SNP loci highly associated with cattle cross section and their application

Abstract

The present invention provides a SNP site associated with cattle cross height and its application, wherein the site of the SNP marker is a site on chromosomes 1, 3, 4, 5, 7, 10, 13, 15, 20, and 28 of the cattle reference genome ARS-UCD1.3 version. The present invention can increase the dominant allele frequency from generation to generation by optimizing the dominant allele of the SNP, improve the cross height of cattle, accelerate the progress of cattle genetic improvement, and thus effectively improve the economic benefits of beef cattle breeding.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a SNP site highly correlated with a cross section on a bovine chromosome and an application thereof. Background Art

[0002] The beef cattle industry is an important part of my country's animal husbandry. At present, the prices of domestic live cattle and beef are rising steadily, the number of beef cattle on hand continues to decline, and the supply of cattle is extremely tight. From the perspective of market supply, there is still a huge gap in my country's beef demand. From the perspective of beef consumption, my country's beef demand is still rigid. With the improvement of people's living standards, my country's beef demand has entered an era of both quality and quantity. People's consumption demand for high-end beef is growing. Due to the high technical requirements for the breeding, feeding and production of high-end beef cattle, the market share of domestic high-end beef is still very low.

[0003] Cross height is one of the traits with high heritability among body sizes. When calves of different body sizes are fed to the same body weight, their weight gain rate and feeding efficiency are also different. In addition, body size is greatly affected by genetics and can directly affect body weight. Therefore, cross height has high economic value. In order to accelerate the molecular breeding process of this trait, genome-wide association analysis can be used to identify significant loci that affect the cross height of Chinese cattle. Studies have shown that cross height is a trait with high heritability. Therefore, it is feasible to improve the cross height of resource cattle by genetic means. At present, many QTLs and a large number of single nucleotide polymorphisms (SNPs) related to cross height have been identified on the cattle genome using the genome-wide association study (GWAS) method. However, cross height is a quantitative trait controlled by multiple genes, and there are still a large number of undiscovered quantitative trait loci (QTLs) affecting cross height on the cattle genome. Identifying new molecular markers that affect cross height and adding molecular markers with significant effects to marker assisted selection (MAS) and genomic selection (GS) can accelerate the genetic improvement of cross height, thereby improving the cross height of future generations of beef cattle and increasing the economic benefits of breeding enterprises. Summary of the invention

[0004] In view of the problems existing in the prior art, the present invention provides a method for identifying significant SNPs that affect the height of the cross section of cattle through a GWAS analysis strategy, and using the SNPs in molecular marker-assisted selection and genomic selection to select genotypes that are beneficial to increasing the height of the cross section for breeding, thereby increasing the gene frequency of the dominant allele from generation to generation, which can accelerate the process of breeding and improvement of cattle and bring huge economic benefits to beef cattle breeding.

[0005] The primary purpose is to determine the SNP molecular markers that are highly correlated with the cross section of cattle. The SNP markers are any site on the reference sequence of the ARS-UCD1.3 version of the cattle genome as follows:

[0006] A>C mutation at position 2776850 on chromosome 1;

[0007] C>A mutation at position 14343331 on chromosome 3;

[0008] C>T mutation at position 117584718 on chromosome 4;

[0009] A>G mutation at position 66589147 on chromosome 5;

[0010] T>C mutation at position 1579250 on chromosome 7;

[0011] C>A mutation at position 26471248 on chromosome 7;

[0012] C>T mutation at position 84699323 on chromosome 10;

[0013] C>T mutation at position 69897502 on chromosome 13;

[0014] A>G mutation at position 69914824 on chromosome 13;

[0015] G>A mutation at position 75652089 on chromosome 13;

[0016] T>A mutation at position 4231029 on chromosome 15;

[0017] C>T mutation at position 23205273 on chromosome 20;

[0018] C>T mutation at position 17991101 on chromosome 28;

[0019] T>G mutation at position 34817449 on chromosome 28

[0020] In a preferred embodiment, the SNP markers include all the molecular marker sites mentioned above.

[0021] Another object of the present invention is to provide a method for detecting the above-mentioned SNP molecular marker, characterized in that it comprises the following steps:

[0022] 1) Extracting sample genomic DNA;

[0023] 2) sequencing the above genomic DNA to obtain sequencing reads;

[0024] 3) Aligning the above sequencing reads to the bovine genome;

[0025] 4) Based on the comparison results, determine the genotype of the above SNP site.

[0026] In a preferred embodiment, the sequencing is first-generation sequencing, second-generation sequencing, or third-generation sequencing.

[0027] Another object of the present invention is to provide an application of the above molecular markers in screening cattle individuals with high cross parts, specifically, detecting the genotype of the above SNP molecular marker site, and retaining individuals with CC genotype at position 2776850 on chromosome 1 as breeding cattle in a breeding program.

