Method for identifying reproduction performance of Tibetan pigs, primer pair and application

Abstract

The present application relates to the field of biotechnology, in particular to a method for identifying the reproductive performance of Tibetan pigs, primer pairs and application. The method of the present application uses Ssc14:8587185C>T, Ssc13:21904604C>T and Ssc16:75075241A>G three loci to identify the reproductive performance of Tibetan pigs, which improves the accuracy of breeding selection, shortens the breeding cycle of Tibetan pigs, and improves the production efficiency.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a method, primer pair, and application for identifying the reproductive performance of Tibetan pigs. Background Technology

[0002] A normal sow consumes about 450 kg of feed in a 153-day reproductive cycle. The feed, facility, labor, and utilities consumed are all initial investments for piglet production. Tibetan pigs are no exception. Although they are smaller and consume slightly less feed, their reproductive cycle is longer, generally reaching 190-200 days. Therefore, the reproductive performance of sows, including Tibetan pigs, is a key factor affecting the overall economic benefits of sows.

[0003] Since reproductive performance is inherently a low-heritability trait, it is difficult to achieve good breeding results using traditional breeding methods. Therefore, molecular selection of reproductive performance using modern biological techniques is a crucial issue that urgently needs to be addressed. To conduct molecular selection, selecting a limited number of molecular markers and performing marker-assisted selection (MAS) is an effective method to improve the breeding results of low-heritability traits. Screening for the genetic markers of these traits is a prerequisite for effective MAS. However, for local pig breeds, due to the unique genetic background, conventional methods of finding effective SNPs are less effective.

[0004] How to effectively identify or screen Tibetan pigs with high reproductive performance is an urgent problem to be solved. Summary of the Invention

[0005] To shorten the breeding cycle of Tibetan pigs and improve production efficiency, this invention provides a method, primer pair, and application for identifying the reproductive performance of Tibetan pigs.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] This invention provides a method for identifying the reproductive performance of Tibetan pigs. The method includes: extracting genomic DNA from Tibetan pigs and identifying the reproductive performance of Tibetan pigs by identifying the genotypes at three loci: Ssc14:8587185C>T, Ssc13:21904604C>T, and Ssc16:75075241A>G.

[0008] Individuals with Ssc14:8587185C>T genotype CC, Ssc13:21904604C>T genotype CC and Ssc16:75075241A>G genotype GG had higher total litter size and live litter size than those with other genotypes.

[0009] The Ssc14:8587185C>T, Ssc13:21904604C>T, and Ssc16:75075241A>G sites are all genomic locations and specific mutations in SusScorfa version 11.1.

[0010] This invention provides a primer pair for identifying the reproductive performance of Tibetan pigs, the primer pair comprising the following primers:

[0011] Primer pair 1, used to amplify the sequence containing the Ssc14:8587185C>T site (as shown in SEQ ID NO:1):

[0012] F1: 5'TGCTGCCCTATGAGT 3' (SEQ ID NO: 2),

[0013] R1: 5'TGAGGCTATCTGAGTTT 3' (SEQ ID NO: 3);

[0014] Primer pair 2 is used to amplify the sequence containing the Ssc13:21904604C>T site:

[0015] F2: 5'GCTGGAGGATTGGTAGG 3' (SEQ ID NO: 4),

[0016] R2: 5'GAGTGGGCTGTGCTTCT 3' (SEQ ID NO: 5);

[0017] Primer pair 3 is used to amplify the sequence containing the Ssc16:75075241A>G site:

[0018] F3: 5'TGTCGAGGTTTATGCA 3' (SEQ ID NO: 6),

[0019] R3: 5'GGAGTTTCCTGGTTGAT 3' (SEQ ID NO:7).

[0020] The present invention also provides a kit for identifying the reproductive performance of Tibetan pigs, the kit comprising the primer pairs described above.

[0021] This invention also provides the application of the primer pairs or kits described above in the identification and / or breeding of the reproductive performance of Tibetan pigs.

