A SNP molecular marker combination for identifying Jianbai Xiang pigs and an identification method

Abstract

The present invention discloses a SNP molecular marker combination for identifying Jianbai Xiang pigs and an identification method. The method provided by the present invention is to detect the 108,621,869th deoxyribonucleotide, the 108,622,346th deoxyribonucleotide, and the 108,622,414th deoxyribonucleotide on chromosome 9 of the pig reference genome Sscrofa11.1 of the pig to be tested, and determine that the genotype combination of Jianbai Xiang pigs is CC-CC-AA, and the genotype combination of deoxyribonucleotides of other pig breeds is non-CC-CC-AA. This method can effectively screen whether the breed of pigs is Jianbai Xiang pigs, effectively reduce the cost of identifying Jianbai Xiang pigs, and improve the efficiency of identifying the breed of Jianbai Xiang pigs. The detection method of the present invention is simple to operate, low in cost, high in accuracy, and can achieve automated direct detection. The present invention will play a huge role in the work of identifying the breed of Jianbai Xiang pigs.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular genetics, and in particular, to a SNP molecular marker combination for identifying Jianbai Xiang pigs and an identification method therefor. Background Art

[0002] There are 83 local pig breeds in China, which is the country with the largest number of local pig breeds in the world. In recent years, with the continuous improvement of people's living standards, high-quality pork mainly sourced from local pig breeds has become increasingly popular among consumers. Therefore, the sales of local breed pigs in the breeding pig market have also become increasingly booming. However, the identification of local pig breeds mainly relies on body shape and appearance, with low accuracy. With the progress of molecular technology, variety molecular identification based on whole-genome SNP chip genotyping technology has become the main method for current local pig breed identification (Liu R, Xu Z, Teng J, Pan X, Lin Q, Cai X, Diao S, Feng X, Yuan X, Li J, Zhang Z. Evaluation of six machine learning classification algorithms in pig breed identification using SNPs array data. Anim Genet. 2023, 54(2): 113-122.). However, the above identification method requires a large reference population of the breed, and the testing cost of the chip is relatively high. Therefore, it is particularly important to mine the unique marker combinations of pig breeds based on the genomic information of local pig breeds and develop a simpler and less costly detection method. Summary of the Invention

[0003] The purpose of the present invention is to provide a SNP molecular marker combination for identifying Jianbai Xiang pigs and an identification method therefor. Specifically, the present invention provides a method for determining the genotype by detecting the deoxyribonucleotide (DNA) sequence variation at specific gene loci of pigs, and a special kit therefor.

[0004] A SNP molecular marker combination for identifying Jianbai Xiang pigs: the homozygote of the 108,621,869th deoxyribonucleotide being C on chromosome 9 of the pig reference genome Sscrofa11.1, the homozygote of the 108,622,346th deoxyribonucleotide being C, and the homozygote of the 108,622,414th deoxyribonucleotide being A; the genotype combination of the deoxyribonucleotides at the three loci of Jianbai Xiang pigs is CC-CC-AA, and the genotype combination of the deoxyribonucleotides at these three loci of other pig breeds is non-CC-CC-AA.

[0005] A method for detecting SNP molecular marker combinations in Jianbai Xiang pigs: Based on the nucleotide sequences at the 108,621,869th, 108,622,346th, and 108,622,414th sites on chromosome 9 of the reference sequence of the international pig genome version 11.1, primer pairs are designed. Using the genomic DNA of Jianbai Xiang pigs as a template for PCR amplification, and the amplified products are subjected to sequencing analysis to convert the nucleotide sites at the 108,621,869th, 108,622,346th, and 108,622,414th sites on chromosome 9 of the reference sequence of the international pig genome version 11.1 into molecular markers; the genotype combinations of Jianbai Xiang pigs at the three sites are CC-CC-AA.

[0006] Preferably, the primer pair used for PCR amplification consists of the nucleotide shown in Sequence 1 in the sequence listing and the nucleotide shown in Sequence 2 in the sequence listing:

[0007] U (upstream primer): 5’-TTCCTACCTGCTCCCATCCC-3’ (Sequence 1 in the sequence listing) D (downstream primer): 5’-CCCTCACGTAACACAGGACC-3’ (Sequence 2 in the sequence listing).

[0008] Preferably, the detection includes the following steps:

[0009] (1) Take the tissue sample of the pig to be tested and extract the total DNA;

[0010] (2) Using the extracted genomic DNA of pigs as a template, perform PCR amplification with the primer pair consisting of the nucleotide shown in Sequence 1 in the sequence listing and the nucleotide shown in Sequence 2 in the sequence listing;

[0011] (3) Sequence the amplified products, analyze the sequencing results, and determine the genotype combinations of the 108,621,869th deoxyribonucleotide, 108,622,346th deoxyribonucleotide, and 108,622,414th deoxyribonucleotide on chromosome 9 of the pig reference genome Sscrofa11.1.

