Application of CD47 gene in detection of pig litter size phenotype and application of SNP site in gene expression regulation

Abstract

The invention provides application of a CD47 gene in detection of pig litter size phenotype and application of an SNP site of the CD47 gene in gene expression regulation. It is found that the CD47 gene is regulated and controlled by the SNP site rs323354626, it is further found that the rs323354626 site and the phenotype of the total litter size of sows show the highest significance, and it is indicated that the CD47 gene and the total litter size of the sows have large correlation. In three main commercial varieties of pigs, namely Duroc, Landrace and Yorkshire, the CD47 expression level in testis tissues is negatively correlated with the litter size, which indicates that the expression mode of the CD47 gene may influence the reproductive performance of the pigs, and the litter size phenotype of the pigs can be detected by detecting the CD47 gene expression level in the testis of the breeding pigs or somatic cells of the sows. Or the litter size of the pigs is influenced by regulating and controlling the expression level of the CD47 gene through the rs323354626 site, and the discovery lays a foundation for improving the breeding efficiency of the pigs, especially for a breeding method for increasing the litter size.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and particularly to the application of the CD47 gene in detecting the phenotypic litter size of pigs and its SNP sites in gene expression regulation. Background Art

[0002] Pigs (Sus scrofa) are one of the important agricultural and economic animals globally and play an important role in meat production. Improving the production performance of pigs, especially reproductive performance, is of great significance for enhancing the economic efficiency of the pig industry. Among them, litter size is one of the key indicators for measuring the reproductive performance of sows.

[0003] High litter size can not only improve the production efficiency of individual sows but also reduce the production cost throughout the feeding cycle. However, litter size is a complex quantitative trait that is jointly affected by multiple genes and environmental factors. Recent studies have shown that litter size is related to multiple physiological processes, including not only aspects of the mother such as ovarian function, uterine capacity, and embryo survival rate, but also closely related to the sperm quality and quantity of boars.

[0004] CD47 (Cluster of Differentiation 47) is an integrin membrane protein encoded by the CD47 gene and is also known as Integrin Associated Protein (IAP). CD47 belongs to the immunoglobulin superfamily and is widely expressed on the surfaces of various tissue cells. As an important cell surface marker, CD47 mainly participates in various biological processes such as cell recognition, immune regulation, cell adhesion, and migration through its interaction with its receptor signal regulatory protein α (SIRPα). However, the research on the CD47 gene in livestock breeding is relatively limited, especially its association with pig reproductive traits, especially litter size, has not been reported. Summary of the Invention

[0005] The present invention provides the application of the CD47 gene in detecting the phenotypic litter size of pigs and its SNP sites in gene expression regulation. The purpose of the present invention is to determine the variant sites that can be used to analyze the association between the CD47 gene and litter size traits by comparing the transcript usage differences of the CD47 gene between different cell types. This study aims to provide a scientific basis for the development of molecular marker-assisted selection based on the CD47 gene, thereby laying a foundation for improving the reproductive efficiency of pigs, especially for breeding methods to increase litter size.

[0006] In the first aspect, the present invention provides the application of the CD47 protein in detecting the phenotypic litter size of pigs. The CD47 protein sequence is as shown by the UniProtKB database number UP00008227.

[0007] Preferably, the application is completed by detecting the expression level of CD47 protein in boar testis or sow somatic cells.

[0008] In a second aspect, the present invention provides a method for comparing the litter size of pigs, specifically, detecting the relative expression level of CD47 protein in samples of breeding pigs and / or sow somatic cells, and comparing the litter-bearing potential of breeding pigs and / or sows according to the level of the relative expression of CD47 protein, wherein breeding pigs and / or sows with a low relative expression level of CD47 protein have a higher litter-bearing potential.

[0009] In a third aspect, the present invention provides the application of a kit for detecting the expression level of CD47 protein in the comparison of pig litter size.

[0010] In a fourth aspect, the present invention provides a method for increasing the litter size of breeding pigs, specifically, reducing the expression level of CD47 protein in breeding pigs and / or sows by means of genetic engineering or protein engineering.

[0011] Preferably, by changing the genotype of the SNP locus rs323354626 within the cell type-specific region of the CD47 gene, the expression level of CD47 protein is reduced, thereby increasing the litter size of breeding pigs; The SNP locus rs323354626 includes a nucleotide sequence as shown in SEQ ID NO 1, and the polymorphism at position 51 of the nucleotide sequence is C / T.

