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Association of Endogenous Avian Viral and Endogenous Viral Genes with Feed Conversion and Six-Week Body Weight in Broilers
MOLECULAR BIOLOGY Association of Endogenous Avian Viral and Endogenous Viral Genes with Feed Conversion and Six-Week Body Weight in Broilers H.J.M. AARTS1-2 and F. R. LEENSTRA DLO-Institute for Animal Science and Health, Branch Beekbergen, Spelderholt 9, 7361 DA Beekbergen, The Netherlands
1995 Poultry Science 74:1022-1028
White Leghorns (Aarts et al, 1991) to 7.3 in meat-type chickens (Sabour et ah, 1992) are Endogenous avian viral (eav) and en- found. Within populations of noninbred dogenous viral (ev) genes are retrovirus- chickens, the individual ev genes are like elements that are found in the chicken found in low to intermediate frequencies. genome. The ev genes have been studied Consequently, the ev restriction fragment extensively (see reviews by Smith, 1987; length polymorphism (RFLP) patterns of Crittenden, 1991). They are thought to be individuals vary significantly within and randomly distributed in the chicken ge- between lines (Aarts et al, 1991, 1992; nome and are found in low to moderate Boulliou et al, 1991; Ronfort et al, 1991; copy numbers. On average, 2.5 copies in and Sabour et al, 1992). The eav genes, which are distantly related to ev genes, were first detected and isolated by Dunwiddie and Farras (1985) Received for publication September 19, 1994. and Dunwiddie et al (1986) by using Accepted for publication February 6, 1995. probes derived from gene-specific restric1 To whom correspondence should be addressed. 2 Present address: DLO-State Institute for Quality tion fragments of the avian sarcoma virus Control of Agricultural Products, Bomsesteeg 45,6708 (ASV). These eav genes represent a more conserved and more ancient retrovirus PD Wageningen, The Netherlands. INTRODUCTION
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ABSTRACT The consistency of the effect of selection on the frequencies of endogenous avian viral (eav) and endogenous viral (ev) specific restriction fragment length polymorphism (RFLP) bands was studied in two broiler lines selected from a single base population and in an F2 population derived from a reciprocal cross of both lines. One broiler line (FC line) was selected for low feed conversion ratio and the other line (GL line) was selected for high 6-wk body weight. In the F2 population, the band frequencies were determined in groups representing separate tails of the distribution of two production traits, namely, low feed conversion ratio between 29 and 42 d of age and body weight at 42 d of age. The F2 population consisted of 288 females belonging to 24 full-sib families. To rule out family effects, the tails for these production traits were composed by either the best or by the worst female performer for each trait in each full-sib family. In total, 29 Hindlll-eav, 34 Mspl-eav, and 21 BamHl-ev bands could be distinguished by RFLP analysis. This report describes the influence of selection on 11 potentially interesting bands. Two bands, the 9.5-kb HmdIII-eaz; and the 15-kb Mspl-eav band, which were found both in higher frequencies in the parental FC line, were also found in higher (P £ .05) frequencies in the F2 tail with a favorable feed conversion ratio. A third band, the 6.5-kb HindUL-eav band, present in lower frequencies in the parental GL line, was also present in lower (P < .05) frequencies in the F2 tail of birds with heavy body weight. (Key words: chicken, endogenous viral genes, endogenous avian viral genes, restriction fragment length polymorphism, production traits)
ENDOGENOUS VIRAL GENES AND PRODUCTION TRAITS
MATERIALS AND METHODS Chicken Lines and Crosses The FC and GL lines used in our experiments were selected from a broiler sire strain. This foundation strain descends from a commercial broiler sire flock (Leenstra et ah, 1986). The selection criterion for the FC line was low feed conversion ratio between 3 and 6 wk of age of individually housed chickens that consumed feed ad libitum. The GL line was selected for high 6-wk body weight of chickens that ate ad libitum and were housed in groups. Both lines were derived from the same 50 sires and 200 dams. In each generation, 30 to 50 sires and 100 dams were selected. In the 12th generation, FC and GL chickens were reciprocally mated to generate an Fj population. The number of parents involved was: 8 GL line males, 8 FC line males, 14 GL line females, and 13 FC line females. From the progeny, six males each of the GL x FC and FC x GL types were mated to two females each, alternating GL x FC and FC x
GL to form the F2. Four hatches were produced. The animals were reared on litter until 19 d of age and then they were housed in individual cages. The F 2 population consisted of 576 (288 females and 288 males) chickens equally distributed over 24 full-sib families. Measurements All F 2 animals were weighed individually at hatch and at 19 and 42 d of age. At 19 and 42 d they were weighed after a feed withdrawal of approximately 10 h. Feed intake was recorded from Day 19 to 42. Blood samples were taken at 29 d of age via the brachial vein, mixed with EDTA anticoagulant, and stored at -20 C for DNA analysis. For the parental lines, blood samples were taken only from the parents of the reciprocal cross. Composition of the Tails of the Distribution of the F2 Population It was decided to use only females for this experiment, as female broilers have in general fewer leg problems and metabolic disorders than male broilers. Consequently, in females, body weights and feed efficiencies (especially those in the light body weight and unfavorable feed conversion groups) are less confounded with disorders that restrict inherent performance. To eliminate family effects, the two extreme groups for each production trait were composed by either the best or worst female performer of each of the 24 full-sib families. These four extreme groups of birds will be referred to as: Tail GL(+) = best performers in body weight; tail GL(-) = worst performers in body weight; tail FC(+) = best performers in feed conversion; tail FC(-) = worst performers in feed conversion. The feed conversion ratios of the FC(+) tail ranged from 2.046 to 1.655 and of the FC(-) tail from 2.343 to 1.856. The 6-wk body weight of the GL(+) tail ranged from 1,821 to 2,183 g and of the GL(-) tail from 1,227 to 1,818 g. DNA Isolation Fifty microliters of blood was lysed for 5 min on ice in 2 mL of Lysis buffer (10 mM
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family and exist at 40 to 100 copies per haploid chicken genome (Dunwiddie et ah, 1986; Resnick et ah, 1990). The eav genes are characterized by unusually short long terminal repeats (Boyce-Jacino et ah, 1989). Although there is some evidence for deleterious (Crittenden et ah, 1982) or advantageous (Robinson et ah, 1981) effects of ev genes to the fowl, they are assumed to be neutral under normal conditions (Bumstead et ah, 1987). In lines selected for production traits, disease resistance, or immune response, however, changes in gene frequency in comparison to control lines were observed Kuhnlein et ah, 1989a,b; Gavora et ah, 1991; Lamont et ah, 1992). This suggests that ev genes could be used as molecular markers in poultry breeding. The advantages and disadvantages of the use of ev gene probes compared to the usual diallelic single-copy RFLP and fingerprint probes have been reviewed by Crittenden (1991). In this report, we describe the use of two multilocus probes, each specific for a different class of ev genes, to find evidence for association of ev or eav genes with the production traits feed conversion and 6-wk body weight in broilers.
