Application of a SNP molecular marker associated with height traits of beef cattle at 3 months of age

Abstract

The present invention belongs to the technical field of molecular marker-assisted selection of beef cattle, and particularly relates to the application of a SNP molecular marker related to the height trait of beef cattle at 3 months of age. The SNP molecular marker is at the base of the 8674692th site of chromosome 21 in the beef cattle genome GCF_002263795.1_ARS-UCD1.2_genomic.fna, and the SNP site of the molecular marker has a G / C polymorphism, wherein C is a favorable allele variation of the height trait of beef cattle at 3 months of age. By detecting the base type of the molecular marker SNP site, it is used to detect or assist in detecting the height trait of beef cattle at 3 months of age, improve the genetic breeding of the height trait of beef cattle at 3 months of age, and accelerate the breeding process of beef cattle varieties.

Description

Technical Field

[0001] The invention relates to the technical field of molecular marker-assisted selection of beef cattle, and in particular to the application of a SNP molecular marker associated with the height trait of 3-month-old beef cattle. Background Art

[0002] With the continuous improvement of the living standards of Chinese residents, traditional dietary concepts have changed and the dietary consumption structure has been upgraded. High-protein, low-fat beef has gradually been loved by consumers due to its rich nutritional value, which has greatly promoted my country's beef consumption. Nowadays, the output value of my country's beef cattle industry is close to 200 billion yuan, and the number of slaughtered heads and beef production rank first and third in the world respectively. Although my country is a major beef cattle breeding country, due to the large population base, there is still a large gap in beef supply. Local cattle breeds in my country generally have the disadvantages of small size and low yield (small carcass). The average carcass weight of slaughtered beef cattle is 231.5kg in the world, while in my country it is only 147.8kg. Therefore, increasing the carcass weight of my country's beef cattle can significantly increase beef production and reduce the gap between supply and demand.

[0003] The height of a cow refers to the vertical height from the ground to the highest point of the shoulder blade, which can reflect the height of the cow and the degree of skeleton development. It is one of the important indicators for evaluating the body shape of a cow. The growth and development rate of calves is fastest at 3 months of age. Research on the height traits of beef cattle during this period provides a scientific basis for determining the breeding goals of beef cattle and selecting excellent individuals.

[0004] Molecular marker-assisted selection refers to the association analysis of the genotype and phenotypic traits of molecular markers closely linked to the target gene, so as to make more accurate early selection for specific traits. Single nucleotide polymorphism (SNP) is a type of molecular marker, which refers to the change of DNA sequence caused by the variation of a single nucleotide at the same position in the genome between individuals, thereby affecting gene expression. Using SNP to conduct early assisted selection of beef cattle height will help improve the body size and yield level of beef cattle in my country. Summary of the invention

[0005] The purpose of the present invention is to provide a SNP molecular marker associated with the height trait of beef cattle at 3 months of age, so as to provide guidance for the detection of beef cattle growth and meat production performance or molecular marker-assisted breeding.

[0006] The technical solution of the present invention is as follows:

[0007] The invention provides a beef cattle SNP molecular marker, which is located at the base of the 8674692th position of chromosome 21 in the beef cattle genome GCF_002263795.1_ARS-UCD1.2_genomic.fna. The SNP site of the molecular marker has a G / C polymorphism, wherein C is a favorable allele variation of the height trait of beef cattle at 3 months of age.

[0008] Application of the beef cattle SNP molecular marker and the material for detecting the beef cattle SNP molecular marker in any of the following aspects:

[0009] Used to detect or assist in detecting the height trait of 3-month-old beef cattle;

[0010] Used to improve the genetic breeding of height traits of 3-month-old beef cattle;

[0011] Preferably, the nucleotide sequence of the SNP molecular marker is as shown in SEQ ID NO.1.

[0012] In the present invention, the substance for detecting beef cattle SNP molecular markers includes PCR primers for amplifying genomic DNA fragments including the molecular marker SNP site, or a kit containing the primers.

[0013] Preferably, the nucleotide sequences of the primers are shown in SEQ ID NO.2 and SEQ ID NO.3.

[0014] Preferably, the kit further comprises a PCR amplification reagent, and / or an enzyme cleavage reagent, wherein the enzyme cleavage reagent comprises a restriction endonuclease Hha I.

