WO2022217910A1 - 与鸡体尺性状相关的igf2bp1基因分子标记及其应用和育种方法 - Google Patents

与鸡体尺性状相关的igf2bp1基因分子标记及其应用和育种方法 Download PDF

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WO2022217910A1
WO2022217910A1 PCT/CN2021/130299 CN2021130299W WO2022217910A1 WO 2022217910 A1 WO2022217910 A1 WO 2022217910A1 CN 2021130299 W CN2021130299 W CN 2021130299W WO 2022217910 A1 WO2022217910 A1 WO 2022217910A1
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chicken
genotype
body size
nucleotide sequence
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French (fr)
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康相涛
李文婷
王克君
田亚东
孙桂荣
韩瑞丽
蒋瑞瑞
李东华
李国喜
李转见
刘小军
李红
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河南农业大学
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Priority to US18/479,837 priority Critical patent/US20240018603A1/en

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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

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  • the invention belongs to the technical field of biological breeding, and in particular relates to an IGF2BP1 gene molecular marker related to chicken body size traits, its application and a breeding method.
  • IGF2BP1 gene the full name of insulin-like growth factor 2 binding protein, has the functions of regulating cell proliferation, differentiation, morphogenesis and metabolism. Its mechanism of action is usually to regulate the localization, stability and translation of target gene mRNA.
  • IGF2BP1 has been confirmed as a recognition protein of m6A methylation, and it is likely to widely act on target genes through the regulation mode of m6A methylation.
  • IGF2BP1 knockout mice exhibited a reduced body weight phenotype.
  • the upstream of IGF2BP1 gene was significantly correlated with body weight, head weight, breast width, leg weight and other carcass and body size traits.
  • the causative mutation of the IGF2BP1 gene affecting chicken body size traits has not been elucidated.
  • the purpose of the present invention is to provide a molecular marker of IGF2BP1 gene related to chicken body size traits.
  • the second object of the present invention is to provide the application of the above molecular markers in the breeding of chicken body size traits improvement.
  • the third object of the present invention is to provide primers and kits for detecting the genotypes of the above molecular markers.
  • the fourth object of the present invention is to provide a breeding method for improving chicken body size traits.
  • a kind of IGF2BP1 gene molecular marker related to chicken body size traits its nucleotide sequence is as shown in SEQ ID NO: 1, the 1210-4443th position from the 5' end or the 2993-4547th position from the 5' end is inserted or deleted ;
  • the nucleotide sequence of the 1210-4443th position from the 5' end of the SEQ ID NO:1 is shown in SEQ ID NO:2, and the 5' end of the SEQ ID NO:1 is from the 2993rd-4547th position.
  • the nucleotide sequence is shown in SEQ ID NO:3.
  • the nucleotide sequence shown in SEQ ID NO: 1 is detected at the 1210-4443rd position from the 5' end or the 2993-4547th position from the 5' end of the nucleotide sequence shown in SEQ ID NO: 1, and the nucleotide shown in SEQ ID NO: 1 is selected. Homozygous chicken individuals with deletions at positions 1210-4443 from the 5' end of the sequence are reserved.
  • the primer for detecting the molecular marker genotype of claim 1, its nucleotide sequence is shown in SEQ ID NO: 4-6.
  • kits for detecting the molecular marker genotype of claim 1 comprises the primers shown in SEQ ID NOs: 4-6.
  • the kit further includes one or more of dNTPs, PCR reaction buffer, and DNA polymerase.
  • a method for improving breeding of chicken body size traits comprising the following steps:
  • the DNA is extracted by phenolic extraction and diluted to 60ng/ ⁇ L.
  • the nucleotide sequences of the designed primers are shown in SEQ ID NO: 4-6; the band size of the PCR amplification product is detected by agarose gel electrophoresis, and it is judged that the nucleotide sequence shown in SEQ ID NO: 1 starts from the 5' end 1210-4443 or 2993-4547 insertion or deletion and genotype identification; it is divided into six genotypes: WW genotype shows a band of 2344bp; L2W genotype shows two bands of 2344bp and 790bp; L1W genotype shows Two bands of 2344bp and 290bp; one band of 790bp for L2L2 genotype; two bands of 290bp and 790bp for L1L2 genotype; one band of 290bp for L1L1 genotype.
