CN113215277B

CN113215277B - SNP genetic marker associated with pig semen quality traits and application

Info

Publication number: CN113215277B
Application number: CN202110550301.1A
Authority: CN
Inventors: 李凤娥; 白嵘; 宋惠彬; 李家连; 陈大可; 杨鑫鹏; 汤金华; 冯越
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2021-05-19
Filing date: 2021-05-19
Publication date: 2022-12-13
Anticipated expiration: 2041-05-19
Also published as: CN113215277A

Abstract

The invention belongs to the field of pig molecular marker screening and application, and relates to an SNP genetic marker associated with pig semen quality traits and application thereof. The genetic marker is cloned from a 7 th exon of a SPAG6 gene, the quality traits of the pig semen are the amount of the pig semen, the sperm density, the sperm motility and the sperm aberration rate, the breeds of the pigs are boars of Duroc pigs, white pigs and long white pigs, the nucleotide sequence of the genetic marker is shown as SEQ ID NO 1, a base substitution of T or C exists at 395 th site of the sequence, the substitution causes ClaI-RFLP polymorphism, and the site is the site for detecting the sperm motility of the Duroc pigs, the white pigs and the long white pigs respectively; detecting the locus of the sperm density of the Changbai pig and the Dabai pig; and detecting the site of the semen volume of the long and white pig. The CC genotype is the dominant gene of individual sperm motility character. The invention also provides application of the primer combination in auxiliary selection of the quality traits of the pig semen.

Description

SNP genetic marker associated with pig semen quality traits and application

Technical Field

The invention belongs to the technical field of pig genetic marker screening and application, and particularly relates to an SNP genetic marker associated with pig semen quality traits and application. The SNP genetic marker is cloned from the SPAG6 gene. The genetic marker can be used for auxiliary selection of boar sperm motility.

Background

In the pig raising production, the boar semen quality directly influences the breeding efficiency, and is one of the important indexes for measuring the production benefit of boars. The breeding of the boars is regulated and controlled by various factors, such as genetic improvement, disease control, feeding management level, production environment and the like, wherein the genetic improvement has a leading role, and the improvement of the boar semen quality has important significance for large-scale pig breeding and the genetic improvement of the boars. The boar semen quality mainly comprises four indexes of semen volume, semen concentration, sperm motility, sperm aberration rate and the like. Wherein, the sperm motility is regulated by the motor protein arms at two sides of the duplex microtubules in the axis wire structure of the sperm flagellum '9+2', so the sperm motility has great influence on the quality of the boar semen. It is generally accepted that the normal motility of intact sperm is the basic condition for ensuring successful sperm fertilization. Therefore, the improvement of the semen quality of the boars is beneficial to improving the economic benefit of large-scale breeding, and the marker-assisted selection is an effective method for improving the semen quality of the boars.

Sperm-associated antigen 6 (sphere-associated antigen 6, SPAG6), the full-length transcript consists of 7 conserved Armadillo (ARM) domains. The SPAG6 protein is a kind of scaffold protein of flagella, is highly homologous with Chlamydomonas reinhardtii PF16 and PF20 (Zhang et al, 2002), is positioned at the tail of sperm, participates in the formation of sperm flagellum axis filaments, and plays an important role in sperm movement and maintaining mature sperm structure (Sapiro et al, 2002). Further studies have shown that SPAG6 is localized to the central axon regulating ciliary movement and determines the localization of other related proteins (e.g., SPAG16, SPAG 17) on the central axon

2011). Wu et al found that SPAG6 gene deletion in both human and mouse showed severe impairment of sperm flagellum structure and significant sperm motility defect (Wu et al, 2020). The above studies indicate that the SPAG6 protein has a major impact on the spermatogenesis process. The invention identifies the correlation between the SNP molecular marker and the boar semen quality character through correlation analysis, thereby laying a foundation for further researching the function candidate gene and the genetic improvement of the semen quality.

Disclosure of Invention

The invention aims to provide an SNP genetic marker associated with the boar semen quality traits and application thereof, wherein the semen quality traits are the semen volume, the sperm density, the sperm motility and the sperm aberration rate of boars, and the breeds of the boars are Duroc, dabai and Changbai boars. The genetic marker is cloned from SPAG6 gene, and the nucleotide sequence of the genetic marker is shown in a sequence table SEQ ID NO. 1. Based on the RNA-seq result, the genetic marker specific primer is designed, the mutant genotype is identified, the existence of T > C mutation at the 7 th exon of the SPAG6 gene is verified, and then favorable alleles which affect the boar semen quality can be screened out, so that a new marker resource is provided for the assistant selection of boars.

