CN117587141A - SNP locus combination for identifying open-eye goose variety and identification method thereof - Google Patents

SNP locus combination for identifying open-eye goose variety and identification method thereof Download PDF

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CN117587141A
CN117587141A CN202311551633.7A CN202311551633A CN117587141A CN 117587141 A CN117587141 A CN 117587141A CN 202311551633 A CN202311551633 A CN 202311551633A CN 117587141 A CN117587141 A CN 117587141A
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eye
open
geese
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徐文娟
朱春红
刘宏祥
章双杰
李慧芳
王晓峰
陶志云
王志成
宋卫涛
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Jiangsu Institute Poultry Sciences
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Abstract

The invention discloses SNP locus combinations for identifying an open-eye goose variety and an identification method thereof, and belongs to the technical field of biology. According to the invention, HY1P to HY15P 15 SNP loci are screened, the primer combination for amplifying the SNP loci is utilized to determine the genotype of each locus, and the probability that the individual belongs to the open-eye goose is judged according to the Bayesian theorem. Through experimental verification, the combined use of 15 SNP loci is proved to judge whether an individual to be detected belongs to the open-eye goose, and when the probability is more than 0.95, the individual to be detected is judged to be the open-eye goose, and the accuracy rate can reach 100%. The method for identifying the variety of the open-eye geese is simple and convenient to operate and high in accuracy, and can effectively strike the degree of flooding of the counterfeit open-eye geese in the market.

Description

SNP locus combination for identifying open-eye goose variety and identification method thereof
Technical Field
The invention relates to the technical field of biology, in particular to SNP locus combinations for identifying an open-eye goose variety and an identification method thereof.
Background
The open-eye geese are local varieties in Shandong province, belong to small-sized geese, have the characteristics of coarse feeding resistance, high growth speed, no nest holding, more egg laying and the like, and have a wide development and utilization prospect. At present, more counterfeit open-eye geese and hybrid individuals of the open-eye geese and other varieties exist in the market for impersonation sales, and certain impact is caused to the pure-bred individual market of the open-eye geese, so that the brand of the open-eye geese is seriously influenced. Therefore, the method and the product for effectively and accurately identifying the variety of the geese with the open eyes are very necessary.
The existing method for identifying the open-eye geese in the market mainly depends on appearance observation. The observation subjectivity is strong, the identification error rate is high, and incorrect classification and identification are easy to cause particularly when the hybrid offspring of the geese with other varieties are identified. And the local variety geese are similar when just hatching, and the identification error rate is higher.
The single nucleotide polymorphism (Single nucleotide polymorphism, SNP) mainly refers to DNA sequence polymorphism caused by single nucleotide variation at genome level, and has the characteristics of rich sites, wide distribution, high genetic stability, representativeness, convenience and rapidness in detection and the like. SNP markers, which utilize specific SNP loci with large variety variability in the genomic DNA of individuals to carry out variety identification through genotypes of different locus combinations, and the identification result is more objective and accurate. In view of the above, development of a product and a method for effectively and accurately identifying the variety of the geese in the open eyes is urgently needed in scientific research and practice, and the method is applied to the variety identification work of the geese in the open eyes.
Disclosure of Invention
The invention aims to provide an SNP locus combination for identifying the variety of the open-eye geese and an identification method thereof, so as to solve the problems in the prior art, and the genotype of 15 SNP loci of the open-eye geese is detected to jointly judge whether an individual to be detected belongs to the open-eye geese.
