Sheep whole genome 50K SNP chip and application

Abstract

The invention discloses a sheep whole genome SNP molecular marker, which comprises 50000 SNP molecular markers, the SNP molecular marker is composed of 5235 functional SNP molecular markers and 44765 SNP molecular markers, the functional SNP molecular markers mainly select variation sites related to economic traits (such as growth, slaughtering, meat quality, breeding, lamb and the like) of sheep, and the variation sites are selected from the variation sites. And the background SNP molecular marker is used for improving the typing precision and polymorphism coverage of the chip, so that the applicability of the chip in a large-scale group is ensured.

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 application thereof. Background Art

[0002] SNP (Single Nucleotide Polymorphism) refers to the variation of a single base at the genome level, including base deletion, insertion, conversion and transversion, etc. It is a molecular marker widely used in genome research. SNP has a wide range of characteristics in quantity and distribution, and can reflect the genetic differences between individuals. Therefore, it has important application value in animal genetic breeding and genetic variation research of complex traits. SNP chip technology fixes fluorescently labeled DNA probes on the surface of silicon wafers and realizes genotyping through hybridization of probes and target DNA. SNP chips can fix the DNA of a large number of individuals on a chip for analysis, greatly improving the efficiency of analysis.

[0003] Genomic selection (GS) is a new method for selective breeding using high-density markers covering the entire genome. Compared with traditional phenotypic selection, it can significantly improve the accuracy of breeding value estimation, enhance selection intensity, and accelerate genetic progress, thereby significantly shortening the generation interval and reducing breeding costs. Genome-wide association study (GWAS), as an important means of mining candidate loci for economic traits of livestock and poultry, is the most commonly used method for obtaining prior information at the genomic level. The results of studies on complex human traits, important economic traits of livestock and poultry, and simulation data have shown that integrating GWAS prior information can improve the accuracy of GS.

[0004] China is a large sheep-breeding country, but the yield per unit area of ​​mutton sheep is low, and there is a lack of commercial mutton sheep breeds with high growth performance. At the same time, the high price of high-density SNP chips on the market limits the widespread application of GS in actual breeding, and there is no dedicated medium-density SNP chip for key economic traits of sheep, which cannot comprehensively evaluate the key traits of sheep and cannot meet the needs of efficient and low-cost breeding.

[0005] Therefore, there is an urgent need for a sheep whole-genome SNP chip. Summary of the invention

[0006] On one hand, the present invention provides a sheep whole genome 50K SNP chip to solve the problem that the existing sheep SNP chip cannot effectively evaluate the complex traits of sheep, and the traditional phenotypic selection and existing non-specific SNP chip evaluation methods are time-consuming and inefficient. On the other hand, the present invention provides an application of a sheep whole genome SNP chip.

[0007] A first aspect of the present invention provides a sheep whole genome SNP molecular marker, including at least one of 5000 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 muscle area, GR value, back fat thickness, perirenal fat weight, greater 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] The seventh 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 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 very small. Genome selection can be directly performed 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 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 amount of information contained in the sites is large, and the population scale referenced 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 genotype of individual sheep. At the depth of dp5, the maximum and minimum individual detection rates are 99.47% and 98.98% respectively, and the average SNP detection rate is 99.21%; and the genotyping stability is good, and the genotype consistency can reach 99.3%. It can be seen that the sheep 50K SNP chip of the present invention can be accurately used for the positioning of sheep trait-related genes, sheep genetic diversity analysis, dairy cow genome-wide association analysis, sheep genotyping detection, and sheep selection breeding. The present invention not only helps to shorten the breeding cycle and improve breeding efficiency and quality, but also detects the genotype of newborn sheep individuals, and can predict their growth rate, slaughter rate, fat content and other production performance indicators, thereby optimizing the production process 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 The chromosome distribution map of the SNP site of the present invention;

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

[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] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of 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.

[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] Combining the 7 SNP sites related to lamb skin traits discovered in the sheep population by typing mass spectrometry in the early stage, and searching the "FecB" gene in the China National Knowledge Infrastructure and PubMed databases, 2 possible SNP sites related to sheep fertility were found.

[0036] S2. Use historical sheep phenotype and genotype data to perform 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 filling 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 the 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, GLIMPSE2 software is used to fill the low-depth sequencing data to reach the data volume of high-depth data, and finally the data is 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 genotype missing rates 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 the 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, as well as the age, sex, and feeding environment of the sheep 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 weight gain from birth to weaning, KR value from birth to weaning, weight at 6 months of age, daily weight gain from weaning to 6 months, daily weight 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 analyzed by 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. Obtaining a 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 SNP.

