Endogenous Viral Gene Distribution in Populations of Meat-Type Chickens1 M. P. SABOUR,2 J. R. CHAMBERS, A. A. GRUNDER, U. KUHNLEIN,3 and J. S. GAVORA Centre for Food and Animal Research, Agriculture Canada, Ottawa, Ontario, K1A 0C6, Canada
1992 Poultry Science 71:1259-1270
INTRODUCTION
Virtually all current populations of chickens, including ancestral red jungle fowl, contain DNA sequences homologous to the avian leukosis virus (ALV) genome (Astrin, 1978; Tereba et al, 1979; Smith, 1986). These endogenous viral (ev) sequences are inherited as stable genes.
Received for publication January 17, 1992. Accepted for publication March 31, 1992. 1 Centre for Food and Animal Research Publication Number 2018. 2 To whom correspondence should be addressed. 3 Department of Animal Science, Macdonald College of McGill University, Ste Anne de Bellevue, PQ, H9X 1C0, Canada.
White Leghorns have over 22 ev loci (Smith, 1986; Kuhnlein et al, 1989a), some of which have been mapped to chromosomes (Tereba and Astrin, 1980, 1982). Noninbred Leghorns usually have one to five ev loci (Rovigatti and Astrin, 1983; Aarts et al, 1991), but some have none (Astrin et al, 1979; Gavora et al, 1989). Some of the ev genes in White Leghorns were shown to be associated with reduced egg production rate, egg weight, and egg quality (Gavora et al, 1991). These reductions were similar to those observed in infections with exogenous ALV (Gavora, 1987). Also, ev genes can increase the rate of ALV infection and subsequent mortality (Crittenden et al, 1982).
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ABSTRACT The present study was designed to document the complexity of endogenous viral (ev) genes and seek evidence for their association with production traits in selected and control strains of meat-type chickens. Three populations were studied, each consisting of a control strain and one to three strains selected for various production traits. The ev genes were revealed by digesting genomic DNA with restriction enzymes and detecting DNA fragments on Southern blots using radioactive probes from nucleotide sequences of the avian leukosis virus genome. A total of 31 polymorphic ev loci were identified in these populations from a Sacl digest, with an average of 7.3 ev genes per bird. There were no significant differences in ev genes per bird between strains within populations or between selected and control strains overall. Thirty of 62 comparisons in the three populations indicated ev gene frequency differences (P < .05). Within populations, 13 of 93 comparisons of ev gene frequencies between control and selected strains and 8 of 62 between three selected strains of a sire population showed such differences (P < .05). Selection for body weight and feed efficiency had been observed to reduce gene frequencies of the slow-feathering gene, which usually contains the eu21 locus; however, these effects were not detected (.05 < P < .06) between strains of the dam population in the current study. Such differences suggested possible associations between ev genes and production traits in meat-type chickens. (Key words: meat-type chickens, ev genes, selected populations, control populations, body weight)
1260 Year
SABOUR ET AL.
Event
STR 90 and 80
STR20. 21.23. and 25
1955
Combination of three commercial and one experimental strains commences
Four strains \l/ Synthesis
1958
STR 90 and three strains for selection produced
STR 90 (unselected)
1972
Combination of strains A, B, and C commences
A,B,C \l/ Synthesis
1974
STR 80 produced
STR 80 (unselected)
1978
Combination of nine Cornish sire stocks; seven White Rock dam stocks;commences
1981
STR 20, 21, 23, 25, 30, and 31 produced
—
STR 30 and 31
A,B,C (BW) Selection
I
STR 20 — 2 1 , (Unselected) I
23,
25,
STR 30 — -31 (Unselected)
I (BW.FE) I (BW.LEA) l (BW.LEA.FE) 1990
Blood samples for DNA
/ (Unselected)
(Unselected)
\ I\
(BW.FE.HEP)
T T T (Unselected) (Selected 10 generations) (Selected nine generations)
FIGURE 1. Origin of three genetic sets of strains with primary selection criterion in parenthesis: LEA = leanness; FE = feed efficiency; HEP = hatching egg production; STR = strain.
Significant changes in ev gene frequencies were observed in strains selected for egg production and related traits compared with unselected controls (Crittenden et al, 1979; Kuhnlein et al, 1989a,b). Association of gene e»21 with increased susceptibility to ALV (Bacon et al, 1988) is of particular importance because this locus is closely associated with the sex-linked slow-feathering gene K, widely used in commercial chickens to sex day-old chicks on the basis of primary wing feather development. Although most knowledge of the distribution of ev genes and their effects on production traits is based on studies in White Leghorns, substantial ev gene polymorphisms in birds of other breeds have been reported (Gudkov et al, 1981, 1986; Rovigatti and Astrin, 1983; Aarts et al, 1991; Boulliou et al, 1991). The present study investigated the distribution of ev genes in meat chicken strains and sought evidence for association of ev genes with production traits through examination of
differences among the strains. A secondary objective was to determine the relationship of optical density measurements on autoradiograms from Southern blots of composite DNA samples from groups of chickens and the frequency of a locus in a population.
MATERIALS AND METHODS Chicken Stocks The strains of meat-type chickens used in the current study represented meat chickens from two time periods and three major sources (Figure 1). The Ottawa Meat Control (OMC) Strain 90 was synthesized in 1955 at Ottawa from four strains representative of meat chickens of that period (Merritt and Gowe, 1962). There has been no artificial selection of Strain 90 following the completion of its synthesis in 1958. Strain 80 was formed by combining three strains originally derived from Strain 90 and selected 14 generations for increased
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Seven dam stocks \l/ Synthesis
Nine sire stocks \l/ Synthesis
ENDOGENOUS VIRAL GENES IN MEAT-TYPE CHICKENS
Deoxyribonucleic Acid Isolation
Genomic DNA was isolated either from blood cells of individual chickens or from pooled blood cells of a group of individuals. Blood cells (25 ^L) collected in 4 mM EDTA were separated from serum by centrifugation and lysed in lysis buffer [100 mM NaCl, 20 mM Tris-HCl (pH 8.0), 10 mM EDTA] containing .5% (wt/vol) SDS and proteinase K (20 units/mL). After overnight incubation with mild agitation at room temperature, the lysates were extracted twice with phenol-chloroform-isoamyl alcohol (25:24:1) and precipitated with ethanol, then air-dried and dissolved in 5 mM Tris-HCl (pH 7.5) and .1 mM EDTA. In some instances, equal volumes of blood cells from 15 individual chickens from a strain were mixed and DNA extracted as above. Comparison of combined samples can be used to rapidly identify ev genes that differ in their frequency between populations (Murray et al, 1987). Analysis of Endogenous Viral Loci
Genomic DNA was digested with BamHl or Sad restriction enzyme (Rovigatti and Astrin, 1983; Smith, 1986). Fragments were separated by agarose gel electrophoresis and blotted onto nylon membrane (Biotrace)4 according to Reed and Mann (1985). Endogenous viral sequences were localized by hybridization with 32P-labeled pRAV-25 and autoradiographed. The pRAV-2 probe is a pBR322 plasmid containing most of the DNA equivalent to the exogenous ALV genome (Smith and Crittenden, 1986). For detection of ep21, the pEV21-int5 probe specific for this locus (Levin and Smith, 1990) was used. Experimental procedures were as described previously (Kuhnlein et al, 1989a). The DNA fragments observed were assumed to contain a part of the ev sequence along with a flanking region of chicken genomic DNA. The size of such fragments obtained after digestion of genomic DNA with the Sacl and BamHl restriction enzymes was used to identify some of the ev loci as equivalent to previously described ev 4 Gelman Sciences, Ann Arbor, MI 48106. loci. In most instances, the loci were 5 Donated by E. J. Smith, Avian Disease and identified only by the size of Sacl DNA Oncology Laboratory, East Lansing, MI 48823.
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63-day body weight (Merritt et al, 1966). After this strain was formed by combining the three selected strains in 1974, there was no further artificial selection. These strains will be further referred to as the OMC strains. Two other sources were strains from the sire and dam populations of meat-type chickens developed at Ottawa from commercial broiler parent stocks (Chambers et al, 1984). Strain 20, the broiler sire, or "Cornish" control strain has been maintained without artificial selection since it was created. It was also the source of three strains, 21, 23, and 25, selected for high 28-day body weight and either low abdominal fatness of 47-day-old siblings or high feed efficiency from 28 to 42 days, or both, respectively. Strain 30, the broiler dam, or "White Rock" control strain, has been maintained without artificial selection since it was created. It is the source of Strain 31, selected for high 28-day body weight, high feed efficiency from 28 to 42 days, and high hatching egg production to 40 wk of age in females. There have been 10 and 9 generations of parental selection for sire and dam population selected strains, respectively. These two sources together will be further referred to as "modern strains". Performance of the strains used in the present study, based on tests of 1990 generations, is shown in Table 1. Strains 20, 21, 23, 25, 80, and 90 were assumed to be homozygous for the fastfeathering (k) gene based upon their ancestry. In past generations, casual observation of feathering development of these chicks between 1 and 6 wk of age supported this belief. Strains 30 and 31 are known to segregate for the slow-feathering (K) gene. Frequencies of the K gene in these strains in the generation of ev loci testing were: .49 (273 females) and .47 (144 males) for Strain 30; .08 (499 females) and .07 (502 males) for Strain 31. Classification of these chicks in earlier generations indicates that the frequency of K is stable in Strain 30 and declining in Strain 31 at an average rate of .045 per generation of selection.
