A sheep whole-genome 50K SNP chip and its application

Abstract

The present invention discloses a sheep whole-genome SNP molecular marker, including 50,000 SNP molecular markers. The SNP molecular markers are composed of 5,235 functional SNP molecular markers and 44,765 SNP molecular marker backgrounds. The functional SNP molecular markers are mainly selected from variant sites related to sheep economic traits (such as growth, slaughter, meat quality, reproduction, lamb skin, etc.), while the background SNP molecular markers are used to improve the typing accuracy and polymorphism coverage of the chip, ensuring the applicability of the chip in a wide range of populations.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a sheep whole genome 50K SNP chip and its application. Background Art

[0002] SNPs (Single Nucleotide Polymorphism) refer to variations in a single base at the genomic level, including base deletions, insertions, transitions, and transversions. They are molecular markers widely used in genomic research. SNPs have a wide range of characteristics in both quantity and distribution, reflecting genetic differences between individuals. Therefore, they hold important application value in animal genetics and breeding, as well as in the study of genetic variation in complex traits. SNP chip technology immobilizes fluorescently labeled DNA probes on the surface of a silicon wafer, achieving genotyping through hybridization between the probes and target DNA. SNP chips can analyze the DNA of numerous individuals on a single chip, significantly improving analysis efficiency.

[0003] Genomic selection (GS) is a novel breeding method that utilizes high-density markers covering the entire genome. Compared with traditional phenotypic selection, it can significantly improve the accuracy of breeding value estimates, enhance selection intensity, and accelerate genetic progress, thereby significantly shortening generation intervals and reducing breeding costs. Genome-wide association studies (GWAS), as an important means of discovering candidate loci for economic traits in livestock and poultry, are the most commonly used method for obtaining prior information at the genomic level. Studies of complex human traits, important economic traits in livestock and poultry, and simulated data have shown that incorporating GWAS prior information can improve the accuracy of GS.

[0004] China is a major sheep producer, but its meat yields per unit area are low, and there is a lack of commercially available meat sheep breeds with high growth performance. Furthermore, the high cost of high-density SNP arrays on the market limits the widespread application of GS in practical breeding. The lack of specialized medium-density SNP arrays targeting key economic traits in sheep makes it impossible to comprehensively assess these traits and fails to meet the demand for efficient and low-cost breeding.

[0005] Therefore, a sheep whole-genome SNP chip is urgently needed. Summary of the Invention

[0006] On one hand, the present invention provides a sheep whole-genome 50K SNP chip to address the inability of existing sheep SNP chips to effectively assess complex traits in sheep, and the time-consuming and inefficient evaluation methods of traditional phenotypic selection and existing non-specific SNP chip evaluation methods. Another aspect of the present invention provides an application of a sheep whole-genome SNP chip.

[0007] A first aspect of the present invention provides a sheep genome-wide SNP molecular marker, comprising at least one of 5,000 SNP molecular markers, and the SNP molecular markers are specifically shown in Table 1.

[0008] The second aspect of the present invention provides a molecular probe, which is used to specifically identify the SNP molecular marker.

[0009] The third aspect of the present invention provides a sheep liquid phase capture chip, comprising the molecular probe.

[0010] The sheep liquid phase capture chip, wherein the SNP molecular marker is associated with at least one of economic traits such as growth, slaughter, meat quality, reproduction, and lamb skin;

[0011] Preferably, the growth traits include: birth weight, weaning weight, daily weight gain from birth to weaning, KR value from birth to weaning, weight at 6 months, daily weight gain from weaning to 6 months, and daily weight gain from birth to 6 months;

[0012] Preferably, the slaughter traits include: live weight before slaughter, carcass weight, bone weight, meat weight, eye loin area, GR value, backfat thickness, perirenal fat weight, omental fat weight, tail fat weight, subcutaneous fat weight, slaughter rate, net meat rate, and meat-to-bone ratio;

[0013] Preferably, the meat quality traits include: 24h drip loss rate, L * 45min 、a * 45min 、b * 45min , L * 24h 、a * 24h 、b * 24h , pH 45min , pH 24h , shear force, moisture, crude protein, crude fat, crude ash, fatty acids;

[0014] Preferably, the reproductive traits include: number of siblings, testis weight, epididymis weight;

[0015] Preferably, the lambskin properties include: lambskin area and pattern.

