Molecular markers for predicting pig live-born litter size traits and use thereof

Abstract

The application relates to a molecular marker for evaluating a pig live litter size character and application thereof. The molecular marker is a C>A single nucleotide polymorphism (SNP) at a site of 7,604,530 of a chromosome 6 of a pig genome Sscrofa11.1 version, and the site is located in a 3' untranslated region (UTR3) of a CDYL2 gene. After whole genome association analysis (GWAS) is carried out on a population containing 268 pigs, it is found that the site is extremely significantly related to an individual live litter size character, wherein an individual carrying a C allele shows higher breeding value, can be used as an important marker for molecular assisted breeding, and can accelerate the formation of a population with excellent growth performance and improve the efficiency of good seed selection.

Description

Technical Field

[0001] This invention relates to the field of molecular marker technology, specifically to a molecular marker for predicting the live piglet count trait and its application. Background Technology

[0002] The hog industry is one of the pillar industries of my country's agriculture, and its stable development is directly related to the national economy, people's livelihood, and food security. Against the backdrop of continuously rising hog production costs, sow reproductive efficiency has become a core factor determining the economic benefits and competitiveness of hog farms. Live piglet count, as a key economic trait for measuring sow reproductive performance, directly affects production efficiency and economic benefits. Therefore, increasing the live piglet count of sows is a key focus and challenge in current hog breeding work.

[0003] However, the number of live piglets is a low-heritability trait, greatly influenced by environmental, nutritional, and management factors, posing a significant challenge to traditional phenotypic-based breeding methods. Traditional breeding methods suffer from limitations such as long cycles, high costs, and low accuracy, severely restricting the rate of genetic progress. Currently, molecular marker research in pigs mainly focuses on economic traits such as backfat thickness, meat quality, and disease resistance, while reports on markers directly related to reproductive performance, such as the number of live piglets, are relatively few. This, to some extent, limits the application of molecular breeding techniques in improving sow reproductive performance.

[0004] With the rapid development of high-throughput sequencing and genome-wide association studies (GWAS), some key gene regions related to sow reproductive performance have been gradually identified. In this study, association analysis between the live piglet count phenotype and genome-wide SNP data in a pig population revealed significant associated loci in the chromosome Y-like 2 (CDYL2) gene. CDYL2 belongs to a family of chromatin regulation-related genes, primarily involved in epigenetic regulation, transcriptional regulation, and the maintenance of genome stability. Its encoded protein contains a chromatin-binding domain, which can recognize histone modifications and regulate chromatin state. It may exert a regulatory effect on sow reproductive performance by influencing chromatin remodeling and gene expression. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a molecular marker for predicting the live piglet count trait in pigs and its application. Based on resequencing data from 286 pigs and live piglet count phenotypic data, this invention identified a SNP locus significantly associated with this trait through genome-wide association analysis (GWAS). This SNP locus is located in the 3' untranslated region (UTR3) of the CDYL2 gene, specifically at position 301 of the nucleotide sequence shown in SEQ ID NO.1, where a C / A mutation exists. This locus is named c.301C>A. Genotyping of this locus was performed using Sanger sequencing, and association analysis was conducted using phenotypic data. The results showed a significant correlation between this locus and the live piglet count trait in pigs; therefore, it can be used as a novel molecular marker for molecular-assisted breeding of pigs.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: An application of a molecular marker associated with the live piglet count trait in pigs, characterized in that the live piglet count level of an individual pig is identified by determining the alleles of the molecular marker. The molecular marker site is located at position 301 of the nucleotide sequence shown in SEQ ID NO.1 (position 7,604,530 on chromosome 6 of the pig genome Sscrofa11.1 version), and the polymorphism of this site is C / A: if the genotyping result is CC, it is determined that the pig individual has a large number of live piglets; if the genotyping result is CA or AA, it is determined that the pig individual has a small number of live piglets.

