Long non-coding RNA molecular markers related to bovine semen quality and their applications

Abstract

The present invention discloses long non-coding RNA molecular markers related to bovine semen quality and their applications. The long non-coding RNA includes the promoter sequence of LNC68393 and the gene sequence of LNC68393. The molecular marker SNP1 is located on the promoter sequence, and the molecular markers SNP2-5 are located on the gene sequence. Different polymorphisms of the molecular markers are significantly correlated with bull semen quality, and can be applied to detect bull semen quality, thereby early evaluating the reproductive performance of bulls, breeding excellent bulls, shortening the breeding time, improving efficiency and reducing costs, and having important application value for improving the reproductive performance of the entire cattle herd.

Description

Technical Field

[0001] The present invention relates to the technical field of bull reproductive performance evaluation and genetic breeding, and specifically relates to long non-coding RNA molecular markers related to bull semen quality and their applications. Background Art

[0002] Bull semen quality is an important indicator to measure the reproductive ability of breeding bulls. It directly affects the success rate of artificial insemination and the health of offspring, and has a significant impact on the genetic improvement and production efficiency of the entire cattle herd.

[0003] Currently, the indicators for evaluating bull semen quality mainly include appearance, ejaculate volume, semen density, sperm motility, and malformation rate, etc. Evaluating these indicators requires collecting semen after the bull reaches sexual maturity for detection. At this time, if it is found that the bull has poor semen quality and the bull is then culled, it will cause relatively serious economic losses. Therefore, using genetic molecular markers to identify bull semen quality early is of great significance for improving breeding efficiency, reducing costs, and improving the reproductive performance of the entire cattle herd.

[0004] LncRNA (Long non-coding RNA) can affect gene expression through multiple mechanisms, including chromatin modification, transcriptional activation, transcriptional interference, and nuclear transport, etc. In the field of reproductive biology, lncRNA may be involved in regulating testicular development and spermatogenesis processes. Patent CN109750106A discloses a group of 14 lncRNAs that can be used to evaluate the high or low sperm motility of bulls, which are distributed on multiple chromosomes, and lncRNA markers for evaluating the high or low sperm motility are developed. Multilocus identification faces problems such as many components in detection reagents and high costs in practical applications, and its use is restricted to a certain extent. Summary of the Invention

[0005] The purpose of the present invention is to provide long non-coding RNA molecular markers related to bull semen quality and their applications. The SNP molecular markers provided by the present invention have polymorphisms significantly related to semen density and can be used as genetic markers for evaluating bull semen quality, which have important application values for early evaluation of bull reproductive performance and the cultivation of excellent breeding bulls.

[0006] The technical solution of the present invention is described in detail as follows:

[0007] In the first aspect, the present invention provides long non-coding RNA molecular markers related to bull semen quality, and the long non-coding RNA includes the LNC68393 promoter shown in SEQ ID NO:1 and the LNC68393 gene shown in SEQ ID NO:2;

[0008] The molecular markers include:

[0009] SNP1 locus: Located at the 2462nd position of the sequence shown in SEQ ID NO:1, with polymorphisms of G or A;

[0010] SNP2 locus: Located at the 216th position of the sequence shown in SEQ ID NO:2, with polymorphisms of G or T;

[0011] SNP3 locus: Located at the 535th position of the sequence shown in SEQ ID NO:2, with polymorphisms of A or T;

[0012] SNP4 locus: Located at the 610th position of the sequence shown in SEQ ID NO:2, with polymorphisms of T or G;

[0013] SNP5 locus: Located at the 1507th position of the sequence shown in SEQ ID NO:2, with polymorphisms of T or C.

[0014] For the above molecular markers, when containing any one or more combinations of the following advantageous genotypes, cattle have a higher sperm density:

[0015] The advantageous genotype at the 2462nd position is AA;

[0016] The advantageous genotype at the 216th position is GG;

[0017] The advantageous genotype at the 535th position is AA;

[0018] The advantageous genotype at the 610th position is TT;

[0019] The advantageous genotype at the 1507th position is CC.