[0049] Step S302, determine the functional sites for the final chip customization according to the Compson probe design system score. Perform typing verification on the candidate sites to be selected, compare the ref base with the reference genome provided by the present invention, remove duplicates, retain the correctly typed sites, and submit the site set to the Compson probe design system for scoring.

[0050] Step S303, evaluate the functional sites of the final chip customization. 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 chip customization SNP sites.

[0051] The evaluation criteria are: mainly evaluate the complexity of the upstream and downstream sequences of the target site, GC content, etc., give priority to placing the target site in the middle of the probe, and the designed probe is 120bp in length. Generally, the probe performance is comprehensively evaluated from the aspects of GC content (20%-80%), Tm value (60-80°C), number of alignments on the reference genome (≤5), etc.

[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] Example 2

[0097] Sheep whole genome chip development

[0098] In this example, the chip sites obtained in Example 1 were confirmed to be correct and 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 had a DNA nucleotide sequence modified 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 3As shown in the figure, the loci of each chromosome are evenly distributed, indicating that the loci contained in this chip can cover each chromosome well. Figure 4 As shown in the figure, 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 compared with other low-density chips. Figure 5 As shown in the figure, this chip covers a variety of mutation types and has important significance for genetic mechanism research. Through the above steps, the sheep 50K SNP chip obtained can be widely used in genetic evaluation, genome selection and precision breeding of sheep populations, providing technical support for the rapid development of the sheep industry and high-quality breeding.

[0099] The molecular probe is used to specifically identify the SNP molecular marker obtained in Example 1.

[0100] The sheep liquid phase SNP capture chip comprises the molecular probe.

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

[0102] Example 3

[0103] Evaluation of the quality of sheep 50K SNP chip genotyping

[0104] 42 sheep were randomly selected for genotyping using the sheep 50K SNP chip obtained in Example 1, and the following quality evaluation was performed.

[0105] 1. Detection rate

[0106] SNP detection rate and individual detection rate are important indicators for measuring chip quality, and are generally measured using autosomal and X chromosome loci.

[0107] The sheep 50K SNP chip obtained in Example 2 has a 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. .22% and 98.24%, the average SNP detection rate was 98.78%, and the standard deviation was 0.21825; at the depth of dp15, the maximum and minimum individual detection rates were 98.95% and 97.20%, respectively, the average SNP detection rate was 98.22%, and the standard deviation was 0.38858; at the depth of dp20, the maximum and minimum individual detection rates were 98.62% and 95.84%, respectively, the average SNP detection rate was 97.50%, and the standard deviation was 0.65122. It can be seen that the quality of sheep 50K SNP chip genotyping is very good.

[0108] 2. Genotyping stability

[0109] Stability is generally measured by the consistency and correlation coefficient of the results of two genotyping tests of repeated samples. Three of the 42 sheep were randomly selected to test the sheep 50K SNP chip obtained in Example 2. The genotyping consistency of the two genotyping tests of the three repeated samples was 99.3%. It can be seen that the stability of the genotyping test of the sheep 50K SNP chip of the present invention is very good.

[0110] 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 embodiments of the present invention.

Claims

1. A sheep whole genome SNP molecular marker, characterized in that: It includes at least one of 50,000 SNP molecular markers, and the SNP molecular markers are specifically shown in Table 1.

2. A molecular probe, characterized in that The molecular probe is used to specifically identify the SNP molecular marker described in claim 1.

3. A sheep liquid phase capture chip, characterized in that: Comprising the molecular probe as claimed in claim 2.

4. The sheep liquid phase capture chip according to claim 3, characterized in that: The SNP molecular marker is associated with at least one of economic traits such as growth, slaughter, meat quality, reproduction, lamb skin, etc.; 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; The slaughter traits include: live weight before slaughter, carcass weight, bone weight, meat weight, eye muscle area, GR value, back fat thickness, perirenal fat weight, omental fat weight, tail fat weight, subcutaneous fat weight, slaughter rate, net meat rate, and meat-to-bone ratio; 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; The reproductive traits include: number of siblings, testis weight, epididymis weight; The lambskin properties include: lambskin area and pattern.

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

6. Use of at least one of the SNP molecular marker according to claim 1, the molecular probe according to claim 2, the sheep liquid phase capture chip according to claim 3, and the kit according to claim 4 in sheep genotyping detection, molecular marker-assisted breeding, or whole genome association analysis.

7. Use of at least one of the SNP molecular markers according to claim 1, the molecular probes according to claim 2, the sheep liquid phase capture chip according to claim 3, and the kit according to claim 4 in sheep genetic diversity analysis, economic trait identification or improvement, or whole genome selection breeding.

8. Use of at least one of the SNP molecular marker according to claim 1, the molecular probe according to claim 2, the sheep liquid phase capture chip according to claim 3, and the kit according to claim 4 in cluster analysis, kinship identification, or identification of sheep breeds.

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