Application of gene chip and SNP loci combination in analysis of sheep growth-related traits

Abstract

The application discloses a gene chip and application of a SNP site combination of sheep in analysis of growth-related traits of sheep, and relates to the field of biotechnology.The SNP site combination of the sheep comprises 5131 SNP sites, and physical position information of the SNP site combination of the sheep is determined based on comparison of a sheep v4.0 genome sequence.The SNP site information provided by the application can be used to quickly and accurately realize evaluation of growth-related traits of the sheep, evaluation of germplasm resources, mining of excellent germplasm resources, variety breeding, market meat quality detection, variety identification and tracing, and is beneficial to research, protection and improvement of sheep germplasm genetic resources in China.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to the application of gene chips and combinations of SNP loci in sheep in the analysis of growth-related traits in sheep. Background Technology

[0002] my country's native sheep breeds possess advantages such as tolerance to roughage, strong adaptability, and high feed conversion rates; however, their economic traits, such as cold resistance and meat quality, vary considerably. Although China currently ranks first in the world in terms of sheep population and mutton production, it still cannot effectively meet the ever-increasing domestic demand for mutton. While my country's sheep farming scale, meat production, and industrialization level have continuously improved, these increases are primarily attributed to technological advancements rather than technological efficiency. For example, artificial insemination and estrus synchronization techniques have significantly improved sheep reproductive rates. One major reason for these problems is that my country's sheep genetic breeding technology still relies mainly on traditional phenotypic selection, resulting in long breeding cycles and low accuracy. Furthermore, long-term chaotic introduction of breeds and disorderly crossbreeding have not only failed to stably improve native breeds but have also led to a serious loss of native sheep genetic resources. Therefore, leveraging the advantages of my country's native sheep breeds and utilizing modern molecular genetic breeding methods to cultivate native sheep breeds with excellent economic traits is a pressing issue that my country's sheep industry needs to address.

[0003] SNPs are genetic variations in DNA sequence caused by single nucleotide variants in the genome. They are abundant, highly representative, and stable, and the detection technology is mature, playing an important role in the identification of plant and animal germplasm resources, genetic evolution analysis, and marker-assisted breeding. GBS liquid-phase microarray technology is based on simplified genome sequencing to obtain partial information of the target genome to represent the whole genome information, reducing the sequencing sequence and lowering the sequencing cost per individual sample, while obtaining a large amount of SNP information. It is more suitable for the needs of large-scale population genotyping and has great advantages in molecular genetic improvement. It has been widely used in the genetic selection and breeding of crops such as wheat and livestock such as cattle and pigs in China.

[0004] Commercially available SNP chips for sheep are primarily developed and launched by Illumina and Affymetrix. These include the Illumina Sheep HD Genotyping Beadchip (680K), the Illumina Ovine SNP50 Beadchip (50K), and the Affymetrix Ovis600K Genotyping BeadChip. The Illumina 50K chip, in particular, includes 54,241 SNP loci covering the entire sheep genome and can be widely used in genome-wide association studies, QTL mapping, gene optimization, and comparative genomics research, making it widely applied in academic research. Although there are many sheep SNP chip products, existing sheep SNP chips are mainly based on genetic data of Western sheep breeds, lacking SNP data combining Chinese sheep breeds and foreign sheep breeds. They also have many problems such as scattered functional loci and regional distribution, excessive locus density, low locus detection frequency in Chinese sheep populations, and high cost of promotion and application in actual production. Therefore, it is of great significance to design an SNP chip suitable for the mining of excellent germplasm resources of Chinese sheep breeds and for the rapid and effective detection of multiple core economic traits such as cold resistance, meat quality, and prolificacy.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of this invention is to develop molecular probe combinations, gene chips, and reagent kits for analyzing sheep growth-related traits based on SNP locus combinations of core growth-related traits in sheep. Utilizing the SNP locus information provided by this invention, rapid and accurate evaluation of sheep growth-related traits, assessment of germplasm resources, discovery of superior germplasm resources, breed selection, market meat quality testing, breed identification, and traceability can be achieved, which is beneficial for the research, protection, and improvement of sheep germplasm genetic resources in my country.