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To determine the frequencies of the ev and eav genes, the occurrence of these genes was examined in individuals. Examples of the autoradiograms containing the individual samples are shown in Figure 1. Due to the vagueness of the bands, the lower molecular weight areas of the HmdIII and Mspl gels were difficult to score reliably. Therefore, only bands larger than 3.8 kb of the HmdIII gels and 3.0 kb of the Mspl gels were examined. Overall, the frequency of 29 HmdIII, 34 Mspl, and 21 BamHI (the internal 1.4-kb and 1.8-kb bands not included) bands was determined both within the FC and GL tails and in the parental GL and FC lines. Statistical Analysis
DNA Analysis
The significance of the differences in Endogenous Avian Viral Genes. Tofrequency between the tails of the distribuvisualize the eav genes, 7 /xg of chro- tion of the F2 population for the eav and ev mosomal DNA was digested with the gene bands were tested per production trait restriction enzyme HmdIII or Mspl and and per individual band by chi-square fractionated on .8% TAE-agarose gels (TAE analysis. To investigate the consistency of buffer: 40 mM Tris-acetate, 2 mM EDTA, altered eav or ev band frequencies possibly pH 8.2). After electrophoresis the gels were associated with feed conversion or 6-wk blotted to nylon filters (Hybond-N4), body weight, the frequency of occurrence of ultraviolet cross-linked, and hybridized the eav and ev gene bands within the with an eav probe. This probe (Benkel and parental (pure line) birds was compared Gavora, 1993) was kindly provided by B. with those observed within the F2 groups of Benkel (Centre for Food and Animal Re- birds that were divided according the same search, Agriculture Canada, Ottawa, ON, phenotypes. Canada, K1A 0C6) and consists of the downstream region of the eav gene. Hybridization conditions were according to RESULTS the nylon filter manufacturer's recommendations. The filters were washed twice with 2 x SSC/.1% SDS at 65 C for 30 min and Comparison of Endogenous once with .2 x SSC/.1% SDS at 65 C for 30 Avian Viral Band Frequencies min. Hindlll-Endogenous Avian Viral Endogenous Viral Genes. To visualize Bands. In the birds examined the average the ev genes, 7 ng of chromosomal DNA frequency of the 29 HmdIII bands larger was digested with the restriction enzyme than 3.8 kb was .65. A mean of 19 scorable BamHI and analyzed as described above. bands per bird was found. Seven HmdIII Vector RCAS, a proviral vector derived bands were present in all birds investigated. from Rous sarcoma virus, which detects all In Table 1, the frequency of five HmdIII ev sequences, was used as probe (Hughes et bands observed within the parental GL and al, 1987; Aarts et al, 1991). Hybridization FC line and within the GL and FC selected and washing conditions were identical to tails are given. These bands were chosen the conditions used for the eav probe. because they showed large differences in frequency between the two parental lines and are therefore potentially interesting for association studies. 3 Beckmann GPR Centrifuge, Beckmann InstruThe five bands were all present in higher ments, Mydrecht, The Netherlands. 4 frequencies in the parental FC line than in Amersham, Houten, The Netherlands.
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Tris-HCl pH 8.0, 1 mM MgCL., 150 mM NaCl, 1% NONIDET P-40). After centrifugation (10 min, 4000 rpm3) 1 mL of nucleilysis buffer (50 mM Tris-HCl pH 9.5, 100 mM EDTA, 1% SDS) containing 200 ng proteinase K was added to the pellet. After brief and gentle vortexing, the tube was incubated overnight at 37 C. The DNA was precipitated by adding 9 mL of a dimethylformamide/70% acetone mix (5:95 vol/vol), rinsed in 80% and then 96% ethanol, and finally dissolved in 1 mL TE (10 mM Tris-HCl pH 7.5,1 mM EDTA). The DNA solution was precipitated for a second time by adding 100 pL ammonium acetate (3 M, pH 5.6) and 3 mL 96% ethanol. The DNA clump was rinsed in 96% ethanol and dissolved in 1 mL H 2 0.
ENDOGENOUS VIRAL GENES AND PRODUCTION TRAITS
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the GL line (Table 1). Only the frequencies of the 6.5-kb and the 9.5-kb Hindlll-eav band were different (P < .05) between the extreme groups. The 9.5-kb Hmdlll-eai; band was found in higher frequencies in the tail with favorable feed conversion ratios. The 6.5-kb Hindlll-eav band was found in lower frequencies in the GL(+) F2 tail.
DISCUSSION
Comparison of the Endogenous Viral Band Frequencies
The use of eav and ev genes as "multiBamHI-Endogenous Viral Bands. In locus" probes have some advantages over the parental and F2 birds, 21 different the usual fingerprint probes such as those
TABLE 1. Endogenous avian viral (eav) and endogenous viral (ev) band frequencies in the parental Feed Conversion (FC) and Growth Line (GL) line and in the selected F2 tails 1 F 2 tail
Parental lines Band
FC
GL
FC(+)
FC(-)
GL(+)
GL(-)
HindW-eaiP12.0 kb 9.5 kb 6.5 kb 5.7 kb 4.0 kb Mspl-eav1 15.0 kb 7.2 kb 6.5 kb 3.3 kb
(19)3 .65 .50 .45 .65 .40
(20) .36 .10 .21 .42 .21
(21) .19 .09* .42 .28 .23
(13) .31 .62 .67 .08 (20) .30 .25
(19) .11 .37 .37 .53 (19) .21 .68
(24) .41 .33 .16 .50 .50 (24) .29 .50 .54 .29 (23) .21 .60
(22) .40 .27 .18 .45 .31 (21) .33 .19 .50 .57 (20) .20 .45
(23) .39 .43 .47* .47 .52 (22) .32 .40 .38 .36 (20) .15 .55
BamHl-ev2 5.8 kb 5.1 kb
(21) .04* .23 .33 .47 (21) .04 .47
'Tail FC(+) is composed by the best female performer in feed conversion of each of the 24 full-sib families; tail FC(-) by the worst female performer in feed conversion of each full-sib family. Tail GL(+) is composed by the best female performer in body weight of each of the 24 full-sib families; tail GL(-) by the worst female performer in body weight of each full-sib family. 2 Hindlll-eav bands, endogenous avian viral specific Hmdin bands; Mspl-eav bands, endogenous avian virus specific Mspl bands; BamHl-ev bands, endogenous viral specific BamHl bands. 3 Number of birds analyzed. *P <, .05.
