CN112430674A - Molecular marking method for detecting goose egg qualification rate - Google Patents
Molecular marking method for detecting goose egg qualification rate Download PDFInfo
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Abstract
The invention relates to the technical field of SNP molecular markers, and provides a molecular marker method for detecting the qualification rate of goose eggs, which comprises the steps of obtaining a whole genome SNP locus by comparing whole genome re-sequencing data to a goose reference genome sequence, and obtaining 7 candidate SNP molecular markers of the qualification rate character of the goose eggs by a whole genome association analysis method; detecting the polymorphism of 7 SNP molecular markers by using a time-of-flight mass spectrometry method and counting the genotype frequency of the polymorphism. The 1 haplotype block was also very significantly associated with egg yield. The polymorphic SNP molecular markers and the primer pairs comprise at least one of 7 SNP molecular markers. The polymorphic SNP molecular marker universal for Sichuan white geese obtained by the screening method provides 7 effective SNP molecular markers, supplements a library of the SNP molecular markers of the Sichuan white geese, can quickly detect individuals with high or low hatching egg qualification rate by using the method, saves the breeding time and cost of the traditional breeding of geese, and provides references for developing variety resources of the Sichuan white geese and industrial development.
Description
Technical Field
The invention relates to a molecular marking method for detecting the qualification rate of goose eggs, and mainly relates to the field of molecular biology.
Background
Single Nucleotide Polymorphism (SNP) refers to a polymorphism in a DNA sequence caused by a variation of a single nucleotide in a genomic sequence. Is the most common variation in the heritable variation of animal and plant genomes and widely exists in the genomes. The SNP has low mutation rate, high accuracy and stability, and is widely applied to one of molecular markers of animals and plants. The method is widely applied to the aspects of positioning target genes of diseases, individual screening in the breeding process of animals and plants, analysis in the evolution process of species, identification of genetic relationship and the like.
China is the most goose-raising country, but for various reasons, the development speed of goose-raising industry is slow, especially the benefit of large-scale goose-raising is poor, and even loss occurs, and the low reproductive performance is one of important factors restricting the goose-raising production development. The breeding of most geese has the characteristics of seasonality, nestability and the like, which causes the female geese to have low egg yield, increases the breeding cost of breeding geese, improves the production cost of commercial geese and severely restricts the development of industry. The goose reproductive traits are greatly influenced by environmental factors, and correct genotypes are difficult to obtain by phenotype selection, so that the application of the traditional quantitative genetics in reproductive trait improvement is limited, and the traditional breeding progress is very little. The molecular genetic marker provides a good opportunity for solving the problem and is a key for effectively developing the molecular breeding of the high-yield new variety goose.
The Sichuan white goose is one of seasonal breeding waterfowls, has the characteristics of strong stress resistance, good adaptability, coarse feeding resistance, strong disease resistance, coarse feeding resistance and the like, and provides meat, eggs, down feather, fat liver and the like for wide consumers. Is widely raised in various parts of China and is further used for improvement and creation of other varieties. The yield traits of hatching eggs are important economic traits and also complex quantitative traits, and are all influenced by heredity, nutrition, feeding modes, external environment and the like. The research is carried out by using methods such as biology, molecular markers such as duck body size, egg shell color and the like are found and are widely applied to molecular marker-assisted selection, and the research progress in the aspects of poultry hatching egg qualification rate and the like is greatly promoted. Sichuan white geese are bred seasonally, egg is produced from September to May of the next year every year, and the rest of the time is spent. The unique habit causes the female goose to have lower egg yield, increases the breeding cost of the breeding geese, improves the production cost of commercial geese, seriously influences the economic benefit and restricts the development of the goose breeding industry. And because the selection of the qualified rate character of the hatching egg is a long-time manual breeding process, the selection character, a detection index method, cost, nutrition level, a character detection technical means, environment and other reasons are used, the difficulty of the qualified rate character breeding of the hatching egg is greatly improved, and the possibility of further utilization of the goose industry is further limited.