[0016] The fourth aspect of the present invention provides a kit comprising the molecular probe or the sheep liquid phase capture chip.

[0017] The fifth aspect of the present invention provides an application of at least one of the SNP molecular marker, the molecular probe, the sheep liquid phase capture chip and the kit in sheep genotyping detection, molecular marker-assisted breeding or whole genome association analysis.

[0018] The sixth aspect of the present invention provides an application of at least one of the SNP molecular marker, the molecular probe, the sheep liquid phase capture chip and the kit in sheep genetic diversity analysis, economic trait identification or improvement or whole genome selection breeding.

[0019] A seventh aspect of the present invention provides a use of at least one of the SNP molecular marker, the molecular probe, the sheep liquid phase capture chip and the kit in sheep cluster analysis, kinship identification or sheep breed identification.

[0020] The beneficial effects are:

[0021] The SNP marker sites provided by the present invention are evenly distributed on the chromosomes of the sheep genome and can be filled into the original sites using family information. The loss of genotype information is minimal. Genome selection can be performed directly using chip sequencing data, or genome selection can be performed using the filled data to obtain acceptable evaluation accuracy.

[0022] The SNP marker sites provided by the present invention have a high MAF in the detection population, with sites with a minimum allele frequency > 0.1 accounting for 97.39%, most of which are concentrated in the range of 0.4-0.5. Therefore, compared with other low-density chips, they have higher information entropy and genotype filling accuracy.

[0023] The 50K chip prepared with the SNP molecular marker combination of the present invention contains a moderate number of sites, the sites contain a large amount of information, and the reference population size during the design process is large, the collected traits cover a wide range, and it has high reliability and universality.

[0024] The sheep 50K SNP chip provided by the present invention can quickly and accurately detect the genotypes of individual sheep. At a depth of 5 dp, the maximum and minimum individual detection rates are 99.47% and 98.98%, respectively, and the average SNP detection rate is 99.21%. Genotyping stability is excellent, with genotype consistency reaching 99.3%. Therefore, the sheep 50K SNP chip described in the present invention can be accurately used for mapping genes related to sheep traits, analyzing sheep genetic diversity, conducting genome-wide association studies, performing sheep genotyping, and performing selective breeding. This invention not only helps shorten breeding cycles and improve breeding efficiency and quality, but also allows genotyping of newborn sheep to predict production performance indicators such as growth rate, slaughter rate, and fat content, thereby optimizing production processes and reducing breeding costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a schematic diagram of the research and development process of the present invention;.

[0026] Figure 2 This is the chromosome distribution map of the SNP sites of the present invention;

[0027] Figure 3 This is a marker distribution density map of the SNP site on the chromosome of the present invention;

[0028] Figure 4 This is the MAF distribution diagram of the SNP sites of the present invention;

[0029] Figure 5 This is a statistical diagram of the SNP site variation types of the present invention. DETAILED DESCRIPTION

[0030] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0031] Example 1

[0032] Sheep whole genome chip SNP molecular marker combination detection

[0033] like Figure 1 As shown, the SNP molecular marker combination described in Example 1 is mainly obtained by the following steps:

[0034] S1. SNP sites screened from sheep population experiments and obtained from existing reports.

[0035] Combined with the 7 SNP sites related to lamb skin traits discovered in the sheep population in the early stage using typing mass spectrometry, the "FecB" gene was searched in the China National Knowledge Infrastructure and PubMed databases, and 2 SNP sites that may be related to sheep fertility were found.

[0036] S2. Use historical sheep phenotypic and genotypic data to conduct genome-wide association analysis (GWAS) to obtain SNP sites related to economic traits.

[0037] Step S2 specifically includes the following steps:

[0038] Step S201, data acquisition and fill panel construction: 351 sheep were sequenced using the BGI MGISEQ-T7 platform for high-depth (10X) resequencing, while 1,494 sheep were sequenced using the BGI MGISEQ-T7 platform for low-depth (1X) resequencing.

[0039] The filling panel was constructed by using high-depth (10X) resequencing data of 351 sheep.

