SNP (Single Nucleotide Polymorphism) molecular marker related to goat feed conversion rate, growth performance and body size character and application of SNP molecular marker

Abstract

The invention belongs to the technical field of molecular markers and genetic breeding, and particularly relates to an SNP (Single Nucleotide Polymorphism) molecular marker related to goat feed conversion rate, growth performance and body size character and application of the SNP molecular marker. The SNP molecular marker provided by the invention corresponds to 21816208bp of a ninth chromosome of a goat genome, and a basic group is C or T; the information version number of the genome sequence of the goat is ASM170441v1. The SNP molecular marker provided by the invention is obviously associated with the body size (chest circumference and chest depth) character of the goat, and the body size character of the goat is the external reflection of the growth development and internal physiological structure of the goat, and has an obvious positive correlation with the feed conversion rate, especially the growth performance; the SNP molecular marker provided by the invention can be used for identifying the feed conversion rate, the growth performance and the body size character of the goat, and lays a foundation for selection and breeding of goat individuals with excellent body size character, high growth performance and high feed utilization rate.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of molecular markers and genetic breeding, and particularly relates to a SNP molecular marker related to feed conversion rate, growth performance and body size traits of a goat and application thereof. BACKGROUND

[0002] The feed conversion rate, growth performance and body size are core evaluation parameters in goat breeding, and there is a close interaction among the three. The body size of a goat is an external reflection of its growth and development and internal physiological structure, and is significantly positively correlated with the feed conversion rate, especially the growth performance. A good body size index (such as chest circumference) reflects sufficient muscle deposition, which is directly related to the digestion rate and conversion efficiency of feed. The chest circumference is the most core index for predicting body weight and reflecting the potential of meat production, and the body length, body height and pipe circumference jointly form the body type basis for supporting rapid growth and high yield performance from different angles (body capacity, skeletal support and bone strength). The chest circumference is the most critical index for measuring the chest development of a goat, including the chest cavity volume, heart and lung function, rib length and opening degree and body fullness, muscle and fat deposition. The larger the chest circumference, the wider the chest cavity, the more developed the muscle and the better the development of internal organs, which can support and support a larger body weight. The correlation coefficient between the chest circumference and the live body weight is usually the highest among all body size traits, and is the most reliable body size index for predicting body weight. The correlation coefficient between the chest depth and the live body weight and daily gain is also high, and the correlation coefficient is only second to the chest circumference.

[0003] Single nucleotide polymorphism (SNP) is a base variation on the genome caused by a single nucleotide, which has the characteristics of a large number of distributions and rich polymorphisms, and is an important molecular genetic marker after the microsatellite molecular marker, known as the third generation of molecular genetic markers. The marker has been widely used in animal and plant marker-assisted breeding, gene mapping, genetic diversity research and other fields.

[0004] CAPs technology is also known as PCR-RFLP (restriction fragment length polymorphism polymerase chain reaction technology). The basic principle of PCR-RFLP is to amplify the target DNA by PCR, and then cut the amplified product into different size fragments by specific endonuclease, and then distinguish it directly on the gel electrophoresis. Different alleles have different distribution of restriction enzyme cutting sites, and produce different length of DNA fragment bands. This technology greatly improves the content and relative specificity of the target DNA, and the method is simple, and the typing time is short. Therefore, based on the CAPs technology, screening the molecular marker related to the feed conversion rate, growth performance and body size index of a goat is particularly important for early individual selection and breeding of the goat. SUMMARY

[0005] The purpose of this invention is to provide a SNP molecular marker related to goat feed conversion ratio, growth performance and body size traits and its application, so as to rapidly, stably, efficiently and at low cost identify goat feed conversion ratio, growth performance and body size traits, and lay the foundation for the selection and breeding of goat individuals with excellent body size traits, high growth performance and high feed utilization.

[0006] This invention provides a SNP molecular marker associated with goat feed conversion ratio, growth performance and body size traits. The SNP molecular marker corresponds to chromosome 9, 21816208 bp of the goat genome, and the base is C or T. The goat genome sequence information version number is ASM170441v1.

[0007] The present invention provides a DNA fragment related to goat feed conversion rate, growth performance and body size traits, the DNA fragment comprising the nucleotide sequence shown in SEQ ID NO:1, wherein M is a base C or T.

[0008] This invention provides primer pairs for amplifying the SNP molecular markers or DNA fragments described in the above-mentioned technical solutions, wherein the primer pairs include an upstream primer F and a downstream primer R; The upstream primer F comprises a nucleotide sequence as shown in SEQ ID NO:2; The downstream primer R comprises a nucleotide sequence as shown in SEQ ID NO:3.

