| 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 traitsPCR-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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