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17
16
19
Both
7.1 6.3 7.9 1.2 2.3 2.0 6 to 10 3 to 13I 3 to 13 15 15 30
146
80
363
Control 90 114 2 -.8 18
25
7.6 7.9 6.4 1.7 1.6 1.7 5 to 11 4 to 11 4 to 9 15 15 15
115 4 -.8 19
132 1 -1.0 22
816 47 3.2 22 7.2 1.1 5 to 9 15
23
21
20
Selected
Sire strains (Cornish)
7.3 1.6 4 to 11 60
29
All 84 4 -.8 20
31
26
Both
7.7 7.7 7.7 2.5 2.1 1.9 4 to 13 4 tc) 10 4 to 13 15 30 15
756 45 3.5 22
Control 30
Dam strains (White Rock)
7
WL
7.3 2.7 1.9 1.0 3 to 13 1 to 5 120 38
31
PP
1 Strain 80 was selected for 14 generations for 63-day BW; Strains 21,23, and 25 were all selected for 10 generations for high 28-day BW and low abdominal fat percentage of sibs (Strain 21), 28- to 42-day feed efficiency (Strain 23), or both feed efficiency and low-fat (Strain 25); Strain 31 was selected for high 28-day BW, 28- to 42-day feed efficiency, and high hatching egg production to 40 wk of age of females; Ottawa control Strain 7. Production traits of selected strains were recorded as deviations from the corresponding control strain. 2 Feed efficiency = (weight gained -s- feed consumed) x 100; abdominal fat = (abdominal fat -s- carcass weight x 100).
28-day BW, g Feed efficiency,2 % Abdominal fat,2 % Number of ev loci Mean number of ev loci per chicken SD Range Number tested
Characteristic
OMC strains
TABLE 1. Production trait characteristics and mean number of different endogenous viral (ev) genes per chicken and per strain in Ottawa meat control (OMC), sire and dam meat-type populations, these populations pooled (PP), and Ottawa White Leghorn (WL) control Strain 5 1
c-
XI en
£ BOU
1263
ENDOGENOUS VIRAL GENES IN MEAT-TYPE CHICKENS Dam
WL Wl
f
Sire
T20
D,
21 23
25T30
f
OMC
31Y90
1(20
21
23
25~||30
m
ev18 — —23.1
ev18 — ev1 —
GV *J
iiiiill:
^~* ^IW*
—23.1
* i
ipr * *
ev3 — —
V
s •» «•
—6.6
—6.6
4$jMfc
—
—4.4 —4.0
— a*
ev6 — "" evrntl —
iff 9 In-
—4.4
—
—3.0
FIGURE 2. Autoradiograph of Southern blot of (left) Sad- and (right) BamHI-digested pooled genomic DNA of 15 individuals from each of eight meat-type chicken strains, hybridized with P-labeled pRAV-2 probe. Sire populations analyzed were: Strain 20, unselected control; Strains 21, 23, and 25, selected. Dam populations analyzed were: Strain 30, unselected control; Strain 31, selected; Ottawa Meat Control (OMC) chicken populations: Strain 90, unselected control; Strain 80, selected. WL = a White Leghorn strain carrying endogenous viral loci evl, ev3, ev6, and eul8, used as a reference. M = DNA marker fragment sizes.
fragment without their designation as to published equivalents. Statistical
Analyses
Tests of differences in frequencies of the DNA fragments either individually or in total among populations or among strains within populations were performed. Differences in presence of the 31 DNA fragments in each strain were analyzed using a loglinear model for categorical data and appropriate chi-square. Differences in frequency of specific DNA fragments were tested using the chi-square test of heterogeneity (SAS Institute, 1985). RESULTS Analyses of Pooled Deoxyribonucleic Acid
Samples
Analysis of ev loci in pooled DNA of the three sources and their strains revealed a complex restriction fragment length polymorphism pattern with fragment sizes ranging from 4.4 to 30 kb and 3.4 to 25 kb when DNA was digested with Sad and
BamHl restriction enzyme, respectively (Figure 2). A visual examination of the bands showed differences in the optical density between populations or strains within a population (Figure 2). Photodensitometric measurements of the bands (Figure 3) were positively correlated (correlation coefficient = .86; F < .01) with the number of ev genes that the band of the pooled DNA represented, as determined by analysis of individual chicken DNA samples. Analyses of Individual Deoxyribonucleic Acid
Chicken Samples
A total of 31 different Sacl DNA fragments were found in the populations studied (Table 1). The numbers of ev loci per population (19,29, and 26 for the OMC, sire, and dam populations, respectively) were not significantly different. The average number of different ev loci per chicken in the three populations combined was 7.3, almost three times higher than the 2.7 ev genes observed in a control strain of White Leghorn chickens. The number of ev loci detected in individual meat chickens
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el,1
evmtl —
M
M
80]
ev6
OMC
31 "l[90 8(51
1264
SABOUR £T AL.
selected strains of OMC, dam, and sire populations, respectively (Table 3). Comparison with Previously Characterized Endogenous Viral Loci
No. of ev fragments in individuals
FIGURE 3. Relationship between the number of endogenous viral (ev) fragments in individuals and relative densitometric values of autoradiograms of pooled DNA in a sample of the 15 chickens shown in Figure 2 (y = 8.141 + 11.230% r = .86).
ranged from 3 to 13. This is in contrast to the White Leghorn control line, with a range of 1 to 5 ev loci per chicken. Presence and frequencies of specific ev loci in the three populations varied. Fifteen ev loci were common to all three meat-type populations (Table 2). The 10.5-kb fragment, identified as eul8, was nearly ubiquitous, as it was present in over 86% of all chickens. Certain loci were present in high frequency in specific populations. For example, the 22.6-kb fragment was detected in 97% of the OMC chickens and similarly the 7.5-kb fragment was present in 97% of the dam population chickens. Two ev loci, 12.2 and 8.3 kb, were detected only in the sire population and one, 9.2 kb, only in the dam population (Table 2). The latter cosegregated with a 20-kb BamHI and was genotyped as evil by the use of specific DNA probe pEV21. There was no significant difference in the average number of ev genes per chicken between selected and control strains (Table 1); however, strain differences in certain ev locus frequencies were observed. For example, there were differences of five, two, and six loci between unselected control and
DISCUSSION Endogenous Viral Gene Frequencies of the Pooled Deoxyribonucleic Acid The positive correlation between the number of ev genes observed in individual birds included in a pooled sample and relative densitometric values of corresponding bands on autoradiograms of pooled DNA provided a means to assess ev gene frequency differences. This relationship was used to search for differences in ev gene frequencies between strains using pooled DNA samples from 15 chickens per strain. The usefulness of this approach was also reported in evaluating gene frequency changes in groups within populations (Plotsky et al., 1990). In summary, DNA evaluation of individual chickens of a strain or group will yield more precise results than results from pooled DNA. However, results from pooled DNA are less laborious and therefore more economical and rapid for population comparisons and, on occasion, sufficiently precise to warrant their use in the assessment of genetic distances between populations. Differences in Endogenous Loci Due to Population Origin A large number and different types of ev loci were detected in the three populations. Past results suggest that most meat-type chicken populations contain similar ev loci
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Paired association of restriction fragment length polymorphism (RFLP) pattern of Sacl to its BamHI fragments was used to identify five previously characterized loci (Table 4). Genes ev3, ev6, evl%, evil, and evrc&l, a locus with unknown phenotype showing a 4.4-kb Sacl fragment and a 3.5-kb BamHI fragment, were present in the three populations. Gene evil was detected only in the dam population, which is known to segregate for the sex-linked, slow-feathering gene.
