CN116622858A - SNP locus primer combination for identifying bamboo-rural chicken variety and application thereof - Google Patents
SNP locus primer combination for identifying bamboo-rural chicken variety and application thereof Download PDFInfo
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Abstract
The invention discloses a set of SNP locus primer combination for identifying bamboo parturient chicken varieties and application thereof. The primer combination amplifies any 4 to 8 selected from the ZX1 locus to the ZX8 locus. When the SNP locus primer combination provided by the invention is adopted for identifying the chicken variety in the Zhuxiang province, the individual can be completely excluded from belonging to the Zhuxiang chickens as long as one SNP locus genotype does not accord with the corresponding genotype of the Zhuxiang chickens; optionally 4 SNP locus combinations, and the judging probability conforming to the genotype of the Zhuxiang chicken can reach more than 98 percent. The method is simple and convenient to operate, has high accuracy and can effectively strike counterfeit bamboo-country chicken products in the market.
Description
Technical Field
The invention relates to a poultry variety identification method, in particular to a set of SNP locus primer combination for identifying a bamboo-country chicken variety and application thereof.
Background
The Zhuxiang chicken is one of the local varieties with excellent characteristics in Guizhou, and is produced in the red water city of Nanzhu in northern Guizhou, and belongs to the black-bone chicken series. The main characteristics of the bamboo-country chicken are black feather, black skin, black meat and black bone of the whole body, and the bamboo-country chicken is rich in eighteen amino acids, and the bamboo-country chicken is determined to contain 3.60% of glutamic acid, 1.49% of alanine, 1.10% of valine, 0.68% of methionine, 1.93% of leucine and 1.07% of isoleucine, which are 1.7 times higher than common chicken. The bamboo-rural chicken has delicious quality, high medicinal value, coarse feeding resistance and strong adaptability, has obvious effects of resisting fatigue, improving hypoxia resistance, resisting aging and improving immunity, and is deeply favored by vast breeders and consumers. Because of the remarkable economic value of the bamboo-country chickens, a plurality of masquerading varieties of the bamboo-country chickens which are mixed with other varieties at home and abroad appear on the market, the body type and appearance of the varieties are similar to those of the bamboo-country chickens, consumers can hardly distinguish the varieties, the interests of vast farmers are damaged, and meanwhile, the seed preservation and breeding of the stock of the bamboo-country chickens are influenced.
The current method for identifying the authenticity of the Zhuxiang chickens in the market mainly depends on appearance observation, has strong subjectivity, and has high identification error rate due to similar appearance when identifying hybrid offspring of the Zhuxiang chickens and other varieties of chickens.
Disclosure of Invention
Aiming at the problem of high error rate of identifying the Zhuxiang chickens in the appearance observation mode, the invention provides a set of SNP locus primer combination for identifying the Zhuxiang chickens and application thereof, and the genotype of 8 SNP loci of the Zhuxiang chickens is detected to jointly judge whether an individual to be detected belongs to the Zhuxiang chickens. The method is simple and convenient to operate, has high accuracy, can accurately identify the bamboo-country chicken variety, and effectively strikes against counterfeit and inferior products of the bamboo-country chicken local variety on the market.
In order to achieve the above object, the present invention provides, in one aspect, a set of SNP locus primer combinations for identification of Zhuxiang chicken breeds, which amplify any 4 to 8 of the following ZX1 loci to ZX8 loci:
the nucleotide sequence of each SNP site primer is as follows:
the screening method of the SNP locus comprises the following steps: screening and determining 55000 SNP loci of 12 chicken breeds by utilizing a chip, comparing each breeder with other breeds, collecting loci with the largest difference, screening the first 1% of genetic differentiation indexes among groups, the smallest allele frequency being more than 0.4 and the deletion rate being less than 0.2, determining 68 SNP loci, preferentially selecting the allele frequency of a reference genome locus of the Zhuxiang chicken to be 0 or 1, determining the average value of different allele frequencies of each locus of other 11 breeds, comparing the average value with the different allele frequencies of each locus of the Zhuxiang chicken, screening SNP loci with larger allele frequency difference, selecting loci with the allele frequency being close to 1 or close to 0 in other groups, finally determining 8 SNP loci for identifying the Zhuxiang chicken breeds,
wherein the 12 chicken species comprise Zhuxiang chicken, black-bone chicken, xingwu black-bone chicken, wu Mengfeng chicken, jinhu black-bone chicken, silk feather black-bone chicken, qian southeast chicken, low-foot chicken, high-foot chicken, weining chicken, hubert chicken and Lavender chicken.
