Transcriptome-based analysis of key genes and pathways affecting the linoleic acid content in chickens

Linoleic acid is an essential polyunsaturated fatty acid that cannot be synthesized by humans or animals themselves and can only be obtained externally.The amount of linoleic acid present has an impact on the quality and flavour of meat and indirectly affects consumer preference.However,the molecular mechanisms influencing the deposition of linoleic acid in organisms are not clear.As the molecular mechanisms of linoleic acid deposition are not well understood,to investigate the main effector genes affecting the linoleic acid content,this study aimed to screen for hub genes in slow-type yellow-feathered chickens by transcriptome sequencing(RNA-Seq)and weighted gene coexpression network analysis(WGCNA).We screened for candidate genes associated with the linoleic acid content in slow-type yellow-feathered broilers.A total of 399 Tiannong partridge chickens were slaughtered at 126 days of age,fatty acid levels were measured in pectoral muscle,and pectoral muscle tissue was collected for transcriptome sequencing.Transcriptome sequencing results were combined with phenotypes for WGCNA to screen for candidate genes.KEGG enrichment analysis was also performed on the genes that were significantly enriched in the modules with the highest correlation.A total of 13310 genes were identified after quality control of transcriptomic data from 399 pectoral muscle tissues.WGCNA was performed,and a total of 26 modules were obtained,eight of which were highly correlated with the linoleic acid content.Four key genes,namely,MDH2,ATP5B,RPL7A and PDGFRA,were screened according to the criteria|GS|>0.2 and|MM|>0.8.The functional enrichment results showed that the genes within the target modules were mainly enriched in metabolic pathways.In this study,a large-sample-size transcriptome analysis revealed that metabolic pathways play an important role in the regulation of the linoleic acid content in Tiannong partridge chickens,and MDH2,ATP5B,RPL7A and PDGFRA were screened as important candidate genes affecting the linoleic acid content.The results of this study provide a theoretical basis for selecting molecular markers and comprehensively understanding the molecular mechanism affecting the linoleic acid content in muscle,providing an important reference for the breeding of slow-type yellowfeathered broiler chickens.

Major regulatory factors for reproductive performances of female chickens

The reproductive performance of female chickens is critical for determining the efficiency of production and productivity and thus profitability.Studies have shown that the reproductive performance of female chickens is mainly regulated by the feed,hormones,genes,and light conditions.Herein,we review the major factors regulating female chicken reproductive performance and assess the reproductive organs and their functions.In the current review,we highlight how the interconnections of hormones,candidate genes,and photo-stimulation regulate female chicken reproductive hormones and thus regulate the reproductive organ performance.In this regard,the roles of main hormones[gonadotropinreleasing hormone(GnRH)and genes(GnRH-Ⅰ)]in regulating sexual maturation and ovarian development and maintenance by influencing the survival and function of follicular granulosa cells were also reviewed.In addition,the current review also highlights how feeding female chickens with diets and artificial light-emitting diodes(LEDs)support the effective functioning of their reproductive capacity through the stimulation of sexual maturity at an appropriate age and regeneration of aged reproductive organs.

Advances in Genetic Engineering Vaccine against Chicken Coccidiosis

Coccidiosis is a protozoan disease that seriously threatens the poultry in-dustry. At present, the prevention and treatment of chicken coccidiosis mainly rely on chemical drugs and coccidia live vaccine. However, drug control has problems of drug resistance and drug residue, and the coccidia live vaccine has drawbacks of pathogenicity, high production costs and virulence enhancement. Instead, genetic en-gineering vaccine is easy to operate, stable, safe and efficient, so it has become a hot spot in the prevention of chicken coccidiosis. In this paper, the research status, immune mechanism, immune effect＇s influencing factors and cytokine adjuvant appli-cation of genetic engineering vaccine against chicken coccidiosis were reviewed to provide some reference for the research and application of chicken coccidiosis gene vaccine.

