A lanping u-shaped bone mutton sheep whole genome molecular probe combination, gene chip and application thereof

Abstract

The present application relates to the field of animal molecular breeding technology, and particularly relates to a Lanping black-bone sheep whole genome molecular probe combination, a gene chip and application thereof.The chip comprises 50000 polymorphic SNP markers, and covers the whole genome of the Lanping black-bone sheep.Among them, the functional sites closely related to the black-bone traits and important economic traits such as reproduction, growth and disease resistance of the Lanping black-bone sheep are collected.The chip adopts a liquid chip platform design of targeted sequence capture, and has high flexibility and cost-effective advantage.The present application solves the problem that the prior art cannot early select and breed the Lanping black-bone sheep, can realize genotype identification of the black-bone traits at the lamb stage, improves the breeding efficiency, promotes the cultivation of new varieties (lines) of the Lanping black-bone sheep, and has important innovation value and application prospect.

Description

Technical Field

[0001] This invention relates to the field of animal molecular breeding technology, specifically to a whole genome molecular probe combination, gene chip, and their applications for Lanping black-boned sheep. Background Technology

[0002] Lanping Black-boned Sheep is a rare local sheep breed unique to Yunnan Province, my country, characterized primarily by its black or dark-colored body tissues (bones, periosteum, muscles, internal organs, etc.). It is the only mammal discovered globally to date that contains a large amount of melanin and possesses a stable genetic trait of blackness, and is the second animal discovered after the Silkie Chicken to exhibit the heritable trait of "black bones and black meat." Lanping Black-boned Sheep possesses unique nutritional and medicinal value; its meat is tender and has a unique flavor. The high melanin content is believed to have antioxidant and immune-enhancing health benefits. Due to its outstanding tonic and medicinal value, it is known as the "Medicinal Sheep," "King of Sheep," and "Golden Sheep," possessing high added value and development potential in the market. In 2009, this breed was included in the National List of Livestock and Poultry Genetic Resources and is protected by the state. Overall, the Lanping Black-boned Sheep is a rare sheep genetic resource that is "unique in the world, exclusive in China, and unique to Yunnan," possessing significant value for conservation and utilization.

[0003] However, the development, utilization, and breeding of Lanping Black-boned Sheep still face numerous technical challenges. First, the black trait is controlled by multiple genes, resulting in variations in the degree of black pigment deposition among individuals, and segregation of black trait expression occurs in conventional crossbreeds. Lanping Black-boned Sheep are visually indistinguishable from ordinary sheep, making it difficult to differentiate lambs carrying the black trait early on by sight alone. Often, confirmation of the black trait in bones and tissues is only possible after slaughter or dissection. This delayed expression and concealment of the trait makes it difficult to identify and select individuals carrying the black trait using traditional breeding methods. Second, the current population of Lanping Black-boned Sheep is small, raising concerns about inbreeding risks and population diversity loss. To expand the Lanping Black-boned Sheep population and maintain its uniqueness, scientific kinship management and early selection are necessary during the breeding process. Existing breeding methods mainly rely on phenotypic observation and empirical matching, which are inefficient and time-consuming, failing to meet the demands of industrial breeding for purification, preservation, and accelerated generational replacement. Third, there is a current lack of molecular breeding tools specifically for local breeds like the Lanping Black-boned Sheep. Existing commercial sheep SNP gene chips (such as the Illumina OvineSNP50 50K chip and the OvineHD600K chip) are mostly developed based on high-yielding meat sheep breeds from abroad. Although they have been applied to genome selection and breed identification in some breeding units, their information efficiency is low when directly applied to Lanping Black-boned Sheep because most loci contained in the chips lack polymorphism in local Chinese sheep and are not designed for specific traits such as blackness. Furthermore, the high density of these solid-phase chips leads to high costs, making them difficult to use on a large scale for the preservation and breeding of local breeds. Therefore, existing technologies cannot meet the actual needs of early selection, breed identification, and new breed development for Lanping Black-boned Sheep, and there is an urgent need to develop a highly efficient molecular breeding tool specifically for the blackness trait of this breed. Summary of the Invention

[0004] To address the challenges of early selection for the black-boned trait in Lanping black-boned sheep and the limitations of existing molecular breeding techniques, this invention completed whole-genome resequencing of 379 individual Lanping black-boned sheep. Simultaneously, resequencing data from 411 other sheep of different breeds were downloaded from public databases (NCBI, Ensembl), covering multiple breeds of wild and domestic sheep. This resulted in a set of 50,000 SNP loci, and based on this, 50,000 polymorphic SNP markers were developed, covering the entire genome of Lanping black-boned sheep. Key functional loci closely related to the black-boned trait and important economic traits such as reproduction, growth, and disease resistance in Lanping black-boned sheep were included.