[0012] More preferably, the above method is to make the genotype of the SNP locus rs323354626 be T / T or C / T by means of genetic engineering.

[0013] The present invention further provides the application of CD47 protein, or a biological material containing the protein, or the above method in any one of the following: (1) Screening pigs with high litter-bearing ability; (2) Improving the litter-bearing ability of pigs; (4) Variety improvement of high-yield piglets.

[0014] In a fifth aspect, the present invention provides the application of an inhibitor for reducing the content or activity of CD47 protein in breeding pigs and / or sows in increasing the litter size after breeding of breeding pigs.

[0015] Preferably, the inhibitor inhibits its expression or activity by inhibiting CD47 protein translation, inhibiting transcriptional processing, or inhibiting the function after transcriptional processing, and the inhibitor is an oral drug, an injection drug, or a patch.

[0016] After predicting the PSI values (3'UTR usage scores) of various cell types in pigs, it was found that the PSI value of the CD47 gene was significantly low in testicular tissue, and the coefficient of variation (CV) was relatively high. This result indicates that there is significant heterogeneity in the length of the 3'UTR of this gene in testicular tissue. Further overlapping analysis of the differential 3'UTR of the CD47 gene with the eQTLs and sQTLs results of PigGTEx identified the SNP locus rs323354626 within the differential 3'UTR region. This locus can regulate the post-translational expression of CD47, and CD47 is lowly expressed when its genotype is T / T or C / T, while it is highly expressed when the genotype is C / C.

[0017] The present invention further found that the genotype of the SNP locus rs323354626 in sows is also associated with all the economically important traits collected in the PigBiobank database. We found that the genotype of the rs323354626 locus showed the highest significance with reproductive-related phenotypes, especially the "total number of piglets born" indicator. Therefore, the CD47 protein regulated by it is very likely related to the total number of piglets born in pigs.

[0018] After comparative analysis of the expression levels of the CD47 gene in the main commercial pig breeds Landrace, Yorkshire, and Duroc, it was found that Yorkshire pigs with the lowest expression level of the CD47 gene had stronger reproductive performance than Duroc and Landrace pigs, indicating that the CD47 expression pattern is related to the genetic background of different pig breeds and thus affects their reproductive performance.

[0019] Through comparative analysis of the expression levels of the CD47 gene in sows of the main commercial pig breeds, it was found that the difference in CD47 expression between the Landrace and Yorkshire breeds was statistically significant (P<0.05), with the Landrace breed showing a higher CD47 expression level. Although the Duroc breed showed a medium level of CD47 expression, the differences from the Landrace and Yorkshire breeds did not reach a statistically significant level. As the maternal female parent in the production of three-way crossbred pigs, Yorkshire pigs have stronger reproductive performance than Duroc and Landrace pigs, and the expression level of the CD47 gene can indeed affect their reproductive performance.

[0020] Further analysis of the relationship between the alleles of the rs323354626 locus and the total number of piglets born in pigs found that the presence of the C and T alleles was statistically correlated with the total number of piglets born. After controlling variables such as breed, parity, and age, individuals carrying the T allele generally showed a higher potential for litter size, but this effect showed different degrees in different breeds, possibly interacting with the genetic background of each breed.

[0021] In the analysis of the allele frequencies at the rs323354626 locus in three major pig breeds, Duroc, Landrace, and Yorkshire, it was found that the frequency of the C allele was the highest (90.5%) and the frequency of the T allele was the lowest (9.5%) in the Duroc breed; the frequency of the C allele was (82.3%) and the frequency of the T allele was (17.7%) in the Landrace breed; in contrast, in the highly fecund Yorkshire pigs, the T allele was dominant (67.2%) and the frequency of the C allele was relatively low (32.8%); indicating that the T allele may be the dominant allele affecting the litter size trait in pigs. This distribution pattern is consistent with the CD47 gene expression levels and litter size characteristics of each breed, suggesting that the allelic variation at the rs323354626 locus is one of the genetic factors affecting the differences in reproductive performance among different pig breeds.