Characterization of Genetic Polymorphism of Novel MHC B-LBⅡ Alleles in Chinese Indigenous Chickens

Genetic polymorphism of the major histocompatibility complex （MHC） B-LBⅡ gene was studied by amplification of exon 2 using PCR, followed by cloning and DNA sequencing in eight indigenous Chinese chicken populations. To reveal the genetic variation of the B-LB Ⅱ gene, 37 types of patterns detected by PCR-SSCP were investigated first, which would be used to screen novel B-LB Ⅱsequences within the breeds. The types of PCR-SSCP patterns and final sequencing allowed for the identification of 31 novel MHC B-LBⅡ alleles from 30 unrelated individuals of Chinese chickens that were sampled. These are the first designators for the alleles of chicken MHC B-LBⅡ gene based on the rule of assignment for novel mammalian alleles. Sequence alignment of the 31 B-LB Ⅱ alleles revealed a total of 68 variable sites in the fragment of exon 2, of which 51 parsimony informative and 17 singleton variable sites were observed. Among the polymorphic sites, the nucleotide substitutions in the first and second positions of the codons accounted for 36.76% and 35.29%, respectively. The sequence similarities between the alleles were estimated to be 90.6%-99.5%. The relative frequencies of synonymous and nonsynonymous nucleotide substitutions within the region were 2.92%±0.94% and 14.64%±2.67%, respectively. These results indicated that the genetic variation within exon 2 appeared to have largely arisen by gene recombination and balancing selection. Alignment of the deduced amino acid sequences of the β1 domain coded by exon 2 revealed 6 synonymous mutations and 27 nonsynonymous substitutions at the 33 disparate sites. In particular, the nonsynonymous substitutions at the putative peptide-binding sites are considered to be associated with immunological specificity of MHC B-LB Ⅱ molecule in Chinese native chickens. These results can provide a molecular biological basis for the study of disease resistance in chicken breeding.

Correlation between Polymorphism of Neuropeptide Y Gene and Traits at First Laying of Wenchang Chicken

[ Objective] The paper presents the polymorphism of neuropeptide Y gene （NPY） and its relationship with traits at first laying in Wen- chang chicken in order to provide information for breeding of high-yielding Wenchang chicken. [Method] The polymorphism of NPYgene at Dra I site was detected by PCR-RFLP, and the genotypes and the genetic stability were determined. The correlation between the polymorphism and traits at first laying （age, body weight and egg weight） was also analyzed. [Result] There were three kinds of genotype of NPYgene at Dra I site in Wenchang chicken, designated as AA, AB and BB; the distribution of these genotypes was consistent with the Hardy -Weinberg law; age and egg weight were not significantly different among the three genotypes （ P 〉0.05）, and body weight was extremely significantly different （ P 〈0.01 ）. [ Conclusion] The NPYgene of Wenchang chicken is in Hardy -Weinberg equilibrium at Dra I site, and its genetic polymorphism affects body weight at first laying.

Immunological Adjuvant Function of Aluminium Phosphate and Chicken IL-18 in NDV F Gene Vaccine

[Objective] This study aimed to investigate the immunological adjuvant function of aluminium phosphate and chicken IL-18 in NDV F gene vaccine. [Method] The vaccine （0.2 ml） containing aluminum phosphate adjuvant （90 μg）, pcDNA/F （200μg）, and pcDNA/chlL-18 （200 μg） was prepared. The 7 d old chick- ens to be tested were randomly divided into six groups （12 chickens in each group） and immunized through intramuscular injection with inactivated Newcastle disease vaccines, pcDNA/F＋pcDNA/chlL-18＋phosphate aluminum, pcDNA/F, pcDNA/F.＋pcDNA/ chlL-18, pcDNA/F＋aluminum phosphate, and physiological saline respectively; the secondary immunization was conducted with the same dose when the chickens were 21 d old. Their blood was sampled 0, 7, 14, 21, 28 d after first immunization. Anti- body titer was detected with ELISA and T cell transformation rate was measured with MIT. Experimental chicken will be challenged with 30 LD50 NDV virulence 28 d after first immunization. [Result] The survival rate of the chickens immunized with pcDNA/F＋aluminium phosphate＋pcDNA/chlL-18 achieved 8/12, higher than that of those immunized with pcDNA/F 4/12 and pcDNA/F＋pcDNA/chlL-18 （6/12）. The NDV antibody titer of the chickens immunized with pcDNA/F＋ aluminum phosphate, pcD- NA/F＋pcDNA/chlL-18 and pcDNA/F＋pcDNA/chlL-18＋aluminum phosphate is not differ- ent （P〉0.05）, but significantly lower than that of the chickens immunized with tradi- tional vaccine （P〈0.05）. The T cell transformation rate of the chickens immunized with pcDNA/F＋pcDNA/chlL-18＋aluminium phosphate was obviously higher than that of the chickens immunized with pcDNA/F （P〈0.05）. The T cell transformation rates of chickens immunized with pcDNA/F and the traditional vaccine showed no signifi- cant difference （P〉0.05）. [Conclusion] Combination of aluminium phosphate and pcD- NA/chlL-18 can significantly enhance the immune effect of NDV F gene vaccine.

