CN114574596A - SNPs molecular marker g.43851G & gtA and application thereof in Hu sheep molecular marker-assisted breeding - Google Patents
SNPs molecular marker g.43851G & gtA and application thereof in Hu sheep molecular marker-assisted breeding Download PDFInfo
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Abstract
The invention belongs to the technical field of Hu sheep molecular marker assisted breeding, and particularly relates to a Hu sheep body length trait related SNPs molecular marker g.43851G & gtA, a primer pair, a kit and application thereof in Hu sheep molecular marker assisted breeding. Wherein, the body length of the AA type individual is obviously higher than that of the GA type individual, and the difference of the GG type individual and the GA and AA type individual is not obvious. By adopting the molecular marker, the primer pair and the kit provided by the invention, the body length character of the Hu sheep can be screened out by adopting a related detection method, and an auxiliary breeding effect is achieved.
Description
Technical Field
The invention belongs to the technical field of Hu sheep molecular marker assisted breeding, and particularly relates to a Hu sheep body length trait related SNPs molecular marker g.43851G & gtA, a primer pair, a kit and application thereof in Hu sheep molecular marker assisted breeding.
Background
The Hu sheep is a famous sheep variety in China, and is famous for the characteristics of strong fecundity, high early growth speed, suitability for barn feeding, high humidity and high heat environment and the like. However, the meat performance of the Hu sheep is greatly different from that of foreign meat sheep varieties such as Dorper sheep, German merino sheep and the like. Therefore, it is necessary to breed the growth performance of the Hu sheep and improve the meat production performance of the Hu sheep.
The applicant has granted Chinese invention patents (publication numbers: CN109251986A, CN109182553A, CN109251987A, CN109251985A, CN109182554A and CN109055579A) which disclose candidate functional genes and SNPs of Hu sheep body size characters screened by GWAS technology, and SNPs which can be used for molecular marker assisted breeding are obtained through group verification of the SNPs, and can be used for Hu sheep meat character molecular marker assisted breeding and early breeding of Hu sheep meat line core group individuals.
The ARHGAP24 gene is a member of the Rho gtpase activator protein family, acts as a negative regulator of Rho gtpase and influences actin remodeling, cell polarity, cell migration, differentiation and development. ARHGAP24 (also known as p73-RHOGAP or FilGAP) is located at the cytoplasmic and cellular junction and plays an important regulatory role in the angiogenic process.
It has now been found that the ARHGAP24 gene is involved in the proliferation, cell cycle, apoptosis, migration and invasion of cancer cells in the kidney, where the effect is that breast cancer cells induced by activated ARF6 cause pseudopodogenesis and promote tumor growth of glioblastoma by increasing Rac1 activity. The expression of ARHGAP24 is reduced in lung adenocarcinoma, and the taxol resistant phenotype of cancer cells can be generated.
The Rho GTP family is involved in a variety of cellular functions in mammals, including actin cytoskeleton reorganization, cell growth control, transcriptional regulation and membrane trafficking, and in animal growth and development, lung development, respiratory system development, angiogenesis, cardiac development and transcriptional regulation. ARHGAP24 has been found to be significantly associated with production traits in GWAS analysis of production traits in duroc pigs. In conjunction with previous studies, mutations in ARHGAP24 may be involved in certain functions in cell growth regulation, further affecting the growth characteristics of the hu sheep from some unknown pathway regulation.
Disclosure of Invention
In order to solve the defects of the prior art and the requirements of practical needs in molecular marker breeding, the invention aims to provide an SNPs molecular marker which is obviously related to the Hu sheep body length character, a detection primer pair and a kit thereof, and a screening method of the molecular marker and application of the molecular marker in Hu sheep molecular marker breeding.
In order to achieve the above object, the present application adopts the following technical solutions:
the molecular marker of SNPs is related to the body length of Hu sheep obviously and is located in g.43851G & gtA.
Further, the application provides a primer pair for detecting the SNPs molecular markers.
Further, the application provides an amplification product obtained by amplifying the primer pair, and the sequence of the amplification product is shown as SEQ ID NO. 1.
Further, the present application provides a primer pair for amplifying the amplification product.
Preferably, the sequences of the primer pair are shown as SEQ ID NO. 2 and SEQ ID NO. 3.
Further, the present application provides a kit comprising the primer pair.
