SNP (Single Nucleotide Polymorphism) site combination for sheep genotyping, liquid chip and application

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) site combination for sheep genotyping, a liquid chip and application, and belongs to the technical field of molecular breeding. The invention provides an SNP (Single Nucleotide Polymorphism) site combination for sheep genetic typing. The SNP site combination comprises 58859 SNP sites. The SNP locus combination covers a plurality of important economic characters, covers important genetic loci related to the production performance, reproductive performance, disease resistance character and adaptive capacity of sheep, and is beneficial to improving the breeding efficiency and the production performance. The efficient, low-cost and high-throughput sheep whole genome 50K SNP liquid chip is designed by combining functional site selection and a liquid chip capture sequencing technology, and genetic markers related to important economic traits of sheep can be accurately screened; and a reliable molecular marker tool is provided for whole genome association analysis, genome selection, genetic improvement and phenotype regulation of sheep.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular breeding, and in particular relates to a SNP site combination, a liquid phase chip and an application thereof for sheep genotyping. Background Art

[0002] With the continuous development of molecular breeding technology, genomic methods are increasingly used in animal genetic improvement and genomic selection, and genotype analysis has become a key technology to improve breeding efficiency. Single nucleotide polymorphism (SNP) is the most widely distributed genetic marker in the genome, with high genetic stability. It has shown strong application potential in fields such as genome-wide association study (GWAS) and genomic selection (GS), and has become the most commonly used marker type in animal genetics research.

[0003] Although the application of SNP markers in genomic research has been extensive and in-depth, existing genotyping technologies, especially those based on solid-phase chips, have some limitations. Solid-phase chips detect SNP sites by fixing specific DNA probes on the chip surface and hybridizing with sample DNA fragments, but their flexibility is poor and they can only detect fixed SNP sites included in the design, and cannot add or delete sites at any time. In addition, the typing cost of solid-phase chips is high and cannot meet the needs of large-scale, multi-sample genetic analysis. In addition, existing sheep chips also have the defects of incomplete coverage of functional sites and low effectiveness. Therefore, it is particularly important to provide a SNP site combination for sheep genotyping and a sheep whole genome liquid phase chip, which covers comprehensive functional sites and has a wide range of applications, making the detection efficient, low-cost and high-throughput. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a SNP site combination for sheep genotyping, covering important genetic sites related to sheep production performance, reproductive performance, disease resistance and adaptability, which is helpful to comprehensively evaluate the growth, reproduction and adaptability of livestock, thereby improving breeding efficiency and production performance.

[0005] Another object of the present invention is to provide a probe combination for detecting SNP site combinations for sheep genotyping.

[0006] Another object of the present invention is to provide a kit for detecting sheep genotyping.

[0007] Another object of the present invention is to provide an application of the SNP site combination or the probe combination or the kit in the preparation of a sheep whole genome chip.

[0008] Another object of the present invention is to provide a sheep whole genome 50K SNP liquid phase chip.

[0009] Another object of the present invention is to provide an application of the SNP site combination or the probe combination or the kit or the liquid phase chip in sheep breeding.

[0010] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0011] The present invention provides a SNP site combination for sheep genotyping, the SNP site combination comprising 58859 SNP sites, the position information of the SNP sites is shown in Table 2; the SNP site combination comprises SNP sites located in the sheep reference genome Oar v4.0.

[0012] The present invention also provides a probe combination for detecting SNP site combinations for sheep genotyping, wherein each SNP site in the SNP site combination is provided with a group of probes for detecting the corresponding SNP site to form a probe combination.

[0013] Preferably, the design parameters of the probe are: the probe length is 120 nt; the GC content is 30% to 70%; the copy number of the entire probe in the genome is 1; the copy number of any 25 bp kmer sequence in the genome does not exceed 5.

[0014] The present invention also provides a kit for detecting sheep genotyping, and the kit comprises the probe combination.

[0015] The present invention also provides an application of the SNP site combination or the probe combination or the kit in the preparation of a sheep whole genome chip.