Identification and characterization of genes that control fat deposition in chickens

Background： Fat deposits in chickens contribute significantly to meat quality attributes such as juiciness, flavor, taste and other organoleptic properties. The quantity of fat deposited increases faster and earlier in the fast- growing chickens than in slow-growing chickens. In this study, Affymetrix Genechip~ Chicken Genome Arrays 32773 transcripts were used to compare gene expression profiles in liver and hypothalamus tissues of fast-growing and slow-growing chicken at 8 wk of age. Real-time RT-PCR was used to validate the differential expression of genes selected from the microarray analysis. The mRNA expression of the genes was further examined in fat tissues. The association of single nucleotide polymorphisms of four lipid-related genes with fat traits was examined in a F2 resource population. Results： Four hundred genes in the liver tissues and 220 genes hypothalamus tissues, respectively, were identified to be differentially expressed in fast-growing chickens and slow-growing chickens. Expression levels of genes for lipid metabolism （SULTIB1, ACSBG2, PNPLA3, LPL, AOAH） carbohydrate metabolism （MGAT4B, XYLB, GBE1, PGM1, HKDCl）cholesttrol biosynthesis （FDPS, LSS, HMGCR, NSDHL, DHCR24, IDI1, MEI） HSD17B7 and other reaction or pro- cesses （CYPIA4, CYP1A1, AKR1BI, CYP4V2, DDO） were higher in the fast-growing White Recessive Rock chickens than in the slow-growing Xinghua chickens. On the other hand, expression levels of genes associated with multicellular organism development, immune response, DNA integration, melanin biosynthetic process, muscle organ develop- ment and oxidation-reduction （FRZB, DMD, FUT8, CYP2C45, DHRSX, and CYP2C18） and with glycol-metabolism （GCNT2, ELOVL d, and FASN）, were higher in the XH chickens than in the fast-growing chickens, fiT-PCR validated high expression levels of nine out of 12 genes in fat tissues. The G1257069A and T1247123C of the ACSBG2 gene were significantly associated with abdominal fat weight. The G4928024A of the FASN gene were significantly associ- ated with fat bandwidth, and abdominal fat percentage. The C4930169T of the FASN gene was associated with ab- dominal fat weight while the A59539099G of the ELOVL 6 was significantly associated with subcutaneous fat. The A8378815G of the DDT was associated with fat band width. Conclusion： The differences in fat deposition were reflected with differential gene expressions in fast and slow growing chickens.

Expression of Sex-Related Genes in Chicken Embryos During Male-to-Female Sex Reversal Exposure to Diethylstilbestrol

Sex emerges out of a delicate dance between a variety of promale, anti-male, and possibly profemale genes. To investigate the role that sex-related genes play in sex determination and gonadal differentiation of fowl, we constructed a male-to- female sex-reversal model of chick induced by diethylstilbestrol （DES） at onset of incubation （E0）. The results of semi- quantitative PCR showed that the expression of Sf1, the orphan nuclear receptor steroidogenic factor-1 gene, was put forward from E7d to E5d and up-regulated during E5-7d; the Dmrt1, the double sex and the Mab-3 related to transcription factor 1 gene, was down-regulated during E3-7d. Meanwhile, anti-Müllerian hormone gene （Amh） expressed at a similar level in the genetic females and sex-reversal females before E7d, while no expression products of the three female-specific genes Wpkci, Fet1 and Foxl2 were detected in male-to-female embryos. These findings suggest that the expression of some certain sex-related genes, induced by the exogenous estrogen during period of sex determination and gonadal differentiation, results in the male-to-female sex reversal. Moreover, high activity of Sf1 gene during E5-7d might be related to the profemale process, while low activity of Dmrt1 gene during E3-5d might be anti-male. The expression activity of Amh gene might only contribute to the promale process after E7d, however, it is possibly not an anti-female gene in chick embryos.