The invention also provides application of the SNP locus primer combination in identification of Zhuxiang chicken varieties.
Specifically, the authentication method includes the steps of:
(1) Extracting total DNA of a chicken genome to be detected;
(2) Respectively carrying out PCR amplification by using corresponding primers according to the selected SNP combination;
(3) Sequencing the PCR amplification product and judging the genotype;
(4) And judging whether the individual belongs to the Zhuxiang chicken variety or not according to genotype results.
The specific judging method in the step (4) is as follows: if the genotype corresponding to the selected SNP combination does not accord with the genotype corresponding to the Zhuxiang chicken, judging that the individual does not belong to the Zhuxiang chicken; if the genotypes corresponding to the selected at least 4 SNP locus combinations all accord with the genotypes corresponding to the Zhuxiang chickens, the probability that the individual belongs to the Zhuxiang chickens is judged according to the Bayesian theorem.
Preferably, the genomic DNA of the chicken to be tested in the step (1) is obtained by blood sampling from the individual ptera veins of the chicken species.
In the step (2), the lengths of the PCR products of ZX 1-ZX 8 are 171 bp, 192 bp, 200 bp, 222bp, 235bp, 217 bp, 200 bp and 205 bp respectively.
Preferably, genotypes of 8 SNP loci ZX1 to ZX8 in the Zhuxiang chicken are AA, AA, AA, GG, GG, CC, AA, AA in sequence.
Through the technical scheme, the invention has the following beneficial effects:
when the SNP locus primer combination is adopted to identify the chicken variety in Zhuxiang, the probability of judging the genotype of any 1 SNP locus as the chicken in Zhuxiang is 72.53 percent and the probability is 83.31 percent at most; the probability of judging the chicken in the bamboo country by combining genotypes of any 2 SNP loci reaches more than 85%; the probability of judging the combination genotype of any 3 SNP loci to be the bamboo-homed chicken reaches more than 94%; the probability of judging the combination genotype of any 4 SNP loci to be the bamboo-homed chicken reaches more than 98.88 percent; the probability of judging the chicken in the bamboo country by combining genotypes of any 5 SNP loci reaches more than 99.60 percent; the probability of judging the combination genotype of any 6 SNP loci to be bamboo-homed chickens reaches more than 99.92%; the probability of judging the combination genotype of any 7 SNP loci to be bamboo-homed chickens reaches more than 99.98 percent; the probability of judging the combined genotype of all 8 SNP loci to be the bamboo partridge chicken is 100%. The probability of excluding the chicken in the bamboo county by any SNP locus genotype reaches 100 percent.
Therefore, as long as one SNP locus genotype does not accord with the corresponding genotype of the Zhuxiang chicken, the individual can be completely excluded from belonging to the Zhuxiang chicken; optionally 4 SNP locus combinations, and the judging probability conforming to the genotype of the Zhuxiang chicken can reach more than 98 percent. The method is simple and convenient to operate, has high accuracy and can effectively strike counterfeit bamboo-country chicken products in the market.
Detailed Description
Aiming at the problem of difficult identification of the bamboo partridge chicken variety, the invention provides a set of SNP locus primer combination for identifying the bamboo partridge chicken variety and application thereof, wherein the primer combination amplifies any 4 to 8 kinds of the primer combination from ZX1 locus to ZX8 locus.
