一种肉用绵羊繁殖性能SNP位点组合及其应用

一种肉用绵羊繁殖性能snp位点组合及其应用
技术领域
1.本发明涉及生物技术领域，尤其涉及一种肉用绵羊繁殖性能snp位点组合及其应用。


背景技术：

2.绵羊是最早驯化的家养动物，也是我国最常见的一种养殖动物，随着人类的活动及选择，具有不同优质资源的绵羊品种逐渐形成，然而在实际生产中，国内外绵羊品种种类繁多，个体差异大，要选择具有特定目标性状的品种进行饲养生产，从而获得更高的经济价值或市场效益成为绵羊养殖的重大难题之一。
3.绵羊选择育种的历史中，经历了从经验育种到育种理论和方法的探索，有选择学说，纯系学说，回交育种、轮回育种、诱变育种、单粒传、理想株型；再到标记辅助选择育种，探索了各种各样的标记，比如扩增片段长度多态性标记辅助选择(aflp)、微卫星标记辅助选择(ssr)和单核苷酸多态性标记辅助选择(snp)。
4.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。
5.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 illuminaovine 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繁殖功能进行快速有效检测snp芯片，有着十分重要的意义。


技术实现要素：

6.为满足我国当前育种生产上对繁殖性能方向的芯片位点功能检测、功能研究方面的需求，本发明提供了一种基于世界范围内的16个野羊（亚洲摩弗伦）和232个家羊个体的高深度全基因组重测序数据，以绵羊 v4.0基因组为参考，结合已有的与绵羊繁殖功能相关
的研究，获得了一种检测准确，使用方便，市场前景广阔的一种肉用绵羊繁殖性能snp位点，所述位点能够用于绵羊品种的筛选、绵羊品种的鉴定、绵羊的育种、种质资源的保护以及种质资源的改良。
7.为实现本发明的技术目的，本发明第一方面提供一种肉用绵羊繁殖性能snp位点组合，所述snp涵盖在ube2q1、dgcr8、bmpr1b、pappa、igf1的基因片段中。
8.其中，所述snp位点组合包括2917个snp位点，其物理位置信息基于绵羊 v4.0基因组序列比对确定，具体如表1所示。
9.表1 本申请提供的snp位点组合的物理位置信息
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其中，所述繁殖性能包括产羔率指标或/和多胎性指标或/和胎产羔数指标。
10.为实现本发明的技术目的，本发明第二方面提供一种肉用绵羊繁殖性能检测探针组合，所述探针组合检测第一方面所述的肉用绵羊繁殖性能snp位点组合。
11.为实现本发明的技术目的，本发明第三方面提供一种肉用绵羊繁殖性能基因芯片，所述基因芯片负载有第二方面所述的探针组合。
12.为实现本发明的技术目的，本发明第四方面提供一种用绵羊繁殖性能检测试剂
盒，包括第二方面所述的检测探针组合或第三方面所述的基因芯片。
13.为实现本发明的技术目的，本发明第五方面提供第一方面所述的snp位点组合或第二方面所述的检测探针组合或第三方面所述的基因芯片或第四方面所述的试剂盒具有如下任一所述的用途：（1）在绵羊品种筛选的应用；（2）在绵羊品种鉴定中的应用；（3）在绵羊育种中的应用；（4）在种质资源保护中的应用；（5）在种质资源改良中的应用。
14.有益效果:1、 本发明基于对多个国外肉羊绵羊品种和中国地方肉用绵羊品种的遗传资源研究，提供了一种国内外通用性好，与繁殖性能相关的肉用绵羊的仅由2917个snp位点组成的snp位点组合，利用本发明提供的位点组合能够在基因组水平上对个体的繁殖能力进行遗传评估，以获得更准确的育种评估值，对早期难以度量的繁殖性状进行选择，缩短世代间隔，加速育种进程，从而节约大量的育种成本。
15.2、基于本发明提供的繁殖性能snp位点组合形成的探针、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
16.图1
‑
5是实施例1对不同组别的繁殖性状的曼哈顿图；图6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
17.以下实施例进一步说明本发明的内容，但不应理解为对本发明的限制。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改或替换，均属于本发明的范围。下述实施例中所使用的实验方法如无特殊说明，均为常规方法。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。实施例1 肉用绵羊繁殖性能snp位点组合1.总snp集合的获取对覆盖亚洲、欧洲、非洲和中东的16个野羊亚洲摩弗伦、172个地方品种和60个培育品种共248个肉用绵羊个体进行高深度测序，平均测序深度为~25.7
×
，应用重测序分析流程，通过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）进行比对，两种方式比对获得的共同结果形成一个snp集合，共包含2836万个snp位点。具体的，对多个肉用绵羊个体进行高深度测序由生物测序公司完成，生物测序公司完成的测序结果均能够实现本发明的技术目的，本发明不做限制。本申请将测序公司返回的 fastq 文件通过 bwa 软件比对到参考基因组绵羊v4.0后得到 bam 文件，利用 samtools和gatk 软件对样本 bam 文件分析得到包含群体 snp 分型信息的 vcf 文件，将两种方式获得的vcf结果合并，经过质量筛选后获得了包含2836万个snp位点的 snp 集合。
