CN110331211B - Molecular marker SNP732 of Hu sheep MC4R gene and application thereof - Google Patents
Molecular marker SNP732 of Hu sheep MC4R gene and application thereof Download PDFInfo
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Abstract
The invention relates to the field of molecular markers, in particular to a molecular marker SNP732 of a Hu sheep MC4R gene and application thereof, and discloses an SNP molecular marker of a Hu sheep MC4R gene, a related primer pair, a kit and application thereof in Hu sheep molecular marker assisted breeding, and further relates to a method for screening Hu sheep body size traits and application thereof in Hu sheep molecular marker assisted breeding.
Description
Technical Field
The invention relates to the field of molecular markers, in particular to an SNP molecular marker, a related primer pair and a kit of Hu sheep MC4R gene, and application thereof in Hu sheep molecular marker assisted breeding, and further relates to a method for screening Hu sheep body size traits and application thereof in Hu sheep molecular marker assisted breeding.
Background
Melanocortin receptor-4 (MC 4R) is a peptide secreted from the ventral medial nucleus of the hypothalamus, involved in the regulation of body weight and energy balance, and expressed by multiple neuronal populations in the central nervous system. Unlike other members of the MCR family, MC4R is not present in the adrenal cortex, hair follicles, and placenta, but is present in large numbers in various regions of the central nervous system, including the cerebral cortex, thalamus, hypothalamus, brainstem, and spinal cord. The MC4R of mammals such as human, pig, cattle, sheep and dog is composed of 332 amino acid residues, has 7-time transmembrane structure, and is one member of transmembrane G protein coupled receptor family.
Leptin and insulin are the most important peripheral regulating factors for animal feeding regulation, and MC4R can be used as a downstream medium of a leptin and insulin feeding regulation pathway to realize the regulation of animal feeding. Therefore, the protein plays an important role in the central nervous system in regulating glucose homeostasis. Mutations in the mammalian MC4R gene disrupt the adrenocortical pathway within the central nervous system, increase appetite and reduce satiety, indirectly resulting in obesity. The mutation research of the MC4R gene of mammals such as human, dog, pig, cow, sheep and the like shows that the gene can be used as a candidate functional gene of the weight or growth traits of the mammals, and a plurality of SNPs related to the growth traits of the animals exist in the gene. Based on the phenotypic effect caused by the mutation of the MC4R gene, the mutation of the gene can be divided into five types: firstly, the synthesis defect of the MC4R protein is caused, or the degradation of the MC4R protein is accelerated, which shows that the amount of the MC4R protein is obviously reduced; ② errors in protein folding or intracellular transport are caused, leading to protein retention in cells; ③ the protein can be correctly integrated in the cell membrane, but the binding capacity with the ligand is reduced; inducing signal transmission obstacle; the effect due to the mutation is not clear.
The MC4R gene is taken as a candidate gene of the classical growth traits of mammals, the SNP research of the MC4R gene is widely concerned, and candidate functional SNPs which can be used for subsequent breeding are obtained in multiple species. Therefore, by utilizing the influence of the SNP of the MC4R gene in the livestock group on the body weight and the body size of livestock, the SNP which is beneficial to the body weight growth, the growth acceleration, the fattening and the slaughtering traits of the Hu sheep is screened out in the gene, and purposeful seed selection and matching are carried out on the genotype individuals which are beneficial to the weight increase and the meat quality scoring, so that the Hu sheep group with high growth speed and good carcass quality can be cultured, and the economic benefit is improved.
Disclosure of Invention
The invention takes 202 female individuals of the third-generation core group of the new group for Hu sheep as research objects, adopts a PCR and direct product sequencing mode to determine SNPs of the MC4R gene of the third-generation core group of the new group for Hu sheep, performs correlation analysis with the growth traits of the individuals, screens the SNPs correlated with the growth traits of Hu sheep and provides a useful method for molecular breeding of the system for Hu sheep.
To this end, it is an object of the present invention to provide a molecular marker for SNPs associated with the Hu sheep body size trait, characterized in that the molecular marker comprises a SNP706, which is C or A, at position 706 corresponding to SEQ ID NO:1 in alignment with SEQ ID NO: 1. Preferably, the sequence of the molecular marker is shown in SEQ ID NO. 1, and the 706 th position is C or A.
Another object of the present invention is to provide a molecular marker for SNP associated with the Hu sheep body size trait, characterized in that the molecular marker comprises SNP732, which is C or G, at position 732 corresponding to SEQ ID NO. 1, aligned with SEQ ID NO. 1. Preferably, the sequence of the molecular marker is shown in SEQ ID NO. 1, and the position 732 is C or G.
Another object of the present invention is to provide a primer set for detecting the SNP706 or the SNP732 described above. Preferably, the sequence is shown in SEQ ID NO 2 and SEQ ID NO 3.
Another object of the present invention is to provide a kit comprising the above primer pair.
The invention also aims to provide application of the SNP molecular marker, the primer pair or the kit in screening Hu sheep body size characters or Hu sheep molecular marker assisted breeding.