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BamHl bands were identified. Most of the ev genes have two internal BamHl cleavage sites. Therefore most ev genes generate, besides the specific 3' flanking fragment, two internal fragments. These internal fragments, 1.4 and 1.8 kb in size, were excluded from our analysis. The majority of the Mspl-Endogenous Avian Viral Bands. BamHl bands were present in low to The average frequency of the 34 analyzed intermediate frequencies. Only two bands, Mspl bands was .46 (= 16 bands per birds). the 23.0- and 11.8 kb-bands, were found in Six of the Mspl bands were found in all high frequencies (.75). For the BamHl bands investigated birds. The 3.3-, 6.5-, 7.2-, and the average frequency of occurrence was 15.0-kb Mspl bands (Table 1) were chosen .26, which corresponds with an average of for further analysis because the frequency 5.5 BamHl ev gene bands per bird. Two of these individual Mspl bands clearly BamHl bands were chosen for further differed between the two parental lines. The analysis. For these two bands the frequency 15.0-kb band, with higher incidence in the parental FC line than in the GL line, was differed clearly between the parental lines, found in only one FC(-) tail bird. A higher however, no differences (at the level of 5%) (P < .05) number of FC(+) birds carried this were found between the F2 tails of both distributions (Table 1). band.
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AARTS AND LEENSTRA
S
:Sssl—i«r' -•• * «
HindIII-eav
1
_5.8 _5.1
i llslii I Mspl-eav
FIGURE 1. Southern blots of genomic DNA from female F2 birds. Left panel: the DNA was digested with the restriction enzyme HmdIII and hybridized with the endogenous avian viral (eav) specific probe. Middle panel: the DNA was digested with the restriction enzyme Mspl and also hybridized with the eav specific probe. Right panel: the DNA was digested with the restriction enzyme BamHl and hybridized with the endogenous viral (ev) specific probe. The length of the fragments mentioned in the text are indicated.
originally described by Jeffreys et al. (1985). In general, retroviral elements are randomly distributed within the chicken genome and there is evidence for the integration of retroviruses nearby active genes (Vijaya et al, 1986). They can influence the expression of adjacent genes by the promotor activity of their long terminal repeats (Cullen et al, 1984) or influence the effect of genes on the production trait by insertional mutagenesis (Soller and Beckmann, 1987). Linkage or association of ev genes with production traits, disease resistance, and immune response was described by Bacon et al. (1988), Kuhnlein et al (1989a,b), Gavora et al. (1991), Smith et al. (1991), and Lamont et al. (1992). The pleiotropic effect of ev genes, particularly evl\, on production traits was reviewed by Chambers et al (1993). Therefore, eav and ev genes can act as molecular markers, can influence traits, and can act as quantitative trait loci themselves. These three features together could increase the possibility of finding differences in eav and ev band frequencies associated with production traits in differentially selected lines.
With the eav probe, the number of scorable bands per DNA sample approximates the number of bands found with the minisatellite DNA fingerprint probes (Hillel et al, 1989; Dunnington et al, 1990; Kuhnlein et al, 1990). In the case of the ev probe, the average number of bands found per individual is 5.5, instead of 16 and 19 as found within the Mspl and Hindlll eav gene patterns, respectively. The number of different ev bands, however, is at least 60% of the number of different bands observed with the eav probe. We did not examine the eav or ev gene distribution within the founder population. However, the GL and FC line descended from the same population of 50 sires and 200 dams and thus an equal gene distribution at the beginning of the divergent selection of both lines might be expected. The differences in band frequencies observed after 12 generations of divergent selection might be due to random drift or could be the consequence of the selection criteria. To discriminate between the two possibilities we chose an experimental design in which the eav and
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r* * - .
iiis
I 0
ENDOGENOUS VIRAL GENES AND PRODUCTION TRAITS
The results presented here show that selection for feed conversion ratios and body weight may have altered the frequency of eav and ev bands. Our results also show that the chosen experimental design is useful for finding candidate genes or markers associated with production traits. The availability of markers for commercially interesting traits is still a limiting step for marker-assisted selection. The three eav-b&nds mentioned above are probably good candidates for further study for their usefulness as genetic
marker in marker-assisted selection for feed conversion or 6-wk body weight. ACKNOWLEDGMENTS The authors thank B. Benkel (Centre for Food and Animal Research, Agriculture Canada, Ottawa, ON, Canada, K1A 0C6) for providing the eav gene-specific probe; Christine Gerritsen and Reint Pit for breeding, care of birds, and collecting the production data; and Ria van der Hulst for collecting blood samples.
REFERENCES Aarts, H.J.M., M. C. van der Hulst-van Arkel, G. Beuving, and F. R. Leenstra, 1991. Variations in endogenous viral (ro)-gene patterns in White Leghorn, medium heavy, White Plymouth Rock and Cornish type chickens. Poultry Sri. 70: 1281-1286. Aarts, H.J.M., M. C. van der Hulst-van Arkel, and F. R. Leenstra, 1992. Different endogenous viral loci in Cornish and White Plymouth Rock chickens. Theor. Appl. Genet. 85:325-330. Bacon, L. D., E. Smith, L. B. Crittenden, and G. B. Havenstein, 1988. Association of the slow feathering (K) and an endogenous viral {evil) gene on the Z chromosome of chickens. Poultry Sri. 67:191-197. Benkel, B. F., and J. S. Gavora, 1993. A novel molecular fingerprint probe based on the endogenous avian retroviral element (EAV) of chickens. Anim. Genet. 24:409-413. Boulliou, A., J. P. Le Pennec, G. Hubert, R. Donal, and M. Smiley, 1991. Restriction fragment length polymorphism analysis of endogenous Avian Leukosis Viral loci: Determination of frequencies in commercial broiler lines. Poultry Sci. 70:1287-1296. Boyce-Jacino, M. T., R. Resnick, and A. J. Farras, 1989. Structural and functional characterization of the unusually short long terminal repeats and their adjacent regions of a novel endogenous avian retrovirus. Virology 173:157-166. Bumstead, N., L. I. Messer, and N. G. Greenwood, 1987. Use of ev loci as a measure of inbreeding in domestic fowls. Br. Poult. Sci. 28:717-725. Chambers, J. R., E. J. Smith, E. A. Dunnington, and P. B. Siegel, 1993. Sex-linked feathering (K, k+) in chickens: A review. Poult. Sci. Rev. 5:97-116. Crittenden, L. B., 1991. Retroviral elements in the genome of the chicken: Implications for poultry genetics and breeding. Crit. Rev. Poult. Biol. 3: 73-109. Crittenden, L. B., A. M. Fadly, and E. J. Smith, 1982. Effect of endogenous leukosis virus genes in response to infection with avian leukosis and reticuloendotheliosis viruses. Avian Dis. 26: 279-294. Cullen, B. R., P. T. Lomedico, and G. Ju, 1984. Transcriptional interference in avian retroviruses—implications for the promoter insertion model of leukomogenesis. Nature 307: 3128-3129.
