WO2023001235A1 - 猪生长速率相关基因RPS27L的InDel标记及其应用 - Google Patents

猪生长速率相关基因RPS27L的InDel标记及其应用 Download PDF

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WO2023001235A1
WO2023001235A1 PCT/CN2022/107042 CN2022107042W WO2023001235A1 WO 2023001235 A1 WO2023001235 A1 WO 2023001235A1 CN 2022107042 W CN2022107042 W CN 2022107042W WO 2023001235 A1 WO2023001235 A1 WO 2023001235A1
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pig
molecular marker
growth rate
pigs
indel
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唐中林
杨亚岚
范新浩
闫君宇
陈慕雅
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中国农业科学院深圳农业基因组研究所
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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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  • the invention belongs to the technical field of pig breeding, and in particular relates to an InDel marker of a pig growth rate-related gene RPS27L and an application thereof.
  • my country is the largest pig breeding and pork consumption country in the world, and the production and consumption of pork account for more than 50% of the world.
  • my country has abundant pig breed resources, accounting for 1/3 of the world's total, but more than 90% of the commercial pigs raised in my country are Western lean breeds. Therefore, excavating and utilizing high-quality and characteristic genetic resources of local pig breeds, cultivating high-quality pig breeds with independent intellectual property rights in my country, and realizing "localization of fine breed pigs" are not only the needs of the healthy and sustainable development of my country's pig industry, but also the foundation of ensuring a safe supply of pork . Breeding must rely on a solid theoretical basis. Therefore, studying the genetic basis of important economic traits such as pig growth rate is the need for the development of my country's pig industry and has important economic value and strategic significance.
  • InDel refers to the insertion or deletion of a certain number of nucleotides in the genome.
  • InDel provides a large amount of biological information for genetic breeding applications.
  • InDel has the advantages of SNP. And compared with other variants, InDel is easier to be directly identified by PCR amplification and agarose gel electrophoresis.
  • the present invention provides the application of InDel molecular markers or substances for detecting InDel molecular markers in identification or auxiliary identification of pig growth rate.
  • the InDel molecular markers are DNA molecules whose nucleotide sequence is shown in SEQ ID No.1.
  • the molecular marker can be a 13 bp insertion and/or deletion polymorphic site located at the Sscrofa11.1 version sequence Chr1:108,880,114-108,880,126 bp of the porcine reference genome.
  • the insertion and/or deletion sequence at Chr1:108,880,114-108,880,126 bp is SEQ ID NO.1, which is located in the promoter region of the RPS27L gene.
  • the substance may contain PCR primers for amplifying the porcine genomic DNA fragment including the InDel molecular marker.
  • the PCR primers can be composed of a forward primer and a reverse primer, the sequence of the forward primer is as shown in SEQ ID No.2, and the sequence of the reverse primer is as shown in SEQ ID No.3 shown.
  • the above-mentioned application can specifically be a method for identifying or assisting in identifying the growth rate of pigs.
  • the method may include detecting whether the pig's genome contains the InDel molecular marker, and identifying the growth rate of the pig according to whether the pig's genome contains the InDel molecular marker, and the pig's genome containing the InDel molecular marker has a higher growth rate than the pig without the InDel molecular marker. Pigs containing the InDel molecular marker.
  • the PCR primers can be used to identify whether the pig genome contains the InDel molecular marker.
  • the genomic DNA of the pig to be identified can be used as a template, and the PCR primers are used to perform PCR amplification to obtain a PCR product, and the genome of the pig containing the InDel molecular marker in the PCR product contains the InDel molecular marker.
  • the invention provides PCR primers for identifying or assisting in identifying the growth rate of pigs, and the PCR primers can be used for amplifying the pig genome DNA fragments including the above-mentioned InDel molecular markers.
  • the PCR primer can be composed of a forward primer and a reverse primer, the sequence of the forward primer is shown in SEQ ID No.2, and the sequence of the reverse primer is shown in SEQ ID No.3.
