Long-chain non-coding RNA molecular marker related to bovine semen quality and application of long-chain non-coding RNA molecular marker

Abstract

The invention discloses a long-chain non-coding RNA molecular marker related to bovine semen quality and application of the long-chain non-coding RNA molecular marker, long-chain non-coding RNA comprises an LNC68393 promoter sequence and an LNC68393 gene sequence, a molecular marker SNP1 is located on the promoter sequence, and a molecular marker SNP2-5 is located on the gene sequence. Different polymorphisms of the molecular marker are significantly related to the bull semen quality, and the molecular marker can be applied to detection of the bull semen quality, further early evaluation of the bull reproductive performance, cultivation of excellent bulls, shortening of the breeding time, improvement of the efficiency and reduction of the cost, and has important application value in improvement of the reproductive performance of the whole bull herd.

Description

Technical Field

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

[0002] The quality of bull semen 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] At present, the indicators for evaluating the quality of bull semen mainly include appearance, ejaculation volume, semen density, sperm motility and deformity rate. These indicators can only be evaluated after the semen is collected after the bull reaches sexual maturity. At this time, if the bull is found to have poor semen quality and then eliminated, it will cause serious economic losses. Therefore, the use of genetic molecular markers to identify the quality of bull semen early is of great significance for improving breeding efficiency, reducing costs, and improving the reproductive performance of the entire herd.

[0004] lncRNA (Long non-coding RNA) can affect gene expression through a variety of mechanisms, including chromatin modification, transcriptional activation, transcriptional interference, and nuclear transport. In the field of reproductive biology, lncRNA may be involved in regulating testicular development and spermatogenesis. Patent CN109750106A discloses a group of 14 lncRNAs that can be used to evaluate the motility of bull sperm, distributed on multiple chromosomes, and developed lncRNA markers for evaluating sperm motility. In practical applications, multi-site identification faces problems such as many detection reagent components and high costs, and its use is subject to certain restrictions. Summary of the invention

[0005] The purpose of the present invention is to provide a long non-coding RNA molecular marker related to bovine semen quality and an application thereof. The SNP molecular marker polymorphism provided by the present invention is significantly correlated with semen density and can be used as a genetic marker for evaluating the semen quality of bulls. It has important application value for early evaluation of bull reproductive performance and breeding of excellent breeding bulls.

[0006] The technical solution of the present invention is described in detail as follows: In a first aspect, the present invention provides a long non-coding RNA molecular marker associated with bovine semen quality, wherein the long non-coding RNA comprises a LNC68393 promoter as shown in SEQ ID NO: 1 and a LNC68393 gene as shown in SEQ ID NO: 2; The molecular markers include: SNP1 site: located at position -55 of the sequence shown in SEQ ID NO: 1, the polymorphism is G or A; SNP2 site: located at position 46 of the sequence shown in SEQ ID NO: 2, the polymorphism is G or T; SNP3 site: located at position 365 of the sequence shown in SEQ ID NO: 2, the polymorphism is A or T; SNP4 site: located at position 440 of the sequence shown in SEQ ID NO: 2, the polymorphism is T or G; SNP5 site: located at position 1377 of the sequence shown in SEQ ID NO: 2, the polymorphism is T or C.

[0007] When the above molecular markers contain any one or more of the following advantageous genotypes, the cattle have a higher sperm density: The dominant genotype at position -55 is AA; The dominant genotype at position 46 is GG; No. 365 has the dominant genotype of AA; The dominant genotype at position 440 is TT; The dominant genotype at position 1377 was CC.

[0008] In a second aspect, the present invention provides a long non-coding RNA molecular marker haplotype combination associated with bovine semen quality, wherein the long non-coding RNA comprises a LNC68393 promoter as shown in SEQ ID NO: 1 and a LNC68393 gene as shown in SEQ ID NO: 2; The molecular markers include: SNP1 site: located at position -55 of the sequence shown in SEQ ID NO: 1, the polymorphism is G or A; SNP2 site: located at position 46 of the sequence shown in SEQ ID NO: 2, the polymorphism is G or T; SNP3 site: located at position 365 of the sequence shown in SEQ ID NO: 2, the polymorphism is A or T; SNP4 site: located at position 440 of the sequence shown in SEQ ID NO: 2, the polymorphism is T or G; SNP5 site: located at position 1377 of the sequence shown in SEQ ID NO: 2, the polymorphism is T or C; The polymorphisms of the above SNP1 to 5 in the haplotype combination are AGTGC / AGTGC respectively.