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ev gene specific bands present in the parental lines were analyzed in F2 birds representing the phenotypic tails for the production traits feed conversion and 6-wk body weight. A comparison between the parental and the groups selected from the F2 distribution should reflect eav and ev gene frequency differences due to selection or random drift. In tail analysis across a population it can be expected that birds in each tail of a distribution tend to be more related than randomly selected birds, whereas birds in different tails of a distribution will be less related than random birds. Consequently, differences in markers between the tails of a distribution might reflect differences between families, i n d e p e n d e n t of linkage with production traits. When birds are compared within families, this family effect is excluded. Therefore we chose to produce the tail groups from the best and worst performer within each family. Some of the differences observed between the frequencies of the eav and ev bands present in the parental lines were consistent within the selected F 2 tails. For example, the 9.5-kb HindlU-eav band was found both in relative higher frequencies in the parental FC line and in the F 2 FC(+) tail than in the parental GL line and FC(-) tail. Similar consistencies of the effect of selection were also noticed for the 6.5-kb HindlU-eav and 15.0-kb Mspl-eav bands (Table 1). The 9.5-kb HmdIII-eai> and 15.0-kb Mspl-eav bands might be associated with feed conversion. The 6.5-kb HmdIII fragment is probably negatively associated with high 6-wk body weight, as it was found in low frequencies in the parental GL line and in the F 2 GL(+) tail.
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AARTS AND LEENSTRA calibration curve defined strains of chickens. Genetics 125:161-165. Lamont, S. J., Y. Chen, H.J.M. Aarts, M. C. van der Hulst-van Arkel, G. Beuving, and F. R Leenstra, 1992. Endogenous viral genes in thirteen highly inbred chicken lines and in lines selected for immune response traits. Poultry Sci. 71:530-538. Leenstra, F. R, P.F.G. Vereijken, and R Pit, 1986. Fat deposition in a broiler sire strain. I. Phenotypic and genetic variation in, and correlations between, abdominal fat, body weight and feed conversion. Poultry Sci. 65:1225-1235. Resnick, R. M., M. T. Boyce-Jacino, Q. Fu, and A. J. Farras, 1990. Phylogenetic distribution of the novel avian endogenous provirus family EAV-0. J. Virol. 64:4640-4653. Robinson, H. L., S. M. Astrin, A. M. Senior, and F. H. Salazar, 1981. Host susceptibility to endogenous viruses: defective, glycoprotein-expressing proviruses interfere with infection. J. Virol. 40: 745-751. Ronfort, C, M. Afanassieff, Y. Chebloune, G. Dambrine, V. M. Nigon, and G. Verdier, 1991. Identification and structure analysis of endogenous proviral sequences in a brown leghorn chicken strain. Poultry Sci. 70:2161-2175. Sabour, M. P., J. R. Chambers, A. A. Grunder, U. Kuhnlein, and J. S. Gavora, 1992. Endogenous viral gene distribution in populations of meattype chickens. Poultry Sci. 71:1259-1270. Smith, E. J., 1987. Endogenous avian leukemia viruses. Pages 101-120 in: Avian Leukosis. G. F. de Boer, ed. Martinus Nijhoff Publishing, Boston, MA. Smith, E. J., A. M. Fadly, I. Levin, and L. B. Crittenden, 1991. The influence of ez>6 on the immune response to avian leukosis virus infection in rapid-feathering progeny of slow- and rapid-feathering dams. Poultry Sci. 70: 1673-1678. SoUer, M., and J. S. Beckmann, 1987. Cloning quantitative trait loci by insertional mutagenesis. Theor. Appl. Genet. 74:369-378. Vijaya, S., D. L. Steffen, and H. L. Robinson, 1986. Acceptor sites for retroviral integrations map near DNAse I-hypersensitive sites in chromatine. J. Virol. 60:683-688.
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Dunnington, E. A., O. Gal, P. B. Siegel, A. Haberfeld, A. Cahaner, U. Lavi, Y. Plotsky, and J. Hillel, 1990. Deoxyribonucleic acid fingerprint comparisons between selected populations of chickens. Poultry Sci. 70:463^167. Dunwiddie, C. T., and A. J. Farras, 1985. Presence of retrovirus reverse transcriptase-related gene sequences in avian cells lacking endogenous avian leukosis viruses. Proc. Natl. Acad. Sci. USA 82: 5097-5101. Dunwiddie, C. T., R. Resnick, M. Boyce-Jacino, J. N. Alegre, and A. J. Farras, 1986. Molecular cloning and characterization of gag-, pol-, and envrelated gene sequences in the eir chicken. J. Virol. 59:669-675. Gavora, J. S., U. Kuhnlein, L. B. Crittenden, J. L. Spenser, and M. P. Sabour, 1991. Endogenous viral genes: Association with reduced egg production rate and egg size in White Leghorns. Poultry Sci. 70:618-623. Hillel, J., Y. Plotsky, A. Haberfeld, U. Lavi, A. Cahaner, and A. J. Jeffreys, 1989. DNA fingerprints of poultry. Anim. Genet. 20:145-155. Hughes, S. H., J. J. Greenhouse, C. J. Petropoulos, and P. Sutrave, 1987. Adapter plasmids simplify the insertion of foreign DNA into helperindependent retroviral vectors. J. Virol. 61: 3004-3012. Jeffreys, A. J., V. Wilson, and S. L. Thein, 1985. Hypervariable "minisatellite" regions in human DNA. Nature 314:67-73. Kuhnlein, U., J. S. Gavora, J. L. Spencer, D. E. Bernon, and M. Sabour, 1989a. Incidence of endogenous viral genes in two strains of White Leghorn chickens selected for egg production and susceptibility or resistance to Marek's disease. Theor. Appl. Genet. 77:26-32. Kuhnlein, U., M. Sabour, J. S. Gavora, W. Fairfull, and D. E. Bernon, 1989b. Influence of selection for egg production and Marek's disease resistance on the incidence of endogenous viral genes in White Leghorns. Poultry Sci. 68: 1161-1167. Kuhnlein, U., D. Zadworny, Y. Dawe, R. W. Fairfull, and J. S. Gavora, 1990. Assessment of inbreeding by DNA fingerprinting: Development of a
1995 Poultry Science 74:1022-1028
White Leghorns (Aarts et al, 1991) to 7.3 in meat-type chickens (Sabour et ah, 1992) are Endogenous avian viral (eav) and en- found. Within populations of noninbred dogenous viral (ev) genes are retrovirus- chickens, the individual ev genes are like elements that are found in the chicken found in low to intermediate frequencies. genome. The ev genes have been studied Consequently, the ev restriction fragment extensively (see reviews by Smith, 1987; length polymorphism (RFLP) patterns of Crittenden, 1991). They are thought to be individuals vary significantly within and randomly distributed in the chicken ge- between lines (Aarts et al, 1991, 1992; nome and are found in low to moderate Boulliou et al, 1991; Ronfort et al, 1991; copy numbers. On average, 2.5 copies in and Sabour et al, 1992). The eav genes, which are distantly related to ev genes, were first detected and isolated by Dunwiddie and Farras (1985) Received for publication September 19, 1994. and Dunwiddie et al (1986) by using Accepted for publication February 6, 1995. probes derived from gene-specific restric1 To whom correspondence should be addressed. 2 Present address: DLO-State Institute for Quality tion fragments of the avian sarcoma virus Control of Agricultural Products, Bomsesteeg 45,6708 (ASV). These eav genes represent a more conserved and more ancient retrovirus PD Wageningen, The Netherlands. INTRODUCTION
