CN109750106A - A kind of combination of long-chain non-coding RNA and its detection method and application for evaluating bull sperm vigor height - Google Patents
A kind of combination of long-chain non-coding RNA and its detection method and application for evaluating bull sperm vigor height Download PDFInfo
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Abstract
The present invention provides a kind of combination of long-chain non-coding RNA and its detection method and application for evaluating bull sperm vigor height, develop lncRNA label and Molecular Detection and the evaluation method for assessing bull sperm vigor, which comprises the acquisition and storage of semen sample；RNA is extracted, library construction and transcript profile sequencing；Transcript profile assembling；The identification of candidate lncRNA and Differential expression analysis；The chromosome mapping of differential expression lncRNA；The functional lncRNA for evaluating bull sperm vigor is screened to combine；Special primer is designed, lncRNA real-time fluorescence quantitative PCR detection method is established.Detection method design is rationally, conveniently, recruit's label-functionality lncRNA of sperm motility is effectively influenced in detection sperm, this has great significance for the development of the molecular breeding of the prolificacy bull based on lncRNA and the Molecular Evaluation technology of bull semen quality and practical value.
Description
Technical Field
The invention belongs to the technical field of animal molecular breeding, and particularly relates to a functional lncRNA combination for evaluating sperm motility of a bull, and a detection method and application thereof.
Background
The fertility of bulls is the most important factor influencing the genetic quality and the economic benefit of cows, and plays a key role in the genetic improvement of modern cows. In recent decades, the breeding of the breeding character is neglected because people pay more attention to the breeding of the characters such as milk yield of the milk cows and the like. With the large-scale application of artificial insemination in cow breeding, the evaluation of semen quality is particularly important. Semen quality is commonly assessed by several indicators, such as ejaculate volume, semen density, sperm motility, and teratogenesis. The sperm motility is a main index for evaluating the quality of the bull semen and is also a main index for determining whether fresh semen can produce frozen semen. Insufficient sperm motility is one of the key causes of low fertility or infertility in bulls (Guzick et al, 2001). Failure of semen production due to poor sperm motility can cause severe economic losses.
The bull sperm motility refers to the percentage of the total sperm count of the advancing movement sperm under the environment of 37 ℃. The heritability of the sperm motility of the breeding bull is about 0.43 (Druet et al, 2009), belongs to the character of moderate heritability, and the breeding of the bull with high sperm motility by using the conventional breeding technology is very difficult. In the national standard GB4143-2008 frozen bovine semen, the sperm motility of fresh semen is required to be more than or equal to 65%, and the sperm motility of frozen semen after unfreezing is required to be more than or equal to 35%. The most outstanding problem in the breeding practice of the bulls is that the quality of the semen of the bulls is difficult to evaluate and predict in the early stage of the bulls, because the high and low sperm motility can be judged only by normal semen collection after the sexual maturity of the bulls, and if the sperm motility is found to be low and then eliminated, great loss of manpower and material resources is caused. Another outstanding problem is that although relevant detection methods and evaluation indexes have been developed to measure semen quality, the existing semen quality evaluation techniques are basically based on phenotypic evaluation, especially in the general evaluation technique of sperm motility microscope, and the accuracy and reliability of detection need to be improved. To date, no mature molecular detection index has been applied to the assessment of bull sperm motility. In some genome-wide association studies, candidate genes and Single Nucleotide Polymorphisms (SNPs) have been identified that correlate with bull sperm motility (Qin et al, 2016). In addition, high throughput technologies, including transcriptomics and proteomics, also reveal many differences between high and low fertility bull sperm (petdinti et al, 2008). However, little is still known about which regulatory mechanisms affect the difference in bull sperm motility. In production practice, we found that although the fully sibling bulls had similar genetic background, there was a great difference in bull sperm motility. Therefore, the potential reasons influencing the sperm motility difference of the bull are urgently needed to be understood, the molecular marker capable of evaluating the sperm motility is identified, the molecular marker has important scientific significance for disclosing the molecular mechanism of the high fertility of the breeding bull, the early genetic evaluation of the breeding performance of the breeding bull is carried out, the prediction and accurate evaluation of the semen quality are carried out, and the application value is great.
Current RNA sequencing studies indicate that some long non-coding RNAs (lncrnas) may regulate testicular development and spermatogenesis by currently unknown mechanisms (Anguera et al, 2011). IncRNA refers to noncoding RNA with the length of more than 200 nucleotides, and is a key regulator in the process of transcription and translation. So far, no research report about functional lncRNA influencing the sperm motility of bulls exists. According to the scheme, the semen transcriptome of the full sibling paired bull with different sperm activities is subjected to high-throughput sequencing, 14 functional lncRNAs influencing the sperm activities of the bull are obtained, lncRNA markers capable of being used for evaluating the sperm activities of the bull are developed, and the method has important significance and practical value for the molecular evaluation technologies of genetic evaluation, early breeding, semen quality and the like of the high-sperm activity bull based on lncRNA.
Disclosure of Invention
In order to solve the problems, the invention provides a functional lncRNA combination for evaluating the sperm motility of bulls, a detection method and application thereof.
The invention is realized by the following technical scheme:
aiming at the prior art, the invention aims to provide a functional lncRNA marker combination for sperm motility assessment, which can perform molecular assessment on bull sperm motility and fill the blank of bull semen quality molecular assessment.