  • step 1) the primers shown in SEQ ID NO: 4 and 6 and the primers shown in SEQ ID NO: 5 and 6 are respectively used for PCR amplification reaction, and the reaction system is as follows:
  • the PCR reaction procedure in step 1) pre-denaturation at 95°C for 5 min; denaturation at 95°C for 15s, annealing at 60°C for 15s, extension at 72°C for 5s, 30 cycles; extension at 72°C for 5 min.
  • the present invention provides an IGF2BP1 gene molecular marker related to chicken body size traits.
  • the inventors found for the first time that there are two deletion genotypes upstream of the IGF2BP1 gene, and it was confirmed by PCR and sequencing that the deletion genotypes were completely consistent with the results of high-throughput sequencing analysis.
  • the present invention analyzes the correlation between the deletion mutation in the promoter region of the chicken IGF2BP1 gene and the body size traits of 23 kinds of chickens, and it is proved that the allele L1 in the variation site is related to the high body size traits (paw weight, shin length, sternum length, 23 traits such as double wing weight) were significantly positively correlated; allele W was significantly positively correlated with chicken low body size traits.
  • the IGF2BP1 gene molecular marker provided by the invention can be applied to the improvement breeding of chicken body size traits.
  • the present invention further provides a method for improving chicken body size traits based on the molecular marker of IGF2BP1 gene.
  • the chicken DNA to be tested is extracted, the gene fragment corresponding to the molecular marker is amplified by PCR, and the band of the PCR amplified product is detected by agarose gel electrophoresis.
  • Chicken germplasm resources with high body size traits can improve the body size traits of chickens.
  • the method can predict the body size traits of chickens after growth in an early, rapid, low-cost and effective manner, shorten the breeding time and speed up the breeding process, and has extremely high economic value and broad application prospects.
  • Fig. 1 is the distribution frequency of 1-2kb deletion and 2-3kb deletion in chicken IGF2BP1 promoter region
  • Fig. 2 is the correlation analysis of 1-2kb and 2-3kb structural variation in chicken IGF2BP1 promoter region
  • Figure 3 is an allele structure diagram of the IGF2BP1 promoter
  • Figure 4 is the genome comparison result of three alleles in the promoter region of IGF2BP1;
  • Fig. 5 is the detection result of allele-specific PCR in the promoter region of IGF2BP1;
  • lane M is the DM2000 marker, and the sizes of the maker bands from bottom to top are: 100bp, 250bp, 500bp, 750bp, 1000bp, 2000bp, 3000bp, and 5000bp.
  • Fig. 6 is IGF2BP1 primer PCR amplification conditions
  • Figure 7 shows the frequency of allele distribution of chromosome structure revealed by PCR sequencing
  • Figure 8 is an analysis of the association between structural variation in the promoter region of IGF2BP1 and body size traits.
  • Example 1 IGF2BP1 gene molecular marker and application related to chicken body size traits
  • This example provides IGF2BP1 gene molecular markers related to chicken body size traits, the nucleotide sequence of which is shown in SEQ ID NO: 1, 1210-4443 from the 5' end or 2993-4547 from the 5' end Insertion or deletion; the nucleotide sequence of positions 1210-4443 from the 5' end of the SEQ ID NO: 1 is shown in SEQ ID NO: 2, and the 5' end of the SEQ ID NO: 1 starts from the 2993rd- The nucleotide sequence at position 4547 is shown in SEQ ID NO:3.
  • the molecular marker can be applied to chicken body size trait improvement breeding: detection of insertion or deletion at positions 1210-4443 from the 5' end or 2993-4547 positions from the 5' end of the nucleotide sequence shown in SEQ ID NO: 1, Homozygous chicken individuals with deletions at positions 1210-4443 (SEQ ID NO: 2) from the 5' end of the nucleotide sequence shown in SEQ ID NO: 1 were selected for breeding. Thereby improving the body size of the flock.