[0005] One objective of this invention is to provide a whole-genome molecular probe array for Lanping black-boned sheep, which detects SNP molecular marker combinations in the whole genome of Lanping black-boned sheep.

[0006] The whole genome SNP molecular marker assemblage of Lanping black-boned sheep consists of 50,000 SNP molecular markers in the whole genome of Lanping black-boned sheep. The SNP molecular marker information is shown in Table 1. The reference genome for the physical location is GCF_002742125.1_Oar_rambouillet_v1.0_genomic.

[0007] Table 1

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[0287] The second objective of this invention is to provide an application of a combination of molecular probes for the whole genome of Lanping black-bone sheep in the preparation of a whole genome gene chip for Lanping black-bone sheep.

[0288] The third objective of this invention is to provide a whole genome gene chip for Lanping black-boned sheep, wherein the chip is loaded with the aforementioned molecular probe combination for the whole genome of Lanping black-boned sheep.

[0289] The fourth objective of this invention is to provide a kit for genotyping of Lanping black-boned sheep, the kit comprising the Lanping black-boned sheep whole genome molecular probe combination or the Lanping black-boned sheep whole genome gene chip.

[0290] The fifth objective of this invention is to provide a molecular probe combination for the whole genome of Lanping black-boned sheep, or the whole genome gene chip for Lanping black-boned sheep, or the kit for genotyping of Lanping black-boned sheep, for the application of Lanping black-boned sheep breed identification, breed tracing, or kinship identification.

[0291] The sixth objective of this invention is to provide a combination of molecular probes for the whole genome of Lanping black-boned sheep, or the whole genome gene chip of Lanping black-boned sheep, or the kit for genotyping of Lanping black-boned sheep, for use in whole genome association analysis or trait-related gene localization of Lanping black-boned sheep.

[0292] The seventh objective of this invention is to provide a combination of molecular probes for the whole genome of Lanping black-boned sheep, or the whole genome gene chip of Lanping black-boned sheep, or the kit for genotyping of Lanping black-boned sheep, for the application of genetic diversity analysis in Lanping black-boned sheep.

[0293] The eighth objective of this invention is to provide a combination of molecular probes for the whole genome of Lanping Black-boned Sheep, or the aforementioned gene chip for the whole genome of Lanping Black-boned Sheep, or the aforementioned kit for genotyping of Lanping Black-boned Sheep, for the application in germplasm resource analysis, germplasm resource improvement, germplasm resource protection, or pedigree reconstruction of Lanping Black-boned Sheep.

[0294] To achieve the above objectives, the present invention adopts the following technical solution:

[0295] Based on the deep resequencing data of 379 samples, the comparison and variant detection were completed through standard procedures and strict quality control was implemented (including sequencing depth, genotype quality, deletion rate, HWE test, repetitive sites and strong LD redundancy removal). Combined with the GWAS of the genotype, population comparison and selection signals (such as FST, XP-EHH, π-ratio) and functional annotation / eQTL and QTL literature information, a set of candidate functional sites for traits such as genotype, reproduction, growth and disease resistance was formed. Finally, 50,000 target SNPs were identified, as shown in Table 1.

[0296] Compared with the prior art, the present invention has the following beneficial effects:

[0297] (1) The Lanping black-bone sheep 50K liquid phase SNP gene chip provided by the present invention utilizes the domestic liquid phase capture sequencing platform to achieve efficient capture and detection of target SNPs. The chip probe design fully considers the uniformity of the distribution and polymorphism of the markers on the chromosome. The liquid phase capture technology used has the advantages of being flexible, scalable, low cost and highly adaptable. The target sites can be flexibly added or removed according to breeding needs, and the cost of large-scale genotyping can be significantly reduced.