  • the present invention also provides a kit for identifying or assisting in identifying pig growth rate traits, which is characterized in that the kit contains the aforementioned PCR primers.
  • the present invention provides a pig breeding method, said method may comprise adopting the pig of RPS27L homozygous genotype to carry out breeding as a parent, and the pig of described RPS27L homozygous genotype is the InDel molecule of the nucleotide sequence shown in the above-mentioned SEQ ID No.1 Marked homozygous genotyped pigs.
  • the purpose of the pig breeding may include breeding pigs with high growth rate.
  • the pig can be a hybrid offspring of a Large White pig and a Min pig, such as the F2 generation.
  • the present invention also provides the application of the above-mentioned PCR primers and/or the above-mentioned kit in identifying or assisting in identifying pig growth rate and/or pig breeding.
  • the growth rate of the above pigs can be reflected in the body weight at 240 days of age.
  • InDel molecular marker is also within the protection scope of the present invention.
  • Figure 1 shows the InDel allele frequency of the insertion/deletion site (InDel) in the promoter region of the pig RPS27L gene in different European and Asian pig breeds in the present invention.
  • Figure 2 is the sequence diagram of PCR amplification products upstream and downstream of the insertion/deletion site (InDel) in the promoter region of the RPS27L gene of Tongcheng pig and Landrace pig; wherein, the part marked by the black box represents the 13-bp insertion sequence.
  • the nucleotide sequence in the upper figure is SEQ ID No.6, and the nucleotide sequence in the lower figure is SEQ ID No.7.
  • Figure 3 shows the correlation analysis between the genotype of the InDel site in the promoter region of the RPS27L gene and the body weight at 240 days in the F2 population of 557 large white pigs crossed with Min pigs.
  • the invention provides a molecular marker related to pig growth rate and application thereof.
  • the molecular marker is that there is a 13 bp insertion/deletion polymorphic site at the Sscrofa11.1 version sequence Chr1:108,880,114-108,880,126 bp of the porcine genome.
  • the nucleotide sequence of the insertion/deletion polymorphic site at the Chr1:108,880,114-108,880,126bp is 5'-GCCACCGGCCTAC-3' (SEQ ID NO.1), which is located in the promoter region of the RPS27L gene.
  • the molecular marker is characterized in that the qq of the 13bp insertion/deletion polymorphic site, that is, the deletion/deletion genotype, can be used as a DNA molecular marker for improving the growth rate of pigs.
  • Validation method for the application of molecular markers related to pig growth rate the steps are as follows:
  • the method uses a primer pair designed with reference to the whole pig genome as primers, uses pig genomic DNA as a template, and detects the insertion/deletion polymorphic site in the promoter region of the pig RPS27L gene through sequence amplification, electrophoresis identification, and Sanger sequencing.
  • the genotype in the population, the sequence amplification primer pair is P1.
  • the primer pair P1 consists of the following upstream primers and downstream primers:
  • Upstream primer P1-F 5'-AGATTTGACAGAGCTGGTTGGT-3' (SEQ ID NO.2);
  • Downstream primer P1-R 5'-CCCATTGTGGCTCAGTGATTA-3' (SEQ ID NO.3).
  • primer pair P1 designed with reference to the whole pig genome as primers, using pig genomic DNA as a template, through sequence amplification, electrophoresis identification, and Sanger sequencing, efficient, accurate, and low-cost detection of insertion/deletion polymorphisms in the promoter region of porcine RPS27L gene Genotypes of sex loci (Chr1:108,880,114-108,880,126bp) in pig populations.
  • Genotype and gene frequency analysis of the insertion/deletion polymorphic site of the pig RPS27L gene promoter, and genetic analysis of the insertion/deletion polymorphic site in the slow-growing fat-type and fast-growing lean-meat pig populations Frequency analysis shows that the 13-bp insertion/deletion polymorphic site detected by the present invention can be used as a molecular marker of pig growth rate, which is beneficial to speed up the breeding speed of fine breed pigs.
