In Silico Biology 5, 0053 (2005); ©2005, Bioinformation Systems e.V.  

Identification of candidate genes at quantitative trait loci on chicken chromosome Z using orthologous comparison of chicken, mouse, and human genomes


Georgina A. Ankra-Badu and Samuel E. Aggrey*




Poultry Genetics and Biotechnology Laboratory, Department of Poultry Science and Institute of Bioinformatics
University of Georgia, Athens, GA 30602-2772, USA



* Corresponding author

   Department of Poultry Science, University of Georgia, Athens, GA 30602-2772
   Email: saggrey@uga.edu
   Phone: +1-706-542 1354; Fax: +1-706-542 1827





Edited by E. Wingender; received June 28, 2005; revised August 25/October 27, 2005; accepted October 27, 2005; published January 04, 2006



Abstract

This study was undertaken to identify novel candidate genes at quantitative trait loci (QTL) on chicken chromosome Z (GGAZ) by comparing orthologous regions of chicken, human and mouse genomes. Primer sequences from marker flanking QTL positions (https://acedb.asg.wur.nl/) were obtained from www.iastate.edu/chickmap and blasted against the chicken genome (www.ensembl.org) using BLASTN. The best matches were those with the highest score, lowest E-values and highest percent identity. Orthologous regions in mice and humans, together with genes located on or around those loci were identified using the Ensembl website. Forty-six chicken genes, 91 mouse genes and 60 human genes associated with QTL on GGAZ were identified in the current study. Among the most promising candidate genes for egg production and egg shell quality are annexin A1 (ANXA1), osteoclast stimulating factor (OSF), thrombospondin-4 (THBS4), programmed cell death proteins (PDCD), follistatin (FST), growth hormone receptor (GHR), interferon (IFN) α and β. The chicken IFN α and β were located on GGAZ around position 13,000,000 bp on the draft chicken sequence map. The neuronal nicotinic acetylcholine receptor (nAChR) is located at a QTL region for abdominal fat (GGAZ 25483091 bp). Nicotine is an agonist at the nAChRs and has been shown to decrease lipolysis and triglyceride uptake, thereby reducing net storage in adipose tissue. Therefore, the nAchRs could be used as therapeutic targets for regulating feed intake and obesity. This study has identified 197 putative candidate genes in probable QTL regions of chicken chromosome Z.

Keywords: candidate genes, quantitative trait loci, chicken chromosome Z, comparative mapping



Introduction

Traditional methods for genetic improvement in farm animals and poultry usually depend on Mendelian principles and quantitative genetics. With these methods the breeding value of an individual is determined either by its phenotype or by the average performance of its relatives [Falconer, 1960; Falconer and Mackay, 1996]. The expected genetic gain would depend on the additive genetic variance associated with the trait. When the heritability of a trait is high, the phenotype becomes a good predictor of an animal's breeding value and artificial selection results in rapid gains. However, genetic gains in lowly heritable traits have been limited. Breeding values on sires for sex-limited traits such as egg or milk production can only be determined from data from female progeny, and that requirement can prolong the generation interval.

Knowledge about genes that affect traits allows the breeder to manipulate the genome of an animal to ensure that the best combinations of alleles in the parental population are transmitted to the progeny. Marker assisted selection, which involves the use of molecular markers for genetic improvement, is one method to achieve this. A genetic marker is a phenotypically recognizable trait that can be used to identify a genetic locus, linkage group or recombination event [Aggrey and Okimoto, 2003]. Molecular markers can also be used to generate genetic maps of many species and combined with trait measurements to determine locations on chromosomes harboring major genes or quantitative trait loci (QTL) that affect traits of economic importance [van Kaam et al., 1998]. Trait loci generated by genome scans do not provide information on the linkage phase between the marker alleles and QTL, the number of genes within the QTL location nor the nature of gene interactions at that location. Therefore, selection on QTL or markers associated with QTL could result in less then expected gains. To gain insight into the molecular basis of QTL, it is desirable to identify the actual genes responsible for the QTL. Candidate gene analysis has been used as an alternative method in characterizing genes with a known function and their association with traits of economic importance [Kuhnlein et al., 1997; Aggrey et al., 1999; Causse et al., 2004]. Generally, the functional candidate gene approach is not adequate without the use of positional information from gene mapping studies [Haley, 1999]. Examination of genes at QTL locations could lead to the identification of novel genes and could also narrow the number of genes to be analyzed.