In order to achieve the above object, the present invention provides the following solutions:
the invention provides a primer combination for identifying an eye-open goose variety, which is used for amplifying SNP locus combinations shown in HY1P-HY15P, wherein the SNP locus combinations shown in HY1P-HY15P are as follows:
HY1P is located at 8149755 on NW_025927674.1 chromosome, and the polymorphism is T or C;
HY2P is located at 8203321 on NW_025927674.1 chromosome, and the polymorphism is A or G;
HY3P is located at 2351173 on NW_025927679.1 chromosome, and the polymorphism is T or C;
HY4P is located at 3059353 on NW_025927682.1 chromosome, and the polymorphism is C or T;
HY5P is located at 6435171 on NW_025927697.1 chromosome, and the polymorphism is A or G;
HY6P is located at 5160223 on NW_025927698.1 chromosome, and the polymorphism is G or A; HY7P is located at 2291555 on NW_025927702.1 chromosome, and the polymorphism is C or A; HY8P is located at 2770782 on NW_025927731.1 chromosome, and the polymorphism is A or C; HY9P is located at 1509689 on NW_025927732.1 chromosome, and the polymorphism is A or G; HY10P is located at 2111483 on NW_025927766.1 chromosome, and the polymorphism is G or A; HY11P is located at 847200 on NW_025927777.1 chromosome, and the polymorphism is T or C; HY12P is located at 847596 on NW_025927777.1 chromosome, and the polymorphism is A or G; HY13P is located at 2195673 on NW_025927786.1 chromosome, and the polymorphism is C or T; HY14P is located at 1174874 on NW_025927805.1 chromosome, and the polymorphism is C or A; HY15P is located at 1194180 on NW_025927847.1 chromosome, and the polymorphism is T or C. Preferably, the amplification primer combination of the SNP locus combination shown in HY1P-HY15P is as follows: HY1F:5'-ACAGCTGGAAGTCACGAAGG-3' (SEQ ID NO: 1);
HY1R：5’-ATGGGAAGCAGGCAGTTACC-3’(SEQ ID NO：2)；
HY2F：5’-CCTCTTCATCTCCTGCCAGC-3’(SEQ ID NO：3)；HY2R：5’-GATGCTTTCTGCACCAGGGA-3’(SEQ ID NO：4)；HY3F：5’-CTGGTAACTGCCTAGCAGATGG-3’(SEQ ID NO：5)；
HY3R：5’-AGCAGAATTACAGCTTCTGGGTTT-3’(SEQ ID NO：6)；
HY4F：5’-CTAACAATGCGTGCCTCAGC-3’(SEQ ID NO：7)；HY4R：5’-TAACACCTTCCCCCAGACCA-3’(SEQ ID NO：8)；HY5F：5’-ACCACCCTTCCCCTACAGAT-3’(SEQ ID NO：9)；
HY5R：5’-CGCAGAGCAGCAGCTACA-3’(SEQ ID NO：10)；HY6F：5’-AAGAGGATCCGCTGAGGTCT-3’(SEQ ID NO：11)；
HY6R：5’-AGTTGCCTGCTAGGTCATCG-3’(SEQ ID NO：12)；
HY7F：5’-TTGCAAGGACTGGCTAACAAC-3’(SEQ ID NO：13)；
HY7R：5’-AATCATTAAGCACAGGTGCGG-3’(SEQ ID NO：14)；
HY8F：5’-TGCTCGACATGTCTCGTCTG-3’(SEQ ID NO：15)；HY8R：5’-GAAAGCTGGACTGAGGGAGTC-3’(SEQ ID NO：16)；
HY9F：5’-TGAGGGGTGAGGAAGGGAAT-3’(SEQ ID NO：17)；
HY9R：5’-CTAAATCGCGCTGCTGTTCC-3’(SEQ ID NO：18)；
HY10F：5’-AGTTTCCATGGCTACGTCACA-3’(SEQ ID NO：19)；
HY10R：5’-GTCAGGCCAATCAAAGATGCC-3’(SEQ ID NO：20)；
HY11F：5’-GAACAGGATGCACCCAAGGA-3’(SEQ ID NO：21)；
HY11R：5’-GCTCATCAGAGCAGTGGTCA-3’(SEQ ID NO：22)；
HY12F：5’-GAGGCTTCAGGTGAGACGAG-3’(SEQ ID NO：23)；
HY12R：5’-CTGTTGCTGAACCTCCCACT-3’(SEQ ID NO：24)；
HY13F：5’-GCTCCTTCACAGAACAGCCT-3’(SEQ ID NO：25)；
HY13R：5’-TGCCAAGCAGATAGCCAGTC-3’(SEQ ID NO：26)；
HY14F：5’-AGACTGTGAAGACAACAAGGGA-3’(SEQ ID NO：27)；
HY14R：5’-GTACTTGCCTAGCAGTCGATGT-3’(SEQ ID NO：28)；
HY15F：5’-TGCATCAATTCTGCACTCACTCAT-3’(SEQ ID NO：29)；
HY15R：5’-GCAGATATGGATGAGGACCCG-3’(SEQ ID NO：30)。
the invention also provides a method for identifying the variety of the open-eye geese, which comprises the following steps:
extracting total DNA of a goose genome to be detected;
taking the total DNA as a template, and carrying out PCR amplification by using the primer combination to obtain an amplification product;
sequencing the amplification product, and judging genotypes of sites shown in HY1P to HY 15P;
and judging whether the goose to be detected belongs to the open-eye goose or not according to the genotype determination result.