The goose egg yield is one of important indexes for evaluating the fertility of the goose, and means that the number of eggs which meet the requirements of the variety and the strain and are produced by the female goose within 70 weeks of age accounts for the percentage of the total number of eggs. The hatching egg qualification rate directly influences the hatching rate of the geese and the quality of the goslings, the economic benefit of the goose industry is directly increased, and the feeding cost is obviously reduced. The yield of hatching eggs is influenced by heredity, environment, diseases and the like. However, no corresponding detection means is available for judging the qualified rate of the goose eggs. The traditional method consumes a large amount of manpower and material resources to detect the qualified rate of hatching eggs, and has higher cost; and the breeding difficulty of the corresponding female geese is higher aiming at the egg yield of the geese.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides the molecular marking method for detecting the qualification rate of the goose eggs, the female goose with higher qualification rate of the goose eggs is bred by using the molecular marking method, the individual with the target character can be bred quickly and accurately in the gosling stage, the breeding efficiency is greatly improved, and the feeding cost is reduced.
In order to achieve the purpose, the technical scheme of the invention is as follows: the method comprises the following steps: s1: comparing the whole genome retest data of the goose to be detected with a reference genome sequence, combining the qualification rate data of the goose eggs to be detected, and screening to obtain 7 candidate SNP sites of the qualification rate of the goose eggs to be detected through whole genome association analysis; s2: the distribution frequency of 7 SNP loci (SNP289, SNP290, SNP292, SNP294, SNP296, SNP297 and SNP298) and 1 haplotype module (constructed by 5 SNPs such as SNP290, SNP292, SNP294, SNP296 and SNP297) in Sichuan white goose individuals are tested by the flight time mass spectrometry technology, and are subjected to correlation analysis with the hatching egg qualification status, and the results show that the SNP loci and the haplotype module are obviously or extremely obviously related to the hatching egg qualification status.
Preferably, D1: collecting blood of the wing vein of a female goose to be detected in the early stage of laying, and extracting whole genome DNA; d2: the DNA obtained in step D1 was subjected to PCR reaction in a 5. mu.l: 10 buffer 0.5. mu.l, Mg2+0.4. mu.l, dNTP 0.1. mu.l, Hotstar 0.2. mu.l, upstream and downstream primer mixture 1. mu.l, triple distilled water 1.8. mu.l, DNA sample to be detected 1. mu.l (20ng-50 ng); PCR amplification procedure: pre-denaturation at 95 ℃ for 2min, 45 cyclesRing (denaturation at 95 ℃ for 30s, annealing at 56 ℃ for 30s, and elongation at 72 ℃ for 60s) was elongated at 72 ℃ for 5min and stored at 25 ℃.
Preferably, D3: SAP enzyme digestion reaction: the total volume of SAP enzyme Mix reaction was 2 x 460 μ l, triple distilled water 1.53 x 460 μ l, sapplus 0.17 x 460 μ l, SAPEnzyme0.3 x 460 μ l, prepared in the following order, and SAP enzyme digestion was performed in a PCR instrument according to the following procedure: storing at 37 deg.C for 40min, 85 deg.C for 5min, and 25 deg.C.
Preferably, D4: single base extension reaction: the single base extension reaction Mix was prepared in the following order: reactant system 2 x 460. mu.l, triple distilled water 0.619 x 460. mu.l, 10 x iplex buffer0.2 x 460. mu.l, Terminatormix0.2 x 460. mu.l, single base extension probe 0.94 x 460. mu.l, single base extension enzyme 0.041 x 460. mu.l; the single base extension reaction was performed in a PCR instrument according to the following procedure: pre-denaturation at 94 deg.C for 30s, 94 deg.C for 5s, 52 deg.C for 5s, 80 deg.C for 5s, 72 deg.C for 3min, and storing at 25 deg.C.
Preferably, D5: resin purification: adding 16 mu l of triple distilled water into a 384-well plate of the reaction product, and centrifuging for 3min at 2000 revolutions in a centrifuge; adding resin, performing resin purification reaction for 35min on a reverse shaking apparatus, and desalting; centrifuging for 3min at 2000 revolutions in a centrifuge after the reaction is finished; and (4) spotting the sample subjected to desalting treatment on a sample target, and naturally crystallizing.