[0028] Or in a breeding program, keep individuals with AA genotype at position 14343331 on chromosome 3 as breeding cattle,

[0029] Or in a breeding program, keep individuals with TT genotype at position 117584718 on chromosome 4 as breeding cattle,

[0030] Or in a breeding program, keep individuals with AG genotype at position 66589147 on chromosome 5 as breeding cattle,

[0031] Or in a breeding program, keep individuals with CC genotype at position 1579250 on chromosome 7 as breeding cattle,

[0032] Or in a breeding program, keep individuals with CA genotype at position 26471248 on chromosome 7 as breeding cattle,

[0033] Or in a breeding program, keep individuals with CC genotype at position 84699323 on chromosome 10 as breeding cattle,

[0034] Or in a breeding program, keep individuals with CC genotype at position 69897502 on chromosome 13 as breeding cattle,

[0035] Or in a breeding program, keep individuals with AA genotype at position 69914824 on chromosome 13 as breeding cattle,

[0036] Or in a breeding program, keep individuals with AA genotype at position 75652089 on chromosome 13 as breeding cattle,

[0037] Or in a breeding program, keep individuals with AA genotype at position 4231029 on chromosome 15 as breeding cattle,

[0038] Or in a breeding program, keep individuals with TT genotype at position 23205273 on chromosome 20 as breeding cattle,

[0039] Or in a breeding program, keep individuals with TT genotype at position 17991101 on chromosome 28 as breeding cattle,

[0040] Or retain individuals with the GG genotype at position 34817449 on chromosome 28 as breeding cattle in a breeding program.

[0041] In a preferred embodiment, individuals having the above genotypes are retained as breeding cattle.

[0042] Further, the cattle population includes Angus cattle and synthetic lines thereof.

[0043] In a preferred embodiment, detecting the genotype specifically comprises the following steps:

[0044] 1) Extracting genomic DNA from the cattle to be tested;

[0045] 2) sequencing the above genomic DNA to obtain sequencing reads;

[0046] 3) Aligning the above sequencing reads to the bovine genome;

[0047] 4) Based on the comparison results, determine the genotype of the above SNP markers.

[0048] Compared with the prior art, the present invention has the following advantages and effects:

[0049] First, new molecular markers affecting cattle cross height were identified and corresponding detection methods were developed.

[0050] Second, the SNP molecular markers determined by the present invention are applied to the genetic improvement of the cross height of breeding cattle, which can improve the cross height of offspring cattle and thus increase the market competitiveness of breeding enterprises. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] The method of the present invention and its beneficial effects are described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0052] Figure 1 It is the QQ diagram of MLM model analysis of Angus cattle at each stage.

[0053] Figure 2 This is the Manhattan plot of the MLM model analysis of Angus cattle at different stages; Figures a to f are the data for newborn, weaning, 6 months, 12 months, 18 months and 24 months of age respectively.

[0054] Figure 3 It is GO functional enrichment analysis.

[0055] Figure 4 It is KEGG functional enrichment analysis.

[0056] Figure 5 The reads detected by Geneious software were aligned to the results of chromosome 3 site 14343331; Figures a and b are CC genotypes, c and d are CA genotypes, and e and f are AA genotypes.

[0057] Figure 6 The reads detected by Geneious software were aligned to the results of chromosome 13, site 69914824; Figures a and b are AA genotypes, and c and d are AG genotypes.

[0058] Figure 7 The reads detected by Geneious software were aligned to the results of chromosome 7 site 26471248; Figures a and b are CC genotypes, and c and d are CA genotypes. DETAILED DESCRIPTION

[0059] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not 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.

[0060] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by ordinary technicians in the technical field to which this application belongs.

[0061] Example 1 GWAS analysis

[0062] 1. Experimental Animals

[0063] The experimental cattle herd used in the present invention all came from 211 Angus cattle in the Ningxia Angus cattle core farm, which was a core herd selected and bred by the Ningxia Animal Husbandry Station, and all the selected individuals were female individuals.

[0064] The core herd breeding project for Angus cattle was implemented in 2016. Staff members are organized to conduct production performance tests on Angus cattle every month. The test traits include weight, height, cross height, body length, chest circumference, abdominal circumference and tube circumference. The test stages include birth, weaning, 6 months, 12 months, 18 months and 24 months. As of the end of 2021, a large amount of growth trait phenotypic data has been accumulated, and genotypic data have been obtained using ddRAD sequencing technology.

[0065] 2. Sample collection

[0066] 50 ml of venous blood from all individuals in the above cattle group was collected using blood collection tubes and stored in a -80°C refrigerator for later use.

