5k liquid phase chip for variety and paternity test of dairy cattle and dairy-meat cattle and application of 5k liquid phase chip

Abstract

The invention discloses a preparation method and application of a 5k liquid phase chip for cattle variety and paternity test. Based on original genome data of cattle varieties in the target cattle variety set and the amplified cattle variety set, position information of genome SNP sites of all the varieties is obtained, and then sites with variety specificity or high genetic stability are screened through Delta and FST value methods; and classifying varieties by combining a machine learning classification model to obtain a specific site combination meeting requirements as a target SNP site combination. The obtained SNP site combination can achieve 100% identification accuracy when variety identification is carried out on a target variety, and efficient, low-cost, large-scale and automatic variety and paternity identification is realized; the production type of the detected cattle can be effectively judged, a pasture can be better assisted to select the cattle for both dairy and meat, the overall milk yield and the milk quality are increased, and the income is increased.

Description

Technical Field

[0001] The present invention belongs to the field of biological breeding, and relates to bovine breed identification and paternity testing. Specifically, it relates to a method for screening SNP loci for dairy and dual-purpose dairy and beef cattle breeds and paternity testing, a 5k liquid chip and its applications. Background Art

[0002] Single nucleotide polymorphisms (SNPs), as the third-generation molecular genetic markers, have the characteristics of high genetic stability, large quantity, wide distribution, and being conducive to automated genotyping. They are commonly used in fields such as biological population identification, genetic structure analysis, and functional gene mapping. With the rapid development of gene chip technology and genomic selection technology, SNP genotyping chips have been widely applied to livestock and poultry genetic breeding work and have brought remarkable results. Currently, SNP genotyping chips are mainly divided into two categories: solid-phase chips and liquid-phase chips. The former is based on the principle of nucleic acid hybridization, while the latter is based on targeted capture sequencing technology. Comparatively speaking, liquid-phase chips not only have the advantages of high throughput, rapidity, high sensitivity, and high degree of automation, but also have the advantage of being able to flexibly adjust SNP markers in a timely manner according to the actual population situation due to their flexibility in locus design.

[0003] In recent years, the importance of protecting livestock and poultry breed resources has become increasingly prominent. As the core means of breed identification, SNP genotyping chip technology has become the research focus in this field. The core of this technology lies in using an SNP selection method to accurately screen out a group of SNP loci with high information scores as breed tags, and then using an efficient machine learning classification algorithm to accurately classify individuals based on these tagged SNPs. Using the same principle, SNP genotyping chips are also widely used in the field of paternity testing. Compared with traditional morphological or biochemical marker identification methods, SNP chip technology not only greatly improves the accuracy of identification but also shows excellent efficiency advantages when dealing with large-scale populations.

[0004] At present, my country has made significant progress in the field of SNP genotyping chips, which has filled the gap in this field and effectively narrowed the technological gap with foreign animal breeding industries. In the past two years, many research institutes have successively released different types of SNP chips. For example, the Beijing Institute of Animal Husbandry of the Chinese Academy of Agricultural Sciences has successfully developed a liquid capture chip "Cattle 110k" for genomic selection breeding of beef cattle (Chen et al., 2024), and Northwest Agriculture and Forestry University has developed my country's first high-density gene chip for yellow cattle (Xia et al., 2023). However, in dairy and dual-purpose cattle, especially in breed and parentage identification, the development of gene chips is still insufficient. Dual-purpose cattle have many advantages in production. One of them is that the milk is rich in nutrition and high in quality, even exceeding many specialized dairy cattle. If the cattle breed can be accurately identified through gene chips to determine its production type, it will effectively improve the efficiency of milk production, promote food health, and enhance regional social welfare. Therefore, we developed a liquid-phase SNP genotyping chip based on the characteristics of dairy and dual-purpose dairy and meat cattle in my country, which is specialized and can be used for both breed and parentage identification, aiming to expand the boundaries of this field and provide strong technical support for breed and parentage identification. This chip is specially designed and developed for the seven dairy and dual-purpose dairy and meat cattle breeds in my country to meet actual needs and promote the development of germplasm resource protection and breeding process. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides a method for preparing a 5k liquid phase chip for dairy and dual-purpose cattle breeds and paternity identification and its application, and specifically provides a SNP site screening method for dairy and dual-purpose cattle breeds and paternity identification, a 5k liquid phase chip and its application. The present method can solve the problems of existing identification methods such as high cost, low efficiency, poor flexibility and inability to be used on a large scale in my country's cattle farms.