[0012] The application of the primer pair consisting of the nucleotide shown in Sequence 1 in the sequence listing and the nucleotide shown in Sequence 2 in the sequence listing in the work of identifying Jianbai Xiang pigs.

[0013] A kit for assisting in identifying the molecular characteristics of Jianbai Xiang pigs, including the primer pair consisting of the nucleotide shown in Sequence 1 in the sequence listing and the nucleotide shown in Sequence 2 in the sequence listing.

[0014] The method for identifying Jianbai Xiang pigs provided by the present invention can determine the genotype of the pig to be tested at this specific locus by using known molecular biology methods, such as sequencing analysis, PCR-SSCP method, etc. Among them, sequencing analysis includes that the products of PCR amplification contain the 108,621,869th deoxyribonucleotide, the 108,622,346th deoxyribonucleotide, and the 108,622,414th deoxyribonucleotide on chromosome 9 of the pig reference genome Sscrofa11.1. By determining whether the genotype combination of the 108,621,869th deoxyribonucleotide, the 108,622,346th deoxyribonucleotide, and the 108,622,414th deoxyribonucleotide on chromosome 9 of the pig reference genome Sscrofa11.1 of the pig to be tested is CC-CC-AA or other genotype combinations, it can be determined whether the pig to be tested is of the Jianbai Xiang pig breed. In the steps of PCR amplification and sequencing of the PCR amplification products, the primer pair used for PCR amplification meets the following conditions: the product of PCR amplification using the genomic DNA of pigs as a template contains the 108,621,869th deoxyribonucleotide, the 108,622,346th deoxyribonucleotide, and the 108,622,414th deoxyribonucleotide on chromosome 9 of the pig reference genome Sscrofa11.1. The primer pair used for PCR amplification can specifically be a primer pair composed of the nucleotide shown in sequence 1 of the sequence listing and the nucleotide shown in sequence 2 of the sequence listing. When PCR amplification is carried out using the said primer pair, depending on the genomic DNA of the pig to be tested, the amplified DNA fragment is the nucleotide sequence shown in sequence 3 or the nucleotide sequence shown in sequence 4.

[0015] The present invention also includes a kit for assisting in the identification of the Jianbai Xiang pig breed. This kit includes a primer pair composed of the nucleotides shown in sequence 1 and sequence 2 in the sequence listing. In addition, the present invention also protects the application of these primer pairs in the preparation of a kit for assisting in the detection and identification of the Jianbai Xiang pig breed. By using the above methods and kits, it can effectively identify whether it is a Jianbai Xiang pig, so as to be applied to the identification of pig breeds.

[0016] The present invention uses gene sequencing methods to detect three deoxyribonucleotide sites. Only PCR reaction and sequencing are required to determine the genotype of an individual. The genotype determination is very accurate, the detection cost is not high, and it can accurately identify whether the pig breed is Jianbai Xiang pig, which has high practical application value.

[0017] Applying the method provided by the present invention to identify pig breeds can effectively screen whether the pig breed is Jianbai Xiang pig, effectively reduce the cost of identifying Jianbai Xiang pig, and improve the efficiency of identifying the breed of Jianbai Xiang pig. The detection method of the present invention is simple to operate, low in cost, high in accuracy, and can achieve automated direct detection. The present invention will play a huge role in the work of identifying the breed of Jianbai Xiang pig.

[0018] In summary, the present invention has broad application prospects in the actual production of identifying the breed of Jianbai Xiang pig, and can effectively solve the problems of long time and high cost in identifying the breed of Jianbai Xiang pig. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a reverse sequencing map of two homozygotes for the 108621869th deoxyribonucleotide on chromosome 9;

[0020] Figure 2 It is a reverse sequencing map of two homozygotes for the 108622346th deoxyribonucleotide on chromosome 9;

[0021] Figure 3 It is a reverse sequencing map of two homozygotes for the 108622414th deoxyribonucleotide on chromosome 9. DETAILED DESCRIPTION OF THE INVENTION

[0022] The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all obtained from regular biochemical reagent stores unless otherwise specified. The primers in the following examples are all synthesized by Beijing Liuhe Huada Gene Technology Co., Ltd.

[0023] Jianbai Xiang pig: This pig is produced by farmers in Jianhe County, Qiandongnan Miao and Dong Autonomous Prefecture, Guizhou Province.

