SNP molecular marker related to growth and development traits of wadong cattle and application thereof

Abstract

The present application relates to the technical field of animal molecular breeding, and particularly relates to SNP molecular markers related to growth and development traits of Wandong cattle and application thereof. The SNP molecular markers related to growth and development traits of Wandong cattle include at least one of a first SNP molecular marker, a second SNP molecular marker, a third SNP molecular marker, a fourth SNP molecular marker and a fifth SNP molecular marker. The first SNP molecular marker, the second SNP molecular marker, the third SNP molecular marker, the fourth SNP molecular marker and the fifth SNP molecular marker are located in a TNNI3 gene on a chromosome 18 of a bovine genome. A primer pair for detecting the above-mentioned SNP markers includes a T3S2 primer pair or / and a T3S4 primer pair. The sequence of the T3S2 primer pair is shown as SEQ ID NO:1 and SEQ ID NO:2, and the sequence of the T3S4 primer pair is shown as SEQ ID NO:3 and SEQ ID NO:4.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of animal molecular breeding, and particularly relates to a SNP molecular marker related to growth and development traits of Wandong cattle and application thereof. BACKGROUND

[0002] Wandong cattle is a kind of cattle breed with local characteristics and excellent genetic resources, which not only performs outstandingly in meat performance, but also has significant advantages in environmental adaptation and genetic diversity. These characteristics make Wandong cattle a valuable livestock genetic resource in China, and it has important development and utilization value for high-quality beef production.

[0003] Therefore, exploring the growth-related genes of Wandong cattle plays a positive role in the development of Chinese cattle industry. Studies have shown that the SNP site of TNNI3 gene provides important information for the muscle quality and myocardial tissue function of Tianfu mutton sheep, but there is no result showing that muscle quality is related to growth traits. SUMMARY

[0004] The present application relates to the technical field of animal molecular breeding, and particularly relates to a SNP molecular marker related to growth and development traits of Wandong cattle and application thereof.

[0005] One of the purposes of the present application is to provide a SNP molecular marker related to growth and development traits of Wandong cattle, which comprises at least one of a first SNP molecular marker, a second SNP molecular marker, a third SNP molecular marker, a fourth SNP molecular marker and a fifth SNP molecular marker; the first SNP molecular marker, the second SNP molecular marker, the third SNP molecular marker, the fourth SNP molecular marker and the fifth SNP molecular marker are all located in the TNNI3 gene of the 18th chromosome of the cattle genome.

[0006] The TNNI3 gene can be referred to NCBI Reference Sequence: AC_000175.1.

[0007] Preferably, the first SNP molecular marker is located at the base of the 62720648 site on the chromosome 18 of the bovine genome, and the SNP molecular marker site has a C / T polymorphism; the second SNP molecular marker is located at the base of the 62720925 site on the chromosome 18 of the bovine genome, and the SNP molecular marker site has a C / G polymorphism; the third SNP molecular marker is located at the base of the 62719179 site on the chromosome 18 of the bovine genome, and the SNP molecular marker site has an A / G polymorphism; the fourth SNP molecular marker is located at the base of the 62719284 site on the chromosome 18 of the bovine genome, and the SNP molecular marker site has a G / C polymorphism; and the fifth SNP molecular marker is located at the base of the 62719317 site on the chromosome 18 of the bovine genome, and the SNP molecular marker site has a G / A polymorphism.

[0008] Preferably, the 6-month-old Wadong cattle with the CC genotype and the CT genotype of the polymorphism site of the first SNP molecular marker have a significantly higher body weight than the TT genotype.

[0009] Preferably, the 24-month-old Wadong cattle with the CC genotype of the polymorphism site of the second SNP molecular marker have a significantly higher body length than the GG genotype.

[0010] Preferably, the 12-month-old Wadong cattle with the GG genotype of the polymorphism site of the third SNP molecular marker have a significantly higher wither height and body length than the AG genotype.

[0011] Preferably, the 12-month-old Wadong cattle with the CC genotype of the polymorphism site of the fourth SNP molecular marker have a significantly higher body length than the GG genotype and the GC genotype.

[0012] Preferably, the 18-month-old Wadong cattle with the GG genotype of the polymorphism site of the fifth SNP molecular marker have a higher body height and a higher body weight than the GA genotype, the 24-month-old Wadong cattle with the GG genotype have a significantly higher body height, body length and body weight than the GA genotype, but the 6-month-old Wadong cattle with the GA genotype have a significantly higher body weight than the GG genotype.