[0006] The present invention discloses a method for preparing the above-mentioned 5k liquid phase chip for dairy and dual-purpose dairy and meat cattle breed and parentage identification, comprising the following steps: S1. Obtain the genome information of each variety in the target variety set through 30x sequencing; obtain the genome information of each variety in the amplified variety set through a public database; and process the raw data obtained above; S2, eliminating the sex chromosome information in the data obtained in S1 above, and testing and quality controlling 29 autosomal SNPs of 16 cattle breeds; S3, screening the SNP loci obtained in S1 above for those with variety specificity or high genetic stability by using the Delta and FST value methods; S4. Screening specific site combinations that meet the requirements as target SNP site combinations. The specific site combinations include sites with variety specificity or high genetic stability in the target variety set and the amplified variety set.

[0007] Furthermore, the breeds included in the target breed set of S1 are the target objects of the 5k liquid phase chip for dairy and dual-purpose dairy and meat cattle breeds and parentage identification in the present invention, including Figure 1 The Holstein cattle, Jersey cattle, Sanhe cattle, Xinjiang brown cattle, Simmental cattle, Shuxuanhua cattle, and Dengchuan cattle are shown, a total of 7 cattle breeds; the role of the expanded breed set is to enhance the accuracy of breed-specific site mining for target breeds, including Angus cattle, Red Angus cattle, Hereford cattle, Swiss brown cattle, Flaviecht cattle, German cattle, Charolais cattle, Nanyang cattle, and Luxi cattle, a total of 9 breeds.

[0008] Furthermore, S1 processes the obtained raw data by using BWA software to align the FASTQ file of the obtained raw data to the reference genome ARS-UCD1.2 of cattle, and then uses samtools to process it to obtain a bam file, and removes the data of individuals with missing sites.

[0009] Furthermore, S1 performs 30x sequencing on the genome of each variety in the target variety set and removes individuals with missing sites, with the aim of improving the detection rate to ensure the precision and accuracy of the data.

[0010] Furthermore, the screening method of S4 is: using multiple machine learning classification models to classify varieties, and at the same time, each model will screen the SNP site combination to obtain different SNP site combination schemes, and finally select a scheme that meets the requirements as the target SNP site combination. Figure 2 The SNP loci corresponding to the physical positions shown in .

[0011] The present invention discloses a 5k liquid chip for detecting the above-mentioned dairy and dual-purpose dairy and beef cattle breeds and parentage identification, wherein the liquid chip comprises a set of probes for target SNP site combinations obtained by the above-mentioned SNP site screening method.

[0012] The present invention discloses the application of the 5k liquid phase chip for identification of dairy and dual-purpose dairy and meat cattle breeds in the identification of multiple cattle breeds. In one embodiment, it includes the application in the identification of seven target breeds, including Holstein cattle, Jersey cattle, Sanhe cattle, Xinjiang brown cattle, Simmental cattle, Shuxuanhua cattle and Dengchuan cattle, with an identification accuracy rate of 100%.

[0013] Compared with the prior art, the SNP site screening method for dairy and dual-purpose dairy and meat cattle breeds and parentage identification, 5k liquid phase chip and application thereof provided by the present invention have the following beneficial effects: The present invention integrates and screens the breed-specific and highly genetically stable SNP sites of 7 dairy and dual-purpose cattle breeds, and adds the breed-specific and highly genetically stable SNP sites of 9 cattle breeds, thereby improving the accuracy of specific site mining for the 7 target breeds, and quickly obtaining the optimal SNP site combination using a machine learning classification model.

[0014] The present invention uses 30x sequencing for all seven target varieties and eliminates individuals with missing sites, eliminating the site filling step, simplifying the analysis process, and effectively improving the detection rate, accuracy, and rigor of SNP sites.

[0015] Dairy and beef cattle have high production value, and their milk quality is better than many dairy cattle. However, there are almost no gene chips for dairy and beef cattle on the current market, resulting in a market gap. The 5k liquid phase chip of the present invention is specially designed and developed for dairy and beef cattle, aiming to help ranches accurately identify and reasonably cultivate dairy and beef cattle.

[0016] Compared with the chips in the prior art, the 5k liquid phase chip of the present invention has a high identification accuracy rate of 100%. The above chip can not only distinguish the seven target breeds, but also identify the production type bias of the hybrid cattle to be tested, such as the bias towards dairy or dairy and meat.

