A SNP marker related to growth traits of chongbai goats and application thereof

Abstract

The application discloses a SNP marker related to growth traits of Chongbai goats, comprising a SNP_1 marker, a SNP_2 marker, a SNP_3 marker and a SNP_4 marker, the SNP_1 marker is located at the 373396th base on a chromosome 17 of a goat reference genome, the base is C or T, the SNP_2 marker is located at the 372464th base on the chromosome 17 of the goat reference genome, the base is C or T, the SNP_3 marker is located at the 372892th base on the chromosome 17 of the goat reference genome, the base is A or G, and the SNP_4 marker is located at the 372853th base on the chromosome 17 of the goat reference genome, the base is A or G. Compared with the prior art, the molecular marker assisted selection can effectively break through the limitation of traditional breeding, and significantly improve the accuracy and efficiency of growth performance selection of the Chongbai goats.

Description

Technical Field

[0001] This invention belongs to the field of animal molecular breeding technology, specifically relating to a SNP marker related to the growth traits of Chongming white goats and its application. Background Technology

[0002] The purpose of meat production in livestock and poultry is to produce meat. The larger the individual's weight, the more meat is produced. Therefore, growth traits are key indicators for evaluating the productivity and economic benefits of meat livestock and poultry (including goats). The phenotype of growth traits is controlled by genes. Therefore, molecular marker-assisted selection based on the genes controlling growth traits has the advantages of being simple to operate and allowing for early selection, which can effectively shorten the breeding cycle and improve breeding efficiency.

[0003] The Chongming Goat of Shanghai is one of China's superior local goat breeds and a national geographical indication product. Originating in Chongming District, Shanghai, its breeding history dates back to the Ming Dynasty, over 600 years ago. This breed is renowned for its tender meat, mild muttony flavor, and strong adaptability, making it a typical representative of meat goats in the hot and humid climate of southern my country. However, compared to domestic and international meat goat breeds (such as the Boer goat), its growth rate is relatively inferior. Therefore, by utilizing modern selective breeding techniques and incorporating molecular markers with significant effects into marker-assisted selection and genomic selection, the genetic improvement of growth traits in Chongming Goats can be significantly enhanced, thereby increasing the meat yield of offspring goats. This is of great significance for promoting the efficient development of my country's local goat industry.

[0004] Currently, there are no reports of single nucleotide polymorphism (SNP) molecular genetic markers significantly associated with growth traits in Chongming white goats. Based on this, our team previously used our self-developed 50K liquid-phase chip (Chongxin-1) to perform genotyping on over 800 core Chongming white goats in Shanghai. Through genome-wide association studies (GWAS) with important growth traits such as birth weight, 180-day weight, body height, and body length, we discovered a set of SNP molecular genetic markers significantly associated with growth traits in Chongming white goats. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide an SNP marker that is significantly related to the growth traits of Chongming white goats.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A SNP marker associated with growth traits of Chongming white goats, comprising SNP_1, SNP_2, SNP_3, and SNP_4; wherein SNP_1 is located at position 373396 of chromosome 17 of the goat reference genome (ARS1.2), and the base at this position is C or T; SNP_2 is located at position 372464 of chromosome 17 of the goat reference genome, and the base at this position is C or T; SNP_3 is located at position 372892 of chromosome 17 of the goat reference genome, and the base at this position is A or G; and SNP_4 is located at position 372853 of chromosome 17 of the goat reference genome, and the base at this position is A or G; the growth traits include body weight, body height, and body length.

[0007] Furthermore, the birth weight of individuals with the CC and CT genotypes marked by SNP_1 was higher than that of individuals with the TT genotype; the 180-day weight of individuals with the TT and CT genotypes marked by SNP_2 was higher than that of individuals with the CC genotype; the 180-day height of individuals with the GG and AG genotypes marked by SNP_3 was higher than that of individuals with the AA genotype; and the 180-day body length of individuals with the AA or AG genotypes marked by SNP_4 was higher than that of individuals with the GG genotype.

