genome data was compared to the goose genome using BWA software, and the GATK method was used for SNP extraction and filtering. After merging, 9,279,339 SNPs and 209 individuals passed the quality control.
本发明对上述209只鹅的种蛋合格率性状与全基因组SNP位点进行全基因组关联分析(GWAS)，并使用GEMMA软件基于混合线性模型(Mixedlinearmodel,MLM)的方法完成该分析，混合线性模型如下：利用GEMMA软件利用线性公式进行全基因组关联分析(GWAS)，y＝Wα+xβ+ε,其中α为协变量矩阵(W)相对应的系数向量，β为SNP的效应大小，ε表示随机残差。GWAS结果分别筛选到7个种蛋合格率性状相关SNP分子标记，如图2。The present invention performs genome-wide association analysis (GWAS) on the above-mentioned 209 geese's egg qualification rate traits and genome-wide SNP sites, and uses GEMMA software to complete the analysis based on a mixed linear model (Mixed linear model, MLM) method. The mixed linear model is as follows : Genome-wide association analysis (GWAS) using linear formula using GEMMA software, y=Wα+xβ+ε, where α is the coefficient vector corresponding to the covariate matrix (W), β is the effect size of the SNP, and ε is the random residual Difference. The GWAS results screened 7 SNP molecular markers related to egg qualification rate traits, as shown in Figure 2.
(2)飞行时间质谱方法检测种蛋合格率候选SNP在四川白鹅中分布频率(2) Time-of-flight mass spectrometry method to detect the distribution frequency of candidate SNPs for egg qualification rate in Sichuan white geese
选择上述209只鹅全基因组DNA为实验材料，针对上述SNP位点进行飞行时间质谱验证。The whole genome DNA of the above 209 geese was selected as the experimental material, and time-of-flight mass spectrometry was performed for the above SNP loci.
1、引物设计：根据SNP位点通过Agena公司的AssayDesign3.1软件进行引物设计，如图1所示。1. Primer design: According to the SNP site, primer design is carried out by the AssayDesign3.1 software of Agena Company, as shown in Figure 1.
2、合成引物及质检：将引物合成公司合成的引物，通过基质辅助激光解吸电离飞行时间质谱仪(MALDI-TOF)进行质检，检测实际分子量与理论分子量是否一致，引物纯度是否达到实验要求。具体操作是：将合成的每条延伸引物吸取2ul配成Mix，从延伸引物Mix中吸取2ul加到40μl ddH2O中，进行质谱检测，所得峰图分子量应与理论值一致，无杂峰。2. Synthesis of primers and quality inspection: Synthesize the primers synthesized by the company, and conduct quality inspection by matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF) to check whether the actual molecular weight is consistent with the theoretical molecular weight, and whether the purity of the primers meets the experimental requirements. . The specific operation is: draw 2ul of each extension primer synthesized into Mix, draw 2ul from the extension primer Mix and add it to 40μl ddH2O, carry out mass spectrometry detection, the molecular weight of the obtained peak map should be consistent with the theoretical value, no impurity peaks.
3、样品提取及浓度纯度质检：通过琼脂糖凝胶电泳检测DNA浓度、纯度 及降解程度，检测结果判读标准：电泳检测胶图中基因组条带单一、清晰、无杂质、无弥散拖尾现象。3. Sample extraction and quality inspection of concentration and purity: DNA concentration, purity and degree of degradation are detected by agarose gel electrophoresis. Standard for the interpretation of test results: single, clear, free of impurities and no diffusion tailing phenomenon in the genomic band in the gel image detected by electrophoresis .
4、将上述全基因组DNA进行PCR反应，体系5μl：10*buffer 0.5μl，Mg 2+ 0.4μl，dNTP 0.1μl，Hotstar 0.2μl，上下游引物混合物1μl，三蒸水1.8μl，待检测DNA样品1μl(20ng-50ng)。PCR扩增程序：预变性95℃2min，45个循环(95℃变性30s，56℃退火30s，72℃延伸60s，)72℃延伸5min，25℃保存。 4. Carry out the PCR reaction of the above whole genome DNA, the system is 5 μl: 10*buffer 0.5 μl, Mg 2+ 0.4 μl, dNTP 0.1 μl, Hotstar 0.2 μl, upstream and downstream primer mix 1 μl, triple distilled water 1.8 μl, DNA sample to be detected 1 μl (20ng-50ng). PCR amplification procedure: pre-denaturation at 95°C for 2 min, 45 cycles (denaturation at 95°C for 30s, annealing at 56°C for 30s, extension at 72°C for 60s), extension at 72°C for 5 min, and storage at 25°C.