[0017] Since the number of SNP sites involved in the present invention is relatively small, the design of the gene chip is more streamlined, and the production cost is reduced. At the same time, the application of gene chip technology makes it possible to perform variety and paternity identification efficiently, at low cost, on a large scale, and automatically.

[0018] The above SNP site combination can not only accurately identify the target breed, reduce the misjudgment rate, and improve the reliability of breed identification, but also detect the production type of the tested cattle. See the specific comparative explanation of the advantages of the present invention in the following examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 These are 7 dairy and dairy-beef dual-purpose cattle breeds in the target breed set used in the present invention.

[0020] Figure 2 It is the physical location corresponding to the SNP site combination used for dairy and dual-purpose dairy and meat cattle breed and parentage identification in the present invention.

[0021] Figure 3 This is a line graph of identification accuracy obtained by using four schemes generated by four machine learning classification models in the present invention.

[0022] Figure 4 This is a line graph of identification accuracy obtained when the present invention is applied to the identification of 7 target cattle breeds. DETAILED DESCRIPTION

[0023] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention so that people in the technical field can better understand the present application scheme. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0024] Embodiment 1, The screening method of the SNP locus combination for dairy and dual-purpose dairy and meat cattle breeds and parentage identification of this embodiment comprises the following steps: S100, obtaining genome information of each variety in the target variety set through 30x sequencing; obtaining genome information of each variety in the amplified variety set through a public database; and processing the raw data obtained above. Specifically, the above content includes the following steps: S110, obtaining genome information of each breed in the target breed set by 30x sequencing: collecting the buffy coat of bovine blood, and obtaining original genome data of 7 bovine breeds by 30x sequencing; S120. Obtain the genome information of each breed in the expanded breed set through public databases: The original genome data of 9 cattle breeds were obtained by downloading the National Center for Biotechnology Information (NCBI) database and searching existing literature data.

[0025] The breeds included in the target breed set are the target objects of the 5k liquid phase chip for dairy and dual-purpose dairy and meat cattle breeds and parentage identification in the present invention, including Figure 1 The Holstein cattle, Jersey cattle, Sanhe cattle, Xinjiang brown cattle, Simmental cattle, Shuxuanhua cattle, Dengchuan cattle, a total of 7 cattle breeds; the role of the expanded breed set is to enhance the accuracy of breed-specific site mining for target breeds, including Angus cattle, Red Angus cattle, Hereford cattle, Swiss brown cattle, Fleveich cattle, German cattle, Charolais cattle, Nanyang cattle, Luxi cattle, a total of 9 breeds. A total of 16 cattle breeds were obtained.

[0026] S130, processing the raw data: using BWA software to align the obtained FASTQ file of the raw data to the reference genome ARS-UCD1.2 of cattle, and then using samtools to process it to obtain a bam file, and excluding the data of individuals with missing sites.

[0027] S200. Exclude the sex chromosome information from the data obtained by excluding the above S100, and detect and quality control the 29 autosomal SNPs of 16 cattle breeds. The above content includes the following steps: S210. Detect the 29 autosomal SNPs of 16 cattle breeds: Use BaseQuality Score Recalibration (BQSR) in the GATK toolset to perform base quality correction; Apply Variant Filtration to filter SNPs and remove low-quality variants.

[0028] S220. Perform quality control on SNP sites: According to the criteria of "maximum site deletion rate max-missing is 0%, minimum allele frequency (MAF) threshold is 0.3, and the number of alleles is 2", use vcftools to perform preliminary quality control on SNP sites; Use plink software to perform LD pruning on the sites after the above preliminary accusation, and the LD pruning parameter is set to "LD(r2) > 0.2". After the above steps, 814,205 high-quality sites are screened out from 16 breeds. These 814,205 sites constitute the basic dataset for the subsequent screening work.

[0029] S300. Screen the SNPs obtained above in S1 for sites with breed specificity or high genetic stability by the Delta and FST value methods. The above content includes the following steps: S310. Use the vcftools tool to perform pairwise comparisons between the SNP sites of 16 breeds, calculate the FST value, and use it to screen for sites with breed specificity or high genetic stability. The command for the above step is: Run the command, use vcftools (vcftools); Specify the input VCF file (-vcf / file name / ); Specify the sample file of the first breed (-weir-fst-pop Fleckvieh.txt); Specify the sample file of the second breed, where ${i} is a variable that will be replaced by the sample file of a certain breed (-weir-fst-pop ${i}.txt); Output the generated result (-outFst_Fleckvieh_${i}). After calculation, there are 614 SNP sites that meet "FST >= 0.25", and 6,435 SNP sites that meet "FST >= 0.2".