One of the objectives of the present invention is to provide a method for identifying functional lncRNA affecting bull sperm motility.
The invention also aims to provide a functional lncRNA combination which can be used for evaluating the sperm motility of bulls and a detection method thereof.
A combination of long-chain non-coding RNAs for evaluating bull sperm motility, which comprises one or more of the following lncRNA positioning chromosomes:
TCONS _00006462, chr11: 20027589-;
TCONS-00006445, chr11: 16735802-;
TCONS _00052267, chr7:23688218-23688432, and the nucleotide sequence is shown as SEQ ID NO. 3;
TCONS-00005210, chr11:23353411-23353642, the nucleotide sequence of which is shown in SEQ ID NO. 4;
TCONS _00043365, chr4:49814880-49815099, the nucleotide sequence is shown in SEQ ID NO. 5;
TCONS-00037129, chr27:22465453-22465676, the nucleotide sequence is shown as SEQ ID NO. 6;
TCONS-00006488, chr11:22929103-22929335, the nucleotide sequence is shown as SEQ ID NO. 7;
TCONS _00050707, chr7:28508257 and 28508488, and the nucleotide sequence is shown as SEQ ID NO. 8;
TCONS _00039078, chr29:50150762 and 50153142, and the nucleotide sequence is shown as SEQ ID NO. 9;
TCONS _00028106, chr21:54789843-54790435, the nucleotide sequence is shown in SEQ ID NO. 10;
TCONS _00008791, chr12:83016641-83016844, and the nucleotide sequence is shown as SEQ ID NO. 11;
TCONS _00055082, chr8:80024030 and 80024244, wherein the nucleotide sequence is shown as SEQ ID NO. 12;
TCONS _00043041, chr4:6923803 and 6924026, the nucleotide sequence is shown as SEQ ID NO. 13;
TCONS _00039303, chr29:20825977 and 20826212, the nucleotide sequence is shown in SEQ ID NO. 14.
According to the long-chain non-coding RNA combination for evaluating the sperm motility of the bull, respectively designing specific primers,
TCONS _00039078 forward primer: 5'-ATGCCTGGGACTTGGAAC-3', as shown in SEQ ID NO. 15;
TCONS — 00039078 reverse primer: 5'-GTGGGTCGTCCGTTCCTTTA-3', as shown in SEQ ID NO. 16;
TCONS _00028106 forward primer: 5'-GATGCCTAAACTTTCCTGA-3', as shown in SEQ ID NO. 17;
TCONS — 00028106 reverse primer: 5'-TTCTGCTTTGAGTGTCTGC-3', as shown in SEQ ID NO. 18;
TCONS _00050707 forward primer: 5'-CACAAATGCGTGACTGAACTG-3', as shown in SEQ ID NO. 19;
TCONS — 00050707 reverse primer: 5'-GTCCTATGGCAAAGCAGAA-3', as shown in SEQ ID NO. 20;
TCONS _00039303 forward primer: 5'-TGGTTACGCAGCAAAAGCA-3', as shown in SEQ ID NO. 21;
TCONS — 00039303 reverse primer: 5'-CTGTCTCTGAAGTAGGTGGCA-3', as shown in SEQ ID NO. 22.
The application of the long-chain non-coding RNA combination in bull sperm motility evaluation and the application of the kit prepared from the primer combination in bull sperm motility evaluation are disclosed.
In a first aspect of the invention, there is provided a method of identifying functional incrnas that affect sperm motility, the method comprising:
s1, collecting and storing semen samples;
s2, RNA extraction, library construction and transcriptome sequencing;
s3, transcriptome assembly;
s4, identifying candidate lncRNA and analyzing differential expression;
s5 chromosomal mapping of differentially expressed lncRNA.
In step S1, the specific method includes:
s1.1 measuring the sperm motility of the homozygote bull by using a sperm analysis system (AndrVision, Minitube, Germany), selecting three pairs of homozygote bull with different sperm motility according to the average sperm motility in the production records from 2011 to 2015, assigning the three bull to high sperm motility groups (H: H1, H2 and H3), and assigning the remaining three bull to low sperm motility groups (L: L1, L2 and L3);
s1.2 fresh semen was collected separately from each bull and immediately stored frozen in liquid nitrogen until RNA extraction.
In step S2, the method specifically includes:
s2.1, extracting total RNA from the semen sample;
s2.2, evaluating the RNA quality;
s2.3, carrying out random fragmentation treatment on the total RNA;
s2.4, reverse transcription of the cleaved RNA fragments to generate a final RNA-seq library;
s2.5 paired-end sequencing was performed.
Wherein,
in step S2.1, RNA extraction is carried out by using Animal Tissue RNA Purification Kit TRK-1002 (LC Sciences, Houston, Texas);
in step S2.2, RNA quality was performed using an Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and a Nano Drop ND-2000 spectrophotometer (Nano-Drop, Wilmington, DE, USA)Evaluating; RNA integrity values in excess of 7.0, OD are required260 / 280A value greater than 1.8;
in step S2.3, the size of the obtained fragment is 200-600 bp;
in step S2.5, the sequencer used is an Illumina HiSeq sequencerTM4000。
In step S3, the method specifically includes:
s3.1, checking the obtained original sequence data in the FASTAQ file format by using FastQC software;
s3.2 the obtained clean data were mapped to the bovine reference genome (UMD 3.1) database using Tophat2 software (v2.0.9). The sequencing fragments for each sample were then assembled, located using a Cufflinks (v2.1.1) package;
s3.3 all transcriptomes from six samples were pooled using the Cuffmerge program to reconstruct a comprehensive transcriptome profile.
