CN116732198A - Sheep fertility-related molecular marker, primer set and application thereof - Google Patents
Sheep fertility-related molecular marker, primer set and application thereof Download PDFInfo
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Abstract
The invention provides a sheep fertility-related molecular marker, a primer group and application thereof, wherein the molecular marker is positioned at G1 and G4 sites in GDF9 genes positioned on chromosome 5 of a Pishanhong sheep, and the sites are T/C polymorphic sites, namely CC, CT and TT respectively. The method aims to provide a theoretical basis for improving the genetic improvement aspect of sheep high propagation, quicken the cultivation of high-quality mutton sheep new varieties with high fertility and shorten the cultivation process.
Description
Technical Field
The invention relates to the field of sheep breeding identification and breeding, in particular to a molecular marker related to sheep breeding characters, a specific primer and application thereof.
Background
Chinese is one of large sheep raising countries, and mutton is rich in nutrition, easy to digest, high in protein content, low in cholesterol and the like, and is deeply favored by consumers. The sheep with red skin and mountain is a newly discovered sheep variety in Xinjiang area, has gentle temperament and coarse feeding resistance, can be well suitable for the ecological environment of the place, and has high breeding rate as the breeding advantage. Growth differentiation factor 9 (growth differentiation factor, GDF 9), a GDF9 gene, also known as FecG, which is located in the 5 th autosomal of sheep and plays an important role in follicular development, is composed of 2 exons separated by an intron 1126bp in length, encodes 456 amino acids, and its active mature protein is composed of 135 amino acid residues, as a high fertility candidate gene, which can significantly affect ovulation rate or lambing number in different sheep breeds.
However, there has not been any report on molecular markers related to sheep reproductive traits, which relate to specific sites of polymorphisms.
The present invention has been made in view of the above-mentioned circumstances.
Disclosure of Invention
The invention aims to provide a molecular marker related to sheep fertility, a primer group and application, and the correlation between different genotypes and sheep reproductive traits is discussed by sequencing and analyzing GDF9 genes, so that theoretical basis is provided for improving the genetic improvement of sheep high-reproduction, the cultivation of high-quality mutton sheep new varieties with high fertility is quickened, and the cultivation process is shortened.
The second object of the invention is to provide a kit comprising the above specific primer combination, which is convenient to use and can extract reagents by a corresponding detection method.
In order to achieve the above object of the present invention, the following technical solutions are specifically adopted:
the invention provides a molecular marker related to sheep fertility, which is positioned at G1 and G4 sites in GDF9 genes on chromosome 5 of Pishan red sheep, wherein the sites are T/C polymorphic sites, namely CC, TC and TT respectively.
The molecular marker is obtained after sheep GDF9 gene amplification, and the nucleotide sequence of the molecular marker is shown as SEQ ID NO. 1. The DNA sequence of the sheep GDF9 gene is amplified and sequenced, polymorphic sites of the GDF9 gene are screened, the correlation between different genotypes and sheep reproduction traits is analyzed, a molecular marker detection method containing the polymorphic sites is established, and the molecular marker can be applied to the work of cultivating new strain mutton sheep. The sheep GDF9 gene has 8 mutation sites, which are respectively: the G1, G4 and FecGE, fecGA, fecGV, fecGH, fecGF, fecGT have polymorphism in the Pichia pastoris only at the G1, G4 and FecGA sites, the other sites are homozygote, and the G1 and G4 sites have three genotypes in the Pichia pastoris respectively, namely CC, CT and TT; fecGA locus has two genotypes of CC and CT at the midpoint of the skin mountain red sheep. Sheep GDF9 gene mRNA sequence published on NCBI, given sequence gene number: NM_001142888.2, the sequence of which is shown as SEQ ID NO. 1.
Wherein, SEQ ID NO.1:
the invention also provides a specific primer group for detecting the molecular marker, which consists of a first primer pair and a second primer pair, wherein each primer pair consists of an upstream primer, a downstream primer and an extension primer, the upstream primer has a base sequence shown in SEQ ID No.2, the downstream primer has a base sequence shown in SEQ ID No.3, the extension primer has a base sequence shown in SEQ ID No.4, the second primer pair has a base sequence shown in SEQ ID No.5, the downstream primer has a base sequence shown in SEQ ID No.6, and the extension primer has a base sequence shown in SEQ ID No. 7.