18.具体的，本发明所使用的肉用绵羊品种包括来自中国山东的小尾寒羊、泗水裘皮
羊、大尾寒羊、洼地绵羊，来自中国江苏的湖羊，来自中国宁夏的滩羊，来自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，来自芬兰的芬兰羊（finnsheep）、来自荷兰的威桑岛羊（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），来自埃塞俄比亚的邦加羊（bonga sheep）、阿法尔羊（afar sheep），来自尼日尔的姆博罗罗羊（mbororo sheep），来自尼日利亚的扬卡羊（yankasa sheep），来自非洲乍得的西非矮羊（west african dwarf sheep）、乌达羊（uda sheep），来自非洲布基纳法索的贾隆凯羊（djallonk
éꢀ
sheep）、摩西羊（mossi sheep）、萨赫勒羊（sahelian sheep），来自西非喀麦隆的喀麦隆羊（cameroon sheep），来自伊拉克的阿华西绵羊（awassi sheep）、哈姆达尼羊（hamdani sheep），来自阿塞拜疆的马泽克羊（mazekh sheep），来自伊朗的灰设拉子羊（grey
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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），来自巴基斯坦的卡拉库尔羊（karakul sheep），来自伊朗舍赫尔科德的亚洲摩弗伦（asiatic mouflon）。
19.2.候选基因及所在功能区域的筛选根据国内绵羊品种和国外绵羊品种在繁殖性状上的表现出来的显著差异进行分组，本发明使用xp
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clr扫荡每组绵羊之间的多基因座等位基因频率差异扫描出繁殖相关的功能区域（如图1所示），同时利用π ratio（即π值）挖掘每组中的绵羊品种中与繁殖相关的功能区域，然后取两种结果的交集，筛选出繁殖相关的功能区域。然后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个与繁殖相关的、功能十分确定的候选基因ube2q1、dgcr8、bmpr1b、pappa、igf1，进而通过perl脚本确定这些候选基因对应的功能区域。
20.具体的，在本发明的一个实施例中，根据国内绵羊品种和国外绵羊品种在繁殖性状上的表现出来的显著差异进行分组，按照以下步骤进行：由于芬兰羊和滩羊繁殖性状上的表现出来的显著差异，湖羊和滩羊繁殖性状上的表现出来的显著差异，小尾寒羊和滩羊繁殖性状上的表现出来的显著差异，哥特兰岛羊和滩羊繁殖性状上的表现出来的显著差异，洼地绵羊和滩羊繁殖性状上的表现出来的显著差异，因此对上述组别进行xp
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clr扫荡形成图1所示的芬兰羊vs 滩羊(fin vs tan)的曼哈顿图，图2所示的湖羊vs 滩羊(hus vs tan) 的曼哈顿图，图3所示的小尾寒羊vs 滩羊(sxw vs tan) 的曼哈顿图，图4所示的哥特兰岛羊vs 滩羊(got vs tan) 的曼哈顿图，图5所示的洼地绵羊vs 滩羊(wds vs tan)的曼哈顿图。
21.3.功能基因对应snp位点的提取利用bedtools在总的snp集合中寻找步骤2中确定的候选基因所在功能区域对应的snp位点，得到与ube2q1、dgcr8、bmpr1b、pappa、igf1共5个繁殖相关的功能基因相关联，且仅包含2917个snp位点的位点组合。
22.实施例2 肉用绵羊繁殖性能的snp基因芯片的制备基于实施例1获得的snp位点组合，本领域技术人员可以采用任一一种方式制备肉用绵羊繁殖性能的snp基因芯片，同样也可以委托生物公司制备，本发明不作限制，例如illumina公司、affymetrix 公司或者agilent公司，需要说明的是，基于本发明实施例1提
供的snp位点制备的基因芯片均属于本发明的保护范围。
23.在本发明的一个实施例中，可以采用illumina公司的方法制备本申请的肉用绵羊繁殖性能的snp芯片，该芯片包括一个玻璃基片以及位于基片小孔中的2917个磁珠，所述每个磁珠上偶联有分别检测实施例1中snp0001
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snp2917所示的位点的探针，用于检测目标样本，其中，磁珠上偶联在珠子的这一端的23个碱基的序列为address序列，也是dna片段的5'端，它是标识微珠的标签序列。离珠子较远端的50个碱基为 3'端的序列，成为probe序列，其与目标dna进行互补杂交，能够识别实施例1中snp0001
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snp2917所示的位点。