[0067] 3. ddRAD simplifies genome sequencing

[0068] 50ml of venous blood was collected from each of the 211 cattle in the above resource population, and the whole genome DNA was extracted using the standard phenol-chloroform method. The DNA concentration and OD ratio (OD260 / 280, OD260 / 230) of each sample were accurately determined by the Nanodrop2000 / 2000C nucleic acid protein detector. The qualified DNA samples were tested by the NanoDrop2000 / 2000C nucleic acid protein detector, and the DNA was diluted to about 50ng / μL according to the test concentration. Then 6μl of the extracted DNA sample to be tested was mixed with 2μl Loading Buffer, loaded into 1% agarose gel, electrophoresed at 150V for 25min, and observed and photographed under a UV spectrophotometer and gel imaging equipment to observe the integrity of the DNA.

[0069] DNA samples were sent to a sequencing company for sequencing, and the whole genome ddRAD sequencing of cattle was performed according to the company's standard process for genotyping. The European common cattle (Bos taurus, assembly ARS-UCD1.3) was used as the reference genome. The reads obtained by sequencing were aligned to the reference genome using bwa software (version: 0.7.17-r1188), and SNP detection was performed using GATK (version 4.1.4.1) based on the positioning results of CleanReads on the reference genome. The average sequencing depth was greater than or equal to 5; the minimum allele frequency was greater than or equal to 0.05; the information completeness of the sample SNP was greater than or equal to 0.70; the SNP quality Q was greater than or equal to 30; and the final SNP site was obtained after filtering.

[0070] 4. Genome-wide association study (GWAS)

[0071] This study used a mixed linear model (MLM) to conduct genome-wide association analysis of traits and variant sites, and screened SNPs based on the significance of the association (P-value).

[0072] The MLM association analysis model is as follows:

[0073] y=Xα+Zβ+Wμ+e

[0074] Note: Y is the phenotypic trait, X, Z and W are the indicator matrices of genotype (fixed effect), population genetic structure and individual kinship, α is the estimated parameter of fixed effect; β is the effect of SNP; μ is the predicted random individual; e is the random residual, which obeys e~(0, δe2).

[0075] The MLM model was used to conduct association analysis between cross height traits and SNPs in different stages of Angus cattle. After MLM association analysis, 1481 SNPs were identified, of which 860 SNPs were significantly associated, 621 SNPs were potentially associated, and 622 genes were annotated for the significantly associated SNPs. The distribution of SNPs is shown in Table 1. The MLM model identified the most SNPs at 12 months of age, including 369 in the intergenic region, 196 in the intron region, 7 in the upstream region of the gene, 3 in the downstream region of the gene, 2 in the exon region, 2 in the UTR3, and 1 in the intron region of non-coding RNA. There were the fewest SNPs in the weaning stage, only 2, located in the intergenic region and in the intron region respectively. The results of MLM model analysis showed that 428 SNPs were identified to be significantly associated with the cross section of Angus cattle at 18 months of age, while no SNPs were identified to be significantly associated with the cross section of Angus cattle at the weaning stage (Table 2).

[0076]

[0077]

[0078] From the QQ graph of the MLM model analysis, we can see that the MLM analysis model is reasonable and relatively ideal ( Figure 1 ). The MLM model also takes the kinship matrix into account, which can reduce the probability of false positives. Although some significantly associated sites may be eliminated, in order to ensure that the selected sites are more real and effective, the MLM model is chosen for association analysis. Figure 2This is the Manhattan plot of the cross-height trait analysis at each stage of the MLM model. Each SNP site that exceeds the red line threshold represents a significant correlation with the cross-height trait, and those that exceed the blue threshold indicate a potential correlation.

[0079] 5. SNP functional annotation

[0080] For the SNP sites with significant significance after the whole genome association analysis, the functional genes within 1 Mb upstream and downstream of the SNP sites were annotated by combining the SNP annotation information and the genome annotation file, and the obtained genes were subjected to functional enrichment analysis, including Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and the functional genes were preliminarily screened. The MLM model analysis annotated 622 genes, which are mainly involved in various biological processes related to growth and development and muscle development, including striated muscle tissue development, positive regulation of glycolytic process, skeletal system morphogenesis, positive regulation of Notch signaling pathway, and negative regulation of apoptotic process ( Figure 3 ). And it was significantly enriched in Wnt signaling pathway, TGF-β signaling pathway, Hedgehog signaling pathway, Notch signaling pathway,

