WO2023001212A1 - 分析绵羊产奶性能的基因芯片、分子探针组合、试剂盒及应用 - Google Patents

分析绵羊产奶性能的基因芯片、分子探针组合、试剂盒及应用 Download PDF

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WO2023001212A1
WO2023001212A1 PCT/CN2022/106907 CN2022106907W WO2023001212A1 WO 2023001212 A1 WO2023001212 A1 WO 2023001212A1 CN 2022106907 W CN2022106907 W CN 2022106907W WO 2023001212 A1 WO2023001212 A1 WO 2023001212A1
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sheep
milk production
production performance
application
snp
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PCT/CN2022/106907
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English (en)
French (fr)
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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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Publication of WO2023001212A1 publication Critical patent/WO2023001212A1/zh
Priority to ZA2024/03950A priority Critical patent/ZA202403950B/en

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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
    • 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
    • C12Q2600/124—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 invention relates to the field of biotechnology, in particular to the field of biodetection technology, and more specifically to the combination of SNP sites for milk production performance of sheep and its application.
  • Multiparous sheep usually cannot secrete enough milk to meet the growth and development needs of lambs, resulting in a decrease in the survival rate of lambs.
  • the use of milk substitutes can alleviate the plight of insufficient milk production in ewes to a certain extent, the fundamental solution to the problem is still Increase milk production of ewes.
  • sheep's milk has higher total fat, milk protein and lactose content, and the nutritional content of goat's milk is close to that of human's milk.
  • One of the sources of food is among the 158 million sheep in our country, the number of milk sheep is almost zero. Therefore, the development of meat and milk sheep is the development direction of our sheep industry. For meat and milk sheep breeds Breeding is becoming more and more urgent.
  • SNP As a genetic molecular marker in biological genomes, SNP plays an increasingly important role in the analysis of genetic evolution of animals and plants, screening of important economic shapes, and molecular breeding.
  • SNP-based SNP chip is a convenient and efficient tool for modern genetic breeding. Because it is easy to realize high-throughput and automatic detection of SNP, it can detect the change of each base pair on genomic DNA, including insertion, deletion, inversion, conversion, etc. , has become a very ideal SNP detection technology, and it is used more and more in the field of sheep breeding.
  • 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 genes Locus mapping, gene optimization and comparative genomics research.
  • the Illumina Sheep HD Genotyping Beadchip (680K) is also a SNP chip produced by Illumina.
  • the present invention provides gene chips, molecular probe combinations, and reagents for analyzing sheep milk production performance based on the SNP site collection of milk production performance. Box and application, using the site information provided by the present invention, can quickly and accurately realize sheep milk production performance evaluation, breed screening, breed identification, breed traceability, and sheep breeding, which is beneficial to the protection of germplasm resources and the improvement of germplasm resources. Time-consuming Short, low cost, broad market benefits.
  • A analyze the molecular probe combination of sheep milk production performance, described molecular probe combination detects the SNP site combination as shown in table 1 in the sample to be tested:
  • the physical location information of the site combinations in Table 1 is determined based on the sequence alignment of the sheep v4.0 genome.
  • the method for analyzing the milk production performance of sheep is to compare the 2030 SNP site genotypes of the genomic DNA of the sheep to be tested with the 2030 SNP site genotypes of the control sheep genomic DNA; the 2030 SNP site genotypes are compared; 2030 SNP sites shown in Table 1.
  • a combination of molecular probes for analyzing milk production performance of sheep which detects the combination of SNP sites described in Table 1 in the sample to be tested.
  • a gene chip for analyzing the milk production performance of sheep which is loaded with the molecular probe combination of the above 3.
  • a kit for analyzing milk production performance of sheep which has the molecular probe combination of the above 3 or the gene chip of the above 4.
  • the method for analyzing milk production performance of sheep is to use 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 to detect the sample to be tested.
  • 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 provides a combination of SNP sites consisting of only 2030 SNP sites that can reflect the milk production performance of sheep. Evaluate the milk production performance of individual sheep to obtain more accurate breeding evaluation information, select the milk production performance traits that are difficult to measure in the early stage, shorten the generation interval, accelerate the breeding process, and save a lot of breeding costs; in addition, the invention provides The milk production performance SNP combination can also realize the identification and traceability of sheep breeds from the perspective of milk production performance, and provide technical support for the protection and improvement of germplasm resources of sheep in my country.
