CN114574596B - SNPs molecular marker g.438513G & gtA and application thereof in Hu sheep molecular marker assisted breeding - Google Patents
SNPs molecular marker g.438513G & 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 molecular marker assisted breeding of Hu sheep, and particularly relates to a molecular marker g.43851G & gtA of SNPs related to Hu sheep body length characters, a primer pair, a kit and application of the molecular marker g.43851G & gtA in molecular marker assisted breeding of Hu sheep. Wherein, the body length of the AA type individual is obviously higher than that of the GA type individual, and the difference between the GG type individual and the GA and AA type individual is not obvious. The molecular marker, the primer pair and the kit provided by the invention can screen the body length characters of Hu sheep by adopting a related detection method, and play a role in auxiliary breeding.
Description
Technical Field
The invention belongs to the technical field of molecular marker assisted breeding of Hu sheep, and particularly relates to a molecular marker g.438511G & gtA of SNPs related to Hu sheep body length characters, a primer pair, a kit and application of the molecular marker g.438511G & gtA in molecular marker assisted breeding of Hu sheep.
Background
Hu sheep is a well-known sheep variety in China, and is distinguished by the characteristics of strong fertility, high early growth speed, suitability for house feeding, high-humidity high-heat environment and the like. However, the Hu sheep has a great difference in meat performance from foreign meat sheep such as Dupox sheep and German merino sheep. Therefore, the breeding of the Hu sheep growth performance is very necessary, and the meat production performance of the Hu sheep is improved.
The applicant has issued Chinese patent (publication numbers: CN109251986A, CN109182553A, CN109251987A, CN109251985A, CN109182554A and CN 109055579A) discloses Hu sheep body ruler character candidate functional genes and SNPs screened by using a GWAS technology, and SNPs which can be used for molecular marker assisted breeding are obtained through population verification of the SNPs, and can be used for character molecular marker assisted breeding for Hu sheep meat and early breeding of Hu sheep meat line core group individuals.
The ARHGAP24 gene is a member of the Rho GTPase activator family, can act as a negative regulator of Rho GTPase, and affects 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.
The ARHGAP24 gene is currently found to be involved in proliferation, cell cycle, apoptosis, migration and invasion of cancer cells in the kidney, where it acts to cause pseudopodogenesis by activated ARF 6-induced breast cancer cells, and to promote tumor growth of glioblastoma by increasing Rac1 activity. Downregulation of ARHGAP24 expression occurs in lung adenocarcinoma, and the paclitaxel resistant phenotype of the cancer cells will occur.
The Rho GTP family is involved in a variety of cellular functions in mammals, including actin cytoskeletal recombination, cell growth control, transcriptional regulation and membrane trafficking, and in the growth and development of animals, lung development, respiratory system development, vascular morphogenesis, cardiac development and transcriptional regulation. ARHGAP24 has been found to be significantly associated with production traits in GWAS analysis of production traits in dolac pigs. In connection with previous studies, mutations in ARHGAP24 may be associated with certain functions in cell growth regulation, thereby further affecting the growth characteristics of Hu sheep from some unknown pathway regulation.
Disclosure of Invention
In order to solve the defects and the actual requirements of the prior art in molecular marker breeding, the invention aims to provide an SNPs molecular marker which is obviously related to Hu sheep body length characters, a detection primer pair and a kit thereof, 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:
SNPs molecular markers which are obviously related to Hu sheep body length characters are positioned as g.438511G & gtA.
Further, the present 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 pairs are shown as SEQ ID NO. 2 and SEQ ID NO. 3.
Further, the application provides a kit comprising the primer pair.
Further, the application provides application of the SNPs molecular markers, the primer pairs and the kit in screening Hu sheep body length characters 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 between the GG type individual and the GA and AA type individual is not obvious.
Further, the application also provides a method for screening the Hu sheep body weight and body size characteristics, which comprises the following steps: extracting genomic DNA of peripheral blood of the Hu sheep, carrying out PCR (polymerase chain reaction) amplification by adopting the primer pair, and detecting SNPs molecular markers 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 between 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 2min; denaturation at 98 ℃,10sec, annealing at 53 ℃,30sec, elongation at 68 ℃,30sec,35 cycles; preserving at 4 ℃ and infinity; the PCR amplification system is as follows:
further, the application of the method in screening Hu sheep body length characters and Hu sheep molecular marker breeding is also provided; wherein, the body length of the AA type individual is obviously higher than that of the GA type individual, and the difference between the GG type individual and the GA and AA type individual is not obvious.
