CN116144786B - FAM151B gene molecular marker related to Hu sheep testis character and application thereof - Google Patents
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Abstract
The invention discloses a FAM151B gene molecular marker related to the testis character of Hu sheep and application thereof, wherein the molecular marker is obtained by amplifying FAM151B gene sequence through a specific primer, and BsaJI-RFLP enzyme cleavage polymorphism is caused by C/T base mutation at 306bp of a nucleotide sequence SEQ ID NO. 1. The result of the association analysis of the FAM151B genotyping and testicle characteristics of the Hu sheep shows that the genotypes of the Hu sheep individuals are classified into CC genotypes, TT genotypes and TC genotypes, and the weight, the major and minor diameters, the epididymal weights and the sperm numbers of the CC genotype individuals are obviously superior to those of the TT type and TC type genotype individuals. The molecular marker provides a new marker source for marker-assisted selection of the testis-related characters of the Hu sheep, and can select individuals with higher sperm yield during the young of the Hu sheep, so that early breeding of the Hu sheep variety is possible, and the breeding period is shortened.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a FAM151B gene molecular marker related to the testis character of Hu sheep.
Background
In recent years, with the rapid development of economy and the improvement of the living standard of people, the demand of people for mutton is increasing, and the mutton market is in short supply. The Hu sheep has the excellent characteristics of early maturing, high reproduction, tender meat quality, large ecological amplitude, suitability for large-scale house feeding and the like, is widely popularized in house feeding and semi-grazing and semi-house feeding sheep areas, and is the mutton sheep variety with the highest market share in the prior art. Therefore, by breeding new varieties or strains of high-fertility sheep, the urgent requirements of the current mutton market stress are relieved. Testes are the specific reproductive organs of male mammals, whose primary functions are to produce sperm and secrete androgens. The number of ram sperms and the semen quality are directly related to the breeding capacity and the conception rate of the ram, and are important for improving the production performance to the maximum extent. It was found that testis size is mainly determined by the number of testis supporting cells, which in turn determine the number of sperm produced, and thus the testis size reflects to some extent the reproductive capacity of male animals. The breeding potential of the stud ram is often judged through the size of the testis in production, but the testis character of the size of the testis is measured after the stud ram is adult, so that the selection time is prolonged, and the breeding progress is slow. Along with the development of molecular marking technology, the testicle characters are marked by a molecular biological method so as to carry out auxiliary selection of early breeding, reduce the blindness of selection in the breeding process, shorten the breeding time and greatly improve the breeding efficiency.
The FAM151B (FAMILY WITH sequence similarity 151, 151 member B) gene family is widely distributed in animals and the encoded proteins belong to the phosphodiesterase superfamily members (FINDLAY AMY S et al 2020). Phosphodiesterases have the function of hydrolysing intracellular second messengers by degrading intracellular cAMP or cGMP, thereby terminating the biochemical actions of cAMP or cGMP in the cell (Preedy mej et al 2020). Studies have shown that cAMP regulates the ability of support cells to develop sperm by regulating the secretion of follitropin during spermatogenesis (Sun Yan et al, 2006). At present, research on the relation between FAM151B genes and the fecundity of Hu sheep and sheep has not been reported yet.
Disclosure of Invention
The invention aims to provide a FAM151B gene molecular marker related to the testis character of Hu sheep, and provides a molecular marker for auxiliary selection for detecting the testis character of Hu sheep so as to improve the accuracy of breeding high-fertility rams.
The invention aims at realizing the following technical scheme:
The FAM151B gene molecular marker related to the testis character of Hu sheep is obtained by amplifying a FAM151B gene sequence through a designed specific primer, the nucleotide sequence of the FAM151B gene molecular marker is shown as SEQ ID NO.1, y represents a C/T base mutation at 306bp of the sequence SEQ ID NO.1, and the mutation leads to BsaJI-RFLP polymorphism;
The primer pair for amplifying the FAM151B gene molecular marker is as follows:
forward primer F: GATTTTATTTAATTCAAGCAT;
reverse primer R: TTCTCTCCATTAGGCTCT.
The FAM151B gene molecular marker related to the testis character of the Hu sheep can be used for molecular marker assisted selection of the Hu sheep, in particular for molecular marker assisted selection of Hu sheep ram related to testis weight, testis long diameter, testis short diameter, epididymal weight or sperm number.
According to the invention, through collecting testis tissues of the Hu sheep, extracting genome DNA, designing a specific primer pair, carrying out PCR amplification on the genome of the Hu sheep, carrying out genotyping on SNP loci of FAM151B genes of the Hu sheep by adopting a PCR-RFLP method, finally analyzing the correlation between the FAM151B genotypes of the Hu sheep and testis-related characters, the result shows that the genotypes of Hu sheep individuals comprise TT genotypes, CC genotypes and TC genotypes, and the testis size, epididymal weight and sperm number of the CC genotype Hu sheep individuals are obviously superior to those of the TT genotype individuals, so that the CC genotype individuals can be selected in early stage seed selection of the Hu sheep, thereby realizing early stage breeding of the Hu sheep with higher sperm yield.
