Application of molecular marker located in OAR1164254640.1 gene in Hu sheep molecular marker assisted breeding

Abstract

The invention belongs to the technical field of Hu sheep molecular marker-assisted breeding, and particularly relates to application of a molecular marker located in an OAR1164254640.1 gene in Hu sheep molecular marker-assisted breeding. According to the invention, Hu sheep is taken as a research object, PCR amplification, direct sequencing of products and sequence analysis are adopted, CADM2 gene polymorphism is analyzed, and correlation between different genotypes of polymorphic sites and different growth traits is comprehensively analyzed. An analysis result shows that downstream 192bp T-C and 235bp T-C of OAR1164254640.1 are remarkably related to birth weight, weaning weight and six-month-old body weight of Hu sheep, and birth weight, weaning weight and six-month-old body weight of CC genotype are remarkably higher than those of individuals of TT and TC genotypes.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular marker-assisted breeding of Hu sheep, and specifically relates to the application of 192bp T→C downstream of molecular marker OAR1_164254640.1 and 235bp T→C downstream of OAR1_164254640.1 in molecular marker-assisted breeding of Hu sheep. Background Art

[0002] Huyang is a rare sheep breed in my country. It is native to Taihu Lake. It is famous for its beautiful lamb skin patterns. It is a unique lamb skin sheep breed in my country and one of the few white lamb skin breeds in the world. It enjoys a high reputation in the international market and is known as a "soft gem". Huyang meat is delicious. Huyang is large in size and produces a high amount of meat. The meat is delicious and nutritious. Compared with other meats, Huyang meat has the advantages of high protein, low fat and low cholesterol. It is juicy and has little smell. However, there is still a big difference in the performance of Huyang meat from foreign meat sheep breeds such as Dorper sheep and German Merino sheep. Therefore, it is very necessary to select and breed Huyang for growth performance to improve its meat production performance.

[0003] The applicant has authorized Chinese invention patents (publication numbers: CN109251986A, CN109182553A, CN109251987A, CN109251985A, CN109182554A and CN109055579A) that disclose candidate functional genes and SNPs for body size traits of Hu sheep screened using GWAS technology, and through population verification of SNPs, obtained SNPs that can be used for molecular marker-assisted breeding, which can be used for molecular marker-assisted breeding of Hu sheep meat traits and early selection of individuals in the core group of Hu sheep meat lines.

[0004] The applicant has also applied for Chinese invention patent applications (CN114381531A, CN114438232A, CN114574596A, CN114480676A) to disclose that there are 15 SNPs in the intron 1 part of the CAPN6 gene, among which there are 4 SNPs sites significantly related to the weight and body size traits of Hu sheep: 1. g.43756G>A is significantly related to the body length of Hu sheep, and 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, AA type individuals is not significant (P>0.05). 2. g.43815G>A is significantly related to the height of Hu sheep, and the height of 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). 3. g.43851G>A was significantly correlated with the body length of Hu sheep. The body length of AA type individuals was significantly higher than that of GA type individuals (P<0.05), and there was no significant difference between GG type individuals and GA and AA type individuals (P>0.05). 4. g.43917A>G was significantly correlated with the body length and daily weight gain of Hu sheep. The body length of AA and GG type individuals was significantly higher than that of AG type individuals (P<0.05), and there was no significant difference between AA and GG (P>0.05). The daily weight gain of GG type individuals from weaning to six months of age was significantly higher than that of AA type individuals, and there was no significant difference between AG type individuals and AA and GG.