[0007] This invention is implemented as follows:

[0008] The application of a sheep SNP locus combination in the analysis of growth-related traits in sheep. The sheep SNP locus combination includes 5131 SNP loci. The physical location information of the sheep SNP locus combination was determined based on sheep v4.0 genome sequence alignment. The sheep SNP locus combination is shown below:

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[0046] Primers were designed and probes synthesized using targeted capture sequencing technology to target the locations of the aforementioned 5131 SNP sites on the OAR4.0 genome, along with 200 bp upstream and downstream of these sites, resulting in a sheep 5K liquid-phase microarray. Compared to traditional liquid-phase microarrays that can detect 5131 fixed-site mutation base types, this invention designs primers and probes targeting the upstream and downstream base sequences of the sites to detect the sequences of the target mutation sites. A high-throughput DNA sequencing library is constructed to capture DNA fragments containing the target sites. The obtained DNA fragments are amplified and purified, and then subjected to high-throughput sequencing. The sequencing results are compared with the sheep reference genome to obtain the genomic genotyping of the sample. This invention's detection method can detect more SNP sites, is flexible in design, and allows for iterative updates by adding marker sites of interest at any time.

[0047] The combination of the above 5131 SNP loci can quickly evaluate the core economic traits of individual sheep at the gene level, obtain more accurate breeding assessment information, select individuals with excellent core economic traits, shorten the generation interval of breeding, accelerate the breeding process, and save breeding costs.

[0048] The above-mentioned 5131 SNP locus combinations were obtained as follows: based on the trait-related loci discovered and verified by the inventors in sheep populations in the early stage, economic trait-related SNP loci reported in journals and other literature, genetic information collection of 3966 sheep individuals from 13 sheep breeds worldwide, human and mouse SNP loci in the NCBI database SNP sub-library, and other databases, after deduplication and screening, a total SNP set of growth-related traits of sheep whole genome was formed; further, 200 bp sequences upstream and downstream of all loci in this set were selected and compared with the NCBI sheep v4.0 reference genome, and loci with sequence locus coverage >88% were screened, totaling 5131 loci, which were finally used as the growth-related trait SNP loci of this invention.

[0049] This SNP locus combination has high coverage and strong representativeness.

[0050] Furthermore, by utilizing the SNP locus combination of growth-related traits provided by this invention, it is also possible to identify and trace sheep breeds from the perspectives of meat quality and lipid deposition, providing technical support for the protection of sheep germplasm resources and genetic improvement in my country.

[0051] This invention provides a molecular probe combination for analyzing growth-related traits in sheep, which detects the above-mentioned SNP site combination in the sample to be tested. The physical location information of the SNP site combination is determined based on sheep v4.0 genome sequence alignment.

[0052] Those skilled in the art can readily design corresponding primers and / or probes upstream and downstream of the SNP site using existing primer design and analysis software based on the SNP site provided by this invention, thereby facilitating subsequent detection.

[0053] For example, detection markers can be set on the probe, such as a fluorescent reporter group at the 5' end and a fluorescent quencher group at the 3' end. The fluorescent reporter group could be HEX, FAM, TET, CF532, JOE, TAMRA, ROX, CY3, CY5, Texas Red, NED, Alexa Flour, or VIC, and the quencher group could be MGB, TAMRA, BHQ1, BHQ2, BHQ3, or QSY.

[0054] Probe types include, for example, Taqman or Taqman-MGB probes.

[0055] The present invention also provides a gene chip for analyzing growth-related traits in sheep, wherein the gene chip is loaded with the above-mentioned combination of molecular probes.

[0056] Gene chips include, but are not limited to, liquid phase chips and solid phase microbead chips.

[0057] The present invention also provides reagents for analyzing growth-related traits in sheep, which include the above-described molecular probe combination.

[0058] In addition, the above-mentioned reagents may also include stabilizers, buffer solutions, etc. in practical applications.