  • the present invention is based on the combination of the above-mentioned SNP sites. Compared with the existing high-density chips, the probe combination, gene chip, and kit formed by the SNP sites of sheep milk production performance provided by the present invention have small throughput, low cost, and more efficient analysis. Easy, wide applicability, broad market prospects.
  • Figure 1 is the Manhattan map of the East Frisian sheep Vs Finnish sheep (EFR versus FIN) group
  • Figure 2 is the Manhattan diagram of the East Frisian sheep Vs Suffolk sheep (EFR versus SFK) group
  • Figure 3 is the Manhattan map of the East Frisian sheep Vs Dorper sheep (EFR versus DPS) 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 combination of sites provided in the present invention.
  • milk production performance of sheep referred to in the present invention reflects its milk production performance from four angles: milk production, milk fat rate, milk protein rate, and lactation period. According to actual production needs, those skilled in the art can set it by themselves. Milk production threshold, so as to judge the pros and cons of milk production performance.
  • 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.
  • East Friedan sheep The genetic information of Vs Finnish sheep (EFR versus FIN) is divided into one group, the genetic information of East Frisian sheep Vs Suffolk sheep (EFR versus SFK) is divided into one group, the genetic information of East Frisian sheep Vs Dorper sheep (EFR The genetic information versus DPS) is grouped into one group.
  • Use XP-CLR to scan the multi-locus allele frequency difference between each sheep group, and scan out the functional areas related to milk production performance (the scanned Manhattan map is shown in Figure 1-3, and use ⁇ ratio (ie ⁇ value) to mine the functional areas related to milk production performance in the sheep breeds in each group, and then take the intersection of the two results to screen out the functional areas related to milk production performance.
  • the genes in the region were screened with reference to the published gene research results, and finally 8 candidate genes related to milk production performance, ALDH9A1, ARL4A, ATF6, NSMAF, PPP1R12A, SLC16A10, SLC40A1, and SND1 were identified, and then passed The perl script determines the functional regions corresponding to the above candidate genes.
  • 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 milk production performance SNP detection kit 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 milk production performance SNP detection kits configured based on the combination of milk production performance SNP sites provided by this application all belong to the present invention protected range.
  • Example 1 of the present application was used to analyze the milk production performance of 18 ewe lambs raised on a certain pasture in Mongolia, specifically:
  • peripheral blood of 18 lambs was collected by conventional methods in the art, and the peripheral blood was an isolated biological sample taken from the lamb, and the whole genome DNA in the sample was extracted using a conventional DNA extraction kit to obtain a whole genome DNA sample.
  • Thermo Scientific NanoDrop2000 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 18 ewe lambs, and obtain the typing result of each site in each ewe lamb (that is, each Whether the locus is homozygous, heterozygous, mutant homozygous or the result of base deletion), calculate the frequency value of the typing result of each locus, and compare it with the population threshold.
  • the comparison results show that 72.2% of the mothers Lambs have better milk production performance, that is, sample numbers M01, M03, M04, M05, M06, M09, M10, M11, M12, M13, M15, M16, M18, and others have weak milk production performance.
  • 18 lambs were bred by conventional methods. After the ewes gave birth, the milk production, milk fat rate, milk protein rate, and lactation period of the 18 ewes were observed. The observation results showed that sample numbers M01, M03, M04, M05, The milk production of M06, M09, M10, M11, M12, M13, M15, M16, M18 sheep can reach more than 200kg, the highest can reach 800kg, the milk fat rate can reach more than 5.8%, the highest can reach 7.5%, and the milk protein rate can reach More than 58%, the lactation period is 82-300 days. The highest milk yield of ewes with other sample numbers is only 164kg, which is consistent with the genetic analysis results.
  • group threshold value in this application is obtained by analyzing groups with better milk production performance and groups with weak milk production performance, and the method is the same as above.
  • the application has carried out a significance test (independent sample Mann-Whitney U test) on the determination results of the analysis of the group with better milk production performance and the group with weak milk production performance.
  • the results are shown in Figure 4. According to the results in the figure, it can be It can be seen that P ⁇ 0.01, the difference is extremely significant, and it can be seen that the result of judging by the method of the present invention is accurate and effective.
  • sheep milk production performance SNP molecular markers based on the sheep milk production performance SNP site combination consisting of only 2030 SNP sites provided by this application, SNP probe combinations and SNPs for detecting sheep milk production performance Microarray, to evaluate the milk production performance of individual sheep at the genome level, or genetic evaluation or breed screening or breed identification to obtain higher breeding value estimation accuracy. Select the milk production 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 breed traceability, sheep germplasm resource protection, and sheep germplasm resource improvement.

