Application of CNGA3 gene molecular marker g.39045A > C 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 CNGA3 gene molecular marker g.39045A > C 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, CNGA3 gene polymorphism is analyzed, and correlation between different genotypes of polymorphic sites and different growth traits is comprehensively analyzed. An analysis result shows that g.39045A > C is remarkably related to the body length of the Hu sheep, and the body length of the AA genotype and the body length of the CC genotype are remarkably higher than the body length of the AC genotype (P > 0.05).

Description

Technical Field

[0001] This invention belongs to the field of molecular marker-assisted breeding technology for Hu sheep, specifically, it relates to the application of the CNGA3 gene molecular marker g.39045A>C in molecular marker-assisted breeding of Hu sheep. Background Technology

[0002] The Hu sheep is an important local sheep breed in my country, characterized by good reproductive performance, rapid growth, and suitability for indoor feeding, making it highly valuable in meat sheep production. Current Hu sheep breeding practices primarily rely on phenotypic indicators such as body weight, height, body length, and chest circumference for selection. However, these methods typically suffer from long testing cycles, limited early selection efficiency, and susceptibility to environmental factors. With the development of molecular marker-assisted breeding technology, identifying stable and detectable single nucleotide polymorphisms (SNPs) from candidate genes or genomic loci related to growth traits and using them for early selection has become an important technological direction for improving the efficiency of Hu sheep breeding.

[0003] Reicher S et al. published in Genomics The paper "A mutation in gene CNGA3 is associated with day blindness in sheep" reports an association between CNGA3 gene mutations and day blindness phenotype in sheep, indicating that the CNGA3 gene has a clear genetic basis for variation and is detectable in sheep. While this paper confirms that the CNGA3 gene can be considered a candidate gene for important sheep traits, its focus is on mutations related to visual dysfunction, and it does not address the correspondence between specific loci in the CNGA3 gene and the body length trait of Hu sheep, nor does it establish a molecular marker detection system for Hu sheep growth screening. Therefore, further research is needed on the relationship between the CNGA3 gene and the body length trait of Hu sheep to obtain specific loci and detection methods that can be used for early screening of growth traits and molecular marker-assisted breeding in Hu sheep. Summary of the Invention

[0004] To investigate the correlation between CNGA3 gene polymorphism and growth traits in Hu sheep and to obtain genetic markers associated with these traits, this invention uses Hu sheep as the research subject. It employs PCR amplification, direct sequencing of the products, and sequence analysis to analyze CNGA3 gene polymorphism and comprehensively analyze the correlation between different genotypes at polymorphic sites and different growth traits.

[0005] One object of the present invention is to provide a reagent for detecting and screening molecular markers for body length in Hu sheep, wherein the molecular marker site is g.39045A>C; wherein the SNP sites are all referenced to the CNGA3 gene position on the Mianyang reference genome GeneID=100233164, NC_056056.1:c102877356-102835910, i.e., 509A>C in the genome shown in SEQ ID NO:1; In the molecular marker g.39045A>C, the body length of the AA and CC genotypes was significantly longer than that of the AC genotype.

[0006] Preferably, the reagent includes primer pairs for identifying the molecular marker.

[0007] Preferably, the nucleotide sequences of the primer pair are as follows: F (SEQ ID NO:2): 5'-GGAAGCACTACACCAAGACC-3'; R (SEQ ID NO:3): 5'-CCTCCTTCTCATCCACTGTC-3'.

[0008] Furthermore, an object of the present invention is to provide a kit comprising the said reagent.

[0009] Furthermore, one object of the present invention is to provide the application of the reagent or the kit in screening for body length-assisted breeding of Hu sheep; when the genotype is AA or CC, the Hu sheep to be tested is determined to be a candidate individual with body length dominance; when the genotype is AC, the Hu sheep to be tested is determined to be a candidate individual without body length dominance.

[0010] Furthermore, an objective of this invention is to provide an application of an amplified product in screening for body length-assisted breeding of Hu sheep. The amplified product is obtained by amplification using the aforementioned reagent, and its nucleotide sequence is shown in SEQ ID NO:1, wherein position 509 is A or C. When the genotype is AA or CC, the Hu sheep to be tested is determined to be a candidate individual with body length dominance; when the genotype is AC, the Hu sheep to be tested is determined to be a candidate individual without body length dominance.