Cloning of Chicken Anemia Virus vp3 Gene and Apoptosis Inductive Effect of vp3 Gene In Vitro

Using PCR technique, the vp3 gene of chicken anemia virus (CAV) was cloned into the eukaryotic expression vector pcDNA3 to construct a recombinant pcDNA vp3. Restriction enzyme digestion and sequencing analysis revealed that CAV vp3 gene was correctly inserted into the blank vector pcDNA3. After LipofectAMINE TM mediated transfection in vitro with pcDNA vp3 and pcDNA3 respectively, the total mRNA was extracted from liver carcinoma cell lines HepG2 and diploid cell line L 02, and RT PCR was performed afterward. The results of RT PCR suggested that vp3 gene was expressed in these two cell lines. At the same time, using in situ apoptotic detection assay, TUNEL kits, the apoptotic cells were found in pcDNA vp3 transfected HepG2, but not in mock transfected cell lines. VP3 could induce cell death by apoptosis in cancer cell lines, but not in diploid cell lines. All the results indicated that CAV vp3 gene, a potential therapeutic agents, has the potential of being used for cancer treatment.

Relationship Between Combined Genotypes of UCP Gene and Growth Traits in Chickens

The uncoupling protein （UCP） is a member of the mitochondrial membrane transporter family, which plays an important role in energy metabolism. In the present study, the UCP gene was considered as a candidate gene for chicken growth traits, and the association of UCP gene SNPs with growth rate was investigated in the eighth generation of NEAUHLF broiler lines. Two SNPs were found in chicken UCP gene, and the association analysis results showed that both the individual and combination of chicken UCPgene SNPs were significantly associated with body weight of 7 weeks （BW7） and carcass weight （CW） （P〈0.05）, and the combination had much significant effects than the single SNP. This research suggested that the UCP gene could be a candidate gene or linked to a major gene which affected growth traits in chicken

Reference Gene Selection for Normalization of PCR Analysis in Chicken Embryo Fibroblast Infected with H5N1 AIV

Chicken embryo fibroblasts (CEFs) are among the most commonly used cells for the study of interactions between chicken hosts and H5N1 avian influenza virus (AIV).In this study,the expression of eleven housekeeping genes typically used for the normalization of quantitative real-time PCR (QPCR) analysis in mammals were compared in CEFs infected with H5N1 AIV to determine the most reliable reference genes in this system.CEFs cultured from 10-day-old SPF chicken embryos were infected with 100 TCID50 of H5N1 AIV and harvested at 3,12,24 and 30 hours post-infection.The expression levels of the eleven reference genes in infected and uninfected CEFs were determined by real-time PCR.Based on expression stability and expression levels,our data suggest that the ribosomal protein L4 (RPL4) and tyrosine 3-monooxygenase tryptophan 5-monooxygenase activation protein zeta polypeptide (YWHAZ) are the best reference genes to use in the study of host cell response to H5N1 AIV infection.However,for the study of replication levels of H5N1 AIV in CEFs,the β-actin gene (ACTB) and the ribosomal protein L4 (RPL4) gene are the best references.

PGC-1α differentially regulates the mRNA expression profiles of genes related to myofiber type specificity in chicken

Previous studies on mammals showed that peroxisome proliferator-activated receptor gamma coactivator-1α(PGC-1α)played a prominent role in regulating muscle fiber type transition and composition.However,the role of PGC-1αin chicken muscle has seldom been explored.To investigate the effect of PGC-1αon chicken skeletal muscles in this study,the PGC-1αgene was overexpressed or silenced in chicken primary myoblasts by using lentivirus,and then the effects of the PGC-1αgene overexpression and knockdown on the mRNA expression profile of genes related to myofiber type specificity were examined during fiber formation.The results showed that overexpression of PGC-1αfrom proliferation to differentiation was accompanied by the up-regulated expression of Pax7,MyoD,and CnAα,which was significantly(P<0.01)increased after one day of transfection(1 I).The enhancement of MyoG,MEF2 c,and MyHC SM expression lagged,which was improved significantly(P<0.01)after four days of transfection(1 I3 D).Overexpression of PGC-1αdecreased(P<0.01)the MyHC FWM expression after four days of transfection(1 I3 D),and it had no significant impact(P>0.05)on the expression of CnB1,NFATc3,and MyHC FRM during myofiber formation.The effective silence(P<0.01)of PGC-1αby lentivirus mediating short hairpin RNA(shRNA)was detected after four days of transfection(1 I3 D)in cultures,and the lack of its function in chicken primary myoblasts significantly(P<0.01)down-regulated the expression of Pax7,MyoD,CnAα,MyoG,MEF2 c,and MyHC SM,significantly(P<0.01)up-regulated the expression of MyHC FWM,and had no significant impact(P>0.05)on the expression of CnB1,NFATc3,and MyHC FRM.These results indicated that the role of PGC-1αin regulating the fiber type specificity of chicken skeletal muscles might be similar to that in mammals,which interplayed with key genes related to myocyte differentiation and calcineurin signaling pathway.