The nucleotide sequence is as follows:
G1：
SEQ ID NO.2:ACGTTGGATGAGGTTCTGTATGATGGGCAC
SEQ ID NO.3:ACGTTGGATGAGCGTATGCCTTATAGAGCC
SEQ ID NO.4:TTTACAGATGACAGAGCTTTGC
G4：
SEQ ID NO.5:ACGTTGGATGATGTTAAACAGGCTGTCCCG
SEQ ID NO.6:ACGTTGGATGCCCACAAGAGGAATATTCAC
SEQ ID NO.7:CCGCTGCAGCTGGTCTT
in general, the primer specificity increases by a factor of 4 per one nucleotide increase, so that the shortest primer length for most applications is 18 nucleotides. The upper limit of the primer length is not very important and is mainly related to the reaction efficiency.
In general, the sequence of the primers has a G+C content of 40% -60%, and the GC content and Tm value of a pair of primers should be coordinated. The primer G+C content in the primer combination is controlled in a proper range, and the base distribution is random, so that the primer combination has a moderate melting temperature and is convenient for amplification.
In a word, the primer combination has good specificity and high amplification efficiency, and can be well applied to detection of the molecular markers related to sheep reproductive traits. The primer group is used for amplifying and detecting the genomic DNA of sheep to determine the genotype of the GDF9 gene of a sample to be detected, so that a sheep variety producing multiple lambs can be bred from the sample.
The invention also provides a method for screening productive sheep, which comprises the following steps:
extracting DNA of a sample to be detected as a template, carrying out PCR amplification on sheep GDF9 genes by using a primer combination, analyzing PCR amplification products after the amplification reaction is finished, judging specific genotypes of G1 and G4 loci in the amplification products, and selecting individuals with genotypes TC and TT as breeding sheep. The numbers of TC and TT lambings are extremely higher than those of CC, which indicates that the G1 and G4 loci are suitable for the Pishan red flocks.
Preferably, in the above method for screening productive sheep, the PCR amplification reaction procedure is as follows: pre-denaturation at 94℃for 120s, denaturation at 94℃for 20s, annealing at 56℃for 30s, extension at 72℃for 60s,48 cycles, extension at 72℃for 180s.
As the reaction proceeds, the enzyme becomes inactive gradually, the raw materials such as dNTPs become consumed gradually, and amplification of some nonspecific products increases accordingly. Thus, although the number of reaction cycles increases, the number of cycles is still not excessive, and the present invention is limited to 48 cycles.
The invention also comprises a kit comprising the primer combination, and the kit has good application in screening of sheep colony multi-lamb characters.
In a word, the invention discovers that C/T polymorphism exists at G1 and G4 sites of amplified fragments by carrying out PCR amplification and sequencing on GDF9 genes of the pimenta, and determines a molecular marker related to sheep reproductive traits by detecting 376 pimenta polymorphisms and a built general linear model, and the molecular marker can be used for cultivating productive sheep, provides an effective genetic engineering means for genetic improvement of sheep reproductive traits, and has great practical application value.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 is a gel electrophoresis diagram of a sheep GDF9 gene fragment for use as a molecular marker in the present invention.
FIG. 2 shows SNP typing results of G1 mutation site of sheep GDF9 gene according to the present invention.
FIG. 3 shows the SNP typing results of G4 mutation site of sheep GDF9 gene according to the present invention.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The reagents and consumables used in the following examples were: DNA extraction kit, 5 XTBE Buffer, 6 XScale Buffer, D2000, beijing Tiangen Bio-company; nucleic acid dyes, bomeid organisms; agarose, beijing Fubo biotechnology Co., ltd; ice absolute, 75% absolute, tianjin Xin platinum specialty Co.
