Application of FABP3 gene molecular marker g.2596A > C in Hu sheep molecular marker assisted breeding
By screening the FABP3 gene g.2596A>C site using PCR amplification and sequencing technology, the problem of early identification of birth weight in Hu sheep breeding was solved, enabling early identification of high-quality breeding sheep and improving breeding efficiency and genetic improvement effects.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
- Filing Date
- 2026-04-13
- Publication Date
- 2026-05-12
AI Technical Summary
Traditional breeding methods for Hu sheep involve long selection cycles, are easily affected by the environment, have low early identification efficiency, and are difficult to effectively screen molecular markers closely related to birth weight, thus affecting breeding efficiency and genetic improvement.
PCR amplification and direct sequencing technologies were used to analyze the FABP3 gene polymorphism, and the g.2596A>C site was screened as a molecular marker. Individuals with birth weight advantage were identified by detecting the AA genotype, and corresponding reagents and kits were provided for assisted breeding of Hu sheep body length.
This enables early and accurate identification of birth weight traits in Hu sheep, improving breeding efficiency, shortening the breeding cycle, and enhancing the level of genetic improvement in the population.
Smart Images
Figure CN122012749A_ABST
Abstract
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 FABP3 gene molecular marker g.2596A>C in molecular marker-assisted breeding of Hu sheep. Background Technology
[0002] Hu sheep is an important local sheep breed in my country, characterized by high fertility, strong adaptability, and rapid early growth, making it highly valuable in meat sheep production and breed improvement. Birth weight is a crucial indicator for evaluating the early growth and development of lambs, closely related not only to their body condition and survival rate at birth but also influencing subsequent growth performance and the effectiveness of selective breeding. Traditional breeding methods rely primarily on phenotypic records and pedigree information for selection, typically requiring full expression of the target trait before a decision can be made. This approach suffers from drawbacks such as a long selection cycle, susceptibility to changes in feeding environment and management conditions, and low efficiency in early identification. Therefore, identifying molecular markers closely related to and stably detectable at birth weight in Hu sheep and using them for early assisted selection is of great significance for improving breeding efficiency, shortening the selection cycle, and enhancing the genetic improvement of the population.
[0003] Chinese invention patent CN111485026B discloses a SNP locus, its application, molecular marker, and primers related to sheep birth weight. This patent reveals that the G / A polymorphic locus at position 81799821 of sheep chromosome 8 is associated with birth weight and can be used for early marker-assisted selection of sheep birth weight. Additionally, Chinese invention patent CN114381531B discloses the SNP molecular marker g.43756G>A and its application in marker-assisted breeding of Hu sheep. This patent uses a locus related to body length traits in Hu sheep for the construction of primer pairs, kits, and screening methods, indicating that the use of specific SNP loci for marker-assisted breeding of growth-related traits in Hu sheep has a certain technical foundation. Given that birth weight is a quantitative trait influenced by multiple genes and genetic background, it is still necessary to further screen new candidate loci and establish more targeted and convenient molecular markers and their application schemes to meet the practical needs of marker-assisted breeding of Hu sheep.
[0004] FABP3 (Fatty Acid Binding Protein 3), also known as heart-type fatty acid binding protein (H-FABP), is a member of the fatty acid binding protein family. It is primarily expressed in cardiac and skeletal muscle, with lower expression in brown adipose tissue, the nervous system, and the placenta. It plays a crucial role in the transport and metabolism of long-chain fatty acids within cells, providing a large amount of energy for life activities. Studies have shown that knocking out the FABP3 gene in mice leads to disorders in plasma long-chain fatty acid metabolism. However, there are currently no studies on the relationship between the FABP3 gene and the body length trait of Hu sheep. Further research is needed to explore the application of the FABP3 gene in early screening for growth traits and molecular marker-assisted breeding in Hu sheep. Summary of the Invention
[0005] To investigate the correlation between FABP3 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 FABP3 gene polymorphism and comprehensively analyze the correlation between different genotypes at polymorphic sites and different growth traits.
[0006] One object of the present invention is to provide a reagent for detecting and screening molecular markers for body length in sheep, wherein the molecular marker site is g.2596A>C; wherein the SNP sites are all referenced to the FABP3 gene position on the sheep reference genome GeneID=100913166, NC_056055.1:236035335-236042973, i.e., 293A>C in the genome shown in SEQ ID NO:1; In the molecular marker g.2596A>C, the birth weight of the AA genotype was significantly higher than that of the CC genotype.