Although the chicken genome is estimated to have about 20,000-23,000 genes [Hillier et al., 2004], only about 400 human gene orthologues have been mapped [Burt and Hocking, 2002]. In spite of the tremendous achievement of a first draft, there are still some gaps, and these are particularly important to the Z chromosome. Comparative mapping of the chicken with other vertebrates has the potential to unravel information about new potential genes and to identify homologous chromosomal segments in distantly related species [Johansson et al., 1995]. Through comparative mapping, potential candidate genes for hypertension in rats were uncovered for the same disease in humans [Rapp et al., 1989; Jacob et al., 1991]. O'Brien and Nash, 1982), mapped 31 cat genes whose homologs have been previously mapped in humans and mice.

The chicken genome has been found to have high levels of synteny with the human and mouse genomes even though these species diverged from each other more than 300 million years ago [Burt and Hocking, 2002]. Information derived from the human genome can be used to resolve unanswered questions involving the chicken genome [Suchyta et al., 2001]. The chicken double sex and mab related transcription factor 1 (DMRT1), for example, was isolated by identifying its homolog in humans [Nanda et al. 2000]. Ladjali-Mohammedi et al., 2001, localized four homeobox genes in a study that identified new sections of conservation between humans and chicken. Smith et al., 2000, mapped the chicken rip associated ICH-1 homolog protein with a death domain (RAIDD) by identifying its homolog in the mouse. Therefore, additional chicken genes can be identified through comparative mapping with other species.

The objective of this study was to identify novel candidate genes at QTL locations on Gallus gallus chromosome (chr) Z (GGAZ) by comparing orthologous regions of chicken, human and mouse genomes. Genetic markers in such novel genes could be used to aid in the genetic improvement of sex-limited traits or traits with low heritability.



Materials and methods

Information on QTL on GGAZ was obtained from https://acedb.asg.wur.nl/. Data on loci and markers on GGAZ were selected by generating a list of all the markers on the chromosome using information from www.genome.iastate.edu/chickmap. The consensus 2000 chicken linkage map (www.genome.iastate.edu/chickmap), which is a combination of mapping data from the East Lansing, Compton and Wageningen chicken populations, was used to locate the position of each marker. Comparative mapping was done by blasting the primer sequence of the markers against the chicken genome sequences in the Sanger Institute website (www.ensembl.org) using the basic local alignment search tool (BLASTN) for comparing a nucleotide query sequence against a nucleotide sequence database. A score is given for matching and mismatching nucleotides and gaps. The total score is given by obtaining the sum of all matches, mismatches and gap penalties for sequence. The E-value or expect score is the number of different values that are equivalent to or better than the score that are expected to occur in a database by chance. The percent identities refer to the extent to which sequences are invariant (www.ncbi.nlm.nih/gov). A score of more than 45, a percentage identity of greater than 70%, and an E-value less than 0.05 are considered to be significant [Pertsemlidis and Fondon, 2001; Jiang and Michal, 2003]. The best matches were those regions that had the highest scores, lowest E-values and highest percentage identities. Each matching sequence was then compared with the mouse and human genome sequences to identify regions of homology. Information on genes at or around the QTL location on GGAZ and their respective homologs in humans and mouse were obtained from www.ensembl.org by identifying genes within homologous regions.



Results and discussion

Quantitative trait loci and their flanking markers on GGAZ are presented on Table 1. Loci for growth, body weight and abdominal fat were found at positions 22 [Kerje et al., 2003], 96 [Sasaki et al., 2004], and 127cM (95% CI: 56-127 cM) [Ikeobi et al., 2002], respectively on the genetic map. QTL for age at first egg were located on positions 22 [Sasaki et al., 2004] and 63-104 cM (90% C.I.: 65-137cM) [Tuiskula-Haavisto et al., 2002]. Quantitative trait loci for egg shell strength and thickness were also identified by Sasaki et al., 2004, at position 36 and 47 cM, respectively. Loci for other egg production traits (egg shell thickness, egg shell weight, egg number and egg weight) were identified at positions 63-104 cM [Tuiskula-Haavisto et al., 2002]. Zhou et al., 2003, found a QTL for antibody response to Brucella (B) abortus on position 28.

