WO2023001209A1 - 分析绵羊脂尾的基因芯片、分子探针组合、试剂盒及应用 - Google Patents

分析绵羊脂尾的基因芯片、分子探针组合、试剂盒及应用 Download PDF

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WO2023001209A1
WO2023001209A1 PCT/CN2022/106904 CN2022106904W WO2023001209A1 WO 2023001209 A1 WO2023001209 A1 WO 2023001209A1 CN 2022106904 W CN2022106904 W CN 2022106904W WO 2023001209 A1 WO2023001209 A1 WO 2023001209A1
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sheep
tail
application
combination
snp
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李孟华
李心
罗凌云
杨继
吕锋骅
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中国农业大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
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    • C40—COMBINATORIAL TECHNOLOGY
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    • C40B40/00—Libraries per se, e.g. arrays, mixtures
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    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/124—Animal traits, i.e. production traits, including athletic performance or the like
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

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  • the present invention relates to the field of biological technology, specifically to the technical field of biological detection, and more specifically to biological products such as molecular probe combinations, gene chips, and kits for analyzing suet tail, and their applications.
  • Sheep tail fat can be used as a form of energy storage, which can provide energy for sheep to migrate or survive the severe winter of lack of forage, so as to maintain their survival needs; on the other hand, it can be used as a high-energy food for human use.
  • Sheep tail fat can be used as a form of energy storage, which can provide energy for sheep to migrate or survive the severe winter of lack of forage, so as to maintain their survival needs; on the other hand, it can be used as a high-energy food for human use.
  • the feed required by livestock to deposit 1kg of fat is equivalent to the feed required by 2kg of lean meat in the body, so the deposition of fat in the tail of sheep will greatly reduce the economic benefits of sheep farming, so excessive tail fat deposition will increase the feeding rate instead. cost.
  • short fat-tailed sheep (such as small-tailed sheep) are easier to breed and increase the conception rate of ewes. Therefore, breeding corresponding fat-tailed sheep breeds according to market demand is an important guide for the sheep industry In addition, suet tail is also of great significance in the traceability of sheep breeds and the protection, development and utilization of germplasm resources.
  • SNP single nucleotide polymorphisms
  • the existing commercial SNP chips for sheep include Illumina Ovine SNP50 Beadchip (50K), Illumina Sheep HD Genotyping Beadchip (680K) and Illumina Ovine LD (5K).
  • the former two are the most commonly used in academic production research.
  • the Illumina Ovine SNP50 Beadchip is jointly developed by Illumina and UCSC, CSIRO, USDA, Agresearch and other research teams and institutions, including more than 54K SNP sites covering the whole sheep genome, which can be used for genetic breeding, genome-wide association analysis, and quantitative trait loci Mapping, gene selection and comparative genomics research.
  • the Illumina Sheep HD Genotyping Beadchip (680K) is also a SNP chip produced by Illumina.
  • the present invention provides a combination of SNP sites for analyzing sheep fat tails, and a combination of molecular probes prepared based on the combination of SNP sites, Biological products and applications such as gene chips and kits can quickly and accurately detect the fat tail traits of sheep to be detected by using the site information provided by the present invention, and realize fat deposition evaluation, variety screening, variety identification, variety traceability, and sheep breeding in sheep tails. It is beneficial to the reconstruction of sheep pedigree, the protection of germplasm resources and the improvement of germplasm resources, with short time-consuming, low cost and broad market benefits.
  • the present invention adopts the following technical means to realize:
  • the 3000 SNP sites are the above-mentioned 3000 SNP sites.
  • a gene chip for analyzing suet tail is loaded with the molecular probe combination of the above 3.
  • a kit for analyzing suet tail which has the molecular probe combination of the above 3 or the gene chip of the above 4.
  • the molecular probe combination of the above-mentioned 3 or the gene chip of the above-mentioned 4 or the kit of the above-mentioned 5 has the purposes described in any of the following: (1) application in the evaluation of fat deposition in the tail of sheep; (2) screening of sheep breeds (3) Application in sheep breed identification; (4) Application in sheep breed traceability; (5) Application in sheep breeding; (6) Application in germplasm resource protection; (7) Application in germplasm resource improvement; (8) application in sheep pedigree reconstruction.
  • the present invention is based on the research on the genetic resources of many sheep at home and abroad, and provides a combination of sheep fat tail SNP sites consisting of only 3000 SNP sites.