The screening method of the 8 SNP loci comprises the following steps: selecting and determining 55000 SNP loci of different chicken varieties (Zhuxiang chicken, wuMongolian black-bone chicken, xingwu black-bone chicken, wu Mengfeng chicken, jinhu black-bone chicken, silk feather black-bone chicken, qian southeast chicken, low-foot chicken, high-foot chicken, weining chicken and other 10 domestic representative chicken varieties and foreign representative introduction of Habodied and Lato-be-hybridized chicken varieties, adding 12 varieties) by using Beijing core first chip (55K, beijing Kang Pusen biotechnology limited company), comparing each variety with other varieties, taking a set of loci with the largest difference, selecting 1% of the Minimum Allele Frequency (MAF) >0.4 before the inter-population genetic differentiation index (Fst), determining 68 SNP loci, preferentially selecting the allele frequency of a reference genome locus of the Zhuxiang chicken to be 0 or 1 in order to further select the core locus information of the Zhuxiang chicken, then determining the allele frequency of each of other 11 varieties to be close to the average value of the N, comparing the average value of the allele with the average value of the N1 of the other 11 varieties, and determining that the average value of the allele frequency of each of the other varieties is close to the average value of the N or the N1, and the average value of the N is 8. The information of 8 SNP loci is shown in the following table.
Based on the physical location of 8 SNP loci, based on the red chicken reference genome sequence (Galgal 5), 8 SNP locus DNA sequences were obtained using UCSC website https:// genome-asia. UCSC. Edu/cgi-bin/hggateway wayhgsild=793043_MvNmFj6QPMRQsauOxQgq3gPtEUU, and primers were designed using Primer 5.0. The sequence of each SNP primer, the length of PCR product and annealing temperature are shown in the following table:
the specific method for identifying the Zhuxiang chicken variety by using the SNP primer combination comprises the following steps:
(1) Extracting total DNA of a chicken genome to be detected;
(2) Respectively carrying out PCR amplification by using corresponding primers according to the selected SNP combination;
(3) Sequencing the PCR amplification product and judging the genotype;
(4) And judging whether the individual belongs to the Zhuxiang chicken variety or not according to genotype results.
In the identification method of the present invention, it is preferable to extract genomic DNA from whole blood obtained by taking a blood sample from a fin vein of a chicken to be tested, the extraction method of genomic DNA is not particularly limited, and a conventional extraction method in the art may be adopted, and in the embodiment of the present invention, a phenol-chloroform method is preferable to extract genomic DNA.
In the invention, one or a plurality of sites of ZX 1-ZX 8 are adopted for identifying the bamboo partridge chicken variety during application, and corresponding upstream and downstream primers are adopted for PCR amplification. PCR amplification is carried out by methods conventional in the art.
In the invention, genotypes of 8 SNP loci ZX1 to ZX8 in the Zhuxiang chicken are AA, AA, AA, GG, GG, CC, AA, AA in sequence.
Comparing the genotypes corresponding to the selected SNP combination by sequencing the PCR amplified products of the chickens to be detected and judging the genotypes, and judging that the individual does not belong to the Zhuxiang chickens if one genotype does not correspond to the Zhuxiang chickens; if all genotypes corresponding to the selected SNP combination accord with the genotypes corresponding to the Zhuxiang chickens, judging the probability that the individual belongs to the Zhuxiang chickens according to the Bayesian theorem. The calculation formula is as follows:
wherein,,Piindicating the first item in SNP combinationiThe frequencies of the genotypes of the other varieties in the SNPs.
The genotype frequencies of the SNP loci in Zhuxiang chickens and other varieties are as follows:
for example, if the combined genotype is AAGGCCAA, the probability that the individual belongs to a bamboo-homed chicken is p=1/(1+0.28×0.30×0.38×0.20) = 0.9936, with a total of 4 SNP loci selected from ZX2, ZX4, ZX6, and ZX 8.
The genotype frequencies of other varieties refer to genotype frequency average values of each site obtained by combining and calculating 11 domestic and foreign representative varieties such as silky fowl, mongolian fowl, xingwu fowl, wu Mengfeng chicken, southeast Qian chicken, habert and Lato chicken.
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
Randomly selecting 30 Zhuxiang chickens, 30 Mongolian black-bone chickens, 30 Xingwu black-bone chickens, 30 Wu Mengfeng chickens, 30 silk feather black-bone chickens, 30 golden lake black-bone chickens and 30 Hubert chickens which are preserved in a key laboratory poultry germplasm resource genetic material library in Jiangsu province, taking blood from the veins of the wings, extracting genome DNA by a phenol-chloroform method, and selecting primers of 4 SNP loci in total of ZX1, ZX3, ZX5 and ZX7 for PCR amplification.