[0016] Preferably, the chip comprises a solid phase chip or a liquid phase chip.

[0017] The present invention also provides a sheep whole genome 50K SNP liquid phase chip, and the liquid phase chip comprises the probe combination.

[0018] The present invention also provides an application of the SNP site combination, the probe combination, the kit or the liquid phase chip in sheep breeding.

[0019] Preferably, the sheep breeding includes one or more of sheep genome selection, positioning of sheep trait-related genes, sheep genetic diversity analysis, sheep whole genome association analysis, sheep breed identification, sheep kinship identification, sheep population classification, sheep phenotypic regulation, sheep germplasm resource improvement and sheep germplasm resource protection.

[0020] Preferably, the traits in the positioning of the sheep trait-related genes include one or more of vision, wool, immunity, reproductive ability, fat deposition, horns, teat morphology, high altitude adaptability, muscle development, nerves, growth performance, lactation performance, carcass traits, hypoxia adaptation, daily weight gain, birth weight, cold adaptation, lactose content, blood, bones, ovarian development, fertilization, litter size, weight, body length, abdominal circumference, chest circumference, chest depth, cross height, cross width, hip width, hip height, shoulder width and body height.

[0021] Beneficial effects of the present invention:

[0022] The SNP locus combination used for sheep genotyping of the present invention is evenly distributed on each chromosome, and the minimum allele frequency is greater than 0.1. The SNP locus combination of the present invention covers multiple important economic traits, including important genetic loci related to sheep production performance, reproductive performance, disease resistance and adaptability, etc., which helps to comprehensively evaluate the growth, reproduction and adaptability of livestock, thereby improving breeding efficiency and production performance.

[0023] The present invention combines functional site selection and liquid chip capture sequencing technology to design an efficient, low-cost and high-throughput sheep whole genome 50K SNP liquid chip. Compared with traditional solid-phase chips with the same number of probes, the liquid chip of the present invention can detect more SNP sites. In addition, the chip design of the present invention is flexible, and newly discovered breeding-related marker sites can be added at any time. The chip of the present invention can accurately screen genetic markers related to important economic traits of sheep, and provide a reliable molecular marker tool for whole genome association analysis, genome selection, genetic improvement and phenotypic regulation of sheep. It can be used for large-scale genotyping of sheep and has obvious price advantages compared with whole genome resequencing. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The distribution density diagram of the SNP sites on each chromosome in Example 1;

[0025] Figure 2 The minimum allele frequency (MAF) statistics of the SNP loci in Example 1;

[0026] Figure 3 The data comparison rate of the sheep whole genome 50K SNP liquid phase chip test sample in Example 2, wherein the numbers on the horizontal axis represent the numbers of different sheep blood DNA samples;

[0027] Figure 4 The sample capture efficiency of the sheep whole genome 50K SNP liquid phase chip test in Example 2, wherein the numbers on the abscissa represent the numbers of different sheep blood DNA samples;

[0028] Figure 5 The redundancy rate of the sample data of the sheep whole genome 50K SNP liquid phase chip test in Example 2, wherein the numbers on the abscissa represent the numbers of different sheep blood DNA samples;

[0029] Figure 6 The SNP detection rate of the sheep whole genome 50K SNP liquid phase chip test sample in Example 2, wherein the numbers on the abscissa represent the numbers of different sheep blood DNA samples;

[0030] Figure 7 is the genetic clustering tree in Example 3;

[0031] Figure 8 It is the Manhattan plot of the whole genome association analysis of sheep in Example 3, wherein A is body weight, B is abdominal circumference, C is shoulder width, D is hip height, E is hip width, F is cross height, G is body height, H is body oblique length, I is cross width, J is chest depth, and K is chest circumference. DETAILED DESCRIPTION

[0032] The present invention provides a SNP site combination for sheep genotyping, the SNP site combination comprising 58859 SNP sites, the position information of the SNP sites is shown in Table 2; the SNP site combination comprises SNP sites located in the sheep reference genome Oar v4.0.