[0081] Growth and development-related signaling pathways include MAPK signaling pathway, cell adhesion molecules (CAMs), thyroid hormone signaling pathway, and regulation of actin cytoskeleton. Figure 4). Among them, PSEN1 gene is enriched in Wnt signaling pathway; PSEN1 gene and MAML1 gene are both enriched in Notch signaling pathway; PDGFD gene and IGF1 gene are both enriched in MAPK signaling pathway and P13K / Akt signaling pathway; SHH gene is enriched in Hedgehog signaling pathway; IGF1 gene and IL6ST are enriched in the pluripotency pathway of stem cells (Signaling pathways regulating pluripotency of stem cells), and the pluripotency pathway of stem cells is closely related to the understanding of other pathways such as Wnt signaling pathway, Notch signaling pathway, BMP signaling pathway and P13K / Akt signaling pathway; ZMIZ1 gene is enriched in TGF-β signaling pathway; MEF2D gene is enriched in thyroid hormone synthesis, secretion and action pathway. Preliminary analysis screened out 13 genes including IGF1, PSEN1, SHH, MEF2D, PAXBP1, MEGF10, ZHX3, EYA2, IL6ST, ZMIZ1, PDGFD, MAML1 and ARID5B, which may be related to the growth and development of Angus cattle and affect the cross height traits of Angus cattle.

[0082] 6. Analysis of the association between different genotypes and the cross-height phenotype.

[0083] For the genes selected after functional annotation, according to the typing results of their corresponding SNP sites, combined with the cross height traits of 211 Angus cows, association analysis was performed to predict the effect of SNP site mutation on the cross height traits of Angus cattle. 13 genes were initially screened out through functional enrichment analysis. The 13 genes selected were used to associate the SNP sites corresponding to each gene through gene annotation, and the genotypes corresponding to each SNP site were counted. The corresponding genotypes were associated with the phenotypic data of the cross height trait. In the association analysis results, 5 SNP sites were genotyped and then uniformly calculated and analyzed according to two genotypes. Among them, there was a mutation from base C to A at 26471248bp on chromosome 7. The number of cattle with CC genotype, 33 cattle with CA genotype, and 1 cattle with AA genotype were detected. At 66589147bp on chromosome 5, there were 175 cattle with AA genotype, 34 cattle with AG genotype, and only one cattle with GG genotype; at 69897502bp on chromosome 13, there were 185 cattle with CC genotype, 24 cattle with CT genotype, and only one cattle with TT genotype; at 84699323 on chromosome 10, there were 164 cattle with CC genotype, and 47 cattle with CT genotype.; at 117584718bp on chromosome 4, there were 183 cattle with TT genotype, 26 cattle with CT genotype, and only one cattle with CC genotype. Because the number of homozygous genotypes after mutation was too small, they were not used as a group of genotypes for association analysis. The results of association analysis showed that only the mutations at 84699323bp on chromosome 10 and 69897502bp and 69914824bp on chromosome 13 would lead to a significant decrease in the cross height trait, while the mutations at the other sites would significantly increase the cross height trait (Table 3). In summary, the identified sites all have a significant effect on the cross height trait of Angus cattle.

[0084]

[0085]

[0086] Example 2 Verification of polymorphic sites

[0087] In order to verify the authenticity of the SNP sites obtained by Qualcomm sequencing and not caused by sequencing errors or alignment errors, three sites were randomly selected and two sequencing samples with different genotypes were selected. The Geneious software was used to re-align the complete reads of the target SNP sites and check the base types and quality values ​​of the corresponding SNP sites.

[0088] The comparison results are as follows Figure 5-Figure 7 As shown in the figure, the base types of each site are accurate. The sequencing quality value of the corresponding site reaches SNP quality Q greater than or equal to 30, that is, the base accuracy is greater than 99.9%, and the sequencing and alignment data are accurate and reliable.

[0089] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to the above embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A breeding method for increasing the cross height of Angus cattle, characterized in that: The method comprises the following steps: detecting the genotype of a SNP site on chromosome 1, wherein the SNP site is located at position 2776850 of chromosome 1 of the cattle genome ARS-UCD1.3 version reference genome, and selecting individuals with CC genotype at position 2776850 on chromosome 1 as breeding cattle.

2. The breeding method for increasing the cross height of Angus cattle according to claim 1, characterized in that: The genotype detection of the SNP site on chromosome 1 comprises the following steps: 1) Extracting genomic DNA from the cattle to be tested; 2) sequencing the above genomic DNA to obtain sequencing reads; 3) aligning the above sequencing reads to the bovine genome; 4) Based on the comparison results, determine the genotype of the SNP site in claim 1.

3. The breeding method for increasing the cross height of Angus cattle according to claim 2, characterized in that: The sequencing is first generation sequencing, second generation sequencing, or third generation sequencing.

4. The application of SNP loci as the detected object in improving the high quality of Angus cattle cross section, characterized in that: The SNP site is located at position 2776850 of chromosome 1 of the cattle genome ARS-UCD1.3 version reference genome, wherein the cross section of cattle with CC genotype is the highest and the cross section of cattle with AC genotype is the shortest.

Images