[0011] Furthermore, one objective of this invention is to provide a method for screening the body length trait of Hu sheep. This method includes the following steps: extracting Hu sheep genomic DNA, performing PCR amplification using primer pairs, and detecting molecular markers in the amplification products to screen for the body length trait in Hu sheep. The molecular marker site is g.39045A>C in the CNGA3 gene, corresponding to site 509 of SEQ ID NO:1, which is either A or C. When the genotype is AA or CC, the Hu sheep to be tested is determined to be a candidate individual with a dominant body length; when the genotype is AC, the Hu sheep to be tested is determined to be a candidate individual without a dominant body length.

[0012] Preferably, the nucleotide sequences of the primer pair are as follows: F: 5'-GGAAGCACTACACCAAGACC-3'; R: 5'-CCTCCTTCTCATCCACTGTC-3'.

[0013] Preferably, the PCR reaction system is 25.0 μL, containing KOD One. TM PCR Master Mix - Blue 12.5 μL, forward and reverse primers 0.2 μL each, template 1 μL, ddH2O 11.1 μL.

[0014] As a preferred option, the PCR reaction program is as follows: denaturation at 98 ℃ for 10 s, annealing at 61 ℃ for 30 s, extension at 68 ℃ for 30 s, for 34 cycles, and storage at 4 ℃ after PCR.

[0015] This invention uses Hu sheep as the research subject and employs PCR amplification, direct sequencing of the products, and sequence analysis to analyze the polymorphism of the CNGA3 gene. It also comprehensively analyzes the correlation between different genotypes at polymorphic sites and different growth traits. The results show that g.39045A>C is significantly correlated with the body length of Hu sheep, and the body length of the AA genotype and CC genotype is significantly different. P >0.05) is higher than that of the AC genotype. Attached Figure Description

[0016] Figure 1 This is a gel electrophoresis image of PCR amplification of SNPs in the CNGA3 gene of Hu sheep, with M being the DL2000 DNA Marker. Detailed Implementation

[0017] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the protection scope of the present invention.

[0018] 1. Materials and Methods 1.1 Sample Collection Blood samples were collected from 130 Hu sheep (ewes), anticoagulated with EDTA, and stored at -20 ℃. Samples were collected from Hangzhou Pangda Agricultural Development Co., Ltd. Growth traits were measured including height, chest circumference, body length, birth weight, weaning weight, six-month weight, and one-year weight.

[0019] 1.2 Primer Design The sheep CNGA3 gene sequence (GeneID=100233164, NC_056056.1:c102877356-102835910) was retrieved from the NCBI Gene database. Primers were designed using DNAMAN 8.0, and the primer sequences are shown in Table 1. The primers were then sent to Hangzhou Youkang Biotechnology Co., Ltd. for synthesis.

[0020] Table 1 Primers for PCR amplification of CNGA3 gene in Hu sheep

[0021] 1.3 PCR amplification and sequence analysis The PCR reaction mixture consisted of 25 μL of KOD One™ PCR Master Mix-Blue (TOYOBO), 12.5 μL of forward and reverse primers, 0.2 μL each of forward and reverse primers, 1 μL of template (whole blood), and 11.1 μL of ddH2O. The reaction conditions were: 98 °C denaturation for 10 s, 61 °C annealing for 30 s, and 68 °C extension for 30 s, for 34 cycles. After PCR, the mixture was stored at 4 °C.

[0022] The amplified products were sent to Anhui General Biotechnology for Sanger sequencing. Mutation Surveyor 5.02 (Softgenetics, USA) software was used to analyze the sequencing peaks of each individual to determine the mutation location and mutation mode.

[0023] 1.4 Data Statistics and Analysis 1.4.1 Polymorphic Information Content Analysis (PIC) The Little Programe software was used to calculate the PIC value, and the PopGen 32 software was used to calculate the effective alleles, average heterozygosity, gene frequency, and genotype frequency of SNPs.