[0005] In addition, the applicant has also applied for Chinese invention patent applications (CN117757958A, CN117701736A, CN117844943A, CN117683908A) to disclose that there are 4 SNPs in the 2nd part of the intron of the ARHGAP24 gene, among which there are 4 SNPs sites significantly associated with the daily weight gain and / or annual weight of Hu sheep from six months to one year old: 1. g.14979C>T is significantly associated with the daily weight gain and annual weight of Hu sheep from six months to one year old, and the daily weight and annual weight of individuals with TT genotype from six months to one year old are significantly higher than those with CT and CC genotypes. 2. g.15042C>T is significantly associated with the daily weight gain and annual weight of Hu sheep from six months to one year old, and the daily weight gain and annual weight of individuals with TT genotype from six months to one year old are significantly higher than those with CT and CC genotypes. 3. g.15255T>C was significantly correlated with the daily weight gain and annual weight of Hu sheep from six months to one year old. The daily weight gain and annual weight of CC genotype individuals were significantly higher than those of TC and TT genotype individuals. 4. g.15599G>T was significantly correlated with the annual weight of Hu sheep. The TT genotype individuals were significantly higher than GT and GG individuals.

[0006] CADM (Cell adhesion molecule) is a family of cell adhesion molecules composed of proteins, whose functions include maintaining cell polarity and tumor suppression. Low expression of CADM2 gene can be observed in several cancers including hepatocellular carcinoma (HCC). CADM2 gene is inhibited by DNA promoter methylation and / or loss of heterozygosity in human renal cell carcinoma. A genome-wide association study using microsatellites in the Japanese population (561 cases and 561 controls) identified CADM2 as a candidate gene for psoriasis. Some scholars have identified CADM2 as a powerful regulator of whole-body energy homeostasis. Reducing CADM2 expression can reverse a variety of metabolic syndrome-related symptoms including obesity, insulin resistance, and impaired glucose homeostasis. Studies have found that CADM2 gene copy number variation is associated with growth traits of five goat breeds in China, and the expression level of CADM2 gene in different tissues of goats is different. Summary of the invention

[0007] In order to study the correlation between CADM2 gene polymorphism and growth traits of Hu sheep and obtain genetic markers related to growth traits, the present invention uses Hu sheep as the research object, adopts PCR amplification, direct product sequencing, and sequence analysis methods to analyze CADM2 gene polymorphism, and comprehensively analyzes the correlation between different genotypes of polymorphic sites and different growth traits.

[0008] One object of the present invention is to provide a reagent for detecting molecular markers that are extremely significantly correlated with the birth weight, weaning weight, and six-month-old weight of Hu sheep, wherein the molecular markers include molecular marker I and molecular marker II, and molecular marker I and molecular marker II are interlocking genes; Molecular marker I was located at 192bp T→C downstream of OAR1_164254640.1, located at the 330th position of SEQ ID NO:1, which was C or T; among them: the birth weight, weaning weight, and six-month-old weight of the CC genotype were significantly higher than those of the TT and TC genotypes; Molecular marker II was located at 235bp T→C downstream of OAR1_164254640.1, located at the 373rd position of SEQ ID NO:1, which was C or T; among them: the birth weight, weaning weight and six-month-old weight of CC genotype were significantly higher than those of TT and TC genotype individuals.

[0009] Furthermore, an object of the present invention is to provide a primer pair for identifying the molecular marker.

[0010] Preferably, the nucleotide sequences of the primer pair are shown in SEQ ID NO:2 and SEQ ID NO:3.

[0011] Furthermore, an object of the present invention is to provide a kit comprising the reagent or the primer pair.

[0012] Furthermore, an object of the present invention is to provide the use of the reagent or the primer pair or the kit in screening the birth weight, weaning weight and six-month-old weight of Hu sheep for assisted breeding.

[0013] Furthermore, an object of the present invention is to provide an application of an amplification product in screening the birth weight, weaning weight and six-month-old weight of Hu sheep for assisted breeding. The amplification product is obtained by amplification using the reagent described, and the nucleotide sequence is shown in SEQ ID NO: 1.

