Pig lactation-related snp molecular marker and application thereof

Abstract

This invention discloses a porcine lactation-related SNP molecular marker and its application. The SNP site of the molecular marker is located on the X chromosome, with RS number rs331986593. Compared to sows carrying the CC genotype, sows carrying the CT or TT genotypes have higher lactation capacity. This molecular marker can accurately predict differences in lactation trait among sows of different genotypes, enabling accurate and efficient selection of superior pig breeds with excellent lactation performance. It has outstanding advantages such as simplicity, speed, high sensitivity, and good specificity. In pig breeding, this molecular marker can serve as a reliable marker for lactation trait, facilitating early selection, thereby shortening the generation interval, increasing selection intensity, and improving selection efficiency and accuracy.

Description

Technical Field

[0001] This invention belongs to the field of molecular marker technology, specifically relating to molecular markers related to porcine lactation capacity and their applications. Background Technology

[0002] Sow lactation capacity is an important economic trait, directly affecting piglet growth and survival, as well as the economic benefits of farms. It is also a crucial indicator of sow reproductive performance. Sow lactation performance is mainly reflected in milk yield, udder health, and milk composition (e.g., milk fat percentage, milk protein percentage), with milk yield being one of the most commonly used indicators. Genetic improvement of lactation capacity to enhance sow lactation performance is an effective way to improve the economic benefits of livestock farming.

[0003] The HS6ST2 gene (heparin-6-O-sulfotransferase 2) is a member of the HS6ST genomic family, located on the X chromosome. Heparin sulfate (HS) is a linear sulfated glycosaminoglycan widely distributed on the cell surface, extracellular matrix, and basement membrane. It interacts with various ligands and participates in regulating physiological functions such as cell growth, differentiation, adhesion, and migration. This gene is a member of the heparin sulfate (HS) sulfotransferase gene family, catalyzing the transfer of sulfate to HS. Different family members and subtypes are thought to be able to synthesize heparin sulfate with tissue-specific structures and functions. HS6ST2 has been reported to be associated with the occurrence and growth of several invasive tumor cells. For example, the lncRNA FAM83H-AS1 inhibits the proliferation and metastasis of non-small cell lung cancer by regulating the miR-545-3p / HS6ST2 axis. In gastric cancer tissues, HS6ST2 expression is significant, and because HS6ST2 is closely related to certain clinicopathological variables and disease prognosis, it is considered a novel biomarker for gastric cancer. Overexpression of HS6ST2 in colorectal cancer (CRC) tissues is often associated with poor prognosis. Furthermore, downregulation of HS6ST2 gene expression can inhibit the proliferation and migration of breast cancer cells. Recent studies have shown a significant association between the HS6ST2 gene and the milk protein and fat content in dairy cows. However, compared to dairy cows, the lactation performance of sows cannot be directly measured due to technological limitations. Moreover, it remains unclear whether genes affecting milk composition are directly related to the lactation performance traits of sows.

[0004] In this invention, we screened a SNP locus in the HS6ST2 gene of pigs that was highly significantly associated with the lactation trait. Therefore, this SNP locus can be used as a molecular marker for herd breeding. In the improvement of lactation trait, early selection of pig herds can be carried out, thereby shortening the generation interval, improving selection intensity, selection efficiency and accuracy, and thus improving the lactation capacity of sows. Summary of the Invention

[0005] The purpose of this invention is to provide a SNP marker related to sow lactation capacity, and to develop a kit and method for detecting sow lactation capacity based on this SNP marker. This molecular marker can accurately predict differences in lactation capacity traits among sows of different genotypes, enabling accurate and efficient selection of superior pig breeds with excellent lactation performance. It has outstanding advantages such as simplicity, speed, high sensitivity, and good specificity. In pig breeding, this molecular marker can serve as a reliable marker for lactation traits, facilitating early selection, thereby shortening generation intervals, increasing selection intensity, and improving selection efficiency and accuracy.

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

[0007] Association analysis identified a SNP locus in the HS6ST2 gene that is significantly associated with sow lactation. This SNP locus is located at base 108880461 on the X chromosome (reference genome version Sscrofa11.1), with SNP ID WU_10.2_X_124874052 and RS number rs331986593. The sequence of the gene containing the SNP marker is shown in SEQ ID NO.1 or 2, with base T or C at position 201.

[0008] A kit for detecting lactation capacity in sows, the kit containing reagents for detecting the rs331986593 site, including primers for amplifying the rs331986593 site, preferably having nucleotide sequences as shown in SEQ ID NO. 3 and 4.

[0009] A method for predicting sow lactation capacity includes in vitro detection of the genotype at the rs331986593 locus in the sow, and prediction of sow lactation capacity based on the genotype. Specifically, sows carrying the CT or TT genotypes have higher lactation capacity than sows carrying the CC genotype. In a specific embodiment of the invention, the method for detecting sow lactation capacity includes: extracting genomic DNA from the sow; performing PCR amplification using primers shown in SEQ ID NO. 3 and 4 to obtain PCR amplification products; sequencing the PCR amplification products; and predicting the sow's lactation capacity based on the sequencing results. Attached Figure Description

[0010] Figure 1 The flowchart shows the technical process for screening SNP loci associated with sow lactation capacity.