Table 1: Probable quantitative trait loci locations on chicken GGAZ and their flanking markers.
Trait Location1 (bp) Location2(cM) Flanking markers Reference
Growth (112-200d) 1,880,642 22 MCW0055 Kerje et al., 2003
Body weight (200d) 11,070,329 22 ADL0273 Kerje et al., 2003
Age at first egg (day) 14,783,516-15,457,996 28 ADL0201-MCW0241 Sasaki et al., 2004
Antibody response to Brucella abortus 14,783,516-17,635,971 28 ADL0201-ADL0250 Zhou et al., 2003
Egg shell thickness 16,538,085 36 LEI0229 Sasaki et al., 2004
Egg shell strength 16,770,587-19,749,500 47 MCW0154-LEI0254 Sasaki et al., 2004
Age at first egg (day) 5,209,429-15,297,279 63-104 MCW258-MCW246 Tuiskula-Haavisto et al., 2002
Egg weight (40 wk) 5,209,429-15,297,279 63-104 MCW258-MCW246 Tuiskula-Haavisto et al., 2002
Egg weight (40-60 wk) 5,209,429-15,297,279 63-104 MCW258-MCW246 Tuiskula-Haavisto et al., 2002
Egg shell strength (40 wk) 5,209,429-15,297,279 63-104 MCW258-MCW246 Tuiskula-Haavisto et al., 2002
Egg number (41- 60 wk) 5,209,429-15,297,279 63-104 MCW258-MCW246 Tuiskula-Haavisto et al., 2002
Body weight (239 d) UN3 96 LEI0075-LEI0123 Sasaki et al., 2004
Abdominal fat weight 22,950,349-31,399,653 127 LEI0111-LEI0075 Ikeobi et al., 2002
Abdominal fatness 22,950,349-31,399,653 127 LEI0111-LEI0075 Ikeobi et al., 2002
1Sequence map
2Genetic map
3Unassigned

Table 2 shows homologous regions between the QTL and the mouse and human genomes. The QTL regions on GGAZ show conserved synteny with mouse (MMU) chr 4, 13 and 19, and human (HSA) chr 5 and 9. The synteny between chicken and human for this QTL region is consistent with Nanda et al., 2002, who indicated that GGAZ and HSA 5 and 9 must have diverged from a common ancestor. Table 3 provides a list of the genes at probable chicken QTL regions and their homologs in mice and humans. Forty-six chicken genes, 91 mouse genes and 60 human genes were identified in this study and selected genes of interest were categorized by Gene Ontology (GO) annotation [Ashburner et al., 2000].

Table 2: Orthologous comparison of probable quantitative trait loci locations on GGAZ with mouse and human genomes.
Trait Location
GGA MMU HSA
Growth 22 13D2.1 5q11.2
Body weight 22 13D1 5q11.2-q14.3
Age at first egg 28 4C3 9p23-p21.3
Antibody response to Brucella Abortus 28 19C1 9p21.3
Egg shell thickness 36 NH NH
Egg shell strength 47 19B 9q21.13
Age at first egg(day) 63-104 4C3-C4 5p12
Egg weight (40 wk) 63-104 4C3-C4 5p12
Egg weight (40-60 wk) 63-104 4C3-C4 5p12
Egg shell strength 63-104 4C3-C4 5p12
Egg number (18-40 wk) 63-104 4C3-C4 5p12
Egg number (41-60 wk) 63-104 4C3-C4 5p12
Body weight (239d) 96 NH NH
Abdominal fat weight 127 4B3 5q14.3
Abdominal fatness 127 4B3 5q14.3
NH = No homology