In step S4, the method specifically includes:
s4.1 annotating the assembled transcript using the Cuffcompare program in the Cufflinks software package;
s4.2, using Coding Potential Corrector (CPC) and Coding-Non-Coding-index (CNCI) to estimate the Coding Potential of the transcript with the length of more than or equal to 200bp, the mapping reading coverage of more than or equal to 3 and the exon number of more than or equal to 1, and considering the transcript with the CPC score of-1 and the CNCI score of less than 0 as a candidate lncRNA;
s4.3 Using the Cuffdiff program in the Cufflinks software package, the expression level of lncRNA was estimated by the FPKM method.
In step S5, the method specifically includes:
analyzing the distribution of the differentially expressed lncrnas on the chromosome;
in a second aspect of the present invention, there is provided a functional lncRNA combination capable of being used for bull sperm motility assessment and a detection method thereof, the method comprising:
s6, screening a functional lncRNA combination for evaluating the sperm motility of the bull;
s7, real-time fluorescent quantitative PCR verification.
In step S6, the specific method includes:
s6.1, positioning the lncRNA with differential expression to a Quantitative Trait Locus (QTLs) of the cattle, and analyzing the lncRNA related to sperm motility;
s6.2 besides the lncRNA on the bovine QTL related to the sperm motility, 6 lncRNA with obvious differences are screened according to the function annotation and the differential expression quantity of the genes adjacent to the lncRNA.
In step S7, the specific method includes:
aiming at the lncRNA combination, a specific primer is designed to carry out Real-Time fluorescent quantitative PCR verification on a LightCycler 480II Real-Time PCR system. 25 μ L reaction: 12.5 μ L of 2 XSSYBR Green Real-time PCRMaster Mix, 1 μ L each of forward and reverse primers, 2 μ L of cDNA, 8.5 μ L H2And O. Reaction conditions are as follows: 3min at 95 ℃; 95 ℃, 15s, 57 ℃, 15s, 72 ℃, 20s, 40 cycles. Each reaction was run in triplicate.
A kit for use in bull sperm motility assessment, said kit comprising: one or more of SEQ ID NO.1 to SEQ ID NO.14, and an upstream primer and a downstream primer for amplifying gene segments; PCR amplification reagents.
Advantageous effects
The detection method is reasonable in design, and can effectively detect functional lncRNA affecting sperm motility in semen;
the invention screens 14 functional lncRNAs which influence the vitality of bull sperms by carrying out high-throughput sequencing on a bull semen transcriptome, and then identifies lncRNAs which can be used for evaluating the vitality of the sperms by carrying out functional annotation and analysis on potential candidate lncRNAs; and finally, an lncRNA marker for evaluating the sperm motility is developed for the first time by establishing a qPCR method for detecting lncRNA, so that the method has important significance and practical value for the molecular breeding of the dairy cattle based on lncRNA and the development of the molecular evaluation technology of the sperm quality of bulls.
Drawings
FIG. 1: classification and density distribution of the novel lncrnas identified. (A) The four types of distributions of "u", "o", "x" and "j". (B) Density distributions of "j", "o", "u" and "x" lncRNA species on bovine chromosomes;
FIG. 2: the expression level of lncRNA in the high sperm motility group and the low sperm motility group;
FIG. 3: distribution of differentially expressed lncrnas on chromosomes;
FIG. 4: localization of differentially expressed lncRNA on bovine QTLs associated with sperm motility;
FIG. 5: the main flow chart of the invention.
Detailed Description
The following examples are given for the detailed implementation and specific operation of the present invention, but the scope of the present invention is not limited to the following examples.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
The invention discloses a functional lncRNA for detecting sperm motility in semen. 14 functional lncRNA combinations affecting the sperm motility of bulls were identified using this method.
The design idea of the invention is as follows: first, total RNA was extracted from bull semen and paired-end sequencing was performed. Next, transcriptome assembly was performed on the sequencing data. Then, lncRNA combinations correlated with sperm motility were selected. And finally, identifying the expression quantity of the lncRNA by adopting real-time fluorescent quantitative PCR.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The test methods in the following examples, which are not specified under specific conditions, are generally carried out under conventional conditions.
1. Semen sample collection and storage
(1) The sperm motility of the homonas was determined by using a sperm analysis system (AndroVision, Minitube, Germany), three pairs of homonas with different sperm motility were selected according to the average sperm motility in the production records from 2011 to 2015, three bulls were assigned to the high sperm motility group (H: H1, H2 and H3), and the remaining three bulls were assigned to the low sperm motility group (L: L1, L2 and L3).
(2) Fresh semen was collected separately from each bull and immediately stored frozen in liquid nitrogen until RNA extraction.
2. RNA extraction, library construction and transcriptome sequencing
(1) Total RNA was extracted from semen samples using the Animal Tissue RNA Purification Kit TRK-1002 (LC Sciences, Houston, Texas).
