A consensus linkage map of the chicken genome.

A consensus linkage map has been developed in the chicken that combines all of the genotyping data from the three available chicken mapping populations. Genotyping data were contributed by the laboratories that have been using the East Lansing and Compton reference populations and from the Animal Breeding and Genetics Group of the Wageningen University using the Wageningen/Euribrid population. The resulting linkage map of the chicken genome contains 1889 loci. A framework map is presented that contains 480 loci ordered on 50 linkage groups. Framework loci are defined as loci whose order relative to one another is supported by odds greater then 3. The possible positions of the remaining 1409 loci are indicated relative to these framework loci. The total map spans 3800 cM, which is considerably larger than previous estimates for the chicken genome. Furthermore, although the physical size of the chicken genome is threefold smaller then that of mammals, its genetic map is comparable in size to that of most mammals. The map contains 350 markers within expressed sequences, 235 of which represent identified genes or sequences that have significant sequence identity to known genes. This improves the contribution of the chicken linkage map to comparative gene mapping considerably and clearly shows the conservation of large syntenic regions between the human and chicken genomes. The compact physical size of the chicken genome, combined with the large size of its genetic map and the observed degree of conserved synteny, makes the chicken a valuable model organism in the genomics as well as the postgenomics era. The linkage maps, the two-point lod scores, and additional information about the loci are available at web sites in Wageningen (http://www.zod.wau.nl/vf/ research/chicken/frame_chicken.html) and East Lansing (http://poultry.mph.msu.edu/).

Enhancement of spontaneous bursal lymphoma frequency by serotype 2 Marek's disease vaccine, SB-1, in transgenic and non-transgenic line 0 white leghorn chickens.

A significant incidence of bursal lymphomas with long latencies was noted in transgenic breeders carrying a benign defective subgroup A avian leukosis provirus, ALVA6, in their germline and maintained free of exposure to avian retroviruses. Serotype 2 Marek's disease (MD) vaccine virus, strain SB-1, a component of the bivalent MD vaccine used to vaccinate the breeders, was suspected as a contributory factor in the increased bursal lymphoma incidence. Although these bursal lymphomas had several characteristics similar to retroviral-induced bursal lymphomas, we found no evidence of retroviral influence based on many virological, immunological and molecular tests that were performed on plasma and tumour cells. These tumours were therefore classified as spontaneous bursal lymphomas, similar to those reported for some specific pathogen-free (SPF) chicken lines. Long-term in vivo experiments in plastic isolators and carefully maintained pens with homozygous and hemizygous ALVA6 and ALVA6-free female chickens (line 0) that were either non-vaccinated, serotype 3 (herpesvirus of turkeys [HVT]; monovalent)-vaccinated, or HVT/SB-1 (bivalent) vaccinated, demonstrated that the incidence of spontaneous bursal lymphomas were significantly higher in those chickens that were vaccinated with the bivalent MD vaccine (P ⩽0.05). In addition, this incidence did not depend on the ALVA6 proviral insert since there was no significant difference in spontaneous bursal lymphoma incidence between bivalent vaccinated hemizygous ALVA6 and ALVA6-free line 0 female chickens. Thus, the increased incidence of spontaneous bursal lymphomas is correlated solely with the presence of SB-1 and is not dependent on the presence of ALVA6.

Genetic mapping of the cloned subgroup A avian sarcoma and leukosis virus receptor gene to the TVA locus.

A chicken gene conferring susceptibility to subgroup A avian sarcoma and leukosis viruses (ASLV-A) was recently identified by a gene transfer strategy. Classical genetic approaches had previously identified a locus, TVA, that controls susceptibility to ASLV-A. Using restriction fragment length polymorphism (RFLP) mapping in inbred susceptible (TVA*S) and resistant (TVA*R) chicken lines, we demonstrate that in 93 F2 progeny an RFLP for the cloned receptor gene segregates with TVA. From these analyses we calculate that the cloned receptor gene lies within 5 centimorgans of TVA, making it highly probable that the cloned gene is the previously identified locus TVA. The polymorphism that distinguishes the two alleles of TVA in these inbred lines affects the encoded amino acid sequence of the region of Tva that encompasses the viral binding domain. However, analysis of the genomic sequence encoding this region of Tva in randomly bred chickens suggests that the altered virus binding domain is not the basis for genetic resistance in the chicken lines analyzed.

Response of chickens carrying germline insert ALVA11 to challenge with a field strain of subgroup A avian leukosis virus.