  • the SNP site combination provided by the invention has good versatility at home and abroad. It can quickly evaluate the tail fat deposition of individual sheep from the genetic level to obtain more accurate breeding evaluation information, select the fat tail traits that are difficult to measure in the early stage, control the breeding process, and save a lot of breeding costs; in addition, using this
  • the fat tail SNP site combination provided by the invention can also realize the identification and traceability of sheep breeds from the perspective of fat deposition in sheep tails, and provide technical support for sheep pedigree reconstruction, germplasm resource protection and germplasm resource improvement.
  • Bio products such as probe combinations, gene chips, and kits made based on the suet tail SNP sites provided by the present invention also have the characteristics of small throughput, low cost, and easier analysis, wide applicability, and market prospects broad.
  • Figure 1 is the Manhattan diagram of Altay sheep and Shetland sheep (ALS versus SHE) group
  • Figure 2 is the Manhattan diagram of the Bashbai sheep and Shetland sheep (BSB versus SHE) group;
  • Figure 3 is the Manhattan map of the Big Tail Han sheep and Shetland sheep (HDW versus SHE) group
  • Fig. 4 is a result diagram of the significance test performed on the judgment result of the population threshold analysis in the present application.
  • biological products referred to in the present invention include but are not limited to primers, probes, gene chips, kits, etc. made based on the site information provided in the present invention.
  • suet tail referred to in the present invention is divided according to the fat deposition in the tail, including two types of tails: fat tail and thin tail.
  • SNP in the present invention refers to single nucleotide polymorphism (Single Nucleotide Polymorphism), mainly refers to the DNA sequence polymorphism caused by the variation of a single nucleotide on the genome level, the single nucleotide Acid variations include those resulting from single base transitions, transversions, insertions, or deletions.
  • the molecular markers referred to in the present invention are all heritable and detectable DNA sequences or proteins, including but not limited to molecular markers based on molecular hybridization, such as RFLP, MinisatelliteDNA; molecular markers based on PCR technology, such as RAPD, STS, SSR and SCAR; DNA markers based on restriction enzyme digestion and PCR technology; molecular markers based on DNA chip technology, such as SNP; analytical marker technology based on EST database development, etc.
  • the molecular markers provided by the invention can be used for genome mapping, gene location research, map-based gene cloning, species relationship and system classification, and the like.
  • the probe referred to in the present invention is a nucleic acid sequence (DNA or RNA) with a detection label and known sequence that is complementary to the target gene, such as Taqman-MGB probe.
  • the kit referred to in the present invention is any kit routinely used in the art that contains reagents for detection or experimentation, so that operators can get rid of the burdensome reagent preparation and optimization process.
  • it includes primers for amplifying the site information provided by the present invention, molecular markers or probes or gene chips for detecting the site information provided by the present invention, and enzymes and gene chips used for amplification. buffer, or also detect with a fluorescent label.
  • Samples carrying genetic information in individual sheep in Step 1 are collected by conventional methods in the art, including but not limited to blood, cells, tissues, skin, hair, excrement, and the like. Extract the genetic information (such as DNA) in the sample for high-depth sequencing, use SAMtools and GATK to compare with the sheep 4.0 reference genome (obtained from NCBI) released in 2015, and combine the two methods to obtain a common result to form a SNP
  • genetic information referred to in the present invention refers to the information that organisms pass from parent to offspring, or from cell to cell each time each cell divides, in order to replicate the same thing as itself.
  • the extraction of genetic information (such as DNA) in the sample for high-depth sequencing can be done by biological companies, such as Huada Gene Company, Illumina Company, etc.
  • the high-depth sequencing method adopts conventional methods in the field or methods of biological companies.
  • the average sequencing depth is ⁇ 25.7 ⁇ , and the resequencing analysis process is used for high-depth sequencing.
  • Figures 1-3 show the scanned Manhattan map in Figures 1-3, of which Figure 1 is Altay Sheep and Shetland sheep (ALS versus SHE) group, Figure 2 shows the Bashbai sheep and Shetland sheep (BSB versus SHE) group, Figure 3 shows the big-tailed Han sheep and Shetland sheep (HDW versus SHE) group , while using ⁇ ratio (i.e. ⁇ value) to mine the functional regions related to lipid tails in the sheep breeds in each group, and then take the intersection of the two results to screen out the functional regions related to lipid tails.