[0040] Step S202, data filling and merging: Using the filling panel constructed with high-depth sequencing data, the low-depth sequencing data were filled using GLIMPSE2 software to reach the data volume of high-depth data. Finally, the data were merged to obtain a total of 26,291,382 SNP sites.

[0041] Step S203, data quality control: routine quality control is performed on the 26,291,382 SNP sites obtained by merging in step S202. The quality control standards include allele frequency (MAF>0.05), hard balance test (HWE>1e-6), genotyping accuracy (call rate>95%), etc.

[0042] Individuals with a genotype missing rate higher than 10% were deleted, and sites with too high or too low heterozygosity rates (e.g., sites with a minimum allele frequency (MAF) lower than 0.05, sites with a Hardy-Weinberg equilibrium (HWE) P value < 1e-6, and sites on sex chromosomes) were removed.

[0043] Step S204: Genome-wide association analysis of sheep economic traits: Beagle 5.0 software was used to fill in sparse missing genotype data and perform population structure analysis, including principal component analysis (PCA) and kinship analysis. The top three PCA and kinship matrices with the largest explained variance, along with sheep age, sex, and housing environment, were included as covariates.

[0044] Based on the data after S203 quality control, the LMM model in Gemma software was used to analyze 39 traits (birth weight, weaning weight, daily gain from birth to weaning, KR value from birth to weaning, weight at 6 months, daily gain from weaning to 6 months, daily gain from birth to 6 months, live weight before slaughter, carcass weight, bone weight, meat weight, eye muscle area, GR value, backfat thickness, perirenal fat weight, omental fat weight, tail fat weight, subcutaneous fat weight, slaughter rate, net meat rate, meat-to-bone ratio, 24h drip loss rate, L * 45min 、a * 45min 、b * 45min , L * 24h 、a* 24h , 2b * 24h , pH 45min , pH 24h , shear force, moisture, crude protein, crude fat, crude ash, fatty acids, number of siblings, testis weight, and epididymis weight) were subjected to genome-wide association analysis.

[0045] The screening criteria for functional SNP sites were adjusted based on the chromosome significance level (P_wald < 5e-6) and false discovery rate (FDR < 0.05), and 7,000 SNP sites significantly associated with key economic traits of sheep were found.

[0046] S3. Acquisition of 50K SNP molecular marker combination in sheep.

[0047] Preferably, in step S3, the steps of obtaining the sheep 50K SNP set are as follows:

[0048] Step S301 , combining the SNP data obtained in steps S1 and S2 , removing duplicate sites, removing sites in linkage disequilibrium, and screening and optimizing all SNP sites based on the polymorphism, functionality, and genetic background information of the SNPs.

[0049] Step S302: Determine the functional sites for the final chip customization based on the Compson Probe Design System scores. The candidate sites are typed and verified, and the ref bases are compared with the reference genome provided by the present invention. Duplicates are removed, and correctly typed sites are retained. The site set is then submitted to the Compson Probe Design System for scoring.

[0050] Step S303: Evaluate the functional sites for the final customized chip. Probe design is performed based on the evaluation results of the upstream and downstream sequences of the target site. Based on the design results, 5,235 functional SNP sites and 44,765 background SNP sites are screened, totaling 50,000 high-quality customized SNP sites for the chip.

[0051] The evaluation criteria primarily assess the complexity of the upstream and downstream sequences of the target site, as well as the GC content. Prioritizing the target site in the middle of the probe, the probe was designed to be 120 bp in length. Generally, probe performance is comprehensively evaluated based on factors such as GC content (20%-80%), Tm value (60-80°C), and the number of alignments to the reference genome (≤5).

[0052] The background SNP sites are used to improve the typing accuracy and polymorphism coverage of the chip, ensuring the applicability of the chip in a wide range of populations. The positions of the SNP molecular markers on the sheep reference genome Oar_v4.0 are shown in Table 1.