Preferably, the probability that the goose to be detected belongs to the open-eye goose is judged according to the Bayesian theorem, and the calculation formula is as follows:
the probability that the goose to be detected belongs to the non-open eye goose is
Wherein p is i Representing the frequency of genotypes corresponding to the variety of the open-eye geese in the ith SNP in the SNP locus combination; q i The average frequency of genotypes corresponding to other varieties in the ith SNP in the SNP locus combination is shown.
Preferably, in order to ensure accuracy according to the calculation result, taking 0.95 as a threshold, and judging that the goose belongs to the eye-opening goose if the probability of the goose is more than or equal to 0.95; if the probability of the non-open-eye goose is more than or equal to 0.95, judging that the non-open-eye goose is present; if less than 0.95, only one possibility can be determined, for example, the probability of belonging to an open-eye goose is 0.85, and only 85% of the possibility is considered to belong to the open-eye goose.
Preferably, the genotypes of the sites HY1P to HY15P in the geese and other varieties and the average genotype frequencies are as follows:
HY1P site: when the genotype is TT, the genotype frequency of the open-eye geese is 0.64, and the genotype frequency of other varieties is 0; when the genotype is TC, the genotype frequency of the open-eye geese is 0.32, and the genotype frequency of other varieties is 0.12; when the genotype is CC, the genotype frequency of the open-eye geese is 0.04, and the genotype frequency of other varieties is 0.88;
HY2P site: when the genotype is AA, the genotype frequency of the open-eye geese is 0.56, and the genotype frequency of other varieties is 0.00; when the genotype is AG, the genotype frequency of the open-eye geese is 0.38, and the genotype frequency of other varieties is 0.08; when the genotype is GG, the genotype frequency of the open-eye geese is 0.06, and the genotype frequency of other varieties is 0.92;
HY3P site: when the genotype is TT, the genotype frequency of the open-eye geese is 0.47, and the genotype frequency of other varieties is 0.00; when the genotype is TC, the genotype frequency of the open-eye geese is 0.43, and the genotype frequency of other varieties is 0.05; when the genotype is CC, the genotype frequency of the open-eye geese is 0.10, and the genotype frequency of other varieties is 0.95;
HY4P site: when the genotype is CC, the genotype frequency of the open-eye geese is 0.51, and the genotype frequency of other varieties is 0.00; when the genotype is CT, the genotype frequency of the open-eye geese is 0.41, and the genotype frequency of other varieties is 0.17; when the genotype is TT, the genotype frequency of the open-eye geese is 0.08, and the genotype frequency of other varieties is 0.83;
HY5P site: when the genotype is AA, the genotype frequency of the open-eye geese is 1.00, and the genotype frequency of other varieties is 0.09; when the genotype is AG, the genotype frequency of the open-eye geese is 0.00, and the genotype frequency of other varieties is 0.42; when the genotype is GG, the genotype frequency of the open-eye geese is 0.00, and the genotype frequency of other varieties is 0.49;
HY6P site: when the genotype is GG, the genotype frequency of the open-eye geese is 0.49, and the genotype frequency of other varieties is 0.01; when the genotype is GA, the genotype frequency of the open-eye geese is 0.42, and the genotype frequency of other varieties is 0.13; when the genotype is AA, the genotype frequency of the open-eye geese is 0.09, and the genotype frequency of other varieties is 0.86;
HY7P site: when the genotype is CC, the genotype frequency of the open-eye geese is 0.60, and the genotype frequency of other varieties is 0.02; when the genotype is CA, the genotype frequency of the open-eye geese is 0.35, and the genotype frequency of other varieties is 0.26; when the genotype is AA, the genotype frequency of the open-eye geese is 0.05, and the genotype frequency of other varieties is 0.72;
HY8P site: when the genotype is AA, the genotype frequency of the open-eye geese is 0.69, and the genotype frequency of other varieties is 0.03; when the genotype is AC, the genotype frequency of the open-eye geese is 0.28, and the genotype frequency of other varieties is 0.29; when the genotype is CC, the genotype frequency of the open-eye geese is 0.03, and the genotype frequency of other varieties is 0.68;
HY9P site: when the genotype is AA, the genotype frequency of the open-eye geese is 0.72, and the genotype frequency of other varieties is 0.03; when the genotype is AG, the genotype frequency of the open-eye geese is 0.26, and the genotype frequency of other varieties is 0.28; when the genotype is GG, the genotype frequency of the open-eye geese is 0.02, and the genotype frequency of other varieties is 0.69;