(2) Using Agilent 2RNA quality was assessed with a 100 bioanalyzer (Agilent Technologies, Santa Clara, Calif., USA) and a Nano Drop ND-2000 spectrophotometer (Nano-Drop, Wilmington, DE, USA). RNA integrity number over 7.0, OD260/280Samples with values greater than 1.8 were available for subsequent experiments.
(3) The total RNA is subjected to fragmentation treatment to obtain a small fragment of 200-600 bp.
(4) The cleaved RNA fragments are reverse transcribed to generate the final RNA-seq library.
(5) Using Illumina HiSeqTMPaired end sequencing was performed on platform 4000.
3. Transcriptome assembly
(1) The raw sequence data obtained in the form of a FASTAQ file was examined using FastQC software. The raw data was then filtered through the following procedure: 1) the linker sequence is removed. 2) Fragments containing more than 5% of the unknown bases are discarded. 3) Fragments with a quality score Q of 10 or less in which more than 20% of the bases are deleted. The clean data obtained (table 1) was used for subsequent analysis.
TABLE 1 summary of statistical data for RNA-seq sequenced fragments in six samples
(2) The obtained clean data were mapped to a bovine reference genomic database (ftp:// ftp. ensembl. org/pub/release-86/fasta/bos _ taurus/dnas /) using Tophat2 software (v2.0.9), see table 2. The sequencing fragments for each sample were then assembled, located using a Cufflinks (v2.1.1) package.
TABLE 2 clean fragments mapping to bovine reference genome
(3) All transcriptomes from six samples were pooled using the Cuffmerge program to reconstruct a comprehensive transcriptome profile.
4. Identification and differential expression analysis of candidate lncRNA
(1) The assembled transcripts were annotated using the Cuffcompare program in the Cufflinks software package.
(2) The Coding Potential of transcripts with a length of 200bp or more, a mapped read coverage of 3 or more and an exon count of 1 or more was estimated using Coding Potential Calculator (CPC) and Coding-Non-Coding-index (CNCI). Prediction results transcripts with CPC score < -1 and CNCI score <0 were considered candidate lncrnas. 11,561 novel candidate lncRNAs were identified. See fig. 1.
(3) The expression level of lncRNA was estimated by FPKM method using the Cuffdiff program in the Cufflinks software package. The FPKM values for approximately 80% of lncRNA transcripts were less than 2, and only approximately 10% were greater than 10, as shown in FIG. 2.
(4) Of the 11,561 lncRNA, 528 were differentially expressed between the H and L groups, with 22 up-regulated genes and 506 down-regulated genes in the high-sperm viability group (FPKM value ≥ 50, FDR ≤ 0.05, and | log2 (fold change) | ≥ 1).
5. Chromosomal mapping of differentially expressed lncRNA
The distribution of differentially expressed lncRNA on chromosomes was analyzed. Differentially expressed lncRNAs are located mostly on chromosomes 1,6 and 14, see fig. 3.
6. Functional lncRNA combination for evaluating sperm motility of bulls
(1) And (3) positioning the lncRNA with differential expression to a bovine Quantitative Trait Locus (QTLs), and analyzing the lncRNA related to sperm motility. Of these 8 are located on bovine QTLs associated with sperm motility, see lncRNA nos. 1-8 in table 3, and the location on bovine QTLs associated with sperm motility is shown in fig. 4.
(2) Besides the lncRNA on the bovine QTL related to sperm motility, 6 lncRNA with obvious differences can be screened according to the function annotation and differential expression quantity of the genes adjacent to the lncRNA, and the lncRNA is shown as No. 9-14 lncRNA in the table 3. The functional lncRNA combination for evaluating the sperm motility of the bull is formed.
TABLE 3 functional lncRNA combinations for evaluation of bull sperm motility
7. Real-time fluorescent quantitative PCR detection
Aiming at the lncRNA combination, the lncRNA combination for high sperm motility screening and the lncRNA combination for low sperm motility screening are selected, and specific primers (shown in table 4 and table 5) are respectively designed to carry out Real-time fluorescent quantitative PCR detection on a LightCycler 480II Real-time PCR system. 25 μ L reaction: 12.5 μ L of 2 XSSYBR Greenreal-time PCR Master Mix, 1 μ L each of forward and reverse primers, 2 μ L of cDNA, 8.5 μ L H2And O. Reaction conditions are as follows: 3min at 95 ℃; 95 ℃, 15s, 57 ℃, 15s, 72 ℃, 20s, 40 cycles. Each reaction was run in triplicate.
TABLE 4 high-sperm-activity-group real-time fluorescent quantitative PCR primers
TABLE 5 real-time fluorescent quantitative PCR primers for low sperm motility groups
A kit for use in bull sperm motility assessment, said kit comprising: one or more of SEQ ID NO.1 to SEQ ID NO.14, and an upstream primer and a downstream primer for amplifying gene segments; PCR amplification reagents. The kit can be well supplemented and used in combination with direct sequencing or used independently, and provides technical and theoretical guidance for sperm motility evaluation of breeding bulls.