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Abstract

一种分析绵羊产奶性能的基因芯片、分子探针组合、试剂盒及应用，涉及生物技术领域，提供能够分析绵羊产奶性能特征的2030个SNP位点组合，其物理位置信息基于绵羊v4.0基因组序列比对确定，利用2030个SNP位点组合以及基于该位点组合形成的分子探针组合、基因芯片、试剂盒能够对绵羊个体进行遗传评估，对早期难以度量的产奶性能性状进行个体选择，控制育种进程，节约育种成本。

Description

分析绵羊产奶性能的基因芯片、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，更具体的涉及绵羊产奶性能SNP位点组合及其应用。
背景技术
多胎绵羊通常不能分泌足够的奶满足羔羊生长发育的需求，导致羔羊的存活率降低，尽管使用代乳料可以在一定程度上缓解母羊产奶不足的困境，但实际上解决的根本途径仍是提高母羊的产奶量。此外，绵羊奶与山羊奶、牛奶相比，总脂肪、乳蛋白、乳糖含量更高，且羊奶的营养成分臂架接近人奶，是生产婴儿代乳粉的理想奶源，也是人类重要的膳食来源之一，然而我国存栏量在1.58亿只左右绵羊中，奶绵羊的存栏量几乎为零，因此发展肉乳兼用型绵羊，是我们养羊行业发展的方向，对于肉乳兼用型绵羊品种的选育也愈来愈迫切。
传统的品种选择方法值基于对动物或其亲缘动物的表型直接测定，因此完成育种策略需要保持广泛的表型记录，然而一些表型只能在绵羊发育到一定程度才能够显现，而且还不易观察到，养殖周期长，养殖投入成本高。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的性能，而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，尤其是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 SNP50 Beadchip(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 SNP50 Beadchip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产奶性能进行快速有效检测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和市场上对绵羊产奶性能上的检测需求，本发明基于产奶性能的SNP位点集合提供分析羊产奶性能的基因芯片、分子探针组合、试剂盒及应用，利用本发明提供的位点信息，能够快速准确的实现绵羊产奶性能评价、品种筛选、品种鉴定、品种溯源、绵羊育种，有利于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，本发明通过以下方式实现：
a、分析绵羊产奶性能的分子探针组合，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：
Figure PCTCN2022106907-appb-000001
Figure PCTCN2022106907-appb-000002
Figure PCTCN2022106907-appb-000003
Figure PCTCN2022106907-appb-000004
Figure PCTCN2022106907-appb-000005
Figure PCTCN2022106907-appb-000006
Figure PCTCN2022106907-appb-000007
Figure PCTCN2022106907-appb-000008
Figure PCTCN2022106907-appb-000009
Figure PCTCN2022106907-appb-000010
Figure PCTCN2022106907-appb-000011
Figure PCTCN2022106907-appb-000012
Figure PCTCN2022106907-appb-000013
Figure PCTCN2022106907-appb-000014
Figure PCTCN2022106907-appb-000015
Figure PCTCN2022106907-appb-000016
所述表1中的位点组合的物理位置信息基于绵羊v4.0基因组序列比对确定。
2、分析绵羊产奶性能的方法，将待测绵羊的基因组DNA的2030个SNP位点基因型与对照绵羊基因组DNA的所述2030个SNP位点基因型进行比较；所述2030个SNP位点为表1所示的2030个SNP位点。
3、分析绵羊产奶性能的分子探针组合，其检测待测样品中表1所述的SNP位点组合。
4、分析绵羊产奶性能的基因芯片，其负载有上述3的分子探针组合。
5、分析绵羊产奶性能的试剂盒，其具有上述3的分子探针组合或上述4的基因芯片。
6、分析绵羊产奶性能的方法，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绵羊的遗传资源研究，提供一种仅由2030个SNP位点组成的能够体现绵羊产奶性能的SNP位点组合，上述SNP位点组合能快速从基因水平上对绵羊个体的产奶性能进行评价，以获得更准确的育种评估信息，对早期难以度量的产奶性能性状进行选择，缩短世代间隔，加速育种进程，节约大量的育种成本；此外，利用本发明提供的产奶性能SNP位点组合，还能够从产奶性能的角度实现绵羊品种的鉴定和溯源，为我国绵羊的种质资源保护和种质资源的改良提供技术支持。
2、本发明基于上述SNP位点组合，提供的绵羊产奶性能SNP位点形成的探针组合、基因芯片、试剂盒与现有的高密度芯片相比，通量小、成本低，分析更容易，普适性广，市场前景广阔。
附图说明
图1是东弗里生羊Vs芬兰羊(EFR versus FIN)组的曼哈顿图；
图2为东弗里生羊Vs萨福克羊(EFR versus SFK)组的曼哈顿图；
图3为东弗里生羊Vs杜泊绵羊(EFR versus DPS)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。
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组合制成的引物、探针、基因芯片、试剂盒等。
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制成的引物、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，本发明所称的绵羊产奶性能是从产奶量、乳脂率、乳蛋白率、泌乳期四个角度体现其产奶性能，根据实际生产需要，本领域技术人员可自行设置产奶阈值，从而判断产奶性能的优劣。