Further, the application provides the application of the SNPs molecular marker, the primer pair and the kit in screening the Hu sheep body length character and Hu sheep molecular marker breeding; wherein, the body length of the AA type individual is obviously higher than that of the GA type individual, and the difference of the GG type individual and the GA and AA type individual is not obvious.
Further, the application also provides a method for screening the lake sheep weight and size characters, which comprises the following steps: extracting Hu sheep peripheral blood genome DNA, carrying out PCR amplification by adopting the primer pair, and detecting the SNPs molecular markers in the amplified product so as to screen out the Hu sheep body length character; wherein, the body length of the AA type individual is obviously higher than that of the GA type individual, and the difference of the GG type individual and the GA and AA type individual is not obvious.
Preferably, the PCR amplification procedure is: pre-denaturation at 94 ℃ for 2 min; denaturation at 98 ℃, 10sec, annealing at 53 ℃, 30sec, extension at 68 ℃, 30sec, 35 cycles; storing at 4 deg.C for infinity; the PCR amplification system is as follows:
further, the application also provides application of the method in screening Hu sheep body length traits and Hu sheep molecular marker breeding; wherein, the body length of the AA type individual is obviously higher than that of the GA type individual, and the difference of the GG type individual and the GA and AA type individual is not obvious.
The invention takes 214 Hu sheep as test material, uses ARHGAP24 gene as candidate gene, adopts PCR amplification, direct product sequencing and sequence analysis technology to screen SNPs, and screens SNP sites which obviously affect the weight and size characters of Hu sheep through correlation analysis.
The invention discovers that 15 SNPs exist in the 1 part region of an ARHGAP24 gene intron, wherein the SNPs which are obviously related to the weight and body size traits of Hu sheep have 4 sites: the g.43756G & gtA is obviously related to the body length of Hu sheep, the body length of AA type individuals is obviously higher than that of GA type individuals (P & lt 0.05), and the difference between GG type individuals and GA and AA type individuals is not significant (P & gt 0.05). The body height of the Hu sheep is obviously related to that of the G.43815G & gtA, the body height of the AA type individual is obviously higher than that of the GG type individual (P & lt 0.05), and the difference between the GA type individual and the GG and AA type individual is not significant (P & gt 0.05). The body length of the Hu sheep is obviously related to that of the G43851G & gtA, the body length of the AA type individual is obviously higher than that of the GA type individual (P & lt 0.05), and the difference of the GG type individual and the GA type individual is not significant (P & gt 0.05). The g.43917A & gtG is obviously related to the body length and daily gain of Hu sheep, the body length of AA and GG individuals is obviously higher than that of AG individuals (P is less than 0.05), the difference between AA and GG is not obvious (P is more than 0.05), the daily gain of GG individuals from weaning to six months of age is obviously higher than that of AA individuals, and the difference between AG individuals and AA and GG is not obvious.
Drawings
FIG. 1: the invention detects the PCR amplification sequence of the primer, wherein gray is the site of the molecular marker.
FIG. 2: PCR amplification of Hu sheep ARHGAP24 gene SNPs detection primers (M: DL5000, 5000bp, 3000bp, 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp from top to bottom, and lanes 1-8 are target fragment amplification results).
Detailed Description
The invention is further illustrated by the following examples.
1. Test materials
1.1 sources of test animal samples
The test animals are from a core group of a new Hu sheep meat class of Hangzhou huge agricultural development limited company, and the number of the test animals is 213, and the test animals are raised according to a standard mutton sheep raising management method. 10mL of jugular venous blood was collected from each individual, placed in an EDTA anticoagulation tube, and stored at-20 ℃.
1.2 measurement of body ruler index
The measurement of the Hu sheep body ruler comprises the body (oblique) length, the body height and the chest circumference. Each sheep was measured at least 3 times and the average was taken as the final measurement. The specific properties and methods are shown in Table 1.
TABLE 1 determination of Properties and determination method
2. Test method
2.1 extraction of genomic DNA from peripheral blood, according to a known conventional method.
2.2 DNA concentration and purity determination, according to known conventional methods.
2.3 Hu sheep ARHGAP24 gene PCR amplification and SNPs detection.
The primers were designed based on the Acc:101119904 partial region of intron 1, and were synthesized by Hangzhou Kongkang Biotechnology Ltd, and the sequences of the primers, the lengths of the amplified fragments and the annealing temperatures are shown in Table 2.