  • Example 2 Correlation analysis between pig growth rate-related InDel molecular markers and natural population phenotypes
  • PCR method was used to detect the insertion/deletion polymorphism of the pig RPS27L gene promoter region (Chr1:108,880,114-108,880,126bp) variation in the pig population, and its genotype distribution in different breeds of pigs was analyzed. To verify whether it can be used as a molecular marker for assisted selection in molecular breeding.
  • Taq DNA polymerase purchased from Takara Company
  • proteinase K purchasedd from Huamei Bioengineering Company
  • Marker I purchasedd from Tiangen Biochemical Technology (Beijing) Co., Ltd.
  • agarose purchased from Aikerui Biology
  • TAE Buffer purchasedd from Beijing Ruibo Xingke Biotechnology Co., Ltd.
  • animal genome DNA extraction kit purchasedd from Tiangen Biochemical Technology (Beijing) Co., Ltd.).
  • Primer pair P1 is:
  • Upstream primer P1-F 5'-AGATTTGACAGAGCTGGTTGGT-3' (SEQ ID NO.2);
  • Downstream primer P1-R 5'-CCCATTGTGGCTCAGTGATTA-3' (SEQ ID NO.3);
  • the target fragment of the promoter region of porcine RPS27L gene was amplified by PCR.
  • Ear tissue DNA was extracted according to the instructions of Tiangen Tissue Genomic DNA Extraction Kit.
  • the released whole genome resequencing data a total of 903 individuals, the number of samples of each breed is shown in Table 1.
  • the above resequencing data was compared with the pig reference genome (Sscrofa11.1), and the InDel site information was extracted, which was found in Chr1: There is a 13bp insertion/deletion (InDel) site at 108,880,114-108,880,126bp, and the InDel is located in the promoter region of the RPS27L gene.
  • P Q represents the frequency of allele Q
  • N QQ represents the number of individuals with the QQ genotype in the population
  • N Qq represents the number of individuals with the Qq genotype in the population
  • N represents the total number of individuals
  • the PCR reaction system includes 10 ⁇ L of 2 ⁇ Taq PCR SuperMix (including Taq DNA polymerase, dNTPs and optimized reaction buffer, the concentration is 2 ⁇ ); the upstream primers P1-F 1 ⁇ L in Example 1; the downstream primers in Example 1 P1-R 1 ⁇ L (concentration of upstream and downstream primers is 10 pmol/ ⁇ L); genomic DNA (concentration of 30 ng/ ⁇ L porcine genomic DNA) 1 ⁇ L; deionized water 7 ⁇ L; PCR amplification system with a total volume of 20 ⁇ L.
  • step 2) Extend at 72.0°C for 30s, enter step 2), a total of 34 cycles;
  • the PCR amplification primer pair P1 was used as the sequencing primer for Sanger sequencing.
  • the sequence information obtained by the sequencing is shown in Figure 2.
  • the sequencing genotype of the Tongcheng pig was Q, and the sequencing genotype of the Landrace pig was q, which was consistent with the whole genome resequencing results.
  • the nucleotide sequence in the upper figure of Fig. 2 is 5'-TTTAGCCACCGGCCTACGCCACAGC-3' (SEQ ID No.6), and the nucleotide sequence in the lower figure is 5'-TTTAGCCACAGCAAGGAGGGATCAG-3' (SEQ ID No.7) .
  • Table 2 Correlation analysis between the genotype of the InDel site in the promoter region of the RPS27L gene and the body weight at 240 days in the F2 population of 557 large white pigs crossed with Min pigs
  • the present invention uses modern molecular biology and bioinformatics technology to conduct in-depth research on pig genetic information, and obtains a molecular marker related to pig growth rate, which is the sequence Chr1 of pig genome Sscrofa11.1 version : There is a 13bp insertion/deletion polymorphic site at 108,880,114-108,880,126bp. Pigs whose genotype is deletion at this locus have a higher growth rate than pigs whose genotype is insertion. The growth traits of pigs can be judged and improved by identifying different genotypes of this molecular marker, shortening the breeding time. cycle, accelerate the improvement process, and increase the economic benefits and social value of pig farming.