The ALVA11 germline insert in chickens is a defective subgroup A avian leukosis virus (ALV) proviral insert that expresses a low-to-moderate level of subgroup A ALV envelope glycoprotein. Chicks carrying or lacking ALVA11 were evaluated for response to challenge by RPL-42, a pathogenic field strain of subgroup A ALV, by either exposure to chicks shedding RPL-42 or direct injection with various doses of RPL-42. Chicks carrying ALVA11 were significantly more resistant, as measured by infectious virus and viral antibody status, to horizontal infection and direct injection of RPL-42 than chicks lacking ALVA11.

Development of an alloantiserum (R2) that detects susceptibility of chickens to subgroup E endogenous avian leukosis virus.

An alloantiserum, termed R2, specifically agglutinates red blood cells (RBC) from line 100B chickens that are susceptible to avian leukosis viruses (ALV) belonging to subgroups B and E, but does not agglutinate RBC from congenic inbred line 7(2) chickens that are resistant to ALV B and E. The R2 antigen was also detected on lymphocytes and thrombocytes. Using chickens from a special cross, it was found that R2 reactivity requires that the chickens must: (1) be susceptible to infection by ALV-E; and (2) express a viral envelope gene with subgroup E specificity. With R2 antiserum, a nearly perfect association was observed between agglutination and susceptibility to ALV-B in F2 chickens containing endogenous viral genes ev2 and/or ev3. These results support earlier evidence that ALV-B and ALV-E share receptors. Moreover, the R2 antiserum was shown to neutralize ALV-E. The R2 antigen showed Mendelian segregation in chickens of a commercial White Leghorn strain-cross containing ev3, ev6 and ev9. However, commercial chickens with or without the R2 antigen did not differ in susceptibility to lymphoid leukosis induction or immune response on infection with ALV of subgroup A for complex reasons we discuss.

RFLP mapping of expressed sequence tags in the chicken.

Chicken cDNA probes were used to map 10 loci by restriction fragment length polymorphism (RFLP) analysis in progeny of the Jungle Fowl x White Leghorn chicken backcross gene mapping reference population. The cDNA probes were from a subset of a T-cell library whose sequence tags showed significant homology to other avian or mammalian sequences in the EMBL database. Placement of loci generated by Compton cDNA clones on the East Lansing linkage map contributes to development of a consensus from the two linkage maps and will help provide a framework to evaluate the phenotypic effects of these genes.

Genetic and physical mapping of the chicken IGF1 gene to chromosome 1 and conservation of synteny with other vertebrate genomes.

The chicken insulin-like growth factor 1 gene has been assigned to the short arm of chromosome 1 near the centromere by fluorescence in situ hybridization and genetic linkage analysis. Comparison of physical and genetic linkage maps locates the centromere between the IGF1 and GAPD loci. Comparison of the genetic maps of chicken and other vertebrates reveals a highly conserved syntenic group, including the GAPD-IGF1 loci.

Development of a genetic map of the chicken with markers of high utility.

Microsatellites are tandem duplications with a simple motif of one to six bases as the repeat unit. Microsatellites provide an excellent opportunity for developing genetic markers of high utility because the number of repeats is highly polymorphic, and the assay to score microsatellite polymorphisms is quick and reliable because the procedure is based on the polymerase chain reaction (PCR). We have identified 404 microsatellite-containing clones of which 219 were suitable as microsatellite markers. Primers for 151 of these microsatellites were developed and used to detect polymorphisms in DNA samples extracted from the parents of two reference populations and three resource populations. Sixty, 39, 46, 49, and 61% of the microsatellites exhibited length polymorphisms in the East Lansing reference population, the Compton reference population, resource population No. 1 (developed to identify resistance genes to Marek's disease), resource population No. 2 (developed to identify genes involved in abdominal fat), and resource population No. 3 (developed to identify genes involved in production traits), respectively. The 91 microsatellites that were polymorphic in the East Lansing reference population were genotyped and 86 genetic markers were eventually mapped. In addition, 11 new random amplified polymorphic DNA (RAPD) markers and 24 new markers based on the chicken CR1 element were mapped. The addition of these markers increases the total number of markers on the East Lansing genetic map to 273, of which 243 markers are resolved into 32 linkage groups. The map coverage within linkage groups is 1,402 cM with an average spacing of 6.7 cM between loci. The utility of the genetic map is greatly enhanced by adding 86 microsatellite markers. Based on our current map, approximately 2,550 cM of the chicken genome is within 20 cM of at least one microsatellite marker.

Chicken genome mapping: a new era in avian genetics.