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产奶性能性状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对不同产奶性能的绵羊遗传信息样本处理
筛选不同产奶性能的绵羊品种，并根据不同绵羊品种在产奶性能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，将东弗里生羊Vs芬兰羊(EFR versus FIN)的遗传信息分为一组、东弗里生羊Vs萨福克羊(EFR versus SFK)的遗传信息分为一组、东弗里生羊Vs杜泊绵羊(EFR versus DPS)的遗传信息分为一组。
3.2对分组后不同产奶性能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，扫描出与产奶性能相关的功能区域(扫描得到的曼哈顿图如图1-3所示，同时利用πratio(即π值)挖掘每组中的绵羊品种中与产奶性能相关的功能区域，然后取两种结果的交集，筛选出产奶性能相关的功能区域。
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8个与产奶性能相关的、功能十分确定的候选基因ALDH9A1、ARL4A、ATF6、NSMAF、PPP1R12A、SLC16A10、SLC40A1、SND1，进而通过perl脚本确定上述候选基因对应的功能区域。
4、产奶性能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ALDH9A1、ARL4A、ATF6、NSMAF、PPP1R12A、SLC16A10、SLC40A1、SND1共8个产奶性能相关的功能基因相关联，且仅包含2030个snp位点的产奶性能位点组合。
实施例2分析绵羊产奶性能的引物组合及探针组合
本领域技术人员根据本发明提供的产奶性能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，根据本申请提供的产奶性能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，根据本申请提供的产奶性能SNP位点组合获得引物也属于本发明的保护范围。
同样的，利用本发明提供的产奶性能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分析绵羊产奶性能的基因芯片
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产奶性能的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产奶性能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绵羊产奶性能的分析试剂盒
本申请的提供的产奶性能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产奶性能检测的SNP试剂盒，但是基于本申请提供的产奶性能SNP位点组合配置的绵羊产奶性能SNP检测试剂盒均属于本发明的保护范围。
实施例5绵羊产奶性能的分析
基本本申请的实施例1提供的绵羊产奶性能SNP位点组合对蒙古某牧场养殖的18只母羊羔进行产奶性能分析，具体为：
采用本领域常规方法采集18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 NanoDrop2000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18只母羊羔的全基因组DNA进行检测，获得每只母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，72.2％的母羊羔具有较佳的产奶性能，即样本号M01、M03、M04、M05、M06、M09、M10、M11、M12、M13、M15、M16、M18，其他产奶性能较弱。
采用常规方法对18只羊羔进行养殖，当母羊分娩后，观测18只母绵羊的产奶量、乳脂率、乳蛋白率、泌乳期，观测结果显示，样本号M01、M03、M04、M05、M06、M09、M10、M11、M12、M13、M15、M16、M18绵羊的泌乳期产奶可达200kg以上，最高可达800kg，乳脂率达5.8％以上，最高可达7.5％，乳蛋白率达58％以上，泌乳期在82-300天。而其他样本号的母绵羊产奶量最高仅为164kg，这与基因分析结果一致。
需要说明的是，本申请的所述群体阈值是通过对产奶性能较佳的群体和产奶性能弱的群体进行分析获得，方法同上。
本申请对产奶性能较佳的群体和产奶性能弱的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2030个SNP位点组成的绵羊产奶性能SNP位点组合可以制成的绵羊产奶性能SNP分子标记、检测绵羊产奶性能的SNP探针组合及SNP芯片，在基因组水平上对绵羊个体的产奶性能进行评价，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。对早期难以度量的产奶性状进行选择，缩短世代间隔，加速育种进程，从而节约大量的育种成本，还能够应用于绵羊品种溯源、绵羊种质资源保护和绵羊种质资源改良中。
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. 2030个SNP位点组合在分析绵羊产奶性能中的应用，所述2030个SNP位点组合如表1所示的SNP位点组合，所述表1中的位点组合的物理位置信息基于绵羊v4.0基因组序列比对确定。
  2. 分析绵羊产奶性能的方法，将待测绵羊的基因组DNA的2030个SNP位点基因型与对照绵羊基因组DNA的所述2030个SNP位点基因型进行比较；
    其中，所述2030个SNP位点为权利要求1所述的2030个SNP位点。
  3. 分析绵羊产奶性能的分子探针组合，其检测待测样品中如权利要求1所述的SNP位点组合。
  4. 分析绵羊产奶性能的基因芯片，所述基因芯片负载有权利要求3所述的分子探针组合。
  5. 分析绵羊产奶性能的试剂盒，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. 分析绵羊产奶性能的方法，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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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117089633A (zh) * 2023-10-20 2023-11-21 中国农业大学 分析山羊毛用性能的分子标记组合及应用