According to the invention, 214 Hu sheep are used as test materials, ARHGAP24 genes are used as candidate genes, PCR amplification is adopted, SNPs are screened by a direct sequencing and sequence analysis technology of products, and SNP loci which obviously influence the body weight and body size characteristics of the Hu sheep are screened through correlation analysis.
The invention discovers that 15 SNPs exist in the 1 part region of the intron of the ARHGAP24 gene, wherein 4 SNPs loci which are obviously related to the body weight and body size traits of Hu sheep are provided: g.43756G > A is obviously related to the Hu sheep body length, the body length of the AA type individual is obviously higher than that of the GA type individual (P < 0.05), and the difference between the GG type individual and the GA type individual is not obvious (P > 0.05). g.43815G > A is significantly related to Hu sheep body height, the AA type individual body height is significantly higher than GG type individual body (P < 0.05), and the GA type individual body is not significantly different from GG and AA type individual body (P > 0.05). g.438511G > A is significantly related to Hu sheep body length, the body length of AA type individuals is significantly higher than that of GA type individuals (P < 0.05), and the difference between GG type individuals and GA and AA type individuals is not significant (P > 0.05). g.43977A > G is significantly related to Hu sheep body length and daily gain, AA and GG type individuals are significantly higher than AG type individuals (P < 0.05), AA and GG type individuals are not significantly different (P > 0.05), GG type individuals gain significantly higher daily gain from weaning to June age than AA type individuals, AG type individuals are not significantly different from AA, GG type individuals.
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Fig. 1: the invention detects PCR amplified sequences of the primers, wherein grey positions are molecular marked positions.
Fig. 2: PCR amplification of SNPs detection primers of the Hu sheep ARHGAP24 gene (M: DL5000, 5000bp, 3000bp, 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp from top to bottom in sequence, lanes 1-8 are target fragment amplification results).
Detailed Description
The invention is further illustrated by the following examples.
1. Test materials
1.1 test animal sample Source
The experimental animals are from a novel core group for Hu sheep meat of Hangzhou huge agriculture development limited company, 213 animals are fed and managed according to a feeding and managing method of mutton sheep standards. 10mL of jugular blood was collected from each individual and placed in an EDTA anticoagulant tube for storage at-20 ℃.
1.2 measurement of body Scale index
The Hu sheep body ruler measurement includes length, height and chest circumference of the human body (inclined). Each sheep was measured at least 3 times and the average was taken as the final measurement. The specific measurement properties and measurement methods are shown in Table 1.
Table 1 measurement Properties and measurement methods
| Determination of Properties | Measurement method |
| Length of body | Straight line distance from the anterior edge of the scapula to the hip end. |
| Height of body | The vertical distance from the highest point of the chignon to the ground. |
| Chest circumference | A length of one week around the chest along the posterior edge of the scapula. |
2. Test method
2.1 extraction of genomic DNA from peripheral blood was carried out according to a known conventional method.
2.2DNA concentration and purity detection was performed according to a known conventional method.
2.3 PCR amplification of ARHGAP24 gene of Hu sheep and SNPs detection.
The design of the primers was based on the partial region of Acc 101119904 intron 1, primers were synthesized by Hangzhou Kangshen Biotechnology Co., ltd, and the primer sequences, amplified fragment lengths and annealing temperatures were shown in Table 2.
Table 2 PCR amplification primer for ARHGAP24 gene of Hu sheep
The PCR amplification system was 25. Mu.L, and the PCR procedure is shown in Table 3. The specific components are shown in Table 4.
TABLE 3 PCR amplification procedure for ARHGAP24 Gene of Hu sheep
Table 4 lake sheep ARHGAP24 gene PCR amplification system
| Reagent(s) | Volume of |
| 2x PCR buffer for KOD FX | 12.5μL |
| 2mM dNTPs | 5μL |
| F | 0.2μL |
| R | 0.2μL |
| KOD FX | 0.5μL |
| DNA template | 1μL |
| Total system | 25μL |
2.4 detection of PCR products
5 mu L of PCR products are sampled on 1.0% agarose gel, DL5000 is used as Marker mark, electrophoresis is carried out for about 15min under 200V voltage, EB dyeing is carried out for 5min, whether amplified bands exist or not is observed in a gel imager, and photographing is carried out.
2.5 sequencing of PCR products
Each sample was amplified according to the primers listed in table 2, and the amplified products were submitted to the catalpa ovata biological technology limited in the Hangzhou, and were sequenced in both forward and reverse directions until the results of the two-way sequencing were consistent.
2.6 sequence analysis
Forward and reverse sequencing peak patterns were analyzed for each individual using Mutation Surveyor 5.02.02 (Softgenetics, U.S.) software, and the mutation position and mutation pattern of each individual ARHGAP24 gene was determined using the partial region of Acc:101119904 intron 1 as a reference sequence. Individuals with abnormal sequencing results or inconsistent forward and reverse sequencing results need to be subjected to secondary sequencing 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 a lite program.