[0298] (2) The 50K liquid-phase SNP gene chip for Lanping black-boned sheep provided by this invention has significant technical effects and application value in the breeding of black-boned sheep: By performing genomic typing in early lambs, the chip of this invention can accurately identify individuals with black-boned traits at an earlier stage, greatly improving the accuracy of breeding and mating, shortening the generation interval for cultivating superior breeds with black-boned characteristics, and accelerating the process of improving the Lanping black-boned sheep breed. Furthermore, the gene markers of key economic traits such as reproductive capacity, growth rate, and disease resistance covered by the chip provide strong support for comprehensively improving the production performance of this breed.

[0299] (3) The present invention can design specific capture probes for the provided SNP sites, providing efficient genomic typing technology for breeding applications such as genome selection, breed identification, and kinship identification of Lanping black-bone sheep. Attached Figure Description

[0300] Figure 1 The distribution map of functional sites on chromosomes of the 50K liquid phase SNP gene chip for Lanping black-bone sheep provided by this invention.

[0301] Figure 2 The distribution map of functional sites of the 50K liquid phase SNP gene chip provided by the present invention for sites with a minimum allele frequency greater than 0.1 in the Lanping black-bone sheep population.

[0302] Figure 3 The functional site trait classification of the 50K liquid phase SNP gene chip for Lanping black-bone sheep provided by this invention.

[0303] Figure 4 In Example 2 of this invention, principal component analysis of a sheep population was performed using 50,000 SNP loci.

[0304] Figure 5 This is an example of phylogenetic tree analysis of a sheep population using 50,000 SNP loci in Example 2 of the present invention.

[0305] Figure 6 In Example 2 of this invention, 50,000 SNP loci were used to analyze the population structure of a sheep population. Detailed Implementation

[0306] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0307] Example 1: Construction of a 50K liquid-phase SNP gene chip (capture probe combo) for Lanping black-boned sheep

[0308] This embodiment provides a method for constructing a 50K liquid-phase SNP gene chip (capture probe combination) for Lanping black-bone sheep, including the specific procedures for molecular marker development, screening, and chip preparation.

[0309] (1) Sample and data preparation: Whole genome resequencing was completed for 379 Lanping black-boned sheep individuals (sequencing depth approximately 12×; sequencing platform: BGI T7; sequencing company: Beijing Compson Biotechnology Co., Ltd.). At the same time, resequencing data of 411 other sheep of different breeds were downloaded from public databases (NCBI, Ensembl), covering multiple breeds of wild and domestic sheep (see Table 2 for specific breeds and number of individuals).

[0310] Table 2 Sheep Breed Information

[0311]

[0312]

[0313]

[0314] (2) Reference genome determination: In this embodiment, the sheep reference genome version GCF_002742125.1_Oar_rambouillet_v1.0_genomic is used as the reference coordinate system. The FASTA file of this version of the genome is downloaded and its chromosome / scaffold naming is fixed as the sole basis for all subsequent coordinates. Then, an alignment index, FASTA index and sequence dictionary file are established for the reference sequence to form a set of reference files that can be directly used for sequencing read alignment, variant detection, site coordinate localization and upstream and downstream sequence extraction. In all subsequent steps, the CHROM and POS of this version are strictly used as the site coordinate standard to ensure that the probe design and genotyping coordinates are completely consistent.

[0315] (3) Quality control of raw sequencing data: A unified quality control process was performed on the raw FASTQ data of each sample, which sequentially completed adapter sequence removal, low-quality base splicing, removal of reads with too high N base ratio and removal of too short reads. The quality control thresholds were fixed as follows: low quality judgment threshold Q20, upper limit of allowed low-quality base ratio 30%, upper limit of allowed N base number 5, and minimum retention length 50bp. After quality control, pairs of clean reads were output, and the total number of reads, Q20 / Q30 ratio and GC content distribution of each sample were statistically analyzed and saved as quality control records. All subsequent analyses used only these clean reads as input.

[0316] (4) Alignment and redundancy removal: Align the clean reads of each sample to the GCF_002742125.1_Oar_rambouillet_v1.0_genomic reference genome indexed in step (2), obtain the SAM file, convert it to BAM and sort it according to the genome coordinates, then mark the repeated reads in the sorted BAM and generate a repeatability statistical index file, and finally build an index file for the marked repeatability BAM to form the final input file for variant detection; in this process, the alignment rate, average sequencing depth and repeatability rate of each sample are statistically recorded simultaneously, where the average sequencing depth is used as the basis for the subsequent threshold calculation of "site coverage depth is not less than 1 / 2 of the average depth of all samples".