  • ⁇ ratio i.e. ⁇ value
  • probes such as tanqman probes
  • the SNP gene chip of the present application adopts conventional method to immobilize the primers or probes obtained in Example 2 on polymer substrates, such as nylon membranes, nitrocellulose membranes, plastics, silica gel wafers, micro-magnetic beads, etc., or to immobilize the probes
  • polymer substrates such as nylon membranes, nitrocellulose membranes, plastics, silica gel wafers, micro-magnetic beads, etc.
  • the primers or probes obtained in Example 2 are directly synthesized on a glass plate, or on a hard surface such as glass, and the use method of the SNP gene chip of the present application is the same as the conventional method.
  • the lipid tail SNP detection kit provided in the present application includes primers or probes or gene chips obtained based on the combination of SNP sites obtained in Example 1. Depending on the type of use, the corresponding detection reagents are also included, for example, when the Taqman probe is obtained based on the combination of SNP sites obtained in Example 1, it also includes buffers, ligases, AceQUniversal U +Probe Master Mix V2, TaqMan Probe, etc.
  • sheep fat tail SNP detection kits configured based on the combination of fat tail SNP sites provided by this application all belong to the protection scope of the present invention.
  • fat tail detection was performed on 23 lambs purchased, specifically:
  • the peripheral blood of 23 lambs was collected by conventional methods.
  • the peripheral blood is an isolated biological sample taken from the lamb, and the whole genome DNA in the sample was extracted by a conventional DNA extraction kit to obtain a whole genome DNA sample.
  • Thermo Scientific NanoDrop 2000 UV spectrophotometer measures DNA sample concentration and dilutes to 20ng/ ⁇ l for later use;
  • the site information in the SNP site combination provided by the present invention to design primers by conventional methods, detect the whole genome DNA of 23 lambs, and obtain the typing results of each site in each lamb (that is, each site Whether it is the result of homozygosity, heterozygosity, mutant homozygosity or base deletion), calculate the frequency value of the typing result of each site, and compare it with the population threshold.
  • the comparison results show that only T04 and T13, T20 , T01, T02, T03, T17 are fatty tails, others are thin tails.
  • 23 lambs were bred in a conventional way. After 8 months, the 23 lambs were observed as adults according to the standards of GB/T-17237-2008, GB/T-12694-2016, and GB/T-51225-2017.
  • the ketone body weight, tail length, tail width, tail thickness and tail weight of T04, T13 and T02 were as high as 14.2% and the lowest was 12.3%, which were significantly higher than those of other adult sheep.
  • T01 , T02, T03, T17, and T20 have short fat tails, but the proportion of tail weight to ketone body weight is 0.07%, which conforms to the nature of short fat-tailed sheep, and they are fat-tailed sheep like T04, T13, and T02; other adult sheep
  • the weight of the tail only accounts for about 0.01 of the weight of the ketone, and the length of the tail is equivalent to that of the short fat tail, but the width and thickness of the tail are significantly lower than that of the short fat tail. This is consistent with the results of genetic analysis, and it is a thin-tailed sheep.
  • the population threshold in the present application is obtained by analyzing the fat tail population, the long fat tail population, the short fat tail population and the thin tail population, and the method is the same as above.
  • the sheep fat tail SNP molecular marker, the SNP probe combination and the SNP chip for detecting the sheep fat tail that can be made by those skilled in the art based on the combination of the sheep fat tail SNP sites that are only composed of 3000 SNP sites in this application are available in Tail fat deposition of individual sheep is evaluated at the genomic level, or genetic evaluation or breed screening or breed identification for higher accuracy in breeding value estimation. Select traits that are difficult to measure in the early stage, shorten the generation interval, and accelerate the breeding process, thereby saving a lot of breeding costs. It can also be applied to sheep pedigree reconstruction, sheep breed traceability, germplasm resource protection and germplasm resource improvement.