  • the present invention finds that the insertion/deletion site in the promoter region of the pig RPS27L gene can be used as a genetic selection marker for pig molecular breeding, which is conducive to the selection of pig breeds with excellent growth characteristics and speeds up the breeding of improved breeds;
  • the present invention uses the primer pair P1 designed with reference to the whole pig genome as a primer, uses the pig genome DNA as a template, and detects and identifies the activation of the pig RPS27L gene efficiently, accurately and at low cost through sequence amplification, electrophoresis identification, and Sanger sequencing Genotypes of subregion insertion/deletion polymorphism (Chr1:108,880,114-108,880,126bp) in pig populations.

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Abstract

本发明公开了InDel分子标记或检测InDel分子标记的物质在鉴定或辅助鉴定猪生长速率中的应用，所述InDel分子标记是核苷酸序列如SEQ ID No.1所示的DNA分子。本发明所具有的有益效果包括：(1)发现猪RPS27L基因启动子区插入/缺失位点可以作为猪分子育种的遗传选择标记，有利于筛选生长性状优良的猪种，加快良种选育速度；(2)以参照猪全基因组设计的引物对P1为引物，以猪基因组DNA为模板，通过序列扩增、电泳鉴定、Sanger测序，高效、精确、低成本地检测猪RPS27L基因启动子区插入/缺失多态性位点(Chr1:108,880,114-108,880,126bp)在猪群体中的基因型。

Description

猪生长速率相关基因RPS27L的InDel标记及其应用 技术领域
本发明属于猪育种技术领域，具体涉及猪生长速率相关基因RPS27L的InDel标记及其应用。
背景技术
我国是世界上最大的生猪养殖和猪肉消费国，猪肉生产和消费量均占全球50％以上。此外，我国拥有丰富的猪种资源，占全世界的1/3，但我国饲养的商品猪90％以上均为西方瘦肉型品种。因此，挖掘和利用地方猪种优质特色基因资源，培育我国具有自主知识产权的优良猪品种，实现“良种猪国产化”，既是我国生猪产业健康可持续发展的需要，也是保障猪肉安全供给的根本。开展育种必须依赖坚实的理论基础，因此，研究猪生长速率等重要经济性状形成的遗传基础，是我国生猪产业发展的需要，具有重要的经济价值和战略意义。
在基因组水平上影响动物表型特征的关键因素是基因组序列的变异。目前，常见的基因组变异包括插入/缺失(InDels)、单核苷酸多态性(SNPs)和结构变异(SVs)等。InDel是指基因组中有一定数量的核苷酸插入或缺失。随着比较基因组学研究的深入，InDel为遗传育种应用提供了大量的生物信息，其作为新一代的遗传学鉴定标记，兼具SNP的优点。并且与其它变异相比，InDel更容易通过PCR扩增和琼脂糖凝胶电泳技术直接被鉴定。
综上所述，发掘与猪生长速率相关的分子标记，对猪生长性状的改良和优良品种猪的培育具有重要意义。
发明公开
本发明提供InDel分子标记或检测InDel分子标记的物质在鉴定或辅助鉴定猪生长速率中的应用，所述InDel分子标记是核苷酸序列如SEQ ID No.1所示的DNA分子。
