Holstein cow breeding probe combination, gene chip, kit and application

Abstract

The application discloses a Holstein cow breeding probe combination, a gene chip, a kit and application, the probe combination can detect 140290 SNP sites based on the reference genome ARS-UCD1.2. The site detected by the Holstein cow breeding probe combination is obtained from 77887 sites after further screening based on 4600 Holstein cow genome sequencing data, in combination with 62387 SNP sites related to yield, reproduction and body shape and 16 defect-related SNP sites, and can effectively reflect the structural heterogeneity of the genome itself. The site detected by the Holstein cow breeding probe combination has high polymorphism information content, and fully covers high polymorphism sites. Meanwhile, the interpolation accuracy is high, the genotyping consistency is equivalent to that of a high-density chip, the cost is significantly lower, and the economy is better, and the application provides an independent technical tool for efficient and accurate genome breeding of Holstein cows.

Description

Technical Field

[0001] This invention relates to bovine molecular breeding, and more particularly to a Holstein bovine breeding probe assembly, gene chip, reagent kit, and its applications. Background Technology

[0002] Molecular breeding is a core technological paradigm in modern agricultural breeding. By utilizing molecular marker information at the DNA level, it enables precise assessment and selection of an individual's genetic potential, significantly surpassing traditional breeding methods that rely on phenotypic observation. In this technological system, genomic selection has become a key pathway. Its core lies in using tens of thousands of single nucleotide polymorphism markers covering the entire genome to construct a genomic breeding value prediction model. This allows for accurate prediction of an individual's breeding value early in life, significantly shortening generation intervals and accelerating genetic progress.

[0003] SNPs, as third-generation molecular markers, refer to variations in a single base in the genome. They are abundant, widely distributed, and highly stable, making them ideal markers for constructing gene chips. By detecting the genotypes of tens of thousands of SNP loci throughout an individual's genome, a detailed genetic map can be obtained, providing a data foundation for genomic selection.

[0004] However, existing commercial bovine SNP chips have significant limitations in application. Current mainstream chips are primarily designed based on the genetic background of breeds such as Holstein cattle in Europe and America, and their SNP loci selection and allele frequencies reflect the characteristics of Western breeding populations. When directly applied to native Holstein cattle herds, due to differences in population genetic structure, breeding history, and environmental adaptability, problems such as a reduced proportion of polymorphic loci and insufficient effective information often occur, leading to decreased accuracy in genome prediction and hindering further improvements in dairy cattle breeding efficiency.

[0005] Existing technologies such as CN118726604A and CN117431324A provide liquid-phase microarrays with approximately 44,000 loci and medium-to-high density microarrays with 120,000 loci, respectively, playing a significant role in advancing molecular breeding of dairy cattle. However, with the continuous expansion of Holstein cattle breeding in my country and the refinement of breeding objectives, higher demands are placed on microarrays in terms of marker density, locus structure adaptability, and coverage of key traits. For example, the former's locus quantity and coverage are mainly geared towards specific target regions, making it difficult to meet the needs of high-density genomic selection models; the latter relies more on existing international commercial microarrays in its locus system construction, and there is still room for further optimization between its locus frequency structure and the genetic characteristics of the Chinese Holstein cattle population. Furthermore, neither microarray fully utilizes the large-scale whole-genome sequencing data accumulated in my country in recent years for systematic mining of high-information-content SNPs, making it difficult to maintain high predictive efficacy in complex trait selection, reference group expansion, and long-term iterative breeding. Summary of the Invention

[0006] Purpose of the invention: The purpose of this invention is to provide a Holstein cattle breeding probe combination that is developed based on a large sample of Chinese Holstein cattle data, has more comprehensive loci, richer information, and higher adaptability; the second purpose is to provide gene chips and reagent kits containing this Holstein cattle breeding probe combination; the third purpose is to provide applications of the above products.

[0007] Technical solution: The Holstein cattle breeding probe combination of the present invention is characterized in that the probe combination detects a combination of Holstein cattle SNP molecular markers, the combination of molecular markers consists of 140,290 SNP sites, and its location information based on the reference genome ARS-UCD1.2 is shown in Table 1.

[0008] Table 1. SNP molecular marker site information in Holstein cattle

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[0344] In Table 1, the site information is formatted as: chromosome number_location.

[0345] Preferably, the molecular marker combination includes: 77,887 sequencing-derived high genetic information SNP loci, 62,387 trait-related loci, and 16 defect-related loci.