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Abstract

一种分析绵羊脂尾的基因芯片、分子探针组合、试剂盒及应用。提供了3000个分析绵羊脂尾性状的SNP位点组合，所述位点组合如表1所示；以及基于上述位点组合制备的分子探针组合、基因芯片、试剂盒等生物产品；利用提供的位点组合物以及相关生物产品，能够从基因水品对绵羊个体进行遗传评估，品种筛选、鉴定，在早期难以度量阶段，即可对绵羊脂尾性状进行个体选择，控制育种进程，节约育种成本，并应用于绵羊品种溯源、绵羊谱系重构、种质资源保护和种质资源改良中。

Description

分析绵羊脂尾的基因芯片、分子探针组合、试剂盒及应用 技术领域
本发明涉及生物技术领域，具体涉及生物检测技术领域，更具体的涉及分析绵羊脂尾的分子探针组合、基因芯片、试剂盒等生物产品及应用。
背景技术
绵羊尾部脂肪能够作为能量储存的形式，可为绵羊在迁徙或度过饲草缺乏的严寒冬天提供能量，使之维持生存需要；另一方面可作为一种高能量的食物供给人类使用。但是，随着牧区或农区绵羊饲养条件的不断提高或人类生活水平的不断改善，绵羊尾部脂肪对绵羊自身或人类的作用逐渐变小，人们对高脂类羊肉需求的减少。且有研究表明，家畜沉积1kg脂肪所需要的饲料相当于体内2kg瘦肉所需的饲料，因此绵羊尾部脂肪的沉积会使养羊业的经济效益大大降低，因此过度尾脂沉积反而会增加饲养成本。此外，在繁殖上，短脂脂尾型的绵羊(例如小尾绵羊)更便于配种，提高母羊的受胎率，因此，根据市场需求选育相应脂尾的绵羊品种对养羊业具有重要的指导作用，此外，绵羊脂尾在绵羊品种溯源及种质资源的保护、开发利用也具有重要意义。
随着科技的发展，已经有越来越多的科研工作者利用基因分析研究绵羊脂尾，目前，分子标记技术由于其准确度高、可操作性强等优点越来越受到重视，其中基于单核苷酸多态性(single nucleotide polymorphisms,SNP)的分子标记技术应用得越来越广泛。SNP作为生物基因组中的一种遗传分子标记，在动植物遗传进化分析、重要经济形状筛选、分子育种等方面起着越来越重要的作用。基于SNP的SNP芯片是进行现代遗传育种便捷高效的工具，由于其容易实现SNP高通量、自动化检测，可以检测出基因组DNA上每个碱基对的变化，包括插入、缺失、颠倒、转换等，已经成为非常理想的SNP检测技术，在绵羊育种领域中的运用也越来越多。
绵羊现有的商业化SNP芯片有Illumina Ovine SNP50Beadchip(50K)、Illumina Sheep HD Genotyping Beadchip(680K)和Illumina Ovine LD(5K)，前两者是在学术生产研究中最为常用的。其中Illumina Ovine SNP50Beadchip是Illumina公司与UCSC、CSIRO、USDA及Agresearch等研究团队和机构共同研制，包括覆盖绵羊全基因组的超过54K个SNP位点，可用于遗传育种、全基因组关联分析、数量性状基因座定位、基因优选及比较基因组学等研究。而Illumina Sheep HD Genotyping Beadchip(680K)同为Illumina公司生产的SNP芯片，是由国际绵羊基因组协会(ISGC)在Ovine SNP50的基础上进行扩增的高密度芯片。但是现有的绵羊SNP芯片主要依据西方品种绵羊的数据，缺乏中国绵羊品种与国外绵羊品种结合的SNP数据，存在位点均匀性不足、对功能性位点和区域的体现不足、超过10％的位点在中国绵羊群体为极低频位点等诸多问题，设计一款适用于中国绵羊群体且能对脂尾进行快速有效检测SNP芯片，有着十分重要的意义
发明内容
为满足我国当前绵羊品种研究以及农业生产上对绵羊脂尾的检测的需求，本发明提供了一种分析绵羊脂尾的SNP位点组合，以及基于SNP位点组合制得的分子探针组合、基因芯片、试剂盒等生物产品及应用，利用本发明提供的位点信息，能够快速准确检测待绵羊的脂尾性状，实现绵羊尾部脂肪沉积评价、品种筛选、品种鉴定、品种溯源、绵羊育种，有利于绵羊系谱重构、种质资源保护以及种质资源改良，耗时短、成本低、市场效益广阔。
为实现本发明的技术目的，本发明采用以下技术手段实现：
1、3000个SNP位点组合在分析绵羊脂尾中的应用，所述3000个SNP位点组合的物理位置如表1所示，其物理位置基于绵羊v4.0基因组序列比对确定。