HY10P site: when the genotype is GG, the genotype frequency of the open-eye geese is 0.31, and the genotype frequency of other varieties is 0.00; when the genotype is GA, the genotype frequency of the open-eye geese is 0.49, and the genotype frequency of other varieties is 0.00; when the genotype is AA, the genotype frequency of the open-eye geese is 0.20, and the genotype frequency of other varieties is 1.00;
HY11P site: when the genotype is TT, the genotype frequency of the open-eye geese is 0.81, and the genotype frequency of other varieties is 0.04; when the genotype is TC, the genotype frequency of the open-eye geese is 0.18, and the genotype frequency of other varieties is 0.31; when the genotype is CC, the genotype frequency of the open-eye geese is 0.01, and the genotype frequency of other varieties is 0.65;
HY12P site: when the genotype is AA, the genotype frequency of the open-eye geese is 0.81, and the genotype frequency of other varieties is 0.04; when the genotype is AG, the genotype frequency of the open-eye geese is 0.18, and the genotype frequency of other varieties is 0.32; when the genotype is GG, the genotype frequency of the open-eye geese is 0.01, and the genotype frequency of other varieties is 0.64;
HY13P site: when the genotype is CC, the genotype frequency of the open-eye geese is 0.90, and the genotype frequency of other varieties is 0.01; when the genotype is CT, the genotype frequency of the open-eye geese is 0.10, and the genotype frequency of other varieties is 0.17; when the genotype is TT, the genotype frequency of the open-eye geese is 0.00, and the genotype frequency of other varieties is 0.82;
HY14P site: when the genotype is CC, the genotype frequency of the open-eye geese is 0.73, and the genotype frequency of other varieties is 0.06; when the genotype is CA, the genotype frequency of the open-eye geese is 0.24, and the genotype frequency of other varieties is 0.35; when the genotype is AA, the genotype frequency of the open-eye geese is 0.03, and the genotype frequency of other varieties is 0.59;
HY15P site: when the genotype is TT, the genotype frequency of the open-eye geese is 0.88, and the genotype frequency of other varieties is 0.03; when the genotype is TC, the genotype frequency of the open-eye geese is 0.12, and the genotype frequency of other varieties is 0.28; when the genotype is CC, the genotype frequency of the open-eye geese is 0.00, and the genotype frequency of other varieties is 0.69.
Preferably, when the probability of judging the open-eye geese is greater than 0.95 by combining SNP loci shown in HY1P to HY15P, the open-eye geese are judged.
Preferably, the other varieties include a long-leaved goose, a mounted goose, a steel goose, a daozhou gray goose, a Lingxian white goose, a lion head goose, a black-mand goose, a Yongkang gray goose and a seed goose.
The invention also provides a kit for identifying the variety of the geese with the open eyes, which comprises the primer combination.
The invention also provides application of the primer combination or the kit in identifying the variety of the geese with the open eyes.
The invention discloses the following technical effects:
according to the Bayesian theorem, the probability that the individual belongs to the open-eye goose can be judged through the 15 SNP combined genotypes, and the open-eye goose is judged according to the calculation formula when 95% of the probability of the individual belongs to the open-eye goose is obtained. The probability of judging the open-eye geese by combining 15 SNP loci can reach 100%, and the method is simple and convenient to operate and high in accuracy, and can effectively strike the degree of flooding of the counterfeit open-eye geese in the market.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
The invention provides a primer combination for identifying an open-eye goose variety, which is used for amplifying SNP locus combinations (see table 1) shown in the following HY1P to HY 15P:
TABLE 1
Sequence number Chromosome of the human body Site(s) Major allele Minor allele
HY1P NW_025927674.1 8149755 T C
HY2P NW_025927674.1 8203321 A G
HY3P NW_025927679.1 2351173 T C
HY4P NW_025927682.1 3059353 C T
HY5P NW_025927697.1 6435171 A G
HY6P NW_025927698.1 5160223 G A
HY7P NW_025927702.1 2291555 C A
HY8P NW_025927731.1 2770782 A C
HY9P NW_025927732.1 1509689 A G
HY10P NW_025927766.1 2111483 G A
HY11P NW_025927777.1 847200 T C
HY12P NW_025927777.1 847596 A G
HY13P NW_025927786.1 2195673 C T
HY14P NW_025927805.1 1174874 C A
HY15P NW_025927847.1 1194180 T C
The screening method of the SNP locus combination comprises the following steps: firstly, determining the average value of different allele frequencies of each site of domestic representative varieties such as Changle geese, mounted geese, daozhou gray geese, steel geese, lin county white geese, lion head geese, black-mand geese, yongkang gray geese, seed geese and the like, and then comparing the average value with the different allele frequencies of each site of the opening geese to screen SNP sites with larger allele frequency difference.
