Genome-wide low-density SNP chip for milk goats and use thereof

Abstract

Disclosed are a genome-wide low-density SNP chip for milk goats and a use thereof. The provided SNP chip is applicable to genetic improvement of economic traits of European milk goat varieties and Chinese native milk goat varieties, can also be applied to a plurality of fields of milk goat genome-wide association study, genome-wide selection breeding, population genetics analysis, trait-related candidate genes, QTL analysis and locating, and the like, and has high value in production application and scientific research.

Description

A dairy goat whole genome low-density SNP chip and its application Technical Field

[0001] The present invention relates to the fields of molecular biology, functional genomics, bioinformatics and genome breeding technology, and in particular to a dairy goat whole genome low-density SNP chip and application thereof. Background Art

[0002] SNP, short for single nucleotide polymorphism, refers to variations in a single nucleotide in the genome, resulting from single-base deletions, insertions, transversions, or substitutions. As important molecular markers, SNPs are ideal for genotyping due to their large number, dense distribution, and ease of detection, making them easy to automate and scale using microarray technology.

[0003] Since its first proposal in 2001, genomic selection (GS) has been widely practiced in animal and plant breeding. The basic principle of GS is to obtain individual genomic information using whole-genome sequencing or SNP chip detection technology. By comparing differences between individual genomes, genetic markers or genes associated with target traits are identified. By screening the genomes of a large number of individuals, individuals with the genomic characteristics required for the target trait are selected for breeding. After multiple generations of selective breeding, the target genomic characteristics gradually accumulate in the offspring, achieving the goal of improving species traits. This is due to the genetic linkage between beneficial genes and beneficial traits, allowing for indirect selection of target traits through selection of genomic characteristics.

[0004] The advantage of genomic selection is that it can accelerate the process of species improvement and improve breeding efficiency. By accurately identifying and selecting target genomic features, genomic selection can more precisely screen for beneficial genes and avoid irrelevant phenotypic variation compared to traditional phenotypic selection.

[0005] Given its advantages of reducing breeding costs and shortening generation intervals, genomic selection has become a routine genetic evaluation strategy for many livestock species. Currently, genomic selection (GS) has been maturely applied in dairy cattle breeding, accelerating the breeding process and reducing breeding costs. However, for animals with short generation intervals and large litters, such as pigs, chickens, and sheep, the current mainstream medium- and high-density SNP chips are still relatively expensive, and therefore GS technology has not been well applied. Currently, the most widely used SNP chip for goats is the Goat 50K SNP chip (GoatSNP50) produced by Illumina. However, as a broad-spectrum chip for goats, it has poor adaptability to dairy goats and is relatively expensive, making it difficult to promote in breeding.

[0006] Therefore, it is necessary to develop a whole-genome chip for dairy goats.

[0007] Summary of the Invention

[0008] In order to solve one of the above technical problems existing in the prior art, the present invention provides a dairy goat whole genome low-density SNP chip and its application.

[0009] In a first aspect, the present invention provides a SNP molecular marker for the whole genome of a dairy goat, wherein the SNP molecular marker is selected from at least one of the markers shown in Table 3.

[0010] In a second aspect, the present invention provides a molecular probe for specifically identifying the SNP molecular marker described in the first aspect.

[0011] In a third aspect, the present invention provides a dairy goat whole genome SNP chip, wherein the SNP chip comprises probes targeting the SNP molecular markers described in the first aspect.

[0012] In some embodiments, the dairy goat whole genome SNP chip provided by the present invention is a dairy goat whole genome low-density 10K SNP chip.

[0013] In some embodiments, the SNP molecular marker is associated with the milk production trait of dairy goats. The milk production trait is a major economic trait of dairy cows, and the milk production trait includes milk yield, milk quality, and melatonin synthesis ability.

[0014] In some embodiments, the milk production is 305 days of milk production.

[0015] In some embodiments, the milk quality includes milk protein rate, milk fat rate, and lactose rate.

[0016] In some embodiments, the SNP molecular marker is associated with reproductive traits of dairy goats, including lambing size and parity interval.

[0017] In the dairy goat whole-genome low-density SNP chip provided by the present invention, the SNP sites include two types of probes: the first type is based on selection signal analysis and whole-genome association analysis, which screens out 1644 SNP sites that are significantly associated with the milk production traits of dairy goats; the second type is SNP sites with good polymorphism screened out using resequencing data of five dairy goat breeds (Saanen dairy goat, Toggenburg goat, Alpine goat, Guanzhong dairy goat, and Laoshan dairy goat), with high polymorphism and uniform genomic distribution as the screening principles, to screen 8269 SNP sites (hereinafter referred to as background sites).

