鸭脂肪组织EST鉴定、脂肪代谢相关基因多态检测及其与肉鸭脂肪、屠体和生长性状的关联分析
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摘要
最近几十年,肉鸭育种在提高生长速度、降低屠宰日龄等方面取得了令人瞩目的成绩,但在高强度选择提高生长速度的同时,出现了体脂、尤其是皮下脂肪和腹脂沉积过多的问题。随着人们生活水平的不断提高,由于脂肪摄入过多而产生的各种疾病已引起广泛关注；另外,过多的脂肪沉积不仅会影响产品的外观以及肉品质、降低生产效率,同时也会增加加工过程中的损耗从而影响经济效益；这些问题在一定程度上制约了肉鸭业的发展。因此,控制脂肪在鸭体内的过多蓄积,选育低脂肉鸭品种是今后肉鸭育种的奋斗目标之一。肉鸭的脂肪性状属于数量性状,受多基因控制,标记辅助选择是一种简便、有效的性状改良方法,通过与性状关联的分子遗传标记,选择控制相关性的候选基因的基因型,可实现标记辅助选择。但目前利用候选基因法在鸭中筛选分子标记,存在缺乏必要的基因序列信息的问题,本研究一方面利用基因组饱和杂交的原理,通过构建鸭脂肪组织均一化cDNA文库、测序获得在鸭脂肪组织中表达的新序列并借助序列分析寻找与鸭脂肪沉积相关的候选基因,为在鸭中开展新基因鉴定和基因功能研究提供序列基础；另一方面运用PCR-SSCP,DNA测序和PCR-RFLP结合的方法寻找和鉴定鸭脂肪性状候选基因LPL、ADP和GH基因的SNP,并借助SNP分析这些基因与鸭脂肪、屠体和生长体重等性状间的关系,为寻找可用于鸭标记辅助选择育种的分子遗传标记奠定基础,为进行高效的低脂鸭育种提供参考。主要研究结果如下：
1.鸭脂肪组织EST的鉴定
(1)首先构建了鸭皮脂和腹脂的两个独立cDNA文库,腹脂独立文库所含克隆数目约为4.8×106,文库重组率约为97%；皮脂独立文库所含克隆数目约为2.48x106,文库重组率约为94%,独立文库质量较好,满足均一化的要求。
(2)采用基因组DNA饱和杂交法,构建了鸭脂肪组织均一化cDNA文库。均一化后文库插入片段约700bp；在测序所得的234条EST序列中,单一序列有188条,占80%；在NCBI核酸数据库中无同源的新序列有93条,占40%；对测序所得EST序列的BLASTN分析发现共有141条序列在数据库中有同源序列,其中79条序列有同源基因的信息。
(3)分析与这些EST同源的基因功能发现,它们包括DNA或RNA水平上的相关调节因子、结构蛋白、细胞分化与凋亡因子、转座子、免疫因子、性别决定相关因子、信号转导因子、线粒体基因和原癌基因,以及一些蛋白质代谢、视觉和肌肉发育、脂质代谢相关基因等,还有一些功能未知的同源基因。这些功能基因的表达也暗示脂肪组织并不仅仅只是一个储存和释放能量的组织,而是一个重要而且活跃的器官,广泛地参与了机体的生理活动过程。
(4)获得了与脂质代谢相关的鸭LPL基因1326bp的cDNA序列(GenBank accessionno.FJ859348),该序列基本包括了鸭LPL基因的CDS区域,可用于下一步的SNP筛选。
2.鸭脂肪代谢相关候选基因LPL、GH和WADP的多态性检测及关联分析
运用PCR-SSCP,DNA测序和PCR-RFLP结合的方法,对LPL、GH和ADP等三个基因进行SNP筛选或确认。利用来自武汉精武食品工业园有限公司精武优质肉鸭场的白改鸭×连城白鸭F2群体资料,分析了上述SNP部分位点与鸭脂肪、屠体和生长体重等性状间的关系,主要研究结果如下：
(1)通过PCR-SSCP和测序方法,在LPL基因(GenBank accession no.FJ859348)中发现了5个新的SNP：外显子2存在一个C150T点突变,该突变造成Eco24Ⅰ酶切位点的改变；外显子5存在三个点突变,分别为C645T,T708C和G726A突变,其中C645T造成Sac Ⅱ酶切位点改变,G726A造成Mva Ⅰ酶切位点改变；外显子8存在一个C1245T点突变。这5个SNP在鸭LPL基因中的分布位置提示外显子5可能是一个突变多发区域。利用PCR-RFLP方法,对C645T和G726A两个SNP位点进行了酶切分型,并将基因型与鸭脂肪、屠体和体重性状进行了关联分析,结果显示：①在公鸭中,CT(AG)基因型的腹脂重、皮脂重均显著或极显著高于CC(GG)基因型(P<0.05或P<0.01)；在母鸭中,TT(AA)基因型的皮脂重极显著高于CC(GG)基因型(P<0.01),而且TT(AA)基因型的腹脂重也高于CC(GG)基因型,接近显著水平(P=0.06)；②在公鸭中,CT(AG)基因型的屠体性状值均极显著高于CC(GG)基因型(P<0.01)；在母鸭中,除头重差异不显著外(P>0.05),TT(AA)基因型的屠体性状值均显著或极显著高于CC(GG)基因型(P<0.05或P<0.01)；③在公鸭中,前8周体重在基因型间差异不显著(P>0.05),从第8周到第12周,CT(AG)基因型的体重极显著高于CC(GG)基因型；在母鸭中,前4周体重差异不显著(P>0.05),从第4周到第12周,TT(AA)基因型的体重均显著或极显著高于CC(GG)基因型(P<0.05或P<0.01)。另外,从LPL基因不同基因型在公母鸭的分布发现,该基因可能存在于鸭的Z染色体上。
(2)利用PCR-RFLP方法,对鸭GH基因(GenBank accession no. AB158760) C3701T突变位点进行了鉴定,发现本实验群中也存在该突变。酶切分型后将基因型与鸭脂肪、屠体和体重性状进行了关联分析,结果显示：①GH基因三种基因型间腹脂重差异显著(P<0.05),皮脂重差异极显著(P<0.01),基因型间腹脂重与皮脂重从大到小均为COCT>TT。②三种基因型间活重、屠体重、半净膛重、全净膛重、腿肌重、胸肌重、头重、脖子重、翅膀重、鸭掌重、心重、肝重和肌胃重等屠体性状均差异极显著(P<0.01),性状值在基因型间从大到小为COCT>TT。③第2周到第4周、第8周到第12周基因型间体重差异显著或极显著(P<0.05或P<0.01)第六周体重在基因型间的差异接近显著水平(P=0.074)。除初生重外,体重性状值在基因型间从大到小依次为COCT>TT。
(3)利用PCR-RFLP方法,对ADP基因(GenBank accession no. DQ452618.1) C540T和C579T两个突变位点进行了鉴定,发现C579T位点在本实验群中未产生突变,该位点在本实验群中均为CC型；C540T突变位点在本实验群中存在分离。对该突变位点酶切分型后将基因型与鸭脂肪、屠体和体重性状进行了关联分析,结果显示：①基因型间脂肪性状差异不显著(P>0.05)；②活重、胸肌重、头重、肌胃重、心重和肝重等在基因型间差异显著(P<0.05)或极显著(P<0.01),屠体重、半净膛重、全净膛重和翅膀重等屠体性状基因型间的差异均接近显著水平(P=0.054-0.073),腿肌重、脖子重和鸭掌重基因型间差异不显著(P>0.05)；③后期体重(8-10周)在基因型间差异显著；TT基因型体重值从初生重至第10周均高于其他两种基因型的体重值。