[0007] A primer pair for detecting the above-mentioned molecular marker, characterized in that the nucleotide sequence of the upstream primer is shown in SEQ ID NO.2; and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.3.

[0008] A detection product for detecting the above-mentioned molecular markers, characterized in that the detection product comprises the above-mentioned primer pair.

[0009] Based on the above scheme, the testing products include testing reagents, testing kits, and testing chips.

[0010] The above molecular markers, primer pairs, or detection products may be used in any of the following: (1) Used to screen or predict breeding sows with high live piglet counts; (2) Breeding index / breeding value (EBV / GEBV) used to establish or optimize reproductive traits; (3) Used for the selection and mating optimization of breeding sows; (4) Used for early typing in the embryonic or piglet stage, thereby shortening the generation interval; (5) Used for molecular marker-assisted breeding and population genetic monitoring.

[0011] A method for determining the number of live piglets, characterized by comprising the following steps: (1) Using the genomic DNA of the pig to be tested as a template, the template was amplified by PCR using the primer pair described in SEQ ID NO.2-3; (2) Sequencing the obtained amplification products to obtain the genotypes of the above molecular markers; (3) If the genotyping result is CC, the pig is judged to have a large number of live piglets; if the genotyping result is CA or AA, the pig is judged to have a small number of live piglets.

[0012] Based on the above scheme, the PCR reaction system is as follows: 25 μL of 2× EasyTaq PCR SuperMix, 1 μL of upstream primer (10 μM), 1 μL of downstream primer (10 μM), and 2 μL of template DNA. Bring the volume to 50 μL (i.e., 21 μL).

[0013] Based on the above protocol, the PCR cycling conditions are as follows: 94 ℃ pre-denaturation for 3 min; followed by 30 cycles, each cycle consisting of 94 ℃ for 30 s (denaturation), 60 ℃ for 30 s (annealing), and 72 ℃ for 1 min (extension); and finally 72 ℃ for 5 min for final extension.

[0014] A method for breeding pigs with a high number of live piglets, characterized in that pig individuals with the C allele as described in claim 1 are selected for breeding.

[0015] The molecular marker for predicting the live piglet count trait described in this invention and its application have the following beneficial effects: Detection and analysis of the c.301C>A site on the CDYL2 gene revealed a significant correlation between this mutation site and the live piglet count trait in pigs. The molecular markers provided by this invention offer advantages such as high accuracy, high efficiency, and low application cost, enabling effective prediction and selection of sow live piglet count levels. This provides a new technical means for molecular-assisted breeding of reproductive performance, thereby accelerating the breeding process of high-reproductive-performance pig herds. Attached Figure Description

[0016] The present invention includes the following figures: Figure 1 The Manhattan plot shows the genome-wide association analysis results of Example 1.

[0017] Figure 2This is an electrophoresis result of the products after PCR amplification using the specific primers provided in this invention in Example 3. From left to right: Marker, 1-5: CC genotype individuals, 6-10: CA genotype individuals, 11-15: AA genotype individuals.

[0018] Figure 3 The image shows the Sanger sequencing results of a wild-type homozygous individual (genotype CC).

[0019] Figure 4 This is a Sanger sequencing result of a heterozygous individual with a mutated allele (genotype CA).

[0020] Figure 5 This is a Sanger sequencing result of a homozygous mutant individual (genotype AA).

[0021] Figure 6 This is a graph showing the results of the analysis of variance on the different genotypes of the molecular markers described in Example 1 and the number of live offspring. Detailed Implementation

[0022] The present invention will be further described in detail below with reference to the accompanying drawings. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0024] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0025] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0026] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0027] The SNP site mentioned in this invention mainly refers to the SNP variation in the UTR3 of the CDYL2 gene, which is located at position 7,604,530 on chromosome 6 of pig (Sscrofa11.1 reference genome).