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113265476B (zh) * 2021-07-21 2021-11-09 中国农业大学 分析绵羊产奶性能的基因芯片、分子探针组合、试剂盒及应用
CN114391508B (zh) * 2022-01-17 2022-10-21 西北农林科技大学 一种奶绵羊新品种的培育方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040241723A1 (en) * 2002-03-18 2004-12-02 Marquess Foley Leigh Shaw Systems and methods for improving protein and milk production of dairy herds
CN105123621A (zh) * 2015-10-22 2015-12-09 西南大学 山羊资源群体的构建方法
CN106434955A (zh) * 2016-11-04 2017-02-22 中国农业科学院北京畜牧兽医研究所 与中国山羊产奶性状相关的snp分子标记及其应用
CN113265476A (zh) * 2021-07-21 2021-08-17 中国农业大学 分析绵羊产奶性能的基因芯片、分子探针组合、试剂盒及应用

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103468819B (zh) * 2013-09-27 2015-09-09 西北农林科技大学 一种利用双基因聚合效应选育奶山羊产奶性状的方法
CN104087677B (zh) * 2014-07-22 2015-12-02 新疆维吾尔自治区畜牧科学院中国-澳大利亚绵羊育种研究中心 Bmpr-1b或prlr基因snp位点预测小尾寒羊泌乳量的方法
CN105063037B (zh) * 2015-08-25 2022-08-26 新疆畜牧科学院生物技术研究所 湖羊催乳素基因snp位点及其应用
CN112695108B (zh) * 2021-03-23 2021-06-08 中国农业大学 一种肉用绵羊繁殖性能snp位点组合及其应用
CN112695107B (zh) * 2021-03-23 2021-06-08 中国农业大学 一种肉用绵羊生长性能snp位点组合及其应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040241723A1 (en) * 2002-03-18 2004-12-02 Marquess Foley Leigh Shaw Systems and methods for improving protein and milk production of dairy herds
CN105123621A (zh) * 2015-10-22 2015-12-09 西南大学 山羊资源群体的构建方法
CN106434955A (zh) * 2016-11-04 2017-02-22 中国农业科学院北京畜牧兽医研究所 与中国山羊产奶性状相关的snp分子标记及其应用
CN113265476A (zh) * 2021-07-21 2021-08-17 中国农业大学 分析绵羊产奶性能的基因芯片、分子探针组合、试剂盒及应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LI XIAO-KAI, GUI WANG, XIAN QIAO, YI-XING FAN, LEI ZHANG, YU-HAO MA, RUI-XUE NIE, RUI-JUN WANG: "Research Progress on Whole-genome Sequencing on Important Domesticated Animals", BIOTECHNOLOGY BULLETIN, vol. 34, no. 6, 31 December 2018 (2018-12-31), pages 11 - 21, XP093026932, DOI: 10.13560/j.cnki.biotech.bull.1985.2017-0719 *
LI XIN, YANG JI, SHEN MIN, XIE XING-LONG, LIU GUANG-JIAN, XU YA-XI, LV FENG-HUA, YANG HUA, YANG YONG-LIN, LIU CHANG-BIN, ZHOU PING: "Whole-genome resequencing of wild and domestic sheep identifies genes associated with morphological and agronomic traits", NATURE COMMUNICATIONS, vol. 11, no. 1, XP093026940, DOI: 10.1038/s41467-020-16485-1 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117089633A (zh) * 2023-10-20 2023-11-21 中国农业大学 分析山羊毛用性能的分子标记组合及应用
CN117089633B (zh) * 2023-10-20 2023-12-29 中国农业大学 分析山羊绒毛有无的分子标记组合及应用

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