Duck breeding for the harvesting of meat has advanced considerably in recent decades, resulting in reduced slaughter age and greatly increased feed efficiency, meat production, and growth rate. The intensive selection for growth rate, however, has led to increased body fat deposition, which has become one of the main problems facing the duck breeding industry. Excessive fat deposition affects duck carcass appearance and meat quality, and it also causes low feeding efficiency, environmental pollution. To reduce fat deposition and select low-fat meat-type duck is the one of important aims in duck breeding in the future. Fatness traits belongs to quantitative traits and is controlled by ploygenes, they are hard and inefficient to be improved by regular breeding approaches. The molecular marker-assisted selection (MAS) promises a high efficient improvement of quantitative traits in simple and effective way by using reliable molecular makers which obtained by candidate gene approach; anyhow, very limited gene sequence information of duck is currently accumulated in the public database, and it causes that candidate gene approach is difficult to carry out in duck for molecular marker investigation. Therefore, one aim of this research is to identify some express sequence tag (EST) by constructing a normalized cDNA library using duck adipose tissues (abdominal fat and subcutaneous fat). Furthermore, single nucleotide polymorphism (SNP) in genes related with fat metabolism, including LPL, ADP and GH, were investigated or identified by PCR-SSCP, DNA sequencing and PCR-RFLP method, and also, the associations of these genes with body weight, carcass, and fatness traits was studied. Following are the main results:
1. Identification of EST from duck adipose tissue (1) Two ordinary cDNA libraries were first constructed for each of duck abdominal fat and subcutaneous fat. These two cDNA library contained4.8×106and2.48×106clones, respectively; and the recombinant efficiency were97%and94%, respectively, the cDNA library met the requirement of normalization.
(2) A normalized cNDA library of duck adipose tissue was constructed based on the strategy of saturation hybridization with genomic DNA; the average size of inserts was700bp.234EST were obtained by randomly cloning and sequencing from the normalized cDNA library, sequence analysis results showed that:80%EST were unique, include188EST;40%EST were new that have no homologues sequences in NCBI database, include93EST; BLASTN analysis revealed that141EST have homologues sequences in NCBI database which79EST have homologues gene information.