[0022] The beneficial effects of the present invention include: Based on the prediction of the relative 3'UTR usage score PSI values of each cell type using single-nucleus transcriptome data, the present invention found that there is significant heterogeneity in the 3'UTR length of the CD47 gene in domestic pig testicular tissue, and this heterogeneity is regulated by the SNP locus rs323354626.

[0023] Further research found that the rs323354626 locus in sows showed the highest significance with reproductive-related phenotypes, especially the "total litter size" indicator, indicating a strong correlation between the CD47 gene and the total litter size in pigs. In the three major commercial pig breeds, Duroc, Landrace, and Yorkshire, it was also found that the CD47 expression level in testicular tissue was negatively correlated with litter size, suggesting that the expression pattern of the CD47 gene may affect its reproductive performance. Therefore, the litter size phenotype of domestic pigs can be detected by measuring the CD47 gene expression level in the testes of breeding pigs or somatic cells of sows, or the litter size of domestic pigs can be affected by regulating the CD47 gene expression level at the rs323354626 locus. This finding lays a foundation for improving the reproductive efficiency of pigs, especially for breeding methods to increase litter size. Description of the Drawings

[0024] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0025] Figure 1It is the dynamic changes of the 3’UTR of the CD47 gene provided by the present invention during tissue and spermatogenesis. Among them, A is the coefficient of variation and PSI analysis of the CD47 gene in different tissues; B is the PSI heat map of 3’ isoforms in different cell types during spermatogenesis (the upper figure is the gene with shortened 3’UTR, and the lower figure is the gene with extended 3’UTR).

[0026] Figure 2 It is the result diagram showing the expression level, PAS usage and gene coverage of the CD47 gene during spermatogenesis provided by the present invention.

[0027] Figure 3 It is the statistical chart of the correlation between the rs323354626 locus and CD47 provided by the present invention. Among them, A is the relationship between the effect size and statistical significance of the eQTL and sQTL of CD47; B is the PSI distribution of CD47 under different genotypes (CC, CT, TT) at the rs323354626 locus.

[0028] Figure 4 It is the pheWAS result of the rs323354626 locus provided by the present invention. The dots represent the association strength with specific phenotypes (-log 10 (P-value)), and the red dotted line is the significance threshold of P value 0.05.

[0029] Figure 5 It is the comparison of the expression levels of the CD47 gene among different pig breeds provided by the present invention.

[0030] Figure 6 It is the allele frequency distribution of the rs323354626 locus in different pig breeds provided by the present invention.

[0031] Figure 7 It is the relationship between different genotypes of the rs323354626 locus and the total number of piglets born provided by the present invention. Detailed implementation manners

[0032] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below with reference to the accompanying drawings in the present invention. Obviously, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art without creative efforts based on the embodiments in the present invention belong to the scope of protection of the present invention.

[0033] Example 1 Discovery of the relationship between the CD47 gene and the number of piglets born This example is about how to discover the relationship between the CD47 gene and the number of piglets born. The specific steps are as follows: 1. Prediction of relative 3’UTR length based on single-nucleus transcriptome data (1) Data processing: Obtain the original sequencing data from GSE233285. Use CellRanger to align the FASTQ files to the reference genome index constructed based on the Ensembl database. Filter the reads with mapping quality < 30 using SAMtools, and split the BAM file according to cell barcodes. UMI-tools remove duplicate reads. DeepTools convert the deduplicated BAM file to BigWig format for IGV visualization, and the results are as Figure 1 shown.

[0034] As Figure 1 shown in A, after predicting the PSI values of each cell type based on single-nucleus transcriptome data, it can be seen that the PSI value of the CD47 gene in testicular tissue is significantly low, and the coefficient of variation (CV) is relatively high. This result indicates that there is significant heterogeneity in the 3’UTR length of this gene in testicular tissue, and the 3’UTR length can affect the stability, translation efficiency, and subcellular localization of mRNA, thereby affecting gene expression. As Figure 1 shown in B, during spermatogenesis, the 3’UTR of the CD47 gene shows a gradually shortening trend.

[0035] (2) 3’UTR profiling strategy: a. Cell type-based quantification method: Use LABRAT to quantify alternative polyadenylation and predict the PSI value for each gene in each cell type.

[0036] b. Single-cell level quantification method: Use SCAPE to estimate the polyadenylation sites (PASs) of each gene and their corresponding weights, and only retain the PASs expressed in more than 5 cells.