[0015] The present invention also provides a method for detecting the body height trait of beef cattle at 3 months of age, comprising: detecting the base type of position 8674692 of chromosome 21 of the beef cattle genome, the body height of the CC genotype group at 3 months of age is higher than that of the GC genotype group, and the body height of the GC genotype group at 3 months of age is higher than that of the GG genotype group.

[0016] Preferably, the method for detecting the base type at position 8674692 of chromosome 21 of the beef cattle genome includes: designing a primer set for amplifying the nucleotide sequence shown in SEQ ID NO.1, using the primer set to perform PCR amplification on beef cattle genomic DNA, using restriction endonuclease Hha I to digest the obtained amplification product, performing agarose gel electrophoresis on the digestion product, and judging the base type at position 8674692 of chromosome 21 of the beef cattle genome according to the electrophoresis bands: the digestion product is GG type for band 1, CC type for band 2, and GC type for band 3.

[0017] Further preferably, the size of the enzyme digestion product with only one band is 541 bp, the sizes of the enzyme digestion product with two bands are 341 bp and 200 bp respectively, and the sizes of the enzyme digestion product with three bands are 541 bp, 341 bp and 200 bp respectively.

[0018] The present invention also provides a genetic breeding method for optimizing the height trait of beef cattle at 3 months of age, comprising: determining the base type of the 8674692th position of chromosome 21 of the breeding cattle in the core group of beef cattle, and making corresponding selections according to the base type:

[0019] The successive breeding of breeding cattle selects individuals with CC and GC bases at position 8674692 of chromosome 21, and eliminates GG individuals to increase the frequency of gene C at this position generation by generation, thereby optimizing the body height trait of offspring beef cattle at 3 months of age.

[0020] Preferably, the base at position 8674692 of bovine chromosome 21 is as shown in 341 bp of the sequence shown in SEQ ID NO.1.

[0021] Beneficial effects of the present invention:

[0022] The present invention studies and determines the SNP molecular markers that affect the height trait of beef cattle at 3 months of age, and uses the molecular markers for early molecular marker-assisted selection, which can accelerate the breeding process of beef cattle varieties.

[0023] The present invention predicts the height trait of beef cattle at 3 months of age by detecting the base type of the molecular marker SNP site. The height of the CC genotype group at 3 months of age is higher than that of the GC genotype group, and the height of the GC genotype group at 3 months of age is higher than that of the GG genotype group. A primer set for amplifying molecular markers affecting the height trait of beef cattle at 3 months of age is used. Through the molecular markers and primer sets, an efficient and accurate molecular marker-assisted breeding technology for beef cattle is established, which is applied to beef cattle breeding, and can screen calves with excellent height traits at 3 months of age, thereby reducing the breeding cycle and improving the breeding efficiency of cattle farms. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 These are the GWAS results for the height of beef cattle at 3 months of age, where A is the Manhattan plot and B is the QQ plot.

[0025] Figure 2 These are the results of the effect analysis of SNP molecular markers and the height phenotype of beef cattle at 3 months of age.

[0026] Figure 3 Agarose gel electrophoresis results of PCR amplification products.

[0027] Figure 4 The results of agarose gel electrophoresis of the digestion products. DETAILED DESCRIPTION

[0028] The invention performs whole genome resequencing on beef cattle genomic DNA, compares the resequencing data with a beef cattle reference genome (GCF_002263795.1_ARS-UCD1.2_genomic.fna), obtains all high-quality SNPs on the genome, and obtains a molecular marker associated with the body height trait of beef cattle at 3 months of age by analyzing the correlation between each site and the body height trait of beef cattle at 3 months of age. The SNP molecular marker is located at the base of the 8674692th site of chromosome 21 of the cattle genome GCF_002263795.1_ARS-UCD1.2_genomic.fna, and the SNP site of the molecular marker has a G / C polymorphism, wherein C is a favorable allele variation of the body height trait of beef cattle at 3 months of age.

[0029] The nucleotide sequence of the SNP molecular marker of the present invention is shown in SEQ ID NO: 1:

[0030] (SEQ ID NO.1).

[0031] In the sequence SEQ ID NO.1, the base at position 341 has a G / C polymorphism, which is significantly correlated with the body height of beef cattle at 3 months of age. The body height of CC genotype and GC genotype at 3 months of age is extremely significantly higher than that of GG genotype (P<0.01), and the body height of CC genotype at 3 months of age is significantly higher than that of GC genotype (P<0.05).

