Hugu sheep 5k low-density snp chip and application thereof

Abstract

The application belongs to the technical field of molecular markers, and provides a Hu sheep 5K low-density SNP chip and application thereof.The chip comprises 5000 SNP molecular markers, and the SNP molecular markers are shown in Table 1.Based on resequencing data of Hu sheep, the application screens SNP markers with rich polymorphic information content of Hu sheep breeds, integrates functional variation sites such as nonsense mutations and splicing mutations, and customizes the 5K low-density chip of Hu sheep, which can be used for Hu sheep kinship identification and functional harmful mutation screening.

Description

Technical Field

[0001] This invention belongs to the field of molecular marker technology, and in particular relates to a 5K low-density SNP chip for sheep and its application. Background Technology

[0002] The Hu sheep is a unique local sheep breed and one of the world's most reproductively efficient sheep breeds. It is medium-sized, with a full white coat, no horns, a docile temperament, and is tolerant of roughage. Its meat is of high quality, making it a very valuable genetic resource. Currently, some Hu sheep farms in China suffer from problems such as small herd sizes and inadequate pedigree records, which easily lead to inbreeding. Inbreeding results in decreased production performance in Hu sheep, and in severe cases, inbreeding depression, directly causing a decline in the economic benefits of sheep farming.

[0003] Currently, genome-wide genetic variation detection has become an important tool for assessing genetic diversity, identifying kinship, and detecting functional variations in domesticated animals. Commonly used genotyping techniques are mainly divided into two types: whole-genome resequencing and microarray-based genotyping. Whole-genome resequencing can comprehensively capture genetic variation information in the Hu sheep genome, but its high sequencing cost makes it difficult to conduct individual difference studies and batch testing in large-scale Hu sheep farming populations. In contrast, SNP microarrays target specific variant sites for detection, significantly reducing testing costs while ensuring genotyping accuracy.

[0004] Currently, different density gene chips of different types, such as 10K, 25K, 45K and 50K, have been designed based on SNPs of the whole genome of Hu sheep. These chips contain loci related to production traits and polymorphic loci for genetic diversity and population structure analysis. However, the main problem is that the large-scale detection cost of Hu sheep-specific gene chips is high, and there are relatively few functional mutation sites such as nonsense mutations and splicing mutations in the chips. Summary of the Invention

[0005] The purpose of this invention is to provide a Lake Sheep 5K low-density SNP chip and its application, aiming to solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] On the one hand, the present invention provides a Hu sheep 5K low-density SNP chip, which is composed of 5000 SNP molecular markers, as shown in Table 1.

[0008] On the other hand, the present invention provides an application of a combination of SNP molecular markers for the detection of Hu sheep in the preparation of Hu sheep 5K low-density SNP chips, or Hu sheep genome detection reagents or kits.

[0009] On the other hand, the present invention provides a Hu sheep 5K low-density SNP chip, which consists of 5,000 SNP molecular markers, as shown in Table 1.

[0010] On the other hand, the present invention provides a reagent or kit for detecting the genome of a Hu sheep, comprising 5,000 SNP molecular markers, as shown in Table 1.

[0011] Table 1. Chromosomal location and base composition information of SNPs

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[0050] On the other hand, the present invention provides an application of a combination of SNP molecular markers for detecting Hu sheep, or a Hu sheep 5K low-density SNP chip, or a Hu sheep genome detection reagent or kit in the detection of functional mutations and analysis of genetic diversity in Hu sheep.

[0051] Compared with the prior art, the specific beneficial effects of the present invention are as follows:

[0052] This invention, based on resequencing data of Hu sheep, screens out SNP markers with rich polymorphic information of Hu sheep breeds, and integrates functional variant sites such as nonsense mutations and splicing mutations to customize a 5K low-density chip for Hu sheep, which can be used for Hu sheep kinship identification and screening for functional harmful mutations. Attached Figure Description

[0053] Figure 1A heatmap showing the regional distribution of SNP counts within a 0.51 Mb sliding window of chromosomes, provided in an embodiment of the present invention.

[0054] Figure 2 This is a statistical chart of SNP variant types provided in an embodiment of the present invention;

[0055] Figure 3 A statistical chart of functional site distribution provided for embodiments of the present invention. Detailed Implementation

[0056] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0057] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0058] Example 1: A low-density SNP chip for sheep (5K), the construction method of which includes the following steps:

[0059] (1) Sample selection: Resequencing data of 55 Hu sheep were downloaded from the NCBI SRA database (https: / / www.ncbi.nlm.nih.gov / sra / ). The original sequencing data underwent quality control to remove adapter sequences and low-quality reads, resulting in Clean Data. The Clean Data was aligned to the sheep reference genome (GCF_016772045.1) using the MEM algorithm in BWA (v0.7.17) software. The BAM files were then sorted and repetitive sequence markers were applied using Picard software for subsequent analysis. SNP detection and genotyping were performed using the HaplotypeCaller module of GATK (v4.1.8.0) software, and strict filtering was performed using the Variant Filtration module. The SNP criteria were as follows: hardfilter_snpExpression1="QD < 2.0 || FS > 60.0 || MQ < 40.0 || SOR > 3.0" || "MQRankSum < -12.5 || ReadPosRankSum < - 8.0";

[0060] (2) Annotation of SNP sites: The detected SNP sites were annotated using ANNOVAR software to obtain the gene region (exon, intron, UTR, upstream and downstream, etc.) where each variant is located, the effect of exon region variants on the encoded protein (non-synonymous mutation, synonymous mutation, nonsense mutation, etc.), and the mutation of splice site, and selected nonsense mutation, splice site mutation, etc.