The instruments used in the following examples were: a pipette, eppendorf, germany (single-pass adjustable pipette 0.5-10ul;10-100ul;20-200ul;100-1000 ul); ultra-micro spectrophotometers, nanoDrop2000, therpro company; electronic balance, mertrer-tolido instruments limited; DYY-6C electrophoresis apparatus, WD-9403F ultraviolet analyzer, six instrument factories in Beijing; vortex mixer, jiangsu sea door Chemie Limited of Linbell instruments (model QL 901); ABI veriti-384PCR instrument, applied Biosystems; a high-speed low-temperature centrifuge, eppendorf company, germany; a drying oven, a vertical pressure steam sterilizing pot, shanghai Boxun Utility company; galanz microwave oven, grosven microwave oven (Guangdong Grosven company); low temperature refrigerator, ice chest, refrigeration equipment limited in guangdong america; DK-8D digital display constant temperature water bath, jiangsu Jinyi instrument and technology company.
All reagents, instruments, materials selected for use in the present invention are well known in the art and are not limiting of the practice of the invention, and other reagents and apparatus well known in the art may be suitable for use in the practice of the following embodiments of the invention.
Example 1 amplification of GDF9 Gene
(1) Primer design
Using sheep GDF9 gene DNA (GenBank accession number: oar_v3.1) as a template, a primer set of 2 pairs of G1 and G4 was designed by using MassARRAY Assay Design 3.1.3.1 software, and the primer sequences were as follows:
G1：
SEQ ID NO.2:ACGTTGGATGAGGTTCTGTATGATGGGCAC
SEQ ID NO.3:ACGTTGGATGAGCGTATGCCTTATAGAGCC
SEQ ID NO.4:TTTACAGATGACAGAGCTTTGC
G4：
SEQ ID NO.5:ACGTTGGATGATGTTAAACAGGCTGTCCCG
SEQ ID NO.6:ACGTTGGATGCCCACAAGAGGAATATTCAC
SEQ ID NO.7:CCGCTGCAGCTGGTCTT
(2) Amplification and sequencing of GDF9 Gene
Genomic DNA of sheep ear tissues is extracted as a DNA template for PCR amplification.
TABLE 1PCR reaction System
TABLE 2 circulation parameters of PCR reaction System
TABLE 3SAP digestion reaction System
TABLE 4SAP digestion reaction System cycle parameters
TABLE 5 extension reaction System
TABLE 6 elongation reaction conditions
And (3) purifying resin: the resin was spread on an orifice plate, and after it was air-dried for 10min or more, 16ul of water was added to each well and centrifuged. The sample is slowly turned over in the air, put on the orifice plate, then turned over together, and the resin falls into the orifice. Sealing the plate with film and mixing with rotator.
And (3) detecting: after diluting 9uL of the reaction product 3-fold, desalting was performed using a resin; the desalted sample is spotted on a sample target to be naturally crystallized; and carrying out mass spectrum detection and collecting detected genotype data.
(3) DNA sequence homology search identification
The DNA sequence obtained after sequencing was compared for sequence homology with known physiological functional genes published in the GenBank database by means of the BLAST (Basic Local Alignment Search Tool) software of the national center for Biotechnology information (NCBI, national Center for Biotechnology Information, http:// www.ncbi.nlm.nih.gov) website to identify and obtain functional information of the DNA sequence. The search result shows that the homology of the sequence with partial sequence of sheep GDF9 gene DNA (GenBank accession number: oar_v3.1) reaches 99%.
