A whole-genome liquid-phase chip for cattle and its application

Abstract

This invention belongs to the field of whole-genome gene chip technology, specifically relating to a liquid-phase whole-genome chip for cattle and its applications. Based on the SNP molecular marker combination provided by this invention (location information is shown in Table 1 of the specification), genotyping of cattle can be achieved through targeted capture sequencing technology, enabling high-throughput detection of cattle genotypes in a short time, thereby promoting cattle breeding. Furthermore, probes designed based on the cattle SNP molecular marker combination provided by this invention can be used to construct a whole-genome breeding chip, which can achieve high detection rates and stability for large-scale genotyping of cattle, and has broad platform applicability. It can be applied to the identification and mining of germplasm resources, genetic diversity analysis, population structure analysis, kinship identification, and whole-genome selection breeding of cattle breeds, especially local cattle breeds in Anhui Province, which is of great significance for improving the efficiency of independent breeding of core cattle breeds.

Description

Technical Field

[0001] This invention belongs to the field of whole genome gene chip technology, specifically relating to a whole genome liquid phase chip of cattle and its application. Background Technology

[0002] Dabie Mountain cattle, Southern Anhui cattle, and Eastern Anhui cattle are excellent local yellow cattle breeds in Anhui Province. Dabie Mountain cattle are dual-purpose (draft and meat) breeds, mainly distributed on the southern slopes of the Dabie Mountains, including neighboring counties in western Dabie Mountain area of ​​Hubei Province and eastern Dabie Mountain area of ​​Anhui Province. They are listed as local breeds in the National Livestock and Poultry Breed Resources Register. Dabie Mountain cattle have a compact body structure, are relatively small in size, and have fine bones. They are known for their tolerance to roughage, tender meat, and rich flavor. Southern Anhui cattle are also dual-purpose (draft and meat) breeds, mainly produced in Yixian, Shexian, Jixi, and Jingde counties south of the Yangtze River in Anhui, as well as the mountainous areas bordering Anhui and Zhejiang, and Anhui and Jiangxi provinces. Long-term mountain grazing has shaped Southern Anhui cattle into breeds that are resistant to roughage, heat, and humidity, with tender meat and excellent flavor. Eastern Anhui cattle are dual-purpose (meat and draft) breeds, mainly produced in the mountainous areas of Fengyang, Dingyuan, Changfeng, Mingguang, and Lai'an, located on the Jianghuai watershed. The Wandong cattle are docile, relatively large in size, tolerant of roughage, and have strong disease resistance, resulting in good meat quality. Overall, while local breeds such as Dabie Mountain cattle, Southern Anhui cattle, and Wandong cattle may not be as good as introduced breeds in terms of growth and development, slaughter performance, and feed costs, they have advantages such as good meat flavor and high nutritional value.

[0003] Due to breed characteristics and a lack of systematic breeding, local yellow cattle breeds in Anhui Province face problems such as low beef yield, slow growth rate, and low breeding efficiency, which seriously restrict the high-quality development of the beef cattle industry in Anhui Province. Therefore, it is urgent to develop breeding chips suitable for genomic selection of local yellow cattle breeds in Anhui Province, and to promote the transformation of traditional breeding of Anhui cattle into targeted and efficient molecular breeding, which is particularly important for achieving high-quality development of the Anhui cattle industry. Summary of the Invention

[0004] The purpose of this invention is to provide a whole-genome liquid phase chip for cattle and its application, which can be used for population genotyping, germplasm resource identification and mining, genetic diversity analysis, population structure analysis, kinship identification, whole-genome association analysis and whole-genome selection breeding of cattle, especially local cattle breeds in Anhui Province.

[0005] To achieve the above objectives, the present invention provides a bovine SNP molecular marker combinatorial system, which includes 21,608 SNP molecular markers. The location information of the SNP molecular markers on the bovine genome in ARS-UCD 1.2 is shown in Table 1 of the specification.

[0006] The present invention also provides a probe targeting the bovine SNP molecular marker combination described in the above technical solution.