[0014] Furthermore, an object of the present invention is to provide a method for screening birth weight, weaning weight, and six-month-old weight traits of Hu sheep, the method comprising the following steps: extracting Hu sheep genomic DNA, performing PCR amplification using primer pairs, and detecting molecular markers in the amplified products, thereby screening Hu sheep weight traits; the molecular markers include molecular markers I and molecular markers II, and molecular markers I and molecular markers II are interlocking genes; Molecular marker I was located at 192bp T→C downstream of OAR1_164254640.1, located at the 330th position of SEQ ID NO:1, which was C or T; among them: the birth weight, weaning weight, and six-month-old weight of the CC genotype were significantly higher than those of the TT and TC genotypes; Molecular marker II was located at 235bp T→C downstream of OAR1_164254640.1, located at the 373rd position of SEQ ID NO:1, which was C or T; among them: the birth weight, weaning weight and six-month-old weight of CC genotype were significantly higher than those of TT and TC genotype individuals.

[0015] Preferably, the nucleotide sequences of the primer pair are shown in SEQ ID NO:2 and SEQ ID NO:3.

[0016] Preferably, the PCR reaction system is 25.0 μL, KOD One TM PCR Master Mix 12.5μL, upstream and downstream primers 0.2μL each, ddH2O 11.1μL.

[0017] Preferably, the PCR reaction procedure is: denaturation at 98°C for 10 s, annealing at 53°C for 30 s, and extension at 68°C for 30 s, for 34 cycles, and storage at 4°C after the PCR is completed.

[0018] The present invention uses Hu sheep as the research object, adopts PCR amplification, direct product sequencing, and sequence analysis methods to analyze CADM2 gene polymorphism, and comprehensively analyzes the correlation between different genotypes of polymorphic sites and different growth traits. The analysis results show that the 192bp T→C downstream of OAR1_164254640.1 is extremely significantly correlated with the birth weight, weaning weight, and six-month-old weight of Hu sheep, and the birth weight, weaning weight, and six-month-old weight of CC genotype are extremely significantly higher than those of TT and TC genotype individuals. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 PCR amplification of SNPs detection primers for CAPN6 gene of Hu sheep, M: DL2000.

[0020] Figure 2 Figure 2 is the nucleotide sequence diagram of the amplified product of the present invention. DETAILED DESCRIPTION

[0021] The following is a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] 1 Materials and methods 1.1 Test materials The experimental animals were all from Hangzhou Pangda Agricultural Development Co., Ltd., totaling 203 animals, and the selected individuals were healthy and disease-free.

[0023] 1.2 Blood collection and determination of indicators 5 mL of jugular vein blood was collected from all sheep, placed in EDTA anticoagulant tubes, and stored at -20°C. Growth trait indicators measured included: body height, body length, and chest circumference. Other indicators were provided by the sheep farm.

[0024] 1.3 DNA extraction and detection Blood DNA was extracted using a blood genomic DNA extraction kit (Tiangen, Beijing), and DNA concentration and purity were tested using BioTek® EPOCH (USA). The sample DNA concentration was ensured to be greater than 50 ng / μL and OD260 / OD280 was between 1.8 and 2.0.

[0025] 1.4 Primer design and PCR amplification Primers were designed using DNAMAN8.0 based on the sheep CADM2 gene sequence published by NCBI (Gene ID: 101120371). The sequences of the primers are shown in Table 1.

[0026] Table 1 PCR primers for CADM2 gene of Hu sheep

[0027] 1.5 PCR amplification The PCR reaction program was: denaturation at 98°C for 10 s, annealing at 53°C for 30 s, and extension at 68°C for 30 s for 34 cycles. After the PCR was completed, the mixture was stored at 4°C.

[0028] Table 2 PCR amplification system

[0029] 1.6 PCR product sequencing and sequence analysis Each sample was amplified using the primers listed in Table 1, and the amplified products were sequenced by Anhui General Biotechnology. The sequencing peaks of each individual were analyzed using Mutation Surveyor 5.02 (Softgenetics, USA) software to determine the mutation position and mutation mode of each individual.

[0030] 1.7 Data Statistics and Analysis Little Programe was used to calculate the PIC value, and PopGen 32 software was used to calculate the site heterozygosity, Shannon information content, gene frequency, and genotype frequency of each SNP.