24.实施例3 snp检测试剂盒基于实施例1获得的snp位点组合，本领域技术人员可以采用任一一种方式制备肉用绵羊繁殖性能的snp检测试剂盒，同样也可以委托生物公司制备，本发明不作限制，例如illumina公司、affymetrix 公司或者agilent公司，需要说明的是，基于本发明实施例1提供的snp位点制备的检测试剂盒均属于本发明的保护范围。
25.在本发明的一个实施例中，为了能够使用实施例2提供的基因芯片进行对目标样本进行检测，试剂盒中还包括四种带标记的双脱氧核苷酸、聚合酶和生物素标记的抗链霉亲合素的抗体、和dnp标记的，抗异种抗体fc端的抗体。
26.当然不同的基因芯片，应当适配相应的试剂，本发明不作限制。
27.实施例4 肉用绵羊繁殖性能的检测基于实施例1获得的snp位点组合或根据实施例2提供的snp基因芯片或实施例3提供的snp检测试剂盒对肉羊绵羊样品进行检测，在本发明的一个实施例中，当采用illumina公司提供的方法制备的基因芯片进行检测时，加入四种带标记的双脱氧核苷酸和聚合酶。其中a、t两种核苷酸是用dnp（二硝基苯）来进行标记，c、g两种碱基是用生物素来标记，聚合酶会在探针的3’末端加上一个双脱氧核苷酸，并同时捎带连上一个标记物，接着加入绿色荧光标记的链霉亲合素，红色荧光标记的抗dnp的抗体。绿色荧光标记的链霉亲合素与生物素特异地结合，让带生物素的c、g碱基显出绿色。红色荧光标记的抗dnp的抗体与dnp结合，让带dnp的a、t碱基显出红色，并且进一步加入生物素标记的抗链霉亲合素的抗体、和dnp标记的，抗异种抗体fc端的抗体。加入这两种抗体的作用，是使荧光信号得到进一步的级联放大，抗体结合完了之后，经过清洗，把游离的抗体都给洗掉。在扫描仪下进行扫描。如果发出的光是绿光，就说明这个snp结合的位点，是个“g”碱基的纯合子。如果发出的是红光，就说明这个snp位点是个“a”碱基的纯合子。如果既有红光、又有绿光，而且两种颜色的光的光强差不多，就说明这个snp位点是一个“a”和“g”的杂合子，如果没有光，则判定为碱基缺失，最后统计目标样本中所有位点基因型频率的结果值，即统计2917个位点中参考型纯合子、杂合子、突变型纯合子、碱基缺失的频率作为频率值，将每个位点基因型频率值进行累加后，获得目标样本的结果值。
28.当统计的结果值与群体阈值进行比对后，得到该目标样本的判断结果。
29.具体的，本申请的所述群体阈值是通过对高繁群体（fin
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hus
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sxw
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got
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wds）和低繁群体（tan）进行分析获得，方法同上。
30.本申请对高繁群体（fin
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hus
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sxw
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got
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wds）和低繁群体（tan）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6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。
31.应用实施例选取具有较强代表性、推广面积较大的8个未知品种（编号为pz01
‑
pz08）的母绵羊，根据实施例1提供的位点信息制备的实施例2的snp基因芯片，并利用实施例3提供的试剂盒检测8个绵羊品种的繁殖性能，每个待测绵羊品种的每个位点中参考型纯合子、杂合子、突变型纯合子、碱基缺失的结果值与群体阈值进行比较，比较结果显示：编号pz04和pz06、pz08的母绵羊为低繁品种，编号pz01、pz02、pz03、pz05和pz07均为高繁品种。
32.申请人对在同一养殖条件下对上述8个未知品种的母绵羊进行养殖，观察统计近2年的上述8只未知品种的产羔率和胎产羔数，统计结果显示，编号pz04和pz06、pz08只能实现一年一胎，一胎仅有一个羔羊，繁殖能力均弱于其他编号的母绵羊。
33.工业应用本申请提供的基于2917个snp位点制成的snp标记、snp探针及snp芯片在基因组水平上对绵羊个体进行繁殖遗传评估，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。
34.以上详细描述了本发明的优选实施方式，但是，本发明并不限于上述实施方式中的具体细节，在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，这些简单变型均属于本发明的保护范围。
35.另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本发明对各种可能的组合方式不再另行说明。
36.此外，本发明的各种不同的实施方式之间也可以进行任意组合，只要其不违背本发明的思想，其同样应当视为本发明所公开的内容。