Preferably, D6: mass spectrum detection and data analysis: and detecting the reaction results obtained from D1-D5 in a nucleic acid flight mass spectrometer, detecting mass spectrum peaks by using Typer4.0 software, and judging the genotype of target sites of each sample according to the mass spectrum peak diagrams.
Preferably, the primer pairs for detecting 7 SNP molecular markers are:
the technical principle and the beneficial effects of the invention are as follows: according to the technical scheme, the female goose with high hatching egg qualification rate is bred by using a molecular marking method, individuals with target characters can be bred quickly and accurately in a gosling stage, breeding efficiency is greatly improved, and feeding cost is reduced.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only 11 of the embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a flight mass spectrum primer chart of 7 SNP molecular markers according to an embodiment of the invention;
FIG. 2 is a frequency chart of the 7 SNP molecular markers and the haplotype block according to the embodiment of the present invention;
FIG. 3 shows the result of genome-wide association analysis of the breeding egg yield traits and genome-wide SNP sites according to the embodiment of the invention;
FIG. 4 is a diagram showing tightly linked haplotypes located on chromosome 1 in accordance with an embodiment of the present invention;
FIG. 5 shows the flight mass spectrum results of SNP289 of the example. Note: no call: detecting a failed individual; c: a CC genotype; t: the TT genotype.
FIG. 6 shows the flight mass spectrum of SNP290 according to an embodiment of the invention. Note: no call: detecting a failed individual; t: the TT genotype; c: the CC genotype.
FIG. 7 shows the flight mass spectrum of SNP292 according to an embodiment of the present invention. Note: no call: detecting a failed individual; t: the TT genotype; c: the CC genotype.
FIG. 8 shows the flight mass spectrum of SNP294 according to an embodiment of the invention. Note: no call: detecting a failed individual; g: a GG genotype; a: the AA genotype.
FIG. 9 shows the results of a SNP296 flight mass spectrum according to an embodiment of the invention. Note: no call: detecting a failed individual; g: a GG genotype; a: the AA genotype.
FIG. 10 shows the flight mass spectrum of SNP297 according to an embodiment of the present invention. Note: no call: detecting a failed individual; a: an AA genotype; t: the TT genotype.
FIG. 11 shows the flight mass spectrum of SNP298 according to an embodiment of the invention. Note: no call: detecting a failed individual; g: a GG genotype; t: the TT genotype.
Detailed Description
The technical solutions in the present invention will be described clearly and completely with reference to the accompanying drawings, and it is to be understood that the described embodiments are merely preferred embodiments of the present invention, rather than all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Examples
As shown in fig. 1, an embodiment of the present invention includes the steps of:
comparing the whole genome re-sequencing data of Sichuan white geese to a reference genome sequence, and screening to obtain 7 candidate SNP sites of the goose egg qualification rate by a whole genome association analysis (GWAS) method in combination with the protein qualification rate data of Sichuan white geese. The distribution frequency of a haplotype module constructed by 7 SNP loci and 5 SNP loci (SNP290, SNP292, SNP294, SNP296 and SNP297) in Sichuan white goose individuals is tested by the flight time mass spectrometry technology and is subjected to correlation analysis, and the SNP loci and the haplotype module are remarkably or extremely remarkably related to the hatching egg qualification (p <0.05 or p < 0.01).
The primers for detecting 7 SNP molecular markers are shown in figure 1: flight mass spectrum primer
The resequencing is a whole genome resequencing sequence of Sichuan white goose, and the reference genome is a reference genome sequence of Sichuan white goose. The method comprises the following steps:
d1: collecting blood from the wing vein of the Sichuan white goose female goose in the early stage of laying eggs, and extracting whole genome DNA;
d2: the DNA in step S1 was subjected to PCR reaction in a 5. mu.l: 10 buffer 0.5. mu.l, Mg2+0.4. mu.l, dNTP 0.1. mu.l, Hotstar 0.2. mu.l, upstream and downstream primer mix 1. mu.l, triple distilled water 1.8. mu.l, DNA sample to be detected 1. mu.l (20ng-50 ng). PCR amplification procedure: pre-denaturation at 95 deg.C for 2min, 45 cycles (denaturation at 95 deg.C for 30s, annealing at 56 deg.C for 30s, and elongation at 72 deg.C for 60s), extension at 72 deg.C for 5min, and storage at 25 deg.C.