The invention establishes a method for detecting functional lncRNA influencing sperm motility in semen by using transcriptome sequencing. Current RNA sequencing studies indicate that some lncrnas may regulate testicular development and spermatogenesis through currently unknown mechanisms. There has been no report on the research of functional lncRNA affecting the sperm motility of bulls. The invention obtains 14 functional lncRNA influencing the sperm motility of the bull by carrying out high-throughput sequencing on the bull semen transcriptome, fills the blank of the research aspect, and has important significance for the development of the lncRNA-based dairy cow molecular breeding.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Sequence listing
<110> Dairy cow research center of Shandong province academy of agricultural sciences
Shandong Oxok animal husbandry Co Ltd
<120> long-chain non-coding RNA combination for evaluating bull sperm motility and detection method thereof
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acttcctatc taacaaaatg tttcaattta aagattctac ttctgtaatt acaaagcaac 120
aaatagtaag cgctaccttt taaaattaaa atcacttcta acttcactgt gtattagggt 180
gcaaaattca ttatgaaatt ggctttcaat tcttttcaag tagttaatct cc 232
<210>5
<211>220
<212>DNA
<213> cattle (Bos Taurus, bovine)
<400>5
ggagaaaatg cctaagaaat gagaaggaag gtgaagggca agagccactt cctagggtgg 60
tcaaatcaga gcaccagcca tgagcactgg gcaaggcagt tctaaggaga caaagacaga 120
atgtaatcct tgttaccagc ttccctctat tctaggtgag ctaaagactt aatggcagag 180
agctcttatt ggtctctttt ctttgtgcag gttctctgct 220
<210>6
<211>224
<212>DNA
<213> cattle (Bos Taurus, bovine)
<400>6
agataatagg ctttgatcca gccttttggc acagagggct tctaacagcc ccagcaacat 60
gtggagagtg ctcttcctca tggagaaaaa ctaaagacac aatagcacgc ttataattct 120
agatctgtga aatgcaaatt gccctctctc atttctcagc actcaaagag aagggtttga 180
ttttcagtca tcaaaaaatg tttattttca aatataaaat gagc 224
<210>7
<211>233
<212>DNA
<213> cattle (Bos Taurus, bovine)
<400>7
acacatccac cctctgaaaa atgcttcaaa cccatgagtt tcaataatta actgaaacta60
cttttactta aagacttcca gaattggaaa agtttttcta cctcttttat gattatgtgc 120
tagtttttaa gaacttaaga gatcctatat acatatcagg aattactaca gtcaaaaaga 180
aactactacc atcaatatat ttatttgatt gattataagt agatctatca gta 233
<210>8
<211>232
<212>DNA
<213> cattle (Bos Taurus, bovine)
<400>8
acacaaatgc gtgactgaac tgaactatat tttaaagaat ctactttgta gagtcagata 60
gcaagagaat actcatagtt ggaggttaaa taaaattttt tcaggaaaaa aacaatgatt 120
ctaagacaca tttattttct gtttaacaag gtcaattttc tttctttctt tttatttttc 180
tgctttgcca taggacatat gggatcttag ttctctgaca tggaattgaa cc 232
<210>9
<211>2380
<212>DNA
<213> cattle (Bos Taurus, bovine)
<400>9
cccgcaggcc gggtaacatc tgcgtgggag ggtgaaagac ccaagctagc agcgcggaga 60
agaagagggg ggagccaggc attcatcccg gtcaattttg gtttcagatc gtggcgactg 120
gtggacaggg gtcttgacag aggctcgccc tggggctggg gcagcggagg cgtagctggc 180
agccgtgggc aggtgaagac agcgctctgc cggccaggtg agtccccttc tcctcgctgg 240
tccttgctct cctggtctcc tgcagaagga ggttctgggg agcgcgggcc agcgaggtgg 300
agaggctggc ctcccggcag agcaggacgc aggctggtca ggcagcacag gtgatatcgt 360
ctcaggggag ctgagacagg gcgggctggg ggccgcggga tggggtgccg cgggctgccc 420
caggtggcaa gggcatttcc agagagccag gcgggagctt agcaggcagg gctgccagca 480
cgtgtagggg gcactcaggc ccccggggcg agcaggacag ggtggggact caggacagag 540
gggttcccag ccgccacttc acccactgta attcacttag tagtaggtca caggagcggc 600
tccggccagg cgttctcagg cctgagccag agcctcgagg gccggagaat gagagagaag 660
gcagcgcagt gcgcgggcaa ccgcggcggg atgtcatctg gggggcggtg gccgcgggac 720
aggcccggag agatacctgc agcagggagg ctgcgcgggc ccagcccgtg gacgccagcc 780
ccacgcagca ctgcggcccg ggagccacgc gggagggccc cagtgcactt ggggacactg 840
gggacacttg acccgaaccg ctggcccttg ggatgggcat gagagataga acagcaccca 900
ggggagtcaa ggacacgggc gagaccaggc taggtggggc gggtgggggg gcgtgaatgt 960
gcactctcgg gggaagggag gccagggcac aggcaggggc gaggggccag agccgggcac 1020