The invention also aims to provide a method for screening the lake sheep body size character, which comprises the following steps: extracting Hu sheep genome DNA, respectively carrying out PCR amplification by using primer pairs for detecting the SNP706 or the SNP732, and respectively detecting the 706 th site or the 732 th site corresponding to the SEQ ID NO. 1 in an amplification product, thereby screening the Hu sheep body size character. Preferably, wherein the primer pair is the primer pair of claim 4. Preferably, wherein the reaction procedure for PCR amplification is: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30sec, annealing at 58 ℃ for 30sec, extension at 72 ℃ for 1min, 35 cycles; extending for 10min at 72 ℃; the reaction system for PCR amplification is as follows:
another objective of the invention is to provide application of any one of the methods in Hu sheep molecular marker-assisted breeding.
The invention has the following beneficial effects: the molecular marker, the primer pair and the related kit can be used for screening the body size characters of the Hu sheep or the molecular marker-assisted breeding of the Hu sheep.
Drawings
FIG. 1 shows PCR amplification (M: Marker DL5000) of MC4R gene primers 1F +1R (lanes 1-3) and 2F +2R (lanes 4-6).
Detailed Description
The present invention is described in detail below with reference to specific embodiments, which should not be construed as limiting the scope of the present invention.
A large number of studies indicate that the factor of sex has a significant influence on the growth and development of sheep and goats. Since the gender has obvious influence on the body weight and body size traits of the Hu sheep, the SNP which can influence the body weight and body size traits of the Hu sheep is screened in the MC4R gene of female individuals of the Hu sheep meat line. The sex identification of the sample can further avoid errors in individual production record and genome extraction processes from influencing the subsequent correlation analysis result of MC4R gene SNP.
Example 1 test materials
Source of test animal samples
The test animals are from Hu sheep meat new group core group G3 generation group constructed by Hangzhou huge agriculture development limited company, and 202 female individuals are bred according to breeding management method of mutton sheep standard. 10mL of jugular venous blood was collected from each individual, placed in an EDTA anticoagulation tube, and stored at-20 ℃.
Measurement of body ruler index
The body size measurement of Hu mutton with the core group individuals of the new group G3 generation line includes body (oblique) length, body height and 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
Example 2 test method
1. Peripheral blood genomic DNA was extracted according to a known conventional method.
DNA concentration and purity determination, according to known conventional methods.
3. PCR amplification and SNPs detection of Hu sheep MC4R gene.
Hu sheep MC4R gene PCR amplification primer
The primers were synthesized by Hangzhou Optingxi Biotechnology Limited, and the primer sequences, the amplified fragment lengths and the P annealing temperatures are shown in Table 2.
TABLE 2 Hu sheep MC4R gene PCR amplification primers
PCR amplification
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 MC4R Gene PCR amplification program
TABLE 4 Hu sheep MC4R gene PCR amplification system
Detection of PCR products
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.
Sequence analysis
The forward and reverse sequencing peak patterns of each individual were analyzed using the Mutation Surveyor 5.02(Softgenetics, usa) software to determine the Mutation position and Mutation pattern of the MC4R gene of each individual with NM _01126370 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.
Data statistics and analysis
The degree of linkage disequilibrium was analyzed using Haploview 4.2 software.
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:
pi and Pj are the ith and jth allele frequencies, respectively; n is the number of alleles
Degree of 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:
pi represents the ith allele frequency
Information content of Shannon
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) the genotype frequency of the gene is the genotype/population × 100%
② the frequency of the gene is homozygous genotype frequency +1/2 Xheterozygous genotype frequency
Hardy-Weinberg equilibrium assay
And (3) identifying whether the genotype frequency and the gene frequency of each locus conform to Hardy-Weinberg balance by using chi-square test (chi 2), wherein the formula is as follows:
wherein m represents the number of genotypes; fi represents the number of individuals of the ith genotype observed; n represents the total number of samples; pi represents the theoretical genotype frequency of the ith genotype.
Association 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.
Bioinformatics analysis
Prediction of secondary and tertiary structures of Hu sheep MC4R protein
The secondary structure of the target protein is predicted by DNASTA software, and the tertiary structure of the target protein and the transmembrane structure of the MC4R protein are predicted on line by Phre2(http:// www.sbg.bio.ic.ac.uk/phyre2/htmL/page.
Example 3 test results
PCR amplification of Hu sheep MC4R gene fragment
The Hu sheep MC4R gene PCR amplification result shows that when primers 1F and 1R, 2F and 2R are used for amplification, specific amplification bands can appear in the range of 750-1000bp, and are consistent with the expected molecular weight (862 bp for primer 1F and 1R products, 670bp for primer 2F and 2R products respectively) (FIG. 1).
Hu sheep MC4R gene PCR amplification product mutation analysis
The results of analysis of mutation of PCR amplification product of Hu sheep MC4R gene are shown in Table 5.