[0018] In the dairy goat whole genome low-density SNP chip provided by the present invention, the average distance between adjacent SNP marker sites on the chip is 259 kb.

[0019] In the dairy goat whole genome low-density SNP chip provided by the present invention, the linkage disequilibrium degree r2 between adjacent SNP sites of the SNP chip is 0.467.

[0020] In the dairy goat whole genome low-density SNP chip provided by the present invention, the average minimum allele frequency (MAF) of the SNP chip is 0.27.

[0021] In a fourth aspect, the present invention provides a kit comprising the molecular probe described in the second aspect, or the SNP chip described in the third aspect.

[0022] In a fifth aspect, the present invention provides use of the molecular probe, or the SNP chip, or the kit in whole genome breeding of dairy goats.

[0023] In a sixth aspect, the present invention provides use of the molecular probe, or the SNP chip, or the kit in identifying kinship of dairy goats.

[0024] In a seventh aspect, the present invention provides use of the molecular probe, or the SNP chip, or the kit in identifying European dairy goat and Chinese dairy goat breeds.

[0025] In an eighth aspect, the present invention provides use of the molecular probe, or the SNP chip, or the kit in genotyping detection of dairy goats.

[0026] In a ninth aspect, the present invention provides use of the molecular probe, or the SNP chip, or the kit in genetic diversity analysis of a dairy goat population.

[0027] In a tenth aspect, the present invention provides use of the molecular probe, or the SNP chip, or the kit in the location analysis of QTL (quantitative trait loci) of target traits in dairy goats.

[0028] Compared with the prior art, the present invention has the following advantages:

[0029] The SNP marker sites provided by the present invention are evenly distributed on the 29 autosomes of the dairy goat genome and the two contigs of the X chromosome. The distribution is uniform and the coverage is high, which ensures the accuracy of the genetic evaluation of the dairy goat genome. The sites included in the chip of the present invention are all large-effect SNP sites obtained during the experiment, which are strongly associated with economic traits, increasing the practicality of the chip. The chip sites of the present invention are derived from SNP sites with good polymorphism in imported dairy goat varieties and local fine breeds widely raised in my country, which not only meets the scientific research needs of scientific researchers but is also suitable for whole-genome molecular breeding.

[0030] The SNP chip provided by the present invention is suitable for use in the genetic improvement of economic traits of European dairy goat breeds and Chinese native dairy goat breeds. It can also be used in multiple fields such as whole-genome association analysis, whole-genome selective breeding, population genetics analysis, and analysis and positioning of trait-related candidate genes and QTL sites in dairy goats. It has high value in both production applications and scientific research. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] FIG1 shows the distribution of SNP sites of the present invention across the entire genome.

[0032] FIG2 shows the distribution of SNP sites of the present invention at the whole genome level.

[0033] FIG3 shows a diagram of the intervals between adjacent SNP sites of the present invention.

[0034] FIG4 shows the r2 frequency distribution diagram between adjacent SNP sites.

[0035] FIG5 shows the cluster analysis results of Chinese dairy goats and European dairy goats in Example 3. FIG. DETAILED DESCRIPTION

[0036] To make the objectives, technical solutions, and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the following examples. The specific embodiments described herein are intended only to illustrate the present invention and are not intended to limit the present invention in any way. In addition, in the following description, descriptions of known structures and technologies are omitted to avoid unnecessary confusion about the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0037] definition

[0038] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0039] Unless the context clearly dictates otherwise, as used herein, the expressions "a" and "an" include plural references. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth.

[0040] As used herein, the term "about" refers to a range of ±20% of the value that follows. In some embodiments, the term "about" refers to a range of ±10% of the value that follows. In some embodiments, the term "about" refers to a range of ±5% of the value that follows.

[0041] The following examples and accompanying drawings are provided to facilitate understanding of the present invention. However, it should be understood that these examples and accompanying drawings are intended to illustrate the present invention only and are not intended to limit the present invention in any way. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and variations may be made without departing from the spirit of the present invention.

[0042] The technical solution of the present invention is not limited to the specific embodiments, and all technical variations made based on the technical solution of the present invention fall within the protection scope of the present invention.

[0043] Example 1 Preparation of a low-density 10K SNP chip for the whole genome of dairy goats

[0044] 1. Functional site screening

[0045] (1) Phenotypic data collection and quality control

[0046] The study population consisted of Saanen, Toggenburg, Alpine, Guanzhong, and Laoshan dairy goats from the core breeding farm of Inner Mongolia Shengjian Biotechnology Co., Ltd. 305-day milk production records, Dairy Herd Improvement (DHI) records, and morning and evening serum melatonin levels were collected. After quality control, 310 dairy goats with complete records were selected.