[0009] This invention provides a kit for detecting the SNP molecular markers or DNA fragments described in the above-mentioned technical solutions, the kit comprising the primer pairs described in the above-mentioned technical solutions.

[0010] This invention provides the application of reagents for detecting the SNP molecular markers described in the above-described technical solutions, or DNA fragments described in the above-described technical solutions, or primer pairs described in the above-described technical solutions, or kits described in the above-described technical solutions, in screening and / or identifying one or more of the following traits in goats: feed conversion ratio, growth performance, and body size.

[0011] This invention provides the application of reagents for detecting the SNP molecular markers described in the above-mentioned technical solutions, or DNA fragments described in the above-mentioned technical solutions, or primer pairs described in the above-mentioned technical solutions, or kits described in the above-mentioned technical solutions in goat breeding; The goat breeding includes selecting goat breeds with one or more of the following characteristics: high feed utilization, high growth performance, and excellent body size traits.

[0012] Preferably, the growth performance includes live weight and / or daily weight gain; the body size characteristics include chest circumference and / or chest depth.

[0013] Preferably, the goat includes a black goat.

[0014] Preferably, the black goat includes the Leizhou goat.

[0015] This invention provides a method for identifying feed conversion ratio, growth performance, and body size traits in goats, comprising the following steps: Using the DNA of the goat to be tested as a template, PCR amplification was performed using the primer pairs described in the above technical solution to obtain PCR amplification products; use TaqI The PCR amplification product was digested with restriction endonucleases to obtain the digested product. Based on the size of the DNA band in the enzyme digestion product, the genotype of the goat to be tested corresponding to the SNP molecular marker described in the above technical solution is identified, and the feed conversion ratio, growth performance and body size traits of the goat to be tested are determined: if the size of the DNA band in the enzyme digestion product is 444bp and 280bp, then the genotype of the goat to be tested corresponding to the SNP molecular marker described in the above technical solution is TT, and the goat to be tested is an individual with high feed utilization, high growth performance and excellent body size traits.

[0016] Beneficial effects: The SNP molecular markers provided by this invention correspond to chromosome 9, 21816208 bp of the goat genome, with bases of C or T; the goat genome sequence information version number is ASM170441v1. The SNP molecular markers described in this invention are significantly associated with goat body size (chest circumference and chest depth) traits. Given that goat body size traits are an external reflection of their growth, development, and internal physiological structure, and have a significant positive correlation with feed conversion ratio, especially growth performance, the SNP molecular markers provided by this invention can be used to identify goat feed conversion ratio, growth performance, and body size traits, laying the foundation for the selection and breeding of goat individuals with superior body size traits, high growth performance, and high feed utilization. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0018] Figure 1 This is a graph showing the gel electrophoresis results of Leizhou goat genomic DNA detection in step 2 of Example 1; Figure 2 This is a graph showing the gel electrophoresis results of the PCR amplification products in step 4 of Example 1; Figure 3 This is a PCR-RFLP gel electrophoresis image of the SNP molecular marker described in this invention; Figure 4 This is a diagram showing the sequencing results of the homozygous CC genotype of the SNP molecular marker described in this invention; Figure 5 This is a diagram showing the sequencing results of the homozygous CT genotype of the SNP molecular marker described in this invention; Figure 6 This is a diagram showing the sequencing results of the homozygous TT genotype of the SNP molecular marker described in this invention. Detailed Implementation

[0019] This invention provides a SNP molecular marker associated with feed conversion ratio, growth performance, and body size traits in goats. The SNP molecular marker corresponds to 21816208 bp on chromosome 9 of the goat genome, with bases C or T. The goat genome sequence information version number is ASM170441v1. The sequence information of goat chromosome 9 described in this invention has the NCBI accession number NC_030816.1.

[0020] The present invention provides a DNA fragment related to goat feed conversion rate, growth performance and body size traits, the DNA fragment comprising the nucleotide sequence shown in SEQ ID NO:1, wherein M is a base C or T.

[0021] The nucleotide sequence shown in SEQ ID NO:1 of this invention is specifically 5'--3'; where M is the base C / T.

[0022] This invention provides primer pairs for amplifying the SNP molecular markers or DNA fragments described in the above-mentioned technical solutions. The primer pairs include an upstream primer F and a downstream primer R. The upstream primer F includes a nucleotide sequence as shown in SEQ ID NO:2. The downstream primer R includes a nucleotide sequence as shown in SEQ ID NO:3.