[0317] (5) Variation detection and joint typing: Using the final BAM file of all samples obtained in step (4) as input, the full sample SNP detection and typing are completed under the GCF_002742125.1_Oar_rambouillet_v1.0_genomic reference coordinate system, and the typing VCF file containing the genotype information of all samples is output; then, only SNP variants are retained from the typing results as the source of candidate sites for the chip, and the consistency of the reference alleles of the VCF is checked to ensure that the REF bases of each site in the VCF are completely consistent with the bases of the corresponding coordinates of the reference genome.

[0318] (6) Locus / Sample Quality Control and Filtering: For the SNP genotyping VCF obtained in step (5), a fixed threshold of sample and locus quality control filtering was performed. The filtering indicators and thresholds were determined as follows: locus HWE test threshold 0.0001, kinship threshold 0.25, locus MAF ≥ 0.05, locus genotype deletion rate ≤ 0.2, and locus read coverage depth threshold of 1 / 2 of the average sequencing depth of all samples. Outlier samples were removed based on the PCA results. The locus read coverage depth was based on the average sequencing depth of all samples as the baseline depth D. - The average coverage depth of the site is lower than D. - All sites with a phylogenetic relationship greater than 0.25 were removed. Sample pairs with a phylogenetic relationship greater than 0.25 were processed according to the phylogenetic coefficient from high to low and only those with higher sequencing quality were retained. Outliers were judged based on PCA coordinates that were significantly deviated from the main cluster and were all removed. After the above filtering was completed, a high-quality SNP set was obtained. In this embodiment, a total of 19,504,130 high-quality SNP sites were identified under the filtering conditions.

[0319] (7) Candidate site set construction and priority strategy: Based on the high-quality SNP set obtained in step (6), a candidate site set for chip design is constructed. The candidate site set satisfies both the conditions of "functional site coverage" and "uniform coverage of the whole genome". First, all QTL / GWAS sites, sites within candidate gene regions, and sites with functional influence categories related to traits such as meat quality, growth, reproduction, meat quality, disease resistance and stress resistance are included in the functional site candidate set. Second, the number of sites is allocated according to chromosome length in the whole genome and the site spacing is controlled according to density constraints to form a uniform coverage candidate set. Finally, the two types of candidate sets are merged to obtain the final candidate site set, and the size of the candidate sites is fixed and expanded to 2-4 times the number of target sites to ensure that there is sufficient selection space for subsequent probe design.

[0320] (8) Site scoring and probe sequence design: The candidate site set obtained in step (7) is scored for probe designability and probe sequence design is completed. The probe design rules are as follows: the upstream and downstream sequences are extracted from the reference genome with the target SNP site as the center and a probe sequence of length of 120bp is generated. The target SNP is placed in the middle of the probe. The complexity and repetitive sequence interference, GC content and potential non-specific binding risk are evaluated for each probe sequence and a scoring result is generated accordingly. All sites that fail the scoring are directly removed. Sites that pass the scoring are retained and a corresponding list of site-probe sequence-scoring result is output to ensure that each subsequent production stage can be traced back to the specific probe sequence.

[0321] (9) Target locus set confirmation: After scoring and removing undesignable loci in step (8), the remaining loci are determined according to the established constraints to determine the final 1 locus set. The confirmation process is to first meet the functional locus coverage requirements and then meet the uniform coverage requirements: loci that have passed the scoring among all functional loci are included first, and on the premise of ensuring that the chromosome distribution is uniform and the local density constraints are not destroyed, the uniform coverage candidate loci are supplemented to a precise 50,000 loci from the scoring from high to low; finally, the list of 50,000 loci is locked and the customized locus set shown in Table 1 is formed.

[0322] (10) Liquid-phase capture chip preparation and quality control: Based on the list of 50,000 site probe sequences locked in step (9), oligonucleotide probes were synthesized and mixed according to the site list to form a liquid-phase capture probe pool covering 50,000 target sites for subsequent liquid-phase hybridization capture genotyping; the integrity and consistency of the site set were checked, and the quality control content was to verify that the number of sites covered by the probe pool was 50,000 and corresponded one-to-one with the site list in Table 1, and to statistically verify the site chromosome distribution, SNP type distribution, MAF distribution, functional site distribution and whole genome density distribution. The statistical results are as follows. Figure 2 , Figure 3 As shown, this confirms that the probe pool provides uniform coverage across the entire genome while also covering functional sites. Ultimately, the site list, probe sequence list, and distribution statistics are used as the basis for chip fabrication and quality control.