[0009] In a third aspect, the present invention provides primers for detecting the quality of bovine semen, including primers for amplifying the LNC68393 promoter sequence, the nucleotide sequence of which is shown in SEQ ID NO:3~4, and primers for amplifying the LNC68393 gene sequence, the nucleotide sequence of which is shown in SEQ ID NO:5~6.

[0010] Promoter upstream primer: 5'-GTCTTAACTGTGCAAGGCTGG-3' (SEQ ID NO: 3), Promoter downstream primer: 5′-GATACCCGGAGGAGCGAGTG-3′ (SEQ ID NO: 4); Gene upstream primer: 5'-GGTCACTCCTCAAGGCTCTCAA-3' (SEQ ID NO: 5), Gene downstream primer: 5'-CTTCTCTCACCCACACACATTC-3' (SEQ ID NO: 6).

[0011] In a fourth aspect, the present invention provides a product for detecting the quality of bovine semen, comprising the above-mentioned primers.

[0012] In a fifth aspect, the present invention provides the use of the above-mentioned molecular markers, or the above-mentioned haplotype combination, or the above-mentioned primers, or the above-mentioned products in screening cattle with excellent semen quality.

[0013] In a sixth aspect, the present invention provides a method for screening cattle with high-quality semen, extracting cattle semen genomic DNA as a template, designing primers according to the LNC68393 promoter as shown in SEQ ID NO:1 and the LNC68393 gene as shown in SEQ ID NO:2 as described in claim 1, performing PCR amplification, sequencing the amplified products to detect the above-mentioned 5 SNP sites, constructing haplotype combinations, and retaining individuals with a haplotype combination of AGTGC / AGTGC, i.e., cattle with high-quality semen.

[0014] Compared with the prior art, the present invention has the following beneficial effects: The present invention identified a total of 5 SNP sites located in the promoter and gene regions of long non-coding RNA-LNC68393, and their different polymorphisms were significantly correlated with bull semen quality. The sperm densities corresponding to GG type individuals at g.46 T>G site (SNP2), TT type individuals at g.365 A>T site (SNP3), GG type individuals at g.440 G>T site (SNP4), CC type individuals at g.1337 T>C site (SNP5) and AA type individuals at g.-55 G>A site (SNP1) were significantly higher than those of TT type individuals at g.46 T>G site (SNP2), AA type individuals at g.365 A>T site (SNP3), TT type individuals at g.440 G>T site (SNP4), TT type individuals at g.1337 T>C site (SNP5) and GG type individuals at g.-55 G>A site (SNP1). The most dominant haplotype combination of SNP1~5 is AGTGC / AGTGC.

[0015] The molecular markers of the present invention can be applied to detect the quality of bull semen, and then to evaluate the reproductive performance of bulls at an early stage, cultivate excellent bulls, shorten the breeding time, improve efficiency and reduce costs, and have important application value for improving the reproductive performance of the entire cattle herd. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Statistical results of the expression level of lncRNA-LNC68393 in different tissues of adult cattle.

[0017] Figure 2 Statistical results of the expression level of lncRNA-LNC68393 in the testes of newborn calves and adult cattle.

[0018] Figure 3 The sequence structures of the two transcripts LNC68393 identified after RACE were compared with the existing reference sequence structures.

[0019] Figure 4 The SNP sites obtained by screening and identification on the promoter and gene sequences of LNC68393.

[0020] Figure 5 The results of the association analysis between different SNP loci and bovine semen quality. The numbers in brackets represent the number of bovine individuals.

[0021] Figure 6 The results of the correlation analysis between different haplotype combinations and semen quality. The numbers in brackets represent the number of cattle individuals.

[0022] Figure 7The figure is the promoter sequence of LNC68393 (SEQ ID NO: 1). The underlined bold mark site is the SNP site SNP1 on the promoter; the underlined italic mark site is the gene transcription start site, which is the +1 site.

[0023] Figure 8 The LNC68393 gene sequence (SEQ ID NO: 2) is shown in FIG. 2 . The underlined bold mark sites are SNP sites SNP2-SNP5 on the promoter; the underlined italic mark sites are gene transcription start sites, which are +1 sites. DETAILED DESCRIPTION

[0024] In order to make the technical personnel in this field better understand the present application scheme, the present application will be clearly and completely described below in conjunction with the embodiments and drawings. Obviously, the described embodiments are only embodiments of a part of the present application, rather than all embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work should belong to the scope of protection of this application. The instruments and reagents used in the embodiments are all derived from commercial channels unless otherwise specified.