The amplification primer combinations (see Table 2) of the sites HY1P to HY15P are as follows:
TABLE 2
The invention also provides a method for identifying the variety of the open-eye geese, which comprises the following steps:
extracting total DNA of a goose genome to be detected;
using the total DNA as a template, and carrying out PCR amplification by using the primer combination to obtain an amplification product;
sequencing the amplification product, and judging genotypes of sites shown in HY1P to HY 15P;
and judging whether the goose to be detected belongs to the open-eye goose or not according to the genotype determination result.
In the identification method of the present invention, it is preferable to collect the genomic DNA from the fin vein of the goose to be tested, the extraction method of the genomic DNA is not particularly limited, and the genomic DNA is extracted by a conventional extraction method in the art, and in the embodiment of the present invention, a phenol-chloroform method is preferable.
In the invention, the identification of the variety of the eye-open goose is carried out by adopting the locus combination in HY1P-HY15P during application, and the PCR amplification is carried out by adopting the corresponding upstream and downstream primers. The PCR amplification method is not particularly limited, and conventional PCR amplification methods in the art can be adopted.
According to the invention, after sequencing the PCR amplification product of the goose to be detected, judging the genotype, comparing the genotype corresponding to the selected SNP combination, judging the probability that the goose to be detected belongs to the open-eye goose according to the Bayesian theorem, and calculating the following formula:
the probability that the goose individuals to be detected belong to non-open eye geese is that
Wherein p is i Representing the frequency of genotypes corresponding to the variety of the open-eye geese in the ith SNP in the SNP locus combination; q i The average frequency of genotypes corresponding to other varieties in the ith SNP in the SNP locus combination is shown. Considering the extreme cases of genotyping error rate and the product of frequency multiplication being 0 when the genotyping frequency is 0, the genotyping frequency smaller than 0.05 is uniformly adjusted to be 0.05, and meanwhile, the maximum genotyping frequency is adjusted to ensure that the sum of the three genotyping frequencies is 1.
According to the calculation result, in order to ensure the accuracy, 0.95 is taken as a threshold value, and the probability of belonging to the open-eye goose is greater than or equal to 0.95, and the open-eye goose is judged; if the probability of the non-open-eye goose is more than or equal to 0.95, judging that the non-open-eye goose is present; if less than 0.95, only one possibility can be determined, for example, the probability of belonging to an open-eye goose is 0.85, and only 85% of the possibility is considered to belong to the open-eye goose.
The genotype and genotype frequency of the SNP locus in the open-eye geese and other varieties are shown in the following table 3:
TABLE 3 Table 3
For example, selecting the 15 SNP sites HY1P to HY15P, if the combined genotype is TT AA TC CC AA GG CC AA AA GA TT AA CC CC TT, the probability that the individual belongs to an open-eye goose is:
the probability that the goose individuals to be detected belong to non-open eye geese is that
The genotype frequencies of the other varieties refer to the genotype frequency average value of each locus obtained by combining and calculating the different varieties, wherein the genotype frequencies of the other varieties are selected from domestic representative varieties such as Changle geese, daozhou gray geese, steel geese, linxian white geese, lion head geese, black-mand geese, yongkang gray geese, and seedgeese. The open-eye geese individually calculated the genotype frequency for each site.
The invention can also be prepared into a kit for identifying the variety of the geese in the eye, and the kit comprises the primer combination and other conventional reagents for detection.
The invention also provides application of the primer combination or the kit in identifying the varieties of the geese in the eye.
The present invention will be described in detail below with reference to examples for the purpose of making the objects, technical solutions and advantages of the present invention more apparent, but they should not be construed as limiting the scope of the present invention.
Example 1
10 geese with open eyes and 10 geese with other varieties are randomly selected, the wing vein is used for blood collection, the genome DNA is extracted by a phenol-chloroform method, and PCR amplification is carried out according to primers of 15 SNP loci in table 2.
The PCR system (20. Mu.L) was: 2. Mu.L of DNA template, 1.5. Mu.L (10 ng/. Mu.L) of forward and reverse primers, 10. Mu.L of 2 XPCR reagent in the day root PCR kit, and the balance of the kit were made up with ultrapure water.