The technical scheme of the invention is as follows:

the application of a genetic marker in the auxiliary selection of the pig semen quality traits is the semen volume, the semen density, the sperm motility and the sperm aberration rate, the breeds of the pig are boars of Duroc, big white pig and long white pig, and the nucleotide sequence of the genetic marker is as follows:

AGTCTGACCTCGCCTTTCAGCATTCAGTGATTAACCCATGCCTTGTGATGAGGGGTTTCATCAGTTAGCTAGTTATGAACTAGCC CTCAGCTTAGAGTTTAGGTAAGAACTTTTGACCTCCATTATGATAGGGGTAAAAGTTTTACATGATAAATTGGACCACAGAGAG GCTCCTCACAGTGGGCGAATTCTTTGATAAATGTCATAATTTCACTAAATGATATTTCACATTTTACCAGATTTTTTTTTAGGTCCT AAAATTTAAAATCTGGCAGTGCTCATATAATATTGAAAGTTCCTTTACACTATAGGGTCATGGTTATCTAGCTCTCCTTCTTTTCTC TTAGCTTTCACAGTTGATAGTTAATGCGGGCGGAGTGGCTGCGGTGATCGAR(T/C) TGCATCGGGTCCTGCAAAGGAAACATAAGGCTGCCAGGCATCATGATGCTGGGCTATGTGGCAGCTCATTCTGAGAACCTGGC CATGGCAGTCATCATCTCCAAGGTTCGTTCTTGCTTCATTTTCCTCTAGTTATGCAACGAGATGCACCAAGA，

the base R at 395 in the sequence is T or C, the mutation causes ClaI-RFLP polymorphism, the site is used for detecting the sperm motility of Duroc pigs, large white pigs and long white pigs, and the CC genotype is a dominant genotype; is a site for detecting the sperm density of the Changbai pig and the big white pig, and the TC gene type is a dominant gene type; is a site for detecting the amount of semen of the long and white pig, and the CC genotype is a dominant genotype.

The invention provides application of a primer combination in auxiliary selection of pig semen quality traits, wherein the pig semen quality traits comprise semen volume, sperm density, sperm motility and sperm aberration rate, the pig breeds comprise Duroc, white pig and long white pig, and the sequence of the primer combination is shown as SEQ ID NO:2 and SEQ ID NO:3, respectively.

The pig reference sequence related by the invention is an international pig genome version 10.2 reference sequence.

The invention obtains the SNP genetic marker related to the sperm motility of the boar.

The invention provides a specific primer of the SNP genetic marker.

Based on the 60-day-old and 180-day-old large white pig testis tissue RNA-SEQ results, the invention discovers that the SPAG6 gene No. 7 exon has T > C mutation, and the mutation corresponds to the nucleotide sequence table SEQ ID NO:1, the mutation results in a PCR-ClaI-RFLP polymorphism.

The genetic marker can be applied to the evaluation of the quality of the boar semen, and can be used for assisting the breeding of high-quality boars and the like.

The applicant provides a method for screening genetic markers of the quality traits of porcine semen, comprising the following steps:

obtaining the genomic DNA of the porcine semen tissue. The Ensembl database is logged in, a pig SPAG6 gene genome sequence (with the login number of ENSSSCG 00000011080) is downloaded to be used as a target sequence, a Primer 5.0 software is used for designing a specific Primer combination of SNP site sequences obtained by an RNA-SEQ result (the sequences of the specific primers are shown as SEQ ID NO:2 and SEQ ID NO: 3), and genomic DNAs of Duroche, white pig and long white pig are respectively used as templates to amplify specific target fragments (shown in figure 2). Bioinformatics predicts that restriction enzyme ClaI can specifically recognize the base site (AT ↓ CGAT), PCR-ClaI-RFLP (restriction enzyme fragment length polymorphism) is used for detecting the T > C mutation, and the detection of the obtained SNP site is shown in FIG. 3.