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ABSTRACT The consistency of the effect of selection on the frequencies of endogenous avian viral (eav) and endogenous viral (ev) specific restriction fragment length polymorphism (RFLP) bands was studied in two broiler lines selected from a single base population and in an F2 population derived from a reciprocal cross of both lines. One broiler line (FC line) was selected for low feed conversion ratio and the other line (GL line) was selected for high 6-wk body weight. In the F2 population, the band frequencies were determined in groups representing separate tails of the distribution of two production traits, namely, low feed conversion ratio between 29 and 42 d of age and body weight at 42 d of age. The F2 population consisted of 288 females belonging to 24 full-sib families. To rule out family effects, the tails for these production traits were composed by either the best or by the worst female performer for each trait in each full-sib family. In total, 29 Hindlll-eav, 34 Mspl-eav, and 21 BamHl-ev bands could be distinguished by RFLP analysis. This report describes the influence of selection on 11 potentially interesting bands. Two bands, the 9.5-kb HmdIII-eaz; and the 15-kb Mspl-eav band, which were found both in higher frequencies in the parental FC line, were also found in higher (P £ .05) frequencies in the F2 tail with a favorable feed conversion ratio. A third band, the 6.5-kb HindUL-eav band, present in lower frequencies in the parental GL line, was also present in lower (P < .05) frequencies in the F2 tail of birds with heavy body weight. (Key words: chicken, endogenous viral genes, endogenous avian viral genes, restriction fragment length polymorphism, production traits)
ENDOGENOUS VIRAL GENES AND PRODUCTION TRAITS
MATERIALS AND METHODS Chicken Lines and Crosses The FC and GL lines used in our experiments were selected from a broiler sire strain. This foundation strain descends from a commercial broiler sire flock (Leenstra et ah, 1986). The selection criterion for the FC line was low feed conversion ratio between 3 and 6 wk of age of individually housed chickens that consumed feed ad libitum. The GL line was selected for high 6-wk body weight of chickens that ate ad libitum and were housed in groups. Both lines were derived from the same 50 sires and 200 dams. In each generation, 30 to 50 sires and 100 dams were selected. In the 12th generation, FC and GL chickens were reciprocally mated to generate an Fj population. The number of parents involved was: 8 GL line males, 8 FC line males, 14 GL line females, and 13 FC line females. From the progeny, six males each of the GL x FC and FC x GL types were mated to two females each, alternating GL x FC and FC x
GL to form the F2. Four hatches were produced. The animals were reared on litter until 19 d of age and then they were housed in individual cages. The F 2 population consisted of 576 (288 females and 288 males) chickens equally distributed over 24 full-sib families. Measurements All F 2 animals were weighed individually at hatch and at 19 and 42 d of age. At 19 and 42 d they were weighed after a feed withdrawal of approximately 10 h. Feed intake was recorded from Day 19 to 42. Blood samples were taken at 29 d of age via the brachial vein, mixed with EDTA anticoagulant, and stored at -20 C for DNA analysis. For the parental lines, blood samples were taken only from the parents of the reciprocal cross. Composition of the Tails of the Distribution of the F2 Population It was decided to use only females for this experiment, as female broilers have in general fewer leg problems and metabolic disorders than male broilers. Consequently, in females, body weights and feed efficiencies (especially those in the light body weight and unfavorable feed conversion groups) are less confounded with disorders that restrict inherent performance. To eliminate family effects, the two extreme groups for each production trait were composed by either the best or worst female performer of each of the 24 full-sib families. These four extreme groups of birds will be referred to as: Tail GL(+) = best performers in body weight; tail GL(-) = worst performers in body weight; tail FC(+) = best performers in feed conversion; tail FC(-) = worst performers in feed conversion. The feed conversion ratios of the FC(+) tail ranged from 2.046 to 1.655 and of the FC(-) tail from 2.343 to 1.856. The 6-wk body weight of the GL(+) tail ranged from 1,821 to 2,183 g and of the GL(-) tail from 1,227 to 1,818 g. DNA Isolation Fifty microliters of blood was lysed for 5 min on ice in 2 mL of Lysis buffer (10 mM
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family and exist at 40 to 100 copies per haploid chicken genome (Dunwiddie et ah, 1986; Resnick et ah, 1990). The eav genes are characterized by unusually short long terminal repeats (Boyce-Jacino et ah, 1989). Although there is some evidence for deleterious (Crittenden et ah, 1982) or advantageous (Robinson et ah, 1981) effects of ev genes to the fowl, they are assumed to be neutral under normal conditions (Bumstead et ah, 1987). In lines selected for production traits, disease resistance, or immune response, however, changes in gene frequency in comparison to control lines were observed Kuhnlein et ah, 1989a,b; Gavora et ah, 1991; Lamont et ah, 1992). This suggests that ev genes could be used as molecular markers in poultry breeding. The advantages and disadvantages of the use of ev gene probes compared to the usual diallelic single-copy RFLP and fingerprint probes have been reviewed by Crittenden (1991). In this report, we describe the use of two multilocus probes, each specific for a different class of ev genes, to find evidence for association of ev or eav genes with the production traits feed conversion and 6-wk body weight in broilers.