[0024] Polymorphism information content (PIC) analysis is used to determine and analyze the information content expressed by a genetic marker. It represents the probability that a progeny inherits a particular allele from the same allele as its parent, and is an ideal indicator for measuring allele polymorphism. A PIC > 0.5 indicates a highly polymorphic site, 0.25 < PIC < 0.5 indicates a moderately polymorphic site, and PIC < 0.25 indicates a lowly polymorphic site. A higher PIC value indicates a greater number of valid alleles and higher heterozygosity.

[0025] The calculation formula is: , P i and P j , i and j are the frequencies of the i-th and j-th alleles, respectively; n is the number of alleles.

[0026] Heterozygosity (He) refers to the average frequency of heterozygous individuals at each locus. Heterozygosity objectively reflects the level of genetic variation in a population. A higher average heterozygosity value indicates greater genetic diversity and higher genetic potential within the population, making it more effective for animal genetic breeding research. Conversely, a lower value indicates higher genetic homogeneity, suggesting less genetic variation and lower genetic potential within the population. The calculation formula is: , p i This represents the frequency of the i-th allele.

[0027] Shannon's information content SIC (Shannon Information Content) is calculated using the following formula: SIC = -C * logP i Where: P i Let be the frequency of the i-th allele in the population, and C be a constant.

[0028] Gene frequency and genotype frequency ① Genotype frequency = (Number of individuals with a genotype / Total number of individuals in the population) × 100% ② Gene frequency = homozygous genotype frequency + 1 / 2 × heterozygous genotype frequency 1.4.2 Correlation Analysis We used a general linear model (GLM; SPSS) to mine SNPs associated with the weight and body size traits of Hu sheep.

[0029] Since all individuals analyzed came from the same farm, had the same feeding environment and management conditions, and were all ewes, field effects and sex effects were not included in the data modeling.

[0030] The specific model is: Y = Xβ + e Wherein, Y: the phenotypic value vector of body size and weight traits of Hu sheep; β: Phenotypic mean, SNP, and other fixed effects vector; e: Residual effect vector; X is the incidence matrix of β.

[0031] 2. Experimental Results 2.1 PCR amplification results of CNGA3 gene SNPs in Hu sheep The PCR product was bright, with a single band and no nonspecific amplification. Figure 1 The actual PCR product size is consistent with the expected PCR amplification product, and subsequent PCR products can be directly sequenced.

[0032] 2.2 Sequencing Results Analysis The sequencing results were compared using Mutation Surveyor 5.02 software. Two SNPs were found in the CNGA3 gene: g.38931C>T and g.39045A>C. Both of them have three genotypes: CC / CT / TT and AA / AC / CC, respectively.

[0033] Table 2. Location of mutation sites on amplified sequences

[0034] 2.3 Population genetic analysis of CNGA3 gene Table 3 shows that the effective alleles were 1.4307 and 1.4208, respectively, with average heterozygosities of 0.3011 and 0.2523, and Shannon information contents of 0.4783 and 0.4726, respectively. Both SNPs were moderately polymorphic (0.25 < PIC < 0.50) and were in Hardy-Weinberg disequilibrium. P <0.05).

[0035] Table 3 Population genetic analysis of CNGA3 gene SNPs

[0036] 2.4 Association analysis of CNGA3 gene SNPs and growth traits Using the association analysis in section 1.4.2, the SNP g.39045A>C of the CNGA3 gene was significantly correlated with the body length of Hu sheep, as shown in Table 4. The body length of the AA genotype and the CC genotype were significantly different.P >0.05) was higher than that of the AC genotype, while there was no significant difference between the AA and CC genotypes. P >0.05).

[0037] Table 4 Association analysis of CNGA3 gene SNPs with growth traits in Hu sheep

[0038] Note: Data with different superscript letters in the same column and at the same position show significant differences. P <0.05).

[0039] 3. Results Analysis: 3.1 Correlation analysis of SNP site g.38931C>T with growth traits of Hu sheep From the statistical significance level ( P (Value) Looking at the values, g.39045A>C corresponds to the following among various trait indicators: P The values ​​were all greater than 0.05, indicating that this locus had no significant impact on the growth traits of Hu sheep in this sample population. The differences among genotypes (CC, CT, TT) in body height, chest circumference, body length, birth weight, weaning weight, six-month weight, and one-year weight were small and not statistically significant. Therefore, g.38931C>T is not a suitable target locus for marker-assisted breeding.