[0059] The present invention also provides a kit for analyzing growth-related traits in sheep, which includes the above-described molecular probe combination or the above-described gene chip.

[0060] The present invention also provides a method for analyzing growth-related traits in sheep, comprising the following steps: comparing the genotypes of 5131 SNP sites in the genomic DNA of the sheep to be tested with the genotypes of 5131 SNP sites in the genomic DNA of the control sheep, wherein the 5131 SNP sites in the genomic DNA of the control sheep are the SNP site combinations of claim 1, and the physical location information of the SNP site combinations is determined based on sheep v4.0 genome sequence alignment.

[0061] This invention also provides a method for analyzing growth-related traits in sheep, using the aforementioned molecular probe combination, gene chip, reagents, or kits to detect the sample to be tested.

[0062] In a preferred embodiment of the present invention, the above-mentioned sheep growth-related traits are selected from at least one of the following: wool production traits, growth traits, reproductive traits, meat quality traits, lactation, prolificacy, cold resistance, lipid deposition capacity, and meat yield of sheep.

[0063] The wool production trait is selected from at least one of the following: wool yield, clean wool yield, wool quality, fur quality, and lamb quality;

[0064] Reproductive traits include, for example, number of litters, birth weight, litter weight, and litter size.

[0065] The above-mentioned molecular probe combinations, gene chips, reagents, or kits may be used for any of the following purposes:

[0066] (1) Application in evaluating growth-related traits in sheep; (2) Application in sheep breed screening; (3) Application in sheep breed identification; (4) Application in sheep breed tracing; (5) Application in sheep germplasm resource evaluation; (6) Application in sheep germplasm resource research; (7) Application in sheep germplasm resource improvement; (8) Application in sheep germplasm resource protection; (9) Application in sheep assisted breeding; (10) Application in sheep population genetic pedigree construction; (11) Application in sheep genotyping.

[0067] This invention also provides the application of molecular probe combinations or gene chips in the preparation of kits for analyzing growth-related traits in sheep.

[0068] The present invention has the following beneficial effects:

[0069] The combination of 5131 SNP loci provided by this invention can quickly evaluate the growth-related traits of individual sheep at the gene level, obtain more accurate breeding assessment information, select individuals with excellent core economic traits, shorten the breeding generation interval, accelerate the breeding process, and save breeding costs.

[0070] Furthermore, by utilizing the SNP locus combination of growth-related traits provided by this invention, it is also possible to identify and trace sheep breeds from the perspectives of wool production traits, growth traits, reproductive traits, meat quality traits, lactation, prolificacy, cold resistance, lipid deposition capacity, and meat yield, and to detect meat quality, thus providing technical support for the protection of sheep germplasm resources and genetic improvement in my country.

[0071] Based on the above-mentioned SNP site combinations, the present invention provides probe combinations, gene chips, and reagent kits for sheep core economic trait SNP sites. Compared with existing high-density chips, these have lower throughput, lower cost, simpler and faster analysis, wider applicability, and broader market prospects. Attached Figure Description

[0072] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0073] Figure 1 The distribution of SNP sites on sheep chromosomes;

[0074] Figure 2 This is the result of the amplified sequence alignment. Detailed Implementation

[0075] The present invention will be further described below with reference to specific embodiments. However, the following embodiments are merely illustrative and should not be construed as limiting the scope of the present invention. All technologies implemented based on the content of the present invention fall within the scope of the present invention. For those skilled in the art, the present invention can have various modifications and variations. Various alterations or modifications made by those skilled in the art based on this invention without departing from its spirit should also fall within the scope of the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art, and the bioinformatics software and products used are all commercially available. Various processes and methods not described in detail are conventional methods known in the art.

[0076] It should be noted that the site combinations and applications provided by this invention were achieved through the inventors' arduous creative work and optimization efforts.

[0077] It should be noted that the biological products referred to in this invention include, but are not limited to, primers, probes, gene chips, reagent kits, etc., formed based on the site combinations provided by this invention.