More than 460 loci representing either expressed or anonymous sequences have been mapped on to the first comprehensive molecular genetic linkage map of the chicken genome. Here, we review the current status of poultry genome mapping and discuss some of the new opportunities this provides.

Influence of the alv6 recombinant avian leukosis virus transgene on production traits and infection with avian tumor viruses in chickens.

The biological costs of the alv6 recombinant transgene that in chickens induces dominant resistance to the subgroup A avian leukosis virus (ALV), in terms of effects on production traits, were studied. Four generations of White Leghorn chickens of Line TR, segregating for alv6 but free of endogenous viral genes, as well as two generations of crosses between TR and Ottawa Line WG (WGTR) were tested under a specific-pathogen-free environment. In the birds studied, the transgene appeared unchanged compared to the original alv6: No major changes in alv6 DNA were detected by restriction analysis, the transgene did not express the group-specific antigen of ALV, and its presence was associated with absence of immune response to ALV. In most test years, and both TR and WGTR genomic backgrounds, alv6 was associated with delayed sexual maturity by 4 to 6 d, reduced egg production to 497 d of age by 20 to 46 eggs, and a 3.6 to 15% decline in egg production rate. No consistent effects on other traits, including mortality, were detected. When inoculated with the AC-1 isolate of Marek's disease virus in a separate experiment, TR birds with alv6 had a significantly lower body weight gain to 10 d of age than their sibs without the transgene. Thus, transgenesis has biological costs that have to be assessed against desirable effects of transgenes.

Microsatellite markers for genetic mapping in the chicken.

Microsatellite markers have been found to be abundant, evenly distributed, and highly polymorphic in a number of eukaryotic genomes. The objective of this study was to determine the utility of (TG)n microsatellites in the chicken. A chicken library enriched for (TG)n repeats was generated and 42 unique clones containing (TG)n microsatellites were identified and sequenced. The number of uninterrupted TG repeats ranged from 4 to 14 with an average of 7.8, which was considerably less than the number of repeats found in mammalian species. When primers designed to amplify across the (TG)n microsatellites were used in polymerase chain reactions (PCR) containing genomic chicken DNA, 19 of the 33 primer sets examined yielded polymorphisms in at least one of the three sets of chicken families: 15, 11, and 11 primer sets detected polymorphisms in the East Lansing (EL) reference population, the Compton (C) reference family, and between Line 63 and Line 72 chickens, respectively. The polymorphic microsatellite markers in the EL and C reference families were genetically mapped. Nine and seven mapped markers in the EL and C reference families, respectively, are polymorphic between Line 63 and Line 72, indicating that microsatellite markers will greatly enhance the ability to genotype specific loci of any chicken population.

Mapping DNA polymorphisms using PCR primers derived from the sequence of an avian CR1 element.

Primers complementary to the chicken middle repetitive sequence element CR1 were used to generate and simultaneously map polymorphic polymerase chain reaction products (CR1-PCR) with various chicken DNAs as templates. Ten primers were prepared using the sequence of a single CR1 element as a guide. These 10 primers generated 23 polymorphic CR1-PCR products. The average number of polymorphic CR1-PCR products generated using single primers (1.1 per primer) was significantly higher than the average number observed using combinations of two primers (0.3 per primer combination). The polymorphic CR1-PCR products were mapped in a subset of a reference backcross population designed for the genetic linkage analysis of the chicken. Nineteen of the polymorphic CR1-PCR products identified were assigned to 13 of 19 linkage groups characterized thus far in this population; three have yet to be linked to a specific map location. One of the CR1-PCR markers mapped to the chicken Z chromosome. There was no evidence for a significant clustering of CR1-PCR markers within the map, even at the site of the CR1 element whose sequence was used for primer design.

An autosomal genetic linkage map of the chicken.

We have developed an autosomal genetic linkage map of the chicken genome using a subpanel of 52 DNAs from a previously described reference backcross mapping population. The population derived from a cross of an inbred Red Jungle Fowl male and a highly inbred White Leghorn female. The backcross subpanel used was made up of offspring of a single F1 male with four White Leghorn females. Ninety-eight markers consisting of classical and erythrocyte antigen genes, restriction fragment length polymorphisms, random amplified polymorphic DNA, and chicken CR1 repeat-element polymorphisms were typed. Seventy-two of these markers were resolved into 19 linkage groups. Four of the linkage groups were assigned to chromosomes 1, 4, and 17. Four linkage groups were associated with linkage groups published earlier. Linkages within approximately 27 cM can be detected with a lod score of 3 with the panel used. The preliminary map contains approximately 590 cM within the linkage groups, and approximately 70% of the randomly selected markers fell in one of the groups; however, a considerable portion of the genome may remain outside of the existing linkage groups. These markers greatly expand the existing linkage map of the chicken genome.