[0040] 3.2 Correlation analysis of SNP site g.39045A>C with growth traits of Hu sheep From the statistical significance level ( P (Value) , g.39045A>C body length (cm) corresponding to P = 0.02, reaching a statistically significant level. The AA genotype (86.66±5.37) and CC genotype (88.17±6.3) were significantly higher than the AC genotype (82.27±6.33). The letter labels (a, b) also indicate that AA / CC>AC, indicating a statistically significant stratification.

[0041] 3.3 Summary of the application value of molecular breeding Locus g.39045A>C is the only SNP in this invention significantly associated with growth traits (body length), with individuals of the AA / CC genotype exhibiting superior growth potential. This locus has moderate polymorphism (PIC = 0.2523), possessing population screening power and can serve as a molecular marker for core breeding populations. It can be used to develop commercial molecular breeding tools, such as SNP detection kits, to guide farms in early identification of high-quality breeding sheep.

[0042] The foregoing description of embodiments of the present invention, through which those skilled in the art are able to implement or use the present invention, will be readily apparent to those skilled in the art. 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. Therefore, the present invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novelty disclosed herein.

Claims

1. A reagent for detecting and screening molecular markers for body length in Hu sheep, characterized in that, The molecular marker site is g.39045A>C; the SNP sites are all referenced to the CNGA3 gene position on the Mianyang reference genome GeneID=100233164, NC_056056.1:c102877356-102835910, i.e., 509 A>C in the genome shown in SEQ ID NO:1; In the molecular marker g.39045A>C, the body length of the AA and CC genotypes was significantly longer than that of the AC genotype.

2. The reagent for detecting molecular markers used to screen for body length of Hu sheep according to claim 1, characterized in that, The reagents include primer pairs for identifying the molecular markers.

3. The reagent for detecting molecular markers used to screen for body length of Hu sheep according to claim 1, characterized in that, The nucleotide sequences of the primer pair are shown below: F: 5'-GGAAGCACTACACCAAGACC-3'; R: 5'-CCTCCTTCTCATCCACTGTC-3'.

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

5. The application of the reagent according to any one of claims 1-3 or the kit according to claim 4 in the screening of body length-assisted breeding of Hu sheep; the body length of AA genotype and CC genotype is significantly higher than that of AC genotype.

6. The application of an amplification product in screening for body length-assisted breeding of Hu sheep, wherein the amplification product is obtained by amplification using the reagents described in any one of claims 1-3 or the kit described in claim 4, and its nucleotide sequence is shown in SEQ ID NO:1, wherein position 509 is A or C, and the body length of the AA genotype and CC genotype is significantly higher than that of the AC genotype.

7. A method for screening the body length trait of Hu sheep, characterized in that, The method includes the following steps: extracting genomic DNA from Hu sheep, performing PCR amplification using primer pairs, and detecting molecular markers in the amplification products to screen for Hu sheep body length traits; the molecular marker site is g.39045A>C in the CNGA3 gene, corresponding to the 509th site of SEQ ID NO:1, which is A or C. When the genotype is AA or CC, the Hu sheep to be tested is determined to be a candidate individual with body length dominance; when the genotype is AC, the Hu sheep to be tested is determined to be a candidate individual without body length dominance.

8. The method for screening the body length trait of Hu sheep according to claim 6, characterized in that, The nucleotide sequences of the primer pair are shown below: F: 5'-GGAAGCACTACACCAAGACC-3'; R: 5'-CCTCCTTCTCATCCACTGTC-3'.

9. The method for screening the body length trait of Hu sheep according to claim 6, characterized in that, The PCR reaction system consisted of 25.0 μL of KOD One. TM PCR Master Mix - Blue 12.5 μL, forward and reverse primers 0.2 μL each, template 1 μL, ddH2O 11.1 μL.

10. The method for screening the body length trait of Hu sheep according to claim 6, characterized in that, The PCR reaction program was as follows: denaturation at 98℃ for 10 s, annealing at 61℃ for 30 s, extension at 68℃ for 30 s, for 34 cycles. After PCR, the product was stored at 4℃.

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