PCR产物进行SAP酶消化反应：按照下述顺序配制SAP酶Mix反应物总体积2*460μl，三蒸水1.53*460μl，SAPBuffer0.17*460μl，SAPEnzyme0.3*460μl，按照下述程序，在PCR仪中进行SAP酶消化：37℃40min，85℃5min，25℃保存。The PCR product was digested with SAP enzyme: the total volume of the SAP enzyme Mix reaction was prepared in the following order: 2*460μl, three-distilled water 1.53*460μl, SAPBuffer 0.17*460μl, SAPEnzyme 0.3*460μl, according to the following procedures, in PCR Perform SAP enzyme digestion in the instrument: 37°C for 40 min, 85°C for 5 min, and store at 25°C.
产物随后进行单碱基延伸反应：按照下述顺序配制单碱基延伸反应Mix：反应物体系2*460μl，三蒸水0.619*460μl，10*iplexbuffer0.2*460μl，Terminatormix0.2*460μl，单碱基延伸探针0.94*460μl，单碱基延伸酶0.041*460μl。按照下述程序，在PCR仪中进行单碱基延伸反应：预变性94℃30s，94℃5s，52℃5s，80℃5s，72℃3min，25℃保存。The product is then subjected to a single-base extension reaction: prepare a single-base extension reaction Mix in the following order: reaction system 2*460μl, triple distilled water 0.619*460μl, 10*iplexbuffer0.2*460μl, Terminatormix0.2*460μl, single Base extension probe 0.94*460μl, single base extendase 0.041*460μl. The single-base extension reaction was performed in a PCR machine according to the following procedure: pre-denaturation at 94°C for 30s, 94°C for 5s, 52°C for 5s, 80°C for 5s, 72°C for 3 min, and storage at 25°C.
将反应产物进行树脂纯化，放入384孔板中加入16μl三蒸水，在离心机中离心2000转离心3min；加入树脂，在反转摇匀仪上做树脂纯化反应35min，脱盐；反应完成后在离心机中离心2000转离心3min；将脱盐处理后的样品点在样品靶上，自然结晶。The reaction product was subjected to resin purification, put into a 384-well plate, added 16 μl of triple-distilled water, and centrifuged at 2000 rpm in a centrifuge for 3 min; added resin, performed a resin purification reaction on an inversion shaker for 35 min, and desalted; after the reaction was completed Centrifuge at 2000 rpm for 3 min in a centrifuge; spot the desalted sample on the sample target and crystallize naturally.
质谱检测及数据分析：将上述所得的反应结果在核酸飞行质谱仪进行检测，利用Typer4.0软件检测质谱峰，并根据质谱峰图判读各样本目标位点基因型。Mass spectrometry detection and data analysis: The reaction results obtained above were detected on a nucleic acid flight mass spectrometer, the mass spectrometry peaks were detected by the Typer 4.0 software, and the genotype of each sample target site was interpreted according to the mass spectrometry peak map.
结果判读：Interpretation of results:
如图2所示，SNP289的CC基因型个体种蛋合格率显著高于其他基因型个体(p>0.05)；SNP290的TT基因型个体种蛋合格率最高，CC基因型个体种蛋合格率最低；SNP292的CC基因型个体种蛋合格率最高；SNP294的GG基因型个体种蛋合格率最高；SNP296的AA基因型显著高于其他基因型；SNP298的GT基因型个体种蛋合格率显著高于TT基因型。单倍型1(TTCTAGAAAT)个体种蛋合格率最高，单倍型6(CCTTAAGGAA)个体种蛋合格率最低。As shown in Figure 2, the qualified rate of eggs of individuals with CC genotype of SNP289 was significantly higher than that of individuals of other genotypes (p>0.05); the qualified rate of eggs of individuals of TT genotype of SNP290 was the highest, and the qualified rate of individuals of CC genotype was the lowest; Individuals with CC genotype had the highest egg qualified rate; SNP294 had the highest egg qualified rate with GG genotype; SNP296 had AA genotype significantly higher than other genotypes; SNP298 had GT genotype with significantly higher egg qualified rate than TT genotype. Haplotype 1 (TTCTAGAAAT) individuals had the highest egg qualification rate, and haplotype 6 (CCTTAAGGAA) individuals had the lowest egg qualification rate.