[0013] The second object of the present application is to provide the application of the above-mentioned SNP molecular marker in the detection of growth and development traits of Wadong cattle or in the breeding of Wadong cattle.

[0014] The third object of the present application is to provide a primer pair for detecting the above-mentioned SNP marker, comprising: a T3S2 primer pair or / and a T3S4 primer pair.

[0015] The upstream primer of the T3S2 primer pair is shown as SEQ ID NO: 1, and the downstream primer of the T3S2 primer pair is shown as SEQ ID NO: 2.

[0016] The upstream primer of the T3S4 primer pair is shown as SEQ ID NO: 3, and the downstream primer of the T3S4 primer pair is shown as SEQ ID NO: 4.

[0017] The fourth object of the present application is to provide an application of the above-mentioned primer pair in detection of growth and development traits of Wandong cattle or in breeding of Wandong cattle.

[0018] The fifth object of the present application is to provide a kit containing the above-mentioned primer pair.

[0019] The sixth object of the present application is to provide a method for predicting growth and development traits of Wandong cattle, which detects the genotypes of the polymorphic sites of the above-mentioned SNP molecular markers.

[0020] Preferably, the above-mentioned primer pair or the above-mentioned kit is used for detection.

[0021] The seventh object of the present application is to provide a breeding method of Wandong cattle, which extracts genomic DNA of Wandong cattle, uses the primer pair of claim 5 for amplification, and makes corresponding selection according to the above-mentioned SNP molecular markers:

[0022] (1) selecting the CC genotype and the CT genotype of the first SNP molecular marker polymorphic site, and eliminating the TT genotype of the first SNP molecular marker polymorphic site;

[0023] (2) selecting the CC genotype of the second SNP molecular marker polymorphic site, and eliminating the GG genotype of the second SNP molecular marker polymorphic site;

[0024] (3) selecting the GG genotype of the third SNP molecular marker polymorphic site, and eliminating the AG genotype of the third SNP molecular marker;

[0025] (4) selecting the CC genotype of the fourth SNP molecular marker polymorphic site, and eliminating the GG genotype and the GC genotype of the fourth SNP molecular marker polymorphic site;

[0026] (5) if a 6-month-old Wandong cattle is needed, the GA genotype of the fifth SNP molecular marker polymorphic site is selected, and the GG genotype of the fifth SNP molecular marker polymorphic site is eliminated;

[0027] If an 18-month-old or a 24-month-old Wandong cattle is needed, the GG genotype of the fifth SNP molecular marker polymorphic site is selected, and the GA genotype of the fifth SNP molecular marker polymorphic site is eliminated. Advantages

[0028] The SNP molecular marker provided by the application is significantly related to the growth and development traits of Wandong cattle, and can be used for assisted selection of the growth and development traits of Wandong cattle, screening of Wandong cattle potential with excellent growth and development, and has important practical application value for further improving the growth and development of Wandong cattle and using specific Wandong cattle as a material for breed or line breeding, and is convenient for breeding personnel to breed and cultivate Wandong cattle. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 It is a Wandong cattle blood genomic DNA electrophoresis detection result graph.

[0030] Figure 2 It is an electrophoresis detection result graph of PCR amplification by using T3S2 primer pairs.

[0031] Figure 3 It is an electrophoresis detection result graph of PCR amplification by using T3S4 primer pairs.

[0032] Figure 4 It is a g.62720648C>T mutation site different genotype sequencing peak graph.

[0033] Figure 5 It is a g.62720753A>C mutation site different genotype sequencing peak graph.

[0034] Figure 6 It is a g.62720859A>G mutation site different genotype sequencing peak graph.

[0035] Figure 7 It is a g.62720911T>C mutation site different genotype sequencing peak graph.

[0036] Figure 8 It is a g.62720925C>G mutation site different genotype sequencing peak graph.

[0037] Figure 9 It is a g.62719179A>G mutation site different genotype sequencing peak graph.

[0038] Figure 10 It is a g.62719284G>C mutation site different genotype sequencing peak graph.

[0039] Figure 11 It is a g.62719317G>A mutation site different genotype sequencing peak graph. DETAILED DESCRIPTION

[0040] The application will be further described below in combination with specific examples.