  • This example provides primers for detecting the molecular marker genotype of the IGF2BP1 gene associated with chicken body size traits, the nucleotide sequences of which are shown in SEQ ID NOs: 4-6.
  • This example provides a kit for detecting the molecular marker genotype of IGF2BP1 gene related to chicken body size traits.
  • the kit includes the detection primers shown in SEQ ID NOs: 4-6 provided in Example 2, and also includes dNTPs , one or more of PCR reaction buffer and DNA polymerase.
  • the present embodiment provides a method for improving chicken body size traits, comprising the following steps:
  • PCR reaction system 12 ⁇ Rapid Taq PCR Master Mix 10 ⁇ L, ASP-F 0.5 ⁇ L, ASP-R 0.5 ⁇ L, DNA template 1 ⁇ L, ddH 2 O 8 ⁇ L; 22 ⁇ Rapid Taq PCR Master Mix 10 ⁇ L, 2K-F 0.5 ⁇ L, ASP -R 0.5 ⁇ L, DNA template 1 ⁇ L, ddH 2 O 8 ⁇ L.
  • PCR reaction program pre-denaturation at 95 °C for 5 min; denaturation at 95 °C for 15 s, annealing at 60 °C for 15 s, extension at 72 °C for 5 s, 30 cycles; extension at 72 °C for 5 min.
  • the W allele band size is 2344bp
  • the L1 allele band size is 290bp
  • the L2 allele band size is 790bp. It is divided into six genotypes: WW genotype shows one band of 2344bp; L2W genotype shows two bands of 2344bp and 790bp; L1W genotype shows two bands of 2344bp and 290bp; L2L2 genotype shows one band of 790bp; L1L2 genotype shows Two bands of 290bp and 790bp; L1L1 genotype showed one band of 290bp.
  • the present invention analyzes and verifies the correlation between the deletion mutation of the chicken IGF2BP1 gene promoter region and the chicken body size traits.
  • the promoter region of the IGF2BP1 gene of different chicken breeds was detected, and the structural variation was genotyped by PCR sequencing, and a total of 3 alleles were found.
  • the deletion length is 3234bp (that is, the 1210-4443th position from the 5' end of the sequence shown in SEQ ID NO: 1 is deleted)
  • the L2 type has a deletion at the position of chr27: 6083984-6085538, and the deletion length is 1555bp (that is, SEQ ID NO: 1).
  • the three allele distributions were identified using allele-specific PCR technology, primer sequences and conditions are shown in Table 1 and Figure 5.
  • the W and L2 alleles were identified with the 2k-F and Asp-R primer combination; the L1 allele was identified with the Asp-F and Asp-R primer combination, based on the physical location of the primer design and the amplification combination ( Figure 3).
  • the PCR sample is DNA, and the concentration is diluted to 60ng/ ⁇ L. Primer concentrations were all 10 ⁇ M.
  • PCR system use 2K-F&ASP-R, ASP-F&ASP-R for PCR amplification respectively:
  • Detection of agarose gel electrophoresis results Take 5 ⁇ L of two PCR reaction products from the same sample and conduct agarose gel electrophoresis.
  • the W allele band size is 2344bp
  • the L1 allele band size is 290bp
  • the L2 allele band size is 790bp.
  • WW genotype showed one band of 2344bp
  • L2W genotype showed two bands of 2344bp and 790bp
  • L1W genotype showed two bands of 2344bp and 290bp
  • L2L2 genotype showed one band of 790bp
  • L1L2 genotype showed two bands of 290bp and 790bp
  • the L1L1 genotype showed a band of 290 bp ( Figure 5).
  • the molecular marker genotypes of 734 individuals in the Gushi ⁇ Anka F2 generation resource group were identified by the aforementioned allele-specific PCR.
  • input the data into the SPSS software select a general linear model, add the genotype data as a fixed effect, and analyze the Gushi ⁇ Anka F2 generation resource group single-point variation general linear model
  • the result of association analysis if the p value of the analysis result is less than 0.05, it is considered that the variant site is significantly related to the trait.