[0078] It should be noted that the core economic traits of sheep referred to in this invention are reflected from multiple perspectives, such as cold resistance, meat yield, meat quality, growth traits, lipid deposition capacity, and prolificacy. According to actual production needs, those skilled in the art can set the trait thresholds themselves to judge the quality of sheep economic traits.

[0079] The SNP referred to in this invention is Single Nucleotide Polymorphism, which mainly refers to DNA sequence polymorphism caused by variations in a single nucleotide at the genomic level. The variations in the single nucleotide include variations caused by the conversion, transversion, insertion or deletion of a single base, and are one of the important molecular markers in animal and plant research.

[0080] It should be noted that the molecular markers referred to in this invention are all heritable and detectable DNA sequences or proteins, including but not limited to molecular markers based on molecular hybridization, such as RFLP and CISH; molecular markers based on PCR technology, such as RAPD, SSR, and SSCP; molecular markers based on restriction enzyme digestion and PCR technology, such as AFLP and CAPS; and molecular markers based on DNA microarray technology, such as SNP and EST. The molecular markers provided by this invention can be used for genome-wide association studies, germplasm resource identification and genetic evolution, gene mapping studies, and molecular genetic breeding.

[0081] It should be noted that the probe referred to in this invention is a nucleic acid sequence (DNA or RNA) that is complementary to the target gene, has a known sequence and carries a detection marker, such as Taqman or Taqman-MGB probes.

[0082] It should be noted that the kit referred to in this invention is any kit commonly used in the art that contains reagents for detection or experimentation, allowing operators to avoid the cumbersome reagent preparation and optimization process. It also includes primers for amplifying the site information provided by this invention, molecular markers or probes or gene chips for detecting the site information provided by this invention, as well as enzymes and buffers used for amplification.

[0083] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0084] Example 1

[0085] Obtaining the combination of SNP sites for core economic traits.

[0086] 1) SNP sites obtained by screening and verification based on sheep population experiments conducted by the team.

[0087] Based on the trait-related loci identified and validated by the team in sheep populations in the previous study, and by searching the titles of Web of Science, PubMed, and Elservier databases ("SNP", "livestock", "meat quality", "lipid metabolism", "cold exposure", "energy metabolism", or "economic traits"), as well as CNKI and Wanfang Data Knowledge Service Platform, literature with publication dates from January 1, 2010 to January 1, 2021 was obtained. After removing duplicates, a meta-analysis of 1030 target literatures on sheep, cattle, and pigs was conducted, and 951 relevant SNP loci were initially identified after removing duplicates.

[0088] 2) Significant SNP sites were obtained by screening sequencing data from multiple sheep breeds at home and abroad.

[0089] To achieve comprehensive coverage of sheep breeds and genetic information both domestically and internationally, the inventors collected genetic information from 3,966 individual sheep of 13 breeds worldwide, including New Zealand Suffolk, New Zealand Merino, Romney, Corridor, Romney, Polled Dorset, Hu sheep, Tan sheep, Small-tailed Han sheep, Tibetan sheep, Altay sheep, domestic Suffolk sheep, and Dumont-Saëns three-way crossbred sheep. Genetic information (DNA, etc.) was extracted from all sheep samples, including but not limited to blood, tissues, skin, hair, and excrement. Primers were designed for target genes related to multiple core traits, such as ADRB3, UCP1, CAST, SCD, and FecB, for genotyping, and combined with phenotypic data analysis to screen for significantly relevant SNP loci. Furthermore, high-throughput sequencing was used to sequence the sheep population genetic information and compare it with the NCBI sheep reference genome (v4.0), obtaining a partial SNP locus set.

[0090] 3) Derived from the SNP sites of human and mouse SNPs in the SNP sub-library of the NCBI database, which are functional genes of economic traits.

[0091] The genes corresponding to the SNP sites obtained from the literature review were defined as candidate genes for core economic traits. All candidate genes were screened for clinically significant SNP sites in the SNP sub-database of the NCBI database, and a total of 8167 human and mouse SNP sites were obtained.

[0092] Since the functional genes of economic traits are studied more extensively and at a more cutting-edge level in humans and mice, the meta-analysis selected a set of SNP functional genes that are significantly associated with those in humans and mice and aligned them to the sheep Oar4.0 genome.