[0041] Example 1

[0042] In Anhui Province, Wan East Niubao Farm, Chuzhou City (Fengyang County) Daming Farming and Pastoral Development Co., Ltd. took the body size and length data of 220 6-month-old, 12-month-old, 18-month-old and 24-month-old Wan East cattle in multiple cowsheds, and all the cattle were taken 5 mL of blood from the jugular vein or tail vein, then put into the blood vessels with anticoagulant and stored in the refrigerator.

[0043] Example 2

[0044] 2.1 Extracting genomic DNA from blood samples

[0045] The frozen blood sample anticoagulant tube was thawed in advance at 4°C in the refrigerator to prevent the blood sample from being lost due to the large temperature difference causing the blood anticoagulant tube to burst. According to the instructions, the purchased blood DNA extraction kit was used to extract the sample DNA. The extracted 220 blood DNA samples were sealed and stored at -20°C for standby.

[0046] 2.2 Detection of extracted blood DNA samples

[0047] 2.2.1 Agarose gel electrophoresis detection

[0048] After adding a small amount of 1×TAE buffer to the agarose gel plate that has been cooled and completely solidified, slowly pull out the electrophoresis comb, put the electrophoresis gel plate into the electrophoresis tank and pour 1×TAE electrophoresis buffer until the electrophoresis gel plate is submerged. First, extract 5μL DL 2000 DNA Marker and add it to the first sample well of the gel plate, then mix different individual blood DNA samples with loading buffer at a ratio of 1μL:5μL and punch into the corresponding sample wells, electrophorese at 120V for 40min, place the gel on the ultraviolet imager to analyze the electrophoresis results, and take pictures for record.

[0049] 2.2.2 Ultra-micro UV spectrophotometer detection

[0050] Adjust the ultra-micro UV spectrophotometer to DNA measurement mode, wash the sample wells of the ultra-micro UV spectrophotometer with ddH2O multiple times and perform blank correction. After the instrument is calibrated, take 1μL of DNA sample from each sample for detection. After each detection, wash and calibrate the sample wells with ddH2O. Determine the concentration and purity of each sample by Conce and OD 260 / OD 280 values.

[0051] Conce value represents the DNA concentration of the measured sample, with unit ng / mol.

[0052] OD 260 / OD 280Theoretical value is between 1.7-2.2, indicating that the purity of each sample DNA is high; sample OD 260 / OD 280 The value is less than 1.7, and the sample DNA may be contaminated by incomplete digestion of protein and other substances during extraction; if OD 260 / OD 280 The value is greater than 2.2, which may be due to RNA contamination during extraction.

[0053] 2.3 Primer design and synthesis

[0054] For the variation site of TNNI3 gene exon 3 in the experimental Wadong cattle population, different SNP sites in TNNI3 gene were designed by Primer3Plus-PickPrimers software, and named T3S2 and T3S4. The primer information is as follows:

[0055]

[0056] 2.4 PCR amplification

[0057] For the above primers, 2x Taq PCR Master mix was used for amplification, and multiple gradient tests were performed to find the optimal annealing temperature, and the optimal reaction conditions and reaction system of each primer were finally determined.

[0058] (1) The PCR reaction system is as follows:

[0059]

[0060] (2) Different primers were designed according to different SNP sites, and the PCR reaction conditions are as follows. According to the reaction system, each pair of primers was amplified, and 5 μL of the obtained PCR product was extracted for agarose gel (3%) electrophoresis detection. The electrophoresis result of the PCR product was observed by ultraviolet imaging instrument. The successfully amplified TNNI3 primers will be used for large-scale verification test in the later stage.

[0061]

[0062] 2.5 Association analysis

[0063] The GLM process of SPSS 27.0 software was used to analyze the association between the growth and development traits of Wadong cattle and the SNP of the candidate gene. The association analysis model is:

[0064] Y = μ + G + m + e

[0065] In the formula, Y represents the measured value of the phenotypic trait, μ represents the population mean,G represents the genotype effect, m represents the gender effect, e represents the residual effect.

[0066] Example 3

[0067] 3.1 Extraction results of genomic DNA of WanDong cattle

[0068] The DNA of the blood sample was tested by agarose gel electrophoresis, and the results are shown in Figure 1 . It can be observed that the bands are clear and bright without impurity bands. The concentration and OD value of the sample DNA tested by ultramicro spectrophotometer meet the test requirements and can be used for subsequent tests.

[0069] 3.2 PCR amplification with different primers

[0070] PCR amplification was performed with the designed primers, and it was found that the gel electrophoresis of T3S2 and T3S4 primers showed clear bands without impurity bands, and the electrophoresis results are shown in Figure 2 and Figure 3 .