[0032] In the present invention, if the base at position 341 of the molecular marker nucleotide sequence mutates from G to C, it can be recognized and cut by the restriction endonuclease Hha I. If no mutation occurs, it cannot be cut by Hha I.

[0033] The present invention provides a substance for detecting the beef cattle SNP molecular marker, including PCR primers for amplifying genomic DNA fragments including the molecular marker SNP site, or a kit containing the primers. Among them, the primer set for amplifying the SNP molecular marker of the present invention includes an upstream primer F: 5'-GTAGATAGCACCAGTTGCCAC-3' (SEQ ID NO.2) and a downstream primer R: 5'-GTTGCTGATCTTCACCTTTGTGA-3' (SEQ ID NO.3). The kit also includes a PCR amplification reagent, and / or an enzyme cutting reagent, and the enzyme cutting reagent includes a restriction endonuclease Hha I.

[0034] In the present invention, the PCR amplification reagent is preferably 2×Taq FastMaster Mix, which is composed of TaqDNA polymerase, dNTP mixture, MgCl 2 The reaction buffer is pre-prepared into a mixture of 2 times the concentration and the ratio is optimized, so the amplification efficiency is good and the detection sensitivity is high.

[0035] The above primer set is used to perform PCR amplification on the genomic DNA of beef cattle. As an embodiment, the PCR amplification program is 98°C 45s; 98°C 10s, 55°C 30s, 72°C 1min, 35 cycles; 72°C 5min; 12°C 1min. The obtained amplified product is digested with restriction endonuclease Hha I and electrophoresed. If neither of the two DNA chains has mutated, the electrophoresis detection result is one band (GG type: 541bp); if only one single DNA strand has mutated, the electrophoresis detection result is three bands (GC type: 541bp, 341bp, 200bp); if both DNA double strands have mutated, the electrophoresis detection result is two bands (CC type: 341bp, 200bp).

[0036] The standard for judging the height of cattle at 3 months of age is:

[0037] If the enzyme digestion product is 1 band, it is GG type, and the height of the cattle at 3 months of age is poor;

[0038] If the enzyme digestion product is 2 bands, it is CC type, and the height of the cattle is the best at 3 months of age;

[0039] If the enzyme digestion product is 3 bands, it is GC type, and the body height of the cow at 3 months old is good.

[0040] The electrophoresis results showed that the beef cattle with two bands were the tallest at 3 months old, the beef cattle with three bands were the second tallest at 3 months old, and the beef cattle with one band were the shortest at 3 months old.

[0041] The invention uses restriction endonuclease Hha I to digest the target fragment amplified by the primer set, and by detecting the number of bands after the digestion, the genotype of the cattle can be determined, thereby achieving the purpose of determining the quality of the height trait of the 3-month-old beef cattle, and the height trait of the 3-month-old beef cattle can be detected and the genetic breeding of the height trait of the 3-month-old beef cattle can be optimized.

[0042] The SNP molecular marker of the present invention can be applied to the association analysis of high-correlation genotypes or high-correlation traits of 3-month-old beef cattle, providing a new molecular marker resource for molecular marker-assisted selection of growth and meat production performance of beef cattle.

[0043] The technical solution of the present invention will be described in more detail below in conjunction with the embodiments and drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0044] The reagents and consumables used in the present invention are all common commercial products and can be purchased from the market.

[0045] The following uses Charolais cattle as an example to illustrate the technical solution of the present invention, but it is not intended to limit the technical solution of the present invention. Those skilled in the art can apply the technical solution of the present invention to other beef cattle breeds, and detect and optimize the breeding of the height trait of beef cattle at 3 months of age, which also falls within the protection scope of the present invention.

[0046] Example 1

[0047] Screening of molecular markers associated with body height of beef cattle at 3 months of age

[0048] (1) Charolais cattle phenotypic data collection

[0049] A total of 240 Charolais cattle born between 2013 and 2023 were selected as research subjects, and the height trait data of the Charolais cattle group at 3 months of age were collected.