[0093] 4) Obtaining the total SNP set of the entire sheep genome

[0094] The above three methods are combined to form a SNP set, totaling 9491 SNP loci, which serves as the total SNP set for the core economic traits of sheep across the entire genome. 200 bp sequences upstream and downstream of each locus in this set are compared with the NCBI sheep v4.0 reference genome, and loci with a sequence coverage >88% are selected, resulting in 5131 loci that are ultimately used as the SNP loci for growth-related traits in this invention.

[0095] Example 2

[0096] Analyze primer and probe combinations for growth-related traits in sheep.

[0097] Those skilled in the art can select appropriate restriction endonucleases for digestion based on the sequence information of the SNP sites in the combination of growth-related trait SNP sites provided by the present invention, and then ligate the digested fragments using T4 DNA ligase. Primers are designed based on the ligation products, and the designed primers are evaluated for specificity, secondary structure, and Tm value to ultimately obtain primers with good specificity, high sensitivity, and efficient detection of the target site under the same reaction conditions.

[0098] Among them, specificity comparison, secondary structure evaluation, and Tm value evaluation can be performed using any method commonly used in this field.

[0099] The above methods are all conventional methods. According to the site information in the SNP site combination of growth-related traits provided by the present invention, it can be obtained without any creative effort. Therefore, primers obtained according to the SNP site combination of growth-related traits provided by this application also fall within the protection scope of the present invention.

[0100] Similarly, molecular probes prepared using the SNP site combinations of growth-related traits provided by this invention, such as Taqman probes, are also within the scope of protection of this invention.

[0101] Example 3

[0102] Gene chip analysis of growth-related traits in sheep.

[0103] The SNP gene chip of the present invention relies on the highly optimized GenoBaits technology platform to directly synthesize the primers or probes obtained in Example 2 by fixing the primers or probes obtained in Example 2 on the surface of a liquid carrier.

[0104] The GBS method of this invention utilizes physical means or selects restriction enzymes to digest whole-genome DNA, then enriches the digested fragments with tagged sequences and performs high-throughput sequencing. With the assistance of a high-quality reference genome or haplotype map, and leveraging large-scale accumulated simplified sequencing data, genotyping is performed using whole-genome SNP locus information, achieving a high-density molecular marker method covering the entire genome with relatively small sequencing volumes. This technique is simple, requires fewer DNA purification steps, eliminates the need for fragment size selection, and can detect a large number of deleted genotypes. It is particularly advantageous for analyzing species with low polymorphism and high repetitive sequences, and is relatively inexpensive. It has already achieved efficient, large-scale genome sequencing in some plants.

[0105] It should be noted that those skilled in the art can use any method to prepare SNP gene chips for detecting growth-related traits in sheep, or they can entrust a biotechnology company to prepare them. However, SNP gene chips prepared based on the combination of growth-related trait SNP sites provided by this invention are all within the protection scope of this invention.

[0106] Example 4

[0107] Kit for analyzing growth-related traits in sheep.

[0108] The growth-related trait SNP detection kit provided by this invention includes primers, probes, or gene chips obtained based on SNP site combinations from Examples 2 and 3. Depending on the type of use, it also includes corresponding detection reagents, such as: TaqMan probes designed based on the SNP site combinations obtained in Example 1, buffers, ligases, TaqMan Probes, etc., commonly used in quantitative real-time PCR reactions.

[0109] Those skilled in the art can configure different SNP kits for detecting growth-related traits in sheep according to different usage methods, but all detection kits configured based on the SNP locus combinations for growth-related traits provided by this invention are within the protection scope of this invention.

[0110] Experimental Example 1

[0111] The inventors performed genotyping on the target gene UCP1. The genotype combinations and gene mutations are shown in Table 1 below. In the table, AA represents the genotypes CC & GG for the combined loci, AB represents the genotypes CT & GG for the combined loci, BB represents the genotypes TT & GG for the combined loci, and BC represents the genotypes TT & GA for the combined loci.