所述分子标记可为位于猪参考基因组Sscrofa11.1版本序列Chr1:108,880,114-108,880,126bp处存在13bp的插入和/或缺失多态性位点。
所述Chr1:108,880,114-108,880,126bp处插入和/或缺失序列为SEQ ID NO.1，位于RPS27L基因的启动子区。
进一步地，上述应用中，所述物质可含有扩增包含所述InDel分子标记在内的猪基因组DNA片段的PCR引物。
进一步地，上述应用中，所述PCR引物可由正向引物和反向引物组成，所述正向引物的序列如SEQ ID No.2所示，所述反向引物的序列如SEQ ID No.3所示。
上述应用具体可为鉴定或辅助鉴定猪生长速率的方法。该方法可包括检测猪的基因组是否含有所述InDel分子标记，根据猪的基因组是否含有所述InDel分子标记鉴定猪生长速率，猪的基因组中含有所述InDel分子标记的猪的生长速 率高于不含有所述InDel分子标记的猪。
上述方法中，可利用所述PCR引物鉴定猪的基因组是否含有所述InDel分子标记。具体可为以待鉴定猪的基因组DNA为模板，利用所述PCR引物进行PCR扩增，得到PCR产物，所述PCR产物含有所述InDel分子标记的猪的基因组含有所述InDel分子标记。
本发明提供用于鉴定或辅助鉴定猪生长速率的PCR引物，所述PCR引物可用于扩增包含上述的InDel分子标记在内的猪基因组DNA片段。
进一步地，所述PCR引物可由正向引物和反向引物组成，所述正向引物的序列如SEQ ID No.2所示，所述反向引物的序列如SEQ ID No.3所示。
本发明同时提供鉴定或辅助鉴定猪生长速率性状的试剂盒，其特征在于，所述试剂盒含有上述PCR引物。
本发明提供猪育种方法，所述方法可包括采用RPS27L纯合基因型的猪作为亲本进行育种，所述RPS27L纯合基因型的猪是上述SEQ ID No.1所示核苷酸序列的InDel分子标记的纯合基因型猪。
进一步地，所述方法中，所述猪育种的目的可包括培育生长速率高的猪。
所述猪具体可为大白猪与民猪的杂交后代，如F2代。
进一步地，本发明还提供上述PCR引物和/或上述的试剂盒在鉴定或辅助鉴定猪生长速率和/或猪育种中的应用。
上述猪生长速率可体现为240日龄体重。
上述的InDel分子标记也是本发明的保护范围。
附图说明
图1为本发明中猪RPS27L基因启动子区插入/缺失位点(InDel)在欧洲和亚洲不同猪品种中InDel等位基因频率。
图2为通城猪和长白猪RPS27L基因启动子区插入/缺失位点(InDel)上下游PCR扩增产物测序图；其中，黑色方框标出的部分表示13-bp插入序列。上图中的核苷酸序列是SEQ ID No.6，下图中的核苷酸序列是SEQ ID No.7。
图3为大白猪与民猪杂交的557头F2代群体中RPS27L基因启动子区InDel位点的基因型与240日龄体重关联分析。
实施发明的最佳方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南，并不以任何方式构成对本发明的限制。
下述实施例中的实验方法，如无特殊说明，均为常规方法，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。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、猪生长速率相关的InDel分子标记的开发
本发明提供了一种与猪生长速率相关的分子标记及其应用。
其中，所述分子标记为猪基因组Sscrofa11.1版本序列Chr1:108,880,114-108,880,126bp处存在13bp的插入/缺失多态性位点。
所述Chr1:108,880,114-108,880,126bp处插入/缺失多态性位点的核苷酸序列为5’-GCCACCGGCCTAC-3’(SEQ ID NO.1)，位于RPS27L基因的启动子区。
根据本发明一种优选的实施方式，所述分子标记的特征在于，13bp插入/缺失多态性位点的qq即缺失/缺失基因型能够作为提高猪生长速率的DNA分子标记。