The invention provides a ClaI-RFLP (restriction endonuclease fragment length polymorphism) genotyping method for identifying the mutation of the sequence T > C, which utilizes a PCR (polymerase chain reaction) technology to amplify a target fragment of the DNA of a pig genome, and carries out ClaI enzyme digestion genotyping and detection. And determining the application of the correlation analysis between different genotype individuals such as Duroc pigs, big white pigs, boars of long white pigs and the like and the semen quality traits by using SAS 9.1 software. And further selecting the boars with dominant genes for breeding according to the genotypes of the to-be-detected boars.

The more detailed scheme of the invention is described in the detailed description.

Drawings

FIG. 1: the nucleotide sequence (visual sequence and mutation site thereof) of the SNP genetic marker related to the boar semen quality traits screened by the invention.

FIG. 2: the invention relates to agarose gel electrophoresis pattern spectrums of SPAG6 gene specific amplification fragments of Duroc pigs, large white pigs and long white pigs. The agarose gel concentration was 1.0%. Description of reference numerals: lane M is DNA Marker DL 2,000; lanes 1-10 are sequence lists SEQ ID NOs: 2 and SEQ ID NO:3, the amplified fragments of the primers shown in the figure in different pig species have the fragment size of 550bp.

FIG. 3: the invention relates to an SNP locus agarose gel electrophoresis typing map of SPAG6 gene specific amplification fragments of Duroc pigs, large white pigs and long white pigs. Description of reference numerals: the agarose gel concentration was 1.0%. Description of reference numerals: lane M is DNA Marker DL 2,000;

lanes

1, 2, 5, 6, 8, 9, 10 are TT genotypes, fragment sizes 395bp, 155bp, respectively;

lanes

3 and 7 are TC genotypes with fragment sizes of 550bp, 395bp, and 155bp, respectively; lane 4 is CC genotype and the fragment size is 550bp.

Detailed Description

Description of the sequence listing:

sequence listing SEQ ID NO:1: the nucleotide sequence of the SNP genetic marker related to the boar semen quality traits cloned by the invention has a mutation of allele T > C at 395 th base of the sequence, and the mutation causes the nucleotide sequence of SEQ ID NO:1 exhibits a ClaI-RFLP polymorphism. The selected pig breeds are Duroc pigs, large white pigs, long white pigs and other foreign kindered boar groups.

Sequence listing SEQ ID NO:2: is the forward primer sequence of ClaI-RFLP (restriction endonuclease fragment length polymorphism) genotyping method of T > C mutation on exon 7 of SPAG6 gene.

Sequence listing SEQ ID NO:3: is a reverse primer sequence of ClaI-RFLP (restriction endonuclease fragment length polymorphism) genotyping method of T > C mutation on exon 7 of SPAG6 gene.

Example 1: acquisition of pig SPAG6 gene fragment and establishment of polymorphism detection method

Logging in an Ensembl database, downloading a pig SPAG6 gene sequence (the accession number is ENSSSCG 00000026526), finding out the screened SNP locus in the sequence, and utilizing the SNP locusPrimer 5.0 software design SNP specific Primer combinations. Wherein the sequence of the forward primer is shown in a sequence table SEQ ID NO:2, namely SPAG6-ClaI-F: AGT CTG ACC TCG CCT TTC, the reverse primer sequence is shown in sequence table SEQ ID NO:3, namely SPAG6-ClaI-R: TGT CTT GGT GTA TCT CGT. Genome DNAs of Duroc pigs, big white pigs and long white pigs are respectively used as templates, a 550bp target fragment (shown in figure 2) is obtained by amplification, and the nucleotide sequence of the target fragment is shown as SEQ ID NO:1 is shown. PCR reaction (25. Mu.l): 2 XPower Taq Master Mix 12.5. Mu.l, upstream and downstream primers 0.5. Mu.l, template DNA 1. Mu.l, add 10.5. Mu.l ddH ₂ O to a total volume of 25 μ l; PCR reaction procedure: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 54 ℃ for 30s, extension at 72 ℃ for 25s, and passing through 36 cycles; finally, extension is carried out for 10min at 72 ℃.

The SNP genetic marker locus of the invention is a T > C mutation (shown in figure 1) AT 395bp of the specific target fragment, and the mutation causes the polymorphism of a ClaI enzyme cutting site (AT ↓ CGAT).