cgagccagtg ggaggggcat tggacttcga gctcgtggcc aaggcgggcc tctgcgggcg 1080
atgacggagc gggatcggtg cctctgagct cggaacggag acggaggtga gtgtccagcc 1140
cgctgccctc ctgatgccct tggccacctc tgcccaccca gtcagcgccc cccacctctc 1200
tctctctgcc cacagccacg tctccagtgg ggtgttggtg cctgccagcc acggaggccg 1260
ggcggccagg acgcgcacag agggatatga tatggtccgg tgtgatggag agagcaggcg 1320
ggaccgtcca gcctcccagg tgagtgcaga tgcgggcggt gcccccaccc ggggcccgct 1380
gacccgcact gagcctcata ggcgcctctc tcccatagga cactggcccc gcggggagcc 1440
cggatatcga cgttccttta gtctcctgac ggagcagaca caccactgct gctcttcaga 1500
cacccagaac cctcaagatg acaaggtgag agggcccgcc acatgcaggg tggcggggcg 1560
catgggcccc agaggtgggc actcacgctc tccctccccg cagagatggt gctacccagc 1620
tcatgcctgg gacttggaac acggacttct tcaagtcttc tagctctgac tcaagaatat 1680
gctgcatttt ggaaccacta cacaccctaa ctcaggaatc agctctggaa ggtactgtcc 1740
cctgcgaagt gcccccgccc cgccccccgg cccagcctgc cccgccctca cgccttctct 1800
ctgtccgcag gttgacctaa aggaacggac gacccacacc accggaagag cagcccccgc 1860
ggcgacaccc accccggccc ggcgactcca tctctgcggc ccccccctgc ctcagccccg 1920
agaccaccga cagcctccct ccctcctttt cctccttttc tgactctatt ttctgatctt 1980
ctctctgcta tcctttgctc aggagactca gagcctccag agccccgttc tccccgtccc 2040
ccttgaatca atttgcacta aaagtcgttt gcactggttg gggctctgga gccagccctg 2100
gtctctgagc gtgtgtaagc gcgcgtgtgt gcacactgct gaatgtgtag gcctccttgg 2160
cccgtgcccc cgcccagcac ccttcccaca cttgcgggtg cctgggaggg ggcccgagtg 2220
ggggggcccg cgtgtgaggg gccccgcccg cccgcaccga gtctggaggc tccgaacatc 2280
aggagcacac accaagcatg aacgtgtgtc catcttgcct agtgtccctc cctgccaggg 2340
cctcctcaga tgccctggac tcatcaataa actcagttac 2380
<210>10
<211>593
<212>DNA
<213> cattle (Bos Taurus, bovine)
<400>10
aggtactcgg gagctcatta acatgagctc tcctggaggc tgccattgtg ataccaagac 60
ctggccccac ccaacagcct ataagctcca gttctgggac acctcaggtc aaacagccaa 120
gagggtgaga acacaacccc atccatcaac agacaggatg cctaaacttt cctgagctcc 180
cagacacatc taaacaaacc ctttgacatg actctgccca ccagagggac aagatccatt 240
tctactcact agtaggaagg cactagtctc tcctaccagg caggagcctg cagaagcatc 300
ttagagcagc cttactcacc aggtggcaga cactcaaagc agaaggaact agaatcctgc 360
agcctgtgcc acaaagacca caaacacaaa gttagacaaa atgagaagac agaggaacat 420
atttcagaca gaggaacaag ataaaaccac agaagaacaa ctaagtgaag tagagtcagg 480
caatatactg gaaaaataac tgagtaatga tagaaaagac aatccaagat ttcagaaaaa 540
gaaaggaggc acagactggg aagatacaag aaatgtttaa ctaagagcta gaa 593
<210>11
<211>204
<212>DNA
<213> cattle (Bos Taurus, bovine)
<400>11
aggaaggtgt tgcccatgac atcggacgat cggtcgcccg gcacggccgt ctgggcgctg 60
tccccgcgcg ctccgatggg acgcgagacg ccggccccag ggtaacccga ggcgcatcgc 120
gggccgcgcg ctggaagctg tgatcccgga cccgggccgg gctccgccgc gaccgccgcg 180
gccggattcc gccggacagg ccag 204
<210>12
<211>215
<212>DNA
<213> cattle (Bos Taurus, bovine)
<400>12
aagaaataaa aaatattttg aaataaatga aaattgaaac aatacaataa aacttatagg 60
atgcagcaaa agcatcataa cttacatttt gaggaactag aaaaagaaaa gcaaattaaa 120
tccaaagtta gaaaaggaag gaaataacaa agagcagagg agaagtaaat agatagagac 180
taggaagata aaaaatcaat gaaactaaga gctag 215
<210>13
<211>224
<212>DNA
<213> cattle (Bos Taurus, bovine)
<400>13
tttctcactt tgtctctctg tgtcatattt tggtaattct tgcaatatta caaacctttt 60
catttttgtc atatttgtta tggtgatcta tgataagtga tctttggtgt tactattata 120
attgttttgg ggagccacag accacaccct tatatgacct tgaatttaac ctaaaaatgt 180
gtgtgttctg actgctctat ctggcagatg tcccccccgc cccc 224
<210>14
<211>236
<212>DNA
<213> cattle (Bos Taurus, bovine)
<400>14
aaagaccttg gatggttacg cagcaaaagc actgaataca aattaaccca cacatttttt 60
ttcattgttg tttatgatgg gttaggtcta tgaccttgga taagtcaagt aatacatctg 120
agtttcaatt atttgccacc tacttcagag acagattgca tgataaagag atgatgagat 180
cgttaatgag ataacatttg ttgagccctt tgagattctt agatgaaaga tgcccc236
<210>15
<211>18
<212>DNA
<213> Artificial sequence (artiartiartifical sequence)
<400>15
atgcctggga cttggaac 18
<210>16
<211>20
<212>DNA