[0030] S320. Using the same method, pairwise comparisons of SNP loci among 16 varieties were performed in sequence to calculate the Delta value. After calculation, there were 9,613 SNP loci that met the condition of "Delta >= 0.3", and 663 SNP loci that met the condition of "Delta >= 0.35".

[0031] S330. Take the intersection between the sets of SNP loci that meet the conditions of "FST >= 0.2" and "Delta >= 0.3", and finally obtain 5,470 SNP loci with variety specificity or high genetic stability.

[0032] S400. Screen the specific locus combinations that not only meet the requirement of the minimum number of combined loci but also achieve the accuracy requirement for variety identification as the target SNP locus combinations. The specific locus combinations include loci with variety specificity or high genetic stability in the target variety set and the amplification variety set. The SNP locus combinations are composed of Figure 2 the SNP loci corresponding to the physical positions shown in, where the names, chromosomal positions, and mutation types of some SNP loci are shown in Table 1.

[0033] Table 1. Information of some SNP loci obtained by screening

[0034]

[0035]

[0036] Specifically, in the process of selecting a certain number of loci from the loci with variety specificity or high genetic stability to form the target SNP locus combinations in this embodiment, the application of 4 classification models is involved: Naive Bayes (NB), Support Vector Machine (SVM), K-Nearest Neighbor (KNN), and Random Forest (RF). Next, use the "caret" package in R software to construct classification models, and all 4 machine learning models are trained based on the variety-specific locus data obtained in step S300. Each model will screen out the SNP locus combinations that meet the requirements according to the preset criteria, thus obtaining 4 solutions. Finally, select the specific locus combination that not only meets the minimum number of combined loci but also has the highest accuracy for variety and paternity testing as the target SNP locus combination from these 4 solutions. The identification accuracies obtained by the 4 solutions are as Figure 3 shown. According to the requirements of the minimum number of combined loci and the highest accuracy for variety and paternity testing, select the SNP locus combination screened by the SVM model as the target SNP locus combination. The target SNP locus combination contains 4,731 high-quality SNP loci, among which 4,209 loci are used for variety identification and 522 loci are used for paternity testing.

[0037] Based on the above, a 5k liquid phase chip for dairy and dual-purpose cattle breed and parentage identification can be obtained, wherein the liquid phase chip comprises a set of probes for target SNP site combinations obtained by the above SNP site screening method.

[0038] Example 2: Application and effect of the cattle 5k liquid phase chip obtained in Example 1 in the breed identification of 7 target cattle breeds.

[0039] This embodiment provides an application and effect of the above-mentioned 5k liquid phase chip for dairy and dual-purpose dairy and meat cattle breed and parentage identification in breed identification of 7 target cattle breeds.

[0040] The 7 target cattle breeds include Holstein, Jersey, Sanhe, Xinjiang brown, Simmental, Shuxuanhua and Dengchuan. Furthermore, the 5k liquid chip was applied to two groups of the above 7 cattle breeds, which consisted of cows and bulls, respectively, with a total of 200 cows and 80 bulls.

[0041] Specifically, the support vector machine (SVM) method was used to predict the application effect of the obtained 5k liquid phase chip in the two groups. The identification accuracy obtained refers to the consistency between the identification results and the real varieties when the gene chip technology is used to identify the varieties of the tested groups. When the varieties are identified for these two groups, the identification accuracy achieved is as follows Figure 4 As shown, the identification accuracy is 100%.

[0042] Compared with the chips in the prior art, the 5k liquid phase chip of the present invention has outstanding advantages in breed identification, as shown in Table 2. Since there are not many SNP chips for cattle in the prior art, the sheep 3K liquid phase chip is also compared.

[0043] Table 1. Chip information of prior art

[0044] Among them, the 0.3k liquid phase chip in CN202410701238.0 has an identification accuracy rate of 35-100% when identifying 12 target varieties. Only 2 varieties (Limousin cattle and Simmental cattle) have reached 100%, and 4 varieties (Enshi cattle, Yunba cattle, Yiling cattle, Zaobei cattle) have not reached 70%. The identification accuracy of the present invention is 100%, so the 5k liquid phase chip of the present invention has an outstanding advantage in identification accuracy, especially compared with the 0.3k liquid phase chip in CN202410701238.0.