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To determine the frequencies of the ev and eav genes, the occurrence of these genes was examined in individuals. Examples of the autoradiograms containing the individual samples are shown in Figure 1. Due to the vagueness of the bands, the lower molecular weight areas of the HmdIII and Mspl gels were difficult to score reliably. Therefore, only bands larger than 3.8 kb of the HmdIII gels and 3.0 kb of the Mspl gels were examined. Overall, the frequency of 29 HmdIII, 34 Mspl, and 21 BamHI (the internal 1.4-kb and 1.8-kb bands not included) bands was determined both within the FC and GL tails and in the parental GL and FC lines. Statistical Analysis
DNA Analysis
The significance of the differences in Endogenous Avian Viral Genes. Tofrequency between the tails of the distribuvisualize the eav genes, 7 /xg of chro- tion of the F2 population for the eav and ev mosomal DNA was digested with the gene bands were tested per production trait restriction enzyme HmdIII or Mspl and and per individual band by chi-square fractionated on .8% TAE-agarose gels (TAE analysis. To investigate the consistency of buffer: 40 mM Tris-acetate, 2 mM EDTA, altered eav or ev band frequencies possibly pH 8.2). After electrophoresis the gels were associated with feed conversion or 6-wk blotted to nylon filters (Hybond-N4), body weight, the frequency of occurrence of ultraviolet cross-linked, and hybridized the eav and ev gene bands within the with an eav probe. This probe (Benkel and parental (pure line) birds was compared Gavora, 1993) was kindly provided by B. with those observed within the F2 groups of Benkel (Centre for Food and Animal Re- birds that were divided according the same search, Agriculture Canada, Ottawa, ON, phenotypes. Canada, K1A 0C6) and consists of the downstream region of the eav gene. Hybridization conditions were according to RESULTS the nylon filter manufacturer's recommendations. The filters were washed twice with 2 x SSC/.1% SDS at 65 C for 30 min and Comparison of Endogenous once with .2 x SSC/.1% SDS at 65 C for 30 Avian Viral Band Frequencies min. Hindlll-Endogenous Avian Viral Endogenous Viral Genes. To visualize Bands. In the birds examined the average the ev genes, 7 ng of chromosomal DNA frequency of the 29 HmdIII bands larger was digested with the restriction enzyme than 3.8 kb was .65. A mean of 19 scorable BamHI and analyzed as described above. bands per bird was found. Seven HmdIII Vector RCAS, a proviral vector derived bands were present in all birds investigated. from Rous sarcoma virus, which detects all In Table 1, the frequency of five HmdIII ev sequences, was used as probe (Hughes et bands observed within the parental GL and al, 1987; Aarts et al, 1991). Hybridization FC line and within the GL and FC selected and washing conditions were identical to tails are given. These bands were chosen the conditions used for the eav probe. because they showed large differences in frequency between the two parental lines and are therefore potentially interesting for association studies. 3 Beckmann GPR Centrifuge, Beckmann InstruThe five bands were all present in higher ments, Mydrecht, The Netherlands. 4 frequencies in the parental FC line than in Amersham, Houten, The Netherlands.
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Tris-HCl pH 8.0, 1 mM MgCL., 150 mM NaCl, 1% NONIDET P-40). After centrifugation (10 min, 4000 rpm3) 1 mL of nucleilysis buffer (50 mM Tris-HCl pH 9.5, 100 mM EDTA, 1% SDS) containing 200 ng proteinase K was added to the pellet. After brief and gentle vortexing, the tube was incubated overnight at 37 C. The DNA was precipitated by adding 9 mL of a dimethylformamide/70% acetone mix (5:95 vol/vol), rinsed in 80% and then 96% ethanol, and finally dissolved in 1 mL TE (10 mM Tris-HCl pH 7.5,1 mM EDTA). The DNA solution was precipitated for a second time by adding 100 pL ammonium acetate (3 M, pH 5.6) and 3 mL 96% ethanol. The DNA clump was rinsed in 96% ethanol and dissolved in 1 mL H 2 0.
ENDOGENOUS VIRAL GENES AND PRODUCTION TRAITS
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the GL line (Table 1). Only the frequencies of the 6.5-kb and the 9.5-kb Hindlll-eav band were different (P < .05) between the extreme groups. The 9.5-kb Hmdlll-eai; band was found in higher frequencies in the tail with favorable feed conversion ratios. The 6.5-kb Hindlll-eav band was found in lower frequencies in the GL(+) F2 tail.
DISCUSSION
Comparison of the Endogenous Viral Band Frequencies
The use of eav and ev genes as "multiBamHI-Endogenous Viral Bands. In locus" probes have some advantages over the parental and F2 birds, 21 different the usual fingerprint probes such as those
TABLE 1. Endogenous avian viral (eav) and endogenous viral (ev) band frequencies in the parental Feed Conversion (FC) and Growth Line (GL) line and in the selected F2 tails 1 F 2 tail
Parental lines Band
FC
GL
FC(+)
FC(-)
GL(+)
GL(-)
HindW-eaiP12.0 kb 9.5 kb 6.5 kb 5.7 kb 4.0 kb Mspl-eav1 15.0 kb 7.2 kb 6.5 kb 3.3 kb
(19)3 .65 .50 .45 .65 .40
(20) .36 .10 .21 .42 .21
(21) .19 .09* .42 .28 .23
(13) .31 .62 .67 .08 (20) .30 .25
(19) .11 .37 .37 .53 (19) .21 .68
(24) .41 .33 .16 .50 .50 (24) .29 .50 .54 .29 (23) .21 .60
(22) .40 .27 .18 .45 .31 (21) .33 .19 .50 .57 (20) .20 .45
(23) .39 .43 .47* .47 .52 (22) .32 .40 .38 .36 (20) .15 .55
BamHl-ev2 5.8 kb 5.1 kb
(21) .04* .23 .33 .47 (21) .04 .47
'Tail FC(+) is composed by the best female performer in feed conversion of each of the 24 full-sib families; tail FC(-) by the worst female performer in feed conversion of each full-sib family. Tail GL(+) is composed by the best female performer in body weight of each of the 24 full-sib families; tail GL(-) by the worst female performer in body weight of each full-sib family. 2 Hindlll-eav bands, endogenous avian viral specific Hmdin bands; Mspl-eav bands, endogenous avian virus specific Mspl bands; BamHl-ev bands, endogenous viral specific BamHl bands. 3 Number of birds analyzed. *P <, .05.