[0033] In the present invention, the SNP site combination includes SNP sites located in the sheep reference genome Oar v4.0 and sheep whole genome SNP variation sites in the iSheep database. The SNP site combination of the present invention is based on resequencing data of various sheep breeds at home and abroad, with comprehensive data and wide application range. The SNP site combination of the present invention is evenly distributed on each chromosome, and the minimum allele frequency is greater than 0.1. The SNP site combination of the present invention covers multiple important economic traits, including but not limited to: vision, wool, immunity, reproductive ability, fat deposition, horns, teat morphology, high altitude adaptability, muscle development, nerves, growth performance, lactation performance, carcass traits, hypoxia adaptation, daily weight gain, birth weight, cold adaptation, lactose content, blood, bones, ovarian development, fertilization, litter size, weight, body length, abdominal circumference, chest circumference, chest depth, cross height, cross width, hip width, hip height, shoulder width and body height, etc., covering important genetic sites related to sheep production performance, reproductive performance, disease resistance traits and adaptability, etc., which is helpful to comprehensively evaluate the growth, reproduction and adaptability of livestock, thereby improving breeding efficiency and production performance.

[0034] The present invention also provides a probe combination for detecting SNP site combinations for sheep genotyping, wherein each SNP site in the SNP site combination is provided with a group of probes for detecting the corresponding SNP site to form a probe combination.

[0035] In the present invention, the design parameters of the probe are preferably: the probe length is 120 nt; the GC content is 30% to 70%; the copy number of the entire probe in the genome is 1; the copy number of any 25 bp kmer sequence in the genome does not exceed 5. The probe combination of the present invention has high capture efficiency, low redundancy rate, and good effectiveness.

[0036] The present invention also provides a kit for detecting sheep genotyping, and the kit comprises the probe combination.

[0037] The present invention also provides an application of the SNP site combination or the probe combination or the kit in the preparation of a sheep whole genome chip.

[0038] In the present invention, the sheep whole genome chip is preferably a solid phase chip or a liquid phase chip, more preferably a liquid phase chip. The working principle of the liquid phase chip is to perform fixed-point capture based on the complementary binding of specific probes and target sequences, and to elute, amplify, build a library and sequence the captured target sequence to finally obtain the genomic genetic variation information of the target area. Compared with solid phase chips, liquid phase chips have the advantages of high throughput, low cost, strong flexibility, etc., can cover more SNP sites, and are suitable for large-scale and complex genomic studies.

[0039] The present invention also provides a sheep whole genome 50K SNP liquid phase chip, and the liquid phase chip comprises the probe combination.

[0040] The present invention combines functional site selection and liquid chip capture sequencing technology to design an efficient, low-cost and high-throughput gene liquid chip. Compared with traditional solid-phase chips with the same number of probes, the liquid chip of the present invention can detect more SNP sites. In addition, the chip design of the present invention is flexible, and newly discovered breeding-related marker sites can be added at any time. The liquid chip of the present invention covers the above-mentioned multiple important economic traits, can accurately screen genetic markers related to important economic traits of sheep, and provide reliable molecular marker tools for whole genome association analysis, genome selection, genetic improvement and phenotypic regulation of sheep. Compared with whole genome resequencing, the liquid chip of the present invention can accurately identify genetic variations in functional regions of the genome through a smaller amount of sequencing, with lower cost, and can provide important support for the discovery of functional mutations in regulating phenotypic organisms, and provide a more efficient tool for genetic improvement and genome selection of animals and plants.

[0041] The present invention also provides an application of the SNP site combination, the probe combination, the kit or the liquid phase chip in sheep breeding.