D3: SAP enzyme digestion reaction: the total volume of SAP enzyme Mix reaction was 2 x 460 μ l, triple distilled water 1.53 x 460 μ l, saprouffer 0.17 x 460 μ l, sapenzyme0.3 x 460 μ l, prepared in the following order, and SAP enzyme digestion was performed in a PCR instrument according to the following procedure: storing at 37 deg.C for 40min, 85 deg.C for 5min, and 25 deg.C.
D4: single base extension reaction: the single base extension reaction Mix was prepared in the following order: reactant system 2 x 460. mu.l, triple distilled water 0.619 x 460. mu.l, 10 x iplex buffer0.2 x 460. mu.l, terminator mix0.2 x 460. mu.l, single base extension probe 0.94 x 460. mu.l, single base extension enzyme 0.041 x 460. mu.l. The single base extension reaction was performed in a PCR instrument according to the following procedure: pre-denaturation at 94 deg.C for 30s, 94 deg.C for 5s, 52 deg.C for 5s, 80 deg.C for 5s, 72 deg.C for 3min, and storing at 25 deg.C.
D5: resin purification, namely adding 16 mu l of triple distilled water into a 384-well plate of a reaction product, and centrifuging for 3min at 2000 revolutions in a centrifuge; adding resin, performing resin purification reaction for 35min on a reverse shaking apparatus, and desalting; centrifuging for 3min at 2000 revolutions in a centrifuge after the reaction is finished; spotting the sample subjected to desalination treatment on a sample target, and naturally crystallizing;
d6: mass spectrum detection and data analysis: and detecting the reaction results obtained from D1-D5 in a nucleic acid flight mass spectrometer, detecting mass spectrum peaks by using Typer4.0 software, and judging the genotype of target sites of each sample according to the mass spectrum peak diagrams.
The project takes the female goose of Sichuan white goose in laying period (30-60 weeks) as research animal (209), and the female goose is raised in individual laying cage, and the ratio of 1: and (4) breeding male geese and female geese according to the proportion of 4, and counting the hatching egg qualification rate every day in the period of 35-60 weeks.
And performing whole genome re-sequencing on 209 geese (the data volume of each individual is more than 11G, the genome coverage rate is more than 10), comparing the whole genome data to goose genomes by using BWA software, extracting SNPs by using a GATK method, filtering and combining, and obtaining 9,279,339 SNPs and 209 individuals through quality control.
The invention carries out genome-wide association analysis (GWAS) on the hatching egg qualification rate traits of 209 geese and all SNP sites, and uses GEMMA software to complete the analysis based on a Mixed Linear Model (MLM), wherein the mixed linear model is as follows: genome-wide association analysis (GWAS) was performed using GENNA software using a linear formula, where y is W α + x β + e, where α is a coefficient vector corresponding to the covariate matrix (W), β is the effect magnitude of the SNP, and e represents the random residual. The GWAS results are respectively screened to 7 SNP molecular markers related to the hatching egg qualification status, as shown in figure 2.
And (3) utilizing Plink software to construct haplotypes aiming at the SNP loci. And (3) detecting the SNP sites and unit types by using a flight mass spectrometry method to perform genotyping in goose groups. And (3) detecting the genotypes and the frequencies of the 7 SNPs in the Sichuan white goose population by using a flight time mass spectrum technology. Notably, 5 SNP sites (SNP290, SNP292, SNP294, SNP296, and SNP297) were constructed into one haplotype block, as shown in fig. 3.
The method mainly provides molecular markers of 7 hatching egg qualification rates, and the specific detection mode can be mastered according to actual conditions.
Unless otherwise indicated, reagents and materials used in the present invention are commercially available.
The Sichuan white goose samples in the following examples are all from Anfu waterfowl experimental base in Rongchang district, Chongqing, and all individuals were hatched in the same batch and raised in the same environment. After the breeding period, adopting single-cage breeding, and obtaining the Sichuan white goose sample based on recording the yield of the hatching eggs of the Sichuan white goose in 35-60 weeks.