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ENDOGENOUS VIRAL GENES IN MEAT-TYPE CHICKENS
TABLE 2. Number of individuals per strain carrying each endogenous viral locus for eight strains of meat-type chickens OMC 1
Dam
Sire Selected
Control 90
Selected 80
Freq. 2
Control 20
21
23
25
Freq. 2
Control 30
Selected 31
Freq. 2
(kb) 33.5 25.0 22.6 20.9 19.5 18.0 16.5 15.0 14.5 13.0 12.2 11.5 10.5 10.2 10.0 9.5 9.2 9.15 9.1 8.7 8.6 8.3 8.1 7.9 7.7 75 7.3 6.8 6.3 5.4 4.4
1 0 15 3 0 0 0 1 9 2 0 0 11 0 9 6 0 0 9 5 8 0 2 0 14 0 0 0 11 0 13
5 0 14 4 0 0 0 0 2 3 0 1 14 3 1 2 0 0 3 0 1 0 6 0 12 0 2 0 11 0 10
.20 .00 .97 .23 .00 .00 .00 .03 .37 .17 .00 .03 .83 .10 .33 .27 .00 .00 .40 .17 .30 .00 .27 .00 .87 .00 .07 .00 .73 .00 .77
2 0 8 4 3 0 1 1 0 0 5 5 14 0 4 2 0 5 0 4 8 10 1 0 9 0 7 1 2 1 11
3 1 3 0 1 2 8 0 1 0 5 10 11 0 3 7 0 0 0 10 6 3 9 3 10 0 3 3 5 0 5
3 0 7 3 3 0 6 0 0 0 5 10 9 2 8 8 0 0 9 6 2 0 8 0 12 8 2 0 0 0 8
3 0 9 3 2 0 2 0 0 0 7 5 13 1 0 0 0 0 2 6 8 2 9 0 7 0 0 2 6 0 9
.18 .02 .45 .17 .15 .03 .28 .02 .02 .00 .37 .50 .78 .05 .25 .28 .00 .08 .18 .43 .40 .25 .45 .05 .63 .13 .20 .10 .22 .02 .55
0 0 7 8 4 0 1 0 2 2 0 2 14 0 4 6 8 3 4 0 9 0 4 0 7 14 2 2 1 1 10
1 1 9 12 3 1 1 0 0 0 0 2 15 0 2 10 3 3 0 0 11 0 2 2 6 15 9 0 0 0 8
.03 .03 .53 .67 .23 .03 .07 .00 .07 .07 .00 .13 .97 .00 .20 .53 .37 .20 .13 .00 .67 .00 .20 .07 .43 .97 .37 .07 .03 .03 .60
!OMC Freq.
2
Ottawa Meat Control strains. Overall frequency of each locus within population.
at different frequencies and this reflects their different genetic origin (Rovigatti and Astrin, 1983; Aarts et al, 1991; Boulliou et al, 1991). Differences in experimental conditions may also lead to different reported fragment sizes and this creates further difficulties in comparing the loci reported by different laboratories. Nevertheless, for the purpose of this discussion, each distinct observed band size will be considered a separate ev locus. The OMC and the modern populations shared several loci; however, certain loci were observed only in the sire (12.2 and 8.3 kb) or in the dam (9.2 kb) populations, whereas nine others were present in both the sire and dam populations. The sire and
dam populations of 1978 origin had 10 and 7 more ev loci than the OMC strains of the 1950s (Table 2). This may reflect either the broad genetic base of the populations synthesized in 1978 or the elimination of low-frequency loci during generations through which the OMC birds have been maintained as closed, relatively small populations. In the White Leghorn Strain 7, used here for a comparison, that has been maintained closed since 1958, there are only seven different loci and, on the average, 2.7 times fewer loci per bird than in the meattype chickens. As mentioned earlier, the sire and dam populations of 1978 origin were synthesized from multiple commercial sources and can be considered to
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Locus
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SABOUR ET AL.
TABLE 3. Tests of heterogeneity (chi-square) among polymorphic endogenous viral loci of meat-type chickens of the Ottawa Meat Control (OMC), sire, and dam populations Within populations Sire
Between populations
Locus
Sire vs. dam
OMC S vs. C 1
Dam S vs. C
NS
*
444
NS
NS
4*4
*
NS
NS NS NS NS NS NS
4
4
NS NS NS NS NS NS
NS NS
44
44
NS NS NS
NS NS NS
***
NS NS 4*4 4
NS
** *
** 4*4
NS
NS
*** t
NS NS NS NS
*
X-
*
*
NS NS NS NS NS NS NS NS NS
4
NS NS NS
4
4
NS NS NS NS NS NS NS NS NS NS NS
NS NS NS
444
NS NS
NS NS NS NS
44
*
** 444
4*
NS NS NS NS
*4 4 *
NS NS
* *
Strain 21 vs. 23
NS
»
NS 44
Strain 25 vs. 21, 23
NS NS NS NS NS NS NS NS NS NS NS NS NS t NS
4*
4*4
S vs. C
44*
NS NS 4
NS
44
NS NS NS NS
44
NS NS NS NS NS NS NS NS
444
444 44*
NS NS
* NS 4*
**4
NS NS NS NS NS NS NS NS
*
NS NS NS NS
t NS NS NS 4*4
NS NS NS NS NS 4*4
NS NS
*
NS
*S vs. C = selected strain(s) versus control strain. < .06. *P < .05. **P < .01. ***P < .001.
+P
contain samples from most internationally used commercial meat stocks of that time (Chambers et al, 1984). Interestingly, only four segregating ev loci in a sample of 12 birds were observed in the red jungle fowl (Gallus bankiva) (Rovigatti and Astrin, 1983), the progenitor of the domesticated chicken (Gallus domesticus) (Frisby et al, 1979). This would suggest that the numerous ev loci observed in the present study as well as in previous published studies may represent insertions that occurred since domestication of the species. Effects of Selection
A comparison between selected and control strains within populations should
reflect differences due to selection. The frequency of a number of loci differed significantly between selected and unselected control strains. However, such differences may also be due to genetic drift or represent sampling errors. With only 15 chickens per strain, the error associated with the observed ev gene frequencies was large. Calculations of necessary gene frequency difference required for significance (P < .05) between two groups of size 15 based on chi-square (1 df) indicated that differences of at least three chickens at extreme frequencies and up to more than five chickens at intermediate frequencies were required. For groupings of the strains, differences in incidence required for statistical significances were lower. It should also
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(kb) 33.5 22.6 20.9 19.5 16.5 15.0 14.5 13.0 12.2 11.5 10.5 10.0 9.5 9.2 9.15 9.1 8.7 8.6 8.3 8.1 7.7 7.5 7.3 6.8 6.3 4.4
OMC vs. sire and dam
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ENDOGENOUS VIRAL GENES IN MEAT-TYPE CHICKENS TABLE 4. Phenotypes of endogenous viral (ev) genes and their frequencies Populations
Size of specific DNA fragment Sad
Modern
BamHl
Locus
Phenotype 1
7.3 4.4 25.0 20.0 3.5
ev3 ev6
gs + chf" gs" chf+ V-E + V-E + Unknown
H-hl
\XD)
6.3 22.6 10.5 9.2 4.4
eo\% ev215 eumtl 6
OMC 2
Sire
Dam
(n = 30) .73***3
(n = 60) .22*4
(n = 30)
07***3
.45
.83
.7%** .OO***4
.00*3 .77t 3
.55
.03 .63 .97 .37 .60
be kept in mind that some of the production trait differences between selected and control strains and those among strains selected from the same base were small (Table 1). Hence, conclusions on associations of ev locus frequency differences with effects of selection may, in such instances, be questionable. Sire population Strains 21,23, and 25 had been selected for low abdominal fatness of 47-day-old sibs, high feed efficiency between 28 and 42 days, or both of these traits, respectively, in addition to body weight at 28 days. Breeding records revealed that realized selection intensities in total differed for these strains. Effects of genes for feed efficiency and abdominal fatness are largely additive in nature; however, nonadditive genetic effects cannot be ruled out. In addition, genetic drift is contributing at least at minor levels to genetic differences between these strains. Therefore, progeny of Strain 25 were not necessarily intermediate to those of Strains 21 and 23 in feed efficiency and abdominal fatness. Consequently, it is difficult to quantify with precision expected differences in gene frequencies among these selected strains. Comparisons of Strains 21 and 23 are more
likely to reflect differences in frequencies of contributing genes or genes linked to them than comparisons involving Strain 25 due to the greater difference in selected traits. In light of this background, the loci with 7.3- and 9.1-kb fragments appear to be most apt to be associated with selection pressures for high feed efficiency or low abdominal fatness (Table 3). Changes in frequencies of several other ev loci seem attributable to genetic selection procedures: the 16.5-, 9.15-, and 8.3-kb loci in the sire population reveal differences between control and selected strains. Of the ev loci that differed significantly between selected and control OMC strains, all had reduced frequencies in the selected strain. Although the reasons for the increase in the frequency of 7.3-kb or the decrease in the 9.1-kb loci in the selected dam strain compared with the control strain were not clear, the 9.2-kb locus, which was identified as the eu21 gene and is known to be associated with the slow-feathering gene K, decreased in frequency in the selected dam Strain 31. This agrees with past observations that selection for body weight and feed conversion reduces the frequency of the slow-feathering gene (eu21) (Pym et al,
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Summarized by Smith (1986). Phenotypes are abbreviated as follows: gs + = expression of internal viral gag proteins; chf" = expression of the viral envelope protein; V-E + = production of infectious endogenous virus. 2 OMC = Ottawa Meat Control. 3 Significant difference between OMC and modern populations. Significant difference between the sire and dam populations. The presence of evil was confirmed by using a eo21-specific DNA probe on the same Southern blot membranes used for hybridization with pRAV-2. 6 m t l = Unidentified meat-type (Iraqi et al, 1991; Boulliou et al, 1991). t P < .06. *P < .05. **P < .01. ***P < .001.
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SABOUR ET Al.