[0323] Example 2: Application of 50K gene chip in breed identification of Lanping black-boned sheep

[0324] The Lanping Black-boned Sheep, originating from Lanping County, Yunnan Province, is currently the only known livestock breed globally, besides the Black-boned Chicken, with a heritable black trait. However, due to the complex genetic mechanism of this trait, lambs only exhibit obvious black characteristics (such as black gums and eyelids) after 1.5 to 2 years of age. Furthermore, this breed is not significantly different from other common local breeds in terms of body shape, appearance, and coat color, making breed identification difficult. This embodiment utilizes the 50K gene chip constructed in Example 1 to identify the Lanping Black-boned Sheep and common local sheep, aiming to improve the accuracy and efficiency of identification.

[0325] (1) Sample collection and DNA extraction: 237 Lanping black-boned sheep and 123 Lanping local common sheep (non-black-boned) were randomly selected as the breed identification and verification group, totaling 360 individuals. A sample registration form was established for each individual (including individual number, breed category, sampling time, sampling location and sampling personnel, etc.). Jugular venous blood was collected using EDTA anticoagulated vacuum blood collection tubes and stored at -20℃. Genomic DNA was then extracted according to the same batch and the same operating procedure, and a negative extraction control was set up to monitor cross-contamination. After extraction, the DNA quality consistency was checked. The A260 / 280 was determined by spectrophotometry and the purity index was recorded. At the same time, the accurate concentration was obtained by fluorescence quantification, and the DNA integrity and degradation were verified by agarose gel electrophoresis. Finally, all sample DNA was uniformly diluted to the same concentration and aliquoted for storage for library construction and capture.

[0326] (2) Library construction: For each genomic DNA, a sequencing library suitable for liquid phase capture is prepared according to a fixed library construction process. Specifically, DNA fragmentation, end repair, 3' end A addition, adapter ligation, and PCR amplification enrichment are completed in sequence, and quality control and recording are set at each key node: After fragmentation, the fragment distribution is detected and the main peak of the fragment is controlled within the range that matches the capture sequencing. After adapter ligation, the library is purified and free adapters and short fragments are removed. During the PCR amplification stage, the number of cycles is fixed and the amplification products are purified a second time to reduce bias. Finally, the fragment size distribution and concentration of each sample library are detected and the library quality check table is generated. Only libraries with fragment distribution and concentration that meet the uniform standard can proceed to the next capture step.

[0327] (3) Liquid-phase hybridization capture and sequencing: Each sample library that passed the quality inspection was mixed in equal volumes according to a pre-determined mixing scheme (the mixing scheme fixedly recorded the amount of each sample library and the overall concentration after mixing). The 50,000-site probe pool determined in Example 1 was added for liquid-phase hybridization capture. After completing the hybridization reaction strictly according to the predetermined hybridization time and temperature program, the hybridization products were captured using a magnetic bead system and washed multiple times to remove non-specific binding fragments. Subsequently, the captured products obtained by elution were captured and enriched by PCR, and the enriched library was purified. The concentration and fragment distribution of the enriched library were verified. After confirming that it met the requirements for sequencing, high-throughput sequencing was performed. During the sequencing process, the read length type and sequencing strategy were fixed and the run information was saved. Finally, capture sequencing data covering 50,000 target sites were obtained. To ensure reproducibility, blank control and duplicate samples (the same sample was independently constructed / captured) were set up during the capture and sequencing process and processed in the same batch as regular samples to quantify the capture background, cross-contamination risk and genotyping consistency.