表1 3000个位点信息
Figure PCTCN2022106904-appb-000001
Figure PCTCN2022106904-appb-000002
Figure PCTCN2022106904-appb-000003
Figure PCTCN2022106904-appb-000004
Figure PCTCN2022106904-appb-000005
Figure PCTCN2022106904-appb-000006
Figure PCTCN2022106904-appb-000007
Figure PCTCN2022106904-appb-000008
Figure PCTCN2022106904-appb-000009
Figure PCTCN2022106904-appb-000010
Figure PCTCN2022106904-appb-000011
Figure PCTCN2022106904-appb-000012
Figure PCTCN2022106904-appb-000013
Figure PCTCN2022106904-appb-000014
Figure PCTCN2022106904-appb-000015
Figure PCTCN2022106904-appb-000016
Figure PCTCN2022106904-appb-000017
Figure PCTCN2022106904-appb-000018
Figure PCTCN2022106904-appb-000019
Figure PCTCN2022106904-appb-000020
Figure PCTCN2022106904-appb-000021
Figure PCTCN2022106904-appb-000022
Figure PCTCN2022106904-appb-000023
2、分析绵羊脂尾的方法，将待测绵羊的基因组DNA的3000个SNP位点基因型与对照绵羊基因组DNA的所述3000个SNP位点基因型进行比较；
其中，所述3000个SNP位点为上述的3000个SNP位点。
3、分析绵羊脂尾的分子探针组合，所述分子探针组合检测待测样品中如表1所示的SNP位点组合。
4、分析绵羊脂尾的基因芯片，所述基因芯片负载上述3的分子探针组合。
5、分析绵羊脂尾的试剂盒，其具有上述3的分子探针组合或上述4的基因芯片。
6、分析绵羊脂尾的方法，应用上述3的分子探针组合或上述4的基因芯片或上述5的试剂盒对待测样品进行检测。
7、上述3的分子探针组合或上述4的基因芯片或上述5的试剂盒具有如下任一所述的用途：(1)在绵羊尾部脂肪沉积评价中的应用；(2)在绵羊品种筛选的应用；(3)在绵羊品种鉴定中的应用；(4)在绵羊品种溯源中的应用；(5)在绵羊育种中的应用；(6)在种质资源保护中的应用；(7)在种质资源改良中的应用；(8)在绵羊系谱重构中的应用。
有益效果:
1、本发明基于对国内外众多绵羊的遗传资源研究，提供一种仅由3000个SNP位点组成的绵羊脂尾SNP位点组合，本发明提供的SNP位点组合具有国内外通用性好，能快速从基因水平上对绵羊个体的尾部脂肪沉积进行评价，以获得更准确的育种评估信息，对早期难以度量的脂尾性状进行选择，控制育种进程，节约大量的育种成本；此外，利用本发明提供的脂尾SNP位点组合，还能够从绵羊尾部脂肪沉淀的角度实现绵羊品种的鉴定和溯源，为绵羊系谱重构、种质资源保护和种质资源的改良提供技术支持。
2、基于本发明提供的绵羊脂尾SNP位点制成的探针组合、基因芯片、试剂盒等生物产品还具有通量小、成本低，分析更容易的特点，普适性广，市场前景广阔。
附图说明
图1是阿勒泰羊和设德兰羊(ALS versus SHE)组的曼哈顿图；
图2为巴什拜羊和设德兰羊(BSB versus SHE)组的曼哈顿图；
图3为大尾寒羊和设德兰羊(HDW versus SHE)组的曼哈顿图；
图4是本申请对群体阈值分析的判定结果进行了显著性检验的结果图。
具体实施方式
下面参考具体实施方式的详细描述来进一步阐明本发明，但这些实施例仅仅是说明性的，而不能理解为对本发明的限制。若未特别指明，实施例中所采用的技术手段为本领域技术人员所熟知的常规手段，可以参照《生物信息学与功能基因组学》原著第三版或者相关书籍进行，所采用的生物信息软件和产品也均为可商业获得的。未详细描述的各种过程和方法是本领域中公知的常规方法，所用材料来源、商品名以及有必要列出其组成成分者，均在首次出现时标明，其后所用相同试剂如无特殊说明，均以首次标明的内容相同。此外，还需要说明的是，本发明提供的位点组合及应用均是本申请的发明人经过艰苦的创造性劳动和优化工作才得以完成。