Example 1:
(1) whole genome correlation analysis of hatching egg percent of pass
Whole genome association analysis:
2mL of blood of a fin vein is collected by using a vacuum blood collection tube containing heparin sodium when the Sichuan white goose is 20 weeks old, blood genome DNA is extracted by using a blood genome extraction kit (Beijing Tiangen, DP332), the concentration and the integrity of a DNA stock solution are measured by using an electrophoresis experiment and a NanoDrop2000 spectrophotometer, and the DNA stock solution is dissolved in a TE solution and stored at the temperature of-20 ℃ for later use. The genomic DNA is sent to Beijing Nuo He genesis science and technology GmbH for whole genome re-sequencing by means of dry ice transportation.
2mL of blood of a fin vein is collected by using a vacuum blood collection tube containing heparin sodium when the Sichuan white goose is 20 weeks old, blood genome DNA is extracted by using a blood genome extraction kit (Beijing Tiangen, DP332), the concentration and the integrity of a DNA stock solution are measured by using an electrophoresis experiment and a NanoDrop2000 spectrophotometer, and the DNA stock solution is dissolved in a TE solution and stored at the temperature of-20 ℃ for later use. The genomic DNA is sent to Beijing Nuo He genesis science and technology GmbH for whole genome re-sequencing by means of dry ice transportation.
And performing whole genome re-sequencing on 209 geese (the data volume of each individual is more than 11G, the genome coverage rate is more than 10), comparing the whole genome data to goose genomes by using BWA software, extracting SNPs by using a GATK method, filtering and combining, and obtaining 9,279,339 SNPs and 209 individuals through quality control.
The invention carries out genome-wide association analysis (GWAS) on the hatching egg qualification rate traits of 209 geese and genome-wide SNP sites, and completes the analysis by using a method based on a Mixed Linear Model (MLM) by using GEMMA software, wherein the mixed linear model is as follows: and (3) carrying out genome-wide association analysis (GWAS) by utilizing GEMMA software by utilizing a linear formula, wherein y is W alpha + x beta + epsilon, alpha is a coefficient vector corresponding to the covariate matrix (W), beta is the effect size of the SNP, and epsilon represents random residual error. The GWAS results are respectively screened to 7 SNP molecular markers related to the hatching egg qualification status, as shown in figure 2.
(2) Method for detecting distribution frequency of candidate SNP in Sichuan white geese in hatching egg yield by flight time mass spectrometry
And selecting the 209 goose whole genome DNAs as experimental materials, and carrying out flight time mass spectrum verification on the SNP loci.
1. Designing a primer: primer design was performed by the assay design3.1 software of Agena corporation according to the SNP site, as shown in FIG. 1.
2. Synthesizing primers and performing quality inspection: the primers synthesized by a primer synthesis company are subjected to quality inspection by a matrix assisted laser desorption ionization time of flight mass spectrometer (MALDI-TOF), and whether the actual molecular weight is consistent with the theoretical molecular weight and whether the purity of the primers meets the experimental requirements is detected. The specific operation is as follows: sucking 2ul of each synthesized extension primer to prepare a Mix, sucking 2ul of each synthesized extension primer from the Mix and adding the mixture into 40 mul of ddH2O for mass spectrum detection, wherein the molecular weight of the obtained peak pattern is consistent with a theoretical value and has no impurity peak.
3. Sample extraction and concentration purity quality inspection: detecting the concentration, purity and degradation degree of the DNA by agarose gel electrophoresis, wherein the detection result judges the standard: the genome in the electrophoresis detection gel picture has single, clear and non-impurity and non-dispersion trailing phenomena.
4. The whole genome DNA was subjected to PCR reaction in a 5. mu.l: 10 buffer 0.5. mu.l, Mg2+0.4. mu.l, dNTP 0.1. mu.l, Hotstar 0.2. mu.l, upstream and downstream primer mix 1. mu.l, triple distilled water 1.8. mu.l, DNA sample to be detected 1. mu.l (20ng-50 ng). PCR amplification procedure: pre-denaturation at 95 deg.C for 2min, 45 cycles (denaturation at 95 deg.C for 30s, annealing at 56 deg.C for 30s, and extension at 72 deg.C for 60s), extension at 72 deg.C for 5min, and storage at 25 deg.C.