Previously Characterized Endogenous Viral Loci
Due to the complexity of the hybridization pattern, determination of the phenotype of most loci was not possible. However, as mentioned earlier, using chicken DNA with known ev loci and matching restriction fragments produced by Sacl and BamHl enzymes, five loci were tentatively characterized as equivalent to previously described loci. The phenotype of four of these (ev3, ev6, eul8, and ez;21) is known (Smith, 1986) and of one, designated erantl, is unknown. Except for eu21, the frequencies of none of these loci differed significantly between selected and unselected control strains. The frequency of both ev3 and ev6 was significantly higher in the OMC (1955 origin) than the modern (1978 origin) populations (Table 2). In the dam population, only one chicken having ev3 was detected in a sample of 30 chickens and locus ev6 was in intermediate frequency in the 1978 populations (Table 2). Loci ev3 and ev6 are known to express endogenous viral envelope glycoproteins at high levels (Hayward et al, 1980), which interferes with endogenous (Subgroup E) viral infection, presumably by blocking the host cell membrane receptors for the virus (Robinson et al, 1981; Crittenden et al, 1982). Hence, ev3 and ev6 may be expected to provide their hosts with some selective advantage, given the negative effects of endogenous virus on egg production traits (Gavora et al, 1991). The patterns observed in the current study
do not seem to provide much evidence for such a hypothesis. Of particular interest is the distribution of evil, an endogenous virus-producing gene that is associated with the dominant, sex-linked, slow-feathering gene K and was detected in the dam population. Congenital transmission of infectious virus, which is encoded by the evil gene, reduces seroconversion and enhances the incidence of lymphoid leukosis (Smith and Fadly, 1988). The frequency of this gene decreased 2.7-fold (P < .06) in Strain 31 (selected for high hatching egg production, body weight, and feed efficiency) compared with control Strain 30. In this context, it is notable that the frequency of the virus-producing eul8 in the selected strain of the dam population was unchanged and remained high (Tables 2 and 3). The present results are in agreement with the observation by Boulliou et al. (1991) that in commercial broilers, 17 loci appear to contain a complete proviral genome without significant deletion. A large number of replication-competent endogenous viruses were detected in Australian commercial broilers by Ignjatovic (1986), suggesting the presence of multiple complete endogenous proviral genomes. Many of the uncharacterized ev loci observed in the current study may be of this typeOccurrence of ez?18, evil, and perhaps other similar but uncharacterized ev loci may be associated with low egg production in the meat-type chickens (Crittenden et al, 1979; Kuhnlein et al, 1989a,b; Gavora et al, 1991). The observations that most commercial White Leghorn strains lack ev genes that code for endogenous virus (Tereba and Astrin, 1980) may reflect the elimination of such genes during selection for egg production. Observations in the current study and those reported by Boulliou et al. (1991) and Aarts et al. (1991) indicate that this may not be the case in meat chickens. The nature of the erantl locus, which is characterized by 4.4-kb Sacl and 3.5-kb BamHl fragments, is not clear. It was frequently present in the populations studied here. Because both commercial layers (Iraqi et al, 1991) and commercial broilers (Boulliou et al, 1991) have this locus at high frequency, it is unlikely that its presence is
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1984). The selection for broiler weight, feed efficiency, and adult hatching egg production of hens has also been observed to reduce the frequency of the slow feathering gene (J. R. Chambers, unpublished data). In the current study, the reduction in frequency of the evil locus and the associated slow-feathering gene from 8 of 15 chickens of the control strain to 3 of 15 in the selected strain of the dam population (Table 2) only approached significance (P < .06). This difference should be real according to the preceding reports (Pym et al, 1984; Chambers, unpublished data); however, the power of the test in the present study was inadequate for its detection.
ENDOGENOUS VIRAL GENES IN MEAT-TYPE CHICKENS
ACKNOWLEDGMENTS
The expert technical assistance of P. S. Griffin is gratefully acknowledged. The authors thank the staff of the Centre for Food and Animal Research poultry facilities for care of the experimental chickens and collecting the blood samples and also thank M. Boichard for helping in the analysis of eu21 locus. The authors thank L. B. Crittenden and E. J. Smith, USDA, Agricultural Research Service, Avian Disease and Oncology Laboratory, East Lansing, MI 48823 for their generous gifts of the pRAV-2, pEV21, and marker chicken DNA.
REFERENCES Aarts, H.J.M., M. C. van der Hulst-Van Arkel, G. Bouving, and F. R. Leenstra, 1991. Variations in endogenous viral gene patterns in White Leghorn, Medium Heavy White Plymouth-Rock,
and Cornish chickens. Poultry Sci. 70:1281-1286. Astrin, S. M., 1978. Endogenous viral genes of the White Leghorn chicken: common site of residence and sites associated with specific phenotypes of viral gene expression. Proc. Natl. Acad. Sci. USA 75:5941-5945. Astrin, S. M., E. G. Buss, and S. W. Hayword, 1979. Endogenous viral genes are nonessential in the chicken. Nature 281:339-341. Bacon, L. D., E. Smith, and L. B. Crittenden, 1988. Association of the slow feathering (K) and an endogenous viral (eo21) gene on the Z chromosome of chickens. Poultry Sci. 67:191-197. Boulliou, A., J. P. Le Pennec, G. Hubert, R. Donald, 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. Chambers, J. R., D. E. Bernon, and J. S. Gavora, 1984. Synthesis and parameters of new populations of meat-type chickens. Theor. Appl. Genet. 69: 23-30. Crittenden, L. B., A. M. Fadly, and E. J. Smith, 1982. Effect of endogenous leukosis virus genes on response to infection with avian leukosis and reticuloendotheliosis virus. Avian Dis. 26: 279-294. Crittenden, L. B., J. S. Gavora, F. A. Gulvas, and R. S. Gowe, 1979. Complete endogenous RNA tumor virus production by inbred and non-inbred chickens. Avian Pathol. 8:125-131. Frisby, D. P., R. A. Weiss, H. Roussel, and. D. Stehelin, 1979. The distribution of endogenous chicken retrovirus sequences in the DNA of galliform birds does not coincide with avian phylogenetic relationships. Cell 17:623-634. Gavora, J. S., 1987. Influences of avian leukosis virus infection on production and mortality and the role of genetic selection in the control of lymphoid leukosis. Pages 241-260 in: Avian Leukosis. G. F. de Boer, ed. Martinus Nijhoff Publishing, Boston, MA. Gavora, J. S., U. Kuhnlein, L. B. Crittenden, J. L. Spencer, 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. Gavora, J. S., U. Kuhnlein, and J. L. Spencer, 1989. Absence of endogenous viral genes in an inbred line of Leghorn chicks selected for high egg production and Marek's disease resistance. J. Anim. Breed. Genet. 106:217. Gudkov, A. V., E. Korec, M. V. Chernov, A. T. Tikhonenko, I. B. Obukh, and I. Hlozanek, 1986. Genetic structure of the endogenous proviruses and expression of the gag gene in Brown Leghorn. Folia Biol. (Cracow) 32:65-72. Gudkov, A. V., I. B. Obukh, S. M. Serov, and B. S. Naroditsky, 1981. Variety of endogenous proviruses in the genomes of chickens of different breeds. J. Gen. Virol. 57:85-94. Haywood, W. S., S. B. Braverman, and S. Astrin, 1980. Transcriptional products and DNA structure of endogenous avian proviruses. Cold Spring Harbor Symp. Quant. Biol. 44:1111-1121. Ignjatovic, J., 1986. Replication-competent endoge-
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associated with differences in the traits selected in the meat-type chickens. In some chickens analyzed in the present study, a 9.5-kb Sacl fragment was associated with a 5.2-kb BamHl fragment, which is characteristic of locus evl in the egg strains (Smith, 1986). However, due to comigration and possible polymorphism, it was difficult to ascertain the presence of an evl. Using a probe specific for evl, this locus was detected in White Plymouth Rock chickens at low frequency (Aarts et al, 1991). White Plymouth Rock chickens were a major contributing breed to the dam population in the current study. The results of the present study further demonstrate the complexity of ev loci in meat-type chickens. Several of ev loci present in these chickens create problems in identifying specific ev loci, without which an understanding of their possible association with production traits will be very difficult. Unequivocal identification of individual ev genes and loci, presumably by assays using cloned flanking regions of the loci, will be required for future analyses of phenotypes of the as yet uncharacterized loci in meat chickens and their association with production trait differences. Such research is justified by the implication of ev genes as factors that influence economic traits in White Leghorns.