[0037] 2. Obtaining differential PASs of the CD47 gene Input the filtered single-cell level PASs file into Seurat for downstream analysis, and use the script "DifferentialTest.R" (https: / / github.com / LuChenLab / SCAPE / blob / main / SCAPE.R / R / DifferentialTest.R) to identify differential PASs, and the results are as Figure 2 shown.

[0038] As Figure 2It is known that during spermatogenesis, although the CD47 gene has similar expression levels in various cell types, the usage of its 3'UTR shows significant differences. The proximal PAS of CD47 (chr13:151,483,728: +) is only expressed in late spermatocytes and spermatids, while the distal PAS (chr13:151,488,515:+) is preferentially expressed in immature germ cells. This pattern reveals the cell type specificity of the 3'UTR of the CD47 gene.

[0039] 3. Identification of SNPs in the cell type-specific 3'UTR of the CD47 gene The differential 3'UTRs of the CD47 gene were overlapped with the eQTLs and sQTLs results of PigGTEx to identify the eQTLs and sQTLs located within the differential 3'UTR regions. Subsequently, the PheWAS analysis results of the target SNPs in different phenotypes were obtained from PigBiobank, and the results are as Figure 3 shown.

[0040] As Figure 3 shown in A, the rs323354626 locus showed a significant effect in testicular tissue: it had a strong positive regulatory effect on the splicing (sQTL) of CD47 and also had a moderate positive effect on its expression level (eQTL). This indicates that the rs323354626 locus has a substantial impact on the expression and splicing of CD47 in testicular tissue.

[0041] As Figure 3 shown in B, there were significant differences in the PSI values of CD47 among different genotypes (CC, CT, and TT) of the rs323354626 locus, and the PSI values decreased in sequence with the CC, CT, and TT genotypes, indicating that the rs323354626 locus can significantly regulate the expression of CD47.

[0042] 4. Study on the relationship between the rs323354626 locus and the litter size of pigs To explore the potential impact of the rs323354626 locus on phenotypes, we also examined its association with all the economically important traits of sows collected in the PigBiobank database. We found that the rs323354626 locus also showed the highest significance with reproductive-related phenotypes in sows, especially the "total litter size" indicator ( Figure 4 ). Since the rs323354626 locus mainly regulates the post-translational expression of the CD47 protein, CD47 is very likely to be related to the total litter size of pigs, and this correlation is valid in both breeding pigs and sows.

[0043] Example 2 Expression levels of CD47 protein and genotype frequencies at the rs323354626 locus in different pig breeds This example discloses the expression levels of CD47 protein and the genotype frequencies at the rs323354626 locus that regulates CD47 protein expression in different pig breeds.

[0044] 1. Using the transcriptome data of three pig breeds (Landrace, Duroc, Yorkshire) collected previously, analyze the expression data of the CD47 gene (ENSSSCG00000054660) in each breed, including the TPM (transcripts per million) or FPKM (fragments per kilobase per million mapped reads) values after normalization. One-way ANOVA and Tukey's HSD test were used to analyze the significance of differences in CD47 gene expression levels among different breeds. Statistical analysis was completed using R software (version 4.0.3), and the significance level was set at P < 0.05. The ggplot2 package was used to draw the expression comparison graph, and the results are as Figure 5 shown.

[0045] As Figure 5 can be seen, the difference in CD47 expression between the Landrace and Yorkshire breeds was statistically significant (P < 0.05), and the Landrace breed showed a higher CD47 expression level. Although the Duroc breed showed a medium level of CD47 expression, the differences from the Landrace and Yorkshire breeds did not reach a statistically significant level. As the maternal female parent in the production of three-way crossbred pigs, Yorkshire pigs have stronger reproductive performance than Duroc and Landrace pigs, while the expression level of the CD47 gene is the lowest, indicating that the expression pattern of the CD47 gene is related to the genetic background of different pig breeds and may thus affect their reproductive performance.

[0046] 2. Randomly select 100 - 150 unrelated female pig individuals from each breed population (including the three main commercial breeds Duroc, Landrace, and Yorkshire), collect ear tissues and extract DNA. The rs323354626 locus was genotyped using the above method. Calculate the frequencies of the C and T alleles in each breed. The chi-square test was used to analyze the significance of differences in allele frequencies among different breeds. At the same time, the Hardy-Weinberg equilibrium test was used to evaluate the genetic equilibrium state of this SNP in each breed population. POPGENE software (version 1.32) was used for population genetics analysis, including the calculation of polymorphic information content (PIC), and the results are as Figure 6 shown.