[0007] Preferably, the GC content of the probe is between 40% and 60%, and the number of homologous regions of the probe sequence in the whole genome is ≤5.

[0008] The present invention also provides a whole-genome SNP chip for cattle, including the probes described in the above technical solution.

[0009] Preferably, the chip is a liquid phase chip.

[0010] The present invention also provides a whole-genome SNP kit for cattle, including the whole-genome SNP chip of cattle described in the above technical solution.

[0011] This invention also provides the application of reagents for detecting the bovine SNP molecular marker combinations described in the above-mentioned technical solutions, or probes described in the above-mentioned technical solutions, or bovine whole-genome SNP chips described in the above-mentioned technical solutions, or bovine whole-genome SNP kits described in the above-mentioned technical solutions, in one or more of the following:

[0012] (1) Genotyping of yellow cattle breeds;

[0013] (2) Application in cattle breeding;

[0014] (3) Locating genes related to traits in yellow cattle;

[0015] (4) Analysis of genetic diversity in cattle;

[0016] (5) One or more of the following: identification, excavation, improvement and protection of cattle germplasm resources;

[0017] (6) Genome-wide association analysis of cattle;

[0018] (7) Background selection of cattle based on molecular markers.

[0019] Preferably, the identification of cattle germplasm resources includes one or more of the following: identification of cattle breed genetic diversity, analysis of cattle breed population structure, and identification of cattle kinship.

[0020] Preferably, the yellow cattle include Anhui yellow cattle.

[0021] Preferably, the Anhui cattle include one or more of the following: Dabie Mountain cattle, Southern Anhui cattle, and Eastern Anhui cattle.

[0022] Beneficial effects:

[0023] This invention provides a bovine SNP molecular marker combinatorial system comprising 21,608 SNP molecular markers. The location information of these SNP molecular markers on the bovine genome in ARS-UCD version 1.2 is shown in Table 1. Based on the SNP molecular marker combinatorial system provided by this invention, genotyping of bovine cattle can be achieved using targeted capture sequencing technology, enabling high-throughput detection of bovine genotypes in a short time, thereby promoting bovine breeding. Compared to traditional solid-phase microarrays, this invention offers rich functional sites, high trait correlation, and uniform genome distribution, which can promote basic and applied research on bovine cattle.

[0024] Furthermore, this invention designs probes based on the aforementioned combinations of SNP molecular markers in cattle and constructs a whole-genome breeding chip. This chip enables large-scale genotyping of cattle with high detection rates, good stability, and broad platform applicability. Compared to traditional markers such as SSRs based on gel electrophoresis detection, it has significant advantages in throughput and timeliness. It can detect cattle genotypes in a short time with high throughput and can be applied to the identification and exploration of germplasm resources, genetic diversity analysis, population structure analysis, kinship identification, and whole-genome selection breeding of cattle breeds, especially local cattle breeds in Anhui Province. This is of great significance for improving the efficiency of independent breeding of core cattle breeds. Attached Figure Description

[0025] 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.

[0026] Figure 1 This is a map showing the location distribution of SNP sites on chromosomes in the whole genome liquid-phase microarray of cattle, as described in Example 1.

[0027] Figure 2 Example 1: Distribution of SNP loci types on the whole genome liquid-phase microarray of cattle;

[0028] Figure 3 Example 1: Distribution map of SNP sites on liquid-phase microarray in the whole genome of cattle (MAF).

[0029] Figure 4 This is a distribution chart of the detection rate of the whole genome liquid phase chip sample of cattle in Example 2;

[0030] Figure 5 This is a graph showing the genotyping consistency of duplicate samples from the whole genome liquid-phase microarray in Example 2.

[0031] Figure 6 Example 3: LD decay analysis of the Dabie Mountain cattle population;

[0032] Figure 7 Principal component analysis distribution diagram of the Dabie Mountain cattle population in Example 4;

[0033] Figure 8 This is a phylogenetic tree diagram of the Dabie Mountain cattle population in Example 5. Detailed Implementation

[0034] This invention provides a bovine SNP molecular marker combinatorial system comprising 21,608 SNP molecular markers. The location information of the SNP molecular markers on the bovine genome in ARS-UCD 1.2 is shown in Table 1 of the specification.