[0020] In the second aspect, the present invention provides a long non-coding RNA molecular marker haplotype combination related to the semen quality of cattle, and the long non-coding RNA includes the LNC68393 promoter shown in SEQ ID NO:1 and the LNC68393 gene shown in SEQ ID NO:2;

[0021] The molecular markers include:

[0022] SNP1 locus: Located at the 2462nd position of the sequence shown in SEQ ID NO:1, with polymorphisms of G or A;

[0023] SNP2 locus: Located at the 216th position of the sequence shown in SEQ ID NO:2, with polymorphisms of G or T;

[0024] SNP3 locus: Located at the 535th position of the sequence shown in SEQ ID NO:2, with polymorphisms of A or T;

[0025] SNP4 locus: Located at the 610th position of the sequence shown in SEQ ID NO:2, with polymorphisms of T or G;

[0026] SNP5 locus: located at the 1507th position of the sequence shown in SEQ ID NO:2, with polymorphisms of T or C;

[0027] In the haplotype combination described above, the polymorphisms of the above SNPs 1-5 are AGTGC / AGTGC respectively.

[0028] In the third aspect, the present invention provides primers for detecting the quality of bovine semen, including primers for amplifying the promoter sequence of LNC68393, with nucleotide sequences shown in SEQ ID NO:3-4, and primers for amplifying the gene sequence of LNC68393, with nucleotide sequences shown in SEQ ID NO:5-6.

[0029] Promoter upstream primer: 5’-GTCTTAACTGTGCAAGGCTGG-3’ (SEQ ID NO:3),

[0030] Promoter downstream primer: 5’-GATACCCGGAGGAGCGAGTG-3’ (SEQ ID NO:4);

[0031] Gene upstream primer: 5’-GGTCACTCCTCAAGGCTCTCAA-3’ (SEQ ID NO:5),

[0032] Gene downstream primer: 5’-CTTCTCTCACCCACACACATTC-3’ (SEQ ID NO:6).

[0033] In the fourth aspect, the present invention provides a product for detecting the quality of bovine semen, containing the above primers.

[0034] In the fifth aspect, the present invention provides the application of the above molecular markers, or the above haplotype combination, or the above primers, or the above product in screening cattle with excellent semen quality.

[0035] In the sixth aspect, the present invention provides a method for screening cattle with high-quality semen. Extract the genomic DNA of bovine semen as a template, design primers based on the LNC68393 promoter shown in SEQ ID NO:1 and the LNC68393 gene shown in SEQ ID NO:2, perform PCR amplification, sequence the amplification products to detect the above 5 SNP loci, construct a haplotype combination, and retain the individuals with the haplotype combination of AGTGC / AGTGC, which are the cattle with high-quality semen.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] The present invention identified a total of 5 SNP sites located in the promoter and gene region of long non-coding RNA-LNC68393, and their different polymorphisms were significantly correlated with bull semen quality. For individuals with the GG genotype at the g.46 T>G site (SNP2), the TT genotype at the g.365 A>T site (SNP3), the GG genotype at the g.440 G>T site (SNP4), the CC genotype at the g.1337 T>C site (SNP5), and the AA genotype at the g.-55 G>A site (SNP1), the sperm density was significantly higher than that of individuals with the TT genotype at the g.46 T>G site (SNP2), the AA genotype at the g.365 A>T site (SNP3), the TT genotype at the g.440 G>T site (SNP4), the TT genotype at the g.1337 T>C site (SNP5), and the GG genotype at the g.-55 G>A site (SNP1). The most dominant haplotype combination of SNP1-5 is AGTGC / AGTGC.

[0038] The molecular markers of the present invention can be applied to detect bull semen quality, thereby early evaluating the reproductive performance of bulls, cultivating excellent bulls, shortening the breeding time, improving efficiency and reducing costs, and have important application value for improving the reproductive performance of the entire cattle herd. Brief Description of the Drawings

[0039] Figure 1 It is the statistical result of the expression level of lncRNA-LNC68393 in different tissues of adult cattle.