[0112] Table 1 UCP1 genotypes

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[0114] Genotyping

[0115] Blood samples were collected from the jugular vein of experimental sheep and stored on FTA cards. The samples were then taken from the blood collection area of ​​the FTA cards using a punch and placed in PCR tubes. 200 μL of 20 mM NaOH solution was added to the PCR tubes, and the tubes were heated at 60 °C for about 15 min until the blood sample faded. The NaOH was removed by vacuum pump, and then 200 μL of TE (Tris-EDTA) (pH=8) buffer was added. The tubes were allowed to stand at room temperature for 8 min. The TE buffer was removed, leaving only the treated blood sample discs. The PCR tubes were left open overnight and stored after thorough drying. 0.8 μL of upstream and downstream primers, 10 μL of Taq enzyme, and 7.6 μL of ddH2O were added to each DNA tube for PCR amplification. The amplification system was as follows: 95 °C pre-denaturation for 2 min, 95 °C for 30 s (denaturation) - 60 °C for 30 s (annealing) - 72 °C for 30 s (extension), for 35 cycles. The final extension was performed at 72 °C for 10 min, and the tubes were stored at 4 °C. After Sanger sequencing of the amplified products, the sequence alignment results were used for genotyping via MEGA (ver:X,USA). Figure 2 ).

[0116] Correlation analysis was performed between the combined genotypes of two SNP loci and individual meat quality traits. Data were statistically analyzed using a mixed linear model in SPSS 20.0 software (SPSS, Chicago, USA), with genotype and sex set as fixed effects. The chi-square test was used to determine if the data followed a normal distribution. For normally distributed data, the Duncan's test was used for one-way ANOVA to analyze data significance. For non-normally distributed data, the Kruskal-Wallis nonparametric test was used to analyze data significance. A p-value < 0.05 was considered statistically significant.

[0117] The differences in slaughter traits between different genotype combinations are shown in Table 2. Female refers to ewes, and Wether refers to castrated sheep.

[0118] Table 2. Differences in slaughter traits among UCP1 genotype combinations.

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[0120] The results showed that different genotype combinations of UCP1 had a significant impact on sheep slaughter rate, with the BB genotype combination significantly increasing sheep slaughter rate.

[0121] The inventors also conducted a differential analysis of amino acids in different genotype combinations and the longissimus dorsi muscle, as shown in Table 3, with the leftmost side of Table 3 showing the different amino acid types.

[0122] Table 3. Amino acid differences in the longissimus dorsi muscle among UCP1 genotype combinations.

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[0125] The results showed that different genotype combinations of UCP1 had a significant impact on the amino acid content of the longissimus dorsi muscle in sheep, and the BB genotype combination significantly improved the amino acid content of the longissimus dorsi muscle in sheep.

[0126] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of SNP loci in sheep in analyzing growth-related traits in sheep, characterized in that, The SNP loci in the sheep refer to two SNP loci in the UCP1 gene. The physical locations of the two SNP loci are chr17:16831864 and chr17:16831899, respectively. The allele genotype of the SNP locus at chr17:16831864 is TT, CC, or CT, and the allele genotype of the SNP locus at chr17:16831864 is GG or GA. The allele genotype combinations of the two SNP loci are CCGG, CTGG, TTGG, and TTGA. The physical location information of the SNP locus combinations in the sheep is determined based on sheep v4.0 genome sequence alignment. The sheep growth-related traits are selected from at least one of ewe dressing percentage and sheep longissimus dorsi muscle amino acid content.

2. A method for analyzing growth-related traits in sheep, characterized in that, It includes the following steps: comparing the genotypes of two SNP sites in the genomic DNA of the sheep to be tested with the genotypes of two SNP sites in the genomic DNA of the control sheep, wherein the two SNP sites in the genomic DNA of the control sheep are the two SNP sites described in claim 1, the physical location information of the SNP site combination is determined based on sheep v4.0 genome sequence alignment, and the sheep growth-related traits are selected from at least one of ewe dressing percentage and sheep longissimus dorsi muscle amino acid content.

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