The position heterozygosity, shannon information content, gene frequency, genotype frequency of each snp was calculated using the PopGen 32 software and tested for compliance with Hardy-Weinberg equilibrium.
Polymorphism information content analysis:
PIC (Polymorphism information content), which shows the probability that an allele obtained from a offspring is derived from the same allele of its parent, is an ideal indicator for measuring polymorphism of an allele fragment, and is calculated by the following formula:
P i and P j 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 the 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 method is applied to animal genetic breeding research effect; the lower the value, the higher the genetic consistency, which means that the genetic variation in the group is small and the genetic potential is also small. The calculation formula is as follows:
p i indicating 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:
(1) genotype frequency = genotype number of individuals/population number of individuals x 100%;
(2) gene frequency = homozygous genotype frequency +1/2 x heterozygous genotype frequency.
Correlation analysis:
SNPs associated with the body weight and body size traits of the new group core group for Hu mutton were mined using a general linear model (General Linear Model, GLM; SPSS 20).
Since all individuals analyzed were female sheep from the same farm, the same feeding environment and management conditions, no field effect and sex effect were included in the data modeling.
The specific model is as follows: y=xβ+e;
wherein Y: the size character and weight character phenotype value vector of the new group core group for the Hu mutton;
beta: fixed effector vectors such as phenotype mean value and SNP;
e: residual effect vector;
x is the correlation matrix of beta.
When Y is a vector of primary heavy phenotypes of a new group core for a lake-mutton, it is analyzed according to model y=xβ+sα+e.
Wherein Y: the new group core group ruler character phenotype value vector for the Hu mutton;
alpha: a fixed effector vector of the siblings;
beta: SNP effect vector;
e: residual effect vector;
x, S are the correlation matrices of β and α, respectively.
3. Test results
3.1 detection PCR amplification result of SNPs of ARHGAP24 Gene of Hu sheep
The PCR products of each primer have higher brightness and single band, no nonspecific amplification (figure 2), the actual PCR products are consistent with the expected PCR amplification products in size, and the subsequent PCR-product direct sequencing test can be performed.
3.2 mutation analysis of ARHGAP24 Gene amplification products of Hu sheep
The analysis results of PCR amplified products of the ARHGAP24 gene of Hu sheep are shown in Table 5, and the amplification of partial region of the intron 1 of the ARHGAP24 gene (476 bp of amplified product) detects 15 mutations in Hu sheep flock, wherein the number of transversions is 3 and the number of transversions is 12.
TABLE 5 SNPs sites and mutation types and modes of ARHGAP24 genes of Hu sheep
TABLE 6 genetic parameters of SNPs loci of ARHGAP24 genes of Hu sheep
| SNP locus | Effective allelic factors | Shannon information content | Average heterozygosity |
| g.43642T＞C | 2.0000 | 0.6931 | 0.5000 |
| g.43649A＞G | 1.4791 | 0.5049 | 0.3239 |
| g.43654A＞T | 1.3118 | 0.4010 | 0.2377 |
| g.43681G>A | 1.9996 | 0.6930 | 0.4999 |
| g.43699C>G | 0.9979 | 0.6926 | 0.4995 |
| g.43722C＞A＞T | 2.226 | 0.9271 | 0.5508 |
| g.43756G＞A | 1.3648 | 0.4378 | 0.2673 |
| g.43788G＞A | 1.4730 | 0.5017 | 0.3211 |
| g.43815G>A | 1.6060 | 0.5649 | 0.3773 |
| g.43851G＞A | 1.3886 | 0.4531 | 0.2798 |
| g.43914A＞G | 1.4186 | 0.4713 | 0.2951 |
| g.43917A＞G | 1.4186 | 0.4713 | 0.2951 |
| g.43934A＞G | 1.4609 | 0.4952 | 0.3155 |
| g.43955C＞T | 1.0478 | 0.1109 | 0.0456 |
| g.43980G＞A | 1.5276 | 0.5294 | 0.3454 |
TABLE 7 genetic analysis of SNPs loci of Hu sheep
Polymorphism Information Content (PIC) is the information content expressed by a genetic marker for determination and analysis, PIC >0.5 is a highly polymorphic site, 0.25< PIC <0.5 is a moderately polymorphic site, and PIC <0.25 is a lowly polymorphic site.