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1270
SABOUR ET AL. Pym, R.A.E., P. J. NichoUs, E. Thomson, A. Choice, and D. J. Farrell, 1984. Energy and nitrogen metabolism of broilers selected over ten generations for increased growth rate, food consumption and conversion of food to gain. Br. Poult. Sci. 25:529-539. Reed, K. C, and D. A. Mann, 1985. Rapid transfer of DNA from agarose gels to nylon membranes. Nucleic Acids Res. 13:7207-7221. Robinson, H. L., S. M. Astrin, A. M. Senior, and I. H. Salazar, 1981. Host susceptibility to endogenous viruses: defective glycoprotein-expressing proviruses interfere with infections. J. Virol. 40: 745-751. Rovigatti, U. G., and S. M. Astrin, 1983. Avian endogenous viral genes. Curr. Top. Microbiol. Immunol. 103:1-21. SAS Institute, 1985. SAS® User's Guide. Version 5 Edition. SAS Institute Inc., Cary, NC. Smith, E. J., 1986. Endogenous avian leukemia viruses. Pages 101-120 in: Avian Leukosis. G. F. de Boer, ed. Martinus Nijhoff Publishing, Boston, MA. Smith, E. J., and L. B. Crittenden, 1986. Endogenous viral genes in a slow-feathering line of White Leghorn chickens. Avian Pathol. 15:395-406. Smith, E. J., and A. M. Fadly, 1988. Influence of congenital transmission of endogenous virus-21 on the immune response to avian leukosis virus infection and the incidence of tumors in chickens. Poultry Sci. 67:1674-1679. Tereba, A., and S. M. Astrin, 1980. Chromosomal localization of ev-1, a frequently occurring endogenous retrovirus locus in White Leghorn chickens, by in situ hybridization. J. Virol. 35: 888-894. Tereba, A., and S. M. Astrin, 1982. Chromosomal clustering of fine defined endogenous retrovirus loci in White Leghorn chickens. J. Virol. 43: 737-740. Tereba, A., M.M.C. Lai, and K. G. Murti, 1979. Chromosome 1 contains the endogenous RAV-0 retrovirus sequence in chicken cells. Proc. Natl. Acad. Sci. 76:6486-6490.
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nous avian leukosis virus in commercial lines of meat chickens. Avian Dis. 30:264-270. Iraqi, F., M7 Soller, and J. S. Beckmann, 1991. Distribution of endogenous viruses in some commercial chicken layer populations. Poultry Sci. 70:665-679. 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, R. 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. Levin, L, and E. J. Smith, 1990. Molecular analysis of endogenous virus eu21 slow-feathering complex of chickens. 1. Cloning of proviral-cell junction fragment and unoccupied integration site. Poultry Sci. 69:2017-2026. Merritt, E. S., and R. S. Gowe, 1962. Development and genetic properties of a control strain of meat-type fowl. Pages 66-70 in: Proceedings of the 12th World's Poultry Congress, Sydney, Australia. Merritt, E. S., M. Zawalsky, and S. B. Slen, 1966. Direct and correlated responses to selection for 63-day body weight in chickens. Pages 86-91 in: Proceedings of the 13th World's Poultry Congress, Kiev, Russia. Murray, J. C, R. Shiang, L. R. Carlock, M. Smith, and K. H. Buctow, 1987. Rapid RFLP screening procedure identifies new polymorphisms at albumin and alcohol dehydrogenase loci. Hum. Genet. 76:274-277. Plotsky, Y., A. Cahaner, A. Haberfeld, U. Lavi, and J. Hillel, 1990. Analysis of genetic association between DNA fingerprint bands and quantitative traits using DNA mixes. Pages 133-136 in: Proceedings of the 4th World Congress on Genetics Applied to Livestock Production, Volume XIII, Edinburgh, Scotland.
1992 Poultry Science 71:1259-1270
INTRODUCTION
Virtually all current populations of chickens, including ancestral red jungle fowl, contain DNA sequences homologous to the avian leukosis virus (ALV) genome (Astrin, 1978; Tereba et al, 1979; Smith, 1986). These endogenous viral (ev) sequences are inherited as stable genes.
Received for publication January 17, 1992. Accepted for publication March 31, 1992. 1 Centre for Food and Animal Research Publication Number 2018. 2 To whom correspondence should be addressed. 3 Department of Animal Science, Macdonald College of McGill University, Ste Anne de Bellevue, PQ, H9X 1C0, Canada.
White Leghorns have over 22 ev loci (Smith, 1986; Kuhnlein et al, 1989a), some of which have been mapped to chromosomes (Tereba and Astrin, 1980, 1982). Noninbred Leghorns usually have one to five ev loci (Rovigatti and Astrin, 1983; Aarts et al, 1991), but some have none (Astrin et al, 1979; Gavora et al, 1989). Some of the ev genes in White Leghorns were shown to be associated with reduced egg production rate, egg weight, and egg quality (Gavora et al, 1991). These reductions were similar to those observed in infections with exogenous ALV (Gavora, 1987). Also, ev genes can increase the rate of ALV infection and subsequent mortality (Crittenden et al, 1982).
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ABSTRACT The present study was designed to document the complexity of endogenous viral (ev) genes and seek evidence for their association with production traits in selected and control strains of meat-type chickens. Three populations were studied, each consisting of a control strain and one to three strains selected for various production traits. The ev genes were revealed by digesting genomic DNA with restriction enzymes and detecting DNA fragments on Southern blots using radioactive probes from nucleotide sequences of the avian leukosis virus genome. A total of 31 polymorphic ev loci were identified in these populations from a Sacl digest, with an average of 7.3 ev genes per bird. There were no significant differences in ev genes per bird between strains within populations or between selected and control strains overall. Thirty of 62 comparisons in the three populations indicated ev gene frequency differences (P < .05). Within populations, 13 of 93 comparisons of ev gene frequencies between control and selected strains and 8 of 62 between three selected strains of a sire population showed such differences (P < .05). Selection for body weight and feed efficiency had been observed to reduce gene frequencies of the slow-feathering gene, which usually contains the eu21 locus; however, these effects were not detected (.05 < P < .06) between strains of the dam population in the current study. Such differences suggested possible associations between ev genes and production traits in meat-type chickens. (Key words: meat-type chickens, ev genes, selected populations, control populations, body weight)
1260 Year
SABOUR ET AL.
Event
STR 90 and 80
STR20. 21.23. and 25
1955
Combination of three commercial and one experimental strains commences
Four strains \l/ Synthesis
1958
STR 90 and three strains for selection produced
STR 90 (unselected)
1972
Combination of strains A, B, and C commences
A,B,C \l/ Synthesis
1974
STR 80 produced
STR 80 (unselected)
1978
Combination of nine Cornish sire stocks; seven White Rock dam stocks;commences
1981
STR 20, 21, 23, 25, 30, and 31 produced
—
STR 30 and 31
A,B,C (BW) Selection
I
STR 20 — 2 1 , (Unselected) I
23,
25,
STR 30 — -31 (Unselected)
I (BW.FE) I (BW.LEA) l (BW.LEA.FE) 1990
Blood samples for DNA
/ (Unselected)
(Unselected)
\ I\
(BW.FE.HEP)
T T T (Unselected) (Selected 10 generations) (Selected nine generations)
FIGURE 1. Origin of three genetic sets of strains with primary selection criterion in parenthesis: LEA = leanness; FE = feed efficiency; HEP = hatching egg production; STR = strain.
Significant changes in ev gene frequencies were observed in strains selected for egg production and related traits compared with unselected controls (Crittenden et al, 1979; Kuhnlein et al, 1989a,b). Association of gene e»21 with increased susceptibility to ALV (Bacon et al, 1988) is of particular importance because this locus is closely associated with the sex-linked slow-feathering gene K, widely used in commercial chickens to sex day-old chicks on the basis of primary wing feather development. Although most knowledge of the distribution of ev genes and their effects on production traits is based on studies in White Leghorns, substantial ev gene polymorphisms in birds of other breeds have been reported (Gudkov et al, 1981, 1986; Rovigatti and Astrin, 1983; Aarts et al, 1991; Boulliou et al, 1991). The present study investigated the distribution of ev genes in meat chicken strains and sought evidence for association of ev genes with production traits through examination of
differences among the strains. A secondary objective was to determine the relationship of optical density measurements on autoradiograms from Southern blots of composite DNA samples from groups of chickens and the frequency of a locus in a population.
MATERIALS AND METHODS Chicken Stocks The strains of meat-type chickens used in the current study represented meat chickens from two time periods and three major sources (Figure 1). The Ottawa Meat Control (OMC) Strain 90 was synthesized in 1955 at Ottawa from four strains representative of meat chickens of that period (Merritt and Gowe, 1962). There has been no artificial selection of Strain 90 following the completion of its synthesis in 1958. Strain 80 was formed by combining three strains originally derived from Strain 90 and selected 14 generations for increased
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Seven dam stocks \l/ Synthesis
Nine sire stocks \l/ Synthesis
ENDOGENOUS VIRAL GENES IN MEAT-TYPE CHICKENS
Deoxyribonucleic Acid Isolation
Genomic DNA was isolated either from blood cells of individual chickens or from pooled blood cells of a group of individuals. Blood cells (25 ^L) collected in 4 mM EDTA were separated from serum by centrifugation and lysed in lysis buffer [100 mM NaCl, 20 mM Tris-HCl (pH 8.0), 10 mM EDTA] containing .5% (wt/vol) SDS and proteinase K (20 units/mL). After overnight incubation with mild agitation at room temperature, the lysates were extracted twice with phenol-chloroform-isoamyl alcohol (25:24:1) and precipitated with ethanol, then air-dried and dissolved in 5 mM Tris-HCl (pH 7.5) and .1 mM EDTA. In some instances, equal volumes of blood cells from 15 individual chickens from a strain were mixed and DNA extracted as above. Comparison of combined samples can be used to rapidly identify ev genes that differ in their frequency between populations (Murray et al, 1987). Analysis of Endogenous Viral Loci
Genomic DNA was digested with BamHl or Sad restriction enzyme (Rovigatti and Astrin, 1983; Smith, 1986). Fragments were separated by agarose gel electrophoresis and blotted onto nylon membrane (Biotrace)4 according to Reed and Mann (1985). Endogenous viral sequences were localized by hybridization with 32P-labeled pRAV-25 and autoradiographed. The pRAV-2 probe is a pBR322 plasmid containing most of the DNA equivalent to the exogenous ALV genome (Smith and Crittenden, 1986). For detection of ep21, the pEV21-int5 probe specific for this locus (Levin and Smith, 1990) was used. Experimental procedures were as described previously (Kuhnlein et al, 1989a). The DNA fragments observed were assumed to contain a part of the ev sequence along with a flanking region of chicken genomic DNA. The size of such fragments obtained after digestion of genomic DNA with the Sacl and BamHl restriction enzymes was used to identify some of the ev loci as equivalent to previously described ev 4 Gelman Sciences, Ann Arbor, MI 48106. loci. In most instances, the loci were 5 Donated by E. J. Smith, Avian Disease and identified only by the size of Sacl DNA Oncology Laboratory, East Lansing, MI 48823.