在本文前述的位点组合部分中所描述的特征和优点，同样适用于基于位点组合所形成的分子探针组合、基因芯片、试剂盒以及其应用，在此不再赘述。
需要说明的是，本发明所称的生物产品包括但不限于基于本发明提供的位点信息制成的引物、探针、基因芯片、试剂盒等。
需要说明的是，本发明所称的绵羊脂尾是根据尾部脂肪沉积划分，包括脂尾和瘦尾两种尾型。
本发明的所称的SNP是指单核苷酸多态性(Single Nucleotide Polymorphism)，主要是指在基因组水平上由单个核苷酸的变异所引起的DNA序列多态性，所述单个核苷酸的变异包括由单个碱基的转换、颠换、插入或缺失所导致的变异。
需要说明的是，本发明所称的分子标记为一切可遗传的并可检测的DNA序列或蛋白质，包括但不限于基于分子杂交的分子标记，如RFLP、MinisatelliteDNA；基于PCR技术的分子标记，如RAPD、STS、SSR和SCAR；基于限制性酶切和PCR技术的DNA标记；基于DNA芯片技术的分子标记，如SNP；基于EST数据库发展的分析标记技术等。本发明提供的分子标记可以用于基因组作图、和基因定位研究、基于图谱的基因克隆、物种亲缘关系和系统分类等。
需要说明的是，本发明所称的探针是一段带有检测标记，且顺序已知的，与目的基因互补的核酸序列(DNA或RNA)，例如Taqman-MGB探针。
需要说明的是，本发明所称的试剂盒为本领域常规使用的任意一种包含检测或实验所用试剂，便于操作人员能够摆脱繁重的试剂配制及优化过程的盒子。在本发明的一个实施例中，其中包含有扩增本发明提供的位点信息的引物、检测本发明提供的位点信息的分子标记或探针或基因芯片，还包括扩增所用的酶和缓冲液，或者还检测用的荧光标记。
实施例1脂尾性状SNP位点组合的获得
1、绵羊个体的选择
为了实现国内外绵羊品种的更加全面的覆盖，申请人对遍布于亚洲、欧洲、非洲和中东的248个绵羊 个体进行遗传信息采集，其中包括16个野羊亚洲摩弗伦品种、172个地方品种和60个培育品种，具体涉及到中国山东的小尾寒羊、泗水裘皮羊、大尾寒羊、洼地绵羊，中国江苏的湖羊，中国宁夏的滩羊，中国新疆的阿勒泰羊、巴什拜羊、杜泊绵羊、中国美利奴羊(细毛羊)、中国美利奴羊(超细毛羊)、萨福克羊、策勒黑羊、多浪羊、Waggir Sheep羊，芬兰的芬兰羊(Finnsheep)、荷兰的威桑岛羊(Ouessant)、设德兰羊(Shetland)、索洛格诺羊(Solognote)、哥特兰岛羊(Gotland)、德伦特希思羊(Drente Heathen)，埃塞俄比亚的邦加羊(Bonga Sheep)、阿法尔羊(Afar Sheep)，尼日尔的姆博罗罗羊(Mbororo Sheep)，尼日利亚的扬卡羊(Yankasa Sheep)，非洲乍得的西非矮羊(West African Dwarf Sheep)、乌达羊(Uda Sheep)，非洲布基纳法索的贾隆凯羊(DjallonkéSheep)、摩西羊(Mossi Sheep)、萨赫勒羊(Sahelian Sheep)，西非喀麦隆的喀麦隆羊(Cameroon Sheep)，伊拉克的阿华西绵羊(Awassi Sheep)、哈姆达尼羊(Hamdani Sheep)，阿塞拜疆的马泽克羊(Mazekh Sheep)，伊朗的灰设拉子羊(Grey-Shiraz Sheep)、盖泽尔羊(Ghezel Sheep)、阿夫沙里羊(Afshari Sheep)、沙尔羊(Shal Sheep)、马奎羊(Makui Sheep)、莫哈尼羊(Moghani Sheep)，巴基斯坦的卡拉库尔羊(Karakul Sheep)，伊朗舍赫尔科德的亚洲摩弗伦(Asiatic mouflon)。
2、绵羊全基因的总SNP集合的获得
采用本领域常规方法采集步骤1中绵羊个体中载有遗传信息的样本，该样本包括但不限于血液、细胞、组织、皮肤、毛发、排泄物等。提取样本中的遗传信息(例如DNA)进行高深度测序，利用SAMtools和GATK两种方式与2015年发布的绵羊4.0参考基因组(从NCBI获得)进行对比，并将两种方式获得共同结果形成一个SNP集合，共计2836万个SNP位点，作为绵羊全基因的总SNP集合。