[0328] (4) Genotyping and Data Quality Control: The captured sequencing data underwent data quality control according to a unified process and was aligned to the GCF_002742125.1_Oar_rambouillet_v1.0_genomic reference genome determined in Example 1. Genotyping was performed on 50,000 target loci under a unified coordinate system, and a genotyping matrix for all samples was output. Subsequently, the genotyping matrix was converted into a standardized downstream analysis format, and output fields (such as individual ID, locus ID, genotype coding rules, etc.) were fixed to ensure that different operators obtained the same data structure. Based on this, the genotyping results were analyzed. To ensure comparability, the same quality control filtering as in Example 1 was performed, with fixed filtering thresholds: missing locus rate ≤ 0.2, MAF ≥ 0.05, HWE test threshold 0.0001, and kinship threshold 0.25. Outliers were removed based on PCA results. The genotyping success rate (proportion of loci with genotype output) for each sample was statistically analyzed, and samples with low genotyping success rates were removed or the experiment was repeated. After quality control, the final genotyping dataset for variety identification was generated, and the number of samples, number of loci, and key statistical indicators (missing rate distribution, MAF distribution, HWE distribution) before and after each filtering step were saved.

[0329] (5) Population genetic analysis and variety identification: Based on the 50,000-locus genotyping matrix after quality control, population genetic analysis was performed using fixed parameters. First, the genotyping matrix was deredundant using Low Fibre (LD) to obtain a set of independent loci for principal component analysis. Based on this, principal components were calculated and a two-dimensional scatter plot was drawn. Figure 4 ) Verify the clustering separation of the two populations in the principal component space; then construct a phylogenetic tree on the same dataset. Figure 5), using 360 individuals as leaf nodes of a tree and examining whether the two types of individuals form clear branches on the tree and whether the individuals within the branches originate from the same source; then, a population structure analysis is performed again ( Figure 6 The study calculated ancestral composition at different K values ​​and compared the stability and consistency of the differences in ancestral proportions between the two groups. Simultaneously, it calculated kinship or blood relation and checked for individuals with identical or highly consistent kinship compositions between the two groups. Based on four types of evidence—PCA clustering results, phylogenetic tree branch consistency, stability of ancestral differences in group structure, and kinship testing—the study determined that Lanping black-boned sheep and Lanping local common sheep were completely separated at the 50K locus set, thus proving that the 50K liquid-phase capture gene chip can achieve stable and reliable breed identification.

[0330] Example 3: Capture specificity of 50K liquid-phase SNP gene chip in Lanping black-boned sheep

[0331] The capture specificity (i.e., targeted capture effectiveness) of the 50K liquid-phase capture chip for Lanping black-boned sheep was quantitatively evaluated using "target site detection rate and depth attainment." Specifically, based on the capture sequencing data of 360 individuals in Example 2, after aligning the sequencing reads to the reference genome, the detection rate (site detection rate) of each of the 50,000 target sites in the population was statistically analyzed, along with the number and proportion of attainable sites at different depth thresholds (dp0, dp5, dp10, dp15, and dp20 respectively). This indicates the number and percentage of detected loci at sequencing depths greater than 0, 5×, 10×, 15×, and 20×. Statistical results show that under dp0 conditions, 49,228 loci (98.46%) had a detection rate between 90% and 100%; under dp5 conditions, 48,922 loci (97.84%) had a detection rate between 90% and 100%; under dp10 conditions, 48,724 loci (97.45%) had a detection rate between 90% and 100%; and under dp15 conditions, 4,851 loci had a detection rate between 90% and 100%. Four loci (97.03%) were in the 90%-100% range, and under dp20 conditions, 48,306 loci (96.61%) were still in the 90%-100% range, indicating that the vast majority of target loci could be stably captured in the population sample and achieve a high sequencing depth (Table 3). Correspondingly, the sample dimensionality statistics also showed that each sample had sufficient detection coverage of the target loci. For example, sample WB242 detected 49,672 (96.61%) loci under dp0, dp5, dp10, dp15, and dp20 conditions, respectively. The samples WB264 detected 49672 (99.34%), 49491 (98.98%), 49375 (98.75%), 49269 (98.54%), and 49131 (98.26%) loci under the conditions of dp0, dp5, dp10, dp15, and dp20, respectively (Table 4).

[0332] The above-mentioned population locus detection statistics and sample detection statistics together demonstrate that the Lanping black-bone sheep 50K liquid phase capture probe combination of the present invention has high consistency, high depth target achievement rate and high stability in capturing target loci. It has good capture specificity and can achieve full coverage of target loci with less sequencing data, thereby supporting subsequent reliable typing and breed identification analysis.

[0333] Table 3. Statistical results of site detection.