[0061] (3) SNP site screening: PLINK1.9 software was used to filter the genotype data, retaining only valid sites on chromosomes. SNP sites and samples were filtered based on minimum allele frequency (MAF > 0.05), site deletion rate (geno < 0.1), sample deletion rate (mind < 0.2), and kinship test (PI_HAT < 0.25). Unbalanced sites were removed by Hardy balance filtering (P = 1e-6). Finally, 17,535,844 high-quality SNP sites were obtained from 45 samples. Sliding window selection was performed on the dataset. Within each window, the optimal SNP site was selected, such as the site with the largest MAF or the site located in the exon region. Finally, 5,000 SNP sites were evenly distributed on the Hu sheep genome (as shown in Table 1). The chromosome number, location, and base information of the SNP sites are shown in Table 1. The number distribution of these SNP marker sites on each chromosome is as follows. Figure 1 As shown, the SNP mutations include six types: A / T, A / C, A / G, C / G, C / T, and G / T. C / T and A / G are the two most common types, accounting for 36.78% and 35.92% respectively (e.g., ...). Figure 2 (As shown); functional variant sites include nonsense mutations (stopgain), stop codon deletions (stoploss), splicing site mutations, and nonsynonymous mutations, and their distribution is as follows: Figure 3 As shown;

[0062] (4) 5K liquid-phase chip preparation: Probe design was carried out based on the upstream and downstream flanking sequence characteristics of candidate sites, focusing on evaluating key indicators such as sequence complexity and GC content, and prioritizing the location of target SNPs in the core region of the probe; the quality of the probe was evaluated from the following four aspects: GC content of 20-80%, Tm value of 60-80℃, continuous repeat bases not exceeding 25 bp, and alignment times on the reference genome not exceeding 5 times; after optimization based on the above standards, 5000 high-quality SNP sites were finally determined for chip customization; the liquid-phase capture probes used DNA single strands with a length of 120 bp and biotin-labeled 5' end.

[0063] Example 2: Application of the chip constructed in Example 1:

[0064] 1. Library construction and genotyping:

[0065] 1) Genomic DNA extraction:

[0066] Blood was collected from the jugular vein of 50 Hu sheep, and DNA was extracted using a blood genomic DNA extraction kit.

[0067] 2) Construction of liquid phase capture library:

[0068] First, the genomic DNA was fragmented, its ends repaired, and A added. DNA fragments in the range of 200-300 bp were recovered using the magnetic bead method. Adapters (CAGT Universal Adapters) were added to both ends of the DNA fragments. Then, an appropriate amount of ligation product was taken for PCR amplification. The library was quantified using the dsDNA HS Assay Kit for Qubit, and the fragment length range of the library was detected by electrophoresis.

[0069] The whole genome libraries of each sample were pooled, then concentrated and hybridized with probes. Excess probes, hybridization reagents and other reagent components were removed by elution, and the target region captured fragments were recovered. The target region was enriched by PCR amplification to obtain sequencing libraries. Finally, the constructed libraries were sequenced using a DNBSEQ-T7 sequencer.

[0070] 3) Data Analysis:

[0071] The raw data underwent quality control steps such as adapter removal and low-quality filtering to obtain clean data. This clean data was then aligned to the sheep reference genome (GCF_016772045.1) using bwa software. The genotypes of SNP loci were generated using the HaplotypeCaller module of GATK software. 5X sequencing depth filtering was used to mark non-compliant loci as deletions. The chip in this embodiment contains a total of 5000 loci, and all 5000 loci were detected, achieving a detection rate of 100%. Based on minimum allele frequency (MAF > 0.03) and locus deletion rate (geno < 0.05), 4883 loci were obtained.

[0072] 2. Application in population genetic diversity analysis:

[0073] Fifty Hu sheep individuals were genotyped using the 5K SNP chip, and the genetic diversity of the population was assessed based on the obtained high-quality SNP data. The results showed that the observed heterozygosity (Hb) of this population was high. O The expected heterozygosity (H) is 0.472511. EThe value of 0.467769 is relatively close to that of the other two values, indicating that the overall genetic structure of the population is relatively stable. Analysis of the continuous homozygous fragments (ROH) of the genome revealed that the number of ROH fragments in the individuals of this population was small and the overall length was short, indicating that the population had a low degree of inbreeding and a relatively rich genetic background.

[0074] 3. Application in functional variation analysis:

[0075] Gene annotation of the filtered 4883 sites revealed the presence of 15 missense mutations, 91 splice donor site mutations, 79 splice acceptor site mutations, 144 nonsense mutations, and 29 stop codon deletion mutations. These functional mutation sites may have a significant impact on the growth and development of Hu sheep and other important economic traits.

[0076] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A combination of SNP molecular markers for detecting sheep in Huzhou, characterized in that, It consists of 5000 SNP molecular markers, which are shown in Table 1.

2. The application of the SNP molecular marker combination for Hu sheep detection as described in claim 1 in the preparation of Hu sheep 5K low-density SNP chips, or Hu sheep genome detection reagents or kits.

3. A low-density SNP chip for sheep, characterized in that, It consists of 5,000 SNP molecular markers, which are shown in Table 1.

4. The Huyang 5K low-density SNP chip according to claim 3, characterized in that, The SNP chip includes a liquid-phase capture probe corresponding to 5000 SNP molecular markers.

5. A reagent or kit for detecting the genome of a Hu sheep, characterized in that, It consists of 5,000 SNP molecular markers, which are shown in Table 1.

6. The application of the SNP molecular marker combination for detecting Hu sheep as described in claim 1, or the Hu sheep 5K low-density SNP chip as described in claim 3, or the Hu sheep genome detection reagent or kit as described in claim 5, in the detection of functional mutations and analysis of genetic diversity in Hu sheep.

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