TABLE 5 SNPs sites, mutation types and mutation modes of Hu sheep MC4R gene
19 mutations were detected in the Hu sheep meat new bouquet core population G3 individuals using primer pairs 1F and 1R (amplification product: 862bp, see SEQ ID NO:6 in the sequence Listing), 2F and 2R (amplification product: 670bp, see SEQ ID NO:7 in the sequence Listing), wherein 11 mutation sites were present in the amplification products of primer pair 1F and 1R, 8 mutation sites were present in the amplification products of primer pair 2F and 2R, and the mutation pattern, substitution pattern, mutation pattern and mutation position of each site are shown in tables 3-5. The mutation positions of 19 mutation sites are shown, wherein the 5'UTR accounts for 26.3 percent (5/19), the ORF region accounts for 42.1 percent (8/19), and the 3' UTR accounts for 31.6 percent (6/19); the mutation pattern had 52.6% (10/19) and 47.4% (9/19) transitions; synonymous mutations accounted for 75.0% (6/8) and missense mutations accounted for 25.0% (2/8) (Table 5).
Association analysis of Hu sheep MC4R gene polymorphism and weight and body size traits
Correlation analysis of the 19 SNPs of the hu sheep MC4R gene with the body size and weight traits of the female individuals of the G3 generation for meat core group was performed using the GLM model in "correlation analysis" (table 10).
Association analysis of Hu sheep MC4R gene polymorphism and Hu sheep meat new group core group G3 generation female individual weight trait
The analysis results show that 19 SNPs of Hu sheep MC4R gene do not affect the body weight traits (P > 0.05) of Hu sheep meat core group G3 generation female individuals, such as birth weight, weaning weight, 6-month-old body weight and adult weight, and the like (Table 10).
Association analysis of Hu sheep MC4R gene polymorphism and Hu sheep meat new group core group G3 generation female individual adult size character
The analysis result shows that 2 SNPs in 19 detected SNPs, namely g.706C → A and g.732C → G, located on an open reading frame can significantly influence the adult height trait (P <0.05 or P <0.01) of the Hu sheep meat core group G3 female individuals (Table 10).
Wherein the adult height (76.09 +/-3.07 cm) of the g.706C → A wild type CC female Hu sheep is obviously lower than that of a mutation homozygous AA individual (79.50 +/-1.91 cm) (P is less than 0.05), and is obviously not different from that of a CA genotype individual (76.77 +/-2.77 cm) (P is more than 0.05). The bred Hu mutton uses a new class group core group G3 generation female individual g.706C → A mainly takes wild type (186/202). Whereas individuals homozygous for the mutation are only 4/202.
Wherein the adult height (77.57 +/-2.49 cm) of the female of the G wild type CC gene type is obviously higher than that (76.20 +/-2.88 cm) (P <0.05) of the mutant heterozygote CG female, the adult height is extremely obviously higher than that (75.72 +/-3.30 cm) (P <0.01) of the mutant homozygote type GG female, and the difference between the adult height and the GG gene type of the mutant heterozygote type CG female is not obvious (P > 0.05). The bred Hu sheep meat uses a new group core group G3 generation female individual g.732C → G mutation heterozygote is the same as mutation homozygote (86/202). Whereas the wild homozygous individual is only 30/202.
Prediction result of secondary and tertiary structure of Hu sheep MC4R protein
The detected mutations at 222bp, 225bp, 306bp, 706bp, 894bp and 951bp of the MC4R gene were found not to cause the amino acid encoded by the protein to change, and compared with the reference sequence NP-001119842, the mutation at g.548C → T resulted in the conversion of threonine (Thr) encoded at position 112 to methionine (Met), and the mutation at g.732C → G resulted in the conversion of isoleucine (Ile) encoded at position 173 to methionine (Met).
DNASTAR is utilized to predict whether the mutation causes the change of alpha helix (alpha-helix), beta sheet (beta-sheet), beta Turn (beta-Turn) and random coil (coi1) in the secondary structure of the protein, and the site mutation can cause the disappearance of the beta Turn at position 120, the random coil at position 120 and the alpha helix at positions 1170-175 of the protein.
The Phre2 is used for predicting the tertiary structure before and after mutation of the MC4R protein, and the result shows that missense mutation can cause spatial change of alpha helix, beta Turn and random coil of the tertiary structure at the corresponding position, wherein the deep red is alpha helix, the yellow is beta sheet, the light blue is beta Turn (beta-Turn), and the white is other residues.
Prediction result of Hu sheep MC4R protein transmembrane region
The prediction of the transmembrane region of Hu sheep MC4R protein is carried out by Phre2 online software, the Hu sheep MC4R protein has 7 transmembrane structures, and the amino acid sequence number of the transmembrane region is as follows: 46 to 70, 80 to 102, 121 to 145, 167 to 185, 193 to 213, 247 to 267, 283 to 305; the N terminal of the protein is outside the membrane, the C terminal is inside the membrane, g.548C → T (Thr112Met) is positioned at the membrane outer region between the 2 nd transmembrane region and the 3 rd transmembrane region, and g.732C → G (Ile173Met) is positioned at the 4 th transmembrane region.