The invention carries out ClaI-RFLP enzyme digestion typing on the target fragment amplified by PCR. The method comprises the following specific steps: mu.l of PCR product was taken and added with 0.4. Mu.l of restriction enzyme and 1. Mu.l of 10 XBuffer, then 2.6. Mu.l of deionized water was added to constitute 10. Mu.l of digestion system, the digestion was carried out for 3 hours at 37 ℃, 10. Mu.l of digestion product was taken and detected by 1.0% agarose gel electrophoresis, the digestion result was recorded (as shown in FIG. 3), and the size of the amplified fragment was 550bp. When the genotype of the boar to be detected is TT, a ClaI restriction site exists, and two fragments are obtained by restriction enzyme, wherein the lengths of the two fragments are 395bp and 155bp respectively; when the genotype of the boar to be detected is CC, the ClaI enzyme cutting site disappears, and only one fragment with the length of 550bp is obtained; when the genotype of the boar to be detected is TC, carrying out enzyme digestion to obtain three fragments with different lengths of 550bp, 395bp and 155bp respectively;

example 2: polymorphism distribution of genetic markers prepared by the invention in different swineries

The extraction of genomic DNA from swine (samples are shown in Table 1) was performed by the method described in Xiong Yuan, introduction to swine biochemistry and molecular genetics experiments, china agricultural Press, 1999.

The PCR-ClaI-RFLP polymorphism of the SPAG6 gene of the pig is detected in 3 foreign consanguinity pig groups, and the result is shown in a table 1.

TABLE 1 frequency distribution of different alleles and genotypes of the porcine SPAG6 gene in porcine species

Note: the group materials are all the varieties which are publicized and popularized in China.

The results show that: there are 3 genotypes in duroc, white and long-white boars. In the duroc boar population, the C allele frequency and the T allele frequency were 0.37 and 0.63, respectively, and thus the T allele was the dominant allele. In the big white boar population, the C allele frequency and the T allele frequency were 0.19 and 0.81, respectively, and thus the T allele was the dominant allele. In the herd of boars of long white pigs, the C allele frequency and the T allele frequency were 0.28 and 0.72, respectively, and thus the T allele was the dominant allele (see table 1).

Example 3: correlation analysis of genetic marker and semen quality character prepared by the invention

In order to determine whether the PCR-ClaI-RFLP of the pig SPAG6 gene is related to the quality difference of the pig semen, duroc pigs, large white pigs and long white pig boar groups cultivated by a certain company are selected as test materials, the ClaI-RFLP method established in the embodiment 1 is adopted to carry out polymorphism detection, and the related relation between different genotypes of the pig ClaI-RFLP and the quality characters of the pig semen is analyzed. Adopting SAS 9.1 statistical software (SAS Institute Inc, version 9.1) GLM program to carry out single-marker variance analysis, simultaneously adopting REG program to calculate gene additive effect and dominant effect, and carrying out significance test, wherein the adopted model is as follows:

Y _ij ＝μ+G _i +F _j +e _ij

Y _ij is a phenotypic value, μ is the mean value, G _i Is genotype effect (including gene additive effect and dominant effect; additive effect uses 1,0 and-1 to represent CC, TC and TT genotypes respectively, and dominant effect uses 1, -1 and 1 to represent CC, TC and TT genotypes respectively); f _j The pig farm comprehensive effect is achieved; e.g. of the type _ij Is the residual effect.

Correlation analysis between different genotypes and semen quality traits was performed in boars of Duroc pigs, big white pigs and Changbai pigs, and the statistical analysis results are summarized in Table 2.

TABLE 2 statistical analysis table of semen quality traits of pig SPAG6 gene PCR-ClaI-RFLP genotype

Note: the above numerical values are the least square mean value plus or minus standard error; the expression difference of the letters containing the same letters is not obvious, the expression difference of the lower case letters is obvious, and the expression difference of the upper case letters is extremely obvious; positive additive effect values indicate that the C allele increases the phenotypic value of the trait. * Denotes P <0.01, denotes P <0.05.

The results show that in three boar groups, the SNP site at 395 th position of the specific nucleotide sequence is obviously related to the sperm motility (P < 0.05), and CC genotype individuals have the highest sperm motility and are the dominant genotypes. In addition, in the long white pigs and the big white pigs, the site is related to the sperm density, the TC genotype sperm density is the highest, and the dominant effect is obvious. In addition, in the long white pig, the site is related to the amount of spermatic fluid, and the CC genotype has the highest amount of spermatic fluid and has obvious additive effect and dominant effect.