50. Mu.L of the total PCR reaction system: DNA template 2. Mu.L, dNTP (2 mmol/L) 4. Mu.L, mg 2+ （3 mmol·L -1 ) 0.6. Mu.L of 1 XPCR reaction buffer 5. Mu.L of upstream and downstream primers (10. Mu. Mol. L) -1 ) 1. Mu.L of each Taq polymerase (1U. Mu.L) -1 ) 2.5. Mu.L, and ultrapure water was added to 50. Mu.L.
PCR reaction procedure: pre-denaturation at 95 ℃ for 5 min; denaturation at 94℃for 30s, annealing at 55℃for 30s, extension at 72℃for 30s for a total of 35 cycles; extending at 72℃for 5 min. The target fragment amplified by PCR was detected by 1.5% agarose gel electrophoresis.
The PCR products were sent to Bio Inc. for sequencing, and the sequences were subjected to polymorphism detection and genotyping. According to the sequencing result, the probability that 30 individuals with the combined genotype of 4 sites of AAAAGGAA are judged as the Zhuxiang chicken is P=1/(1+0.17x0.23 x0.35 x0.24) = 0.9966, and the 30 individuals can be all identified as the Zhuxiang chicken, and the identification accuracy is 100%. All the other individuals did not fully meet the AAAAGGAA genotype, and therefore were judged as non-bamboo chickens.
Example 2
The other conditions were the same as in example 1, except that PCR amplification was performed using primers for 6 SNP sites corresponding to ZX1, ZX2, ZX3, ZX4, ZX7, and ZX 8. According to the sequencing result, the probability that 25 individuals with 6 loci combined with the genotype AAAAAAGGAAAA are judged as the Zhuxiang chicken is P=1/(1+0.17×0.24×0.23×0.36×0.24×0.23) =0.9998, and 99.98% can identify all 30 individuals as the Zhuxiang chicken, and the identification accuracy is 100%. All the other individuals did not completely meet the AAAAAAGGAAAA genotype, and therefore were judged as non-bamboo chickens.
Example 3
Other conditions were the same as in example 1, except that primers of 8 SNP loci corresponding to ZX1 to ZX8 were used for PCR amplification. According to the sequencing result, the probability that 30 individuals with the combined genotype of 8 sites of AAAAAAGGGGCCAAAA are judged as the Zhuxiang chicken is P=1/(1+0.17×0.24×0.23×0.36×0.35×0.49×0.23) =1.0000, and 25 individuals can be totally identified as the Zhuxiang chicken by 100%, and the identification accuracy is 100%. All the other individuals did not completely meet the AAAAAAGGGGCCAAAA genotype, and therefore were judged as non-bamboo chickens.
The preferred embodiments of the present invention have been described in detail above with reference to the examples, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solutions of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.
In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.
Moreover, any combination of the various embodiments of the invention can be made without departing from the spirit of the invention, which should also be considered as disclosed herein.
Claims (9)
1. A set of SNP locus primer combinations for identifying bamboo-rural chicken varieties, which is characterized in that the primer combinations amplify any 4 to 8 of the following ZX1 loci to ZX8 loci:
ZX1 is located at position 89881165 on NC_006091.4 chromosome 4, which is a C or A polymorphism;
ZX2 is located at position 14963591 on NC_006093.4 chromosome 6, which is a T or A polymorphism;
ZX3 is located at position 56394647 on NC_006092.4 chromosome 5, which is a G or A polymorphism;
ZX4 is located at position 7609934 on chromosome 19 of NC_006106.4, which is an A or G polymorphism;
ZX5 is located at position 91034398 on NC_006090.4 chromosome 3, which is a T or G polymorphism;
ZX6 is located at position 4782894 on chromosome 13 of NC_006100.4, which is a T or C polymorphism;
ZX7 is located at position 47655290 on NC_006091.4 chromosome 4, which is a G or A polymorphism;
ZX8 is located at position 3144758 on chromosome 27 of NC_006114.4, which is a G or A polymorphism.