SNP and detection and analysis method thereof
An SNP is a DNA sequence polymorphism caused by a change in the position of a specific nucleotide on the genomic level. At present, methods for detecting SNPs can be roughly divided into two major categories, one being a gel electrophoresis-based detection method represented by single-strand conformation polymorphism, enzyme digestion amplification polymorphism sequence, ligase detection reaction, and the like; the other type is a high-throughput and high-automation detection method represented by direct sequencing, denaturing high-performance liquid chromatography, high-resolution dissolution curve and the like.
The invention adopts a DNA direct sequencing method and combines with Mutation Surveyor 5.02 software to analyze the SNPs of the complete sequence of the female individuals MC4R gene of the new group G3 generation core group for Hu mutton and co-discover 19 SNPs, wherein 5 SNPs are arranged in the 5' UTR, and the SNPs are g.36G → A, g.73G → A, g.79C → T, g.110C → G and g.139G → A respectively; 8 SNPs within the ORF region, g.222C → T, g.225G → C, g.306G → A, g.548C → T (Thr112Met), g.706C → A, g.732C → G (Ile173 → Met), g.894G → C and g.951G → A, respectively; 8 SNPs in the 3' UTR, g.1229G → A, g.1267G → A, g.1291G → C, g.1337C → A, g.1352G → C and g.1382G → C, respectively. In the sheep population, mutations of MC4R genes g.36G → A, g.73G → A, g.79C → T, g.225G → C, g.1337C → A, g.1352G → C and g.1382G → C have not been reported.
Compared with a reference sequence, in an amplification product with the length of 1464bp, DNA direct sequencing is used, and the Mutation Surveyor 5.02 software is combined to detect 19 SNPs altogether, wherein the detection length of a 5' UTR region is 213bp, 5 SNPs are detected, and 1 SNP appears in each 43bp on average; the ORF region is 999bp in length, 8 SNPs are detected, and 1 SNP appears in each 125bp on average; the detection length of the 3' UTR region is 203bp, 8 SNPs are detected, and 1 SNP appears in every 29bp on average. The density of the SNPs in the 3' UTR region is higher than that of other segments, and the SNPs detected by the invention are higher than the SNPs research results of the published sheep MC4R gene regardless of the quantity and density.
Hu sheep MC4R gene SNPs
Mutant forms and positions of SNPs
The frequency of SNPs occurring in a population is greater than 1%. Including single base transitions, transversions, insertions and deletions. Transitions refer to substitutions between homobases, such as between purines and purines (A → G), pyrimidines and pyrimidines (C → T); transversions refer to substitutions that occur between purines and pyrimidines (A → C, A → T, G → C and G → T). SNP transitions occur most frequently, accounting for approximately 66.7% of the total number of substitutions, and occur at a high frequency as C → T transitions, probably because methylated CpG can deaminate to thymine by spontaneous hydrolysis.
The invention discovers that the conversion ratio of the Hu sheep MC4R gene SNPs is 52.6%, and the conversion is 47.4%, which is a conclusion that the current research discovers that the SNP conversion variation accounts for 66.7%' has a certain difference. And the conversion mode of Hu sheep MC4R gene is mainly G → A and accounts for 70% (7/10) and C → T accounts for 30% (3/10), which is different from the conclusion that the C → T occurrence frequency is higher in SNP conversion found in the prior art, and the mutation form and the number of SNPs of Hu sheep MC4R gene have certain specificity.
The method can be divided into protein coding SNP and non-protein coding SNP according to the influence of the SNP on the biological genetic traits, wherein the protein coding SNP is mainly distributed in a gene coding region, and the mutation is synonymous protein coding SNP, otherwise, the mutation is called non-synonymous protein coding SNP or missense protein coding SNP. Due to selection pressure, SNP distribution is heterogeneous throughout the genome and between populations, with SNPs higher in number in non-coding regions than in coding regions.
The invention discovers that 8 SNPs are discovered in the coding region of the Hu sheep MC4R gene SNPs. Among them, missense mutations account for 25.0% (2/8), g.548C → T (Thr112Met) and g.732C → G (Ile173Met), while synonymous mutations account for 75.0% (6/8). The reason for this is that missense mutations have a low frequency of occurrence because they have a large influence on the physiological functions of individuals, whereas synonymous mutations have a small influence and a high probability of occurrence.
Group genetics analysis of Hu sheep MC4R gene SNPs
Evaluation of Hu sheep MC4R gene SNPs genetic variation degree
Genetic variation is a prerequisite for an organism to adapt to environmental changes. Parameters such as heterozygosity of variation and effective allele factors can be used to measure the degree of genetic variation within a population. For a population, the greater the number of variant heterozygosity and effective allele factors, the poorer the gene identity within the population, the higher the variability and the greater the selection potential, and conversely, the lower the population variation, the lower the selection potential.