[0008] The primer set used to detect the SNP markers related to the growth traits of Chongming white goats includes primer pairs for detecting SNP_3 and SNP_4 markers, primer pairs for detecting SNP_1 markers, and primer pairs for detecting SNP_2 markers. The primer pairs used to detect SNP_3 and SNP_4 markers: GR_1F:5'-AGGAAGCACTGGGACTTGTG-3' GR_1R:5'-GCTGTTTCTACTGGATGCTGC-3' The primer pair used to detect the SNP_1 marker: GR_2F:5'-GCAGCATCCAGTAGAAACAGC-3' GR_2R:5'-GCAGGTGGTGTGGTTTTTCC-3' The primer pair used to detect the SNP_2 marker: GR_3F:5'-CCCTGGAGTTTTGGAGATAGTCA-3' GR_3R: 5'-TGAGGTCATCCCAACAAGATC-3'.

[0009] The application of the above primer set in screening Chongming white goats with excellent growth traits.

[0010] The application of the above primer set in molecular marker-assisted breeding of Chongming white goats.

[0011] A method for screening Chongming white goats with superior growth traits includes the following steps: (1) Extract DNA from the goat sample to be tested; (2) Using the DNA obtained in step (1) as a template, perform PCR amplification using the above primer set to obtain the amplification product; (3) Sequencing the amplification products obtained in step (2), determining the genotype of the SNP markers of the samples based on the sequencing results, and selecting individuals that meet at least one of the following conditions as Chongming white goats with excellent growth traits: ① Individuals with SNP_1 markers of CC or CT genotypes; ② Individuals with SNP_2 markers of TT or CT genotypes; ③ Individuals with SNP_3 markers of GG or AG genotypes; ④ Individuals with SNP_4 markers of AA or AG genotypes.

[0012] Furthermore, in step (3), individuals meeting the following criteria are selected as Chongming white goats with excellent growth traits: ① Individuals with SNP_1 markers of CC or CT genotypes; ② Individuals with SNP_2 markers of TT or CT genotypes; ③ Individuals with SNP_3 markers of GG or AG genotypes; ④ Individuals with SNP_4 markers of AA or AG genotypes. These individuals can be retained as breeding stock in the core group of Chongming white goats with high growth traits.

[0013] The beneficial effects of this invention are: 1) This invention provides four SNP markers related to the growth traits of Chongming goats. These molecular markers provide a scientific basis for marker-assisted selection of Chongming goat populations with high growth traits. Using these molecular markers to assist in breeding can effectively overcome the limitations of traditional breeding and significantly improve the accuracy and efficiency of growth performance selection for Chongming goats.

[0014] 2) By using the four SNP markers related to the growth traits of Chongming goats provided by this invention for joint breeding, the accuracy and effectiveness of selecting high-growth-trait populations of Chongming goats can be greatly increased. Early selection of high-growth traits through marker-assisted technology can accelerate the improvement of the growth performance of Chongming goats and cultivate a higher-quality Chongming goat breed. Attached Figure Description

[0015] Figure 1 The results of a genome-wide association analysis of the birth weight trait in Shanghai Chongming white goats; Figure 2The results of genome-wide association analysis of body weight traits in Shanghai Chongming white goats at 180 days of age; Figure 3 The results of a genome-wide association analysis of the body height trait in Shanghai Chongming white goats at 180 days of age; Figure 4 The results of genome-wide association analysis of body length trait in Shanghai Chongming white goats at 180 days of age; Figure 5 For the statistical analysis of growth phenotypic and SNP genotype data of Chongming white goats; Figure 6 This is to verify the results of Sanger sequencing. Detailed Implementation

[0016] The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings and specific embodiments. Example 1

[0017] 1. Source of laboratory animals The experiment used 863 Chongming white goats, which were obtained from Shanghai Chongming Yuandu Livestock Breeding Co., Ltd. (Chongming white goat breeding farm). The growth phenotypic data of all individuals were determined and provided by Shanghai Chongming Yuandu Livestock Breeding Co., Ltd.