[0334]

[0335]

[0336] Note: dp0: number of sites detected when the depth is greater than 0; dp0%: percentage of sites detected when the depth is greater than 0; dp5: number of sites detected when the depth is greater than 5X; dp5%: percentage of sites detected when the depth is greater than 5X; and so on.

[0337] Table 4 shows the detection statistics for some samples.

[0338] dp0 dp0% dp5 dp5% dp10 dp10% dp15 dp15% dp20 dp20% WB242 49672 99.34 49528 99.06 49439 98.88 49349 98.7 49251 98.5 WB264 49672 99.34 49491 98.98 49375 98.75 49269 98.54 49131 98.26 WB001 49639 99.28 49476 98.95 49335 98.67 49187 98.37 49037 98.07 LPB2306 49694 99.39 49531 99.06 49433 98.87 49328 98.66 49202 98.4 LPB28 49686 99.37 49541 99.08 49456 98.91 49380 98.76 49274 98.55 WB212 49668 99.34 49530 99.06 49439 98.88 49345 98.69 49247 98.49 WB376 49663 99.33 49520 99.04 49394 98.79 49289 98.58 49177 98.35 LPB2307 49664 99.33 49485 98.97 49341 98.68 49215 98.43 49071 98.14 WB208 49683 99.37 49528 99.06 49447 98.89 49361 98.72 49273 98.55

[0339] Note: dp0: number of sites detected when the depth is greater than 0; dp0%: percentage of sites detected when the depth is greater than 0; dp5: number of sites detected when the depth is greater than 5X; dp5%: percentage of sites detected when the depth is greater than 5X; and so on.

Claims

1. A whole-genome molecular probe array for Lanping black-boned sheep, characterized in that: The molecular probe combination was used to detect SNP molecular marker combinations in the whole genome of Lanping black-bone sheep. The whole genome SNP molecular marker assemblage of Lanping black-boned sheep consists of 50,000 SNP molecular markers in the whole genome of Lanping black-boned sheep. The SNP molecular marker information is shown in Table 1. The reference genome for the physical location is GCF_002742125.1_Oar_rambouillet_v1.0_genomic.

2. The application of the whole genome molecular probe combination of Lanping black-bone sheep as described in claim 1 in the preparation of the whole genome gene chip of Lanping black-bone sheep.

3. A whole genome gene chip for Lanping black-boned sheep, characterized in that: The chip is loaded with the whole genome molecular probe combination of Lanping black-boned sheep as described in claim 1.

4. A kit for genotyping of Lanping black-boned sheep, characterized in that, The kit contains the whole genome molecular probe combination of Lanping black-bone sheep as described in claim 1 or the whole genome gene chip of Lanping black-bone sheep as described in claim 3.

5. The application of the whole genome molecular probe combination of Lanping black-boned sheep as described in claim 1, or the whole genome gene chip of Lanping black-boned sheep as described in claim 3, or the kit for genotyping of Lanping black-boned sheep as described in claim 4, in the identification, tracing, or identification of kinship of Lanping black-boned sheep breeds.

6. The application of the whole genome molecular probe combination of Lanping black-boned sheep as described in claim 1, or the whole genome gene chip of Lanping black-boned sheep as described in claim 3, or the kit for genotyping of Lanping black-boned sheep as described in claim 4, in the genome selection of Lanping black-boned sheep.

7. The application of the whole genome molecular probe combination of Lanping black-boned sheep as described in claim 1, or the whole genome gene chip of Lanping black-boned sheep as described in claim 3, or the kit for genotyping of Lanping black-boned sheep as described in claim 4, in the whole genome association analysis or trait-related gene localization of Lanping black-boned sheep.

8. The application of the whole genome molecular probe combination of Lanping black-boned sheep as described in claim 1, or the whole genome gene chip of Lanping black-boned sheep as described in claim 3, or the kit for genotyping of Lanping black-boned sheep as described in claim 4, in the genetic diversity analysis of Lanping black-boned sheep.

9. The application of the whole genome molecular probe combination of Lanping black-boned sheep as described in claim 1, or the whole genome gene chip of Lanping black-boned sheep as described in claim 3, or the kit for genotyping of Lanping black-boned sheep as described in claim 4, in the analysis, improvement, protection, or pedigree reconstruction of Lanping black-boned sheep germplasm resources.

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