[0047] It can be seen from Figure 6 that there are significant differences in the distribution of C and T alleles among different breeds. The frequency of the C allele is the highest (90.5%) and the frequency of the T allele is the lowest (9.5%) in the Duroc breed; the frequency of the C allele is 82.3% and the frequency of the T allele is 17.7% in the Landrace breed; while in the highly fecund Yorkshire pig breed, the situation is reversed, with the T allele being dominant (67.2%) and the frequency of the C allele being relatively low (32.8%); indicating that the T allele may be the dominant allele affecting the litter size trait in pigs. This distribution pattern is consistent with the CD47 gene expression levels and litter size characteristics of each breed, suggesting that allelic variations at the rs323354626 locus can affect the reproductive performance of different pig breeds by regulating the CD47 expression level.

[0048] Example 3 Verification of the relationship between the genotype at the rs323354626 locus and the total litter size of pigs Samples of three major commercial pig breeds, including Duroc, Landrace, and Yorkshire, as well as Luchuan (Luchuan pig, a Chinese local breed), were collected from the Guangxi Bama Miniature Pig Conservation and Breeding Base. A total of 575 sows were collected, and production record data of these sows were collected, including reproductive performance indicators such as total litter size and number of live-born piglets. Ear tag tissues were used to extract genomic DNA, and genotyping of the rs323354626 locus was performed. A mixed linear model was used to analyze the association between the genotypes (CC, CT, TT) at the rs323354626 locus and the total litter size. Fixed effects such as breed, parity, and age were included in the model, and the genetic relationship between individuals was used as a random effect. Bonferroni correction was used for multiple comparisons, and the significance level was set at P < 0.05. The results are as Figure 7 shown.

[0049] It can be seen from Figure 7 that the presence of C and T alleles shows a statistical correlation with the total litter size. After controlling variables such as breed, parity, and age, individuals carrying the T allele generally show a higher litter size potential, but this effect shows different degrees in different breeds, which may interact with the genetic backgrounds of each breed.

[0050] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of CD47 protein in detecting the litter size phenotype of pigs.

2. The application according to claim 1, wherein The above application is completed by detecting the expression level of CD47 protein in somatic cells of boars and / or sows.

3. A method for comparing the litter size of pigs, characterized in that, Detect the relative expression level of CD47 protein in somatic cell samples of breeding pigs and / or sows, and compare the litter-bearing potential of breeding pigs and / or sows according to the high or low relative expression level of the CD47 protein. Among them, breeding pigs and / or sows with a low relative expression level of CD47 protein have a higher litter-bearing potential.

4. Application of a kit for detecting the expression level of CD47 protein in comparing the litter size of pigs.

5. A method for increasing the litter size of pigs, characterized in that, Reduce the expression level of CD47 protein in breeding pigs and / or sows by genetic engineering means.

6. The method according to claim 5, wherein Reduce the expression level of CD47 protein by changing the genotype of SNP locus rs323354626 within the cell type-specific region of the CD47 gene, thereby increasing the litter size of pigs; The SNP locus rs323354626 includes the nucleotide sequence shown in SEQ ID NO 1, and the polymorphism at position 51 of the nucleotide sequence is C / T.

7. The method according to any one of claims 5 or 6, characterized in that Make the genotype of the SNP locus rs323354626 be T / T or C / T by genetic engineering means.

8. Application of CD47 protein, or a biological material containing the protein, or the method according to any one of claims 3, 5 - 7 in any one of the following: (1) Screening pigs with high litter-bearing ability; (2) Improving the litter-bearing ability of pigs; (4) Variety improvement of high-yield piglets.

9. Application of an inhibitor that reduces the content or activity of CD47 protein in breeding pigs and / or sows in increasing the litter size after breeding of breeding pigs.

10. The application according to claim 9, characterized in that, The inhibitor inhibits its expression or activity by inhibiting CD47 protein translation, inhibiting transcriptional processing, and inhibiting the function after transcriptional processing. The inhibitor is an oral drug, an injection drug, or a patch.

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