[0040] Figure 2 It is the statistical result of the expression level of lncRNA-LNC68393 in the testes of neonatal and adult cattle.

[0041] Figure 3 It is the comparison result of the sequence structures of two transcripts LNC68393 identified after RACE and the existing reference sequence structure.

[0042] Figure 4 They are the SNP sites screened and identified on the promoter and gene sequence of LNC68393.

[0043] Figure 5 It is the analysis result of the correlation between different SNP sites and bull semen quality. The numbers in brackets represent the number of cattle individuals.

[0044] Figure 6 It is the analysis result of the correlation between different haplotype combinations and semen quality. The numbers in brackets represent the number of cattle individuals.

[0045] Figure 7It is the promoter sequence of LNC68393 (SEQ ID NO: 1). The site marked in bold with an underline in the figure is the SNP site SNP1 on the promoter; the site marked in italics with an underline is the gene transcription start site, which is the +1 site.

[0046] Figure 8 It is the gene sequence of LNC68393 (SEQ ID NO: 2). The sites marked in bold with an underline in the figure are the SNP sites SNP2 - SNP5 on the promoter; the site marked in italics with an underline is the gene transcription start site, which is the +1 site. Specific implementation manners

[0047] To enable those skilled in the art to better understand the solution of this application, the following will clearly and completely describe this application in combination with the embodiments and the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, rather than all the embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the scope of protection of this application. The instruments and reagents used in the embodiments are all from commercial channels unless otherwise specified.

[0048] Example 1

[0049] 1.1 Transcriptome sequencing

[0050] Use transcriptome sequencing to screen for lncRNAs with differential expression in adult and neonatal bovine testes. The significance of gene differential expression is screened with |log2 (fold change)| > 1 and P < 0.05 as the screening criteria. There are 920 lncRNAs with significant differential expression in the neonatal group and the adult group, among which 556 are significantly up-regulated in the adult group and 364 are significantly down-regulated in adult cattle. According to the principle of screening differentially expressed LncRNAs, screen for differentially expressed mRNAs within 100k upstream and downstream of the lncRNA, and construct cis-regulatory pairs of lncRNA-mRNA. A total of 822 pairs of significantly differentially expressed lncRNA-mRNA cis-regulatory pairs are screened. Perform GO and KEGG functional enrichment analysis on 551 differentially expressed genes targeted by 374 differentially expressed lncRNAs. The results of GO enrichment analysis show that it mainly includes biological processes related to mRNA splicing, cilia, activation of transcription factor binding, activation of transcriptional repressors, etc. In the KEGG pathway analysis, the signal pathways more closely related to testicular development and spermatogenesis mainly include synaptic vesicle cycle, spliceosome, etc.

[0051] 1.2 Sequence identification of LncRNA ENSBTAT00000068393

[0052] According to the principle that the FPKM value of lncRNA is greater than 1 in any group related to the function of cis-target genes and spermatogenesis, male reproduction, meiosis, and mitosis, functional lncRNAs to be further studied were initially determined. A total of 21 pairs of lncRNA-mRNA regulatory pairs were screened. LncRNA ENSBTAT00000068393 (hereinafter referred to as LNC68393) is enriched in bovine testicular tissue, see Figure 1 , and its expression level in adult bovine testicular tissue is significantly higher than that in neonatal calves, see Figure 2 . This spatio-temporal specificity of expression indicates that LNC68393 may play an important role in testicular development and spermatogenesis.