[0047] (2) Genotyping, quality control and site filtering

[0048] Ear tissue samples were collected from 310 dairy goats and total DNA was extracted. Whole genome resequencing was performed on 110 of them and the other 200 were used for Genotyping was performed using the targeted capture chip "Dairy Goat 55K". SNPs with a detection rate <95%, a minimum allele frequency <0.01, or an extreme failure to comply with the Hardy-Weinberg equilibrium test with a P value less than 10 were removed. -6 Individuals with a detection rate of <90% were included. Missing genotypes were imputed using Beagle software (version 4.1). After imputation, quality control was performed again using the same conditions as above. After quality control, 310 dairy goats and 94,194 SNPs were included in subsequent analysis.

[0049] (3) Select signal analysis

[0050] The cross-population composite likelihood ratio test (XP-CLR) was used to analyze the selection signal in Chinese dairy goat breeds (Guanzhong dairy goat, Laoshan dairy goat) and European imported dairy goat breeds (Saanen dairy goat, Toggenburg dairy goat, Alpine dairy goat). The minimum number of SNPs per window was set to 200; the window size was set to 50,000; the sliding window step was set to 2,000; and the weight analysis parameter was set to 0.95. The top 1% of the analysis results were selected as functional loci.

[0051] (4) Genome-wide association analysis model

[0052] A mixed linear model (MLM) was used to conduct genome-wide association analysis for each trait. For each trait, effects that significantly affected the trait were added as fixed effects to the model.

[0053] MLM model equation: y=Xm+Wa+e

[0054] Where y is the phenotype vector, m is the vector of SNP marker effects, and a is a random polygenic effect that conforms to the normal distribution. X and W are the relationship matrices corresponding to m and a, respectively, and e is a random residual that conforms to a normal distribution. After the analysis is completed, the SNP sites are ranked according to their significance in association with the trait, and the top 1% of SNP sites are selected as functional sites.

[0055] (5) 10K chip site preparation

[0056] RStudio (R language) was used to obtain the distribution map of the SNP sites of the present invention across the entire genome, as shown in Figure 1. SNP sites were screened based on the requirements that SNPs are evenly distributed on each chromosome, densely distributed at both ends of each chromosome, and have good polymorphism. Based on the selection signal and the results of the whole-genome association analysis, sites were evenly selected according to a window length of 35kb, as shown in Figure 2, and a low-density chip containing 9,913 SNPs was eventually formed. The adjacent distances of each SNP are shown in Figure 3. The degree of linkage disequilibrium (LD) of adjacent SNP sites is measured by the r2 frequency. The r2 of adjacent SNP sites calculated using PLINK software is shown in Figure 4. It can be seen from the figure that the r2 between adjacent SNP sites is concentrated between 0.2 and 1. The number of SNP chip sites distributed on each chromosome is shown in Table 1.

[0057] Table 1. The number of SNP chip sites on each chromosome of the whole genome of dairy goats of the present invention

[0058] As shown in Table 1, the SNP chip sites of the dairy goat genome of the present invention are evenly distributed, covering 29 autosomes and the contig representing the X chromosome, and having good whole-genome coverage.

[0059] Example 2 Process of whole genome 10K SNP chip detection of dairy goat DNA samples

[0060] (1) Sample collection and preservation

[0061] Samples for chip testing can be freshly collected goat ear tissue or peripheral venous blood. Ear tissue samples should be collected in a size similar to a soybean. Before collection, disinfect the sampling site with iodine and 75% ethanol. After collection, completely immerse the sample in 75% ethanol and store at -20°C. Peripheral venous blood should be collected using anticoagulant blood collection tubes with a volume of no less than 5ml. After collection, store at -20°C.

[0062] (2) DNA extraction gene

[0063] Genomic DNA was extracted from dairy goats using a genomic DNA extraction kit. The specific process was adjusted based on the sampling type (ear margin tissue or peripheral venous blood). The extracted genomic DNA should be stored at -20°C. DNA quality was verified using 1% agarose gel electrophoresis and a microspectrophotometer. Qualified samples had a clear main band, a total DNA (no RNA) content ≥1.0 μg, a concentration ≥20 ng / μL, a volume >50 μl, an OD260:OD280 ≤2.0 of 1.8, an OD260:OD230 ≥1.8, and no or slight degradation.

[0064] (3) Construction of dairy goat DNA library and chip detection

[0065] For dairy goat genomic DNA samples, library samples were prepared according to the high-throughput library development reaction kit process. After quality control, the library samples were ready for microarray testing. Microarray probes hybridized with the samples to capture the target loci. Sequencing was then performed on the sequencing machine, along with basic bioinformatics analysis. The target interval variation information for the sample was analyzed, allowing for genotyping.