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] Using genome-wide microarray data to identify effective SNP loci in local pig breeds is a preferred method for obtaining a large number of accurate genetic markers for reproductive performance. The applicant selected Tibetan pigs from Tibet, Gansu, and Qinghai provinces and conducted genome-wide association analysis using Illumina SNP50 microarray data with total litter size, live litter size, and healthy litter size. This revealed several significantly associated SNP loci. Further validation analysis showed that some SNP loci were less effective when used alone. To better utilize these SNP loci, this invention employs a combined approach using the three loci Ssc14:8587185C>T, Ssc13:21904604C>T, and Ssc16:75075241A>G to identify the reproductive performance of Tibetan pigs. The combination of these three loci with the CCCCGG genotype showed superior breeding performance compared to other genotypes. This invention provides a more accurate and effective method for identifying the reproductive performance of Tibetan pigs. Attached Figure Description

[0024] Figure 1 This is a sequencing peak diagram of the Ssc14:8587185C>T site. Detailed Implementation

[0025] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, 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 pertains.

[0026] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0027] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0028] Example 1

[0029] 126 Tibetan pigs were selected from multiple locations in Tibet, Gansu, and Qinghai, and 89 of them were used for microarray sequencing. The SNP quality control conditions and sample quality control results are described in the patent application filed on the same day, "SNP Locus Combinations, Reagent Kits and Applications for Identifying Reproductive Performance of Tibetan Pigs," and will not be discussed in detail here.

[0030] Association analyses of the total litter size and live litter size traits of Tibetan pigs using GLM, GLM(Q), MLM(K), and MLMQK models revealed several significantly associated SNP loci, including Ssc14:8587185C>T, Ssc13:21904604C>T, and Ssc16:75075241A>G. The association analysis results for Ssc13:21904604C>T and Ssc16:75075241A>G are documented in the patent application filed on the same day, "Molecular Markers, Primer Pairs, and Applications Related to the Fertility Performance of Tibetan Pigs," and will not be described in detail here. The association results for Ssc14:8587185C>T are shown in Table 1 below.

[0031] Table 1. Genome-wide association significance analysis of total litter size and live litter size in Tibetan pigs.

[0032]

[0033] 1 Chr. = chromosome; 2 P-val. = P value

[0034] Genomic DNA was collected from the above 126 Tibetan pigs, and the associations of the Ssc14:8587185C>T, Ssc13:21904604C>T and Ssc16:75075241A>G sites were verified.

[0035] DNA was extracted from pig ear tissue, and PCR amplification was performed using the pig ear DNA as a template and the primers described below.

[0036] F1: 5'TGCTGCCCTATGAGT 3',

[0037] R1: 5'TGAGGCTATCTGAGTTT 3';

[0038] F2: 5'GCTGGAGGATTGGTAGG 3",

[0039] R2: 5'GAGTGGGCTTGTGCTTCT 3';

[0040] F3: 5'TGTCGAGGTTTATGCA 3',

[0041] R3: 5'GGAGTTTCCTGGTTGAT 3'.

[0042] The PCR amplification products were sequenced. Information on the Ssc14:8587185C>T site mutation is shown in Table 2 below. Figure 1 As shown.

[0043] Table 2. Information on mutation site location and mutation type.

[0044]

[0045] The genotype of pigs and the phenotypic values ​​of total litter size and live litter size of Tibetan pigs were analyzed using the least squares method. The specific method has been disclosed in the existing technology and will not be discussed in detail here.

[0046] (1) Unit point association results

[0047] The association results of the Ssc14:8587185C>T site are shown in Table 3 below.

[0048] Table 3. Association Analysis of Ssc14:8587185C>T with Reproductive Traits of Tibetan Pigs

[0049]

[0050] Note: Data in the same column with the same letter or no letter in the superscript indicate no significant difference (P>0.05), while different letters indicate significant difference (P<0.05). The same applies to the following table.

[0051] The total litter size of CT-type individuals at the Ssc14:8587185 locus reached 6.15, with 6.14 live offspring, which can improve production performance.

[0052] The association analysis results of Ssc13:21904604C>T and Ssc16:75075241A>G sites are described in the patent "Molecular Markers, Primer Pairs and Their Applications Related to Fertility Performance of Tibetan Pigs".

[0053] (2) Two-site joint analysis

[0054] The results of the joint analysis of Ssc14:8587185 and Ssc13:21904604 sites are shown in Table 4 below:

[0055] Table 4. Association analysis of Ssc14:8587185C>T and Ssc13:21904604C>T with reproductive traits of Tibetan pigs.

[0056]

[0057] Joint analysis of Ssc14:8587185 and Ssc13:21904604 loci showed good breeding effects for CCCC, CTCC, and CTCT individuals. CCCC breeding increased the total litter size of Tibetan pigs from an average of 5.8 to 8.6, and the number of live piglets from 5.4 to 7.8. CTCC breeding increased the total litter size of Tibetan pigs from an average of 5.8 to 8.0, and the number of live piglets from 5.4 to 8.0. CTCT breeding increased the total litter size of Tibetan pigs from an average of 5.8 to 6.3, and the number of live piglets from 5.4 to 6.3.