[0035] The present invention also provides a probe targeting the bovine SNP molecular marker combination described in the above technical solution.

[0036] In one embodiment, the GC content of the probe of the present invention is between 40% and 60%. In another embodiment, the number of homologous regions of the probe sequence across the entire genome is ≤5. In another embodiment, the length of the probe of the present invention is 120 bp. In yet another embodiment, the capture region of the probe of the present invention is 200-300 bp. The present invention does not impose strict requirements on the preparation method of the probe; it can be synthesized by a biotechnology company using the bovine SNP molecular marker combination provided by the present invention.

[0037] The present invention also provides a whole-genome SNP chip for cattle, including the probes described in the above technical solution.

[0038] As one implementation method, the chip described in this invention is a liquid phase chip.

[0039] The present invention also provides a whole-genome SNP kit for cattle, including the whole-genome SNP chip of cattle described in the above technical solution.

[0040] The bovine SNP molecular marker combination, bovine whole-genome SNP chip, and bovine whole-genome SNP kit provided in this invention can be used for bovine genotyping via targeted capture sequencing technology. Compared to traditional solid-phase chips, this invention can detect more SNP loci and is flexible in design, allowing for the addition of new marker loci later. Compared to whole-genome resequencing, this invention has a significant price advantage, enabling large-scale bovine genotyping and thus promoting bovine breeding. Compared to traditional SSR and other gel electrophoresis-based markers, this invention has significant throughput and time advantages, enabling high-throughput detection of bovine genotypes in a short time, thereby promoting basic and applied research on bovine genotypes.

[0041] Given the advantages of the cattle SNP molecular marker combination, cattle whole genome SNP chip, and cattle whole genome SNP kit provided by this invention, the application of the cattle SNP molecular marker combination, cattle whole genome SNP chip, and cattle whole genome SNP kit described in this invention in one or more of the following is also within the scope of protection of this invention: (1) cattle breed genotyping; (2) application in cattle breeding; (3) cattle trait-related gene localization; (4) cattle genetic diversity analysis; (5) identification, excavation, improvement, and protection of cattle germplasm resources (one or more); (6) cattle whole genome association analysis; (7) cattle marker-based background selection.

[0042] In one embodiment, the identification of cattle germplasm resources according to the present invention includes one or more of the following: identification of cattle breed genetic diversity, analysis of cattle breed population structure, and identification of cattle kinship. In one embodiment, the cattle described in the present invention include Anhui cattle. In one embodiment, the Anhui cattle described in the present invention include one or more of Dabie Mountain cattle, Southern Anhui cattle, and Eastern Anhui cattle.

[0043] To further illustrate the present invention, the following detailed description of a whole-genome liquid phase chip for cattle and its applications, in conjunction with the accompanying drawings and embodiments, is provided but should not be construed as limiting the scope of protection of the present invention.

[0044] Example 1

[0045] Design and fabrication of whole-genome liquid-phase microarray of cattle

[0046] 1. Site screening

[0047] (1) Resequencing was performed on Dabie Mountain cattle, Southern Anhui cattle, and Eastern Anhui cattle at a sequencing depth of 15X. Using the ARS-UCD 1.2 version of the bovine genome as a reference sequence, the resequencing data of different cattle breeds were aligned to the reference genome. Fatty acids were measured in the outer lobe of Dabie Mountain cattle, and transcriptome analysis was performed according to high and low fatty acid groups to identify differentially expressed genes and screen for key candidate gene loci related to beef quality traits of Dabie Mountain cattle. Based on the genome-wide association analysis of candidate loci and related loci within the genome-wide sliding window for body size traits of Dabie Mountain cattle (body weight, height, cross height, straight body length, oblique body length, chest girth, cannon bone girth, abdominal girth, ischial end width, and loin angle width), a total of 50K SNP loci were obtained for subsequent screening.