Performing SAP enzyme digestion reaction on the PCR product: the total volume of SAP enzyme Mix reaction was 2 x 460 μ l, triple distilled water 1.53 x 460 μ l, saprouffer 0.17 x 460 μ l, sapenzyme0.3 x 460 μ l, prepared in the following order, and SAP enzyme digestion was performed in a PCR instrument according to the following procedure: storing at 37 deg.C for 40min, 85 deg.C for 5min, and 25 deg.C.
The product is then subjected to a single base extension reaction: the single base extension reaction Mix was prepared in the following order: reactant system 2 x 460. mu.l, triple distilled water 0.619 x 460. mu.l, 10 x iplex buffer0.2 x 460. mu.l, terminator mix0.2 x 460. mu.l, single base extension probe 0.94 x 460. mu.l, single base extension enzyme 0.041 x 460. mu.l. The single base extension reaction was performed in a PCR instrument according to the following procedure: pre-denaturation at 94 deg.C for 30s, 94 deg.C for 5s, 52 deg.C for 5s, 80 deg.C for 5s, 72 deg.C for 3min, and storing at 25 deg.C.
Purifying the reaction product with resin, placing into 384-well plate, adding 16 μ l of triple distilled water, centrifuging in centrifuge at 2000 rpm for 3 min; adding resin, performing resin purification reaction for 35min on a reverse shaking apparatus, and desalting; centrifuging for 3min at 2000 revolutions in a centrifuge after the reaction is finished; and (4) spotting the sample subjected to desalting treatment on a sample target, and naturally crystallizing.
Mass spectrum detection and data analysis: and detecting the obtained reaction result in a nucleic acid flight mass spectrometer, detecting a mass spectrum peak by using Typer4.0 software, and judging the genotype of each sample target site according to a mass spectrum peak diagram.
And (4) interpretation of results:
as shown in fig. 2, the hatching egg qualification rate of the CC genotype individual of SNP289 is significantly higher than that of other genotype individuals (p > 0.05); the TT genotype individual hatching egg qualification rate of the SNP290 is the highest, and the CC genotype individual hatching egg qualification rate is the lowest; the CC genotype individual hatching egg qualification rate of the SNP292 is the highest; the GG genotype individual hatching egg qualification rate of SNP294 is highest; the AA genotype of SNP296 was significantly higher than other genotypes; the GT genotype individual hatching egg qualification rate of SNP298 is obviously higher than that of TT genotype. Haplotype 1(TTCTAGAAAT) has the highest individual hatching egg qualification rate, and haplotype 6(CCTTAAGGAA) has the lowest individual hatching egg qualification rate. Individuals with the above genotypes or haplotypes can be retained or eliminated according to actual conditions and requirements.
The above description is only a preferred embodiment of the present invention, and should not be taken as limiting the invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (7)
1. The molecular marking method for detecting the qualification rate of the goose eggs is characterized by comprising the following steps:
s1: comparing the whole genome retest data of the goose to be detected with a reference genome sequence, screening and obtaining 7 candidate SNP loci (SNP289, SNP290, SNP292, SNP294, SNP296, SNP297 and SNP298) of the qualification rate of the goose egg to be detected by combining the qualification rate data of the goose egg to be detected and performing whole genome association analysis;
s2: the distribution frequency of 7 SNP loci and 1 haplotype module (constructed by SNP290, SNP292, SNP294, SNP296 and SNP297) in Sichuan white goose individuals is tested by the flight time mass spectrometry technology, and correlation analysis is carried out, and the results show that the 7 SNP molecular markers and the haplotype module are obviously or extremely obviously related to the hatching egg qualification rate.