TABLE 2 Hu sheep ARHGAP24 Gene PCR amplification primers
The PCR amplification system was 25. mu.L, and the PCR reaction program is shown in Table 3. The specific components are shown in Table 4.
TABLE 3 Hu sheep ARHGAP24 Gene PCR amplification program
TABLE 4 Hu sheep ARHGAP24 gene PCR amplification system
2.4 detection of PCR products
2.5 sequencing of PCR products
Each sample is amplified according to the primers listed in Table 2, and the amplification products are handed over to Hangzhou Zhike catalpi Biotechnology Limited to perform forward and reverse bidirectional sequencing until the bidirectional sequencing results are consistent.
2.6 sequence analysis
The forward and reverse sequencing peak patterns of each individual were analyzed by using the Mutation Surveyor 5.02(Softgenetics, USA) software, and the Mutation position and Mutation pattern of ARHGAP24 gene of each individual were determined by using a partial region of Acc:101119904 intron 1 as a reference sequence. And (3) performing secondary sequencing on individuals with abnormal sequencing results or inconsistent forward and reverse sequencing results until the forward and reverse sequencing analysis results of the PCR products are completely consistent.
2.7 data statistics and analysis
PIC values were calculated using little program.
Calculating the site heterozygosity, Shannon information content, gene frequency and genotype frequency of each SNPs by using PopGen 32 software, and detecting whether each site meets Hardy-Weinberg balance.
Polymorphic information content analysis:
pic (polymorphism information content), which indicates the possibility that an allele obtained from one offspring is from the same allele of its parent, is an ideal index for measuring the polymorphism of an allele fragment, and is calculated by the formula:
Piand Pj(ii) the ith and jth allele frequencies, respectively; n is the allelic factor.
Site heterozygosity:
he (heterozygosity) refers to the average frequency of heterozygous individuals present at each locus. The heterozygosity can objectively reflect the genetic variation level of a population, and the larger the average heterozygosity value is, the larger the genetic difference in the population is, the genetic diversity is rich, the genetic potential is large, and the effect of the research on the animal genetic breeding is good; the lower the value, the higher the genetic consistency, which indicates that the genetic variation in the population is small and the genetic potential is also small. The calculation formula is as follows:
piindicates the ith allele frequency.
Shannon information content:
SIC (shannon information content), the calculation formula is:
SIC＝-ClogPi
wherein: pi is the frequency of the ith allele in the population and C is a constant.
Gene frequency and genotype frequency:
(ii) genotype frequency is genotype number/population number × 100%;
② the gene frequency is homozygous genotype frequency +1/2 × heterozygous genotype frequency.
Correlation analysis:
SNPs associated with the body weight and size traits of the core population of the new cluster for Hu sheep meat were mined using a General Linear Model (GLM; SPSS 20).
Since all individuals analyzed were female sheep from the same feedlot, same feeding environment and management conditions, field effect and gender effect were not included in the data modeling.
The concrete model is as follows: y ═ X β + e;
wherein, Y: the core group size character and the body weight character phenotype value vector of the new group for the Hu sheep meat;
beta: fixed effect vectors such as phenotypic mean, SNP, etc.;
e: a residual effect vector;
and X is a correlation matrix of beta.
And when Y is the vector of the birth weight table value of the new cluster core group for the Hu mutton, analyzing according to the model Y which is X beta + S alpha + e.
Wherein Y: the core group size character phenotype value vector of the new group for the Hu sheep meat;
α: fixed effect vector of sibling number;
beta: an SNP effect vector;
e: a residual effect vector;
x, S are the incidence matrixes of beta and alpha respectively.
3. Test results
3.1 Hu sheep ARHGAP24 gene SNPs detection PCR amplification result
The PCR products of the primers have higher brightness and single bands, and have no non-specific amplification (figure 2), and the actual PCR products are consistent with the estimated PCR amplification products in size, so that the subsequent direct sequencing test of the PCR-products can be carried out.
3.2 mutation analysis of amplification product of Hu sheep ARHGAP24 Gene
The results of analysis of mutation in PCR amplification product of Hu sheep ARHGAP24 gene are shown in Table 5, and 15 mutations were detected in Hu sheep population by amplifying a partial region of ARHGAP24 gene intron 1 (amplification product is 476bp), wherein 3 mutations were inverted and 12 mutations were converted.