与猪生长速率相关的分子标记应用的验证方法，步骤如下：
所述方法以参照猪全基因组设计的引物对为引物，以猪基因组DNA为模板，通过序列扩增、电泳鉴定、Sanger测序，检测猪RPS27L基因启动子区插入/缺失多态性位点在猪群体中的基因型，所述序列扩增引物对为P1。
所述引物对P1由下述上游引物和下游引物组成：
上游引物P1-F：5’-AGATTTGACAGAGCTGGTTGGT-3’(SEQ ID NO.2)；
下游引物P1-R：5’-CCCATTGTGGCTCAGTGATTA-3’(SEQ ID NO.3)。
以参照猪全基因组设计的引物对P1为引物，以猪基因组DNA为模板，通过序列扩增、电泳鉴定、Sanger测序，高效、精确、低成本地检测猪RPS27L基因启动子区插入/缺失多态性位点(Chr1:108,880,114-108,880,126bp)在猪群体中的基因型。
对猪RPS27L基因启动子插入/缺失多态性位点进行基因型和基因频率分析，对插入/缺失多态性位点在生长速率慢的脂肪型和生长速率快的瘦肉型猪种群进行基因频率分析，结果表明本发明检测的13-bp插入/缺失多态性位点能够作为猪生长速率的分子标记，有利于加快良种猪的选育速度。
实施例2、猪生长速率相关的InDel分子标记与自然群体表型的关联分析
利用PCR方法对猪RPS27L基因启动子区(Chr1:108,880,114-108,880,126bp)变异在猪群体中产生的插入/缺失多态性进行检测，并对其在不同品种猪中的基因型分布情况进行分析，验证其是否可以作为分子育种中辅助选择的分子标记。
2.1、实验药品与试剂
Taq DNA聚合酶(购自Takara公司)；蛋白酶K(购自华美生物工程公司)；Marker I(购自天根生化科技(北京)有限公司)；琼脂糖(购自艾科瑞生物)；TAE缓冲液(购自北京睿博兴科生物技术有限公司)；动物基因组DNA提取试剂盒(购自天根生化科技(北京)有限公司)。
引物对P1为：
上游引物P1-F：5’-AGATTTGACAGAGCTGGTTGGT-3’(SEQ ID NO.2)；
下游引物P1-R：5’-CCCATTGTGGCTCAGTGATTA-3’(SEQ ID NO.3)；
PCR扩增猪RPS27L基因的启动子区目的片段。
猪耳样品的采集与品种信息收集：
实验所用的动物共计903个样本。采取个体耳组织样，在70％乙醇中保存，冰盒低温带回实验室后置于-80℃冻存，同时记录猪的品种信息。
组织样品基因组DNA的提取与分离：
参照天根组织基因组DNA提取试剂盒说明书提取耳组织DNA。
2.2、InDel位点的筛选
选择巴克夏猪、欧洲野猪、尤卡坦猪、杜洛克猪、大白猪、长白猪、皮特兰猪等欧洲瘦肉型猪种，以及德宝猪、梅山猪、淮猪、保山猪、藏猪、闽北花猪、五指山猪、环江香猪、屯昌猪、二花脸猪、通城猪、滇南小耳猪、陆川猪、皖南猪、巴马猪、贵州猪、高黎贡山猪、金华猪、大花白猪、东山猪、官庄花猪、广东小耳猪、蓝塘猪、莱芜猪、明光小耳猪等亚洲脂肪型猪种，整合本课题组自己测序所得全基因重测序数据以及公共数据库已发布的全基因组重测序数据，共903个个体，各品种样本数见表1，对以上重测序数据与猪参考基因组(Sscrofa11.1)进行比对，提取出InDel位点信息，发现在Chr1:108,880,114-108,880,126bp处存在一个13bp的插入/缺失(InDel)位点，该InDel位于RPS27L基因的启动子区。
2.3、等位基因频率计算：
P Q＝(2N QQ+N Qq)/2N
式中，P Q表示等位基因 Q频率，N QQ表示群体中具有QQ基因型的个体数量，N Qq表示群体中具有Qq基因型个体数量，N表示总个体数量；
Q代表插入型等位基因(13-bp insertion)，PCR产物如SEQ ID No.4所示；q代表缺失型等位基因(13-bp deletion)，PCR产物如SEQ ID No.5所示。
SEQ ID No.4：