Sequence-tagged microsatellite sites as markers in chicken reference and resource populations.

Two chicken genomic libraries were screened for the presence of poly(TG/AC) microsatellite tracts. The number of positive clones was low, confirming the low frequency of such microsatellites in the chicken genome relative to mammalian genomes. Polymorphism of 29 microsatellite tracts, comprising 11 from the library screening and 18 obtained from GenBank, was examined in the East Lansing and Compton reference families, in a resource population formed by a cross between a single White Rock broiler and inbred Leghorn females, and in a panel of birds from five layer stocks. Twenty microsatellites, primarily of the poly(TG/AC) type, were polymorphic in at least one of the populations. Thirteen of the microsatellites were polymorphic in the East Lansing reference family and 13 were also polymorphic in the resource population, confirming that the genetic distance between White Rock and White Leghorn is about as great as between Jungle fowl and White Leghorn. Only six microsatellites were polymorphic in the Compton reference family, formed by a cross between two White Leghorn strains. Twelve of the microsatellites were mapped in the East Lansing and/or Compton reference families. These were well dispersed among the various linkage groups and did not show any indications of terminal clustering.

A new defective retroviral vector system based on the Bryan strain of Rous sarcoma virus.

We have constructed a helper cell line and vector system based on the Bryan high titer (BH) strain of Rous sarcoma virus (RSV). BH-RSV is a defective virus which lacks an env gene; however, if env is supplied in trans, it replicates to a very high titer. Like BH-RSV, the vector contains gag and pol genes and lacks an env gene. The helper cell line supplies env in trans and permits the production of infectious virions. To construct the helper cell line the subgroup A env gene from the Schmidt-Ruppin-A (SRA) RSV was stably transfected into Qt6 cells, a chemically transformed quail fibroblast line. To minimize homology between the vector and helper cell line, transcription of the env gene is driven by a MuLV LTR, and 3' processing is controlled by the simian virus 40 (SV40) polyadenylation signal. This combination of vector and helper cells can be used to produce high-titer viral stocks in which recombinant replication-competent virus have not been detected even when the stocks were used to inoculate chickens. This system should be useful for developing transgenic chickens, studying cell lineage, and introducing genes into cultured cells.

Genetic map of the chicken Z chromosome using random amplified polymorphic DNA (RAPD) markers.

Commercially important traits of domestic animals have often been genetically linked to sex chromosomes, such as the Z chromosome of chickens. Using a backcross mapping population between two divergent, inbred lines and random-amplified polymorphic DNA (RAPD)-PCR markers, a genetic map of the chicken Z chromosome has been generated. Thirteen Z-linked RAPD markers were identified, mapped, and linked to two RFLPs and one phenotypic marker. The protocol used also generated RAPD markers for the W chromosome. The linkage distances obtained suggest that the RAPD markers are widely distributed throughout the Z chromosome and are likely to be linked to most or all traits of interest on this chromosome. The map provides a preliminary estimate of genetic to physical distance of about 0.5 Mb per centimorgan for the Z chromosome in chickens (male-specific recombination). A similar approach should be applicable to facilitate the mapping and analysis of sex-linked traits in other domestic animals.

A transgene, alv6, that expresses the envelope of subgroup A avian leukosis virus reduces the rate of congenital transmission of a field strain of avian leukosis virus.

A major mode of transmission of avian leukosis virus (ALV) is from a dam that is viremic with and immunologically tolerant to ALV, through the egg to the progeny. The authors have produced a line of chickens transgenic for a defective ALV provirus that expresses envelope glycoprotein, but not infectious virus, and is very resistant to infection with Subgroup A ALV. In the present experiment the authors sought to prevent or reduce congenital transmission by mating viremic-tolerant hens to males carrying the inserted provirus, thus introducing a gene for resistance into the progeny. Mature viremic females were mated with males hemizygous for the transgene to produce over 80 progeny each with and without the transgene. The chicks were hatched and maintained for 36 wk and observed for viremia, antibody, and the incidence of bursal lymphomas. Over 90% of the transgene-negative controls remained viremic through 36 wk of age and 51% developed bursal lymphomas. In contrast, 27% of the transgene-positive birds remained viremic and 18% died with bursal lymphomas. Thus, expression of Subgroup A envelope protein in the developing embryo reduced but did not eliminate congenital infection.