[0346] More preferably, the method for establishing high genetic information SNP sites from sequencing sources in the molecular marker set includes:

[0347] (1) Raw filtering and indexing based on the reference genome

[0348] (2) Select suitable Chinese Holstein cattle for whole-genome sequencing;

[0349] (3) Optimize the sequencing results;

[0350] (4) Use software to filter and screen to obtain SNP sites with high genetic information.

[0351] Further preferably, the software in step (4) is a software for selecting high genetic information loci in the process of designing livestock and poultry genome chips, with the software copyright registration number 2024SR1581643.

[0352] Preferably, the probes in the probe assembly use sequences within 20 bases upstream and downstream of the single nucleotide polymorphism molecular marker site as design reference sequences, with a length of 18-30 bases; more preferably, the probes in the probe assembly have a GC content between 40% and 60%, and an annealing temperature between 55 and 65°C.

[0353] The gene chip described in this invention is loaded with the Holstein cattle breeding probe combination as described above.

[0354] Preferably, the chip is a liquid-phase chip or a solid-phase chip.

[0355] The kit of the present invention is characterized in that it contains the aforementioned Holstein cattle breeding probe combination or gene chip.

[0356] The application of the Holstein cattle breeding probe combination, gene chip, or kit described in this invention in the molecular selection of Holstein cattle.

[0357] The application of the Holstein cattle breeding probe combination, gene chip, or kit described in this invention in the analysis of the genetic structure of Holstein cattle populations.

[0358] The application of the Holstein cattle breeding probe combination, gene chip, or kit described in this invention in the analysis of genomic kinship of Holstein cattle.

[0359] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: 1. The sites detected by this Holstein cattle breeding probe combination are obtained by further screening based on the genome sequencing data of 4600 Holstein cattle, combined with SNP sites related to yield, reproduction, and body size, as well as defect-related SNP sites, which can effectively reflect the structural heterogeneity of the genome itself; 2. The sites detected by this Holstein cattle breeding probe combination have high polymorphism information content, fully covering highly polymorphic sites; 3. The site interpolation accuracy of this Holstein cattle breeding probe combination is high, the genotyping consistency is comparable to that of high-density chips, the cost is significantly lower, and it has better economic efficiency, providing an independent technical tool for efficient and precise genome breeding of Holstein cattle. Attached Figure Description

[0360] Figure 1 A schematic diagram of the gene chip kit product "Ningyang No. 1" based on Holstein cattle breeding probe combination;

[0361] Figure 2 A statistical graph showing the number of SNP loci detected by probe combinations for Holstein cattle breeding on 29 autosomes and the X chromosome;

[0362] Figure 3 Density distribution of SNP loci on 29 autosomes and the X chromosome detected by probe combinations for Holstein cattle breeding;

[0363] Figure 4 A statistical diagram showing the distribution of minor allele frequencies at SNP sites detected by a probe combination for Holstein cattle breeding.

[0364] Figure 5 Statistical graph of SNP site mutation types detected by probe combination for Holstein cattle breeding;

[0365] Figure 6 Figure showing the comparison of autosomal genome interpolation accuracy between gene chips based on Holstein cattle breeding probe combinations and commercial 100K chips;

[0366] Figure 7 The figure shows the consistency of genotyping results between the gene chip based on the Holstein cattle breeding probe combination and the 150K commercial chip for hair follicle samples.

[0367] Figure 8 The figure shows the comparison results of genotyping consistency between a gene chip based on a Holstein cattle breeding probe combination and a 150K commercial chip in blood samples.

[0368] Figure 9 The image shows the results of principal component analysis of Holstein cattle using a gene chip based on a combination of Holstein breeding probes.

[0369] Figure 10 The image shows the results of using a gene chip based on a combination of Holstein breeding probes for Holstein bovine pedigree analysis.

[0370] Figure 11 This image shows the results of a gene chip based on a combination of Holstein breeding probes used for Holstein state homology analysis. Detailed Implementation

[0371] The technical solution of the present invention will be further described below.

[0372] Example 1: Establishment of Holstein cattle breeding probe combination and preparation of gene chip

[0373] (1) 4,600 Chinese Holstein cattle were selected from 15 ranches under Ningxia Dairy Group. Genomic DNA was extracted from tail venous blood. The purity of the DNA was detected using NanoDrop2000, the DNA concentration was accurately quantified using Qubit2.0, and the integrity of the genomic DNA was detected by agarose gel electrophoresis. The qualified DNA was sent to Shijiazhuang Borui Biotechnology Co., Ltd. for whole genome sequencing, and 30× depth sequencing data were obtained.