  • the 23 body-scale traits are: claw weight, claw rate, 12-week-old tibia length, 8-week-old sternum length, 12-week-old sternum length, double wing weight, full venom weight, half venom weight, head weight, carcass weight, Leg weight, leg muscle weight, pelvic width at 12 weeks, shin circumference at 8 weeks, shin circumference at 12 weeks, body oblique length at 8 weeks, body oblique length at 12 weeks, muscle stomach weight, body weight at 6 weeks, 10 Body weight at week age, body weight at 12 weeks, body weight after shedding, and growth rate at 0-4 weeks.
  • the wild-type allele W is mainly found in red jungle fowl and local chicken breeds, while the L1 and L2 alleles are mainly found in commercial pure lines and synthetic lines of meat, and the distribution frequency is shown in Figure 7.
  • the allele L1 was associated with chicken height traits (paw weight, shin length, 23 traits such as sternum length and double wing weight) were significantly positively correlated
  • allele W was significantly positively correlated with chicken low body size traits
  • genotype L1L1 was associated with high body size traits (claw weight, shin length, sternum length, double size). 23 traits such as wing weight) were significantly positively correlated, and the results of association analysis are shown in Figure 8.

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Abstract

本发明涉及与鸡体尺性状相关的IGF2BP1基因分子标记及其应用和育种方法。本发明通过基因关联分析发现，鸡IGF2BP1基因启动子区的缺失突变与鸡体尺性状具有显著相关性。

Description

与鸡体尺性状相关的IGF2BP1基因分子标记及其应用和育种方法 技术领域
本发明属于生物育种技术领域，具体涉及与鸡体尺性状相关的IGF2BP1基因分子标记及其应用和育种方法。
背景技术
中国地方鸡品种资源丰富，具有肉质鲜美，抗逆性强等特点，但体型较小，生长速度缓慢，无法满足现代化集约化生产要求。国外的快大型白羽肉鸡具有生长速度快，屠体性能好，导致中国白羽肉鸡种源全部依赖进口。因此，培育具有中国自主知识产权的快大型肉鸡具有重要意义。体尺性状一直是鸡遗传改良的关键经济性状之一。传统育种中通常根据个体测定、同胞测定或者后裔测定的表型性状留种，但传统育种方法无法进行早期选种，增大了世代间隔，降低了选择反应。因此，开发新的分子标记，借助分子标记辅助选择加快育种进程成为鸡种质资源改良的新策略。