The FAM151B gene molecular marker provided by the invention is used for carrying out auxiliary selection of the high sperm yield Hu sheep, the sperm yield of the mature Hu sheep of the young Hu sheep can be predicted by detecting the characteristic amplification strip of the molecular marker, the Hu sheep individual with higher sperm yield can be selected when the Hu sheep is young, the selection target is clear, the selection efficiency is high, the Hu sheep is not influenced by the growth environment, the early breeding of the Hu sheep variety is possible, and the breeding period is shortened.
Drawings
FIG. 1 shows partial sequencing results and SNP loci of FAM151B genes of Hu sheep of the invention;
FIG. 2 shows the detection result of PCR-BsaJI-RFLP of FAM151B gene of Hu sheep of the present invention; agarose concentration in the assay was 3%, lane M in the figure was Trans2K Plus DNA MARKER (containing 8 band types of 100 bp, 200 bp, 500 bp, 750 bp, 1000 bp, 2000 bp, 3000bp and 5000 bp); TT genotype fragment size is 457 bp, CC genotype fragment sizes are 151 bp and 306 bp respectively; the TC genotype fragment sizes were 457 bp, 306 bp and 151 bp, respectively.
Detailed Description
The invention is further described below with reference to the accompanying drawings, which should not be construed as limiting the invention. Any modification or replacement made by the present invention without departing from the spirit and substance of the present invention shall be deemed to fall within the scope of the present invention.
1. Hu sheep testis tissue and semen collection
The Hu sheep testis tissue samples were collected from Min county Defu agricultural biotechnology Co. Taking the left testis tissue of the Hu sheep ram, and freezing and preserving the left testis tissue for DNA extraction. And weighing epididymal tails on the left side of Hu sheep, soaking in 50 mL saline, cutting tissues into small pieces by using sterile scissors, placing in a test tube, incubating for 1 hour in a water bath at 37 ℃, filtering by using a 200-mesh copper net to remove tissue fragments, and counting the number of sperms by using a cell counting plate after semen is obtained.
2. Testis DNA extraction
The testis tissue genome DNA is separated by using a root DNA extraction kit, absolute ethyl alcohol is added into rinsing liquid GD and PW according to the requirement before use, and the main steps are as follows:
(1) 20mg testis tissue-like liquid nitrogen milling;
(2) 200. Mu.L of GA buffer was added, and after suspending the cells with a pipette tip, 20. Mu.L of proteinase K was added, followed by 10 min in a 56℃water bath;
(3) Adding 200 mu L of GB buffer solution, fully and uniformly mixing, and then carrying out water bath at 70 ℃ for 10 min until the solution becomes bright;
(4) Adding 200 mu L of absolute ethyl alcohol, fully and uniformly mixing, and generating flocculent precipitate;
(5) Transferring the solution and the precipitate in the step (4) to an adsorption column CB3, centrifuging at 12000 r/min for 30 s, and discarding the liquid;
(6) Adding 500 mu L of rinsing solution GD to CB3, centrifuging at 12000 r/min for 30 s, and discarding the liquid;
(7) Adding 700 mu L of rinsing liquid PW to CB3, centrifuging at 12000 r/min for 30 s, discarding the liquid, and repeating the process;
(8) Placing CB3 into a collecting pipe, centrifuging at 12000 r/min for 2 min, taking out CB3, opening a cover, and standing at room temperature for 5 min and drying;
(9) Placing CB3 into a clean centrifuge tube, adding 40 mu L of eluent, and placing 2 min and 12000r/min for centrifugation for 2 min, wherein the obtained liquid is the Hu sheep DNA sample;
(10) Electrophoresis was performed on a 1% agarose gel and DNA purity and concentration were determined using Nanodrop 2000.
3. Primer design
Six pairs of primers P1 to P6 (shown in Table 1) were designed based on the DNA sequence of FAM151B (Gene Bank ID: 101120919) using Oligo7, and FAM151B Gene fragments were amplified, respectively.
TABLE 1 Gene polymorphism investigation primers
4. Polymerase Chain Reaction (PCR)
And (3) carrying out equivalent mixing on the randomly selected 20 Hu sheep DNA samples, preparing a DNA mixed pool, and carrying out PCR amplification on target fragments by using the mixed pool DNA as a template by adopting a PCR method. The total PCR amplification reaction system was 20. Mu.L: wherein 1. Mu.L of the DNA template, 2. Mu.L of 10 XBuffer, 0.5. Mu.L of each of the upstream and downstream primers, 0.2. Mu.L of pfu DNA polymerase, and 20. Mu.L of ddH 2 O were added. The PCR amplification reaction conditions were: pre-denaturation at 94 ℃ 4 min; denaturation at 94℃is 30s, annealing for 30s, and extension at 72℃for 5min after 35 cycles. The amplified PCR product was subjected to electrophoresis using a 2% agarose gel.