[0023] This invention provides a kit for detecting the SNP molecular markers or DNA fragments described in the above-mentioned technical solutions, the kit comprising the primer pairs described in the above-mentioned technical solutions.

[0024] In one embodiment, the kit of the present invention further includes Taq DNA polymerase. In another embodiment, the kit of the present invention further includes... TaqI Restriction endonucleases.

[0025] This invention provides the application of reagents for detecting the SNP molecular markers described in the above-described technical solutions, or DNA fragments described in the above-described technical solutions, or primer pairs described in the above-described technical solutions, or kits described in the above-described technical solutions, in screening and / or identifying one or more of the following traits in goats: feed conversion ratio, growth performance, and body size.

[0026] This invention provides the application of reagents for detecting the SNP molecular markers described in the above-mentioned technical solutions, or DNA fragments described in the above-mentioned technical solutions, or primer pairs described in the above-mentioned technical solutions, or kits described in the above-mentioned technical solutions in goat breeding; the goat breeding includes selecting goat breeds with one or more characteristics such as high feed utilization, high growth performance, and excellent body size traits.

[0027] In one embodiment, the body size characteristics described in this invention include chest circumference and / or chest depth. In one embodiment, the growth performance described in this invention includes live weight and / or daily weight gain.

[0028] In one embodiment, the goat described in this invention includes a black goat. In another embodiment, the black goat described in this invention includes a Leizhou goat.

[0029] In this invention, when the genotype of the SNP molecular marker is TT, or when the genotype of the DNA fragment corresponding to the SNP molecular marker is TT, the goat is an individual with high feed utilization, high growth performance and excellent body size traits.

[0030] This invention provides a method for identifying feed conversion ratio, growth performance, and body size traits in goats, comprising the following steps: Using the DNA of the goat to be tested as a template, PCR amplification was performed using the primer pairs described in the above technical solution to obtain PCR amplification products; use TaqI The PCR amplification product was digested with restriction endonucleases to obtain the digested product. Based on the size of the DNA band in the enzyme digestion product, the genotype of the goat to be tested corresponding to the SNP molecular marker described in the above technical solution is identified, and the feed conversion ratio, growth performance and body size traits of the goat to be tested are determined: if the size of the DNA band in the enzyme digestion product is 444bp and 280bp, then the genotype of the goat to be tested corresponding to the SNP molecular marker described in the above technical solution is TT, and the goat to be tested is an individual with high feed utilization, high growth performance and excellent body size traits.

[0031] In one embodiment, the concentration of the goat DNA to be tested in this invention is ≥30 ng / μL; in another embodiment, the concentration of the goat DNA to be tested in this invention is 50~200 ng / μL. In one embodiment, the OD260 / 280 value of the goat DNA to be tested in this invention is 1.8~2.0. This invention does not have strict requirements on the source of the goat DNA to be tested; it can be obtained using conventional methods, such as extracting DNA from the blood of the goat to be tested.

[0032] In one embodiment, the PCR amplification system of the present invention, in 20 μL volume, includes 1 μL of DNA template, 0.4 μL of upstream primer F, 0.4 μL of downstream primer R, 10 μL of Taq DNA polymerase, and the remainder being deionized double-distilled water (ddH2O). In another embodiment, the PCR amplification reaction program of the present invention is as follows: 94℃ pre-denaturation for 2 min; 94℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 45 s, 35 cycles; final extension at 72℃ for 5 min; storage at 4℃.

[0033] As one embodiment, the enzymatic digestion reaction system of the present invention, in 30 μL increments, includes 2 μL of 10×FuniCut@Buffer, FuniCut@ TaqI 1 μL of PCR amplification product, 10 μL of PCR product, and the remainder ddH2O. As one embodiment, the enzyme digestion temperature is 60°C. As one embodiment, the enzyme digestion time is 30 min. As one embodiment, the enzyme digestion product is subjected to agarose gel electrophoresis to determine the size of the DNA bands in the enzyme digestion product.

[0034] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes a SNP molecular marker related to goat feed conversion rate, growth performance, and body size traits, and its applications. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0035] Example 1 1. Sample Collection A total of 275 blood samples from Leizhou goats were used in this embodiment, collected from Leizhou Zhuangyuan Black Goat Breeding Co., Ltd. 5 mL of blood was collected from the jugular vein, treated with EDTK2 anticoagulation, and stored at -20°C for later use. Ear tags and body size data for each individual were also recorded.