[0028] Sequence Description: The nucleotide sequence shown in SEQ ID NO.1 is the gene fragment screened in this invention, serving as a molecular marker for detecting the live piglet count trait. A C / A base mutation exists at position 301 of this sequence, indicating an allele substitution (C / A) at position 301.

[0029] Example 1: Mining of genetic markers for the live piglet count trait in pigs 268 pigs were selected, and their reproductive performance was recorded and the number of live piglets was calculated. Genomic DNA was extracted from ear tissue, and whole-genome resequencing libraries were constructed and sequenced. The sequencing data were filtered for quality control using PLINK software, with the following conditions: (1) SNPs with missing chromosomes and physical location annotations were deleted; (2) Samples with an individual genotype deletion rate greater than 0.05 were deleted; (3) SNPs with a deletion rate greater than 0.05 were deleted; (4) SNPs with a minor allele frequency (MAF) less than 0.01 were deleted; (5) SNPs with Hardy-Weinberg equilibrium test were deleted. P The value is less than 2.2989 × 10 -5 The SNPs obtained after quality control were used for subsequent research.

[0030] GWAS was performed using GEMMA software based on a linear mixture model (LMM). The model is: y = Xα + Yβ + Zγ + e; in It is a phenotypic vector. The SNP additive effect vector (genotypes are encoded as 0 / 1 / 2, corresponding to genotypes CC, CA, and AA, respectively, where A is the minor allele). β is the fixed effect vector, mainly including environmental factors that significantly affect the number of live offspring, such as parity, mating season, and mating batch / farm. For residual multigene random effects, the assumed distribution is as follows: ,in The variance is additive genetic, and G is the marker-inferred kinship matrix (GRM). X, Y, and Z are respectively related to... and The corresponding design matrix. E is the residual. Where I is the identity matrix, This represents the residual variance.

[0031] The significance test was performed using the Wald test, with a significance threshold set to 1. P <2.2989×10 -5 The Manhattan Project results are as follows Figure 1 As shown. The results showed that a highly significant association signal was detected at position 7,604,530 on chromosome 6. P =1.75×10 -5 This site is a C>A SNP located in the 3' untranslated region (UTR3) of the CDYL2 gene. For ease of application and transformation, a flanking sequence of ±300 bp from this site was extracted from the reference genome and designated as SEQ ID NO.1. The position of this site in the sequence is defined as the 301st base, with a C / A allelic substitution, and named c.301C>A.

[0032] 1. DNA extraction This invention uses pigs as experimental material and extracts DNA from pig ear tissue using a genomic DNA extraction kit. The steps are as follows: (1) Add the specified volume of anhydrous ethanol to the washing buffer CB2 and washing buffer WB2 according to the instructions and mix thoroughly.

[0033] (2) Take about 25 mg of ear tissue sample, cut it into small pieces and place it in a 1.5 mL centrifuge tube. Add 100 μL of lysis buffer LB2 and 20 μL of proteinase K, and lyse in a metal bath at 55 °C for 12 h. Then add 20 μL of RNase A, incubate at room temperature for 2 min, centrifuge at 12000g for 5 min, and transfer the supernatant to a new 1.5 mL centrifuge tube.

[0034] (3) Add 500 μL of adsorption buffer BB2, vortex immediately for 5 s, and incubate at room temperature for 10 min.

[0035] (4) Add all the solution to the centrifuge column, centrifuge at 12000 g for 30 s, and discard the effluent.

[0036] (5) Add 500 μL CB2, centrifuge at 12000 g for 30 s, and discard the effluent.

[0037] (6) Add 500 μL WB2, centrifuge at 12000 g for 30 s, and discard the effluent.

[0038] (7) Repeat step (6) once.

[0039] (8) Centrifuge at 12000 g for 2 min to completely remove residual WB2 from the column.

[0040] (9) Place the centrifuge column in a new 1.5 mL centrifuge tube, add 100 μL of EB preheated in a 70 ℃ metal bath to the center of the column, let it stand at room temperature for 1 min, centrifuge at 12000 g for 1 min, and elute the DNA.