[0053] Table 1 SNP site information

[0054]

[0055]

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

[0070]

[0071]

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097] Example 2

[0098] Sheep whole genome chip development

[0099] In this example, the chip sites obtained in Example 1 were confirmed to be correct and then submitted to Compson for chip probe synthesis, including 5,235 functional SNP sites and 44,765 background SNP sites. The probe length was 120bp, and the 5' end was modified with a DNA nucleotide sequence with a biotin group. Subsequently, the sheep liquid phase capture SNP chip and kit were developed to form the final sheep 50K SNP chip. This chip has important application value in accurately evaluating key economic traits of sheep and improving breeding efficiency. Figure 2 As shown in Figure 2, the selected functional sites are evenly distributed on the chromosome and have good coverage. Figure 3 As shown in the figure, the sites of each chromosome are evenly distributed, indicating that the sites contained in this chip can well cover each chromosome. Figure 4 As shown, the sites with the minimum allele frequency > 0.1 account for 97.39%, and most of them are concentrated in 0.4-0.5, so it has higher information entropy and genotype filling accuracy than other low-density chips. Figure 5 As shown, this array covers a wide range of mutation types and has important implications for genetic mechanism research. The resulting sheep 50K SNP array, generated through the above steps, can be widely used in genetic assessment, genomic selection, and precision breeding of sheep populations, providing technical support for the rapid development of the sheep industry and high-quality breeding.

[0100] The molecular probe is used to specifically identify the SNP molecular marker obtained in Example 1.

[0101] The sheep liquid phase SNP capture chip comprises the molecular probe.

[0102] The kit comprises the molecular probe or the sheep liquid phase capture chip.

[0103] Example 3

[0104] Quality evaluation of the sheep 50K SNP chip genotyping

[0105] 42 sheep were randomly selected for genotyping using the sheep 50K SNP chip obtained in Example 1, and the following quality evaluation was performed.

[0106] 1. Detection rate

[0107] SNP detection rate and individual detection rate are important indicators for measuring chip quality, and are generally measured using loci on autosomes and chromosomes.

[0108] The sheep 50K SNP chip obtained in Example 2 has a very high SNP detection rate. At the depth of dp0, the maximum and minimum individual detection rates are 99.82% and 99.53%, respectively, the average SNP detection rate is 99.62%, and the standard deviation is 0.060411; at the depth of dp5, the maximum and minimum individual detection rates are 99.47% and 98.98%, respectively, the average SNP detection rate is 99.21%, and the standard deviation is 0.10628; at the depth of dp10, the maximum and minimum individual detection rates are 99. The maximum and minimum individual detection rates were 98.95% and 97.20%, respectively, with an average SNP detection rate of 98.22% and a standard deviation of 0.21825. At a depth of dp15, the maximum and minimum individual detection rates were 98.95% and 97.20%, respectively, with an average SNP detection rate of 98.22% and a standard deviation of 0.38858. At a depth of dp20, the maximum and minimum individual detection rates were 98.62% and 95.84%, respectively, with an average SNP detection rate of 97.50% and a standard deviation of 0.65122. These results indicate that the sheep 50K SNP chip genotyping quality is excellent.

[0109] 2. Genotyping stability

[0110] Stability is generally measured by the consistency and correlation coefficient of two replicate genotyping results. Three of the 42 sheep randomly selected were tested on the sheep 50K SNP chip obtained in Example 2. The genotypic consistency of the three replicates was 99.3%. This demonstrates the excellent stability of the sheep 50K SNP chip genotyping assay described herein.

[0111] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A molecular probe combination, characterized in that: The molecular probe combination is used to specifically detect 50,000 SNP site combinations, and the positions of the 50,000 SNP site combinations on the sheep reference genome Oar_v4.0 are shown in Table 1.

2. A sheep liquid phase capture chip, characterized in that: The method comprises the molecular probe combination as claimed in claim 1.

3. A kit, characterized in that It comprises the molecular probe combination as claimed in claim 1 or the sheep liquid phase capture chip as claimed in claim 2.

4. Use of at least one of the molecular probe combination according to claim 1, the sheep liquid phase capture chip according to claim 2, and the kit according to claim 3 in sheep genotyping detection.

5. Use of at least one of the molecular probe combination according to claim 1, the sheep liquid phase capture chip according to claim 2, and the kit according to claim 3 in sheep genetic diversity analysis.

6. Use of at least one of the molecular probe combination according to claim 1, the sheep liquid phase capture chip according to claim 2, and the kit according to claim 3 in cluster analysis or kinship identification of sheep.

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