[0036] 2. Extraction and quality testing of blood genomic DNA Genomic DNA was extracted from blood using the Hipure Universal DNA Kit D3018 from Meiji Biotechnology. The concentration and purity of the extracted DNA fragments were determined using a nanodrop spectrophotometer. Buffer AE was used as a blank control to ensure that the extracted DNA sample concentration was not less than 30 ng / μL, the OD260 / 280 value was between 1.8 and 2.0, and the concentration was between 50 and 200 ng / μL. The quality of the extracted DNA was then assessed by 1% agarose gel electrophoresis. The results showed that the electrophoretic bands were clear and densely bright. Figure 1 This indicates that the extracted goat genomic DNA sample is of good quality, with purity and concentration meeting the experimental requirements, and can be used for subsequent experiments.

[0037] 3. Candidate gene primer synthesis Based on candidate gene sequences published in GenBank, primers were designed for the 21,816,208 bp site on goat chromosome NC_030816.1. The upstream primer F and downstream primer R were designed using Premier 5.0 software and synthesized by Sangon Biotech Co., Ltd. The primer sequences are: upstream primer F: 5'-CATGGCATCTGGTCCCATTAC-3' (SEQ ID NO:2), downstream primer R: 5'-TTATGCCAGAATCCCGTACC-3' (SEQ ID NO:3).

[0038] 4. PCR amplification PCR reaction system: The 20 μL system contains 1 μL of genomic DNA template, 0.4 μL each of forward and reverse primers, 10 μL of Taq DNA polymerase, and ddH2O to the final volume.

[0039] PCR reaction procedure: 94℃ pre-denaturation for 2 min; 94℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 45 s, 35 cycles; 72℃ final extension for 5 min; store at 4℃.

[0040] After the reaction was completed, 5 μL of the reaction product was taken and its length and integrity were detected by 1% agarose gel electrophoresis. The results showed that the target band was clear and bright after PCR electrophoresis, without any impurities, and the size of the target fragment was consistent with the expected result, which was 724 bp. Figure 2 ).

[0041] 5. PCR-RFLP analysis (1) DNA samples were extracted from 20 and 50 different individuals of Leizhou goats, respectively, and mixed in equal amounts to form two DNA pools. These pools were then used as templates for PCR amplification according to the method described in Example 1, and the PCR products were sequenced. The sequencing results were organized using DNAMAN software and compared with the genome sequence on NCBI. The nucleotide sequence of the amplified sequence was found to be as shown in SEQ ID NO:1, with a C→T mutation at position 280 bp, which is the SNP molecular marker. SnapGene software was used to find the corresponding restriction endonucleases for the screened SNP molecular markers. The restriction endonucleases corresponding to the SNP mutation sites were obtained as follows: TaqI .

[0042] (2) Utilization TaqI The amplification product was digested with restriction endonucleases in a 30 μL digestion system: 10×FuniCut@Buffer 2 μL, Funicut@ TaqI 1 μL of substrate DNA, 10 μL of ddH2O, and 17 μL of ddH2O were thoroughly mixed and placed in a 65°C water bath for 30 min of enzyme digestion. After digestion, the sample was removed and subjected to 1% agarose gel electrophoresis. The genotypes in each lane were analyzed based on the gel imaging results, which showed three genotypes. Figure 3 : Mutant heterozygous CT (724bp, 444bp and 280bp), mutant homozygous TT (444bp and 280bp) and wild-type homozygous CC (724bp).

[0043] (3) The PCR products of the three different genotypes of the SNP molecular marker were sent for sequencing. Sequence sequencing was performed at Sangon Biotech Co., Ltd., and bidirectional sequencing of gene fragments was conducted. The results are as follows: Figures 4-6 As shown.

[0044] 6. Data Statistics and Analysis (1) Genetic heterozygosity (He), genetic homozygosity (Ho), polymorphism information content (PIC), and effective number of alleles (Ne) are important parameters for evaluating population genetic variation. Different genetic parameters represent the essential genetic differences between populations. The genetic parameters of the SNP molecular markers described in this invention are shown in Table 1.

[0045] Table 1. Analysis of SNP molecular marker genetic parameters in this invention.

[0046] Table 1 shows that the polymorphism information content is 0.34, ranging from 0.25 to 0.5, indicating moderate polymorphism. The homozygosity and heterozygosity are similar, both around 0.5, suggesting a relatively even distribution of these two alleles in the Leizhou goat population. Chi-square results show that the genotype distribution conforms to Hardy-Weinberg equilibrium, indicating that the SNP molecular markers of this invention may not be affected by current breeding measures, and their genetic changes during the breeding process are random.