IGF2BP1基因，全称胰岛素样生长因子2结合蛋白，具有调节细胞增殖、分化、形态发生及代谢的作用。其作用机制通常为调控靶基因mRNA的定位、稳定性及翻译。近年来，IGF2BP1已经被证实为m6A甲基化的识别蛋白，很可能通过m6A甲基化的调控模式广泛作用于靶基因。IGF2BP1基因敲除小鼠表现出体重体尺降低的表型。而在鸡、鸭的全基因组关联分析及QTL定位研究发现位于IGF2BP1基因上游显著与体重、头重、胸宽、腿重等屠体和体尺性状相关。但影响鸡体尺性状的IGF2BP1基因的致因突变仍然未被阐明。
发明内容
本发明的目的在于提供一种与鸡体尺性状相关的IGF2BP1基因分子标记。
本发明的第二个目的在于提供上述分子标记在鸡体尺性状改良育种中的应用。
本发明的第三个目的在于提供用于检测上述分子标记的基因型的引物及试剂盒。
本发明的第四个目的在于提供一种鸡体尺性状改良育种方法。
为了实现上述目的，本发明采用以下技术方案：
一种与鸡体尺性状相关的IGF2BP1基因分子标记，其核苷酸序列如SEQ ID NO：1所示，5’端起第1210-4443位或5’端起第2993-4547位插入或缺失；所述SEQ ID NO：1的5’端起第1210-4443位的核苷酸序列如SEQ ID NO：2所示，所述SEQ ID NO：1的5’端起第2993-4547位的核苷酸序列如SEQ ID NO：3所示。
上述的分子标记在鸡体尺性状改良育种中的应用。
优选的，检测如SEQ ID NO：1所示核苷酸序列5’端起第1210-4443位或5’端起第2993-4547位插入或缺失，选择SEQ ID NO：1所示核苷酸序列5’端起第1210-4443位缺失的纯合型鸡个体留种。
用于检测权利要求1所述分子标记基因型的引物，其核苷酸序列如SEQ ID NO：4-6所示。
用于检测权利要求1所述分子标记基因型的试剂盒，所述试剂盒包含如SEQ ID NO：4-6所示的引物。
优选的，所述试剂盒还包括dNTPs、PCR反应缓冲液、DNA聚合酶中的一种或几种。
一种鸡体尺性状改良育种方法，包括如下步骤：
1)提取待测鸡DNA；根据上述分子标记设计引物，利用设计引物进行PCR扩增；
2)根据PCR扩增结果选择SEQ ID NO：1所示核苷酸序列5’端起第1210-4443位缺失的纯合型鸡个体留种。
优选的，步骤1)中利用酚仿抽提法提取DNA，并稀释到60ng/μL。设计引物的核苷酸序列如SEQ ID NO：4-6所示；利用琼脂糖凝胶电泳检测PCR扩增产物的条带大小，判断SEQ ID NO：1所示核苷酸序列5’端起第1210-4443位或2993-4547位插入或缺失并进行基因型鉴定；共分为六种基因型：WW基因型显示2344bp一条带；L2W基因型显示2344bp和790bp两条带；L1W基因型显示2344bp和290bp两条带；L2L2基因型显示790bp一条带；L1L2基因型显示290bp和790bp两条带；L1L1基因型显示290bp一条带；选择基因型为L1L1的鸡个体留种。
优选的，步骤1)中分别采用SEQ ID NO：4、6所示引物和SEQ ID NO：5、6所示引物进行PCR扩增反应，反应体系如下：
①2×Rapid Taq PCR Master Mix 10μL,ASP-F 0.5μL,ASP-R 0.5μL,待测鸡DNA模板1μL,ddH 2O 8μL；
②2×Rapid Taq PCR Master Mix 10μL,2K-F 0.5μL,ASP-R 0.5μL,待测鸡DNA模板1μL,ddH 2O 8μL。
优选的，步骤1)中PCR反应程序：95℃预变性5min；95℃变性15s，60℃退火15s，72℃延伸5s，30个循环；72℃延伸5min。
本发明取得的有益效果：
本发明提供了一种与鸡体尺性状相关的IGF2BP1基因分子标记。发明人首次发现IGF2BP1基因上游存在两种缺失基因型，经PCR和测序证实缺失基因型与高通量测序分析结果完全一致。本发明对鸡IGF2BP1基因启动子区的缺失突变与23种鸡体尺性状的相关性进行了分析，证明该变异位点中等位基因L1与高体尺性状(爪重、胫长、胸骨长、双翅重等23个性状)显著正相关；等位基因W与鸡低体尺性状呈显著正相关。本发明提供的IGF2BP1基因分子标记能够应用于鸡体尺性状改良育种。