The PCR procedure was: firstly, carrying out pre-denaturation at 94 ℃ for 1min, then carrying out denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s and extension at 72 ℃ for 30s, carrying out 35 cycles, and finally carrying out extension for 5min after 72 ℃ and preserving at 4 ℃. The PCR instrument was a Burley T100 gradient PCR instrument.
The amplified product was sent to the division of biological engineering (Shanghai) for sequence polymorphism detection and genotyping. According to the sequencing result, judging whether the probability of the open-eye goose belongs to the open-eye goose through the joint genotype of 15 SNP loci, and judging that the probability of the open-eye goose is larger than 0.95 by taking 0.95 as a threshold value, wherein the result is that all 10 open-eye geese are judged to be correct; the probability of the non-open-eye geese is greater than 0.95, the non-open-eye geese are judged, and the result is that all 10 non-open-eye geese are judged correctly. The results are shown in Table 4 below.
TABLE 4 Table 4
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.

Claims (9)

1. The primer combination for identifying the eye-open goose variety is characterized in that the primer combination is used for amplifying SNP locus combinations shown in HY1P-HY15P, and the SNP locus combinations shown in HY1P-HY15P are as follows:
HY1P is located at 8149755 on NW_025927674.1 chromosome, and the polymorphism is T or C;
HY2P is located at 8203321 on NW_025927674.1 chromosome, and the polymorphism is A or G;
HY3P is located at 2351173 on NW_025927679.1 chromosome, and the polymorphism is T or C;
HY4P is located at 3059353 on NW_025927682.1 chromosome, and the polymorphism is C or T;
HY5P is located at 6435171 on NW_025927697.1 chromosome, and the polymorphism is A or G;
HY6P is located at 5160223 on NW_025927698.1 chromosome, and the polymorphism is G or A;
HY7P is located at 2291555 on NW_025927702.1 chromosome, and the polymorphism is C or A;
HY8P is located at 2770782 on NW_025927731.1 chromosome, and the polymorphism is A or C;
HY9P is located at 1509689 on NW_025927732.1 chromosome, and the polymorphism is A or G;
HY10P is located at 2111483 on NW_025927766.1 chromosome, and the polymorphism is G or A;
HY11P is located at 847200 on NW_025927777.1 chromosome, and the polymorphism is T or C;
HY12P is located at 847596 on NW_025927777.1 chromosome, and the polymorphism is A or G;
HY13P is located at 2195673 on NW_025927786.1 chromosome, and the polymorphism is C or T;
HY14P is located at 1174874 on NW_025927805.1 chromosome, and the polymorphism is C or A;
HY15P is located at 1194180 on NW_025927847.1 chromosome, and the polymorphism is T or C.
2. The primer combination according to claim 1, wherein the amplification primer combination of the SNP site combination shown in HY1P-HY15P is:
HY1F：5’-ACAGCTGGAAGTCACGAAGG-3’；
HY1R：5’-ATGGGAAGCAGGCAGTTACC-3’；
HY2F：5’-CCTCTTCATCTCCTGCCAGC-3’；
HY2R：5’-GATGCTTTCTGCACCAGGGA-3’；
HY3F：5’-CTGGTAACTGCCTAGCAGATGG-3’；
HY3R：5’-AGCAGAATTACAGCTTCTGGGTTT-3’；
HY4F：5’-CTAACAATGCGTGCCTCAGC-3’；
HY4R：5’-TAACACCTTCCCCCAGACCA-3’；
HY5F：5’-ACCACCCTTCCCCTACAGAT-3’；
HY5R：5’-CGCAGAGCAGCAGCTACA-3’；
HY6F：5’-AAGAGGATCCGCTGAGGTCT-3’；
HY6R：5’-AGTTGCCTGCTAGGTCATCG-3’；
HY7F：5’-TTGCAAGGACTGGCTAACAAC-3’；
HY7R：5’-AATCATTAAGCACAGGTGCGG-3’；
HY8F：5’-TGCTCGACATGTCTCGTCTG-3’；
HY8R：5’-GAAAGCTGGACTGAGGGAGTC-3’；
HY9F：5’-TGAGGGGTGAGGAAGGGAAT-3’；
HY9R：5’-CTAAATCGCGCTGCTGTTCC-3’；
HY10F：5’-AGTTTCCATGGCTACGTCACA-3’；
HY10R：5’-GTCAGGCCAATCAAAGATGCC-3’；
HY11F：5’-GAACAGGATGCACCCAAGGA-3’；
HY11R：5’-GCTCATCAGAGCAGTGGTCA-3’；
HY12F：5’-GAGGCTTCAGGTGAGACGAG-3’；
HY12R：5’-CTGTTGCTGAACCTCCCACT-3’；
HY13F：5’-GCTCCTTCACAGAACAGCCT-3’；
HY13R：5’-TGCCAAGCAGATAGCCAGTC-3’；
HY14F：5’-AGACTGTGAAGACAACAAGGGA-3’；
HY14R：5’-GTACTTGCCTAGCAGTCGATGT-3’；
HY15F：5’-TGCATCAATTCTGCACTCACTCAT-3’；
HY15R：5’-GCAGATATGGATGAGGACCCG-3’。
3. a method for identifying an eye-open goose variety, comprising the steps of:
extracting total DNA of a goose genome to be detected;
performing PCR amplification by using the primer combination according to claim 1 or 2 with the total DNA as a template to obtain an amplification product;
sequencing the amplification product, and judging genotypes of sites shown in HY1P to HY 15P;
and judging whether the goose to be detected belongs to the open-eye goose or not according to the genotype determination result.