[0011] Figure 2 , Figure 3 Manhattan plot and QQ-plot showing the results of genome-wide association analysis of lactation capacity. Detailed Implementation

[0012] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0013] Example 1

[0014] In this embodiment, reference Figure 1 We screened for molecular markers related to sow lactation capacity using the following method.

[0015] 1. Pig data collection

[0016] (1) Source of phenotypic data:

[0017] The experimental sample consisted of 1336 healthy sows from Guangxi Yangxiang Co., Ltd. Ear tag, sample number, birth litter weight, number of piglets, and 21-day weaning litter weight were recorded for each sow. Lactation capacity was assessed using the 21-day weaning litter weight method based on covariate correction. The phenotypic value of lactation capacity for the i-th sow was calculated according to the formula:

[0018]

[0019] Among them, y i L represents the phenotypic value of the lactation capacity trait of the i-th sow. W21 The minimum nest weight at 21 days of age for weaning in the group, y i ′ represents the covariate-corrected litter weight at 21 days of age for the i-th sow, and L represents the litter weight at 21 days of age. y 'R represents the minimum weaning litter weight at 21 days of age after covariate correction within the population.' W21 R is the difference between the maximum and minimum nest weight of weaned infants at 21 days of age in the group. y′ This represents the difference between the maximum and minimum weaning weights at 21 days of age in the population after covariate correction.

[0020] (2) Gene Data Collection

[0021] DNA was extracted from pig ear tissue, with all samples stored at -20°C prior to extraction. DNA was extracted using a kit provided by TIANGEN, following the manufacturer's instructions. DNA samples were extracted from 1336 sows using this method. Genotyping was performed using the Illumina PorcineSNP50 Bead Chip, and all 50,697 SNPs were matched to the entire Sus scrofa genome v11.1.

[0022] (3) Using a mixed linear model (MLM), association analysis was performed on all SNP sites of the HS6ST2 gene and the lactation trait.

[0023] The mixed linear model used is: y = Xa + Ku + e

[0024] y represents the phenotypic value of lactation trait, a represents the fixed effect of SNP gene, u represents the random effect, e represents the residual, and X and K are the design matrices corresponding to a and u.

[0025] Table 1. Association analysis results between the HS6ST2 gene and the lactation trait.

[0026] SNP ID chromosome number Location P-value H3GA0051959 X 108797445 0.570423 WU_10.2_X_124864406 X 108870827 0.85944 WU_10.2_X_124874052 X 108880461 1.02E-06 WU_10.2_X_124928407 X 108884862 0.729204 WU_10.2_X_124931910 X 108888376 0.515794 H3GA0051963 X 108892087 0.522532 WU_10.2_X_124965364 X 108920145 0.94436 WU_10.2_X_124990985 X 108950091 0.440023 WU_10.2_X_125005763 X 108971782 0.541005 WU_10.2_X_125018072 X 108983937 0.757249 WU_10.2_X_125030741 X 108996604 0.82117

[0027] Based on the association results and the significance threshold (P<0.05), among the multiple SNP sites of the HS6ST2 gene, only WU_10.2_X_124874052 (RS number rs331986593) was significantly associated with the lactation trait.

[0028] Depend on Figure 2 The Manhattan plot results show that a SNP on the X chromosome (labeled WU_10.2_X_124874052) exceeds the significance threshold, indicating that this SNP is significantly associated with sow lactation capacity.

[0029] Depend on Figure 3 The QQ plot results show that the SNP on the X chromosome deviates significantly from the red line and exceeds the confidence interval, indicating that the SNP is significantly associated with the sow's lactation capacity.

[0030] 2. Test for significant SNPs

[0031] The association between the rs331986593 locus of the HS6ST2 gene and the lactation trait was analyzed using a mixed linear model with GMAT software, followed by multiple comparisons using the Tukey HSD method.

[0032] Table 2. Significance test of the rs331986593 locus of the HS6ST2 gene with the lactation trait.

[0033]

[0034] As shown in Table 2, the association and multiple comparison results indicate that the rs331986593 locus genotype of the HS6ST2 gene is significantly associated with lactation capacity (P<0.01). The lactation capacity of TT and CT individuals is significantly higher than that of CC individuals (P<0.01), and CT and TT are the dominant genotypes for lactation capacity.

[0035] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. The application of a reagent for detecting SNP marker genotypes in predicting sow lactation capacity, characterized in that, The nucleotide sequence of the SNP marker is shown in SEQ ID NO.1 or SEQ ID NO.

2.

2. The application of the kit in predicting sow lactation capacity, characterized in that, The kit contains reagents for detecting the rs331986593 locus genotype.

3. The application according to claim 2, characterized in that, The reagent used to detect the rs331986593 locus genotype is a primer, the nucleotide sequence of which is shown in SEQ ID NO.3-4.

4. A method for predicting sow lactation capacity, characterized in that, The method includes in vitro detection of the genotype at the rs331986593 locus in the sows to be tested. Sows carrying the CT and TT genotypes have higher lactation capacity compared to sows carrying the CC genotype.

5. The method according to claim 4, characterized in that, The method includes: extracting genomic DNA from the sow to be tested; performing PCR amplification using the primers described in claim 3 to obtain PCR amplification products; and predicting the lactation capacity of the sow to be tested based on the genotype of the 201st base of the amplification product.

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