[0007] Preferably, the reagent includes primer pairs for identifying the molecular marker.
[0008] Preferably, the nucleotide sequences of the primer pair are as follows: F (SEQ ID NO:2): 5'- GCATCTTCTTCATTAGCAGGC-3'; R (SEQ ID NO:3): 5'-AGTGTTGGTCTCTGTTCGG-3'.
[0009] Furthermore, an object of the present invention is to provide a kit comprising the said reagent.
[0010] Furthermore, an object of the present invention is to provide the application of the reagent or kit described herein in the screening of body length-assisted breeding of Hu sheep.
[0011] Furthermore, an object of the present invention is to provide an application of an amplified product in the screening of body length-assisted breeding of Hu sheep, wherein the amplified product is obtained by amplification using the aforementioned reagent, and the nucleotide sequence is shown in SEQ ID NO:1, wherein position 293 is A or C.
[0012] 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 genomic DNA from Hu sheep, performing PCR amplification using primer pairs, and detecting molecular markers in the amplification products to screen for birth weight traits in Hu sheep. The molecular marker site is g.2596A>C in the FABP3 gene, i.e., 293A>C in the genome shown in SEQ ID NO:1. When the genotype is AA, the Hu sheep to be tested is determined to be a candidate individual with dominant birth weight; when the genotype is AC or CC, the Hu sheep to be tested is determined to be a candidate individual without dominant birth weight.
[0013] Preferably, the nucleotide sequences of the primer pair are as follows: F: 5'-GCATCTTCTTCATTAGCAGGC-3'; R: 5'-AGTGTTGGTCTCTGTTCGG-3'.
[0014] As a preferred option, the PCR reaction system consists of 25.0 μL of KOD One™ PCR Master Mix - Blue, 12.5 μL of KOD One™ PCR Master Mix - Blue, 0.2 μL each of forward and reverse primers, 1 μL of template, and 11.1 μL of ddH2O.
[0015] 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.
[0016] 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 FABP3 gene. It also comprehensively analyzes the correlation between different genotypes at polymorphic sites and different growth traits. The results show that g.2596A>C is significantly correlated with the birth weight of Hu sheep, and the AA genotype is significantly associated with a higher birth weight. P >0.05) is higher than that of the CC genotype. Attached Figure Description
[0017] Figure 1 The image shows a gel electrophoresis result of PCR amplification of SNPs in the FABP3 gene of Hu sheep. M is the DL2000 DNA Marker. Detailed Implementation
[0018] 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.
[0019] 1. Materials and Methods 1.1 Sample Collection Blood samples were collected from 172 Hu sheep (ewes) via jugular vein, 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, birth weight, weaning weight, six-month weight, and one-year weight.
[0020] 1.2 Primer Design The sheep FABP3 gene sequence (GeneID=100913166, NC_056055.1:236035335-236042973) 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.
[0021] Table 1 Primers for PCR amplification of the FABP3 gene in Hu sheep
[0022] 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, 68 °C extension for 30 s, for 34 cycles.
[0023] 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.
[0024] 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.
[0025] 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.
[0026] 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.
[0027] 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.
[0028] 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.
[0029] 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.
[0030] 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.
[0031] 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.
[0032] 2. Experimental Results 2.1 Results of PCR amplification of SNPs in the FABP3 gene of 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.
[0033] 2.2 Sequencing Results Analysis The sequencing results were compared using Mutation Surveyor 5.02 software. Two SNPs were found in the FABP3 gene, namely g.2429C>A and g.2596A>C, each with three genotypes: CC / CA / AA and AA / AC / CC, respectively.
[0034] Table 2. Location of mutation sites on amplified sequences
[0035] 2.3 Population genetic analysis of FABP3 gene Table 3 shows that the effective allele counts for g.2429C>A and g.2596A>C are 1.9182 and 1.9377, respectively, with average heterozygosities of 0.4787 and 0.4893, and Shannon information contents of 0.6717 and 0.6770, respectively. Both SNPs are moderately polymorphic (0.25 < PIC < 0.50) and are in Hardy-Weinberg equilibrium. P >0.05).