[0071] Among them, Figure 2 is the PCR result of the mutation site of T3S2 primer pair, and the length of the amplified band is 630bp; Figure 3 is the PCR result of the mutation site of T3S4 primer pair, and the length of the amplified band is 584bp; which is consistent with the length of the designed primer, and the PCR amplification is normal, so these two pairs of primers can continue to the next test operation.

[0072] 3.3 Mutation site sequencing

[0073] After Sanger sequencing, the detection results showed that there were 8 mutation sites in the TNNI3 gene. Through GeneBank search, it was found that 3 SNP sites were in the 5th intron region, and 5 SNP sites were in the 7th intron region. The specific information is shown in the table below.

[0074]

[0075] Thereafter, peak sequencing was performed on the 8 sites, among which:

[0076] The mutation of g.62720648C>T is shown in Figure 4 , which has three genotypes, i.e. CC, CT and TT genotypes. CC is wild homozygous type, TT is mutant homozygous type, and CT is mutant heterozygous type, so the mutant heterozygous type will show as a nested peak state in the sequencing peak graph;

[0077] The mutation of g.62720753A>C is shown in Figure 5As shown, there are three genotypes, namely AA, AC, and CC genotypes, AA is wild homozygous type, GG is mutant homozygous type, and AG is mutant heterozygous type, so in the sequencing peak map, the mutant heterozygous type will show as a nested peak state.

[0078] g.62720859A>G mutation as shown in Figure 6 As shown, there are three genotypes, namely AA, AG, and GG genotypes, AA is wild homozygous type, GG is mutant homozygous type, and AG is mutant heterozygous type, so in the sequencing peak map, the mutant heterozygous type will show as a nested peak state.

[0079] g.62720911T>C mutation as shown in Figure 7 As shown, there are three genotypes, namely TT, TC, and CC genotypes, TT is wild homozygous type, CC is mutant homozygous type, and TC is mutant heterozygous type, so in the sequencing peak map, the mutant heterozygous type will show as a nested peak state.

[0080] g.62720925C>G mutation as shown in Figure 8 As shown, there are three genotypes, namely GG, GC, and CC genotypes, GG is wild homozygous type, CC is mutant homozygous type, and GC is mutant heterozygous type, so in the sequencing peak map, the mutant heterozygous type will show as a nested peak state.

[0081] g.62719179A>G mutation as shown in Figure 9 As shown, there are three genotypes, namely AA, AG, and GG genotypes, AA is wild homozygous type, GG is mutant homozygous type, and AG is mutant heterozygous type, so in the sequencing peak map, the mutant heterozygous type will show as a nested peak state.

[0082] g.62719284G>C mutation as shown in Figure 10 As shown, there are three genotypes, namely GG, GC, and CC genotypes, GG is wild homozygous type, CC is mutant homozygous type, and GC is mutant heterozygous type, so in the sequencing peak map, the mutant heterozygous type will show as a nested peak state.

[0083] g.62719317G>A mutation as shown in Figure 11 As shown, there are two genotypes, namely GG and GA genotypes, GG is wild homozygous type, and GA is mutant heterozygous type, so in the sequencing peak map, the mutant heterozygous type will show as a nested peak state.

[0084] 3.4 Statistical sequencing result analysis of test samples

[0085] The genotype frequency and gene frequency of the 8 mutation sites of TNNI3 gene are shown in the table below. According to the calculated chi-square test χ 2 value analysis shows that the 8 sites are in Hardy-Weinberg equilibrium state.

[0086]

[0087] 3.5 Polymorphism information content statistics of 8 mutation sites of TNNI3 gene in sample population

[0088] Through the statistics of the polymorphism information content of the 8 mutation sites of the TNNI3 gene in the sample population, the statistical results are shown in the following table.

[0089]

[0090] Through the analysis, we can get:

[0091] (1) The polymorphism information content of g.62720648C>T and g.62719317G>A is less than 0.25, so these three sites are low polymorphism, and therefore the g.62720648C>T and g.62719317G>A sites in the TNNI3 gene in the sample population are low-level genetic variation.

[0092] (2) The polymorphism information content of g.62720753A>C, g.62720859A>G, g.62720911T>C, g.62720925C>G, g.62719179A>G, and g.62719284G>C is greater than 0.25 and less than 0.5, so these six sites are moderate polymorphism, and therefore the g.62720753A>C, g.62720859A>G, g.62720911T>C, g.62720925C>G, g.62719179A>G, and g.62719284G>C sites in the TNNI3 gene in the sample population are moderate-level genetic variation.