(3) The function of homologues genes including gene expression regulation, structural proteins, cell differentiation and apoptosis, transposition, immunity, sex determination and differentiation, signal transport, cancer related, mitochondria related, muscle development, protein and fat metabolism, etc; some homologues gene's function were unknown. So many genes expressed in duck adipose tissue hinted that duck adipose tissue not only for storage and provide energy, but an important and active organ involved in lots of physiological and biochemical process.
(4) A1326bp cDNA sequence of duck LPL gene which related to fat metabolism was obtained. This sequence contained almost LPL gene CDS domain and can be used to further research for SNP detection.2. SNP dectecting of duck LPL, GH and ADP gene and their association with fatness, carcass and bodyweight traits
PCR-SSCP, PCR-RFLP and sequencing methods were used to investigate or confirm SNP in the duck LPL, GH and ADP. A White Kaiya×White Liancheng F2population from Jingwu Food Industrial Garden Ltd.(Wuhan, China) was used to study the association between these genes and duck fatness, carcass and bodyweight traits in the present study. The results were as follows:(1) PCR-SSCP and sequencing methods were used to investigate SNP in the duck LPL gene (GenBank accession no.FJ859348). Five new SNPs were discovered in LPL gene: C150T in exon2caused an Eco24Ⅰ restriction enzyme site change; three SNP, C645T, T708C and G726A, were detected in exon5, C645T caused a Sac Ⅱ restriction enzyme site change, G726A caused a Mva Ⅰ restriction enzyme site change; and one SNP, C1245T, was detected in exon8. The locations of these five SNP indicated that exon5of duck LPL gene might be a variable area. C645T and G726A were genotyped using the PCR-RFLP in the population describled above. The association analysis results showed that:①In males, the abdominal fat Weight and subcutaneous fat plus skin weight of genotype CT (AG) were significantly higher than those of CC (GG)(P<0.05or P<0.01); In females, subcutaneous fat plus skin weight of genotype TT (AA) was significantly higher than those of CC (GG)(P<0.01), abdominal fat Weight of genotype TT (AA) was higher than those of CC (GG)(P=0.06);②In males, the carcass traits of genotype CT (AG) were significantly higher than those of CC (GG)(P<0.01); In females, the carcass traits of genotype TT (AA) were significantly higher than those of CC (GG)(P<0.05or P<0.01), except for head weight (P>0.05);③In males, the body weight of genotype CT (AG) was significantly higher than that of CC (GG) from week8to week12(P<0.01); In females, the body weight of genotype TT (AA) was significantly higher than that of CC (GG) from week4to week12(P<0.05or P<0.01). In addition, the distribution of genotypes differed between males and females indicated that the duck LPL gene might be located on Z chromosome.