[0042] In the present invention, the SNP site combination covers the above-mentioned multiple important economic traits, including but not limited to: vision, wool, immunity, reproductive ability, fat deposition, horns, nipple morphology, high altitude adaptability, muscle development, nerves, growth performance, lactation performance, carcass traits, hypoxia adaptation, daily weight gain, birth weight, cold adaptation, lactose content, blood, bones, ovarian development, fertilization, litter size, weight, body oblique length, abdominal circumference, chest circumference, chest depth, cross height, cross width, hip width, hip height, shoulder width and body height, etc., which is helpful to comprehensively evaluate the growth, reproduction and adaptability of sheep, thereby improving breeding efficiency and production performance. The SNP site combination of the present invention or the probe combination or kit for detecting the SNP site combination or the liquid phase chip can accurately screen genetic markers related to important economic traits of sheep, and provide a reliable molecular marker tool for sheep genome selection, positioning of sheep trait-related genes, sheep genetic diversity analysis, sheep whole genome association analysis, sheep breed identification, sheep kinship identification, sheep population classification, sheep phenotypic regulation, sheep germplasm resource improvement and sheep germplasm resource protection.

[0043] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0044] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0045] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0046] Example 1 Design and preparation of sheep whole genome 50K SNP liquid phase chip

[0047] 1. Data preparation and integration

[0048] First, the whole genome resequencing data of 10 East Frisian sheep (accession number PRJNA624020) were obtained from the NCBI database BioProject for analysis, aligned to the Oar v4.0 reference genome, and high-quality SNP data after quality control were screened and obtained. Subsequently, these SNP data were merged with the sheep whole genome SNP variation site information in the iSheep database (https: / / ngdc.cncb.ac.cn / isheep / ) to ensure the comprehensiveness and consistency of the data source.

[0049] 2. Functional area definition

[0050] Based on existing literature (Cao Y, Ai Y, Zhang X, Zhang J, Long X, Zhu Y, Wang L, GuQ, Han H. Genome-wide epigenetic dynamics during postnatal skeletal musclegrowth in Hu sheep. Commun Biol. 2023 Oct 23;6(1):1077. doi: 10.1038 / s42003-023-05439-0. PMID: 37872364; PMCID: PMC10593826), information on elements related to sheep epigenetic regulation was obtained. This information was used to define functional site regions for further optimization of the chip.

[0051] 3. Screening of fixed sites

[0052] According to literature reports, several functional genes or variant sites for the positioning of traits of interest were identified as fixed sites of the chip. These fixed sites mainly cover important genetic sites known to be related to sheep production performance, reproductive performance, disease resistance and adaptability. The functional gene information is shown in Table 1.

[0053] Table 1 Functional gene information

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[0055] 4. Probe design

[0056] The OligoMiner tool was used to design probes for all regions on the genome. The parameters for probe design were: the probe length was set to 120 nt; the GC content was controlled at 30%-70%; the copy number of the entire probe in the genome was 1; and the copy number of any 25 bp kmer sequence in the genome did not exceed 5.

[0057] Through screening and optimization, we ensure that the probe has high specificity and coverage, which is suitable for the detection needs of liquid phase chips.

[0058] 5. Site screening and optimization

[0059] Taking into account multiple indicators such as evolutionary function score, eQTL information and minimum allele frequency (MAF), the initially screened SNP sites were optimized to finally generate a sheep genome-wide 50K SNP liquid phase chip site set. The probes designed for each site were handed over to Shadow Gene Company for probe synthesis and liquid phase chip preparation to obtain a sheep genome-wide 50K SNP liquid phase chip. The location information of 58,859 SNP sites of the present invention is shown in Table 2; the distribution density diagram of SNP sites on each chromosome is shown in Figure 1 As shown in Figure 2, it can be seen that the 58859 core loci are evenly distributed on each chromosome; the minimum allele frequency (MAF) statistics of the SNP loci are as follows: Figure 2 As shown, it can be seen that the MAFs are all greater than 0.1.

[0060] Table 2 Location information of SNP loci

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[0134] Example 2 Sheep Whole Genome 50K SNP Liquid Phase Chip Test Evaluation

[0135] 1. DNA extraction from sheep blood

[0136] 114 sheep (84 East Frisians, 19 Small-tailed Han sheep, and 11 F1 hybrids of East Frisians and Small-tailed Han sheep) were collected. The samples were taken from the jugular vein blood of the sheep. DNA was extracted using a genomic DNA extraction kit and stored at -20°C. The DNA quality was verified using a micro-spectrophotometer and agarose gel electrophoresis. The verification criteria included: total DNA amount ≥ 1.0 μg, concentration ≥ 20 ng / μL, volume ≥ 50 μL; OD 260 / OD 280 The ratio is between 1.8 and 2.0, OD 260 / OD 230 ≥1.8; and the main band of the sample is clear, with no degradation or slight degradation.