本发明进一步提供了基于IGF2BP1基因分子标记的鸡体尺性状改良育种方法，提取待测鸡DNA，通过PCR扩增对应分子标记的基因片段，利用琼脂糖凝胶电泳检测PCR扩增产物的条带大小，判断SEQ ID NO：1所示核苷酸序列5’端起第1210-4443位或2993-4547位插入或缺失并进行基因型鉴定，选择基因型为L1L1的鸡个体留种，从而获得高体尺性状的鸡种质资源，实现鸡群的体尺性状改良。该方法能够早期、快速、低成本、有效的预测鸡成长后体尺性状，缩短育种时间，加快育种进程，具有极高的经济价值和广阔的应用前景。
附图说明
图1为鸡IGF2BP1启动子区1-2kb缺失和2-3kb缺失的分布频率；
图中，(A)IGF2BP1启动子区1-2kb；(B)IGF2BP1启动子区2-3kb。
图2为鸡IGF2BP1启动子区1-2kb和2-3kb结构变异关联分析；
图中，(A)IGF2BP1启动子区1-2kb；(B)IGF2BP1启动子区2-3kb。
图3为IGF2BP1启动子等位基因结构图；
图4为IGF2BP1启动子区三种等位基因基因组比较结果；
图5为IGF2BP1启动子区等位基因特异性PCR检测结果；
图中，泳道M为DM2000marker，从下至上maker条带大小分别为：100bp、250bp、500bp、750bp、1000bp、2000bp、3000bp、5000bp。
图6为IGF2BP1引物PCR扩增条件；
图7为PCR测序揭示染色体结构等位基因分布频率；
图8为IGF2BP1启动子区结构变异与体尺性状关联分析。
具体实施方式
下面结合具体实施方式对本发明作进一步描述，但本发明的保护范围并不仅限于此；实施例及试验例中所用的各类试剂均为市售商品。
实施例1 与鸡体尺性状相关的IGF2BP1基因分子标记及应用
该实施例提供了与鸡体尺性状相关的IGF2BP1基因分子标记，其核苷酸序列如SEQ ID NO：1所示，5’端起第1210-4443位或5’端起第2993-4547位插入或缺失；所述SEQ ID NO：1的5’端起第1210-4443位的核苷酸序列如SEQ ID NO：2所示，所述SEQ ID NO：1的5’端起第2993-4547位的核苷酸序列如SEQ ID NO：3所示。
所述分子标记能够应用于鸡体尺性状改良育种：检测如SEQ ID NO：1所示核苷酸序列5’端起第1210-4443位或5’端起第2993-4547位插入或缺失，选择SEQ ID NO：1所示核苷酸序列5’端起第1210-4443位(SEQ ID NO：2)缺失的纯合型鸡个体留种。从而改良鸡群的体尺性状。
实施例2 检测分子标记基因型的引物
该实施例提供了用于检测与鸡体尺性状相关的IGF2BP1基因分子标记基因型的引物，其核苷酸序列如SEQ ID NO：4-6所示。
用2k-F和Asp-R引物组合鉴定SEQ ID NO：1所示核苷酸序列5’端起第2993-4547位插入或缺失；用Asp-F和Asp-R引物组合鉴定SEQ ID NO：1所示核苷酸序列5’端起第1210-4443位插入或缺失。
实施例3 检测分子标记基因型的试剂盒
本实施例提供了用于检测与鸡体尺性状相关的IGF2BP1基因分子标记基因型的试剂盒，该试剂盒包括实施例2提供的SEQ ID NO：4-6所示的检测引物，还包括dNTPs、PCR反应缓冲液、DNA聚合酶中的一种或几种。
实施例4 鸡体尺性状改良育种方法
本实施例提供了一种鸡体尺性状改良育种方法，包括如下步骤：
1)收集待测鸡群，翅静脉采血，血液放入抗凝管，利用酚仿抽提法提取DNA，并稀释到60ng/μL。
2)PCR扩增含有分子标记的目的基因：
用2k-F(SEQ ID NO：5)和Asp-R(SEQ ID NO：6)引物组合鉴定SEQ ID NO：1所示核苷酸序列5’端起第2993-4547位插入或缺失；用Asp-F(SEQ ID NO：4)和Asp-R(SEQ ID NO：6)引物组合鉴定SEQ ID NO：1所示核苷酸序列5’端起第1210-4443位插入或缺失。
PCR反应体系：①2×Rapid Taq PCR Master Mix 10μL,ASP-F 0.5μL,ASP-R 0.5μL,DNA模板1μL,ddH 2O 8μL；②2×Rapid Taq PCR Master Mix 10μL,2K-F 0.5μL,ASP-R0.5μL,DNA模板1μL,ddH 2O 8μL。