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63-day body weight (Merritt et al, 1966). After this strain was formed by combining the three selected strains in 1974, there was no further artificial selection. These strains will be further referred to as the OMC strains. Two other sources were strains from the sire and dam populations of meat-type chickens developed at Ottawa from commercial broiler parent stocks (Chambers et al, 1984). Strain 20, the broiler sire, or "Cornish" control strain has been maintained without artificial selection since it was created. It was also the source of three strains, 21, 23, and 25, selected for high 28-day body weight and either low abdominal fatness of 47-day-old siblings or high feed efficiency from 28 to 42 days, or both, respectively. Strain 30, the broiler dam, or "White Rock" control strain, has been maintained without artificial selection since it was created. It is the source of Strain 31, selected for high 28-day body weight, high feed efficiency from 28 to 42 days, and high hatching egg production to 40 wk of age in females. There have been 10 and 9 generations of parental selection for sire and dam population selected strains, respectively. These two sources together will be further referred to as "modern strains". Performance of the strains used in the present study, based on tests of 1990 generations, is shown in Table 1. Strains 20, 21, 23, 25, 80, and 90 were assumed to be homozygous for the fastfeathering (k) gene based upon their ancestry. In past generations, casual observation of feathering development of these chicks between 1 and 6 wk of age supported this belief. Strains 30 and 31 are known to segregate for the slow-feathering (K) gene. Frequencies of the K gene in these strains in the generation of ev loci testing were: .49 (273 females) and .47 (144 males) for Strain 30; .08 (499 females) and .07 (502 males) for Strain 31. Classification of these chicks in earlier generations indicates that the frequency of K is stable in Strain 30 and declining in Strain 31 at an average rate of .045 per generation of selection.
1261
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17
16
19
Both
7.1 6.3 7.9 1.2 2.3 2.0 6 to 10 3 to 13I 3 to 13 15 15 30
146
80
363
Control 90 114 2 -.8 18
25
7.6 7.9 6.4 1.7 1.6 1.7 5 to 11 4 to 11 4 to 9 15 15 15
115 4 -.8 19
132 1 -1.0 22
816 47 3.2 22 7.2 1.1 5 to 9 15
23
21
20
Selected
Sire strains (Cornish)
7.3 1.6 4 to 11 60
29
All 84 4 -.8 20
31
26
Both
7.7 7.7 7.7 2.5 2.1 1.9 4 to 13 4 tc) 10 4 to 13 15 30 15
756 45 3.5 22
Control 30
Dam strains (White Rock)
7
WL
7.3 2.7 1.9 1.0 3 to 13 1 to 5 120 38
31
PP
1 Strain 80 was selected for 14 generations for 63-day BW; Strains 21,23, and 25 were all selected for 10 generations for high 28-day BW and low abdominal fat percentage of sibs (Strain 21), 28- to 42-day feed efficiency (Strain 23), or both feed efficiency and low-fat (Strain 25); Strain 31 was selected for high 28-day BW, 28- to 42-day feed efficiency, and high hatching egg production to 40 wk of age of females; Ottawa control Strain 7. Production traits of selected strains were recorded as deviations from the corresponding control strain. 2 Feed efficiency = (weight gained -s- feed consumed) x 100; abdominal fat = (abdominal fat -s- carcass weight x 100).
28-day BW, g Feed efficiency,2 % Abdominal fat,2 % Number of ev loci Mean number of ev loci per chicken SD Range Number tested
Characteristic
OMC strains
TABLE 1. Production trait characteristics and mean number of different endogenous viral (ev) genes per chicken and per strain in Ottawa meat control (OMC), sire and dam meat-type populations, these populations pooled (PP), and Ottawa White Leghorn (WL) control Strain 5 1
c-
XI en
£ BOU
1263
ENDOGENOUS VIRAL GENES IN MEAT-TYPE CHICKENS Dam
WL Wl
f
Sire
T20
D,
21 23
25T30
f
OMC
31Y90
1(20
21
23
25~||30
m
ev18 — —23.1
ev18 — ev1 —
GV *J
iiiiill:
^~* ^IW*
—23.1
* i
ipr * *
ev3 — —
V
s •» «•
—6.6
—6.6
4$jMfc
—
—4.4 —4.0
— a*
ev6 — "" evrntl —
iff 9 In-
—4.4
—
—3.0
FIGURE 2. Autoradiograph of Southern blot of (left) Sad- and (right) BamHI-digested pooled genomic DNA of 15 individuals from each of eight meat-type chicken strains, hybridized with P-labeled pRAV-2 probe. Sire populations analyzed were: Strain 20, unselected control; Strains 21, 23, and 25, selected. Dam populations analyzed were: Strain 30, unselected control; Strain 31, selected; Ottawa Meat Control (OMC) chicken populations: Strain 90, unselected control; Strain 80, selected. WL = a White Leghorn strain carrying endogenous viral loci evl, ev3, ev6, and eul8, used as a reference. M = DNA marker fragment sizes.
fragment without their designation as to published equivalents. Statistical
Analyses
Tests of differences in frequencies of the DNA fragments either individually or in total among populations or among strains within populations were performed. Differences in presence of the 31 DNA fragments in each strain were analyzed using a loglinear model for categorical data and appropriate chi-square. Differences in frequency of specific DNA fragments were tested using the chi-square test of heterogeneity (SAS Institute, 1985). RESULTS Analyses of Pooled Deoxyribonucleic Acid
Samples
Analysis of ev loci in pooled DNA of the three sources and their strains revealed a complex restriction fragment length polymorphism pattern with fragment sizes ranging from 4.4 to 30 kb and 3.4 to 25 kb when DNA was digested with Sad and
BamHl restriction enzyme, respectively (Figure 2). A visual examination of the bands showed differences in the optical density between populations or strains within a population (Figure 2). Photodensitometric measurements of the bands (Figure 3) were positively correlated (correlation coefficient = .86; F < .01) with the number of ev genes that the band of the pooled DNA represented, as determined by analysis of individual chicken DNA samples. Analyses of Individual Deoxyribonucleic Acid
Chicken Samples
A total of 31 different Sacl DNA fragments were found in the populations studied (Table 1). The numbers of ev loci per population (19,29, and 26 for the OMC, sire, and dam populations, respectively) were not significantly different. The average number of different ev loci per chicken in the three populations combined was 7.3, almost three times higher than the 2.7 ev genes observed in a control strain of White Leghorn chickens. The number of ev loci detected in individual meat chickens
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el,1
evmtl —
M
M
80]
ev6
OMC
31 "l[90 8(51
1264
SABOUR £T AL.
selected strains of OMC, dam, and sire populations, respectively (Table 3). Comparison with Previously Characterized Endogenous Viral Loci
No. of ev fragments in individuals
FIGURE 3. Relationship between the number of endogenous viral (ev) fragments in individuals and relative densitometric values of autoradiograms of pooled DNA in a sample of the 15 chickens shown in Figure 2 (y = 8.141 + 11.230% r = .86).