4. A method according to claim 3, wherein the probability that the goose to be tested belongs to an eye-open goose is determined according to bayesian theorem, and the calculation formula is as follows:
the probability that the goose to be detected belongs to the non-open eye goose is calculated as follows:
wherein p is i Representing the frequency of genotypes corresponding to the variety of the open-eye geese in the ith SNP in the SNP locus combination; q i The average frequency of genotypes corresponding to other varieties in the ith SNP in the SNP locus combination is shown.
5. The method of claim 4, wherein, according to the calculation result, in order to ensure the accuracy, taking 0.95 as a threshold, determining that the probability of belonging to the open-eye goose is greater than or equal to 0.95; and judging that the non-open-eye goose is not less than 0.95 in probability of being the non-open-eye goose.
6. A method according to claim 3, wherein the genotypes, average frequencies of genotypes at the sites of HY1P to HY15P in geese and other varieties are as follows:
HY1P site: when the genotype is TT, the genotype frequency of the open-eye geese is 0.64, and the genotype frequency of other varieties is 0; when the genotype is TC, the genotype frequency of the open-eye geese is 0.32, and the genotype frequency of other varieties is 0.12; when the genotype is CC, the genotype frequency of the open-eye geese is 0.04, and the genotype frequency of other varieties is 0.88;
HY2P site: when the genotype is AA, the genotype frequency of the open-eye geese is 0.56, and the genotype frequency of other varieties is 0.00; when the genotype is AG, the genotype frequency of the open-eye geese is 0.38, and the genotype frequency of other varieties is 0.08; when the genotype is GG, the genotype frequency of the open-eye geese is 0.06, and the genotype frequency of other varieties is 0.92;
HY3P site: when the genotype is TT, the genotype frequency of the open-eye geese is 0.47, and the genotype frequency of other varieties is 0.00; when the genotype is TC, the genotype frequency of the open-eye geese is 0.43, and the genotype frequency of other varieties is 0.05; when the genotype is CC, the genotype frequency of the open-eye geese is 0.10, and the genotype frequency of other varieties is 0.95;
HY4P site: when the genotype is CC, the genotype frequency of the open-eye geese is 0.51, and the genotype frequency of other varieties is 0.00; when the genotype is CT, the genotype frequency of the open-eye geese is 0.41, and the genotype frequency of other varieties is 0.17; when the genotype is TT, the genotype frequency of the open-eye geese is 0.08, and the genotype frequency of other varieties is 0.83;
HY5P site: when the genotype is AA, the genotype frequency of the open-eye geese is 1.00, and the genotype frequency of other varieties is 0.09; when the genotype is AG, the genotype frequency of the open-eye geese is 0.00, and the genotype frequency of other varieties is 0.42; when the genotype is GG, the genotype frequency of the open-eye geese is 0.00, and the genotype frequency of other varieties is 0.49;
HY6P site: when the genotype is GG, the genotype frequency of the open-eye geese is 0.49, and the genotype frequency of other varieties is 0.01; when the genotype is GA, the genotype frequency of the open-eye geese is 0.42, and the genotype frequency of other varieties is 0.13; when the genotype is AA, the genotype frequency of the open-eye geese is 0.09, and the genotype frequency of other varieties is 0.86;
HY7P site: when the genotype is CC, the genotype frequency of the open-eye geese is 0.60, and the genotype frequency of other varieties is 0.02; when the genotype is CA, the genotype frequency of the open-eye geese is 0.35, and the genotype frequency of other varieties is 0.26; when the genotype is AA, the genotype frequency of the open-eye geese is 0.05, and the genotype frequency of other varieties is 0.72;
HY8P site: when the genotype is AA, the genotype frequency of the open-eye geese is 0.69, and the genotype frequency of other varieties is 0.03; when the genotype is AC, the genotype frequency of the open-eye geese is 0.28, and the genotype frequency of other varieties is 0.29; when the genotype is CC, the genotype frequency of the open-eye geese is 0.03, and the genotype frequency of other varieties is 0.68;