<213> Artificial sequence (artiartiartifical sequence)
<400>16
gtgggtcgtc cgttccttta 20
<210>17
<211>19
<212>DNA
<213> Artificial sequence (artiartiartifical sequence)
<400>17
gatgcctaaa ctttcctga 19
<210>18
<211>19
<212>DNA
<213> Artificial sequence (artiartiartifical sequence)
<400>18
ttctgctttg agtgtctgc 19
<210>19
<211>21
<212>DNA
<213> Artificial sequence (artiartiartifical sequence)
<400>19
cacaaatgcg tgactgaact g 21
<210>20
<211>19
<212>DNA
<213> Artificial sequence (artiartiartifical sequence)
<400>20
gtcctatggc aaagcagaa 19
<210>21
<211>19
<212>DNA
<213> Artificial sequence (artiartiartifical sequence)
<400>21
tggttacgca gcaaaagca 19
<210>22
<211>21
<212>DNA
<213> Artificial sequence (artiartiartifical sequence)
<400>22
ctgtctctga agtaggtggc a 21
Claims (10)
1. A combination of long non-coding RNAs for evaluating sperm motility of bulls, comprising one or more of the following lncRNA mapping chromosomes:
TCONS _00006462, chr11: 20027589-;
TCONS-00006445, chr11: 16735802-;
TCONS _00052267, chr7:23688218-23688432, and the nucleotide sequence is shown as SEQ ID NO. 3;
TCONS-00005210, chr11:23353411-23353642, the nucleotide sequence of which is shown in SEQ ID NO. 4;
TCONS _00043365, chr4:49814880-49815099, the nucleotide sequence is shown in SEQ ID NO. 5;
TCONS-00037129, chr27:22465453-22465676, the nucleotide sequence is shown as SEQ ID NO. 6;
TCONS-00006488, chr11:22929103-22929335, the nucleotide sequence is shown as SEQ ID NO. 7;
TCONS _00050707, chr7:28508257 and 28508488, and the nucleotide sequence is shown as SEQ ID NO. 8;
TCONS _00039078, chr29:50150762 and 50153142, and the nucleotide sequence is shown as SEQ ID NO. 9;
TCONS _00028106, chr21:54789843-54790435, the nucleotide sequence is shown in SEQ ID NO. 10;
TCONS _00008791, chr12:83016641-83016844, and the nucleotide sequence is shown as SEQ ID NO. 11;
TCONS _00055082, chr8:80024030 and 80024244, wherein the nucleotide sequence is shown as SEQ ID NO. 12;
TCONS _00043041, chr4:6923803 and 6924026, the nucleotide sequence is shown as SEQ ID NO. 13;
TCONS _00039303, chr29:20825977 and 20826212, the nucleotide sequence is shown in SEQ ID NO. 14.
2. The combination of long non-coding RNAs for evaluating sperm motility of bull as claimed in claim 1, wherein the specific primers are designed respectively,
TCONS _00039078 forward primer: 5'-ATGCCTGGGACTTGGAAC-3', as shown in SEQ ID NO. 15;
TCONS — 00039078 reverse primer: 5'-GTGGGTCGTCCGTTCCTTTA-3', as shown in SEQ ID NO. 16;
TCONS _00028106 forward primer: 5'-GATGCCTAAACTTTCCTGA-3', as shown in SEQ ID NO. 17;
TCONS — 00028106 reverse primer: 5'-TTCTGCTTTGAGTGTCTGC-3', as shown in SEQ ID NO. 18;
TCONS _00050707 forward primer: 5'-CACAAATGCGTGACTGAACTG-3', as shown in SEQ ID NO. 19;
TCONS — 00050707 reverse primer: 5'-GTCCTATGGCAAAGCAGAA-3', as shown in SEQ ID NO. 20;
TCONS _00039303 forward primer: 5'-TGGTTACGCAGCAAAAGCA-3', as shown in SEQ ID NO. 21;
TCONS — 00039303 reverse primer: 5'-CTGTCTCTGAAGTAGGTGGCA-3', as shown in SEQ ID NO. 22.
3. The use of the long non-coding RNA combination of claim 1 or 2 in bull sperm motility assessment, and the use of a kit prepared from the primer combination in bull sperm motility assessment.
4. A detection method for evaluating the vitality of bull sperms is characterized by comprising the following steps:
s1, collecting and storing semen samples;
s2, RNA extraction, library construction and transcriptome sequencing;
s3, transcriptome assembly;
s4, identifying candidate lncRNA and analyzing differential expression;
s5 chromosome localization of differentially expressed lncRNA;
s6 screening for functional IncRNA combinations according to claim 1 for use in evaluating sperm motility of bulls;
s7, designing specific primers and establishing a real-time fluorescent quantitative PCR detection method.