PCR反应程序：95℃预变性5min；95℃变性15s，60℃退火15s，72℃延伸5s，30个循环；72℃延伸5min。
3)琼脂糖凝胶电泳结果检测：取同一个样品两个PCR反应产物各5μL，进行琼脂糖凝胶电泳。
W等位基因条带大小为2344bp,L1等位基因条带大小为290bp,L2等位基因条带大小为790bp。共分为六种基因型：WW基因型显示2344bp一条带；L2W基因型显示2344bp和790bp两条带；L1W基因型显示2344bp和290bp两条带；L2L2基因型显示790bp一条带；L1L2基因型显示290bp和790bp两条带；L1L1基因型显示290bp一条带。
4)根据基因判型结果，结合育种程序选择基因型为L1L1的个体留种，从而改良鸡群的体尺性状。
试验例1
本发明对鸡IGF2BP1基因启动子区的缺失突变与鸡体尺性状的相关性进行了分析和验证。
1.高通量测序发现鸡IGF2BP1启动子区结构变异及缺失变异的相关性分析
利用全基因组重测数据，通过比较分析基因组深度和覆盖度的方法以1kb为窗口大小鉴定基因组调控区结构变异，发现IGF2BP1基因的promoter区存在缺失变异(Absence)(突变型和野生型)，且缺失型和野生型在地方鸡和商品鸡品种中存在显著的频率差异(图1)。基于F2群体关联分析发现该结构变异与鸡体尺性状显著关联(图2)。
2.PCR检测鉴定出IGF2BP1启动子区三种结构变异等位基因
对不同鸡品种IGF2BP1基因promoter区进行检测，通过PCR测序对该结构变异进行了基因分型，共发现3个等位基因，突变型(L1型和L2型)：L1型在chr27：6082202-6085435位置存在缺失，缺失长度为3234bp(即SEQ ID NO：1所示序列5’端起第1210-4443位缺失)，L2型在chr27：6083984-6085538位置存在缺失，缺失长度为1555bp(即SEQ ID NO：1所示序列5’端起第2993-4547位缺失)；野生型在该位置不存在缺失(见图3和图4)。
利用等位基因特异性PCR技术鉴定三种等位基因分布，引物序列和条件如表1和图5。根据引物设计的物理位置和扩增组合(图3)，用2k-F和Asp-R引物组合鉴定W和L2等位基因；用Asp-F和Asp-R引物组合鉴定L1等位基因。
表1 IGF2BP1启动子三种等位基因特异性引物
Figure PCTCN2021130299-appb-000001
Figure PCTCN2021130299-appb-000002
PCR样本为DNA，浓度稀释至60ng/μL。引物浓度均为10μM。
PCR体系：分别利用2K-F&ASP-R，ASP-F&ASP-R进行PCR扩增：
①2×Rapid Taq PCR Master Mix 10μL,ASP-F 0.5μL,ASP-R 0.5μL,DNA模板1μL,ddH 2O 8μL；
②2×Rapid Taq PCR Master Mix 10μL,2K-F 0.5μL,ASP-R 0.5μL,DNA模板1μL,ddH 2O 8μL；
2×Rapid Taq PCR Master Mix购买自南京诺唯赞、引物合成自上海生工
PCR反应程序：95℃预变性5min；95℃变性15s，60℃退火15s，72℃延伸5s，30个循环；72℃延伸5min(图6)。
琼脂糖凝胶电泳结果检测：取同一个样品两个PCR反应产物各5μL，进行琼脂糖凝胶电泳。W等位基因条带大小为2344bp,L1等位基因条带大小为290bp,L2等位基因条带大小为790bp。WW基因型显示一条带为2344bp；L2W基因型显示2344bp和790bp两条带；L1W基因型显示2344bp和290bp两条带；L2L2基因型显示790bp一条带；L1L2基因型显示290bp和790bp两条带；L1L1基因型显示290bp一条带(图5)。
3.IGF2BP1启动子区等位基因PCR检测验证结果
利用前述等位基因特异性PCR对固始×安卡F2代资源群734个个体进行分子标记基因型的鉴定，鉴定后与固始×安卡F2代资源群体尺性状一一对应，即一个个体对应一个基因型数据和一组表型数据，将数据输入至SPSS软件中，选择一般线性模型，添加基因型数据为固定效应，分析得到固始×安卡F2代资源群单点变异一般线性模型关联分析的结果，分析结果若p值小于0.05即认为该变异位点与性状显著相关。