[0058] The results of the joint analysis of Ssc14:8587185 and Ssc16:75075241 sites are shown in Table 5 below.

[0059] Table 5. Association analysis of Ssc14:8587185C>T and Ssc16:75075241A>G with reproductive traits of Tibetan pigs.

[0060]

[0061] Joint analysis of Ssc14:8587185 and Ssc16:75075241 loci showed that the breeding effects of CCGG and CTGG individuals were better: CCGG individual breeding increased the total number of piglets born to an average of 5.8 to 6.6, and the number of live piglets born increased from 5.4 to 5.9. CTGG individual breeding increased the total number of piglets born to an average of 5.8 to 6.7, and the number of live piglets born increased from 5.4 to 6.7.

[0062] The combined analysis of Ssc13:21904604C>A and Ssc16:75075241A>G sites showed that the CCGG individual breeding effect was the best among the genotypes. Through CCGG individual breeding, the total number of piglets born to Tibetan pigs could be increased from an average of 5.8 to 8.9, and the number of live piglets born could be increased from 5.4 to 8.1.

[0063] (3) Three-point joint analysis

[0064] The results of the joint analysis of Ssc14:8587185, Ssc13:21904604 and Ssc16:75075241 sites are shown in Table 6 below.

[0065] Table 6. Association analysis of Ssc14:8587185C>T, Ssc13:21904604C>T, and Ssc16:75075241A>G with reproductive traits.

[0066]

[0067] Joint analysis of the Ssc14:8587185, Ssc13:21904604, and Ssc16:75075241 loci showed good breeding effects for individuals with the genotypes CCCCGG, CCCTGG, CTCCGG, CTCTGG, and CTTTGG. The joint analysis revealed that breeding of individuals with the genotype CCCCGG increased the total litter size of Tibetan pigs from an average of 5.8 to 9.5, and the number of live piglets from 5.4 to 8.5. This genotype showed superior breeding performance compared to other genotypes. The three-locus combination of Ssc14:8587185C>T, Ssc13:21904604C>T, and Ssc16:75075241A>G effectively identified the reproductive performance of Tibetan pigs.

[0068] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for identifying the reproductive performance of Tibetan pigs, characterized in that, Genomic DNA was extracted from Tibetan pigs, and their reproductive performance was assessed by identifying the genotypes at three loci: Ssc14:8587185C>T, Ssc13:21904604C>T, and Ssc16:75075241A>G. Individuals with Ssc14:8587185C>T genotype CC, Ssc13:21904604C>T genotype CC and Ssc16:75075241A>G genotype GG had higher total litter size and live litter size than those with other genotypes. The Ssc14:8587185C>T, Ssc13:21904604C>T, and Ssc16:75075241A>G sites are all genomic locations and specific mutations in SusScorfa version 11.

1.

2. A primer pair for identifying the reproductive performance of Tibetan pigs, characterized in that, The primer pair includes the following primers: Primer pair 1, used to amplify the sequence containing the Ssc14:8587185C>T site: F1: 5' TGCTGCCCTATGAGT 3', R1: 5' TGAGGCTATCTGAGTTT 3'; Primer pair 2 is used to amplify the sequence containing the Ssc13:21904604C>T site: F2: 5' GCTGGAGGATTGGTAGG 3'', R2: 5' GAGTGGGCTGTGCTTCT 3'; Primer pair 3 is used to amplify the sequence containing the Ssc16:75075241A>G site: F3: 5' TGTCGAGGTTTATGCA 3', R3: 5' GGAGTTTCCTGGTTGAT 3'.

3. A reagent kit for identifying the reproductive performance of Tibetan pigs, characterized in that, The kit contains the primer pair as described in claim 2.

4. The application of the primer pair of claim 2 or the kit of claim 3 in the identification and / or breeding of the reproductive performance of Tibetan pigs, characterized in that, Individuals with Ssc14:8587185C>T genotype CC, Ssc13:21904604C>T genotype CC and Ssc16:75075241A>G genotype GG had higher total litter size and live litter size than those with other genotypes. The Ssc14:8587185C>T, Ssc13:21904604C>T, and Ssc16:75075241A>G sites are all genomic locations and specific mutations in SusScorfa version 11.1.

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