A larger PIC value indicates a greater effective allele factor and heterozygosity, and a higher variability of the population at this SNP site. The coding region of Hu sheep MC4R gene has 8 SNPs, 2 of which are missense mutations: g.548C → T is low polymorphic, g.732C → G is medium polymorphic, 6 are synonymous mutations: g.222C → T, g.225G → C, g.306A → G, g.706C → A are medium polymorphism, g.894G → C, g.951G → A are low polymorphism, and mutation sites belonging to medium polymorphism in the colony are located in the coding region.
PIC values of g.222T → C, g.225G → C, g.306A → G and g.732C → G sites are all between 0.25 and 0.5, and are moderate polymorphic SNPs, which shows that the 3 sites have higher genetic diversity and high breeding potential.
Evaluation of Hu sheep MC4R gene SNPs
Hardy-weinberg equilibrium is an ideal state. Within a population of organisms, various factors affecting the state of equilibrium are constantly occurring, resulting in changes in the genetic composition of the population, thereby causing the evolution of the organism. If the test population is in equilibrium, it is indicated that the gene frequency of the sample within the population may represent the gene frequency of the normal population. And if the test population is deviated and does not reach the equilibrium state, the population is indicated to have the possibility of affinity, natural selection, random drift, mutation rate increase, migration and the like. As the breeding work of the Hu sheep needs continuous breeding according to the production performance and the body shape of the sheep, the Hardy-weinberg is easy to generate unbalanced state.
The results of the chi 2 test showed that g.139G → A, g.706C → A deviate significantly from Hard-Weinberg equilibrium (P <0.01), and the remaining 17 SNPs in the same population were in Hard-Weinberg equilibrium.
The results of correlation analysis of the body weight traits and body size traits of adult sheep of female individuals of the core group of the G3 generation of the Hu mutton line 706C → A show that the body weight traits (birth weight, weaning weight, six months of age weight and week age weight) of AA genotype individuals are not significantly different from those of CC genotype individuals, but the body height (79.50 +/-1.91 cm) of the AA genotype individuals is significantly higher than that of the CC genotype individuals (76.09 +/-3.07 cm). As the observed individual numbers of the wild CC and the mutation homozygous AA exceed the corresponding theoretical individual numbers, the g.706C → A locus is possibly linked with certain genes meeting the breeding requirement in the breeding process of the new cluster core group for the Hu mutton, so that the locus is not in a Hard-Weinberg balanced state. Of course, this conclusion still requires large sample size, and many generations of observation and validation in female individuals of the new cluster core group for Hu sheep meat.
Hu sheep MC4R gene polymorphic site genotype and weight trait association analysis
The 19 SNPs of the gene are respectively associated with the weight traits for analysis, and the 19 SNPs in the detection population are shown to be irrelevant to the weight. The results of the group prophase research show that the difference of the weight and the slaughter rate of female individuals of the F1 generation hybridized by the female Hu sheep with the age of 6 months and the Dorper X Hu sheep with the age of 6 months is not obvious, and the difference of the weight and the slaughter rate of male individuals of the F1 generation hybridized by the male Hu sheep with the age of 6 months and the Dorper X Hu sheep with the age of 6 months is obvious.
The weight trait is a micro-effect multi-gene controlled QTL trait, and considering that the test objects are Hu mutton new group core group female individuals, the influence of single SNP in a group with similar genetic background on the weight trait of the female individual is far less than the influence level of the variety on the weight. Therefore, it can be explained that although the MC4R gene is an important candidate functional gene related to growth traits, no position related to the body weight of female Hu sheep individuals can be detected in the gene.
Hu sheep MC4R gene polymorphic site genotype and body size character correlation analysis
The 19 SNPs of the gene are respectively associated with the weight traits and analyzed, and the 2 SNPs in the detection population are related to the body size traits of adult female individuals (P < 0.05).
706C → A is located in coding region, and is a synonymous mutation, and 3 genotypes are detected and found. The research result shows that g.706C → A only affects the body size character of adult sheep of female Hu sheep, and the adult height (76.09 +/-3.07 cm) of the wild CC female Hu sheep is obviously lower than that of a mutant homozygous AA individual (79.50 +/-1.91 cm) (P <0.05), but is not obviously different from that of a CA genotype individual (76.77 +/-2.77 cm) (P > 0.05).
732C → G is located in coding region, and has 3 genotypes of CC, CG and GG, which can cause the 173 th amino acid located in the 4 th transmembrane region of protein to be mutated from isoleucine to methionine. Wherein the adult height (77.57 +/-2.49 cm) of a female with a wild CC genotype is obviously higher than that (76.20 +/-2.88 cm) (P is less than 0.05) of a CG individual with a mutation homozygous type, the adult height (75.72 +/-3.30 cm) (P is less than 0.01) of a GG individual with a mutation homozygous type, and the adult height (P is more than 0.05) of the CG female with the mutation heterozygous type is not obviously different from the GG genotype. The new class group G3 generation female individual g.732C → G for the bred Hu sheep meat is mainly homozygous for mutation (86/202). Whereas the wild homozygous individual is only 30/202. The wild type CC individuals are directionally bred at the locus, and the genetic progress of the body size characters can be accelerated.