[0066] Example 3 Principal component analysis based on the low-density 10K SNP chip of the whole genome of dairy goats of the present invention

[0067] The DNA genotype of 23 European dairy goats (EUR) and 77 Chinese dairy goats (CN) was detected using the dairy goat full genome low-density 10K SNP chip provided by the present invention, first eliminating the SNP site with MAF less than 0.01, then respectively utilizing PLINK software to carry out principal component analysis. Principal component analysis result is as shown in Figure 5, and the hollow triangle on the left represents Chinese dairy goat individual, and the solid square on the right represents European dairy goat individual, and the two classes of dairy goats are clearly divided into two classes. Above-mentioned principal component analysis result can well demonstrate the applicability of the SNP chip of the present invention in the identification of Central European dairy goats, and its identification result is accurate and reliable.

[0068] Example 4 Application of the present invention's whole-genome low-density 10K SNP chip in genomic breeding of dairy goats

[0069] This experiment selected 300 Saanen dairy goats from the core breeding farm of Inner Mongolia Shengjian Biotechnology Co., Ltd. as the experimental group, born between 2019 and 2020. These 300 goats were tested with a 55K chip, and the traditional breeding values ​​(EBV) of milk production, milk protein rate and melatonin content in milk at 305 days were calculated using BLUPF90 software. For further verification, we selected the youngest 100 goats as the verification group, which had both 10K and 55K chip information. The remaining 200 goats were used as a filling reference group. Using Beagle 4.1 software, the chip information of the verification group was filled from 10K to 55K.

[0070] EBV is used as the corresponding vector for genomic prediction, and the GBLUP model is used to estimate the genomic breeding value of the target trait. To measure the accuracy of the genomic breeding value prediction, the correlation between EBV and genomic breeding value is used as a criterion; a higher correlation indicates a more accurate genomic breeding value estimate. Generally, the accuracy of different traits varies greatly. Complex traits controlled by multiple genes have an accuracy above 0.3, indicating high accuracy. Furthermore, the unbiasedness of the prediction is measured by the regression of EBV on the genomic breeding value; the closer the regression is to 1, the better the unbiasedness. The calculated results are shown in Table 2:

[0071] Table 2. Accuracy and unbiasedness of 10K and 55K genomic breeding value (GEBV) estimates

[0072] As shown in Table 2, compared to the prediction results based on the 55K chip, the prediction results based on the 10K chip showed only minor variations, with no significant differences. This result demonstrates that genome prediction based on the 10K low-density SNP chip of the present invention achieves performance similar to that of the 55K chip, with high accuracy, good unbiasedness, and a high cost-effectiveness, demonstrating the suitability of the 10K chip of the present invention for early breeding of Saanen dairy goats.

[0073] The dairy goat whole genome 10K SNP chip designed by the present invention contains 9913 SNP sites. The position information of all SNP sites is shown in Table 3.

[0074] Table 3. Location information of SNP sites

Claims

1. A SNP molecular marker for the whole genome of dairy goats, characterized in that: The SNP molecular marker is selected from at least one of the markers shown in Table 3.

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 dairy goat whole genome SNP chip, characterized in that: The SNP chip comprises probes targeting the SNP molecular markers according to claim 1.

4. The dairy goat whole genome SNP chip according to claim 3, characterized in that: The molecular markers are associated with milk production traits and reproductive traits of dairy goats; Preferably, the milk production traits include milk yield, milk protein rate, milk fat rate, lactose rate, and melatonin synthesis capacity; Preferably, the reproductive traits include lambing number and parity interval.

5. A kit, characterized in that: The kit comprises the molecular probe according to claim 2, or the SNP chip according to claim 3 or 4.

6. Use of the molecular probe according to claim 2, or the SNP chip according to claim 3 or 4, or the kit according to claim 5 in whole genome breeding of dairy goats.

7. Use of the molecular probe according to claim 2, or the SNP chip according to claim 3 or 4, or the kit according to claim 5 in identifying the kinship of dairy goats.

8. Use of the molecular probe according to claim 2, or the SNP chip according to claim 3 or 4, or the kit according to claim 5 in identifying European dairy goat and Chinese dairy goat breeds.

9. Use of the molecular probe according to claim 2, or the SNP chip according to claim 3 or 4, or the kit according to claim 5 in genotyping detection of dairy goats.

10. Use of the molecular probe according to claim 2, or the SNP chip according to claim 3 or 4, or the kit according to claim 5 in genetic diversity analysis of dairy goat populations or QTL positioning analysis of target traits.

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