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BamHl bands were identified. Most of the ev genes have two internal BamHl cleavage sites. Therefore most ev genes generate, besides the specific 3' flanking fragment, two internal fragments. These internal fragments, 1.4 and 1.8 kb in size, were excluded from our analysis. The majority of the Mspl-Endogenous Avian Viral Bands. BamHl bands were present in low to The average frequency of the 34 analyzed intermediate frequencies. Only two bands, Mspl bands was .46 (= 16 bands per birds). the 23.0- and 11.8 kb-bands, were found in Six of the Mspl bands were found in all high frequencies (.75). For the BamHl bands investigated birds. The 3.3-, 6.5-, 7.2-, and the average frequency of occurrence was 15.0-kb Mspl bands (Table 1) were chosen .26, which corresponds with an average of for further analysis because the frequency 5.5 BamHl ev gene bands per bird. Two of these individual Mspl bands clearly BamHl bands were chosen for further differed between the two parental lines. The analysis. For these two bands the frequency 15.0-kb band, with higher incidence in the parental FC line than in the GL line, was differed clearly between the parental lines, found in only one FC(-) tail bird. A higher however, no differences (at the level of 5%) (P < .05) number of FC(+) birds carried this were found between the F2 tails of both distributions (Table 1). band.
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S
:Sssl—i«r' -•• * «
HindIII-eav
1
_5.8 _5.1
i llslii I Mspl-eav
FIGURE 1. Southern blots of genomic DNA from female F2 birds. Left panel: the DNA was digested with the restriction enzyme HmdIII and hybridized with the endogenous avian viral (eav) specific probe. Middle panel: the DNA was digested with the restriction enzyme Mspl and also hybridized with the eav specific probe. Right panel: the DNA was digested with the restriction enzyme BamHl and hybridized with the endogenous viral (ev) specific probe. The length of the fragments mentioned in the text are indicated.
originally described by Jeffreys et al. (1985). In general, retroviral elements are randomly distributed within the chicken genome and there is evidence for the integration of retroviruses nearby active genes (Vijaya et al, 1986). They can influence the expression of adjacent genes by the promotor activity of their long terminal repeats (Cullen et al, 1984) or influence the effect of genes on the production trait by insertional mutagenesis (Soller and Beckmann, 1987). Linkage or association of ev genes with production traits, disease resistance, and immune response was described by Bacon et al. (1988), Kuhnlein et al (1989a,b), Gavora et al. (1991), Smith et al. (1991), and Lamont et al. (1992). The pleiotropic effect of ev genes, particularly evl\, on production traits was reviewed by Chambers et al (1993). Therefore, eav and ev genes can act as molecular markers, can influence traits, and can act as quantitative trait loci themselves. These three features together could increase the possibility of finding differences in eav and ev band frequencies associated with production traits in differentially selected lines.
With the eav probe, the number of scorable bands per DNA sample approximates the number of bands found with the minisatellite DNA fingerprint probes (Hillel et al, 1989; Dunnington et al, 1990; Kuhnlein et al, 1990). In the case of the ev probe, the average number of bands found per individual is 5.5, instead of 16 and 19 as found within the Mspl and Hindlll eav gene patterns, respectively. The number of different ev bands, however, is at least 60% of the number of different bands observed with the eav probe. We did not examine the eav or ev gene distribution within the founder population. However, the GL and FC line descended from the same population of 50 sires and 200 dams and thus an equal gene distribution at the beginning of the divergent selection of both lines might be expected. The differences in band frequencies observed after 12 generations of divergent selection might be due to random drift or could be the consequence of the selection criteria. To discriminate between the two possibilities we chose an experimental design in which the eav and
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r* * - .
iiis
I 0
ENDOGENOUS VIRAL GENES AND PRODUCTION TRAITS
The results presented here show that selection for feed conversion ratios and body weight may have altered the frequency of eav and ev bands. Our results also show that the chosen experimental design is useful for finding candidate genes or markers associated with production traits. The availability of markers for commercially interesting traits is still a limiting step for marker-assisted selection. The three eav-b&nds mentioned above are probably good candidates for further study for their usefulness as genetic
marker in marker-assisted selection for feed conversion or 6-wk body weight. ACKNOWLEDGMENTS The authors thank B. Benkel (Centre for Food and Animal Research, Agriculture Canada, Ottawa, ON, Canada, K1A 0C6) for providing the eav gene-specific probe; Christine Gerritsen and Reint Pit for breeding, care of birds, and collecting the production data; and Ria van der Hulst for collecting blood samples.
REFERENCES Aarts, H.J.M., M. C. van der Hulst-van Arkel, G. Beuving, and F. R. Leenstra, 1991. Variations in endogenous viral (ro)-gene patterns in White Leghorn, medium heavy, White Plymouth Rock and Cornish type chickens. Poultry Sri. 70: 1281-1286. Aarts, H.J.M., M. C. van der Hulst-van Arkel, and F. R. Leenstra, 1992. Different endogenous viral loci in Cornish and White Plymouth Rock chickens. Theor. Appl. Genet. 85:325-330. Bacon, L. D., E. Smith, L. B. Crittenden, and G. B. Havenstein, 1988. Association of the slow feathering (K) and an endogenous viral {evil) gene on the Z chromosome of chickens. Poultry Sri. 67:191-197. Benkel, B. F., and J. S. Gavora, 1993. A novel molecular fingerprint probe based on the endogenous avian retroviral element (EAV) of chickens. Anim. Genet. 24:409-413. Boulliou, A., J. P. Le Pennec, G. Hubert, R. Donal, and M. Smiley, 1991. Restriction fragment length polymorphism analysis of endogenous Avian Leukosis Viral loci: Determination of frequencies in commercial broiler lines. Poultry Sci. 70:1287-1296. Boyce-Jacino, M. T., R. Resnick, and A. J. Farras, 1989. Structural and functional characterization of the unusually short long terminal repeats and their adjacent regions of a novel endogenous avian retrovirus. Virology 173:157-166. Bumstead, N., L. I. Messer, and N. G. Greenwood, 1987. Use of ev loci as a measure of inbreeding in domestic fowls. Br. Poult. Sci. 28:717-725. Chambers, J. R., E. J. Smith, E. A. Dunnington, and P. B. Siegel, 1993. Sex-linked feathering (K, k+) in chickens: A review. Poult. Sci. Rev. 5:97-116. Crittenden, L. B., 1991. Retroviral elements in the genome of the chicken: Implications for poultry genetics and breeding. Crit. Rev. Poult. Biol. 3: 73-109. Crittenden, L. B., A. M. Fadly, and E. J. Smith, 1982. Effect of endogenous leukosis virus genes in response to infection with avian leukosis and reticuloendotheliosis viruses. Avian Dis. 26: 279-294. Cullen, B. R., P. T. Lomedico, and G. Ju, 1984. Transcriptional interference in avian retroviruses—implications for the promoter insertion model of leukomogenesis. Nature 307: 3128-3129.