Hu sheep MC4R gene linkage disequilibrium analysis
Linkage disequilibrium is an association of alleles. Linkage disequilibrium is usually used in r2 and D 'measurement, if r2 > 0.33, D' > 0.8, can be considered 2 SNPs sites are closely linked, as a whole to genetic.
The linkage disequilibrium analysis result shows that the mutation sites g.222C → T and g.225C → G, r2 is 1, D' is 0.97, and the mutation sites are in a strong linkage disequilibrium state, but the linkage analysis result shows that g.222C → T and g.225C → G do not influence the weight character and body size character of female Hu sheep, and the two sites seem to have no great significance for the breeding of the production character of Hu sheep. However, the conclusion still needs to expand the population number and determine whether the locus does not affect the body weight and size traits of male Hu sheep individuals as well.
Prediction result of secondary and tertiary structures of Hu sheep MC4R protein and transmembrane structure analysis
The primary structure of a protein determines the higher order structure, which determines the function of the protein. The protein structure prediction and the protein transmembrane region are carried out by utilizing bioinformatics, and then the position of a functional gene is predicted, and data related to the biological function of the functional gene is obtained. Electronic prediction results of the transmembrane region of the Hu sheep MC4R protein show that the Hu sheep MC4R protein also has a seven-pass transmembrane structure, wherein the N end is outside the cell, and the C end is inside the cell.
Two missense mutated sites found in the present invention: g.548C → T may result in the mutation of threonine at position 112 (the 2 nd transmembrane domain and the 3 rd transmembrane domain of the extracellular domain) to methionine. g.C → G at position 732 leads to mutation of isoleucine (transmembrane region 4) at position 173 to methionine. Threonine is a polar, hydroxyl-containing amino acid. Isoleucine is a nonpolar or hydrophobic amino acid, a neutral amino acid with an aliphatic hydrocarbon side chain. Methionine is a nonpolar or hydrophobic amino acid, is a sulfur-containing amino acid, and is also one of α -amino acids, and contains a basic group (-NH2) and an acidic group (-COOH) in its molecule, which is amphoteric. If base mutation occurs, the change of gene structure is likely to occur in the transmembrane structural sequence, thereby affecting the activity or function of the protein.
The result of DNASTAR on Hu sheep MC4R protein structure prediction shows that Ile173Met can cause alpha helix loss at 1700-175 positions of Hu sheep MC4R protein, and then alpha helix change of a fourth transmembrane region of a tertiary structure is caused. Therefore, the missense mutation may affect the protein structure, affect its function, and show a difference in size traits.
TABLE 6 Hu sheep g.732G → A locus and body height correlation analysis
Note: the difference between the upper marks of different capital letters is obvious (P is less than 0.01)
In conclusion, the invention detects 19 SNPs in the Hu mutton new population G3 generation core population, 15 sites are low-degree polymorphism, and 4 sites are medium polymorphism; g.222C → T is strongly linked with g.222C → G; g.306A → G has abundant genetic polymorphisms; except for g.139G → A, g.706C → A, the other 17 SNPs reached Hard-Weinberg equilibrium state.
2 SNPs which are obviously related to the adult height of Hu sheep female individuals exist in the coding region of the MC4R gene, and g.548C → T can cause the mutation of 112 th threonine (the membrane outer region between the 2 nd transmembrane region and the 3 rd transmembrane region) into methionine. g.C → G at position 732 leads to mutation of isoleucine (transmembrane region 4) at position 173 to methionine. Wherein the adult height (76.09 +/-3.07 cm) of the g.706C → A wild type CC female Hu sheep is obviously lower than that of a mutation homozygous AA individual (79.50 +/-1.91 cm) (P is less than 0.05), and is obviously not different from that of a CA genotype individual (76.77 +/-2.77 cm) (P is more than 0.05). g.732C → G wild type CC genotype female has higher adult height (77.57 +/-2.49 cm) than mutation heterozygotic CG individual (76.20 +/-2.88 cm) (P <0.05), higher than mutation homozygotic GG individual (75.72 +/-3.30 cm) (P <0.01), and less difference between adult height and GG genotype (P > 0.05).
The early breeding of Hu mutton family group molecular marker assisted breeding by using the SNP can obviously improve the meat use character of the new Hu mutton family female individual.
The invention has been described in terms of specific embodiments, and equivalent alterations and modifications can be effected thereto, by those of skill in the art, without departing from the spirit of the invention and within the scope of the claims.