5’-CCCAAAGGGGGGTACTTTCATTATTGTATTCGTTTTCTATTTTCACTATAACAAATTGCCATAAAGTTAGTGGCTTTTTTATTTTATTGTCTTTTTGCCATTTCTTGGGCCGCTCCCACGGCACATGGAGGTTCCCAGGTTAGGGGTCTAATCGGAGCTTTAGCCACCGGCCTACGCCACAGCAAGGAGGGATCAGAGCCGCATCTGCAACCTACACTGCAGCTCACCACAACGTCAGATCCTTAACCCACTGAGCAAGGCGAGGGATCAAACCTGCAACCTCATGGTTCCTAGTCAGATTCGTTAATCACTGAGCCACAATGGGCTCGAGTCTAGAGTCGACCTGCAGGCATGCAAGCTGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCGGCCGCTTCGAGCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGGAGATGTGGGA-3’；
SEQ ID No.5：
5’-GGAAGGGGGGATCTTTCATTATTGTATTCGTTTTCTATTTTCACTATAAC AAATTGCCATAAAGTTAGTGGCTTTTTTATTTTATTGTCTTTTTGCCATTTCTTGGGCCGCTCCCACGGCACATGGAGGTTCCCAGGTTAGGAGTCTAATCGGAGCTTTAGCCACAGCAAGGAGGGATCAGAGCCGCATCTGCAACCTACACTGCAGCTCACCACAACGCCAGATCCTTAACCCACTGAGCAAGGCCAGGGATCAAACCTGCAACCTCATGGTTCCTAGTCAGATTCGTTAATCACTGAGCCACAATGGGCTCGAGTCTAGAGTCGACCTGCAGGCATGCAAGCTGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCGGCCGCTTCGAGCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGT-3’。
等位基因频率计算结果如表1所示：
表1.猪RPS27L基因启动子区InDel位点的多态性参数
Figure PCTCN2022107042-appb-000001
Figure PCTCN2022107042-appb-000002
由表1可以看出，插入型等位基因Q主要分布于亚洲脂肪型猪，缺失型等位基因q主要分布于欧洲瘦肉型猪。
2.4、InDel序列信息验证
2.4.1、PCR扩增
选取脂肪型猪通城猪(Tongcheng)和瘦肉型长白猪(Landrace)为代表，利用PCR技术扩增InDel及其侧翼序列。
PCR反应体系包括2×Taq PCR SuperMix(包括Taq DNA聚合酶、dNTPs和优化的反应缓冲液，浓度为2×)10μL；实施例1中的上游引物P1-F 1μL；实施例1中的下游引物P1-R 1μL(上下游引物浓度为10pmol/μL)；基因组DNA(浓度为30ng/μL猪基因组DNA)1μL；去离子水7μL；共20μL体积的PCR扩增体系。
PCR反应的程序：
1)95.0℃预变性5min，然后进入步骤2)；
2)95.0℃变性30s，
3)60.0℃复性30s，
4)72.0℃延伸30s，进入步骤2)，共34个循环；
5)72.0℃延伸10min
2.4.2、琼脂糖凝胶电泳检测
步骤如下：
1)制作2.0％的琼脂糖凝胶，使用核酸染料染色，点样4.5μL，点样后120V电压电泳40-50min；
2)待分子量不同的DNA片段分离清晰时，在BIO-RAD Gel Doc 2000凝胶成像系统成像；
3)根据琼脂糖凝胶电泳结果初步分析InDel多态性。
2.4.3、Sanger测序
以PCR扩增引物对P1为测序引物进行Sanger测序，测序所得序列信息如图2所示，通城猪测序基因型为Q，长白猪测序基因型为q，与全基因组重测序结果一致。图2的上图中的核苷酸序列是5’-TTTAGCCACCGGCCTACGCCACAGC-3’(SEQ ID No.6)，下图中的核苷酸序列是5’-TTTAGCCACAGCAAGGAGGGATCAG-3’(SEQ ID No.7)。