[0024] Example 1: Specific screening of detection site combinations

[0025] I. PCR amplification and sequencing typing of three polymorphic sites on pig chromosome 9

[0026] 1. PCR amplification

[0027] According to the genomic DNA sequence information of the 108621869th deoxyribonucleotide, the 108622346th deoxyribonucleotide, and the 108622414th deoxyribonucleotide on pig chromosome 9, a pair of primers is designed as follows:

[0028] U (upstream primer): 5’-TTCCTACCTGCTCCCATCCC-3’ (Sequence 1 of the sequence listing); D (downstream primer): 5’-CCCTCACGTAACACAGGACC-3’ (Sequence 2 of the sequence listing).

[0029] Three Jianbai Xiang pigs were selected as experimental materials. Using the genomic DNA of Jianbai Xiang pigs as a template, PCR amplification was performed using primers U and D.

[0030] The amplification system was 25 μL: 12.5 μL of 2×Phanta Max Buffer, 1 μL of DNA, 0.5 μL of DNA Polymerase, 0.5 μL of dNTPs, 1 μL each of the upstream and downstream primers, and 8.5 μL of sterile water.

[0031] The PCR reaction conditions were as follows: denaturation at 95°C for 5 min; then denaturation at 95°C for 20 s, annealing at 58°C for 30 s, extension at 72°C for 45 s, for a total of 35 cycles; and finally extension at 72°C for 10 min.

[0032] The PCR products were detected by agarose gel and stored at -20°C. The products amplified using the genomic DNA of three Jianbai Xiang pigs as a template were named Product 1.

[0033] 2. Sequencing and sequence analysis

[0034] This kind of PCR product was sent to Beijing Liuhua Huada Gene Technology Co., Ltd. The sequencing results showed that: Product 1 was Sequence M1, and the length of Sequence M1 was 1333 bp. The genotypes of the 108,621,869th deoxyribonucleotide, the 108,622,346th deoxyribonucleotide, and the 108,622,414th deoxyribonucleotide on chromosome 9 of the pig reference genome Sscrofa11.1 were combined as CC-CC-AA (see Figure 1 ), and the genotype combination of these three loci was named JBX-CCA.

[0035] II. Identification and application of the specific locus combination JBX-CCA in multiple pig breeds

[0036] To determine whether the 108,621,869th deoxyribonucleotide, the 108,622,346th deoxyribonucleotide, and the 108,622,414th deoxyribonucleotide on chromosome 9 of Sscrofa11.1 are related to the identification of Jianbai Xiang pigs, Jianbai Xiang pigs and 13 other pig breeds were used as experimental materials for the following experiment: Genomic DNA was extracted for sequencing analysis, and the method was the same as in Step 1;

[0037] The results are shown in Table 1.

[0038] Table 1 Identification of JBX-CCA polymorphic loci in Jianbai Xiang pigs and 13 other pig breeds

[0039]

[0040]

[0041]

[0042]

[0043]

[0044] The results showed that after detecting the genotype combinations of three polymorphic loci in Jianbai Xiang pigs and multiple indigenous pig breeds such as Duroc pigs, Erhua pigs, and Guanling pigs, only the genotype combination of Jianbai Xiang pigs was CC-CC-AA. Therefore, this method is extremely effective for specifically detecting the Jianbai Xiang pig breed.

Claims

1. A method for identifying Jianbai Xiang pig breed, characterized in that: The steps include: (1) Take the pig tissue sample to be tested and extract total DNA; (2) Using the extracted total DNA as a template, PCR amplification is performed using a primer pair consisting of the nucleotides shown in Sequence 1 and the nucleotides shown in Sequence 2; the sequence of the primer pair is as follows: Upstream primer: 5'- TTCCTACCTGCTCCCATCCC-3', Downstream primer: 5′-CCCTCACGTAACACAGGACC -3′; The amplified product was sequenced, and the sequencing results were analyzed to determine the genotype combination of the 108621869th deoxyribonucleotide, the 108622346th deoxyribonucleotide, and the 108622414th deoxyribonucleotide on chromosome 9 of the pig reference genome Sscrofa11.1; The Jianbai Xiang pig is homozygous for C at deoxyribonucleotide position 108621869, C at deoxyribonucleotide position 108622346, and A at deoxyribonucleotide position 108622414 on chromosome 9 of the pig reference genome Sscrofa11.1 for the Jianbai Xiang pig; the genotype combination of deoxyribonucleotides at positions 108621869, 108622346, and 108622414 on chromosome 9 of the pig reference genome Sscrofa11.1 for the Jianbai Xiang pig is CC-CC-AA, while the genotype combination of deoxyribonucleotides at these three sites of other breeds of pigs is non-CC-CC-AA.

2. The identification method according to claim 1, characterized in that: The sequence length of the amplified product obtained in step (2) is 1333 bp.

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