The invention identifies the SNP polymorphism of the SPAG6 gene in Duroc pigs, big white pigs and long white pigs and analyzes the correlation between the SNP polymorphism and the traits of the sperm amount, the sperm density, the sperm motility and the like. In three different boar groups, the SNP locus and the sperm motility character are all obviously related, the CC genotype may be the genotype beneficial to increasing the sperm motility, and the SNP locus of 395 of the specific nucleotide sequence can be used as a candidate marker locus of the boar semen quality. In the practical working process of breeding boars, the individual is expected to have higher sperm motility characters compared with other individuals according to whether the genotype carried by the boars is the CC genotype or not. Therefore, the method can be used for guiding the auxiliary selection of early screening of the boars, so that the production cost of live pig breeding can be reduced, and the economic benefit can be increased.

Sequence listing

<110> university of agriculture in Huazhong

<120> SNP genetic marker associated with boar semen quality traits and application

<141> 2021-05-19

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 550

<212> DNA

<213> pig (Sus scrofa)

<220>

<221> gene

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agtctgacct cgcctttcag cattcagtga ttaacccatg ccttgtgatg aggggtttca 60

tcagttagct agttatgaac tagccctcag cttagagttt aggtaagaac ttttgacctc 120

cattatgata ggggtaaaag ttttacatga taaattggac cacagagagg ctcctcacag 180

tgggcgaatt ctttgataaa tgtcataatt tcactaaatg atatttcaca ttttaccaga 240

ttttttttta ggtcctaaaa tttaaaatct ggcagtgctc atataatatt gaaagttcct 300

ttacactata gggtcatggt tatctagctc tccttctttt ctcttagctt tcacagttga 360

tagttaatgc gggcggagtg gctgcggtga tcgactgcat cgggtcctgc aaaggaaaca 420

taaggctgcc aggcatcatg atgctgggct atgtggcagc tcattctgag aacctggcca 480

tggcagtcat catctccaag gttcgttctt gcttcatttt cctctagtta tgcaacgaga 540

tgcaccaaga 550

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agtctgacct cgcctttc 18

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tcttggtgca tctcgttg 18

Claims

1. The application of the genetic marker containing the SNP locus in the auxiliary screening of the sperm amount, the sperm density and the sperm motility characters of the pigs is characterized in that the breeds of the pigs are respectively big white pigs and boars of long white pigs, and the nucleotide sequence of the genetic marker is as follows:

AGTCTGACCTCGCCTTTCAGCATTCAGTGATTAACCCATGCCTTGTGATGAGGGGTTTCATCAGTTAGCTAGTTATGAACTAGCCCTCAGCTTAGAGTTTAGGTAAGAACTTTTGACCTCCATTATGATAGGGGTAAAAGTTTTACATGATAAATTGGACCACAGAGAGGCTCCTCACAGTGGGCGAATTCTTTGATAAATGTCATAATTTCACTAAATGATATTTCACATTTTACCAGATTTTTTTTTAGGTCCTAAAATTTAAAATCTGGCAGTGCTCATATAATATTGAAAGTTCCTTTACACTATAGGGTCATGGTTATCTAGCTCTCCTTCTTTTCTCTTAGCTTTCACAGTTGATAGTTAATGCGGGCGGAGTGGCTGCGGTGATCGARTGCATCGGGTCCTGCAAAGGAAACATAAGGCTGCCAGGCATCATGATGCTGGGCTATGTGGCAGCTCATTCTGAGAACCTGGCCATGGCAGTCATCATCTCCAAGGTTCGTTCTTGCTTCATTTTCCTCTAGTTATGCAACGAGATGCACCAAGA，

the base R at position 395 of the above sequence is T or C, and the mutation results inClaI-RFLP polymorphism.

2. The application of the primer combination in the auxiliary selection of the swine semen quality traits is characterized in that the swine semen quality traits are swine semen amount, sperm density and sperm motility, the breeds of swine are white swine and long white swine, and the sequence of the primer combination is shown in a sequence table SEQ ID NO:2 and SEQ ID NO:3, respectively.