(2) PCR-RFLP method was used to confirm the SNP site C3701T in the duck GH gene (GenBank accession no. AB158760), the result showed that the SNP site C3701T was also segregated in our experimental population. C3701T was genotyped using the PCR-RFLP and the association analysis showed that:①The abdominal fat weight and subcutaneous fat plus skin weight were significantly different among genotypes (P<0.05or P<0.01), and the individuals with genotype CC were significantly higher than genotype CT and TT;②The body weight (before slaughter), carcass weight, eviscerated with giblet weight, eviscerated weight, breast muscle weight (right side), leg muscle weight (right side), head weight, neck weight, wing weight (right side), shank plus palma weight, heart weight, liver weight, and muscular stomach weight were all significantly different among genotypes (P<0.01), and the individuals with genotype CC were significantly higher than genotype CT and TT;③the body weight were significantly different among genotypes from2nd to4th and8th to12th weeks (P<0.05or P<0.01), the body weight of6th week was close to significantly different among genotypes (P=0.074), and the individuals with genotype CC were significantly higher than genotype CT and TT except birth weight.
(3) PCR-RFLP method was used to confirm the SNP sites C540T and C579T in the duck ADP gene (GenBank accession no. DQ452618.1), it showed that the C579T was fixed with CC genotype and the C540T was segregated in the experimental population. Genotyping of C540T using the PCR-RFLP method and the association analysis showed that:①there were no significant differences in fatness traits among genotypes (P>0.05);②The body weight (before slaughter), breast muscle weight (right side), head weight, heart weight, muscular stomach weight and liver weight were significantly different among genotypes (P<0.05or P<0.01); the carcass weight, eviscerated with giblet weight, eviscerated weight and wing weight (right side) were close to significantly different among genotypes (P=0.054-0.073); and there were no significant differences in leg muscle weight (right side), neck weight and shank plus palma weight among genotypes (P>0.05);③The body weight were significantly different among genotypes from8th to10th weeks (P<0.05or P<0.01), and the individuals with genotype TT were higher than genotype CT and TT from birth to10th week.