[0018] 2. Development of SNP molecular markers significantly associated with the growth performance of Chongming white goats We used the "Chongxin-1" 50K SNP liquid phase chip, developed jointly by our team and Shijiazhuang Borui Biotechnology Co., Ltd., based on genotyping by target sequencing (GBTS) technology, to perform SNP genotyping on 863 Chongming white goats from Shanghai Chongming Yuandu Livestock Breeding Co., Ltd. We then used a mixed linear model (MLM) in GEMMA (v 0.98.1) software to perform genome-wide association studies (GWAS) on relevant growth phenotypic traits (birth weight, 180-day weight, body height, and body length). We used R to plot Manhattan plots of the Chongming white goat growth trait GWAS results, and the Bonferroni method was used to correct the multiple test thresholds for GWAS. The GWAS results are represented in the Manhattan plot by red horizontal solid lines (significant association threshold) and blue horizontal solid lines (potential association threshold), respectively.

[0019] GWAS results of birth weight traits in Shanghai Chongming white goats are shown in [link to data]. Figure 1 ,in, Figure 1 A is the GWAS Manhattan diagram. Figure 1B is the QQplot; the genome-wide association analysis (GWAS) results of the 180-day-old body weight trait in Shanghai Chongming white goats are shown in [link to GWAS]. Figure 2 ,in, Figure 2 A is the GWAS Manhattan diagram; Figure 2 B is the QQplot; the genome-wide association analysis (GWAS) results of the body height trait of Shanghai Chongming white goats at 180 days of age are shown in [link to GWAS]. Figure 3 ,in, Figure 3 A is the GWAS Manhattan diagram. Figure 3 B is the QQplot; the genome-wide association analysis (GWAS) results of the body length trait of Shanghai Chongming white goats at 180 days of age are shown in [link to GWAS]. Figure 4 ,in, Figure 4 A is the GWAS Manhattan diagram. Figure 4 B is a QQplot. (Used via...) Figures 1 to 4 Four SNPs (SNP_1, SNP_2, SNP_3, and SNP_4) were identified as being most associated with the growth phenotypic trait. Based on the statistical results of the growth phenotypic data, the associations of these four SNPs with the growth performance of Chongming white goats are shown in Table 1. Table 1. Four SNPs and their correlation with the growth performance of Chongming white goats.

[0020]

[0021] Statistical data on growth phenotypic traits and SNP genotypes of Chongming White Goats can be found in [link to relevant data]. Figure 5 ,in, Figure 5 A represents the genotypic data of the SNP located at 373396th base on chromosome 17 of the goat reference genome. Figure 5 B represents the genotypic data of the SNP located at 372464 bases on chromosome 17 of the goat reference genome. Figure 5 C represents the genotypic data of the SNP located at 372892 bases on chromosome 17 of the goat reference genome. Figure 5 D represents the genotypic data of the SNP located at nucleotide 372853 on chromosome 17 of the goat reference genome. Combined with... Figures 1 to 5 As shown in Table 1, this experiment yielded four SNP markers associated with growth traits in Shanghai Chongming white goats: SNP_1, SNP_2, SNP_3, and SNP_4. SNP_1 is located at base 373396 on chromosome 17 of the goat reference genome. GWAS results showed that this marker was significantly correlated with goat birth weight (Table 1 and Table 2). Figure 5A), the birth weight of Chongming goat individuals with the CC and CT genotypes of this SNP marker was significantly higher than that of individuals with the TT genotype (P value < 0.0001). Figure 5 A); The SNP_2 marker is located at base 372464 on chromosome 17 of the goat reference genome. Individuals of Chongming goats with the TT and CT genotypes of this SNP marker had significantly higher 180-day body weights than those with the CC genotype (P value < 0.0001 and P value < 0.01, Table 1 and...). Figure 5 B); The SNP_3 marker is located at nucleotide 372892 on chromosome 17 of the goat reference genome. Individuals with the GG and AG genotypes of Chongming goats with this SNP marker had significantly higher 180-day heights than those with the AA genotype (P value < 0.0001 and P value < 0.05, respectively, see Table 1 and...). Figure 5 C); The SNP_4 marker is located at nucleotide 372853 on chromosome 17 of the goat reference genome. Individuals with the AG genotype of Chongming goats bearing this SNP marker had a significantly longer 180-day body length than those with the GG genotype (P value < 0.0001, Table 1 and...). Figure 5 (D) Individuals with the AA genotype also have a longer body length than individuals with the other two genotypes.