The QTL region for antibody resistance to B. abortus and egg shell strength contained annexin A1 (ANXA1) and nuclear orphan receptor ROR-β (RORB). Annexins are involved in the biological processes of arachidonic acid secretion, cell cycle and signal transduction. They are calcium regulated phospholipid and membrane-binding proteins [Rescher and Gerke 2004]. ANXA1, a member of the annexin family, is an important endogenous modulator of inflammation [Yona et al., 2005] and is actively expressed in lymphoid tissues. However, its direct involvement in resistance against or susceptibility to B. abortus in chicken is not known. ANXA1 could be a candidate gene for egg shell quality since it has calcium binding properties and is secreted in the epithelial and endothelial lining of the endometrium [Bedford et al., 2003]. Other genes found in the QTL region for egg shell strength and other egg quality traits included the osteoclast stimulating factor (OSF1), riboflavin kinase (RFK) and the guanine nucleotide binding protein (GNB). OSF1 is a transcription factor (GO: 0003700). It enhances osteoclast formation and bone absorption through a cellular signal transduction cascade [Kurihara et al., 2001]. In humans, Kurihara et al., 2001, suggested that the OSF interaction with survival motor neuron (SMN) could be important in a novel signaling cascade that induces stimulators of osteoclast formation. Dodds et al., 1995, reported that osteoclasts control the deposition of osteopontin, which is present in the egg shell, bone and other hard tissues [Gautron et al., 2001]. Several chicken genes were found in the QTL region for egg traits, and they mostly control cell differentiation, embryonic development and immune response. They include thrombospondin 4 (THBS4), programmed cell death protein (PDCD), follistatin (FST) precursor and growth hormone receptor (GHR) all of which play key roles in cellular and biological processes. The THBS4 and GHR genes are also involved in molecular function and calcium binding and receptor activity, respectively. Orthologous regions in the mouse and human for the QTL harbor several IFN α genes. THBSs are a family of related calcium binding glycoproteins found in the embryonic extracellular matrix [Tucker et al., 1995] that are associated with tissue genesis and remodeling. The THBS4 promoter is similar to promoters of housekeeping, growth regulating, and other THBS genes which contain multiple GC box sequences and lacks a CAAT box. The presence of multiple E-box motifs is consistent with THBS4 expression in muscle and cartilage, tendon and bone tissue [Newton et al., 1999]. THBS4 could be a candidate gene for egg shell strength because it belongs to a group of calcium binding proteins that regulate tissue genesis [Lawler et al., 1995]. The human PDCD was found in the region homologous to egg related traits in chicken. PDCD is involved in the transcriptional regulation and biological processes of apoptosis. It controls cell death in the female germline of several species and is believed to remove defective cells unable to develop after fertilization [Buszczak and Cooley, 2000]. Programmed cell death ensures that viable eggs will receive nutrients, which could explain why this gene is associated with egg related traits. FST, a gene involved in female gonad development (GO: 0008585) and gametogeneis (GO: 0007276) suppresses the secretion of the follicle stimulating hormone (FSH) and reverses the effect of activin on oxytocin and progesterone thereby preventing degenerating effects on dominant follicles [Michel et al., 1993]. FST has the ability to bind the pleiotropic growth and differentiation factor activin, thereby neutralizing activin action. This glycoprotein is potentially an important regulatory factor, capable of modulating autocrine and paracrine functions which would alter differentiation and development [Farnworth et al., 1995]. GHR is associated with an array of production traits [Kuhnlein et al., 1997] and GHR variants have been shown to be associated with reproduction, growth and immune response [Feng et al., 1998]. These workers reported that selection for egg production in white leghorns had led to a co-selection of a GHR variant.