[0047] (2) The molecular marker loci were statistically analyzed using the GLM (General Linear Model) program in SPSS 26.0 statistical analysis software in conjunction with the constructed unit point effect model. Multiple comparisons between means were performed using the LSD and Dunnett's T3 methods to study the effect of genotype on the body size trait of Leizhou goats. The results are presented in the form of "mean ± standard deviation (Mean ± SD)". The linear model used for statistical analysis is as follows: yjkl=μ+G k +e jkl Where: yjkl is the observed value of the trait; μ is the population mean; Gk is the fixed effect of the k-th genotype; e jkl This represents a random residual effect. The results are shown in Table 2.

[0048] Table 2. Association analysis between the SNP molecular markers of this invention and body size traits of Leizhou goats.

[0049] Note: Different letters in the same column indicate significant differences. P <0.05).

[0050] As can be seen from Table 2, the SNP molecular markers of this invention are significantly associated with chest circumference and chest depth traits. P <0.05. The mean chest circumference of individuals with the TT genotype (74.700 cm) was significantly higher than that of individuals with the CC genotype (71.182 cm). P <0.05. The mean chest depth of individuals with the CC genotype (27.189 cm) was significantly higher than that of individuals with the CT genotype (26.060 cm). P <0.05). Given that body size traits in goats are an external reflection of their growth, development, and internal physiological structure, and are significantly positively correlated with feed conversion ratio, especially growth performance, with chest circumference and chest depth being highly positively correlated with live weight and daily weight gain, Leizhou goats carrying the TT genotype may have greater potential for live weight gain, faster daily weight gain, and higher feed utilization.

[0051] As can be seen from the above, the SNP molecular markers provided by this invention are related to goat feed conversion rate, growth performance and body size traits, laying the foundation for the selection and breeding of goat individuals with excellent body size traits, high growth performance and high feed utilization.

[0052] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A SNP molecular marker associated with goat feed conversion ratio, growth performance, and body size traits, characterized in that, The SNP molecular marker corresponds to chromosome 9, line 21816208 of the goat genome, with bases C or T; the goat genome sequence information version number is ASM170441v1.

2. A DNA fragment associated with goat feed conversion ratio, growth performance, and body size traits, characterized in that, The DNA fragment includes the nucleotide sequence shown in SEQ ID NO:1, where M is a base C or T.

3. A primer pair for amplifying the SNP molecular marker of claim 1 or the DNA fragment of claim 2, characterized in that, The primer pair includes an upstream primer F and a downstream primer R; The upstream primer F comprises a nucleotide sequence as shown in SEQ ID NO:2; The downstream primer R comprises a nucleotide sequence as shown in SEQ ID NO:

3.

4. A kit for detecting the SNP molecular marker of claim 1 or the DNA fragment of claim 2, characterized in that, The kit includes the primer pair as described in claim 3.

5. The use of the reagent for detecting the SNP molecular marker of claim 1, the DNA fragment of claim 2, the primer pair of claim 3, or the kit of claim 4 in screening and / or identifying one or more of goat feed conversion ratio, growth performance, and body size traits.

6. The application of the reagent for detecting the SNP molecular marker of claim 1, the DNA fragment of claim 2, the primer pair of claim 3, or the kit of claim 4 in goat breeding; The goat breeding includes selecting goat breeds with one or more of the following characteristics: high feed utilization, high growth performance, and excellent body size traits.

7. The application according to claim 5 or 6, characterized in that, The growth performance includes live weight and / or daily weight gain; the body size traits include chest circumference and / or chest depth.

8. The application according to claim 5 or 6, characterized in that, The goats mentioned include black goats.

9. The application according to claim 8, characterized in that, The black goats mentioned include Leizhou goats.

10. A method for identifying feed conversion ratio, growth performance, and body size traits in goats, characterized in that, Includes the following steps: Using the DNA of the goat to be tested as a template, PCR amplification was performed using the primer pair described in claim 3 to obtain the PCR amplification product; use TaqI The PCR amplification product was digested with restriction endonucleases to obtain the digested product. Based on the size of the DNA band in the enzyme digestion product, the genotype of the goat to be tested corresponding to the SNP molecular marker of claim 1 is identified, and the feed conversion ratio, growth performance and body size traits of the goat to be tested are determined: if the size of the DNA band in the enzyme digestion product is 444bp and 280bp, then the genotype of the goat to be tested corresponding to the SNP molecular marker of claim 1 is TT, and the goat to be tested is an individual with high feed utilization, high growth performance and excellent body size traits.

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