[0025] Example 1 1.1 Transcriptome sequencing Transcriptome sequencing was used to screen lncRNAs that were differentially expressed in the testes of adult and newborn cattle. The significance of gene differential expression was determined by |log2 (fold change)|>1 and P<0.05. There were 920 significantly differentially expressed lncRNAs in the newborn and adult groups, of which 556 were significantly upregulated in the adult group and 364 were significantly downregulated in the adult group. According to the principle of screening differentially expressed lncRNAs, differentially expressed mRNAs within 100k upstream and downstream of lncRNAs were screened, and lncRNA-mRNA cis-regulatory pairs were constructed. A total of 822 pairs of significantly differentially expressed lncRNA-mRNA cis-regulatory pairs were screened. GO and KEGG functional enrichment analysis was performed on 551 differentially expressed genes targeted by 374 differentially expressed lncRNAs. The results of GO enrichment analysis showed that the main biological processes involved included mRNA splicing, cilia, activating transcription factor binding, and transcriptional repressor activation. In the KEGG pathway analysis, the signaling pathways that are closely related to testicular development and spermatogenesis mainly include synaptic vesicle cycle, spliceosome, etc.

[0026] 1.2 Sequence identification of lncRNA ENSBTAT00000068393 According to the principle that the function of cis target genes is related to spermatogenesis, male reproduction, meiosis, and mitosis, and the FPKM value of lncRNA is greater than 1 in any group, the functional lncRNAs for further study were preliminarily determined. A total of 21 lncRNA-mRNA regulatory pairs were screened. LncRNA ENSBTAT00000068393 (hereinafter referred to as LNC68393) is enriched in bovine testicular tissue, see Figure 1 , and the expression level in testicular tissue of adult cattle is significantly higher than that in newborn cattle, see Figure 2 This temporal and spatial specificity of expression suggests that LNC68393 may play an important role in testicular development and spermatogenesis.

[0027] Due to the incompleteness of the second-generation sequencing, we used the ENSBTAT00000068393 sequence in Ensemble as the reference sequence, and used RACE technology to design 5'GSP, 5'NGSP, 3'GSP and 3'NGSP primers to amplify the complete sequence of the 5' and 3' ends of LNC68393, and obtained the complete LNC68393 sequence. The results showed that LNC68393 has two transcripts. The full-length sequence of transcript 1 is 1135 bp, including 4 exons, and the full-length sequence of transcript 2 is 640 bp, including 3 exons, see Figure 3 The RACE primers used were as follows: Table 1 RACE primers 1.3 Analysis of the association between genetic markers on LNC68393 and semen quality 1.3.1 Extraction of genomic DNA from frozen bull semen using high salt method The high-salt method was used to extract genomic DNA from the frozen semen of 229 bulls; all 229 bulls had complete semen quality records, and the semen quality data, including semen collection volume, sperm motility, semen density, post-freezing motility and deformity rate, were taken from the average values ​​of various semen quality traits from 2018 to 2024.

[0028] 1.3.2 Amplification of the bull LNC68393 gene and promoter sequence PCR primers were designed according to the NCBI reference sequence (NC_037346.1) to amplify the gene sequence and promoter sequence of LNC68393 from 229 bulls.

[0029] Gene upstream primer: 5'-GGTCACTCCTCAAGGCTCTCAA-3' (SEQ ID NO: 5), Gene downstream primer: 5'-CTTCTCTCACCCACACACATTC-3' (SEQ ID NO: 6); Promoter upstream primer: 5'-GTCTTAACTGTGCAAGGCTGG-3' (SEQ ID NO: 3), Promoter downstream primer: 5'-GATACCCGGAGGAGCGAGTG-3' (SEQ ID NO: 4).

[0030] 1.3.3 Identification of SNP sites in the LNC68393 gene and promoter The amplified gene and promoter sequences were sequenced by Sanger sequencing, and then sequence alignment was performed using DNASTAR software to identify the SNP sites on the promoter and gene of LNC68393. 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 was identified on the promoter, g.-55 G>A, see Figure 4 . LNC68393 was genotyped in 229 bulls according to five SNP loci.

[0031] 1.3.4 Correlation analysis between different genotypes and haplotypes and bull semen quality The correlation between different genotypes of LNC68393 and bull semen quality was analyzed using SPASS software. The results showed that all five SNP sites were significantly correlated with sperm density (P < 0.05). Figure 5 The sperm densities of GG individuals at g.46 T>G, TT individuals at g.365A>T, GG individuals at g.440 G>T, CC individuals at g.1337 T>C and AA individuals at g.-55 G>A were significantly higher than those of TT individuals at g.46 T>G, AA individuals at g.365 A>T, TT individuals at g.440 G>T, TT individuals at g.1337 T>C and GG individuals at g.-55 G>A.