[0053] Due to the incompleteness of second-generation sequencing, using the ENSBTAT00000068393 sequence in Ensemble as the reference sequence, and by using the RACE technique, 5’GSP, 5’NGSP, 3’GSP, and 3’NGSP primers were designed respectively to amplify the complete 5’ and 3’ ends of LNC68393, and the complete LNC68393 sequence was obtained. The results showed that LNC68393 has two transcripts. The full-length sequence of transcript 1 is 1135 bp, containing 4 exons, and the full-length sequence of transcript 2 is 640 bp, containing 3 exons, see Figure 3 . The RACE primers used are as follows:

[0054] Table 1 RACE primers

[0055]

[0056] 1.3 Association analysis between genetic markers on LNC68393 and semen quality

[0057] 1.3.1 Extraction of genomic DNA from frozen semen of bulls by high-salt method

[0058] Genomic DNA of frozen semen from 229 bulls was extracted by the high-salt method; all 229 bulls had complete semen quality records, and the semen quality data, including semen volume, sperm motility, semen density, post-thaw motility, and malformation rate, were taken from the average values of each semen quality trait from 2018 to 2024.

[0059] 1.3.2 Amplification of the LNC68393 gene and promoter sequence of bulls

[0060] According to the NCBI reference sequence (NC_037346.1), PCR primers were designed to amplify the gene sequence and promoter sequence of LNC68393 in 229 bulls respectively.

[0061] Upstream primer of the gene: 5’-GGTCACTCCTCAAGGCTCTCAA-3’ (SEQ ID NO:5),

[0062] Downstream primer of the gene: 5’-CTTCTCTCACCCACACACATTC-3’ (SEQ ID NO:6);

[0063] Upstream primer of the promoter: 5’-GTCTTAACTGTGCAAGGCTGG-3’ (SEQ ID NO:3),

[0064] Downstream primer of the promoter: 5’-GATACCCGGAGGAGCGAGTG-3’ (SEQ ID NO:4).

[0065] 1.3.3 Identification of SNP sites on the LNC68393 gene and promoter

[0066] The amplified gene and promoter sequences were subjected to Sanger sequencing, and then sequence alignment was performed using DNASTAR software to identify SNP sites on the LNC68393 promoter and gene. Four SNP sites were identified on the gene, namely: g.46 T>G, g.365 A>T, g.440 G>T, g.1337 T>C, and one SNP site g.-55 G>A was identified on the promoter, see Figure 4 . Genotyping of LNC68393 in 229 bulls was performed according to the 5 SNP sites.

[0067] 1.3.4 Correlation analysis of different genotypes and haplotypes with bull semen quality

[0068] SPASS software was used to analyze the correlation between different genotypes of LNC68393 and bull semen quality. The results showed that all 5 SNP sites were significantly correlated with sperm density (P < 0.05), see Figure 5 . For the GG genotype individuals at the g.46 T>G site, TT genotype individuals at the g.365A>T site, GG genotype individuals at the g.440 G>T site, CC genotype individuals at the g.1337 T>C site, and AA genotype individuals at the g.-55 G>A site, the corresponding sperm densities were significantly higher than those of the TT genotype individuals at the g.46 T>G site, AA genotype individuals at the g.365 A>T site, TT genotype individuals at the g.440 G>T site, TT genotype individuals at the g.1337 T>C site, and GG genotype individuals at the g.-55 G>A site.

[0069] The online software SHEsis was used to perform haplotype analysis on 5 SNP loci, and a total of 3 haplotypes were found (H1 = GTATT, H2 = AGTTC, H3 = AGTGC). The SPASS software was used to analyze the correlation between different haplotype combinations of LNC68393 and the semen quality traits of bulls. The results showed that the semen density of the haplotype combination H3H3 was significantly higher than that of the H1H1 and H1H2 haplotype combinations, which was the dominant haplotype combination, as shown in Figure 6 .

[0070] Example 2

[0071] Semens were collected and their quality was detected from bulls with known genotypes (three haplotype combinations of H1H1, H1H2, and H3H3). Meanwhile, genomic DNA of the semen was extracted, and PCR amplification and SNP locus polymorphism detection were performed using the primers shown in SEQ ID NO: 3-6.

[0072] The results of SNP polymorphism and semen quality detection were consistent with those in Example 1. The semen density of bulls with the H3H3 haplotype combination was significantly higher than the other two combinations.