[0022] Example 2 Validation sequencing of SNP molecular markers: (1) Goat DNA extraction: Blood (2-3 mL) was collected from the anterior vena cava of a goat using a sterile syringe and injected into an anticoagulant blood collection tube pre-added with EDTA. Blood cells were separated from plasma by centrifugation. After washing the blood cell precipitate, lysis buffer was added to disrupt the cell membrane and release DNA. Proteinase K was added to digest cell proteins. Acetic acid and isopropanol were added to coagulate and precipitate the DNA. The DNA precipitate was then separated from the supernatant by centrifugation. The DNA precipitate was washed with acetate buffer to remove impurities and residual proteinase. The DNA was dissolved in sterile double-distilled water (ddH2O) or TE buffer and stored at -20°C for subsequent experiments.

[0023] (2) PCR fragment amplification of target SNP: PCR amplification was performed using the three primer pairs in Table 2 to obtain the amplification product, which was then sent to Shanghai Platinum Biotech Co., Ltd. for direct sequencing of the PCR product.

[0024] Table 2. PCR primer information for SNPs

[0025] Primers GR_1F and GR_1R are primer pairs used to detect SNP_3 and SNP_4 markers, primers GR_2F and GR_2R are primer pairs used to detect SNP_1 markers, and primers GR_3F and GR_3R are primer pairs used to detect SNP_2 markers. The PCR primers were designed by the inventors using the Primer3 Plus online primer design tool (https: / / www.primer3plus.com / ) based on the genomic location sequence of the target SNPs, and primer synthesis was commissioned to Sangon Biotech (Shanghai) Co., Ltd.

[0026] The PCR reaction system was: 25 μL (1 μL upstream primer + 1 μL downstream primer + 12.5 μL Tiangen Biotech PCR premix + 9.5 μL ddH2O + 1 μL goat DNA). The reaction conditions were: A. 94℃ for 3 minutes, (94℃ for 30 seconds, 55℃ for 30 seconds, 72℃ for 1 minute), repeated 30 times, 72℃ for 10 minutes, and stopped at 4℃.

[0027] The target amplified fragment was detected by agarose gel electrophoresis, and the product was recovered for Sanger sequencing.

[0028] Sanger sequencing validation results are shown below. Figure 6 , Figure 6 The correctness of the above four sites (SNP_1, SNP_2, SNP_3 and SNP_4) was verified.

[0029] Example 3 A method for screening Chongming white goats with superior growth traits includes the following steps: (1) Extract DNA from the goat sample to be tested; Blood (2-3 mL) was collected from the anterior vena cava of a goat using a sterile syringe and injected into an anticoagulant blood collection tube pre-laced with EDTA. Blood cells were separated from plasma by centrifugation. After washing the blood cell pellet, lysis buffer was added to disrupt the cell membrane and release DNA. Proteinase K was added to digest cellular proteins, and acetic acid and isopropanol were added to coagulate and precipitate the DNA. The DNA precipitate was then separated from the supernatant by centrifugation. The DNA precipitate was washed with acetate buffer to remove impurities and residual proteins, and dissolved in anhydrous water or TE buffer. The DNA was stored at -20°C for subsequent experiments. DNA obtained from fresh goat tissue samples (such as ear tissue or muscle tissue) using the phenol-chloroform extraction method can also be used as an alternative to goat blood DNA.

[0030] (2) Using the DNA obtained in step (1) as a template, PCR amplification was performed using the three primer pairs in Table 2 to obtain the amplification product. The PCR reaction system and conditions were the same as in Example 2.

[0031] (3) Sequencing the amplification products obtained in step (2), determining the genotype of the SNP markers in the samples based on the sequencing results, and selecting individuals that meet at least one of the following conditions as Chongming white goats with excellent growth traits based on the genotyping results: ① Individuals with SNP_1 markers of CC or CT genotypes; ② Individuals with SNP_2 markers of TT or CT genotypes; ③ Individuals with SNP_3 markers of GG or AG genotypes; ④ Individuals with SNP_4 markers of AA or AG genotypes. It is recommended to select individuals whose genotypes at all four loci simultaneously meet two or more of the above conditions as core breeding individuals for Chongming white goats with high growth traits.