ranged from 3 to 13. This is in contrast to the White Leghorn control line, with a range of 1 to 5 ev loci per chicken. Presence and frequencies of specific ev loci in the three populations varied. Fifteen ev loci were common to all three meat-type populations (Table 2). The 10.5-kb fragment, identified as eul8, was nearly ubiquitous, as it was present in over 86% of all chickens. Certain loci were present in high frequency in specific populations. For example, the 22.6-kb fragment was detected in 97% of the OMC chickens and similarly the 7.5-kb fragment was present in 97% of the dam population chickens. Two ev loci, 12.2 and 8.3 kb, were detected only in the sire population and one, 9.2 kb, only in the dam population (Table 2). The latter cosegregated with a 20-kb BamHI and was genotyped as evil by the use of specific DNA probe pEV21. There was no significant difference in the average number of ev genes per chicken between selected and control strains (Table 1); however, strain differences in certain ev locus frequencies were observed. For example, there were differences of five, two, and six loci between unselected control and
DISCUSSION Endogenous Viral Gene Frequencies of the Pooled Deoxyribonucleic Acid The positive correlation between the number of ev genes observed in individual birds included in a pooled sample and relative densitometric values of corresponding bands on autoradiograms of pooled DNA provided a means to assess ev gene frequency differences. This relationship was used to search for differences in ev gene frequencies between strains using pooled DNA samples from 15 chickens per strain. The usefulness of this approach was also reported in evaluating gene frequency changes in groups within populations (Plotsky et al., 1990). In summary, DNA evaluation of individual chickens of a strain or group will yield more precise results than results from pooled DNA. However, results from pooled DNA are less laborious and therefore more economical and rapid for population comparisons and, on occasion, sufficiently precise to warrant their use in the assessment of genetic distances between populations. Differences in Endogenous Loci Due to Population Origin A large number and different types of ev loci were detected in the three populations. Past results suggest that most meat-type chicken populations contain similar ev loci
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Paired association of restriction fragment length polymorphism (RFLP) pattern of Sacl to its BamHI fragments was used to identify five previously characterized loci (Table 4). Genes ev3, ev6, evl%, evil, and evrc&l, a locus with unknown phenotype showing a 4.4-kb Sacl fragment and a 3.5-kb BamHI fragment, were present in the three populations. Gene evil was detected only in the dam population, which is known to segregate for the sex-linked, slow-feathering gene.
1265
ENDOGENOUS VIRAL GENES IN MEAT-TYPE CHICKENS
TABLE 2. Number of individuals per strain carrying each endogenous viral locus for eight strains of meat-type chickens OMC 1
Dam
Sire Selected
Control 90
Selected 80
Freq. 2
Control 20
21
23
25
Freq. 2
Control 30
Selected 31
Freq. 2
(kb) 33.5 25.0 22.6 20.9 19.5 18.0 16.5 15.0 14.5 13.0 12.2 11.5 10.5 10.2 10.0 9.5 9.2 9.15 9.1 8.7 8.6 8.3 8.1 7.9 7.7 75 7.3 6.8 6.3 5.4 4.4
1 0 15 3 0 0 0 1 9 2 0 0 11 0 9 6 0 0 9 5 8 0 2 0 14 0 0 0 11 0 13
5 0 14 4 0 0 0 0 2 3 0 1 14 3 1 2 0 0 3 0 1 0 6 0 12 0 2 0 11 0 10
.20 .00 .97 .23 .00 .00 .00 .03 .37 .17 .00 .03 .83 .10 .33 .27 .00 .00 .40 .17 .30 .00 .27 .00 .87 .00 .07 .00 .73 .00 .77
2 0 8 4 3 0 1 1 0 0 5 5 14 0 4 2 0 5 0 4 8 10 1 0 9 0 7 1 2 1 11
3 1 3 0 1 2 8 0 1 0 5 10 11 0 3 7 0 0 0 10 6 3 9 3 10 0 3 3 5 0 5
3 0 7 3 3 0 6 0 0 0 5 10 9 2 8 8 0 0 9 6 2 0 8 0 12 8 2 0 0 0 8
3 0 9 3 2 0 2 0 0 0 7 5 13 1 0 0 0 0 2 6 8 2 9 0 7 0 0 2 6 0 9
.18 .02 .45 .17 .15 .03 .28 .02 .02 .00 .37 .50 .78 .05 .25 .28 .00 .08 .18 .43 .40 .25 .45 .05 .63 .13 .20 .10 .22 .02 .55
0 0 7 8 4 0 1 0 2 2 0 2 14 0 4 6 8 3 4 0 9 0 4 0 7 14 2 2 1 1 10
1 1 9 12 3 1 1 0 0 0 0 2 15 0 2 10 3 3 0 0 11 0 2 2 6 15 9 0 0 0 8
.03 .03 .53 .67 .23 .03 .07 .00 .07 .07 .00 .13 .97 .00 .20 .53 .37 .20 .13 .00 .67 .00 .20 .07 .43 .97 .37 .07 .03 .03 .60
!OMC Freq.
2
Ottawa Meat Control strains. Overall frequency of each locus within population.
at different frequencies and this reflects their different genetic origin (Rovigatti and Astrin, 1983; Aarts et al, 1991; Boulliou et al, 1991). Differences in experimental conditions may also lead to different reported fragment sizes and this creates further difficulties in comparing the loci reported by different laboratories. Nevertheless, for the purpose of this discussion, each distinct observed band size will be considered a separate ev locus. The OMC and the modern populations shared several loci; however, certain loci were observed only in the sire (12.2 and 8.3 kb) or in the dam (9.2 kb) populations, whereas nine others were present in both the sire and dam populations. The sire and
dam populations of 1978 origin had 10 and 7 more ev loci than the OMC strains of the 1950s (Table 2). This may reflect either the broad genetic base of the populations synthesized in 1978 or the elimination of low-frequency loci during generations through which the OMC birds have been maintained as closed, relatively small populations. In the White Leghorn Strain 7, used here for a comparison, that has been maintained closed since 1958, there are only seven different loci and, on the average, 2.7 times fewer loci per bird than in the meattype chickens. As mentioned earlier, the sire and dam populations of 1978 origin were synthesized from multiple commercial sources and can be considered to
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Locus
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SABOUR ET AL.
TABLE 3. Tests of heterogeneity (chi-square) among polymorphic endogenous viral loci of meat-type chickens of the Ottawa Meat Control (OMC), sire, and dam populations Within populations Sire
Between populations
Locus
Sire vs. dam
OMC S vs. C 1
Dam S vs. C
NS
*
444
NS
NS
4*4
*
NS
NS NS NS NS NS NS
4
4
NS NS NS NS NS NS
NS NS
44
44
NS NS NS
NS NS NS
***
NS NS 4*4 4
NS
** *
** 4*4
NS
NS
*** t
NS NS NS NS
*
X-
*
*
NS NS NS NS NS NS NS NS NS
4
NS NS NS
4
4
NS NS NS NS NS NS NS NS NS NS NS
NS NS NS
444
NS NS
NS NS NS NS
44
*
** 444
4*
NS NS NS NS
*4 4 *
NS NS
* *
Strain 21 vs. 23
NS
»
NS 44
Strain 25 vs. 21, 23
NS NS NS NS NS NS NS NS NS NS NS NS NS t NS
4*
4*4
S vs. C
44*
NS NS 4
NS
44
NS NS NS NS
44
NS NS NS NS NS NS NS NS
444
444 44*
NS NS
* NS 4*
**4
NS NS NS NS NS NS NS NS
*
NS NS NS NS
t NS NS NS 4*4
NS NS NS NS NS 4*4
NS NS
*
NS
*S vs. C = selected strain(s) versus control strain. < .06. *P < .05. **P < .01. ***P < .001.