需要说明的是，本发明所称的遗传信息(genetic information)是指生物为复制与自己相同的东西、由亲代传递给子代、或各细胞每次分裂时由细胞传递给细胞的信息。
需要说明的是，提取样本中的遗传信息(例如DNA)进行高深度测序可以由生物公司完成，例如华大基因公司，illumina公司等，高深度测序方法采用本领域常规方法或生物公司的方法，在本发明的一个实施例中，采用平均测序深度为～25.7×，应用重测序分析流程进行高深度测序。
3、候选基因及所在功能区域的筛选
3.1对不同脂尾的绵羊遗传信息样本处理
筛选不同脂尾的绵羊品种，并根据不同绵羊品种在脂尾性状上表现出来的显著差异，对相应的遗传信息样本进行分组，在本发明的一个实施例中，发明人将阿勒泰羊和设德兰羊(ALS versus SHE)的遗传信息分为一组、巴什拜羊和设德兰羊(BSB versus SHE)的遗传信息分为一组、大尾寒羊和设德兰羊(HDW versus SHE)的遗传信息分为一组。
3.2对分组后不同脂尾的绵羊遗传信息的处理
应用XP-CLR扫荡每个绵羊组之间的多基因座等位基因频率差异，扫描出与脂尾相关的功能区域(扫描得到的曼哈顿图如图1-3所示，其中，图1为阿勒泰羊和设德兰羊(ALS versus SHE)组，图2为巴什拜羊和设德兰羊(BSB versus SHE)组，图3为大尾寒羊和设德兰羊(HDW versus SHE)组，同时利用πratio(即π值)挖掘每组中的绵羊品种中与脂尾相关的功能区域，然后取两种结果的交集，筛选出脂尾相关的功能区域。
参照已公开的基因研究成果对区域内的基因进行筛选，最终确定5个与脂尾相关的、功能十分确定的候选基因PDGFD、NRIP1、MAP2K3、SGCZ、GBE1，进而通过perl脚本确定上述候选基因对应的功能区域。
4、脂尾SNP位点组合的获得
利用bedtools在总的SNP集合中寻找步骤3中确定的候选基因所在功能区域对应的SNP位点，得到与PDGFD、NRIP1、MAP2K3、SGCZ、GBE1共5个脂尾相关的功能基因相关联，且仅包含3000个snp位点的脂尾位点组合。
实施例2将脂尾SNP位点组合用于制备引物组合及探针组合
本领域技术人员根据本发明提供的脂尾SNP位点组合中的每一个位点的序列信息设计引物，并将设计得到的引物利进行二级结构评估和Tm值评估，最终获得特异性好、灵敏度高，并且可以在同一反应条件下实现检测目的的引物。
其中，二级结构评估及Tm值评估可以采用本领域常用的任一一种方式进行，例如采用DNA folding form评估其二级结构，具体参见(http://unafold.rna.albany.edu/？q＝mfold/DNA-Folding-Form)，然后采用软件RaW-Probe评估其Tm值。
上述方法均为常规方法，根据本申请提供的脂尾SNP位点组合中的位点信息，不再需要付出创造性劳动的情况下就能获得，因此，根据本申请提供的脂尾SNP位点组合获得引物也属于本发明的保护范围。
同样的，利用本发明提供的脂尾SNP位点组合制备探针，例如tanqman探针也属于本发明的保护范围。
实施例3将脂尾SNP位点组合用于制备基因芯片
本申请的SNP基因芯片是采用常规方法将实施例2获得的引物或探针固定在聚合物基片上，例如尼龙膜、硝酸纤维膜、塑料、硅胶晶片、微型磁珠等，或将探针固定在玻璃板上，或在玻璃等硬质表面上直接合成实施例2获得的引物或探针，本申请的SNP基因芯片的使用方法与常规方法相同。
需要说明的是，本领域技术人员可以采用任一一种方式制备检测绵羊脂尾的SNP基因芯片，同样也可以委托生物公司制备，但是基于本申请提供的脂尾SNP位点组合制备的SNP基因芯片均属于本发明的保护范围。
实施例4绵羊脂尾的分析试剂盒
本申请的提供的脂尾SNP检测试剂盒包括基于实施例1获得的SNP位点组合获得的引物或探针或基因芯片。根据使用类型的不同，还包括相应的检测试剂，例如当基于实施例1获得的SNP位点组合获得的为Taqman探针时，还包括荧光定量PCR反应常规使用的缓冲液、连接酶、AceQUniversal U+Probe Master Mix V2，TaqMan Probe等。
本领域技术人员根据使用方式的不同配置不同的绵羊脂尾检测的SNP试剂盒，但是基于本申请提供的脂尾SNP位点组合配置的绵羊脂尾SNP检测试剂盒均属于本发明的保护范围。