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ev gene specific bands present in the parental lines were analyzed in F2 birds representing the phenotypic tails for the production traits feed conversion and 6-wk body weight. A comparison between the parental and the groups selected from the F2 distribution should reflect eav and ev gene frequency differences due to selection or random drift. In tail analysis across a population it can be expected that birds in each tail of a distribution tend to be more related than randomly selected birds, whereas birds in different tails of a distribution will be less related than random birds. Consequently, differences in markers between the tails of a distribution might reflect differences between families, i n d e p e n d e n t of linkage with production traits. When birds are compared within families, this family effect is excluded. Therefore we chose to produce the tail groups from the best and worst performer within each family. Some of the differences observed between the frequencies of the eav and ev bands present in the parental lines were consistent within the selected F 2 tails. For example, the 9.5-kb HindlU-eav band was found both in relative higher frequencies in the parental FC line and in the F 2 FC(+) tail than in the parental GL line and FC(-) tail. Similar consistencies of the effect of selection were also noticed for the 6.5-kb HindlU-eav and 15.0-kb Mspl-eav bands (Table 1). The 9.5-kb HmdIII-eai> and 15.0-kb Mspl-eav bands might be associated with feed conversion. The 6.5-kb HmdIII fragment is probably negatively associated with high 6-wk body weight, as it was found in low frequencies in the parental GL line and in the F 2 GL(+) tail.
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AARTS AND LEENSTRA calibration curve defined strains of chickens. Genetics 125:161-165. Lamont, S. J., Y. Chen, H.J.M. Aarts, M. C. van der Hulst-van Arkel, G. Beuving, and F. R Leenstra, 1992. Endogenous viral genes in thirteen highly inbred chicken lines and in lines selected for immune response traits. Poultry Sci. 71:530-538. Leenstra, F. R, P.F.G. Vereijken, and R Pit, 1986. Fat deposition in a broiler sire strain. I. Phenotypic and genetic variation in, and correlations between, abdominal fat, body weight and feed conversion. Poultry Sci. 65:1225-1235. Resnick, R. M., M. T. Boyce-Jacino, Q. Fu, and A. J. Farras, 1990. Phylogenetic distribution of the novel avian endogenous provirus family EAV-0. J. Virol. 64:4640-4653. Robinson, H. L., S. M. Astrin, A. M. Senior, and F. H. Salazar, 1981. Host susceptibility to endogenous viruses: defective, glycoprotein-expressing proviruses interfere with infection. J. Virol. 40: 745-751. Ronfort, C, M. Afanassieff, Y. Chebloune, G. Dambrine, V. M. Nigon, and G. Verdier, 1991. Identification and structure analysis of endogenous proviral sequences in a brown leghorn chicken strain. Poultry Sci. 70:2161-2175. Sabour, M. P., J. R. Chambers, A. A. Grunder, U. Kuhnlein, and J. S. Gavora, 1992. Endogenous viral gene distribution in populations of meattype chickens. Poultry Sci. 71:1259-1270. Smith, E. J., 1987. Endogenous avian leukemia viruses. Pages 101-120 in: Avian Leukosis. G. F. de Boer, ed. Martinus Nijhoff Publishing, Boston, MA. Smith, E. J., A. M. Fadly, I. Levin, and L. B. Crittenden, 1991. The influence of ez>6 on the immune response to avian leukosis virus infection in rapid-feathering progeny of slow- and rapid-feathering dams. Poultry Sci. 70: 1673-1678. SoUer, M., and J. S. Beckmann, 1987. Cloning quantitative trait loci by insertional mutagenesis. Theor. Appl. Genet. 74:369-378. Vijaya, S., D. L. Steffen, and H. L. Robinson, 1986. Acceptor sites for retroviral integrations map near DNAse I-hypersensitive sites in chromatine. J. Virol. 60:683-688.
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Dunnington, E. A., O. Gal, P. B. Siegel, A. Haberfeld, A. Cahaner, U. Lavi, Y. Plotsky, and J. Hillel, 1990. Deoxyribonucleic acid fingerprint comparisons between selected populations of chickens. Poultry Sci. 70:463^167. Dunwiddie, C. T., and A. J. Farras, 1985. Presence of retrovirus reverse transcriptase-related gene sequences in avian cells lacking endogenous avian leukosis viruses. Proc. Natl. Acad. Sci. USA 82: 5097-5101. Dunwiddie, C. T., R. Resnick, M. Boyce-Jacino, J. N. Alegre, and A. J. Farras, 1986. Molecular cloning and characterization of gag-, pol-, and envrelated gene sequences in the eir chicken. J. Virol. 59:669-675. Gavora, J. S., U. Kuhnlein, L. B. Crittenden, J. L. Spenser, and M. P. Sabour, 1991. Endogenous viral genes: Association with reduced egg production rate and egg size in White Leghorns. Poultry Sci. 70:618-623. Hillel, J., Y. Plotsky, A. Haberfeld, U. Lavi, A. Cahaner, and A. J. Jeffreys, 1989. DNA fingerprints of poultry. Anim. Genet. 20:145-155. Hughes, S. H., J. J. Greenhouse, C. J. Petropoulos, and P. Sutrave, 1987. Adapter plasmids simplify the insertion of foreign DNA into helperindependent retroviral vectors. J. Virol. 61: 3004-3012. Jeffreys, A. J., V. Wilson, and S. L. Thein, 1985. Hypervariable "minisatellite" regions in human DNA. Nature 314:67-73. Kuhnlein, U., J. S. Gavora, J. L. Spencer, D. E. Bernon, and M. Sabour, 1989a. Incidence of endogenous viral genes in two strains of White Leghorn chickens selected for egg production and susceptibility or resistance to Marek's disease. Theor. Appl. Genet. 77:26-32. Kuhnlein, U., M. Sabour, J. S. Gavora, W. Fairfull, and D. E. Bernon, 1989b. Influence of selection for egg production and Marek's disease resistance on the incidence of endogenous viral genes in White Leghorns. Poultry Sci. 68: 1161-1167. Kuhnlein, U., D. Zadworny, Y. Dawe, R. W. Fairfull, and J. S. Gavora, 1990. Assessment of inbreeding by DNA fingerprinting: Development of a