Investigation of Genetic Variation of B-G Gene with PCR-SSCP and RFLP in Chinese Indigenous Chickens

To reveal genetic variation of MHC B-G gene at Chinese native chickens, two PCR primer pairs were designed to hybridize specifically with conserved sequences surrounding hypervariable regions within the B-G gene and used to amplify two DNA fragments in ten Chinese indigenous chicken breeds and one introduced breed. The fragments were cloned and sequenced to assure that the expected sequences of chicken B-G gene were isolated. Of which the 189 bp fragment encompassing the most variable region within exon 2 of B-G gene was employed for PCR-SSCP assay, this method provided evidence for the presence of at least 56 B-G genotypes in the Chinese chickens sampled. It revealed a high degree of diversity in B-G genes of Chinese local breeds; particularly, high variation of B-G gene was confirmed with the presence of 48 B-G genotypes within Tibetan chicken population. Not only can the B-G genotypes be used to preliminarily screen new B-G alleles, but also they would be utilized to investigate MHC haplotypes and matched unrelated donors for bone marrow transplantation in immune researches. Another fragment of 401 bp size spanning over partial intron 1 and exon 2 of B-G gene was employed for PCR-RFLP analysis with two restriction enzymes of Msp Ⅰ and Tas Ⅰ in the breeds sampled. In this part of the gene, three novel SNPs were detected at the two restriction sites. It was more generally found the transition of two nucleotides of A294G and T295C occurred at Tas Ⅰ restriction site, and consequently led to a non-synonymous substitution of asparagine into serine at position 54 within the deduced amino acid sequence of immunoglobulin variable-region-like domain encoded by the exon 2 of B-G gene. It was observed at rare frequency that an alone mutation of A294G occurring at the site, which also caused the substitution of amino acid, asparagine 54-to-serine; and we haven＇t found only single mutation occurred at position 295 of the restriction site. At the Msp Ⅰ site, the transversion of G318C led to a non-synonymous substitution, glutamine 62-to-histidine. The variations at expression level caused by the genetic variability of B-G gene may bring about the changes in immune specificity of B-G antigen finally. Furthermore, two alleles, A and B, were identified at Msp Ⅰ and Tas Ⅰ loci of B-G gene, respectively. The allele frequencies were estimated, which gave a nonsymmetrical distribution either in the eight Chinese local breeds or in the introduced breed. By comparison, allele A at Msp Ⅰ locus was tended to be dominative, while, the allele B at Tas Ⅰ locus was tended to be prevalent in the breeds analyzed. It is concluded that the genetic variability of B-G gene revealed by the PCR-SSCP and RFLP assays in Chinese native chickens provide molecular data for further investigating the varied immune functions of B-G antigen; and the PCR-RFLPs at Msp Ⅰ and Tas Ⅰ loci of B-G gene might be used as genetic markers in selecting for the traits of disease resistance in chicken breeding.

Effect of the C.–1388 A>G polymorphism in chicken heat shock transcription factor 3 gene on heat tolerance

Heat stress is one of the main factors that inlfuence poultry production. Heat shock proteins (HSPs) are known to affect heat tolerance. The formation of HSPs is regulated by heat shock transcription factor 3 (HSF3) in chicken. A DNA pool was established for identifying single nucleotide polymorphisms (SNPs) of the chicken HSF3, and 13 SNPs were detected. The bioinformatic analysis showed that 8 SNPs had the capacity to alter the transcription activity of HSF3. The dual luciferase report gene assay showed that there was a signiifcant difference (P<0.01) in the Firelfy luciferase/Renil a luciferase ratio (F/R) of C.–1 703 A>G (S1) and C.–1 388 A>G (S4) sites at the 5′-untranslated region (UTR) of chicken HSF3. The elec-trophoretic mobility shift assay showed that the S4 site was a transcription binding factor. The analysis of the association of the S1 and S4 sites with heat tolerance index revealed that the S4 site was signiifcantly correlated with the CD3+T cel , corticosterone, and T3 levels in Lingshan chickens and with the heterophil/lymphocyte value in White Recessive Rock. These results showed that the S4 site at the 5′ UTR of chicken HSF3 might have an impact on heat tolerance in summer and could be used as a potential marker for the selection of chicken with heat tolerance in the future.