可以根据实际情况和需求，对具有上述基因型或单倍型个体进行保留或被淘汰。Individuals with the above genotypes or haplotypes can be retained or eliminated according to actual conditions and needs.
以上所述仅为本发明针对四川白鹅的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。The above is only a preferred embodiment of the present invention for Sichuan White Goose, and is not intended to limit the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall include within the protection scope of the present invention.

Claims (7)

  1. 检测鹅种蛋合格率的分子标记方法，其特征在于，包括如下步骤：The molecular marker method for detecting the qualification rate of goose breeding eggs, is characterized in that, comprises the steps:
    S1：以待检测鹅的全基因组重测序数据比对到参考基因组序列，结合待检测鹅种蛋合格率数据，通过全基因组关联分析，筛选获得待测鹅蛋合格率的候选的7个SNP位点(SNP289，SNP290，SNP292，SNP294，SNP296，SNP297和SNP298)；S1: Compare the whole-genome resequencing data of the goose to be tested with the reference genome sequence, combine the data of the pass rate of the goose eggs to be tested, and screen the 7 candidate SNP loci to obtain the pass rate of the goose eggs to be tested through genome-wide association analysis (SNP289, SNP290, SNP292, SNP294, SNP296, SNP297 and SNP298);
    S2：经过飞行时间质谱的技术检验7个SNP位点和1个单倍型模块(由SNP290，SNP292，SNP294，SNP296和SNP297构建)在四川白鹅个体中的分布频率，并且做关联分析，结果显示上述7个SNP分子标记和单倍型模块与种蛋合格率性状显著或极显著相关。S2: The frequency of distribution of 7 SNP loci and 1 haplotype module (constructed by SNP290, SNP292, SNP294, SNP296 and SNP297) in Sichuan white goose individuals was tested by time-of-flight mass spectrometry, and correlation analysis was performed. It was shown that the above 7 SNP molecular markers and haplotype modules were significantly or extremely significantly correlated with hatching egg qualification rate traits.
  2. 根据权利要求1所述的检测鹅种蛋合格率的分子标记方法，其特征在于：The molecular marker method of detecting the qualification rate of goose eggs according to claim 1, is characterized in that:
    D1：对产蛋前期的待检测鹅母鹅的翅静脉采血，并提取全基因组DNA；D1: Collect blood from the wing veins of the goose and female goose to be tested in the early stage of egg laying, and extract the whole genome DNA;
    D2：将步骤D1中的DNA进行PCR反应，体系5μl：10*buffer0.5μl，Mg 2+0.4μl，dNTP 0.1μl，Hotstar 0.2μl，上下游引物混合物1μl，三蒸水1.8μl，待检测DNA样品1μl(20ng-50ng)；PCR扩增程序：预变性95℃2min，45个循环(95℃变性30s，56℃退火30s，72℃延伸60s，)72℃延伸5min，25℃保存。 D2: PCR reaction of DNA in step D1, 5 μl of system: 10*buffer 0.5 μl, Mg 2+ 0.4 μl, dNTP 0.1 μl, Hotstar 0.2 μl, upstream and downstream primer mix 1 μl, triple distilled water 1.8 μl, DNA to be detected 1 μl of sample (20ng-50ng); PCR amplification program: pre-denaturation at 95°C for 2 min, 45 cycles (denaturation at 95°C for 30s, annealing at 56°C for 30s, extension at 72°C for 60s), extension at 72°C for 5min, and storage at 25°C.