[0041] (10) To increase yield, a second elution was performed, and step (9) was repeated. The DNA was stored at -20 °C.

[0042] 2. DNA amplification Using the porcine Sscrofa11.1 genome as a reference, a pair of primers (product length approximately 490 bp) was designed based on the flanking sequence of the chr6:7,604,530 variant position ±300 bp for PCR amplification and Sanger sequencing. The primer sequences used are as follows. SEQ ID NO.2: 5'-GCGGAATTGCTTCTCTCTTGT-3'; SEQ ID NO.3: 5'-TTGGCACCAAACCAGAATGC-3'; The primer pair was used for PCR amplification (reaction system and conditions are shown in Tables 1 and 2), purification of PCR products, cloning and sequencing, and sequence alignment analysis.

[0043] Table 1 PCR reaction system 2×EasyTaq RCR SuperMix 25 Upstream primer (10 μM) 1 Downstream primer (10 μM) 1 Template DNA 2 Nuclease-free Water 21 Total volume 50 ; Table 2 PCR Procedure ; The amplified products were used for electrophoretic identification (see...) Figure 2 After product purification, Sanger sequencing was performed for genotyping. Figure 3 For CC homozygous, Figure 4 It is CA heterozygous. Figure 3 (It is homozygous for AA).

[0044] 3. Analysis of variance of the number of live offspring of individuals with different genotypes Based on the genotype results of this molecular marker, the samples were divided into CC / CA / AA genotypes. Analysis of variance and linear model comparisons were performed within the same covariate framework as in genome-wide association studies (GWAS) (including fixed effects such as batch / farm number). The results are as follows: Figure 6 As shown in the figure. The analysis results show that the number of live offspring of individuals with the CC genotype is significantly higher than that of individuals with the CA and AA genotypes (P <0.05), indicating that CC is a favorable genotype.

[0045] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

[0046] The contents not described in detail in this specification are existing technologies known to those skilled in the art.

Claims

1. The application of a molecular marker related to the live piglet count trait in pigs, characterized in that, The live piglet count of an individual pig can be determined by identifying the alleles of the molecular markers; the criteria for identification are: individuals with the CC genotype have a higher live piglet count than those with the CA and AA genotypes. The molecular marker site is located at position 301 of the nucleotide sequence shown in SEQ ID NO.1, and the polymorphism of this site is C / A.

2. The application of the molecular marker of claim 1, the primer pair for detecting the molecular marker, or the detection product containing the primer pair in screening or predicting breeding sows with high live piglet counts: The primer pairs are shown in SEQ ID NO.2-3; The detection products include detection reagents, detection kits, and detection chips containing the primer pairs.

3. A method for determining the number of live piglets, characterized in that, Includes the following steps: (1) Using the genomic DNA of the pig to be tested as a template, the template is amplified by PCR using the primer pair described in claim 2; (2) Sequencing the obtained amplification products to obtain the genotype of the molecular marker described in claim 1; (3) If the genotyping result is CC, the pig is judged to have a large number of live piglets; if the genotyping result is CA or AA, the pig is judged to have a small number of live piglets.

4. The method according to claim 3, characterized in that, The PCR reaction system consisted of: 25 μL of 2× EasyTaq PCRSuperMix, 1 μL of 10 μM upstream primer, 1 μL of 10 μM downstream primer, and 2 μL of template DNA. Bring the volume to 50 μL.

5. The method according to claim 3, characterized in that, The PCR cycling conditions were as follows: 94 °C pre-denaturation for 3 min; followed by 30 cycles, each cycle consisting of 94 °C for 30 s denaturation, 60 °C for 30 s annealing, 72 °C for 1 min extension, and finally 72 °C for 5 min final extension.

6. A method for breeding pigs with a high number of live piglets, characterized in that, Pig individuals with the CC allele as described in claim 1 were selected for breeding.

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