2. The molecular marking method for detecting the goose hatching egg yield according to claim 1, which is characterized in that:
d1: collecting blood of the wing vein of a female goose to be detected in the early stage of laying, and extracting whole genome DNA;
d2: the DNA obtained in step D1 was subjected to PCR reaction in a 5. mu.l: 10 buffer 0.5. mu.l, Mg2+0.4. mu.l, dNTP 0.1. mu.l, Hotstar 0.2. mu.l, upstream and downstream primer mixture 1. mu.l, triple distilled water 1.8. mu.l, DNA sample to be detected 1. mu.l (20ng-50 ng); PCR amplification procedure: pre-denaturation at 95 deg.C for 2min, 45 cycles (denaturation at 95 deg.C for 30s, annealing at 56 deg.C for 30s, and extension at 72 deg.C for 60s), extension at 72 deg.C for 5min, and storage at 25 deg.C.
3. The molecular marking method for detecting the qualification rate of the goose eggs according to claim 2, wherein the molecular marking method comprises the following steps: d3: SAP enzyme digestion reaction:
the total volume of SAP enzyme Mix reaction was 2 x 460 μ l, triple distilled water 1.53 x 460 μ l, saprouffer 0.17 x 460 μ l, SAPEnzyme0.3 x 460 μ l, prepared in the following order, and SAP enzyme digestion was performed in a PCR instrument according to the following procedure: storing at 37 deg.C for 40min, 85 deg.C for 5min, and 25 deg.C.
4. The molecular marking method for detecting the qualification rate of the goose eggs according to claim 3, wherein the molecular marking method comprises the following steps: d4: single base extension reaction:
the single base extension reaction Mix was prepared in the following order: reactant system 2 x 460. mu.l, triple distilled water 0.619 x 460. mu.l, 10 x iplex buffer0.2 x 460. mu.l, Terminatormix0.2 x 460. mu.l, single base extension probe 0.94 x 460. mu.l, single base extension enzyme 0.041 x 460. mu.l; the single base extension reaction was performed in a PCR instrument according to the following procedure: pre-denaturation at 94 deg.C for 30s, 94 deg.C for 5s, 52 deg.C for 5s, 80 deg.C for 5s, 72 deg.C for 3min, and storing at 25 deg.C.
5. The molecular marking method for detecting the goose hatching egg yield as claimed in claim 4, wherein the molecular marking method comprises the following steps: d6: resin purification:
adding 16 mu l of triple distilled water into a 384-well plate of the reaction product, and centrifuging for 3min at 2000 revolutions in a centrifuge; adding resin, performing resin purification reaction for 35min on a reverse shaking apparatus, and desalting; centrifuging for 3min at 2000 revolutions in a centrifuge after the reaction is finished; and (4) spotting the sample subjected to desalting treatment on a sample target, and naturally crystallizing.
6. The molecular marking method for detecting the goose hatching egg yield as claimed in claim 5, wherein the molecular marking method comprises the following steps: d7: mass spectrum detection and data analysis:
and detecting the reaction results obtained from D1-D5 in a nucleic acid flight mass spectrometer, detecting mass spectrum peaks by using Typer4.0 software, and judging the genotype of target sites of each sample according to the mass spectrum peak diagrams.
7. The molecular marking method for detecting the goose hatching egg yield according to claim 1, which is characterized in that:
the primer pairs for detecting 7 SNP molecular markers are as follows:
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| WO2022134554A1 (en) * | 2020-12-23 | 2022-06-30 | 重庆市畜牧科学院 | Snp molecular marker method and primer for screening quality of goose eggs |
| WO2022134553A1 (en) * | 2020-12-23 | 2022-06-30 | 重庆市畜牧科学院 | Molecular marking method for detecting pass rate of hatching goose eggs |
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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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| CN106636367A (en) * | 2016-11-25 | 2017-05-10 | 浙江省农业科学院 | Molecular genetic marker related with egg laying performance of hens and application |
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| WO2022134554A1 (en) * | 2020-12-23 | 2022-06-30 | 重庆市畜牧科学院 | Snp molecular marker method and primer for screening quality of goose eggs |
| WO2022134553A1 (en) * | 2020-12-23 | 2022-06-30 | 重庆市畜牧科学院 | Molecular marking method for detecting pass rate of hatching goose eggs |
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