The larger PIC value indicates a greater effective allele and heterozygosity, as well as a higher variability of the population at this SNP site. The Hu sheep ARHGAP24 gene has moderate polymorphism of g.43640T > C, g.43649A > G, g.43654A > T, g.436811G > A, g.43699C > G, g.4372C > A > T, g.43788G > A, g.43815G > A, g.43914A > G, g.43972A > G, g.43934A > G, g.43980G > A, g.43970G > A, g.43956G > A, g.438511G > A, and g.43955C > T.
Association analysis of Hu sheep ARHGAP24 gene polymorphism and body weight and body size traits:
the correlation analysis of 15 SNPs of the ARHGAP24 gene and the body size trait and the body weight trait of Hu sheep were performed by using the GLM model in the "correlation analysis" (Table 8).
The analysis result shows that g.43756G > A is obviously related to the Hu sheep body length, the body length of the AA type individual is obviously higher than that of the GA type individual (P < 0.05), and the difference between the GG type individual and the GA and the AA type individual is not obvious (P > 0.05).
43815G > A is significantly associated with increased sheep in Hu sheep, the height of the AA type individuals is significantly higher than that of GG type individuals (P < 0.05), and the difference between GA type individuals and GG, AA type individuals is not significant (P > 0.05).
438511G > A is significantly related to Hu sheep body length, the body length of AA type individuals is significantly higher than that of GA type individuals (P < 0.05), and the difference between GG type individuals and GA and AA type individuals is not significant (P > 0.05).
43917A > G is obviously related to Hu sheep body length and daily gain, the body length of AA and GG type individuals is obviously higher than that of AG type individuals (P is smaller than 0.05), the difference between AA and GG type individuals is not obvious (P is larger than 0.05), the daily gain from weaning to june age of GG type individuals is obviously higher than that of AA type individuals, and the difference between AG type individuals and AA and GG type individuals 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. Various modifications to these embodiments will be readily apparent to those skilled in the art. The generic principles defined herein may be applied to 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.
Sequence listing
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Claims (9)
1, application of SNP molecular markers in screening hu sheep body length character breeding; the SNP molecular marker is characterized in that the nucleotide sequence of the SNP molecular marker is SEQ ID NO:1, and wherein the base at position 271 is A or G; wherein, the body length of the AA type individual is obviously higher than that of the GA type individual, and the difference between the GG type individual and the GA and AA type individual is not obvious.
2. An application of a primer pair for detecting SNP molecular markers in screening hu sheep body length character breeding; the SNP molecular marker is characterized in that the nucleotide sequence of the SNP molecular marker is SEQ ID NO:1, and wherein the base at position 271 is A or G; wherein, the body length of the AA type individual is obviously higher than that of the GA type individual, and the difference between the GG type individual and the GA and AA type individual is not obvious.
3. The use according to claim 2; the primer pair is characterized in that the nucleotide sequences of the primer pair are shown as SEQ ID NO. 2 and SEQ ID NO. 3.
4. Use of a kit comprising a primer pair for detecting SNP molecular markers in screening hu sheep body length trait breeding; the nucleotide sequence of the SNP molecular marker is SEQ ID NO:1, and wherein the base at position 271 is A or G; wherein, the body length of the AA type individual is obviously higher than that of the GA type individual, and the difference between the GG type individual and the GA and AA type individual is not obvious.
5. The use according to claim 4; the primer pair is characterized in that the nucleotide sequences of the primer pair are shown as SEQ ID NO. 2 and SEQ ID NO. 3.
6. A method for screening Hu sheep body length traits, which is characterized by comprising the following steps: extracting the genome DNA of the peripheral blood of the Hu sheep, carrying out PCR (polymerase chain reaction) amplification by adopting a primer pair, and detecting SNP molecular markers in amplification products, thereby screening the Hu sheep body length characters of the Hu sheep; wherein, the body length of the AA type individual is obviously higher than that of the GA type individual, and the difference between the GG type individual and the GA and AA type individual is not obvious;
the primer pair is used for detecting SNP molecular markers, and the nucleotide sequence of the SNP molecular markers is SEQ ID NO:1, and wherein the base at position 271 is A or G.
7. The method of claim 6; the primer pair is characterized in that the nucleotide sequences of the primer pair are shown as SEQ ID NO. 2 and SEQ ID NO. 3.
8. The method of claim 7, wherein the PCR amplification procedure is: 94. pre-denaturation at temperature for 2min; denaturation at 98 ℃,10sec, annealing at 53 ℃,30sec, elongation at 68 ℃,30sec,35 cycles; 4. preserving at a temperature of infinity; the PCR amplification system is as follows:
。
9. Use of the method of claim 6 or 7 or 8 for screening Hu sheep body length traits for assisted breeding; wherein, the body length of the AA type individual is obviously higher than that of the GA type individual, and the difference between the GG type individual and the GA and AA type individual is not obvious.
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