[0036] Table 3 Population genetic analysis of FABP3 gene SNPs
[0037] 2.4 Association analysis of FABP3 gene SNPs and growth traits Association analysis was performed on two SNPs of the FABP3 gene and the growth traits of Hu sheep using the method described in "1.4.2 Association Analysis". As shown in Table 4, g.2596A>C was significantly associated with the birth weight of Hu sheep, and the birth weight of the AA genotype was significantly higher than that of the CC genotype. P <0.05), there was no significant difference between the AC and CC genotypes ( P >0.05).
[0038] Table 4. Association analysis of FABP3 gene SNPs with growth traits in Hu sheep.
[0039] Note: Data with different superscript letters in the same column and at the same position show significant differences. P <0.05).
[0040] 3. Results Analysis: 3.1 Correlation analysis of SNP site g.2429C>A with growth traits of Hu sheep From the statistical significance level ( P Looking at the values, g.2429C>A 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, CA, AA) in body height, chest circumference, birth weight, weaning weight, six-month weight, and one-year weight were small and not statistically significant. Therefore, g.2429C>A is not a suitable target locus for marker-assisted breeding.
[0041] 3.2 Correlation analysis of SNP site g.2596A>C with growth traits of Hu sheep From the statistical significance level ( P (Value) Looking at the birth weight corresponding to g.2596A>C, P = 0.04, reaching a significant difference level. The AA genotype (2.98±0.40) was significantly higher than the CC genotype (2.82±0.34), while the AC genotype (2.92±0.2) was not significantly different from either the AA or CC genotypes.
[0042] 3.3 Summary of the application value of molecular breeding The locus g.2596A>C is the only SNP in this invention that is significantly associated with growth traits (birth weight), with individuals of the AA 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.
[0043] 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 birth weight in Hu sheep, characterized in that, The molecular marker site is g.2596A>C; the SNP sites are all referenced to the FABP3 gene position on the sheep reference genome GeneID=100913166, NC_056055.1:236035335-236042973, i.e., 293A>C in the genome shown in SEQ ID NO:1; In the molecular marker g.2596A>C, the birth weight of the AA genotype was significantly higher than that of the CC genotype.
2. The reagent for detecting and screening molecular markers for birth weight 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 and screening molecular markers for birth weight of Hu sheep according to claim 1, characterized in that, The nucleotide sequences of the primer pair are shown below: F: 5'-GCATCTTCTTCATTAGCAGGC-3'; R: 5'-AGTGTTGGTCTCTGTTCGG-3'.
4. A kit comprising the reagents of 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 screening for birth weight in assisted breeding of Hu sheep; when the genotype is AA, the Hu sheep to be tested is determined to be a candidate individual with birth weight dominance; when the genotype is AC or CC, the Hu sheep to be tested is determined to be a candidate individual without birth weight dominance.
6. The application of an amplification product in screening for birth weight-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 293 is A or C; when the genotype is AA, the Hu sheep to be tested is determined to be a candidate individual with birth weight dominance; when the genotype is AC or CC, the Hu sheep to be tested is determined to be a candidate individual without birth weight dominance.
7. A method for screening the birth weight 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 birth weight traits in Hu sheep; the molecular marker site is g.2596A>C in the FABP3 gene, i.e., 293A>C in the genome shown in SEQ ID NO:1; when the genotype is AA, the Hu sheep to be tested is determined to be a candidate individual with birth weight dominance; when the genotype is AC or CC, the Hu sheep to be tested is determined to be a candidate individual without birth weight dominance.
8. The method for screening the birth weight trait of Hu sheep according to claim 7, characterized in that, The nucleotide sequences of the primer pair are shown below: F: 5'-GCATCTTCTTCATTAGCAGGC-3'; R: 5'-AGTGTTGGTCTCTGTTCGG-3'.
9. The method for screening the birth weight trait of Hu sheep according to claim 7, characterized in that, The PCR reaction system was 25.0 μL, including: 12.5 μL KOD One™ PCR Master Mix - Blue, 0.2 μL each of forward and reverse primers, 1 μL template, and 11.1 μL ddH2O.
10. The method for screening the birth weight trait of Hu sheep according to claim 7, 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 ℃.