TABLE 7 genetic parameters of the SNPs sites of Hu sheep MC4R gene
TABLE 8 group genetics analysis of SNPs loci of Hu sheep MC4R gene
TABLE 9 Hardy-Weinberg equilibrium test of SNPs sites of Hu sheep MC4R gene
Sequence listing
<110> Zhejiang province academy of agricultural sciences
Molecular marker SNP732 of Hu sheep MC4R gene and application thereof
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1788
<212> DNA
<213> Hu sheep (hu sheet)
<220>
<221> mutation
<222> (36)..(36)
<223> s is g or a
<220>
<221> mutation
<222> (73)..(73)
<223> s is g or a
<220>
<221> mutation
<222> (79)..(79)
<223> s is c or t
<220>
<221> mutation
<222> (110)..(110)
<223> s is c or g
<220>
<221> mutation
<222> (139)..(139)
<223> s is g or a
<220>
<221> mutation
<222> (222)..(222)
<223> s is c or t
<220>
<221> mutation
<222> (225)..(225)
<223> s is c or g
<220>
<221> mutation
<222> (306)..(306)
<223> s is g or a
<220>
<221> mutation
<222> (548)..(548)
<223> s is c or t
<220>
<221> mutation
<222> (706)..(706)
<223> s is c or a
<220>
<221> mutation
<222> (732)..(732)
<223> s is c or g
<220>
<221> mutation
<222> (894)..(894)
<223> s is g or c
<220>
<221> mutation
<222> (951)..(951)
<223> s is g or a
<220>
<221> mutation
<222> (1229)..(1229)
<223> s is g or a
<220>
<221> mutation
<222> (1267)..(1267)
<223> s is g or a
<220>
<221> mutation
<222> (1291)..(1291)
<223> s is g or c
<220>
<221> mutation
<222> (1337)..(1337)
<223> s is c or a
<220>
<221> mutation
<222> (1352)..(1352)
<223> s is g or c
<220>
<221> mutation
<222> (1382)..(1382)
<223> s is g or c
<400> 1
cagcctaaga tttccaagtg atgccgacca gagccscacc tgaaagagac tgaaaacttc 60
ctctccagct ccsgagctsg ggacgtttat tcacagcagg catgccacts tccgccgcct 120
aactttcgtt tggggcaast caagactgga gaaaggcgct gaggctgccg gacccgggag 180
gttcagtcag tccagagggg acctgaatcc aagatgaact csacscagcc ccatggaatg 240
cacacctctc tccactcctg gaaccgcagc ggccacgggc tgcccaccaa tgtcagtgag 300
tccccsgcaa aaggctactc ggacgggggg tgctatgagc agctctttgt ctctcccgag 360
gtgtttgtga ctctgggggt catcagcttg ttggagaata ttctggtgat tgtggccatc 420
gccaagaata agaatctgca ctcacccatg tactttttca tctgcagcct ggctgtggct 480
gacatgttgg tgagcgtttc caacgggtcc gaaaccattg tcatcaccct gctgaacagc 540
acagacasgg acgcgcagag cttcacggtg aatattgaca acgtcatcga ctcggtgatc 600
tgcagctcct tgctcgcctc catctgcagc ttgctgtcga tcgcggtgga caggtacttc 660
accatcttct atgcgctcca gtaccatagc atcatgacgg tgcggsgggt ggcgatcacc 720
atcagtgcca tstgggcggc ctgcacggtg tcgggcgtct tgttcatcat ttactcagac 780
agcagcgctg tcatcatctg cctcatcacc gtgttcttca ccatgctggc tctcatggcg 840
tctctctacg tccacatgtt cctcatggcc aggctccaca ttaagaggat cgcsgtcctg 900
ccaggcaccg gcgccatccg ccagggcgcc aacatgaagg gggcgatcac sctgaccatc 960
ctgatcgggg tctttgttgt ctgctgggcc cccttcttcc tgcacctgat attctacatc 1020
tcctgccccc agaaccccta ctgcgtgtgc ttcatgtctc actttaacct gtatctcatc 1080
ctgatcatgt gtaattctgt catcgaccct ctgatctacg ccctgcggag ccaggaactg 1140
aggaaaacct tcaaagagat catttgctgc tctcctctag gtggcctctg tgatttgtct 1200
agcagatatt aaatggggac aaactgcgst gccaaacaca agcttaagag accttctcct 1260
tctcatstgt acaatctgaa cagtctgtat sagccacagc tttttcttct gtgtagggca 1320
tggagtgaaa aattctsttg tatcagttga astttgtgat ttttttctga tgtgaagcag 1380
tscccagtct tgctgtattt ttaatatcat gctactttct ggctgtaaaa tgtgaatcca 1440
catcacaggt tataggcact atggatttat aaaaaaaaaa aagaaacaaa gtccttatga 1500
ggagtttaac agtgtttcct tcttgtgatt tacaaggatg tgacactttg cttgcttttg 1560
taacatggaa atcacagctt ccttaagtat attctcatga gtggatttaa tgttatactt 1620
tacaacactg gagtataaaa tttgattcca gtatttaggg gagaaatatt gagaacatat 1680
tgcttaatca taaaaaaaaa aaccaagctg aaatttcagg taatttaata agacttgctc 1740
attcattctt cctgtgccga agttgaaatg aaacttctac tgggacaa 1788
<210> 2
<211> 25
<212> DNA
<213> primers (Primer)
<400> 2
gcctaagatt tccaagtgat gctga 25
<210> 3
<211> 24
<212> DNA
<213> primers (Primer)
<400> 3
ggaacatgtg gacatagaga gacg 24
<210> 4
<211> 24
<212> DNA
<213> primers (Primer)
<400> 4
catctgcctc atcaccgtgt tctt 24
<210> 5
<211> 23