[0073] Specific examples are used in this article to elaborate on the inventive concept in detail. The descriptions of the above examples are only used to help understand the core idea of the present invention. It should be noted that for those of ordinary skill in the art, any obvious modifications, equivalent replacements, or other improvements made without departing from the inventive concept shall be included in the protection scope of the present invention.

Claims

1. Use of long non-coding RNA molecular markers related to bovine semen quality in screening cattle with high sperm density, characterized in that the long non-coding RNA includes the LNC68393 promoter shown in SEQ ID NO:1 and the LNC68393 gene shown in SEQ ID NO:2; the molecular markers include: SNP1 locus: located at the 2462nd position of the sequence shown in SEQ ID NO:1, with polymorphisms of G or A; SNP2 locus: located at the 216th position of the sequence shown in SEQ ID NO:2, with polymorphisms of G or T; SNP3 locus: located at the 535th position of the sequence shown in SEQ ID NO:2, with polymorphisms of A or T; SNP4 locus: located at the 610th position of the sequence shown in SEQ ID NO:2, with polymorphisms of T or G; SNP5 locus: located at the 1507th position of the sequence shown in SEQ ID NO:2, with polymorphisms of T or C.

2. The application according to claim 1, wherein When a combination contains any one or more of the following advantageous genotypes, the cattle have higher sperm density: The advantageous genotype at the 2462nd position is AA; The advantageous genotype at the 216th position is GG; The advantageous genotype at the 535th position is AA; The advantageous genotype at the 610th position is TT; The advantageous genotype at the 1507th position is CC.

3. Use of long non-coding RNA molecular marker haplotype combinations in screening cattle with high sperm density, characterized in that the long non-coding RNA includes the LNC68393 promoter shown in SEQ ID NO:1 and the LNC68393 gene shown in SEQ ID NO:2; the molecular markers include: SNP1 locus: located at the 2462nd position of the sequence shown in SEQ ID NO:1, with polymorphisms of G or A; SNP2 locus: located at the 216th position of the sequence shown in SEQ ID NO:2, with polymorphisms of G or T; SNP3 locus: located at the 535th position of the sequence shown in SEQ ID NO:2, with polymorphisms of A or T; SNP4 locus: located at the 610th position of the sequence shown in SEQ ID NO:2, with polymorphisms of T or G; SNP5 locus: located at the 1507th position of the sequence shown in SEQ ID NO:2, with polymorphisms of T or C; In the haplotype combination, the polymorphisms of the above SNPs 1-5 are AGTGC / AGTGC respectively.

4. Use of primers for detecting the quality of bovine semen in screening cattle with high sperm density, characterized in that, It includes primers for amplifying the LNC68393 promoter sequence, with nucleotide sequences shown in SEQ ID NO:3-4, and primers for amplifying the LNC68393 gene sequence, with nucleotide sequences shown in SEQ ID NO:5-6; Screening method: Extract the genomic DNA of bovine semen as a template, perform PCR amplification using the primers of SEQ ID NO:3-6, sequence the amplification products to detect the 5 SNP loci of claim 1, construct a haplotype combination, and retain the individuals with the haplotype combination of AGTGC / AGTGC, which are the cattle with high sperm density.

5. Use of a product for detecting the quality of bovine semen in screening bulls with high sperm density, characterized in that, Containing the primers described in claim 4; Screening method: Extract the genomic DNA of bovine semen as a template, perform PCR amplification using the primers of SEQ ID NO: 3-6, sequence the amplification product to detect the 5 SNP sites of Claim 1, construct haplotype combinations, and retain the individuals with the haplotype combination of AGTGC / AGTGC, which are the cattle with high sperm density.

6. A method for screening cattle with high sperm density, characterized in that, Extract the genomic DNA of bovine semen as a template, design primers based on the LNC68393 promoter shown in SEQ ID NO: 1 and the LNC68393 gene shown in SEQ ID NO: 2 according to Claim 1, perform PCR amplification, sequence the amplification product to detect the 5 SNP sites of Claim 1, construct haplotype combinations, and retain the individuals with the haplotype combination of AGTGC / AGTGC, which are the cattle with high sperm density.

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