[0048] (2) Optimize and screen the 50K SNP sites detected above: 1. Selected SNP sites are evenly distributed across all chromosomes; 2. Exclude SNPs with a deletion rate exceeding 5%; 3. Exclude SNPs that do not conform to Hardy-Weinberg equilibrium ( pSNPs with a deletion value less than 0.000001 were excluded; 4. Samples with a deletion rate exceeding 10% were excluded; 5. SNPs with a small allele frequency below 5% were excluded. Finally, a total of 21,608 high-quality SNP loci corresponding to the ARS-UCD 1.2 bovine genome were obtained for microarray preparation. The distribution information of these SNP loci is as follows: Figures 1-3 As shown in Table 1, the coordinates of these SNP sites on the genomes of Dabie Mountain cattle and ARS-UCD 1.2 cattle are shown in Table 1.

[0049] Table 1. Location and variation information of 21,608 high-quality SNP loci.

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[0104] 2. Preparation of whole-genome liquid-phase microarray of cattle

[0105] Probe design was based on CAGT® targeted capture sequencing technology from Beijing Compson Biotechnology Co., Ltd. The target site and upstream / downstream 200bp genomic sequences were aligned to the ARS-UCD 1.2 bovine genome. Primers were designed based on a primer length of approximately 21bp, a TM temperature of 59–64℃, and a GC content of 40%–60%. Non-specific and primer dimer evaluations were performed, filtering out non-specific primers and primer dimers. The GC content, complexity, number of homologous regions, and SSR high repetition of the target site and upstream / downstream genomic sequences were evaluated. Probe sequences with a length of 120bp, a capture region of 200–300bp, a GC content between 40% and 60%, and ≤5 homologous regions across the whole genome were selected to cover the target site, thus designing a whole-genome probe sequence library. Detection rate, depth, and consistency of genotyping of repetitive samples were tested, and the results were delivered to Beijing Compson Biotechnology Co., Ltd. for the preparation of a genome breeding liquid-phase chip.

[0106] Example 2

[0107] Quality assessment of whole-genome liquid-phase chip genotyping in cattle

[0108] 1. Extraction of genomic DNA from cattle

[0109] Blood samples from 10 local yellow cattle (Dabie Mountain cattle) in Anhui Province were selected as test samples and numbered W1 to W10. Whole genomic DNA was extracted from the test samples using a whole blood genomic DNA extraction kit.

[0110] 2. DNA sample quality testing

[0111] DNA purity was determined by 1% agarose gel electrophoresis (A). 260 / A 280 The ratio should be greater than 1.8, A 260 / 230 (Between 1.8 and 2.2) and integrity, the Qubit fluorescence quantitative quantitation instrument measures the concentration of genomic DNA.

[0112] 3. Bovine liquid phase chip detection

[0113] (1) Detection rate detection

[0114] Following the instructions of the MGI library construction kit, DNA libraries were constructed for each sample, and the quality of library construction was evaluated. The bovine whole-genome liquid-phase chip-specific probes obtained in Example 1 were hybridized with genomic DNA to capture and enrich target region sequences, which were then subjected to high-throughput sequencing on the Illumina sequencing platform. The quality of the data was checked using FastQC software, and Illumina adapters and low-quality sequences were removed using Trimmomatic software. The quality-controlled reads were aligned to the ARS-UCD 1.2 bovine genome using BWA software, and genotyping of the genomic breeding liquid-phase chip was performed using GATK software. The results are as follows: Figure 4 As shown. According to Figure 4 As can be seen, the average detection rate can reach over 99.84%, indicating that the whole genome liquid phase chip of cattle obtained in Example 1 has excellent performance and can realize rapid detection and analysis of high-throughput, low-cost genotyping of local cattle populations in Anhui Province.