Table 3: Putative candidate genes around QTL locations on chicken GGAZ and their respective homologues in mouse and human.
Trait Probable location Putative candidate genes
GGA MMU HSA
Growth
Body weight
22 claustrin cAMP-specific 3',5'-cyclic phosphodiesterase 4D
transportin 1
mitochondrial ribosomal protein S27
microtubule-associated protein 1B
scamp 1
serine/threonine-protein kinase PLK2
Ras-related protein
mitochondrial ribosomal protein S2
microtubule-associated protein 1B
pentatricopeptide repeat domain 2
zinc finger protein 266
Antibody response to Brucella abortus 28 proprotein convertase PC6
nuclear orphan receptor ROR-β
tight junction protein
annexin A1 (annexin I) (lipocortin I)
annxexin A1 (lipocortin 1)
RNP particle component
transmembrane cochlear-expressed protein 1
aldehyde dehydrogenase 1A1
zinc finger protein 216
guanine deaminase
long transient receptor potential channel 3
transmembrane protein 2
annexin A1
Egg shell strength 47 annexin A1 (annexin I) (lipocortin I)
nuclear orphan receptor ROR-β
bZIP protein E4BP4
proprotein convertase PC6
transducin-like enhancer of split 4
40S ribosomal protein S15
trypsin II-P29 precursor
endometrial progesterone-induced protein
guanine nucleotide-binding protein, α-14 subunit
riboflavin kinase
IP63 protein
proprotein convertase subtilisin/kexin type 5 precursor
chorea-acanthocytosis homolog
guanine nucleotide-binding protein G (q), α subunit
β-1,3-galactosyl-O-glycosyl- glycoprotein β-1,6-N- acetylglucosaminyltransferase
forkhead box protein B2
transient receptor potential cation channel, subfamily M, member 6
osteoclast stimulating factor 1
vacuolar protein sorting 13A (chorein)
proprotein convertase subtilisin/kexin type 5
guanine nucleotide-binding protein, α-14 subunit
riboflavin kinase
guanine nucleotide binding protein (G protein) (β-1,6-N-acetylglucosaminyltransferase)
phosphoserine aminotransferase 1
Egg weight (40 wk)
Egg weight (40-60 wk)
Egg shell strength (40wk)
Egg number (18-40 wk)
Egg number (41-60 wk)
Age at first egg (day)
63-104
63-104
63-104
63-104
63-104
63-104
centromere protein H
thrombospondin-4
fibroblast growth factor 10
ZOV3 gene product
laminin and collagen receptor
NADH dehydrogenase
3-hydroxy-3-methylglutaryl-CoA reductase
claustrin
sodium/potassium/calcium exchanger 2 precursor
endophilin
growth hormone receptor precursor
hydroxymethylglutaryl-CoA synthase, cytoplasmic p52 pro-apototic protein
insulin protein ISL-1 gene enhancer
follistatin precursor
spindling polymerase (DNA directed) kappa
glycine dehydrogenase [decarboxylating], mitochondrial precursor40S ribosomal protein S6
bZIP protein E4BP4
multiple PDZ domain protein
RNP particle component
nuclear factor 1 B-type
zinc finger DHHC domain containing protein 21
cerberus 1 homolog
small nuclear RNA activating complex, polypeptide 3
lens epithelium-derived growth factor; weakly similar to hypothetical 71.7 kDa protein
basonuclin 2
adipophilin (Adipose differentiation-related protein)
ADAMTS-like protein 1 precursor (punctin)
solute carrier family 24 (sodium/potassium/calcium exchanger)
Ras-related GTP-binding protein
40S ribosomal protein S6 (phosphoprotein NP33)
cancer related gene-liver 1
myeloid/lymphoid or mixed lineage-leukemia translocation to 3 homolog
interferon β precursor
MKIAA1797 protein
α-interferon
interferon α 14
interferon α family, gene 12; interferon α 12
interferon α family, gene 13; interferon α 13;
interferon α 6T
limitin
interferon α family, gene 11
interferon α-1 precursor
interferon τ-1
interferon α-4 precursor
S-methyl-5-thioadenosine phosphorylase
cyclin-dependent kinase 4 inhibitor A
cyclin-dependent kinase 4 inhibitor B
zinc finger protein 352
L1Md-A13 repetitive sequence
RNP particle component (fragment)
5,6-dihydroxyindole-2-carboxylic acid oxidase precursor
growth hormone receptor precursor (GH receptor) (somatotropin receptor)
selenoprotein P precursor
small inducible cytokine A28 precursor
integrin α-1 (laminin and collagen receptor
hydroxymethylglutaryl-CoA synthase, cytoplasmic
NAD(P) transhydrogenase, mitochondrial precursor
annexin II receptor
collagen receptor
fibroblast growth factor-10 precursor
programmed cell death protein 9
potassium/sodium hyperpolarization-activated cyclic -nucleotide-gated channel 1
zinc finger protein 131
gene similar to embigin
insulin gene enhancer protein ISL-1
poly (ADP-ribose) polymerase family
molybdenum cofactor synthesis protein 2 large subunit
myosin tail domain containing protein
small nuclear RNA activating complex, polypeptide
PC4 and SFRS1 interacting protein 1
Abdominal fat weight 127 ORF2 protein
Limb expression 1
mature protein
acetylcholine receptor protein
putative α3 fucotransferase
pro--neuregulin 1 precursor (contains neuregulin which has- acetylcholine receptor inducing activity)
progesterone receptor binding protein
phosphodiesterase 6 β subunit
purpurin precursor
neuronalacetylcholine
receptor
ATP binding fructose biphosphate aldolase B cassette
creatine kinase, sarcomeric mitochondrial precursor
dihydrofolate reductase
cartilage link protein
RNP particle component
olfactory receptor 273
T-cell acute lymphocytic leukemia-2 protein homolog
olfactory receptor 270
cystatin and DUF19 domain-containing protein 1
olfactory receptor 275
fukutin
olfactory receptor 272
Snap3B protein
ATP-binding cassette, sub-family A (ABC1), member 1 gene
UV excision repair protein RAD23 homolog B
zinc finger protein 462
Kruppel-like factor 4
inhibitor of kappa light polypeptide enhancer in B-cells
catenin α-like 1
gene similar to brain protein
protein tyrosine phosphatase
glyceraldehyde 3-phosphate dehydrogenase
A-kinase anchor protein 2
polydomain protein
muscle, skeletal, receptor tyrosine kinase
adapter-related protein complex 3 β1 subunit
lipoma HMGIC fusion partner-like 2
junction-mediating and regulatory protein
single-stranded DNA-binding protein 2
thrombospondin 4 precursor cardiomyopathy associated
secretory carrier-associated membrane protein 1
dimethylglycine dehydrogenase, mitochondrial precursor
AASA9217
membrane-type 1 matrix metalloproteinase cytoplasmic tail binding protein-1
hyaluronan and proteoglycan link protein 1 precursor
Homer protein homolog 1
arylsulfatase B precursor developmentally regulated protein TPO1
zinc finger CCHC domain containing protein 9
cytosolic phosphoprotein DP58
DNA-repair protein XRCC4 (X-ray repair cromethyltransferase 2 cross-complementing protein 4
PAP associated domain containing 4
dihydrofolate reductase
Ras protein-specific guanine nucleotide-releasing factor 2
creatine kinase, sarcomeric mitochondrial precursor
cytoplasmic acetyl-CoA hydrolase 1
APG10 autophagy 10-like protein
ribosomal protein S23
DNA mismatch repair protein Ms
versican core protein precursor (large fibroblast proteoglycan)
developmentally regulated endothelial cell locus 1 protein