5. The detection method according to claim 4, wherein the specific method for collecting and storing the sample in step S1 comprises:
s1.1, determining the sperm motility of the homozygote bull by using a sperm analysis system, screening three pairs of homozygote bull with different sperm motility according to the average sperm motility in the production records from 2011 to 2015, distributing the three bull to a high-sperm motility group, and distributing the remaining three bull to a low-sperm motility group;
s1.2 fresh semen collected from each bull separately should be immediately stored frozen in liquid nitrogen until RNA extraction.
6. The detection method according to claim 4, wherein in step S2, the method specifically includes:
s2.1, extracting total RNA from the semen sample, and extracting the RNA by using Animal Tissue RNA Purification Kit TRK-1002;
s2.2, evaluating the RNA quality by using an Agilent 2100 bioanalyzer and a Nano Drop ND-2000 spectrophotometer; RNA integrity values in excess of 7.0, OD are required260 / 280A value greater than 1.8;
s2.3, carrying out random fragmentation treatment on the total RNA, wherein the size of the obtained fragment is 200-600 bp;
s2.4, reverse transcription of the cleaved RNA fragments to generate a final RNA-seq library;
s2.5 paired end sequencing with Illumina HiSeq sequencerTM4000。
7. The detection method according to claim 4, wherein in step S3, the method specifically includes:
s3.1, checking the obtained original sequence data in the FASTAQ file format by using FastQC software;
s3.2 mapping the obtained clean data to a bovine reference genomic database using Tophat2 software, then assembling the sequencing fragments for each sample using cufflnksv2.1.1package;
s3.3 all transcriptomes from six samples were pooled using the Cuffmerge program to reconstruct a comprehensive transcriptome profile.
8. The detection method according to claim 4, wherein in step S4, the method specifically includes:
s4.1 annotating the assembled transcript using the Cuffcompare program in the Cufflinks software package;
s4.2, using Coding Potential Calculator and Coding-Non-Coding-Index to estimate the Coding Potential of the transcript with the length being more than or equal to 200bp, the mapping reading coverage being more than or equal to 3 and the exon number being more than or equal to 1, and judging the transcript with the CNCI score being less than 0 as a candidate LncRNA according to the prediction result, wherein the CPC score is < -1;
s4.3, estimating the expression level of lncRNA by an FPKM method by using a Cuffdiff program in a Cufflinks software package;
s4.4 screening for differential expression lncRNA between H and L groups requires FPKM value of H or L group to be greater than or equal to 50, FDR to be less than or equal to 0.05 and | log2 (fold change) | to be greater than or equal to 1.
9. The assay of claim 4, wherein the specific method for selecting the functional IncRNA combination for evaluating sperm motility of bulls in step S6 comprises:
s6.1, positioning the lncRNA with differential expression to a bovine Quantitative Trait Locus (QTLs), and screening 8 lncRNA related to sperm motility, wherein the lncRNA is as follows: TCONS-00006462, chr11: 20027589-; TCONS _00006445, chr11: 16735802-; TCONS _00052267, chr7:23688218 and 23688432; TCONS-00005210, chr11: 23353411-23353642; TCONS _00043365, chr4:49814880 and 49815099; TCONS _00037129, chr27:22465453 and 22465676; TCONS _00006488, chr11: 22929103-22929335; TCONS _00050707, chr7:28508257 and 28508488;
s6.2 besides the lncRNA on the bovine QTL related to sperm motility, 6 lncRNA with obvious differences are screened out, and the differences are as follows: TCONS-00039078, chr29: 50150762-; TCONS _00028106, chr21: 54789843-54790435; TCONS _00008791, chr12: 83016641-83016844; TCONS _00055082, chr8:80024030 and 80024244; TCONS-00043041, chr4:6923803 and 6924026; TCONS-00039303, chr29:20825977 and 20826212.
10. The detection method according to claim 4, wherein the real-time fluorescent quantitative PCR detection method in step S7 specifically comprises:
s7.1, selecting lncRNA combination for high sperm motility screening and lncRNA combination for low sperm motility screening, and respectively designing a specific primer, TCONS _00039078 (forward primer): 5'-ATGCCTGGGACTTGGAAC-3' the flow of the air in the air conditioner,
TCONS — 00039078 (reverse primer): 5'-GTGGGTCGTCCGTTCCTTTA-3', respectively;
TCONS — 00028106 (forward primer): 5'-GATGCCTAAACTTTCCTGA-3' the flow of the air in the air conditioner,
TCONS — 00028106 (reverse primer): 5'-TTCTGCTTTGAGTGTCTGC-3', respectively;
TCONS — 00050707 (forward primer): 5'-CACAAATGCGTGACTGAACTG-3' the flow of the air in the air conditioner,
TCONS — 00050707 (reverse primer): 5'-GTCCTATGGCAAAGCAGAA-3', respectively;
TCONS — 00039303 (forward primer): 5'-TGGTTACGCAGCAAAAGCA-3' the flow of the air in the air conditioner,
TCONS — 00039303 (reverse primer): 5'-CTGTCTCTGAAGTAGGTGGCA-3', respectively;
s7.2 reaction system: 12.5 μ L of 2 XSSYBR Green Real-time PCR Master Mix, 1 μ L each of forward and reverse primers, 2 μ L of cDNA, 8.5 μ L H2O; s7.3 reaction conditions: 3min at 95 ℃; 95 ℃ for 15s, 57 ℃ for 15s, 72 ℃ for 20s, 40 cycles.
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