实施例3、InDel分子标记与生长速率的关联分析
以大白猪(欧洲猪)作为父本，民猪(亚洲猪)作为母本杂交获得的557头F2代群体中RPS27L基因启动子区InDel位点的基因型与生长速率(240日龄体重)基因型与生长速率(240日龄体重)关联分析见表2和图3。
Q代表插入型等位基因(13-bp insertion)，PCR产物如SEQ ID No.4所示；q代表缺失型等位基因(13-bp deletion)，PCR产物如SEQ ID No.5所示。
表2：大白猪与民猪杂交的557头F2代群体中RPS27L基因启动子区InDel位点的基因型与240日龄体重关联分析
基因型 样本数 240日龄体重(kg)
qq 411 109.63±15.78
Qq 110 108.48±16.11
QQ 36 103.69±16.15
表2和图3结果表明qq纯合基因型的待鉴定猪的成长速率明显高于QQ纯合基因型的待鉴定猪。
以上对本发明进行了详述。对于本领域技术人员来说，在不脱离本发明的宗旨和范围，以及无需进行不必要的实验情况下，可在等同参数、浓度和条件下，在较宽范围内实施本发明。虽然本发明给出了特殊的实施例，应该理解为，可以对本发明作进一步的改进。总之，按本发明的原理，本申请欲包括任何变更、用途或对本发明的改进，包括脱离了本申请中已公开范围，而用本领域已知的常规技术进行的改变。按以下附带的权利要求的范围，可以进行一些基本特征的应用。
工业应用
为了提高育种效率，本发明利用现代分子生物学和生物信息学技术对猪遗传信息进行了深入研究，得到一种与猪生长速率相关的分子标记，该分子标记为猪基因组Sscrofa11.1版本序列Chr1:108,880,114-108,880,126bp处存在13bp的插入/缺失多态性位点。该位点的基因型为缺失型的猪，相对于基因型为插入的猪具有更高的生长速率，可以通过鉴定该分子标记的不同基因型来对猪的生长性状进行判定和改良，缩短育种周期，加速改良进程，提高生猪养殖的经济效益与社会价值。
本发明所具有的有益效果包括：
(1)本发明发现猪RPS27L基因启动子区插入/缺失位点可以作为猪分子育种的遗传选择标记，有利于筛选生长性状优良的猪种，加快良种选育速度；
(2)本发明以参照猪全基因组设计的引物对P1为引物，以猪基因组DNA为模板，通过序列扩增、电泳鉴定、Sanger测序，高效、精确、低成本地检测、鉴定猪RPS27L基因启动子区插入/缺失多态性位点(Chr1:108,880,114-108,880,126bp)在猪群体中的基因型。

Claims (9)

  1. InDel分子标记或检测所述InDel分子标记的物质在鉴定或辅助鉴定猪生长速率中的应用，其特征在于，所述InDel分子标记是核苷酸序列如SEQ ID No.1所示的DNA分子。
  2. 如权利要求1所述的应用，其特征在于，所述物质含有扩增包含所述InDel分子标记在内的猪基因组DNA片段的PCR引物。
  3. 如权利要求2所述的应用，其特征在于，所述PCR引物由正向引物和反向引物组成，所述正向引物的序列如SEQ ID No.2所示，所述反向引物的序列如SEQ ID No.3所示。
  4. 用于鉴定或辅助鉴定猪生长速率的PCR引物，其特征在于，所述PCR引物用于扩增包含权利要求1所述的InDel分子标记在内的猪基因组DNA片段。
  5. 根据权利要求4所述的PCR引物，其特征在于，所述PCR引物由正向引物和反向引物组成，所述正向引物的序列如SEQ ID No.2所示，所述反向引物的序列如SEQ ID No.3所示。
  6. 鉴定或辅助鉴定猪生长速率性状的试剂盒，其特征在于，所述试剂盒含有权利要求4或5所述的PCR引物。
  7. 猪育种方法，其特征在于，所述方法包括采用RPS27L纯合基因型的猪作为亲本进行育种，所述RPS27L纯合基因型的猪是缺失权利要求1中所述InDel分子标记的纯合基因型猪。
  8. 如权利要求7所述的方法，其特征在于，所述猪育种的目的包括培育生长速率高的猪。
  9. 权利要求1所述的InDel分子标记。
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