[0137] 2. Liquid phase gene chip test evaluation

[0138] (1) Comparison rate

[0139] The matching rate reflects the similarity between the sample sequencing data and the reference genome, and is an overall indicator for measuring the overall sequencing accuracy and the presence of contaminating DNA. The higher the matching rate, the better, indicating high data validity. Ten sheep blood DNA samples were randomly selected for chip matching rate testing. The results are as follows: Figure 3 As shown. Figure 3 The results show that in this evaluation, based on the DNBSEQ-T7 sequencing platform, the sheep whole genome 50K SNP liquid phase chip showed an excellent matching rate between sheep blood samples, and the matching rate of each library exceeded 99.7%.

[0140] (2) Target area capture efficiency

[0141] Capture efficiency refers to the ratio of the measured sequences (valid sequences) aligned to the target region to the total sequences aligned to the reference genome. The capture efficiency does not affect the data quality and is mainly used to evaluate the effectiveness of the probe design. The higher the capture efficiency, the smaller the amount of data required to cover the same depth, and the lower the sequencing cost, indicating that the probe design is highly effective. Nine sheep blood DNA samples were randomly selected for the chip capture efficiency test. The results are as follows: Figure 4 As shown. Figure 4 The results show that in this evaluation, based on the DNBSEQ-T7 sequencing platform, the sheep whole genome 50K SNP liquid phase chip showed excellent capture efficiency, and the capture efficiency of each library was higher than 77%.

[0142] (3) Data redundancy rate

[0143] During the sample library construction process, PCR amplification will lead to inconsistent amplification of different regions of the target sequence, thereby introducing artificial bias and generating some duplicate data, which is data redundancy. Duplicate data not only causes data waste, but also introduces sequencing errors. Therefore, the lower the data redundancy rate, the better. Nine sheep blood DNA samples were randomly selected for chip data redundancy rate testing. The results are as follows: Figure 5 As shown. Figure 5 The results show that in this evaluation, based on the DNBSEQ-T7 sequencing platform, the sheep whole genome 50K SNP liquid phase chip showed a very low redundancy rate of less than 20%.

[0144] (4) SNP detection rate

[0145] The SNP detection rate is an important indicator for measuring chip quality, indicating the proportion of high-quality genotyped core SNPs to all core SNPs. Nine sheep blood DNA samples were randomly selected for SNP detection rate testing. The results are as follows: Figure 6 As shown. Figure 6 The results show that in this evaluation, the SNP detection rate of each sample (typing accuracy is not less than 99%) exceeds 95.98%, indicating that the use of the sheep whole genome 50K SNP liquid phase chip of the present invention can obtain more effective and complete variation typing results.

[0146] Example 3 Application of Sheep Whole Genome 50K SNP Liquid Phase Chip

[0147] 1. Application in genotyping

[0148] The 114 sheep blood DNA samples in Example 2 were processed through a complete process including library construction, target region capture, Illumina sequencing, and bioinformatics analysis, and the target interval variation information analysis of the samples to be tested was completed, and genotyping was performed. The obtained genotyping results were quality controlled, and only SNP sites with chromosome numbers 1 to 26 and chromosome X were retained, and SNP sites with a genotype missing rate greater than 1% and a minimum allele frequency less than 1% were removed.

[0149] 2. Application in group classification

[0150] Cluster analysis is a method of classifying groups based on the degree of correlation or similarity between different individuals. We used the neighbor-joining method (NJ) to construct a genetic clustering tree for the 114 sheep in Example 2 using MEGA software, clustering closely related individuals into a small classification unit and relatively distantly related individuals into a larger classification unit until all samples were clustered. The results are as follows: Figure 7 shown. Figure 7The results clearly demonstrated the grouping effect of different breeds of sheep (East Frisian, Small-tailed Han sheep and their hybrid F1 generation). This study further verified that the liquid phase chip of the present invention can distinguish the genetic background of different breeds of sheep, providing strong support for population classification and genetic research.