实施例5绵羊脂尾的检测
基于本申请的实施例1提供的绵羊脂尾SNP位点组合对采购的23只羊羔进行脂尾检测，具体为：
采用常规方法采集23只羊羔的外周血，该外周血是从羊羔体内取出的离体生物样品，并采用常规的DNA提取试剂盒提取样本中的全基因组DNA，获得全基因组DNA样本，利用Thermo Scientific NanoDrop 2000紫外分光光度计测量DNA样本浓度，并稀释至20ng/μl，备用；
采用常规方法利用本发明提供的SNP位点组合中的位点信息设计引物，对23只羊羔的全基因组DNA进行检测，获得每只羊羔中每个位点的分型结果(即每个位点是否为纯合子、杂合子、突变型纯合子或碱基缺失的结果)，计算每个位点的分型结果的频率值，并与群体阈值进行比较，比较结果显示，只有T04和T13、T20、T01、T02、T03、T17为脂尾、其他均为瘦尾。
对23只羊羔进行养殖，养殖方式为常规方法，8个月后，参照GB/T-17237-2008、GB/T-12694-2016、GB/T-51225-2017标准观测成年后的23只羊羔的酮体重、尾长、尾宽、尾厚和尾重，观测结果显示，T04、T13、T02的尾巴重量占酮体重的比例高达14.2％，最低达到12.3，均显著高于其他成年羊，T01、T02、T03、T17、T20脂尾较短，但尾巴重量占酮体重的比例为0.07％，符合短脂尾羊的性质，其与T04、T13、T02同为脂尾羊；其他成年羊的尾巴重量仅占酮体重的0.01左右，尾长与短脂尾相当，但是尾宽与尾厚均显著低于短脂尾，这与基因分析结果一致，为瘦尾羊。
需要说明的是，本申请的所述群体阈值是通过对肥臀尾群体，长脂尾群体、短脂尾群体以及瘦尾群体进行分析获得，方法同上。
本申请对肥臀尾群体，脂尾群体以及瘦尾群体进行分析的判定结果进行了显著性检验(独立样本曼惠特尼U检验)，结果如图4所示，根据图中结果可以看出，P<0.01，差异极显著，可见采用本发明方法进行判定的结果具有准确性和有效性。
工业应用
本领域技术人员基于本申请提供的仅由3000个SNP位点组成的绵羊脂尾SNP位点组合可以制成的绵羊脂尾SNP分子标记、检测绵羊脂尾的SNP探针组合及SNP芯片，在基因组水平上对绵羊个体的尾部脂肪沉积进行评价，或遗传评估或品种筛选或品种鉴定，以获得更高的育种值估计准确性。对早期难以度量的性状进行选择，缩短世代间隔，加速育种进程，从而节约大量的育种成本，还能够应用于绵羊系谱重构、绵羊品种溯源和种质资源保护和种质资源改良中。
以上所述仅为帮助理解本发明的优选实例，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化，在不违背本发明的思想下，本领域技术人员在此基础上对本发明作出的各种改动或者修改，同样应属于本发明的范围。

Claims (7)

  1. 3000个SNP位点组合在分析绵羊脂尾中的应用，所述3000个SNP位点组合的物理位置如表1所示，其物理位置基于绵羊v4.0基因组序列比对确定。
  2. 分析绵羊脂尾的方法，将待测绵羊的基因组DNA的3000个SNP位点基因型与对照绵羊基因组DNA的所述3000个SNP位点基因型进行比较；
    其中，所述3000个SNP位点为权利要求1所述的3000个SNP位点。
  3. 分析绵羊脂尾的分子探针组合，所述分子探针组合检测待测样品中如权利要求1所述的SNP位点组合。
  4. 分析绵羊脂尾的基因芯片，所述基因芯片负载有权利要求3所述的分子探针组合。
  5. 分析绵羊脂尾的试剂盒，其具有权利要求3所述的分子探针组合或权利要求4所述的基因芯片。
  6. 分析绵羊脂尾的方法，应用权利要求3所述的分子探针组合或权利要求4所述的基因芯片或权利要求5所述的试剂盒对待测样品进行检测。
  7. 权利要求3所述的分子探针组合或权利要求4所述的基因芯片或权利要求5所述的试剂盒具有如下任一所述的用途：
    (1)在绵羊尾部脂肪沉积评价中的应用；
    (2)在绵羊品种筛选的应用；
    (3)在绵羊品种鉴定中的应用；
    (4)在绵羊品种溯源中的应用；
    (5)在绵羊育种中的应用；
    (6)在种质资源保护中的应用；
    (7)在种质资源改良中的应用；
    (8)在绵羊系谱重构中的应用。
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