HY9P site: when the genotype is AA, the genotype frequency of the open-eye geese is 0.72, and the genotype frequency of other varieties is 0.03; when the genotype is AG, the genotype frequency of the open-eye geese is 0.26, and the genotype frequency of other varieties is 0.28; when the genotype is GG, the genotype frequency of the open-eye geese is 0.02, and the genotype frequency of other varieties is 0.69;
HY10P site: when the genotype is GG, the genotype frequency of the open-eye geese is 0.31, and the genotype frequency of other varieties is 0.00; when the genotype is GA, the genotype frequency of the open-eye geese is 0.49, and the genotype frequency of other varieties is 0.00; when the genotype is AA, the genotype frequency of the open-eye geese is 0.20, and the genotype frequency of other varieties is 1.00;
HY11P site: when the genotype is TT, the genotype frequency of the open-eye geese is 0.81, and the genotype frequency of other varieties is 0.04; when the genotype is TC, the genotype frequency of the open-eye geese is 0.18, and the genotype frequency of other varieties is 0.31; when the genotype is CC, the genotype frequency of the open-eye geese is 0.01, and the genotype frequency of other varieties is 0.65;
HY12P site: when the genotype is AA, the genotype frequency of the open-eye geese is 0.81, and the genotype frequency of other varieties is 0.04; when the genotype is AG, the genotype frequency of the open-eye geese is 0.18, and the genotype frequency of other varieties is 0.32; when the genotype is GG, the genotype frequency of the open-eye geese is 0.01, and the genotype frequency of other varieties is 0.64;
HY13P site: when the genotype is CC, the genotype frequency of the open-eye geese is 0.90, and the genotype frequency of other varieties is 0.01; when the genotype is CT, the genotype frequency of the open-eye geese is 0.10, and the genotype frequency of other varieties is 0.17; when the genotype is TT, the genotype frequency of the open-eye geese is 0.00, and the genotype frequency of other varieties is 0.82;
HY14P site: when the genotype is CC, the genotype frequency of the open-eye geese is 0.73, and the genotype frequency of other varieties is 0.06; when the genotype is CA, the genotype frequency of the open-eye geese is 0.24, and the genotype frequency of other varieties is 0.35; when the genotype is AA, the genotype frequency of the open-eye geese is 0.03, and the genotype frequency of other varieties is 0.59;
HY15P site: when the genotype is TT, the genotype frequency of the open-eye geese is 0.88, and the genotype frequency of other varieties is 0.03; when the genotype is TC, the genotype frequency of the open-eye geese is 0.12, and the genotype frequency of other varieties is 0.28; when the genotype is CC, the genotype frequency of the open-eye geese is 0.00, and the genotype frequency of other varieties is 0.69.
7. The method of claim 4 or 6, wherein the other varieties include geese, daozhou gray geese, tinctoria white geese, lion head geese, black-bristled geese, yongkang gray geese and seedgeese.
8. A kit for identifying an open eye goose variety comprising the primer combination of claim 1 or 2.
9. Use of a primer combination according to any one of claims 1-2 or a kit according to claim 8 for identifying an open-eye goose variety.
CN202311551633.7A 2023-11-21 2023-11-21 SNP locus combination for identifying open-eye goose variety and identification method thereof Pending CN117587141A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117802248A (en) * 2024-02-29 2024-04-02 江西科技师范大学 Primer combination and method for identifying Xingguo gray goose variety

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117802248A (en) * 2024-02-29 2024-04-02 江西科技师范大学 Primer combination and method for identifying Xingguo gray goose variety
CN117802248B (en) * 2024-02-29 2024-04-30 江西科技师范大学 Primer combination and method for identifying Xingguo gray goose variety

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