Genetic basis of negative heterosis for growth traits in chickens revealed by genome-wide gene expression pattern analysis

Background:Heterosis is an important biological phenomenon that has been extensively utilized in agricultural breeding.However,negative heterosis is also pervasively observed in nature,which can cause unfavorable impacts on production performance.Compared with systematic studies of positive heterosis,the phenomenon of negative heterosis has been largely ignored in genetic studies and breeding programs,and the genetic mechanism of this phenomenon has not been thoroughly elucidated to date.Here,we used chickens,the most common agricultural animals worldwide,to determine the genetic and molecular mechanisms of negative heterosis.Results:We performed reciprocal crossing experiments with two distinct chicken lines and found that the body weight presented widely negative heterosis in the early growth of chickens.Negative heterosis of carcass traits was more common than positive heterosis,especially breast muscle mass,which was over−40%in reciprocal progenies.Genome-wide gene expression pattern analyses of breast muscle tissues revealed that nonadditivity,including dominance and overdominace,was the major gene inheritance pattern.Nonadditive genes,including a substantial number of genes encoding ATPase and NADH dehydrogenase,accounted for more than 68%of differentially expressed genes in reciprocal crosses(4257 of 5587 and 3617 of 5243,respectively).Moreover,nonadditive genes were significantly associated with the biological process of oxidative phosphorylation,which is the major metabolic pathway for energy release and animal growth and development.The detection of ATP content and ATPase activity for purebred and crossbred progenies further confirmed that chickens with lower muscle yield had lower ATP concentrations but higher hydrolysis activity,which supported the important role of oxidative phosphorylation in negative heterosis for growth traits in chickens.Conclusions:These findings revealed that nonadditive genes and their related oxidative phosphorylation were the major genetic and molecular factors in the negative heterosis of growth in chickens,which would be beneficial to future breeding strategies.

Antibiotic susceptibility and molecular characterization of resistance genes among Escherichia coli and among Salmonella subsp. in chicken food chains

Objective: To investigate the occurrence of resistance genes among Escherichia coli(E. coli) and Salmonella subsp. isolated in chicken food chains in Phnom Penh, 2012–2013.Methods: Six hundred eighty two E. coli and 181 Salmonella Albany, Corvallis, and Kentucky strains were examined for susceptibilities to eight antimicrobials and following resistance genes were identified by PCR: blaTem, Str A, aad A, sul1, sul2, gyr A, Tet(A), and Tet(B).Results: E. coli presented high resistances to tetracycline, amoxicillin, and sulfamethoxazole(63.1%–76.1%). Salmonella Albany and Salmonella Kentucky traduced high resistance percentages to amoxicillin, tetracycline, sulfamethoxazole, and nalidixic acid(84.6%–100%). Among amoxicillin-resistant isolates, blaTemgenes were observed for 62% of E. coli isolates and 20% of 65 Salmonella Kentucky. The Str A gene was prevalent in 36% of 331 aminoglycoside-resistant E. coli and 90% of 40 aminoglycoside-resistant Salmonella Corvallis. The sul2 gene was predominant among sulfamethoxazole-resistant isolates, for 56% of 431 E. coli and 53% of 66 Salmonella Corvallis; the sul1 gene was observed in 54% of Salmonella Albany. The Tet(A) resistance gene was prevalent in E.coli(86%), Salmonella Corvallis(82%), Salmonella Kentucky(84%). High percentages of gyr A genes observed among nalidixic-acid resistant E. coli(91%), Salmonella Albany(92%), Salmonella Corvallis(75%) and Salmonella Kentucky(85%).Conclusions: Important occurrences of resistance gene were observed among E. coli and Salmonella in chicken food chains in Cambodia.

Effects of NPY Gene on Total Number of Eggs at 300 Days of Age in Donglan Black-bone Chicken

In this study, PCR-RFLP technique was employed to detect the genetic polymorphism of NPY gene and analyze the effects of various genotypes on the total number of eggs at 300 days of age in 135 Donglan black-bone chicken. According to the results, there were three genotypes （AA, AB and BB） of NPYgene in Donglan black-bone chicken group. Different genotypes exhibited significant effects （P 〈 0. 05 ） on the total number of eggs at 300 days of age. The total number of eggs at 300 days of age of AA genotype was significantly higher than that of BB genotype （P 〈 0. 05）. Therefore, the polymorphic site of NPY gene could be used as a candidate molecular marker that affects egg laying in Donglan black-bone chicken.