[0374] (2) Based on the bovine assembled genome ARS-UCD1.2, raw filtering and reference genome indexing were performed using NGSQCToolkit (v2.3) and Burrows-WheelerAligner (v0.7.17). File format conversion was performed using Samtools (v1.21), and Picard (v3.0) was used for reordering and marking identical reads, while simultaneously generating a new index. The original .vcf file was generated using Genome Analysis Toolkit (v4.3). SNP and index selection were then performed. Finally, SNP filtering was performed to obtain SNPs from the 30× depth sequencing data. The filtering requirements were as follows: minimum allele frequency greater than 0.05, and a Hardy-Weinberg equilibrium p-value greater than 10. -6 SNP sites with a detection rate greater than 90%;

[0375] (3) Using genome-wide association analysis (GWAS) and consulting the bovine quantitative trait database (https: / / www.animalgenome.org / cgi-bin / QTLdb / BT / index), we screened for Holstein cattle traits related to milk production (milk yield, milk fat percentage, milk protein percentage, somatic cell count), reproduction (calving difficulty, calving interval), and body conformation (the trait measurement process referred to the Technical Specifications for Body Conformation Identification of Holstein Cattle in China (National Standard No.: GB / T)). A total of 62,387 functional SNPs and 16 defect-related SNPs were obtained from the 35568-2017 SNP data and genetic defect-related SNPs. Then, using SNPSelection (v1.0, software copyright registration number 2024SR1581643), a software for selecting high genetic information loci in livestock genome chip design, 77,887 high genetic information SNP loci were selected from the SNPs in the 30× depth sequencing data. Functional loci were then screened using genome-wide association analysis and CattleQTLdb (https: / / www.animalgenome.org / cgi-bin / QTLdb / BT / srchsnp) to establish the whole-genome SNP molecular marker combination for Holstein cattle. The location information based on the reference genome ARS-UCD1.2 is shown in Table 1; the locus composition information is shown in Table 2.

[0376] Table 2. 140K chip site composition

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[0379] Based on the whole genome SNP molecular marker combination of Holstein cattle, probes were designed using Primer3 (v2.6.1) for all 140,290 sites. The design principles are as follows: the sequence within 20 bases upstream and downstream of the single nucleotide polymorphism molecular marker site is used as the reference sequence, with a length of 18-30 base pairs; GC content between 40% and 60%; annealing temperature of 55-65℃; and avoidance of dimers and hairpin structures.

[0380] The designed probes are Holstein cattle breeding probe combinations, which were synthesized by Shijiazhuang Borui Biotechnology Co., Ltd., resulting in a 140K SNP chip. The reagent kit product "Ningyang No. 1" is as follows: Figure 1 As shown.

[0381] Example 2: Genomic distribution characteristics and density assessment of a 140K SNP chip based on a Holstein cattle breeding probe array within a 0.1Mb ​​window.

[0382] Using a window-based statistical method based on physical location, the 140,290 SNP loci obtained in Example 1 were processed into binary files in PLINK format using PLINK software (v2.0) based on their physical coordinates on the bovine reference genome assembly version ARS-UCD1.2. The whole genome was divided into continuous, non-overlapping 0.1Mb ​​windows. The number of SNP loci falling into each window was accurately counted, and the uniformity of SNP distribution and potential clustering regions were visually revealed by plotting using R language (v4.1.0).

[0383] The statistical results of the number of SNP loci on each chromosome detected in a 140K SNP chip based on Holstein cattle breeding probes are as follows: Figure 2 As shown, it exhibits typical non-uniform characteristics. The overall SNP density in the 140K chip ranges from 2,000 to 10,000. Some chromosomes (such as Chr1, Chr2, Chr3, Chr4 and Chr5) have a relatively large number of SNPs, reaching 8,000 to 10,000, indicating that these chromosomes have achieved high label coverage in the chip design.

[0384] The density distribution of SNP sites on each chromosome, detected using a 140K SNP chip based on Holstein cattle breeding probes, is shown below. Figure 3 As shown, some regions have higher density. The SNP distribution pattern of the chip fully reflects the structural heterogeneity of the genome itself, indicating that the chip design can still ensure effective monitoring of the entire genome while prioritizing coverage of polymorphic regions, laying a solid foundation for subsequent high-precision genetic analysis.

[0385] Example 3: Genotyping performance evaluation and polymorphism analysis of a 140K SNP chip based on a Holstein cattle breeding probe combination

[0386] 1. Use PLINK software (v1.9) to calculate the minor allele frequency (MAF) of each SNP locus. Based on the MAF calculation results, statistically analyze the distribution and proportion of loci across different MAF regions in the whole chip.