[0093] 3.6 Association analysis of SNP sites and growth and development traits of Wandong cattle

[0094] SPSS 27.0 software was used to analyze the correlation between TNNI3 gene SNP sites and growth and development traits in the Wandong cattle test population.

[0095] The correlation analysis results of g.62720648C>T site and growth traits of Wandong cattle at each month are shown in the following table:

[0096]

[0097] The correlation analysis results of g.62720753A>C site and growth traits of Wandong cattle at each month are shown in the following table:

[0098]

[0099] g.62720859A>G site and each month of Wan Dong cattle growth traits associated with the results are shown in the following table:

[0100]

[0101] g.62720911T>C site and each month of Wan Dong cattle growth traits associated with the results are shown in the following table:

[0102]

[0103] g.62720925C>G site and each month of Wan Dong cattle growth traits associated with the results are shown in the following table:

[0104]

[0105] g.62719179A>G site and each month of Wan Dong cattle growth traits associated with the results are shown in the following table:

[0106]

[0107] g.62719284G>C site and each month of Wan Dong cattle growth traits associated with the results are shown in the following table:

[0108]

[0109] g.62719317G>A site and each month of Wan Dong cattle growth traits associated with the results are shown in the following table:

[0110]

[0111] In the above table, two groups of data shoulder mark the same letter indicates no significant difference, shoulder mark different letters indicate that the difference is significant. And lower case letters indicate P<0.05, that is, there is a significant difference; capital letters indicate P<0.01, that is, there is a very significant difference.

[0112] The analysis results show that: in the 8 SNP sites of TNNI3 gene, g.62720648C>T, g.62720925C>G, g.62719179A>G, g.62719284G>C, g.62719317G>A have correlation with Wan Dong cattle growth and development traits.

[0113] In summary, the application selects the mutation sites on the TNNI3 gene, analyzes the influence of these mutation sites on the growth and development traits of Wan Dong cattle, and further selects the growth and development traits of Wan Dong cattle from the molecular level.

[0114] The invention is found through experiments and chi-square test shows that: the eight sites of TNNI3 gene g.62720648C>T, g.62720753A>C, g.62720859A>G, g.62720911T>C, g.62720925C>G, g.62719179A>G, g.62719284G>C, g.62719317G>A are all in Hardy-Weinberg equilibrium state (P>0.05), which indicates that the eight sites have good adaptability after long-term selection evolution. The polymorphic information content shows that the six sites of g.62720753A>C, g.62720859A>G, g.62720911T>C, g.62720925C>G, g.62719179A>G, g.62719284G>C are in moderate state (0.25≤PIC<0.5), which indicates that the six sites have strong selection potential in Wandong cattle. The polymorphic information content PIC of the two sites of g.62720648C>T and g.62719317G>A is less than 0.25, so the three sites are low polymorphism, and therefore the two sites of g.62720648C>T and g.62719317G>A in the TNNI3 gene in the sample population are low-level genetic variation.

[0115] The correlation analysis shows that the five sites of g.62720648C>T, g.62720925C>G, g.62719179A>G, g.62719284G>C and g.62719317G>A in the eight SNP sites of TNNI3 gene have correlation with the growth and development traits of Wandong cattle. The five mutation sites are used for molecular breeding of Wandong cattle to breed excellent growth and development traits which can be stably inherited to offspring; the SNP molecular marker provided by the invention is significantly related to the growth and development traits of Wandong cattle, and the SNP marker can be used for assisted selection of the growth and development of Wandong cattle, screening of Wandong cattle with excellent growth and development trait potential, and has important practical application value for further improving the growth and development of Wandong cattle and using specific Wandong cattle as material for breed or strain breeding, which is convenient for breeding personnel to breed and cultivate Wandong cattle.

[0116] The above is only the preferred specific embodiment of the present application, but the protection scope of the present application is not limited thereto, any person skilled in the art can make equivalent replacement or change according to the technical scheme and inventive concept of the present application within the technical range disclosed by the present application, which should be covered in the protection scope of the present application.