[0031] Polymorphism information content analysis PIC (Polymorphism information content) indicates the possibility that an offspring obtains a certain allele from the same allele of its parents. It is an ideal indicator for measuring the polymorphism of allele fragments. The calculation formula is:

[0032] Pi and Pj are the frequencies of the ith and jth alleles, respectively; n is the number of alleles Site heterozygosity He (heterozygosity) refers to the average frequency of heterozygous individuals at each locus. Heterozygosity can objectively reflect the level of genetic variation in a population. The larger the average heterozygosity value, the greater the genetic difference within the population, the richer the genetic diversity, the greater the genetic potential, and the better the effect of animal genetic breeding research; the lower the value, the higher the genetic consistency, indicating that the genetic variation within the population is small and the genetic potential is also small. The calculation formula is:

[0033] pi represents the frequency of the ith allele Shannon Information Content SIC (shannon information content), calculated as: SIC = -ClogPi Where: Pi is the frequency of the i-th allele in the population, and C is a constant.

[0034] Gene frequency and genotype frequency ①Gene frequency = number of individuals with the same genotype / number of individuals in the population × 100% ②Gene frequency = homozygous genotype frequency + 1 / 2 × heterozygous genotype frequency 1.7.1 Correlation Analysis The general linear model (GLM; SPSS 20) was used to explore SNPs associated with the weight and body size traits of the core population of the new meat-producing population of Hu sheep.

[0035] Since all individuals analyzed were female sheep from the same farm, with the same rearing environment and management conditions, farm effects and sex effects were not included in data modeling.

[0036] The specific model is: Y=Xβ+e Among them, Y is the phenotypic value vector of size and weight traits of the core population of the new group of Hu sheep for meat; β: fixed effect vector of phenotype mean, SNP, etc.; e: residual effect vector; X is the incidence matrix of β.

[0037] When Y is the vector of the initial phenotypic values ​​of the core group of the new group for Hu sheep meat, the analysis is based on the model Y = Xβ + Sα + e.

[0038] Where Y: phenotypic value vector of size trait of core population of new group for Hu sheep meat; α: fixed effect vector of the number of siblings; β: SNP effect vector; e: residual effect vector; X and S are the incidence matrices of β and α respectively.

[0039] 2 Test results 2.1 PCR amplification results of SNPs detection of CADM2 gene in Hu sheep The PCR products of each primer are bright, with single bands and no non-specific amplification ( Figure 1 ), the actual PCR product is consistent with the expected PCR amplification product size, and subsequent PCR-product direct sequencing experiments can be performed.

[0040] 2.2 The nucleotide sequence of the PCR amplified product of the Hu sheep CADM2 gene is as follows Figure 2The mutation analysis results are shown in Table 3, where the red parts are SNP sites.

[0041] Table 3 SNPs sites and mutation types and patterns of CADM2 gene in Hu sheep

[0042] 2.3 Population genetics analysis of the CADM2 gene of Hu sheep is shown in Tables 4 and 5.

[0043] Table 4 Genetic parameters of SNPs loci of CADM2 gene in Hu sheep

[0044] Table 5 Population genetics analysis of SNPs loci in Hu sheep

[0045] Polymorphic Information Content (PIC) is used to determine and analyze the information content expressed by a genetic marker. PIC>0.5 is a highly polymorphic site, 0.25<PIC<0.5 is a moderately polymorphic site, and PIC<0.25 is a low polymorphic site. The larger the PIC value, the greater the number of effective alleles and heterozygosity. The four SNP sites in this population are all low polymorphic.

[0046] 2.4 Association analysis of growth traits of Hu sheep with CADM2 gene The GLM model in “1.7 Data Statistics and Analysis - Association Analysis” was used to conduct association analysis between the four SNPs of the CADM2 gene and the growth traits of Hu sheep (Table 6).

[0047] Table 6 Association analysis between CADM2 gene SNPs and growth traits of Hu sheep

[0048] Note: If there is no letter after the same column of data, or if there are the same letters, it means that the difference is not significant ( P＞ 0.05), different small letters indicate significant differences ( P＜ 0.05). Different capital letters indicate extremely significant differences ( P <0.01).