[0387] The results are as follows Figure 4 As shown, in the 140K SNP microarray based on Holstein cattle breeding probes, the minor allele frequency (MAF) is mainly concentrated in the range of 0.1-0.5, with each corresponding locus exceeding 10,000. The MAF peaks highest in the 0.4-0.5 region (approximately 11,500 loci), and the proportion of loci with MAF (>0.1) is 86.91%, indicating that the microarray is enriched with highly polymorphic loci, which is beneficial for subsequent association analysis. This suggests that the loci contained in the microarray have a high polymorphic information content and are highly suitable for subsequent population genetics and genomic selection analysis.

[0388] 2. Based on the bovine reference genome (ARS-UCD1.2), the upstream and downstream bases of each SNP site were determined. Using Python (v3.12.2), each SNP was precisely classified according to its base substitution direction, and the absolute number of each mutation type and its percentage of the total sites were calculated.

[0389] Statistical results of SNP site mutation types are as follows: Figure 5 As shown, six different types of nucleotide substitutions were found in the 150K chip: A / C, A / G, A / T, C / T, and G / T. The chip mutation types were dominated by C / T (49,127) and A / G (48,816) conversions, which together accounted for 69.96% of the total SNPs. The remaining types, including A / C, A / T, C / G, G / T, and InDel, accounted for 30.04%, with a conversion / transversion ratio of approximately 2.33:1, which indirectly confirms the biological rationality of this chip site screening.

[0390] Example 4: Comparison of interpolation accuracy between a 140K SNP chip based on a Holstein cattle breeding probe combination and a commercial 100K chip

[0391] Two hundred Holstein cattle were selected from ranches under the Ningxia Dairy Group, and genomic DNA was extracted from their tail veins after blood collection. DNA purity was assessed using NanoDrop2000, DNA concentration was precisely quantified using Qubit2.0, and genomic DNA integrity was assessed using agarose gel electrophoresis. DNA samples that passed the initial tests were then sent to Shijiazhuang Borui Biotechnology Co., Ltd. for 30× depth sequencing.

[0392] Genotyping of 200 Holstein cattle was performed using the 140K SNP chip obtained in Example 1 or a commercially available 100K chip to simulate different low-density genotyping schemes, specifically including:

[0393] a) 140K chip: The 140K SNP chip obtained in Example 1 was used to genotype 200 Holstein cattle to obtain genotype data;

[0394] b) 100K Commercial Chipset: Genotyping of 200 Holstein cattle was performed using a commercial 100K SNP chip (Neogen GGP Bovine 100K, SKUNo.40) to obtain genomic data;

[0395] c) 140K random group: 140,000 sites were randomly selected without replacement from all SNPs contained in the 30× depth sequencing data using R language (v4.5.2) to form a random SNP list, and the corresponding genotype data were extracted using PLINK (v1.9) based on this list.

[0396] Using the three datasets mentioned above as target datasets, whole-genome genotyping was performed using the Beagle 5.0 interpolation software, and the correlation coefficients (R²) of all genotype combinations were calculated. 2 (This is used to measure the accuracy of interpolation.)

[0397] The results of the interpolation accuracy comparison are as follows: Figure 6 As shown, under the same reference population size, the filling accuracy of the 140K SNP chip based on the Holstein cattle breeding probe combination is better than that of the existing commercial 100K chip, and is also significantly higher than that of the randomly selected 140K SNP set, indicating that its selection strategy is superior.

[0398] Example 5: Comparison of genotyping consistency between 140K SNP chips based on Holstein cattle breeding probe combinations and commercial 150K chips

[0399] Hair follicle samples were collected from 72 Holstein cattle randomly selected from ranches under Ningxia Dairy Group, and blood samples were collected from 178 Holstein cattle. Genotyping was performed independently using either the 140K SNP chip obtained in Example 1 or a commercially available 150K solid-phase chip (Neogen GGPBovine 100K, SKU No. 11), resulting in two raw genotype data files. Quality control was then performed on both datasets (quality control conditions: removal of SNP loci with a detection rate below 95%; removal of individual samples with a detection rate below 90%; exclusion of loci with a minimum allele frequency (MAF) less than 0.01; removal of loci showing significant deviations from Hardy-Weinberg equilibrium (P < 1e-6).