Analysis of the Genetic Effects of Three Genes Involved in de novo Purine Biosynthesis on IMP Content in Baier Chickens

GPAT,AIRC and purH genes are involved in the de novo purine biosynthesis,and they all had significant effects on muscle inosine monophosphate(IMP) content in chickens.This study was designed to investigate the effects of GPAT,AIRC and purH genes on muscle IMP content in a relatively large population of Baier chickens;single genotype effects as well as the combined genotype effects were analyzed.The results further support that GPAT,AIRC and purH genes had effects on muscle IMP content;they might be candidate loci or linked to major genes affecting muscle IMP content.The combined genotypic effects were significantly higher than those of the single genotypes and were even larger when more gene loci were combined.We tentatively conclude that combined genotypes are superior to single genotypes as potential molecular markers for meat quality traits in chicken.

Haplotypes at the 5′-Flanking Region of Peroxisome Proliferator-Activated Receptor γ Gene and Their Association with the Growth and Body Composition Traits in Chickens

Peroxisome proliferator-activated receptor g（PPARg） is an important regulator of chicken preadipocyte proliferation and differentiation.In this study,polymorphisms were detected by DNA sequencing,PCR-RFLP and some other methods and three polymorphisms（g.-1784_-1768del17,c.-1241GA and c.-75GA） were found in the 5＇ flanking region of PPARg gene.Growth and body composition traits were measured in the 8th-10th generation populations of the Northeast Agricultural University broiler lines were divergently selected for abdominal fat content.Polymorphisms among individuals were screened in the above populations.The haplotype-based association analysis on growth and body composition traits was carried out.The association analysis showed that haplotypes based on three polymorphisms at 5＇ flanking region of PPARg gene were significantly associated with abdominal fat weight（AFW）,abdominal fat percentage（AFP,AFW/BW7）,liver weight（LW）,liver weight percentage（LFP,LW/BW7）,shank length（ShL）,femur weight（FeW）,keel length（KeL）,and metatarsus circle（MeC）（P0.05） and suggestive significantly associated with pectoralis major weight（PMaW）,pectoralis minor weight（PMiW）,pectoralis minor weight percentage（PMiWP,PMiW/BW7）,and metatarsus length（MeL）（P0.2）.The least square analysis showed that the birds with BGA haplotype had significantly higher AFW and AFP than the birds with other haplotypes（P0.05）.The birds with AAG haplotype had significantly higher LW and LW/BW than the birds with other haplotypes（P0.05）.The birds with AAG haplotype had significantly higher PMiW and PMiW/BW than the birds with other haplotypes（P0.05）.The birds with AAG haplotype had significantly higher ShL,FeW,MeL,MeC and KeL than the birds with AGG haplotypes（P0.05）.The results in this study revealed that QTL affecting fatness traits may exist in 5＇ flanking region of PPARg gene in chickens and PPARg gene might be one of the genes having important influences on the growth and bone traits in chickens.

Reproductive characteristics of transgenic (TG) chickens caping an exogenous gene

An exogenous gene (lacZ/MiwZ) introduced into the germinal crescent region (GCR) of avian embryos was con-firmed to be successfully transferred to the gonads via the primordial germ cells (PGCs). Following hatching, the chickswere raised until the stage of sexual maturation. The incorporation of MiwZ DNA was detected in male and female trans-genic chickens, respectively. The normal male and female transgenic birds were subjected to artificial insemination ac-cording to routine methods. Fertilized eggs obtained from female transgenic chickens were incubated for 72 h and the em-bryos removed from the yolk were examined by X-gal staining to detect the introduction of MiwZ in the offspring. As aresult, the expression of MiwZ was detected in the offspring. Furthermore, the presence of MiwZ in the extracts fromembryos was also detected by polymerase chain reaction (PCR) analysis. In male transgenic chickens, the presence of in-jected MiwZ in the extracts from sperm was also confirmed. The exogenous gene introduced into the GCR migrated suc-cessfully to the gonad resulting in its incorporation into the offspring and spermatozoa of transgenic chickens. (Asian JAndrol 1999 Sep ; 1: 139 - 144)