5. FAM151B gene single nucleotide polymorphism screening
And (3) sending the PCR product in the mixed pool to Beijing ao Ding-Sheng organism for sequencing, and finding that the PCR product obtained by amplifying the primer P4 has a C/T base mutation after sequencing comparison, wherein the sequencing peak diagram part result is shown in figure 1. The C > T mutation causes a BsaJI cleavage site (C Y [ C/T ] TGG ∈G) polymorphism.
6. Restriction polymorphism analysis
The PCR-RFLP method is adopted to genotype the SNP locus of the FAM151B gene of the Hu sheep by using a primer P4, and the enzyme digestion reaction system is 20 mu L: wherein 10. Mu.L of the PCR product, 2. Mu.L of 10 XBuffer Buffer, 1. Mu.L of BsaJI endonuclease, ddH 2 O were added to 20. Mu.L. After the samples are evenly mixed, the samples are centrifuged, cut at 37 ℃ for 60 min, and then electrophoretically detected by using 3% agarose gel.
Mu.L of the digested product was subjected to 3% agarose gel electrophoresis, followed by genotyping and identification, and the results are shown in FIG. 2. The amplified fragment has the size of 457bp, the restriction polymorphic site is positioned at 306bp of the fragment, when the mutation site is C base, restriction endonuclease BsajI restriction site (C Y [ C/T ] TGG ∈G) is generated, after BsaJI restriction enzyme digestion of PCR product, 2 bands with the sizes of 306bp and 151 bp are generated, and the genotype is CC type; when the site is a T base, a restriction enzyme cleavage site of BsaJI cannot be generated, and the BsaJI enzyme cleavage PCR product is still a 457bp band, and the genotype is TT type; when this position was found to be C/T heterozygous, bsaJI digested with both C and T bases gave 3 bands of sizes 457bp, 306bp and 151 bp, respectively, with the genotype TC.
7. Results of analysis of association of FAM151B genotypes and testicle traits of Hu sheep
The polymorphism of 491 Hu sheep is detected in total, and the genotype detection result shows that the number of CC genotypes in 491 Hu sheep individuals is 164, the number of TC types is 303, and the number of TT types is 24. The frequency distribution of the single nucleotide polymorphism gene on the FAM151B gene of Hu sheep was analyzed, and the results are shown in Table 2.
TABLE 2 FAM151B genotype frequencies and allele frequencies
Correlation analysis between genotype and phenotype was performed using SPSS 19.0 software, normal distribution was checked with One-Sample Kolmogorov-Smirnov, variance alignment was checked with level ne, and differences in genotype phenotype values were checked with One Way ANOVA or Tamhane (non-variance alignment). All data are expressed as "mean ± standard error" and P <0.05 is considered statistically significant.
The genotype and testis character analysis results are shown in Table 3, and the FAM151B gene polymorphism of Hu sheep is obviously related to testis weight, testis long diameter, testis short diameter, epididymal weight and sperm number.
TABLE 3FAM151B Gene different genotypes Hu sheep propagation parameters
Note that: the differences between averages with the same letter shoulder on the same row were insignificant (P > 0.05), and the differences between averages with different letter shoulders were significant (P < 0.05).
The results in table 3 show that the testis weight, the testis major diameter, the testis minor diameter, the epididymal weight on the left side and the sperm count of the Hu sheep CC genotype individuals are significantly higher than those of the TT type individuals (P < 0.05). Therefore, CC genotype individuals can be selected in early breeding of the Hu sheep, so that early breeding of the Hu sheep with higher sperm yield is realized.
Claims (1)
1. The application of the FAM151B gene molecular marker related to the testis character of the Hu sheep in the auxiliary selection of the Hu sheep molecular marker is characterized in that the FAM151B gene molecular marker is used for the auxiliary selection of the molecular marker of the Hu sheep ram related to the testis weight, the testis long diameter, the testis short diameter, the epididymal weight or the sperm number;
The FAM151B gene molecular marker related to the testis character of Hu sheep is obtained by amplifying a FAM151B gene sequence through a designed specific primer, the nucleotide sequence of the FAM151B gene molecular marker is shown as SEQ ID NO.1, y at 306bp of the sequence SEQ ID NO.1 represents a C/T base mutation, and the mutation leads to BsaJI-RFLP polymorphism;
the primer pair for amplifying the FAM151B gene molecular marker is as follows:
forward primer F: GATTTTATTTAATTCAAGCAT;
reverse primer R: TTCTCTCCATTAGGCTCT.
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