[0400] PLINK (v1.9) was used to merge the two quality control datasets into a single merged dataset containing all samples and common SNP loci. The genotypes of each sample at all common SNP loci were then compared to the consistency of the two microarray genotyping results for the same cow at the same loci. The genotyping consistency comparison results for hair follicle samples are shown below. Figure 7 As shown, comparing the data of the 140K SNP chip and the commercial 150K solid phase chip, the average detection consistency rate is 98.85%, with the maximum consistency rate being 99.40% and the minimum consistency rate being 97.88%.

[0401] The results of the genotyping consistency comparison of blood samples are as follows: Figure 8 As shown, comparing data from the liquid phase chip and the commercial 150K solid phase chip, the average detection consistency rate was 98.50%, with a maximum consistency rate of 98.95% and a minimum consistency rate of 96.65%. In conclusion, the 140K SNP chip based on the Holstein cattle breeding probe combination exhibits high genotyping consistency with the commercial 150K chip.

[0402] Example 6: 140K SNP chip of Holstein cattle breeding probe combination for population genetic structure and kinship analysis

[0403] Two hundred Holstein cattle were randomly selected from ranches under Ningxia Dairy Group, and blood samples were collected. Genotyping was performed using the 140K SNP chip obtained in Example 1 to obtain the original genotype data file. Quality control was then performed under the following conditions: SNP loci with a detection rate below 95% were removed; individual samples with a detection rate below 90% were removed; loci with a minimum allele frequency (MAF) less than 0.01 were removed; and loci with significant deviations from Hardy-Weinberg equilibrium (P < 1e-6) were removed. Principal component analysis and phylogenetic analysis (blood homology and state homology analysis) were performed on the quality-controlled genotype data using the "SNPRelate" package in R (v4.5.2).

[0404] The principal component analysis results of 200 Holstein cattle are as follows: Figure 9 As shown, the eigenvalue percentages of the first four principal components are PC1 = 2.9%, PC2 = 2.0%, PC3 = 1.7%, and PC4 = 1.6%, respectively, explaining a total of 8.2% of the total genetic variation. All components are well below the conventional empirical threshold of 5%, suggesting that no significant large-scale stratification signal was detected in the population, and the genetic background is basically homogeneous.

[0405] Bloodline homology analysis results are as follows Figure 10 As shown, the results The values ​​are all close to 0 and the distribution is concentrated, indicating that no dominant kinship signal was found in the 200 Holstein cattle, confirming that the degree of blood homology among the samples is extremely low, indicating that the 200 Holstein cattle are outcrossed individuals.

[0406] The results of the state homology analysis are as follows Figure 11 As shown, the population was not clearly distinguished, indicating that the 200 Holstein cattle belonged to the same genetic group and there was no significant subgroup structure.

Claims

1. A Holstein cattle breeding probe assembly, characterized in that, The probe combination was used to detect a molecular marker combination of Holstein SNPs, which consisted of 140,290 SNP sites, and their location information based on the reference genome ARS-UCD1.2 is shown in Table 1.

2. The Holstein cattle breeding probe assembly according to claim 1, characterized in that, The molecular marker combination includes: 77,887 sequencing-derived high genetic information SNP loci, 62,387 trait-related loci, and 16 defect-related loci.

3. The Holstein cattle breeding probe assembly according to claim 1, characterized in that, The probes in the probe assemblies are designed with a sequence within 20 bases upstream and downstream of the single nucleotide polymorphism molecular marker site as the design reference sequence, and the length is 18-30 bases.

4. The Holstein cattle breeding probe assembly according to claim 3, characterized in that, The probe GC content in the probe assembly is between 40% and 60%, and the annealing temperature is between 55 and 65°C.

5. A gene chip, characterized in that, The chip load is the Holstein cattle breeding probe assembly as described in any one of claims 1-4.

6. The gene chip according to claim 5, characterized in that, The chip is either a liquid-phase chip or a solid-phase chip.

7. A reagent kit, characterized in that, The kit comprises the Holstein cattle breeding probe combination as described in any one of claims 1-4, or the gene chip as described in any one of claims 5-6.

8. The application of the Holstein cattle breeding probe combination according to any one of claims 1-4, the gene chip according to any one of claims 5-6, or the kit according to claim 7 in the molecular selection of Holstein cattle.

9. The application of the Holstein cattle breeding probe combination according to any one of claims 1-4, the gene chip according to any one of claims 5-6, or the kit according to claim 7 in the genetic structure analysis of Holstein cattle populations.

10. The application of the Holstein cattle breeding probe combination according to any one of claims 1-4, the gene chip according to any one of claims 5-6, or the kit according to claim 7 in the analysis of genomic kinship of Holstein cattle.

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