Claims

1. Application of a SNP molecular marker in the detection of growth and development traits of Wandong cattle or in Wandong cattle breeding, characterized in that: The SNP molecular marker includes: at least one of a first SNP molecular marker, a second SNP molecular marker, a third SNP molecular marker, a fourth SNP molecular marker, and a fifth SNP molecular marker; The first SNP molecular marker, the second SNP molecular marker, the third SNP molecular marker, the fourth SNP molecular marker, and the fifth SNP molecular marker are all located in the TNNI3 gene on chromosome 18 of the bovine genome; wherein, the GenBank accession number of the bovine genome is AC_000175.1; The first SNP molecular marker is located at the base of position 62720648 on chromosome 18 of the cattle genome, and the SNP molecular marker site has a C / T polymorphism; the second SNP molecular marker is located at the base of position 62720925 on chromosome 18 of the cattle genome, and the SNP molecular marker site has a C / G polymorphism; the third SNP molecular marker is located at the base of position 62719179 on chromosome 18 of the cattle genome, and the SNP molecular marker site has an A / G polymorphism; the fourth SNP molecular marker is located at the base of position 62719284 on chromosome 18 of the cattle genome, and the SNP molecular marker site has a G / C polymorphism; the fifth SNP molecular marker is located at the base of position 62719317 on chromosome 18 of the cattle genome, and the SNP molecular marker site has a G / A polymorphism; The body weight of 6-month-old Wandong cattle with CC genotype and CT genotype of the first SNP molecular marker polymorphism site was significantly higher than that with TT genotype; The CC genotype of the second SNP molecular marker polymorphism site had a significantly higher body length in 24-month-old Wandong cattle than the GG genotype. The GG genotype of the third SNP molecular marker polymorphism site had significantly higher cross section and body oblique length in 12-month-old Wandong cattle than those with the AG genotype. The CC genotype of the fourth SNP molecular marker polymorphism site had a significantly higher body length in 12-month-old Wandong cattle than those with the GG and GC genotypes. The height and weight of 18-month-old Wandong cattle with the GG genotype at the fifth SNP molecular marker polymorphism site were higher than those with the GA genotype. The height, body length and weight of 24-month-old Wandong cattle with the GG genotype were significantly higher than those with the GA genotype. However, the weight of 6-month-old Wandong cattle with the GA genotype was significantly higher than that with the GG genotype.

2. A primer pair for detecting SNP markers in the application of claim 1 in the detection of growth and development traits of Wandong cattle or in Wandong cattle breeding, characterized in that: The primer pair includes: a T3S2 primer pair and / or a T3S4 primer pair, the upstream primer of the T3S2 primer pair is shown as SEQ ID NO: 1, the downstream primer of the T3S2 primer pair is shown as SEQ ID NO: 2, the upstream primer of the T3S4 primer pair is shown as SEQ ID NO: 3, and the downstream primer of the T3S4 primer pair is shown as SEQ ID NO:

4.

3. A kit for detecting SNP markers in the application of claim 1 for detecting growth and development traits of Wandong cattle or in Wandong cattle breeding, characterized in that: Contains the primer pair for use according to claim 2.

4. A method for predicting the growth and development traits of Wandong cattle, characterized in that: Detect the polymorphic site genotype of the SNP molecular marker used in the application as claimed in claim 1.

5. The method according to claim 4, characterized in that The detection is performed using the primer pair in the application of claim 2 or the kit in the application of claim 3.

6. A method for breeding Wandong cattle, characterized in that: Extract the genomic DNA of Wandong cattle, amplify it using the primer pair described in claim 2, and make corresponding selections with reference to the SNP molecular markers described in claim 1: (1) Select the CC genotype and CT genotype of the first SNP molecular marker polymorphic site, and eliminate the TT genotype of the first SNP molecular marker polymorphic site; (2) Select the CC genotype of the second SNP molecular marker polymorphic site and eliminate the GG genotype of the second SNP molecular marker polymorphic site; (3) Select the GG genotype of the third SNP molecular marker polymorphic site and eliminate the AG genotype of the third SNP molecular marker; (4) Select the CC genotype of the fourth SNP molecular marker polymorphic site and eliminate the GG genotype and GC genotype of the fourth SNP molecular marker polymorphic site; (5) If 6-month-old Wandong cattle are needed, the GA genotype of the fifth SNP molecular marker polymorphic site is selected and the GG genotype of the fifth SNP molecular marker polymorphic site is eliminated; If 18-month-old or 24-month-old Wandong cattle are needed, the GG genotype of the fifth SNP molecular marker polymorphic site is selected, and the GA genotype of the fifth SNP molecular marker polymorphic site is eliminated.

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