1.鸭脂肪组织EST的鉴定
(1)首先构建了鸭皮脂和腹脂的两个独立cDNA文库,腹脂独立文库所含克隆数目约为4.8×106,文库重组率约为97%；皮脂独立文库所含克隆数目约为2.48x106,文库重组率约为94%,独立文库质量较好,满足均一化的要求。
(2)采用基因组DNA饱和杂交法,构建了鸭脂肪组织均一化cDNA文库。均一化后文库插入片段约700bp；在测序所得的234条EST序列中,单一序列有188条,占80%；在NCBI核酸数据库中无同源的新序列有93条,占40%；对测序所得EST序列的BLASTN分析发现共有141条序列在数据库中有同源序列,其中79条序列有同源基因的信息。
(3)分析与这些EST同源的基因功能发现,它们包括DNA或RNA水平上的相关调节因子、结构蛋白、细胞分化与凋亡因子、转座子、免疫因子、性别决定相关因子、信号转导因子、线粒体基因和原癌基因,以及一些蛋白质代谢、视觉和肌肉发育、脂质代谢相关基因等,还有一些功能未知的同源基因。这些功能基因的表达也暗示脂肪组织并不仅仅只是一个储存和释放能量的组织,而是一个重要而且活跃的器官,广泛地参与了机体的生理活动过程。
(4)获得了与脂质代谢相关的鸭LPL基因1326bp的cDNA序列(GenBank accessionno.FJ859348),该序列基本包括了鸭LPL基因的CDS区域,可用于下一步的SNP筛选。
2.鸭脂肪代谢相关候选基因LPL、GH和WADP的多态性检测及关联分析
运用PCR-SSCP,DNA测序和PCR-RFLP结合的方法,对LPL、GH和ADP等三个基因进行SNP筛选或确认。利用来自武汉精武食品工业园有限公司精武优质肉鸭场的白改鸭×连城白鸭F2群体资料,分析了上述SNP部分位点与鸭脂肪、屠体和生长体重等性状间的关系,主要研究结果如下：
(1)通过PCR-SSCP和测序方法,在LPL基因(GenBank accession no.FJ859348)中发现了5个新的SNP：外显子2存在一个C150T点突变,该突变造成Eco24Ⅰ酶切位点的改变；外显子5存在三个点突变,分别为C645T,T708C和G726A突变,其中C645T造成Sac Ⅱ酶切位点改变,G726A造成Mva Ⅰ酶切位点改变；外显子8存在一个C1245T点突变。这5个SNP在鸭LPL基因中的分布位置提示外显子5可能是一个突变多发区域。利用PCR-RFLP方法,对C645T和G726A两个SNP位点进行了酶切分型,并将基因型与鸭脂肪、屠体和体重性状进行了关联分析,结果显示：①在公鸭中,CT(AG)基因型的腹脂重、皮脂重均显著或极显著高于CC(GG)基因型(P<0.05或P<0.01)；在母鸭中,TT(AA)基因型的皮脂重极显著高于CC(GG)基因型(P<0.01),而且TT(AA)基因型的腹脂重也高于CC(GG)基因型,接近显著水平(P=0.06)；②在公鸭中,CT(AG)基因型的屠体性状值均极显著高于CC(GG)基因型(P<0.01)；在母鸭中,除头重差异不显著外(P>0.05),TT(AA)基因型的屠体性状值均显著或极显著高于CC(GG)基因型(P<0.05或P<0.01)；③在公鸭中,前8周体重在基因型间差异不显著(P>0.05),从第8周到第12周,CT(AG)基因型的体重极显著高于CC(GG)基因型；在母鸭中,前4周体重差异不显著(P>0.05),从第4周到第12周,TT(AA)基因型的体重均显著或极显著高于CC(GG)基因型(P<0.05或P<0.01)。另外,从LPL基因不同基因型在公母鸭的分布发现,该基因可能存在于鸭的Z染色体上。
(2)利用PCR-RFLP方法,对鸭GH基因(GenBank accession no. AB158760) C3701T突变位点进行了鉴定,发现本实验群中也存在该突变。酶切分型后将基因型与鸭脂肪、屠体和体重性状进行了关联分析,结果显示：①GH基因三种基因型间腹脂重差异显著(P<0.05),皮脂重差异极显著(P<0.01),基因型间腹脂重与皮脂重从大到小均为COCT>TT。②三种基因型间活重、屠体重、半净膛重、全净膛重、腿肌重、胸肌重、头重、脖子重、翅膀重、鸭掌重、心重、肝重和肌胃重等屠体性状均差异极显著(P<0.01),性状值在基因型间从大到小为COCT>TT。③第2周到第4周、第8周到第12周基因型间体重差异显著或极显著(P<0.05或P<0.01)第六周体重在基因型间的差异接近显著水平(P=0.074)。除初生重外,体重性状值在基因型间从大到小依次为COCT>TT。
(3)利用PCR-RFLP方法,对ADP基因(GenBank accession no. DQ452618.1) C540T和C579T两个突变位点进行了鉴定,发现C579T位点在本实验群中未产生突变,该位点在本实验群中均为CC型；C540T突变位点在本实验群中存在分离。对该突变位点酶切分型后将基因型与鸭脂肪、屠体和体重性状进行了关联分析,结果显示：①基因型间脂肪性状差异不显著(P>0.05)；②活重、胸肌重、头重、肌胃重、心重和肝重等在基因型间差异显著(P<0.05)或极显著(P<0.01),屠体重、半净膛重、全净膛重和翅膀重等屠体性状基因型间的差异均接近显著水平(P=0.054-0.073),腿肌重、脖子重和鸭掌重基因型间差异不显著(P>0.05)；③后期体重(8-10周)在基因型间差异显著；TT基因型体重值从初生重至第10周均高于其他两种基因型的体重值。
Duck breeding for the harvesting of meat has advanced considerably in recent decades, resulting in reduced slaughter age and greatly increased feed efficiency, meat production, and growth rate. The intensive selection for growth rate, however, has led to increased body fat deposition, which has become one of the main problems facing the duck breeding industry. Excessive fat deposition affects duck carcass appearance and meat quality, and