<212> DNA
<213> primers (Primer)
<400> 5
ccatagtgcc tataacctgt gat 23
<210> 6
<211> 862
<212> DNA
<213> Hu sheep (hu sheet)
<400> 6
cagcctaaga tttccaagtg atgccgacca gagccgcacc tgaaagagac tgaaaacttc 60
ctctccagct ccggagctcg ggacgtttat tcacagcagg catgccactg tcggccacct 120
aactttcgtt tggggcaagt caagactgga gaaaggcgct gaggctgccg gacccgggag 180
gttcagtcag tccagagggg acctgaatcc aagatgaact ctacgcagcc ccatggaatg 240
cacacctctc tccactcctg gaaccgcagc ggccacgggc tgcccaccaa tgtcagtgag 300
tccccagcaa aaggctactc ggacgggggg tgctgggagc agctctttgt ctctcccgag 360
gtgtttgtga ctctgggggt catcagcttg ttggagaata ttctggtgat tgtggccatc 420
gccaagaata agagtctgca ctcacccatg tactttttca tctgcagcct ggctgtggct 480
gacatgttgg tgagcgtttc caacgggtcc gaaaccattg tcatcaccct gctgaacagc 540
acagacacgg acgcgcagag cttcacggtg aatactgaca acgtcatcga ctcggtgatc 600
tgcagctcct tgctcgcctc catctgcagc ttgctgtcga tcgcggtgga caggtacttc 660
accatcttct atgcgctcca gtaccatagc atcatgacgg tgcggcgggt ggcgatcacc 720
atcagtgcca tgtgggcggc ctgcacggtg tcgggcgtct tgttcatcat ttactcagac 780
agcagcgctg tcatcatctg cctcatcacc gtgttcttca ccatgctggc tctcatggcg 840
tctctctacg tccacatgtt cc 862
<210> 7
<211> 669
<212> DNA
<213> Hu sheep (hu sheet)
<400> 7
catctgcctc atcaccgtgt tcttcaccat gctggctctc atggcgtctc tctacgtcca 60
catgttcctc atggccaggc tccacattaa gaggatcgcg gtcctgccag gcaccggcgc 120
catccgccag ggcgccaaca tgaagggggc gatcacgctg accatcctga tcggggtctt 180
tgttgtctgc tgggccccct tcttcctgca cctgatattc tacatctcct gcccccagaa 240
cccctactgc gtgtgcttca tgtctcactt taacctgtat ctcatcctga tcatgtgtaa 300
ttctgtcatc gaccctctga tctacgccct gcggagccag gaactgagga aaaccttcaa 360
agagatcatt tgctgctctc ctctaggtgg cctctgtgat ttgtctagca gatattaaat 420
ggggacaaac tgcgatgcca aacacaagct taagagacct tctccttctc atgtgtacaa 480
tctgaacagt ctgtatgagc cacagctttt tcttctgtgt agggcatgga gtgaaaaatt 540
ctcttgtatc agttgaagtt tgtgattttt ttctgatgtg aagcagtgcc cagtcttgct 600
gtatttttaa tatcatgcta ctttctggct gtaaaatgtg aatcctcatc acaggttaca 660
ggcactatg 669
Claims (5)
1. The application of a reagent for detecting SNP molecular markers related to Hu sheep body size traits in screening the Hu sheep body size traits is characterized in that the sequence of the SNP molecular markers is shown as SEQ ID NO. 1, and the 732 th site is C or G; the Hu sheep body size character is the body height of the Hu sheep.
2. The use of claim 1, wherein the reagent is a primer pair for detecting the SNP molecular marker, the sequence of the upstream primer 1F of the primer pair is shown as SEQ ID NO. 2, and the sequence of the downstream primer 1R is shown as SEQ ID NO. 3.
3. The use according to claim 2, wherein the reagent is a kit comprising said primer pair.
4. A method for screening lake sheep body size characters comprises the following steps: extracting Hu sheep genome DNA, performing PCR amplification by using a primer pair, and detecting the 732 th site corresponding to SEQ ID NO. 1 in an amplification product so as to screen the Hu sheep body size character; the sequence of the upstream primer 1F of the primer pair is shown as SEQ ID NO. 2, and the sequence of the downstream primer 1R is shown as SEQ ID NO. 3; the Hu sheep body size character is the body height of the Hu sheep.
5. The method of claim 4, wherein the reaction sequence for PCR amplification is: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30sec, annealing at 58 ℃ for 30sec, extension at 72 ℃ for 1min, 35 cycles; extending for 10min at 72 ℃; the reaction system for PCR amplification is as follows:
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| CA2498264A1 (en) * | 2002-09-09 | 2004-05-13 | Nura, Inc. | G protein coupled receptors and uses thereof |
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| WO2016004387A1 (en) * | 2014-07-02 | 2016-01-07 | H. Lee Moffitt Cancer Center And Research Institute, Inc. | Gene expression signature for cancer prognosis |
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