结果显示该变异位点中基因型L1L1与23个体尺性状均显著正相关。23个体尺性状为：爪重、爪率、12周龄胫长、8周龄胸骨长、12周龄胸骨长、双翅重、全净膛重、半净膛重、头重、屠体重、腿重、腿肌重、12周龄盆骨宽、8周龄胫围、12周龄胫围、8周龄体斜长、12周龄体斜长、肌胃重、6周龄体重、10周龄体重、12周龄体重、褪毛后体重、0～4周龄生长率。
野生型等位基因W主要存在于红原鸡、地方鸡品种，而L1和L2等位基因主要存在于肉商业纯系和合成系，分布频率如图7。基于固始×安卡F2代资源群单点变异一般线性模型关联分析发现，该关联群体中未发现存在等位基因L2型，等位基因L1与鸡高体尺 性状(爪重、胫长、胸骨长、双翅重等23个性状)呈显著正相关，等位基因W与鸡低体尺性状呈显著正相关，基因型L1L1与高体尺性状(爪重、胫长、胸骨长、双翅重等23个性状)显著正相关，关联分析结果如图8。

Claims (10)

  1. 一种与鸡体尺性状相关的IGF2BP1基因分子标记，其特征在于，其核苷酸序列如SEQ ID NO：1所示，5’端起第1210-4443位或5’端起第2993-4547位插入或缺失；所述SEQ ID NO：1的5’端起第1210-4443位的核苷酸序列如SEQ ID NO：2所示，所述SEQ ID NO：1的5’端起第2993-4547位的核苷酸序列如SEQ ID NO：3所示。
  2. 如权利要求1所述的分子标记在鸡体尺性状改良育种中的应用。
  3. 根据权利要求2所述的应用，其特征在于，判断如SEQ ID NO：1所示核苷酸序列5’端起第1210-4443位或5’端起第2993-4547位插入或缺失，选择SEQ ID NO：1所示核苷酸序列5’端起第1210-4443位缺失的纯合型鸡个体留种。
  4. 用于检测权利要求1所述分子标记基因型的引物，其特征在于，其核苷酸序列如SEQ ID NO：4-6所示。
  5. 用于检测权利要求1所述分子标记基因型的试剂盒，其特征在于，所述试剂盒包含如SEQ ID NO：4-6所示的引物。
  6. 根据权利要求5所述的试剂盒，其特征在于，所述试剂盒还包括dNTPs、PCR反应缓冲液、DNA聚合酶中的一种或几种。
  7. 一种鸡体尺性状改良育种方法，包括如下步骤：
    1)提取待测鸡DNA；根据权利要求1所述的分子标记设计引物，利用设计引物进行PCR扩增；
    2)根据PCR扩增结果选择SEQ ID NO：1所示核苷酸序列5’端起第1210-4443位缺失的纯合型鸡个体留种。
  8. 根据权利要求7所述的鸡体尺性状改良育种方法，其特征在于，步骤1)中，设计引物的核苷酸序列如SEQ ID NO：4-6所示；利用琼脂糖凝胶电泳检测PCR扩增产物的条带大小，判断SEQ ID NO：1所示核苷酸序列5’端起第1210-4443位或2993-4547位插入或缺失并进行基因型鉴定；共分为六种基因型：WW基因型显示2344bp一条带；L2W基因型显示2344bp和790bp两条带；L1W基因型显示2344bp和290bp两条带；L2L2基因型显示790bp一条带；L1L2基因型显示290bp和790bp两条带；L1L1基因型显示290bp一条带；选择基因型为L1L1的鸡个体留种。
  9. 根据权利要求7或8所述的鸡体尺性状改良育种方法，其特征在于，步骤1)中分别采用SEQ ID NO：4、6所示引物和SEQ ID NO：5、6所示引物进行PCR扩增反应，反应体系如下：
    ①2×Rapid Taq PCR Master Mix 10μL,ASP-F 0.5μL,ASP-R 0.5μL,待测鸡DNA模板 1μL,ddH 2O 8μL；
    ②2×Rapid Taq PCR Master Mix 10μL,2K-F 0.5μL,ASP-R 0.5μL,待测鸡DNA模板1μL,ddH 2O 8μL。
  10. 根据权利要求9所述的鸡体尺性状改良育种方法，其特征在于，步骤1)中PCR反应程序：95℃预变性5min；95℃变性15s，60℃退火15s，72℃延伸5s，30个循环；72℃延伸5min。
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