TABLE 5 SNPs sites, mutation types and mutation modes of Hu sheep ARHGAP24 gene
TABLE 6 genetic parameters of the SNPs sites of Hu sheep ARHGAP24 gene
TABLE 7 Hu sheep SNPs locus population genetics analysis
Polymorphic Information Content (PIC) is used to determine and analyze the information content expressed by a genetic marker, PIC >0.5 is a high polymorphic site, 0.25< PIC <0.5 is a medium polymorphic site, and PIC <0.25 is a low polymorphic site.
A larger PIC value indicates a greater effective allele factor and heterozygosity, and a higher variability of the population at this SNP site. The genes of Hu sheep ARHGAP24, g.43642T > C, g.43649A > G, g.43654A > T, g.43681G > A, g.43699C > G, g.43722C > A > T, g.43788G > A, g.43815G > A, g.43914A > G, g.43917A > G, g.43934A > G, g.43980G > A are medium polymorphisms, g.43756G > A, g.43851G > A, g.43955C > T are low polymorphisms.
Association analysis of Hu sheep ARHGAP24 gene polymorphism and body weight and body size traits:
the GLM model in association analysis was used to perform association analysis of 15 SNPs of the ARHGAP24 gene with the body size and weight traits of the hu sheep (table 9).
The analysis result shows that g.43756G & gtA is obviously related to the body length of Hu sheep, the body length of AA type individuals is obviously higher than that of GA type individuals (P & lt 0.05), and the difference between GG type individuals and GA and AA type individuals is not significant (P & gt 0.05).
The g.43815G & gtA is obviously related to the improvement of Hu sheep, the height of the AA type individual is obviously higher than that of the GG type individual (P & lt 0.05), and the GA type individual has no obvious difference (P & gt 0.05) compared with the GG and AA type individuals.
g.43851G & gtA is obviously related to the body length of Hu sheep, the body length of AA type individuals is obviously higher than that of GA type individuals (P & lt 0.05), and the difference between GG type individuals and GA and AA type individuals is not significant (P & gt 0.05).
The G of 43917A is more than G, the body length of the AA type individual and the GG type individual is obviously higher than that of the AG type individual (P is less than 0.05), the difference between the AA type individual and the GG type individual is not obvious (P is more than 0.05), the daily gain of the GG type individual from weaning to six months of age is obviously higher than that of the AA type individual, and the difference between the AG type individual and the AA and the GG type individual is not obvious.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention, including any reference to the above-mentioned embodiments. Various modifications to these embodiments will be readily apparent to those skilled in the art. The general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (10)
1. The SNPs molecular marker is characterized in that the molecular marker is positioned as g.43851G & gtA.
2. A primer pair for detecting molecular markers of SNPs according to claim 1.
3. The primer pair of claim 2, wherein the sequence of the amplification product is shown as SEQ ID NO. 1.
4. A primer pair for amplifying the amplification product of claim 3.
5. The primer pair of claim 4, wherein the sequences of the primer pair are shown as SEQ ID NO. 2 and SEQ ID NO. 3.
6. A kit comprising the primer pair of claim 2 or 4 or 5.
7. Use of the SNPs molecular markers of claim 1, the primer pairs of claim 2 or 4 or 5, and the kit of claim 6 for screening Hu sheep body length traits and Hu sheep molecular marker breeding; wherein, the body length of the AA type individual is obviously higher than that of the GA type individual, and the difference of the GG type individual and the GA and AA type individual is not obvious.
8. A method for screening the weight and size characters of lake sheep is characterized by comprising the following steps: extracting Hu sheep peripheral blood genome DNA, carrying out PCR amplification by adopting the primer pair of claim 2, 4 or 5, and detecting SNPs molecular markers of claim 1 in an amplification product so as to screen the Hu sheep body length character; wherein, the body length of the AA type individual is obviously higher than that of the GA type individual, and the difference of the GG type individual and the GA and AA type individual is not obvious.
9. The method of claim 8, wherein the PCR amplification procedure is: pre-denaturation at 94 ℃ for 2 min; denaturation at 98 ℃, 10sec, annealing at 53 ℃, 30sec, extension at 68 ℃, 30sec, 35 cycles; preserving at 4 deg.C for infinity; the PCR amplification system is as follows:
10. the use of the method of claim 8 or 9 in screening for Hu sheep body length traits and Hu sheep molecular marker assisted breeding; wherein, the body length of the AA type individual is obviously higher than that of the GA type individual, and the difference of the GG type individual and the GA and AA type individual is not obvious.
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