it also causes low feeding efficiency, environmental pollution. To reduce fat deposition and select low-fat meat-type duck is the one of important aims in duck breeding in the future. Fatness traits belongs to quantitative traits and is controlled by ploygenes, they are hard and inefficient to be improved by regular breeding approaches. The molecular marker-assisted selection (MAS) promises a high efficient improvement of quantitative traits in simple and effective way by using reliable molecular makers which obtained by candidate gene approach; anyhow, very limited gene sequence information of duck is currently accumulated in the public database, and it causes that candidate gene approach is difficult to carry out in duck for molecular marker investigation. Therefore, one aim of this research is to identify some express sequence tag (EST) by constructing a normalized cDNA library using duck adipose tissues (abdominal fat and subcutaneous fat). Furthermore, single nucleotide polymorphism (SNP) in genes related with fat metabolism, including LPL, ADP and GH, were investigated or identified by PCR-SSCP, DNA sequencing and PCR-RFLP method, and also, the associations of these genes with body weight, carcass, and fatness traits was studied. Following are the main results:
1. Identification of EST from duck adipose tissue (1) Two ordinary cDNA libraries were first constructed for each of duck abdominal fat and subcutaneous fat. These two cDNA library contained4.8×106and2.48×106clones, respectively; and the recombinant efficiency were97%and94%, respectively, the cDNA library met the requirement of normalization.
(2) A normalized cNDA library of duck adipose tissue was constructed based on the strategy of saturation hybridization with genomic DNA; the average size of inserts was700bp.234EST were obtained by randomly cloning and sequencing from the normalized cDNA library, sequence analysis results showed that:80%EST were unique, include188EST;40%EST were new that have no homologues sequences in NCBI database, include93EST; BLASTN analysis revealed that141EST have homologues sequences in NCBI database which79EST have homologues gene information.
(3) The function of homologues genes including gene expression regulation, structural proteins, cell differentiation and apoptosis, transposition, immunity, sex determination and differentiation, signal transport, cancer related, mitochondria related, muscle development, protein and fat metabolism, etc; some homologues gene's function were unknown. So many genes expressed in duck adipose tissue hinted that duck adipose tissue not only for storage and provide energy, but an important and active organ involved in lots of physiological and biochemical process.