The influence of ev6 on the immune response to avian leukosis virus infection in rapid-feathering progeny of slow- and rapid-feathering dams.

Endogenous virus (EV) locus ev6 encodes only virus envelope glycoprotein. The influence of ev6 on the immune response to contact infection with hatchmates infected with avian leukosis virus (ALV) was compared in replicate hatches. The ALV Subgroup E-resistant, rapid-feathering (RF) female chickens produced by slow-feathering (SF) and RF dams with and without ev6 were exposed at hatch to hatchmates infected with ALV Subgroup A (Strain RPL-40). The RPL-40 viremia, shedding, and virus neutralizing antibodies were measured among pullets from two hatches at 22 wk of age. Although significant (P less than .05) differences between hatches in the immune response to contact infection were noted among ev6+ pullets, significantly fewer ev6+ pullets seroconverted than their ev6- hatchmates. At 22 wk of age, significantly more lymphomas were also found among ev6+ pullets than among ev6- hatchmates. In flocks wherein both parents and progeny were homozygous resistant to Subgroup E virus, there was no deterimental maternal effect on RF progeny from SF dams that carried ev21. These results also confirm that selection for genetic cellular resistance to Subgroup E ALV infection eliminates congenital transmission of EV21.

Endogenous viral genes: association with reduced egg production rate and egg size in White Leghorns.

Endogenous viral (ev) genes are DNA sequences residing permanently in the genome of most chickens that have a high degree of homology to avian leukosis viruses (ALV). Association of ev genes with production trait differences was studied in White Leghorns free of exogenous ALV. The Cornell Strain K and S chickens used in Experiment 1 had multiple ev genes. In each of four lines of chickens in Experiment 2, there was a 1:1 segregation of full-sibs free of ev genes and those carrying one ev gene: ev-12 that produces the complete endogenous virus, ev-3 or ev-6 that express certain viral antigens, or ev-1, a silent gene. In Experiment 1, the presence of genes ev-10 or ev-19, known to produce the complete virus, was associated with a 9% reduction in the annual egg production rate (P less than .05) in Strain S. Similarly, the presence of the virus-producing ev-12 in Experiment 2 was associated with an 8% reduction of annual egg production rate (P less than .05), a 2.2-g reduction in egg weight (P less than .01), and a .003 reduction in egg specific gravity (P less than .01). No significant effects of ev genes on age at first egg, Haugh unit score, percentage of eggs with blood spots, and body weight of hens were observed. It was concluded that ev genes producing complete endogenous virus are associated with production trait differences similar to those associated with subclinical infections with exogenous ALV.

Experimentally introduced defective endogenous proviruses are highly expressed in chickens.

We have previously described the experimental introduction of recombinant subgroup A avian leukosis viruses (ALV) with Rous-associated virus 0 long terminal repeats into the germ line of line 0 chickens and the generation of 23 transgenic lines. Two of these transgenic lines, alv6 and alv11, do not produce infectious virus. Both of these lines contain defective proviruses but do express the gag and/or env protein. We have measured viral RNA expression in tissues derived from alv6, alv11, and the parental line 0. Total RNA was prepared from 9-day embryo, 16-day embryo, 1-day chicken, and 28-day chicken tissues. Viral RNA was detected by Northern RNA transfer analysis. The results indicate that both alv6 and alv11 chickens express viral RNA in all tissues tested regardless of the stage of development. No viral transcripts were detected in any line 0 (C/E; ev-negative) tissue. The levels of biologically active env glycoprotein correlates with the env RNA levels in both lines. In an in vivo interference assay, alv6, alv11, and line 0 chickens were infected with Rous-associated virus 1 and monitored for viremia, antibody against Rous-associated virus 1, and ALV-induced pathogenesis from 4 to 21 weeks. None of the 61 alv6 chickens contained detectable virus or produced antibody against subgroup A ALV. Virus and/or antibody against subgroup A ALV was detected in 34 of the 43 alv11 chickens, whereas 51 of 52 line 0 birds were viremic and/or produced antibody. ALV-induced pathogenesis was observed predominantly in line 0 chickens (10 of 59), whereas very little ALV-induced pathogenesis was seen in either alv6 (1 of 62) or alv11 (1 of 44) chickens. Presumably the mechanism for the increased resistance of alv6 and alv11 chickens was subgroup-specific receptor interference. These results clearly demonstrate that experimentally introduced endogenous proviruses can be expressed at high levels in the avian system.