With the exception of IFN-γ and -ω, the entire IFN family located on MMU4 is homologous to the QTL region for egg traits. IFNs are part of the immune system that controls resistance to viral and bacterial infections and are known to exist in chicken embryo [Isaacs and Lindenmann, 1957]. Interferons are involved in extracellular space, defense response and cytokine activities. Kaspers et al., 1994, demonstrated that chicken IFN mediated the induction of MHC class II antigens on peripheral blood monocytes. In a study to determine the effect of interferons on chicks with Marek's disease, Volpini et al., 1996, demonstrated that the Marek's disease antigen is down regulated by IFNs and other cytokines. Nanda et al., 1998, mapped chicken IFN1 and IFN2, which are homologous to IFN-α and IFN-β respectively, to the short arm of the GGAZ at position p2.2-p2.4. Since the chicken orthologs of mouse IFN-α and IFN-β are located between 13,001,112 and 13,001,639 bp on GGAZ, the chicken IFN-α and IFN-β could be located at this region with a high probability. The location of IFN in the QTL region for egg traits could imply that disease resistance traits are also associated with egg related traits. Since some genes of the IFN family are transcription factors that regulate antigen expression, mutations of these genes could affect other genes that control immune response in the chicken. Therefore polymorphisms in these genes could control both egg production and immunity.