(4) A1326bp cDNA sequence of duck LPL gene which related to fat metabolism was obtained. This sequence contained almost LPL gene CDS domain and can be used to further research for SNP detection.2. SNP dectecting of duck LPL, GH and ADP gene and their association with fatness, carcass and bodyweight traits
PCR-SSCP, PCR-RFLP and sequencing methods were used to investigate or confirm SNP in the duck LPL, GH and ADP. A White Kaiya×White Liancheng F2population from Jingwu Food Industrial Garden Ltd.(Wuhan, China) was used to study the association between these genes and duck fatness, carcass and bodyweight traits in the present study. The results were as follows:(1) PCR-SSCP and sequencing methods were used to investigate SNP in the duck LPL gene (GenBank accession no.FJ859348). Five new SNPs were discovered in LPL gene: C150T in exon2caused an Eco24Ⅰ restriction enzyme site change; three SNP, C645T, T708C and G726A, were detected in exon5, C645T caused a Sac Ⅱ restriction enzyme site change, G726A caused a Mva Ⅰ restriction enzyme site change; and one SNP, C1245T, was detected in exon8. The locations of these five SNP indicated that exon5of duck LPL gene might be a variable area. C645T and G726A were genotyped using the PCR-RFLP in the population describled above. The association analysis results showed that:①In males, the abdominal fat Weight and subcutaneous fat plus skin weight of genotype CT (AG) were significantly higher than those of CC (GG)(P<0.05or P<0.01); In females, subcutaneous fat plus skin weight of genotype TT (AA) was significantly higher than those of CC (GG)(P<0.01), abdominal fat Weight of genotype TT (AA) was higher than those of CC (GG)(P=0.06);②In males, the carcass traits of genotype CT (AG) were significantly higher than those of CC (GG)(P<0.01); In females, the carcass traits of genotype TT (AA) were significantly higher than those of CC (GG)(P<0.05or P<0.01), except for head weight (P>0.05);③In males, the body weight of genotype CT (AG) was significantly higher than that of CC (GG) from week8to week12(P<0.01); In females, the body weight of genotype TT (AA) was significantly higher than that of CC (GG) from week4to week12(P<0.05or P<0.01). In addition, the distribution of genotypes differed between males and females indicated that the duck LPL gene might be located on Z chromosome.
(2) PCR-RFLP method was used to confirm the SNP site C3701T in the duck GH gene (GenBank accession no. AB158760), the result showed that the SNP site C3701T was also segregated in our experimental population. C3701T was genotyped using the PCR-RFLP and the association analysis showed that:①The abdominal fat weight and subcutaneous fat plus skin weight were significantly different among genotypes (P<0.05or P<0.01), and the individuals with genotype CC were significantly higher than genotype CT and TT;②The body weight (before slaughter), carcass weight, eviscerated with giblet weight, eviscerated weight, breast muscle weight (right side), leg muscle weight (right side), head weight, neck weight, wing weight (right side), shank plus palma weight, heart weight, liver weight, and muscular stomach weight were all significantly different among genotypes (P<0.01), and the individuals with genotype CC were significantly higher than genotype CT and TT;③the body weight were significantly different among genotypes from2nd to4th and8th to12th weeks (P<0.05or P<0.01), the body weight of6th week was close to significantly different among genotypes (P=0.074), and the individuals with genotype CC were significantly higher than genotype CT and TT except birth weight.
(3) PCR-RFLP method was used to confirm the SNP sites C540T and C579T in the duck ADP gene (GenBank accession no. DQ452618.1), it showed that the C579T was fixed with CC genotype and the C540T was segregated in the experimental population. Genotyping of C540T using the PCR-RFLP method and the association analysis showed that:①there were no significant differences in fatness traits among genotypes (P>0.05);②The body weight (before slaughter), breast muscle weight (right side), head weight, heart weight, muscular stomach weight and liver weight were significantly different among genotypes (P<0.05or P<0.01); the carcass weight, eviscerated with giblet weight, eviscerated weight and wing weight (right side) were close to significantly different among genotypes (P=0.054-0.073); and there were no significant differences in leg muscle weight (right side), neck weight and shank plus palma weight among genotypes (P>0.05);③The body weight were significantly different among genotypes from8th to10th weeks (P<0.05or P<0.01), and the individuals with genotype TT were higher than genotype CT and TT from birth to10th week.
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