Example 2 establishment of genotyping assay
(1) Primer sequence design
Designing primer pairs aiming at two T/C polymorphic sites of the amplified fragment in the example 1 so as to be used for specific detection of the polymorphic sites, delivering the designed primers to Beijing Kang Pusen agricultural technologies Inc., and synthesizing the designed primer pairs, wherein the nucleotide sequences of the designed primer pairs are as follows:
forward primer 1: ACGTTGGATGAGGTTCTGTATGATGGGCAC
Reverse primer 1: ACGTTGGATGAGCGTATGCCTTATAGAGCC
Forward primer 2: ACGTTGGATGATGTTAAACAGGCTGTCCCG
Reverse primer 2: ACGTTGGATGCCCACAAGAGGAATATTCAC
(2) DNA quality control
A5 uL sample of the extracted genomic DNA was extracted and detected by 1% agarose electrophoresis, and the extraction results are shown in FIG. 1. The DNA electrophoresis strip is complete and clear, no front and back tailing phenomenon exists, the degradation and RNA pollution does not exist, the detection concentration of Nano drop2000 finds that the OD260/280 value is about 1.8-2.0, the pollution of proteins or phenols and the like does not exist, and the DNA extracted by the test is complete and has higher purity and can be used for the subsequent amplification of target genes.
(3) Genotyping
Genotyping was performed on 2 SNPs of 376 picomountain red sheep samples from which qualified DNA was extracted by using a mass spectrometry technique, and the typing results were obtained by statistical analysis of mass spectrometry software, and specific results are shown in fig. 2 to 3.
(4) Application of molecular marker in sheep reproductive trait correlation analysis
The association analysis of the lambing number and genotype of the skin red flock in this study was performed using a general linear model of SPSS27.0, which was constructed as follows:
Y＝μ+G+e；
wherein: y is a lambing number record value; mu is the average value of the population; g is genotype effect, e is random residual effect, and specific results are shown in Table 3 below assuming that e is independent of each other and obeys the N (0, σ2) distribution.
TABLE 7 correlation analysis of the lambing numbers of each genotype at 2 loci of GDF9 Gene
Note that: the difference is obvious (P < 0.05) in the same column of data by the difference of lower case letters of the shoulder marks, the difference is extremely obvious (P < 0.01) by the difference of upper case letters of the shoulder marks, and the difference is not obvious (P > 0.05) by the same or no shoulder marks.
The lambing number association analysis in Table 7 shows that the G1 and G4 loci have a certain association between the genotypes of the Pishan red sheep population, the difference of the lambing numbers between the genotypes of the G1 and G4 loci is obvious (P < 0.05), the TC-type lambing number is extremely higher than that of the CC-type (P < 0.05), and the G1 and G4 loci are suitable for breeding of the multi-lambing character of the Pishan red sheep population.
While particular embodiments of the present invention have been illustrated and described, it will be appreciated that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.
Claims (7)
1. A molecular marker related to sheep fertility, which is characterized in that the molecular marker is positioned at G1 and G4 sites in GDF9 genes on chromosome 5 of Pishan red sheep, wherein the sites are T/C polymorphic sites, namely CC, TC and TT respectively.
2. A specific primer set for detecting the molecular marker according to claim 1, characterized by comprising a first primer pair and a second primer pair, wherein each primer pair comprises an upstream primer, a downstream primer and an extension primer, the upstream primer has a base sequence shown in SEQ ID No.2, the downstream primer has a base sequence shown in SEQ ID No.3, the extension primer has a base sequence shown in SEQ ID No.4, the second primer pair has a base sequence shown in SEQ ID No.5, the downstream primer has a base sequence shown in SEQ ID No.6, and the extension primer has a base sequence shown in SEQ ID No. 7.
3. A kit comprising the specific primer set of claim 2.
4. The use of the molecular marker of claim 1 in breeding of a multi-lamb trait of an auxiliary sheep population.
5. Use of the specific primer set of claim 2 and the kit of claim 3 for screening sheep flock for multi-lamb traits.
6. A method of screening productive sheep comprising the steps of:
carrying out PCR amplification on the sheep GDF9 gene by adopting the specific primer set as claimed in claim 2 or the kit as claimed in claim 3 to obtain an amplification product;
judging specific genotypes of the G1 and G4 loci of 2 polymorphic loci in the amplified product, and selecting individuals with genotypes TC and TT as breeding sheep for breeding.
7. The method of screening for productive sheep of claim 6 wherein the PCR amplification procedure is: pre-denaturation at 94℃for 120s, denaturation at 94℃for 20s, annealing at 56℃for 30s, extension at 72℃for 60s,48 cycles, extension at 72℃for 180s.
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