[0032] By detecting the aforementioned SNP markers in the genome sequences of individual Chongming goats, the growth performance and potential of the goats can be evaluated based on the genotype of these SNP markers. Therefore, the SNP markers of this invention are significantly correlated with goat growth traits and can be effectively used for marker-assisted breeding of Chongming goats. This allows for the selection of core Chongming goat populations for breeding or as breeding material based on actual breeding needs, thereby enabling the accurate and efficient selection of superior Chongming goat individuals with high growth performance, improving the efficiency and accuracy of breeding.

Claims

1. A SNP marker associated with growth traits of Chongming white goats, characterized in that, The SNP markers include SNP_1, SNP_2, SNP_3, and SNP_4. The SNP_1 marker is located at position 373396 on chromosome 17 of the goat reference genome, and the base at this position is either C or T. The SNP_2 marker is located at position 372464 on chromosome 17 of the goat reference genome, and the base at this position is either C or T. The SNP_3 marker is located at position 372892 on chromosome 17 of the goat reference genome, and the base at this position is either A or G. The SNP_4 marker is located at position 372853 on chromosome 17 of the goat reference genome, and the base at this position is either A or G. The growth traits include body weight, body height, and body length. The version number of the goat reference genome is ARS1.

2.

2. The SNP markers related to the growth traits of Chongming white goats as described in claim 1, characterized in that, The birth weight of individuals with the CC and CT genotypes marked by SNP_1 was higher than that of individuals with the TT genotype; the 180-day weight of individuals with the TT and CT genotypes marked by SNP_2 was higher than that of individuals with the CC genotype; the 180-day height of individuals with the GG and AG genotypes marked by SNP_3 was higher than that of individuals with the AA genotype; and the 180-day body length of individuals with the AA or AG genotypes marked by SNP_4 was higher than that of individuals with the GG genotype.

3. A primer set for detecting SNP markers related to the growth traits of Chongming white goats as described in claim 1 or 2, characterized in that, The primer set includes primer pairs for detecting SNP_3 and SNP_4 markers, primer pairs for detecting SNP_1 markers, and primer pairs for detecting SNP_2 markers; The primer pairs used to detect SNP_3 and SNP_4 markers: GR_1F:5'-AGGAAGCACTGGGACTTGTG-3' GR_1R:5'-GCTGTTTCTACTGGATGCTGC-3' The primer pair used to detect the SNP_1 marker: GR_2F:5'-GCAGCATCCAGTAGAAACAGC-3' GR_2R:5'-GCAGGTGGTGTGGTTTTTCC-3' The primer pair used to detect the SNP_2 marker: GR_3F:5'-CCCTGGAGTTTTGGAGATAGTCA-3' GR_3R: 5'-TGAGGTCATCCCAACAAGATC-3'.

4. The application of the primer set described in claim 3 in screening Chongming goats with excellent growth traits.

5. The application of the primer set described in claim 3 in molecular marker-assisted breeding of Chongming white goats.

6. A method for screening Chongming white goats with excellent growth traits, characterized in that, Includes the following steps: (1) Extract DNA from the goat sample to be tested; (2) Using the DNA obtained in step (1) as a template, perform PCR amplification using the primer set described in claim 3 to obtain the amplification product; (3) Sequencing the amplification products obtained in step (2), determining the genotype of the SNP markers of the samples based on the sequencing results, and selecting individuals that meet at least one of the following conditions as Chongming white goats with excellent growth traits: ① Individuals with SNP_1 markers of CC or CT genotypes; ② Individuals with SNP_2 markers of TT or CT genotypes; ③ Individuals with SNP_3 markers of GG or AG genotypes; ④ Individuals with SNP_4 markers of AA or AG genotypes.

7. The method for screening Chongming goats with excellent growth traits as described in claim 6, characterized in that, In step (3), individuals that meet the following conditions are selected as Chongming white goats with excellent growth traits: ① Individuals with SNP_1 marked as CC or CT genotype; ② Individuals with SNP_2 marked as TT or CT genotype; ③ Individuals with SNP_3 marked as GG or AG genotype; ④ Individuals with SNP_4 marked as AA or AG genotype.

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