[0151] 3. Application in genome-wide association analysis in sheep

[0152] The whole genome association analysis was performed using the screened SNP sites and the growth trait data of 114 sheep measured in Example 2. The FarmCPU model was used for analysis, and the results were as follows: Figure 8 As shown. Figure 8 The results show that the SNP site with the most significant correlation with body weight is 1_52649252 ( Figure 8 The SNP locus with the most significant correlation with body length is 13_64811000 ( Figure 8 The SNP locus with the most significant correlation with waist circumference is 2_21355515 ( Figure 8 The SNP locus with the most significant correlation with chest circumference is 18_20856334 ( Figure 8 The SNP locus with the most significant correlation with chest depth is 8_19584881 ( Figure 8 The SNP locus with the most significant correlation with the cross section is 4_49499000 ( Figure 8 The SNP locus with the most significant correlation with the cross width is 5_266266 ( Figure 8 The SNP locus with the most significant correlation with hip width is 5_70323169 ( Figure 8 The SNP locus with the most significant correlation with hip height is 9_52090567 ( Figure 8 The SNP locus with the most significant correlation with shoulder width is 10_85395445 ( Figure 8 The SNP site with the most significant correlation with body height is 19_59949787 ( Figure 8 These results indicate that the liquid phase chip of the present invention contains key SNP sites related to multiple growth traits of sheep, can detect a large number of SNP sites at the same time, provide high-throughput and accurate genotype data, and provide important genetic markers for further genetic improvement and gene function research.

[0153] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A probe combination for detecting a combination of SNP sites for sheep genotyping, characterized in that: For each SNP site in the SNP site combination, a group of probes for detecting the corresponding SNP site is set to form a probe combination; the SNP site combination includes 58,859 SNP sites, and the position information of the SNP sites is shown in Table 2 of the specification; the SNP site combination includes SNP sites located in the sheep reference genome Oar v4.

0.

2. The probe combination according to claim 1, characterized in that: The design parameters of the probe are: the probe length is 120 nt; the GC content is 30% to 70%; the copy number of the entire probe in the genome is 1; the copy number of any 25 bp kmer sequence in the genome does not exceed 5.

3. A kit for detecting sheep genotyping, characterized in that: The kit comprises the probe combination according to any one of claims 1 to 2.

4. Use of the probe combination according to any one of claims 1 to 2 or the kit according to claim 3 in the preparation of a sheep whole genome chip.

5. The use according to claim 4, characterized in that: The chip includes a solid phase chip or a liquid phase chip.

6. A sheep whole genome 50K SNP liquid phase chip, characterized in that: The liquid phase chip comprises the probe combination according to any one of claims 1 to 2.

7. Use of the probe combination according to any one of claims 1 to 2, the kit according to claim 3, or the liquid phase chip according to claim 6 in sheep breeding.

8. The use according to claim 7, characterized in that: The sheep breeding includes one or more of sheep genome selection, positioning of sheep trait-related genes, sheep genetic diversity analysis, sheep whole genome association analysis, sheep breed identification, sheep kinship identification, sheep population classification, sheep phenotypic regulation, sheep germplasm resource improvement and sheep germplasm resource protection.

9. The use according to claim 8, characterized in that: The traits located in the sheep trait-related genes include one or more of vision, wool, immunity, reproductive ability, fat deposition, horns, teat morphology, high altitude adaptability, muscle development, nerves, growth performance, lactation performance, carcass traits, hypoxia adaptation, daily weight gain, birth weight, cold adaptation, lactose content, blood, bones, ovarian development, fertilization, litter size, weight, body length, abdominal circumference, chest circumference, chest depth, cross height, cross width, hip width, hip height, shoulder width and body height.

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