[0050] (2) Charolais cattle sample collection and genomic DNA preparation

[0051] A 5 mL blood sample was collected from the jugular vein of Charolais cattle using a veterinary blood collection needle and placed in an EDTA anticoagulant tube and stored at 4°C. After extracting leukocytes from Charolais cattle blood samples, the sample genomic DNA was extracted using the Beijing Tianmo DNA extraction kit according to the manufacturer's instructions and stored in a -80°C refrigerator after passing the quality inspection.

[0052] (3) Whole genome resequencing and SNPs detection and filtering

[0053] The DNA that passed the quality inspection was sent to the BGI platform for whole genome resequencing with a sequencing depth of 10× to obtain the original data. Fastp software was used to filter the raw data, and the filtering criteria were as follows: reads with adapters; reads with a certain proportion of N (N means that the base information cannot be determined) (default 5bp); reads containing more than 40% low-quality bases (quality value ≤ 20); sliding window quality trimming: with a sliding window size of 4bp, trimming with an average quality of less than 20. After quality control in the above steps, clean reads were obtained. BWA software was used to align clean reads to the beef cattle reference genome (GCF_002263795.1_ARS-UCD1.2_genomic.fna). The BAM file of the initial alignment result was obtained, sorted using Samtools software, and Picard was used to mark duplicate reads. Then, the GATK4.2.0.0 software toolkit was used to detect SNPs sites, and the Plink software was used to filter the SNP sites (geno 0.1--maf0.05--hwe 1e-06). Finally, Beagle version 5.4 software was used to fill in the genotypes, and finally 4,088,633 high-quality SNPs were obtained for subsequent analysis.

[0054] (4) GWAS analysis

[0055] The FarmCPU model was used to combine the phenotypic values ​​of the height trait of Charolais cattle at 3 months of age with the SNPs loci for GWAS analysis, and Manhattan plots and QQ plots were generated ( Figure 1 ). Taking P=2.44E-07 as the threshold of significant SNP loci, the SNPs loci significantly associated with the height trait of Charolais cattle at 3 months of age were obtained.

[0056] Example 2

[0057] SNPs variation site effect analysis

[0058] According to the significant SNP sites and sample genotype information obtained by GWAS analysis, the beef cattle population in step (1) of Example 1 was grouped, and individuals with the same genotype were divided into the same group and the phenotypic values ​​of each group were counted, and the mean and standard deviation were calculated. Then, the effect size of each SNP site mutation on the corresponding phenotype was analyzed by GraphPadPrism 8 and plotted ( Figure 2 ).

[0059] The results of effect analysis showed that the variation of locus 8674692 on chromosome 21 of the beef cattle genome GCF_002263795.1_ARS-UCD1.2_genomic.fna could increase the body height of Charolais cattle at 3 months of age. The body height trait of the homozygous mutant CC genotype at 3 months of age was significantly higher than that of the GC type (P<0.05) and extremely significantly higher than that of the GG type (P<0.01). CC was a favorable allele variation of this trait.

[0060] Example 3

[0061] PCR-RFLP genotyping, the specific steps are as follows:

[0062] 1) Using the NCBI website, find the gene sequence containing the SNP site in the cattle genome database, select the region extending 1000 bp upstream and downstream of the SNP site as a template, and use Primer5 software to design a specific amplification primer set.

[0063] 2) Collect the blood sample of Charolais cattle to be tested, extract the white blood cells from the blood sample, and use the Beijing Tianmo DNA extraction kit to extract the sample genomic DNA according to the kit instructions. The samples that pass the quality inspection will be used for the next SNP typing.

[0064] 3) Using the extracted whole genome DNA as a template, the DNA sequence containing the SNP molecular marker was amplified by PCR using the primers described in Table 1. The PCR program was 98°C for 45s; 98°C for 10s, 55°C for 30s, 72°C for 1min, 35 cycles; 72°C for 5min; 12°C for 1min. The PCR system is shown in Table 2.

[0065] Table 1 Primer sets and restriction enzymes used in PCR-RFLP

[0066]

[0067] Table 2 PCR reaction system

[0068]

[0069]

[0070] 3) The PCR amplification product was detected using 2% agarose gel electrophoresis at 100 V and 120 mA. After a single band appeared and the size was in line with expectations, subsequent SNP enzyme digestion and typing were performed.

[0071] 4) The PCR amplification product was digested with restriction endonuclease Hha I from Thermo Fisher Scientific. The enzyme digestion system was prepared and incubated at 37° C. for 1 h. The enzyme digestion system is shown in Table 3.