[0115] (2) Repeatability test

[0116] Following the steps in step (1), the whole-genome liquid phase chip of cattle obtained in Example 1 was used to perform three repeated tests on a sample of cattle DNA for genotyping, and the detection rate was calculated. The results are as follows: Figure 5 As shown. According to Figure 5 It can be seen that the genotypic consistency rate of the duplicated samples can reach over 99.82%, indicating that the whole-genome liquid phase chip of cattle obtained in Example 1 has excellent performance and good reproducibility.

[0117] Example 3

[0118] Application of whole-genome liquid phase chip in the analysis of cattle population structure in Dabie Mountain

[0119] Based on the whole-genome liquid phase chip of cattle obtained in Example 1, genotyping of blood samples from 33 Dabie Mountain cattle was detected according to the steps in Example 2. LD decay analysis of the Dabie Mountain cattle population was performed using Plink software. It was found that when the physical distance was small, the average R² value was high, close to 0.45, indicating a strong linkage disequilibrium between loci. The decrease was rapid in the 0-25 kb range, and then the decrease stabilized with increasing physical distance, indicating a gradual weakening of the linkage disequilibrium between loci. Figure 6 ).

[0120] Example 4

[0121] Application of whole-genome liquid phase chip in kinship identification of cattle population in Dabie Mountains

[0122] Based on the whole-genome liquid phase chip of cattle obtained in Example 1, the genotyping of blood samples from 30 Dabie Mountain cattle was detected according to the steps in Example 2. Principal component analysis (PCA) of the Dabie Mountain cattle population was performed using PLINK software. It was found that the samples were concentrated in the central region, and the first two principal components explained approximately 22.74% of the genetic variation, indicating that there is a certain degree of genetic differentiation within the population, but the overall genetic background is relatively similar. Figure 7 ).

[0123] Example 5

[0124] Application of liquid phase microarray in genetic diversity analysis of cattle

[0125] Based on the whole-genome liquid phase chip of cattle obtained in Example 1, the genotyping of blood samples from 30 Dabie Mountain cattle was detected according to the steps in Example 2, and a phylogenetic tree was constructed. The phylogenetic tree of the 30 Dabie Mountain cattle was found to be clustered into four branches at the molecular level, indicating significant genetic diversity within this population. Each branch represents a group of genetically relatively independent individuals, who may have certain genetic differences. The existence of these families also indicates that Dabie Mountain cattle possess certain evolutionary potential and adaptability. Figure 8 ).

[0126] As can be seen from the above, the technical solution provided by this invention can detect the genotype of cattle in a short time with high throughput. It can be applied to the identification and mining of germplasm resources, genetic diversity analysis, population structure analysis, kinship identification and whole-genome selection breeding of cattle breeds, especially local cattle breeds in Anhui Province. It is of great significance to improve the efficiency of independent breeding of core cattle breeds.

[0127] 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 bovine SNP molecular marker combinatorial system, characterized in that, The bovine SNP molecular marker assemblage includes 21,608 SNP molecular markers, and the location information of the SNP molecular markers on the bovine genome in ARS-UCD 1.2 is shown in Table 1 of the specification.

2. A probe targeting the bovine SNP molecular marker combination as described in claim 1.

3. The probe according to claim 2, characterized in that, The probe has a GC content between 40% and 60%, and the number of homologous regions of the probe sequence across the whole genome is ≤5.

4. A bovine whole-genome SNP chip, characterized in that, Includes the probe described in claim 2 or 3.

5. The bovine whole-genome SNP chip according to claim 3, characterized in that, The chip is a liquid phase chip.

6. A whole-genome SNP kit for cattle, characterized in that, Includes the cattle whole genome SNP chip as described in claim 4 or 5.

7. The application of the reagent for detecting the bovine SNP molecular marker combination of claim 1, the probe of claim 2, the bovine whole genome SNP chip of claim 4 or 5, or the bovine whole genome SNP kit of claim 6 in the identification of bovine germplasm resources; The identification of cattle germplasm resources includes one or more of the following: identification of cattle breed genetic diversity, analysis of cattle breed population structure, and identification of cattle kinship. The cattle in question are Dabie Mountain cattle.

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