A homolog of the zinc finger protein, basonuclin 2 (BNC2), was found on MMU4 which is homologous to the QTL region for egg quality. BNC2 is a transcription factor that maintains cell proliferation and prevents terminal differentiation [Vanhoutteghem and Djian, 2004]. The strong conservation of BNC2 among vertebrates strongly suggests an important function, presumably as a regulatory protein of transcription. Claustrin (MAP1B), a keratin sulphate proteoglycan and collagen receptor (VLA-2 α chain) could also be novel candidate genes for egg shell strength. The eggshell is an ordered structure comprised of calcium carbonate deposits onto an organic matrix. It is made up of a mineralized portion (95%) and the organic phase (3.5%) [Gautron et al., 2001]. The non-mineralized portion of the egg shell matrix is made up of collagen while the mineralized portion contains keratin sulphate proteoglycans [Dennis et al., 2000]. The proteoglycans and egg white proteins are involved in the nucleation of calcite crystals on the outer membrane of egg shell [Gautron, 2001]. Chicken genes found at the QTL region for abdominal fat weight were sarcomeric creatine kinase (CKMT2), creatine kinase (CK), progesterone receptor binding protein (PGRBP) and the neuronal nicotine acetylcholine receptor (nAChR). Mouse genes in the homologous regions included fukutin (FKTN), olfactory receptors (OR), and tyrosine kinase (TXK). Fukutin and TXK are cellular components with TXK having a molecular function as well. CK has molecular functions and is also involved in biological processes. Human genes in the homologous regions included endosome associated protein (EA1) and CK. CK is a muscle-specific enzyme that plays an important role in energy transfer by catalyzing conversion of creatine to creatine phosphate with the expenditure of ATP. CK activity is especially high in tissues with high-energy transfer [Sattler and Furll, 2004] and could affect abdominal fat deposition by increasing energy utilization. Steroid hormones affect adipose tissue metabolism [Pedersen et al., 2003]. Pedersen et al., 2003, showed that progesterone counteracts the action of glucocorticoids, which increase central accumulation of fat tissue. The nAChR is located at position 25483091 bp at the QTL region for abdominal fat. Neuronal nAChRs are distributed throughout the central and peripheral nervous system. In muscle, AChRs are found exclusively at the neuromuscular junctions and are responsible for mediating the effects of nicotine [Li et al., 2003]. Nicotine is metabolized extensively into a series of metabolites. The physiological effects of nicotine are produced through its agonist interaction with the acetylcholine receptor.

Li and Kane, 2003, have shown that nicotine regulates appetite, body fat and weight gain in rats via upregulation of hypocretin (orexin) neuropeptide precursor (HCRT), neuropeptide Y (NPY), and leptin (LEP) in the forebrain areas. In the periphery, LEP is down-regulated while uncoupling protein 1 (UPC1) is up-regulated after nicotine administration. Nicotine has also been found to decrease lipolysis and triglyceride uptake hence reducing net storage in adipose tissue [Sztalryd et al., 1996]. The effect of nicotine and its agonism at both neural and muscular AChRs has led to the assumption that AchRs could be therapeutic targets for regulating feed intake and obesity [Li et al., 2003]. Therefore the chicken it is possible that AChRs could be associated with obesity.

In avian species, the constitution of sex chromosomes is ZZ for males and ZW for females. The genes located on the Z chromosome in females follow a sex-linked inheritance, whereas their male counterparts follow Mendelian inheritance. Evaluation of genes on GGAZ should be performed separately for both sexes to account for sex-linkage because no allele is transmitted by the dam to female progeny. There are limitations in comparing the current genetic and sequence maps, because the resolution of the genetic map is modest. This places wide confidence intervals on marker locations and on recombination rates resulting in a non-linear concordance between location in bp and position in cM.

Despite the limitations of the current genetic and genome sequence maps, the present study has demonstrated that comparative mapping can be utilized to identify novel candidate genes potentially associated with traits of economic importance. One hundred and fifty-one additional genes that could not be extracted from the chicken draft sequence were located through comparative mapping. Furthermore, differentially expressed genes from microarray experiments can potentially sort out the role of candidate genes that may be tightly linked together within the same QTL region. However, before any gene is considered as a candidate for genetic improvement programs, fine mapping must first be performed to ensure that these genes are indeed located within the confidence interval of the QTL position, secondly genetic markers have to be developed and their association with traits must be demonstrated in a population segregating for variants of the candidate gene.



Acknowledgements

This work was supported by a grant from the USDA-IFAFS Program (SEA) (00-52100-9614).




References