  3. 根据权利要求2所述的检测鹅种蛋合格率的分子标记方法，其特征在于：D3：SAP酶消化反应：The molecular marker method for detecting the qualification rate of goose eggs according to claim 2, is characterized in that: D3: SAP enzyme digestion reaction:
    按照下述顺序配制SAP酶Mix反应物总体积2*460μl，三蒸水1.53*460μl，SAPBuffer0.17*460μl，SAPEnzyme 0.3*460μl，按照下述程序，在PCR仪中进行SAP酶消化：37℃40min，85℃5min，25℃保存。Prepare the total volume of SAP enzyme Mix reaction volume 2*460μl, triple distilled water 1.53*460μl, SAPBuffer 0.17*460μl, SAPEnzyme 0.3*460μl in the following order, and perform SAP enzyme digestion in PCR machine according to the following procedure: 37℃ 40min, 5min at 85°C, and stored at 25°C.
  4. 根据权利要求3所述的检测鹅种蛋合格率的分子标记方法，其特征在于：D4：单碱基延伸反应：The molecular marker method for detecting the qualification rate of goose eggs according to claim 3, is characterized in that: D4: single base extension reaction:
    按照下述顺序配制单碱基延伸反应Mix：反应物体系2*460μl，三蒸水0.619*460μl，10*iplexbuffer 0.2*460μl，Terminatormix 0.2*460μl，单碱基延伸探针0.94*460μl，单碱基延伸酶0.041*460μl；按照下述程序，在PCR仪中进行单碱基延伸反应：预变性94℃30s，94℃5s，52℃5s，80℃5s，72℃3min，25℃保存。Prepare single base extension reaction Mix in the following order: reaction system 2*460μl, triple distilled water 0.619*460μl, 10*iplexbuffer 0.2*460μl, Terminatormix 0.2*460μl, single base extension probe 0.94*460μl, single base Base elongase 0.041*460 μl; carry out single-base extension reaction in a PCR machine according to the following procedure: pre-denaturation at 94°C for 30s, 94°C for 5s, 52°C for 5s, 80°C for 5s, 72°C for 3 min, and store at 25°C.
  5. 根据权利要求4所述的检测鹅种蛋合格率的分子标记方法，其特征在于：D6：树脂纯化：The molecular marker method for detecting the qualification rate of goose eggs according to claim 4, is characterized in that: D6: resin purification:
    将反应产物的384孔板中加入16μl三蒸水，在离心机中离心2000转离心 3min；加入树脂，在反转摇匀仪上做树脂纯化反应35min，脱盐；反应完成后在离心机中离心2000转离心3min；将脱盐处理后的样品点在样品靶上，自然结晶。Add 16 μl of triple-distilled water to the 384-well plate of the reaction product, centrifuge at 2000 rpm for 3 minutes in a centrifuge; add resin, perform resin purification reaction on a reversal shaker for 35 minutes, and desalt; after the reaction is completed, centrifuge in a centrifuge Centrifuge at 2000 rpm for 3 min; place the desalted sample on the sample target and crystallize naturally.
  6. 根据权利要求5所述的检测鹅种蛋合格率的分子标记方法，其特征在于：D7：质谱检测及数据分析：The molecular marker method for detecting the qualification rate of goose eggs according to claim 5, is characterized in that: D7: mass spectrometry detection and data analysis:
    将D1-D5所得的反应结果在核酸飞行质谱仪进行检测，利用Typer4.0软件检测质谱峰，并根据质谱峰图判读各样本目标位点基因型。The reaction results obtained from D1-D5 were detected by a nucleic acid flight mass spectrometer, and the mass spectrometry peaks were detected by the Typer 4.0 software, and the genotypes of the target sites of each sample were interpreted according to the mass spectrometry peaks.
  7. 根据权利要求1所述的检测鹅种蛋合格率的分子标记方法，其特征在于：The molecular marker method of detecting the qualification rate of goose eggs according to claim 1, is characterized in that:
    检测7个SNP分子标记的引物对为：The primer pairs for detecting 7 SNP molecular markers are:
    Figure PCTCN2021106541-appb-100001
    Figure PCTCN2021106541-appb-100001
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CN112430674B (en) * 2020-12-23 2023-08-11 重庆市畜牧科学院 Primer for detecting 7 SNP molecular markers of goose egg qualification rate
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