[0032] The haplotype analysis of the five SNP loci was performed using the SHEsis online software, and there were three haplotypes (H1=GTATT, H2=AGTTC, H3=AGTGC). The SPASS software was used to analyze the correlation between different haplotype combinations of LNC68393 and bull semen quality traits. The results showed that the semen density of the haplotype combination H3H3 was significantly higher than that of the haplotype combinations H1H1 and H1H2, and it was the dominant haplotype combination. Figure 6 .

[0033] Example 2 Semen was collected and quality tested for bulls with known genotypes (three haplotype combinations: H1H1, H1H2, and H3H3). Genomic DNA from semen was extracted and PCR amplified and SNP site polymorphism detected using primers shown in SEQ ID NO: 3-6.

[0034] 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 that of the other two combinations.

[0035] The invention concept is described in detail using specific examples herein, and the description of the above embodiments is only used to help understand the core idea of ​​the invention. It should be pointed out that any obvious modification, equivalent substitution or other improvement made by a person of ordinary skill in the art without departing from the invention concept should be included in the protection scope of the present invention.

Claims

1. A long non-coding RNA molecular marker associated with bovine semen quality, characterized in that: The long non-coding RNA includes the LNC68393 promoter as shown in SEQ ID NO: 1 and the LNC68393 gene as shown in SEQ ID NO: 2; The molecular markers include: SNP1 site: located at position -55 of the sequence shown in SEQ ID NO: 1, the polymorphism is G or A; SNP2 site: located at position 46 of the sequence shown in SEQ ID NO: 2, the polymorphism is G or T; SNP3 site: located at position 365 of the sequence shown in SEQ ID NO: 2, the polymorphism is A or T; SNP4 site: located at position 440 of the sequence shown in SEQ ID NO: 2, the polymorphism is T or G; SNP5 site: located at position 1377 of the sequence shown in SEQ ID NO: 2, the polymorphism is T or C.

2. The molecular marker according to claim 1, characterized in that Cattle with any one or more of the following dominant genotypes have higher sperm density: The dominant genotype at position -55 is AA; The dominant genotype at position 46 is GG; No. 365 has the dominant genotype of AA; The dominant genotype at position 440 is TT; The dominant genotype at position 1377 was CC.

3. A long non-coding RNA molecular marker haplotype combination related to bovine semen quality, characterized in that: The long non-coding RNA includes the LNC68393 promoter as shown in SEQ ID NO: 1 and the LNC68393 gene as shown in SEQ ID NO: 2; The molecular markers include: SNP1 site: located at position -55 of the sequence shown in SEQ ID NO: 1, the polymorphism is G or A; SNP2 site: located at position 46 of the sequence shown in SEQ ID NO: 2, the polymorphism is G or T; SNP3 site: located at position 365 of the sequence shown in SEQ ID NO: 2, the polymorphism is A or T; SNP4 site: located at position 440 of the sequence shown in SEQ ID NO: 2, the polymorphism is T or G; SNP5 site: located at position 1377 of the sequence shown in SEQ ID NO: 2, the polymorphism is T or C; The polymorphisms of the above SNP1 to 5 in the haplotype combination are AGTGC / AGTGC respectively.

4. A primer for detecting the quality of bovine semen, characterized in that: It includes primers for amplifying the LNC68393 promoter sequence, the nucleotide sequence of which is shown in SEQ ID NO: 3-4, and primers for amplifying the LNC68393 gene sequence, the nucleotide sequence of which is shown in SEQ ID NO: 5-6.

5. A product for detecting the quality of bovine semen, characterized in that: Containing the primer according to claim 4.

6. Use of the molecular marker according to any one of claims 1 to 2, or the haplotype combination according to claim 3, or the primer according to claim 4, or the product according to claim 5 in screening cattle with excellent semen quality.

7. A method for selecting cattle with high-quality semen, characterized in that: Extract bovine semen genomic DNA as a template, design primers according to the LNC68393 promoter as shown in SEQ ID NO: 1 and the LNC68393 gene as shown in SEQ ID NO: 2 as described in claim 1, perform PCR amplification, sequence the amplified products to detect the 5 SNP sites as described in claim 1, construct a haplotype combination, and retain individuals with a haplotype combination of AGTGC / AGTGC, that is, cattle with high-quality semen.

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