[0049] The analysis results showed that the 192bp T→C downstream of OAR1_164254640.1 was significantly correlated with the birth weight, weaning weight and six-month-old body weight of Hu sheep. The birth weight, weaning weight and six-month-old body weight of CC genotype were significantly higher than those of TT and TC genotype individuals.

[0050] The 235bp T→C downstream of OAR1_164254640.1 was extremely significantly correlated with the birth weight, weaning weight and six-month-old body weight of Hu sheep. The birth weight, weaning weight and six-month-old body weight of CC genotype were extremely significantly higher than those of TT and TC genotype individuals.

[0051] The above is a description of the embodiments of the present invention. Through the above description of the disclosed embodiments, professionals and technicians in the field can implement or use the present invention. Various modifications to these embodiments will be apparent to professionals and technicians in the field. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown in this article, but will conform to the widest range consistent with the principles and novelties disclosed herein.

Claims

1. A reagent for detecting molecular markers that are significantly correlated with birth weight, weaning weight, and six-month-old weight of Hu sheep, characterized in that: The molecular marker includes molecular marker I and molecular marker II, and molecular marker I and molecular marker II are interlocking genes; Molecular marker I was located at 192bp T→C downstream of OAR1_164254640.1, located at the 330th position of SEQ ID NO:1, which was C or T; among them: the birth weight, weaning weight, and six-month-old weight of the CC genotype were significantly higher than those of the TT and TC genotypes; Molecular marker II was located at 235bp T→C downstream of OAR1_164254640.1, located at the 373rd position of SEQ ID NO:1, which was C or T; among them: the birth weight, weaning weight and six-month-old weight of CC genotype were significantly higher than those of TT and TC genotype individuals.

2. The reagent according to claim 1, characterized in that The reagents include a primer pair.

3. The reagent according to claim 1, characterized in that The nucleotide sequences of the primer pair are shown in SEQ ID NO:2 and SEQ ID NO:

3.

4. An amplification product, which is obtained by amplification using the reagent according to claim 3, and the nucleotide sequence is shown in SEQ ID NO:

1.

5. A kit comprising the reagent according to any one of claims 1 to 3.

6. Use of the reagent described in any one of claims 1 to 3, the amplification product described in claim 4, or the kit described in claim 5 in screening Hu sheep for birth weight, weaning weight, and six-month-old weight to assist breeding.

7. A method for screening birth weight, weaning weight, and six-month-old weight traits, characterized in that: The method comprises the following steps: extracting Hu sheep genomic DNA, performing PCR amplification using primer pairs, and detecting molecular markers in the amplified products, thereby screening the weight traits of Hu sheep; the molecular markers include molecular marker I and molecular marker II, and molecular marker I and molecular marker II are interlocking genes; Molecular marker I was located at 192bp T→C downstream of OAR1_164254640.1, located at the 330th position of SEQ ID NO:1, which was C or T; among them: the birth weight, weaning weight, and six-month-old weight of the CC genotype were significantly higher than those of the TT and TC genotypes; Molecular marker II was located at 235bp T→C downstream of OAR1_164254640.1, located at the 373rd position of SEQ ID NO:1, which was C or T; among them: the birth weight, weaning weight and six-month-old weight of CC genotype were significantly higher than those of TT and TC genotype individuals.

8. The method according to claim 7, characterized in that The nucleotide sequences of the primer pair are shown in SEQ ID NO:2 and SEQ ID NO:

3.

9. The method of claim 7, characterized in that: The PCR reaction system was 25.0 μL, KOD One TM PCR MasterMix 12.5μL, upstream and downstream primers 0.2μL each, ddH2O 11.1μL.

10. The method according to claim 8 or 9, characterized in that The PCR reaction program was: denaturation at 98°C for 10 s, annealing at 53°C for 30 s, and extension at 68°C for 30 s for 34 cycles. After the PCR was completed, the mixture was stored at 4°C.

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