[0072] Table 3 Enzyme digestion reaction system

[0073]

[0074] 5) After the enzyme digestion is completed, the above enzyme digestion products are detected using 2% agarose gel electrophoresis under 100V, 120mA conditions.

[0075] 6) According to the results of enzyme typing ( Figure 3-4 ), judge the quality of the height trait of Charolais cattle at 3 months of age, and select the breeding individuals. The judgment criteria are: if the enzyme digestion product is 1 band, it is GG type, and the height of the Charolais cattle at 3 months of age is poor; if the enzyme digestion product is 2 bands, it is CC type, and the height of the Charolais cattle at 3 months of age is the best; if the enzyme digestion product is 3 bands, it is GC type, and the height of the Charolais cattle at 3 months of age is medium.

[0076] In summary, the SNP molecular markers associated with the 3-month-old body height trait of beef cattle provided by the present invention can be used to identify the 3-month-old body height trait of beef cattle according to genotype, thereby achieving early selection and accelerating the breeding process.

[0077] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A use of a beef cattle SNP molecular marker or a substance for detecting the beef cattle SNP molecular marker in any of the following aspects, characterized in that: Used to detect or assist in detecting the height trait of 3-month-old beef cattle; Used to improve the genetic breeding of height traits of 3-month-old beef cattle; The SNP molecular marker is located at the base of the 8674692th position of chromosome 21 in the beef cattle genome GCF_002263795.1_ARS-UCD1.2_genomic.fna. The SNP site of the molecular marker has a G / C polymorphism, wherein C is a favorable allele variation of the height trait of beef cattle at 3 months of age.

2. The use according to claim 1, characterized in that: The nucleotide sequence of the SNP molecular marker is shown as SEQ ID NO.

1.

3. The use according to claim 1, characterized in that: The substance for detecting beef cattle SNP molecular markers includes PCR primers for amplifying genomic DNA fragments including the molecular marker SNP site, or a kit containing the primers.

4. The use according to claim 3, characterized in that: The nucleotide sequences of the primers are shown in SEQ ID NO.2 and SEQ ID NO.

3.

5. The use according to claim 3, characterized in that: The kit also includes a PCR amplification reagent and / or an enzyme cleavage reagent, wherein the enzyme cleavage reagent includes a restriction endonuclease Hha I.

6. A method for detecting the height trait of beef cattle at 3 months of age, characterized in that: The base type of position 8674692 on chromosome 21 in the beef cattle genome GCF_002263795.1_ARS-UCD1.2_genomic.fna was detected. The body height of the CC genotype group at 3 months of age was higher than that of the GC genotype group, and the body height of the GC genotype group at 3 months of age was higher than that of the GG genotype group.

7. The method according to claim 6, characterized in that The method for detecting the base type of the 8674692nd position of chromosome 21 of the beef cattle genome comprises: designing a primer set for amplifying the nucleotide sequence shown in SEQ ID NO.1, performing PCR amplification on beef cattle genome DNA using the primer set, enzymatically digesting the obtained amplification product using restriction endonuclease Hha I, performing agarose gel electrophoresis on the enzymatic digestion product, and judging the base type of the 8674692nd position of chromosome 21 of the beef cattle genome according to the electrophoresis bands: the enzymatic digestion product has a GG type for band 1, a CC type for band 2, and a GC type for band 3.

8. The method according to claim 7, characterized in that The size of the enzyme digestion product with only one band is 541 bp, the sizes of the enzyme digestion product with two bands are 341 bp and 200 bp respectively, and the sizes of the enzyme digestion product with three bands are 541 bp, 341 bp and 200 bp respectively.

9. A genetic breeding method for optimizing the height trait of beef cattle at 3 months of age, characterized in that: Determine the base type of position 8674692 on chromosome 21 in the cattle genome GCF_002263795.1_ARS-UCD1.2_genomic.fna in the core group of beef cattle, and make corresponding choices based on the base type: The successive breeding of breeding cattle selects individuals with CC and GC bases at position 8674692 of chromosome 21, and eliminates GG individuals to increase the frequency of gene C at this position generation by generation, thereby optimizing the body height trait of offspring beef cattle at 3 months of age.

10. The method according to claim 9, characterized in that The base at position 8674692 of the bovine chromosome 21 is C or G.

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