2. The SNP site primer combination of claim 1, wherein the nucleotide sequence of each SNP site primer is:
ZX1-F：5’GGGTTATTGTCCTGGTTTGC 3’
ZX1-R：5’TGTGGAGTCACGTGGAAGTC 3’
ZX2-F：5’ACTGCCCACCAACTGTCTCT 3’
ZX2-R：5’TTCTCCTCACCACCTGTTCC 3’
ZX3-F：5’GCCAGTCTGCAACCCTTAAA 3’
ZX3-R：5’TTGGATGCAGGAGGTAAAGC 3’
ZX4-F：5’ACCAAAGTCAGCCTGGAGAA 3’
ZX4-R：5’CTGAAGCTCACCAACAACCA 3’
ZX5-F：5’CAGCCAATGCTACAGAGCAC 3’
ZX5-R：5’ATCAAAGTGCTGGTCATCCA 3’
ZX6-F：5’AAGGTGACAGCTGGTTTGCT 3’
ZX6-R：5’AGGCTGCATCTGAGAAGTGG 3’
ZX7-F：5’AGAATGAACAGCCTGTGGTG 3’
ZX7-R：5’ATTCCTGCCCTGTCTTCCTT 3’
ZX8-F：5’TCTCCGAGAGGCAGACAGTT 3’
ZX8-R：5’ATTCGTGCTGCAGTGATGAG3’。
3. the SNP site primer combination according to claim 1 or 2, wherein the SNP site screening method is as follows: screening and determining 55000 SNP loci of 12 chicken breeds by utilizing a chip, comparing each breeder with other breeds, collecting loci with the largest difference, screening the first 1% of genetic differentiation indexes among groups, the smallest allele frequency being more than 0.4 and the deletion rate being less than 0.2, determining 68 SNP loci, preferentially selecting the allele frequency of a reference genome locus of the Zhuxiang chicken to be 0 or 1, determining the average value of different allele frequencies of each locus of other 11 breeds, comparing the average value with the different allele frequencies of each locus of the Zhuxiang chicken, screening SNP loci with larger allele frequency difference, selecting loci with the allele frequency being close to 1 or close to 0 in other groups, finally determining 8 SNP loci for identifying the Zhuxiang chicken breeds,
wherein the 12 chicken species comprise Zhuxiang chicken, black-bone chicken, xingwu black-bone chicken, wu Mengfeng chicken, jinhu black-bone chicken, silk feather black-bone chicken, qian southeast chicken, low-foot chicken, high-foot chicken, weining chicken, hubert chicken and Lavender chicken.
4. Use of the SNP locus primer combination as set forth in any one of claims 1 to 3 in the identification of Zhuxiang chicken variety.
5. The use according to claim 4, wherein the authentication method comprises the steps of:
(1) Extracting total DNA of a chicken genome to be detected;
(2) PCR amplification of each of the sets of primers corresponding to claim 2 according to the selected SNP combination;
(3) Sequencing the PCR amplification product and judging the genotype;
(4) And judging whether the individual belongs to the Zhuxiang chicken variety or not according to genotype results.
6. The use according to claim 5, wherein the genomic DNA of the chicken to be tested in step (1) is obtained by taking blood from the individual fin veins of the chicken species.
7. The use according to claim 5, wherein the genotypes of the 8 SNP loci ZX1 to ZX8 in the Zhuxiang chicken are AA, AA, AA, GG, GG, CC, AA, AA in sequence.
8. The method according to claim 5, wherein in step (2), the PCR products of ZX1 to ZX8 have lengths of 171 bp, 192 bp, 200 bp, 222bp, 235bp, 217 bp, 200 bp, 205 bp, respectively.
9. The use according to claim 5, wherein in step (4), the judging method comprises: if the genotype corresponding to the selected SNP combination does not accord with the genotype corresponding to the Zhuxiang chicken, judging that the individual does not belong to the Zhuxiang chicken; if the genotype of the selected combination of at least 4 SNP loci accords with the genotype corresponding to the Zhuxiang chicken, judging the probability that the individual belongs to the Zhuxiang chicken according to the Bayes theorem.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN118166124A (en) * | 2024-04-23 | 2024-06-11 | 江苏省家禽科学研究所 | SNP locus combination, primer combination and method for identifying high-foot chicken variety |
| CN118207342A (en) * | 2024-04-22 | 2024-06-18 | 江苏省家禽科学研究所 | SNP locus combination, primer combination and method for identifying low-foot chicken variety |
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| CN118207342A (en) * | 2024-04-22 | 2024-06-18 | 江苏省家禽科学研究所 | SNP locus combination, primer combination and method for identifying low-foot chicken variety |
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