Claims

1. A method for SNP site screening for dairy and dual-purpose cattle breeds and parentage identification, characterized in that: The steps include: S1. Obtaining genome information of each breed in a target breed set by sequencing, wherein the target breed set includes 7 cattle breeds, namely, Holstein cattle, Jersey cattle, Sanhe cattle, Xinjiang brown cattle, Simmental cattle, Shuxuanhua cattle and Dengchuan cattle; obtaining genome information of each breed in an expanded breed set by using a public database, wherein the expanded breed set includes 9 cattle breeds, namely, Angus cattle, Red Angus cattle, Hereford cattle, Swiss brown cattle, Fleveich cattle, German cattle, Charolais cattle, Nanyang cattle and Luxi cattle; processing the raw data obtained above; S2, eliminating the sex chromosome information in the data obtained in S1 above, and testing and quality controlling 29 autosomal SNPs of 16 cattle breeds; S3, screening the SNP loci obtained in S1 above for those with variety specificity or high genetic stability by using the Delta and FST value methods; S4. Use multiple machine learning classification models to classify varieties. At the same time, each model will screen SNP site combinations to obtain different SNP site combination schemes, and screen and obtain specific site combinations that meet the requirements as target SNP site combinations. The specific site combinations include sites with variety specificity or high genetic stability in the target variety set and the expanded variety set.

2. The screening method according to claim 1, characterized in that The method for obtaining the location information of the SNP site in S3 is: obtaining the original genome data of the cattle breeds described in the target breed set and the amplified breed set, and then processing the original data, aligning it to the cattle reference genome ARS-UCD1.2, and then detecting the SNP of the whole genome, and finally performing quality control on the SNP site.

3. The screening method according to claim 2, characterized in that The quality control in S2 includes: the maximum site missing rate max-missing is 0%, that is, the SNP sites with a detection rate of 100% are retained; at the same time, the minimum allele frequency MAF threshold is set to 0.3, and the number of alleles is limited to 2.

4. The screening method according to claim 1, characterized in that The machine learning classification model used in S4 is selected from Naive Bayes NB, Support Vector Machine SVM, K Nearest Neighbor KNN or Random Forest RF.

5. A 5k liquid phase chip for detecting dairy and dual-purpose dairy and meat cattle breeds and parentage testing, characterized in that: The liquid phase chip comprises a set of probes for a target SNP site combination screened by the method described in any one of claims 1 to 4.

6. The 5k liquid phase chip according to claim 5, characterized in that: The bovine 5k liquid phase chip is composed of independently packaged bovine 5k probe mixture and hybrid capture reagent; the bovine 5k probe is a double-stranded probe, which is designed and synthesized according to the screened SNP site and has a nucleotide sequence that overlaps 100% and covers the combination of the SNP sites; the SNP sites are screened and obtained by the method described in any one of claims 1-4; the design principle of the 5k probe is: the probe length is 100bp, the probe GC content is between 30%-70%, and the selected area does not contain SSR and GAP areas to the maximum extent; for each SNP site, a probe is designed within the range of 100bp upstream and downstream, and probes that cannot be uniquely matched on the genome and contain repetitive sequences in the flanking sequence are removed; the nucleotide sequence designed according to the design principle is subjected to single-stranded nucleotide synthesis, and the synthesized two DNA nucleotide sequences with a length of 100bp and a biotin group modified at the 5' end become bovine 5k probes; the above two synthesized bovine 5k probes are mixed with equimolar mass, and the EDTA and Tris-HCl mixed solution is used to fix the volume to a bovine 5k probe mixture, The bovine 5k probe mixture and hybrid capture reagent are packaged separately to obtain a 5k liquid phase chip for dairy and dual-purpose cattle breed and parentage identification.

7. A cattle breed and parentage identification test kit, characterized in that: The kit comprises the 5k liquid phase chip according to claim 5 or 6.

8. Use of the 5k liquid phase chip according to claim 5 or 6 in cattle breed and paternity identification.

9. The use according to claim 8, characterized in that: The cattle breeds are selected from Holstein cattle, Jersey cattle, Sanhe cattle, Xinjiang brown cattle, Simmental cattle, Shuxuanhua cattle and Dengchuan cattle.

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