+P
contain samples from most internationally used commercial meat stocks of that time (Chambers et al, 1984). Interestingly, only four segregating ev loci in a sample of 12 birds were observed in the red jungle fowl (Gallus bankiva) (Rovigatti and Astrin, 1983), the progenitor of the domesticated chicken (Gallus domesticus) (Frisby et al, 1979). This would suggest that the numerous ev loci observed in the present study as well as in previous published studies may represent insertions that occurred since domestication of the species. Effects of Selection
A comparison between selected and control strains within populations should
reflect differences due to selection. The frequency of a number of loci differed significantly between selected and unselected control strains. However, such differences may also be due to genetic drift or represent sampling errors. With only 15 chickens per strain, the error associated with the observed ev gene frequencies was large. Calculations of necessary gene frequency difference required for significance (P < .05) between two groups of size 15 based on chi-square (1 df) indicated that differences of at least three chickens at extreme frequencies and up to more than five chickens at intermediate frequencies were required. For groupings of the strains, differences in incidence required for statistical significances were lower. It should also
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(kb) 33.5 22.6 20.9 19.5 16.5 15.0 14.5 13.0 12.2 11.5 10.5 10.0 9.5 9.2 9.15 9.1 8.7 8.6 8.3 8.1 7.7 7.5 7.3 6.8 6.3 4.4
OMC vs. sire and dam
1267
ENDOGENOUS VIRAL GENES IN MEAT-TYPE CHICKENS TABLE 4. Phenotypes of endogenous viral (ev) genes and their frequencies Populations
Size of specific DNA fragment Sad
Modern
BamHl
Locus
Phenotype 1
7.3 4.4 25.0 20.0 3.5
ev3 ev6
gs + chf" gs" chf+ V-E + V-E + Unknown
H-hl
\XD)
6.3 22.6 10.5 9.2 4.4
eo\% ev215 eumtl 6
OMC 2
Sire
Dam
(n = 30) .73***3
(n = 60) .22*4
(n = 30)
07***3
.45
.83
.7%** .OO***4
.00*3 .77t 3
.55
.03 .63 .97 .37 .60
be kept in mind that some of the production trait differences between selected and control strains and those among strains selected from the same base were small (Table 1). Hence, conclusions on associations of ev locus frequency differences with effects of selection may, in such instances, be questionable. Sire population Strains 21,23, and 25 had been selected for low abdominal fatness of 47-day-old sibs, high feed efficiency between 28 and 42 days, or both of these traits, respectively, in addition to body weight at 28 days. Breeding records revealed that realized selection intensities in total differed for these strains. Effects of genes for feed efficiency and abdominal fatness are largely additive in nature; however, nonadditive genetic effects cannot be ruled out. In addition, genetic drift is contributing at least at minor levels to genetic differences between these strains. Therefore, progeny of Strain 25 were not necessarily intermediate to those of Strains 21 and 23 in feed efficiency and abdominal fatness. Consequently, it is difficult to quantify with precision expected differences in gene frequencies among these selected strains. Comparisons of Strains 21 and 23 are more
likely to reflect differences in frequencies of contributing genes or genes linked to them than comparisons involving Strain 25 due to the greater difference in selected traits. In light of this background, the loci with 7.3- and 9.1-kb fragments appear to be most apt to be associated with selection pressures for high feed efficiency or low abdominal fatness (Table 3). Changes in frequencies of several other ev loci seem attributable to genetic selection procedures: the 16.5-, 9.15-, and 8.3-kb loci in the sire population reveal differences between control and selected strains. Of the ev loci that differed significantly between selected and control OMC strains, all had reduced frequencies in the selected strain. Although the reasons for the increase in the frequency of 7.3-kb or the decrease in the 9.1-kb loci in the selected dam strain compared with the control strain were not clear, the 9.2-kb locus, which was identified as the eu21 gene and is known to be associated with the slow-feathering gene K, decreased in frequency in the selected dam Strain 31. This agrees with past observations that selection for body weight and feed conversion reduces the frequency of the slow-feathering gene (eu21) (Pym et al,
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Summarized by Smith (1986). Phenotypes are abbreviated as follows: gs + = expression of internal viral gag proteins; chf" = expression of the viral envelope protein; V-E + = production of infectious endogenous virus. 2 OMC = Ottawa Meat Control. 3 Significant difference between OMC and modern populations. Significant difference between the sire and dam populations. The presence of evil was confirmed by using a eo21-specific DNA probe on the same Southern blot membranes used for hybridization with pRAV-2. 6 m t l = Unidentified meat-type (Iraqi et al, 1991; Boulliou et al, 1991). t P < .06. *P < .05. **P < .01. ***P < .001.
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SABOUR ET Al.
Previously Characterized Endogenous Viral Loci
Due to the complexity of the hybridization pattern, determination of the phenotype of most loci was not possible. However, as mentioned earlier, using chicken DNA with known ev loci and matching restriction fragments produced by Sacl and BamHl enzymes, five loci were tentatively characterized as equivalent to previously described loci. The phenotype of four of these (ev3, ev6, eul8, and ez;21) is known (Smith, 1986) and of one, designated erantl, is unknown. Except for eu21, the frequencies of none of these loci differed significantly between selected and unselected control strains. The frequency of both ev3 and ev6 was significantly higher in the OMC (1955 origin) than the modern (1978 origin) populations (Table 2). In the dam population, only one chicken having ev3 was detected in a sample of 30 chickens and locus ev6 was in intermediate frequency in the 1978 populations (Table 2). Loci ev3 and ev6 are known to express endogenous viral envelope glycoproteins at high levels (Hayward et al, 1980), which interferes with endogenous (Subgroup E) viral infection, presumably by blocking the host cell membrane receptors for the virus (Robinson et al, 1981; Crittenden et al, 1982). Hence, ev3 and ev6 may be expected to provide their hosts with some selective advantage, given the negative effects of endogenous virus on egg production traits (Gavora et al, 1991). The patterns observed in the current study
do not seem to provide much evidence for such a hypothesis. Of particular interest is the distribution of evil, an endogenous virus-producing gene that is associated with the dominant, sex-linked, slow-feathering gene K and was detected in the dam population. Congenital transmission of infectious virus, which is encoded by the evil gene, reduces seroconversion and enhances the incidence of lymphoid leukosis (Smith and Fadly, 1988). The frequency of this gene decreased 2.7-fold (P < .06) in Strain 31 (selected for high hatching egg production, body weight, and feed efficiency) compared with control Strain 30. In this context, it is notable that the frequency of the virus-producing eul8 in the selected strain of the dam population was unchanged and remained high (Tables 2 and 3). The present results are in agreement with the observation by Boulliou et al. (1991) that in commercial broilers, 17 loci appear to contain a complete proviral genome without significant deletion. A large number of replication-competent endogenous viruses were detected in Australian commercial broilers by Ignjatovic (1986), suggesting the presence of multiple complete endogenous proviral genomes. Many of the uncharacterized ev loci observed in the current study may be of this typeOccurrence of ez?18, evil, and perhaps other similar but uncharacterized ev loci may be associated with low egg production in the meat-type chickens (Crittenden et al, 1979; Kuhnlein et al, 1989a,b; Gavora et al, 1991). The observations that most commercial White Leghorn strains lack ev genes that code for endogenous virus (Tereba and Astrin, 1980) may reflect the elimination of such genes during selection for egg production. Observations in the current study and those reported by Boulliou et al. (1991) and Aarts et al. (1991) indicate that this may not be the case in meat chickens. The nature of the erantl locus, which is characterized by 4.4-kb Sacl and 3.5-kb BamHl fragments, is not clear. It was frequently present in the populations studied here. Because both commercial layers (Iraqi et al, 1991) and commercial broilers (Boulliou et al, 1991) have this locus at high frequency, it is unlikely that its presence is
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1984). The selection for broiler weight, feed efficiency, and adult hatching egg production of hens has also been observed to reduce the frequency of the slow feathering gene (J. R. Chambers, unpublished data). In the current study, the reduction in frequency of the evil locus and the associated slow-feathering gene from 8 of 15 chickens of the control strain to 3 of 15 in the selected strain of the dam population (Table 2) only approached significance (P < .06). This difference should be real according to the preceding reports (Pym et al, 1984; Chambers, unpublished data); however, the power of the test in the present study was inadequate for its detection.
ENDOGENOUS VIRAL GENES IN MEAT-TYPE CHICKENS
ACKNOWLEDGMENTS
The expert technical assistance of P. S. Griffin is gratefully acknowledged. The authors thank the staff of the Centre for Food and Animal Research poultry facilities for care of the experimental chickens and collecting the blood samples and also thank M. Boichard for helping in the analysis of eu21 locus. The authors thank L. B. Crittenden and E. J. Smith, USDA, Agricultural Research Service, Avian Disease and Oncology Laboratory, East Lansing, MI 48823 for their generous gifts of the pRAV-2, pEV21, and marker chicken DNA.
REFERENCES Aarts, H.J.M., M. C. van der Hulst-Van Arkel, G. Bouving, and F. R. Leenstra, 1991. Variations in endogenous viral gene patterns in White Leghorn, Medium Heavy White Plymouth-Rock,
and Cornish chickens. Poultry Sci. 70:1281-1286. Astrin, S. M., 1978. Endogenous viral genes of the White Leghorn chicken: common site of residence and sites associated with specific phenotypes of viral gene expression. Proc. Natl. Acad. Sci. USA 75:5941-5945. Astrin, S. M., E. G. Buss, and S. W. Hayword, 1979. Endogenous viral genes are nonessential in the chicken. Nature 281:339-341. Bacon, L. D., E. Smith, and L. B. Crittenden, 1988. Association of the slow feathering (K) and an endogenous viral (eo21) gene on the Z chromosome of chickens. Poultry Sci. 67:191-197. Boulliou, A., J. P. Le Pennec, G. Hubert, R. Donald, 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. Chambers, J. R., D. E. Bernon, and J. S. Gavora, 1984. Synthesis and parameters of new populations of meat-type chickens. Theor. Appl. Genet. 69: 23-30. Crittenden, L. B., A. M. Fadly, and E. J. Smith, 1982. Effect of endogenous leukosis virus genes on response to infection with avian leukosis and reticuloendotheliosis virus. Avian Dis. 26: 279-294. Crittenden, L. B., J. S. Gavora, F. A. Gulvas, and R. S. Gowe, 1979. Complete endogenous RNA tumor virus production by inbred and non-inbred chickens. Avian Pathol. 8:125-131. Frisby, D. P., R. A. Weiss, H. Roussel, and. D. Stehelin, 1979. The distribution of endogenous chicken retrovirus sequences in the DNA of galliform birds does not coincide with avian phylogenetic relationships. Cell 17:623-634. Gavora, J. S., 1987. Influences of avian leukosis virus infection on production and mortality and the role of genetic selection in the control of lymphoid leukosis. Pages 241-260 in: Avian Leukosis. G. F. de Boer, ed. Martinus Nijhoff Publishing, Boston, MA. Gavora, J. S., U. Kuhnlein, L. B. Crittenden, J. L. Spencer, 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. Gavora, J. S., U. Kuhnlein, and J. L. Spencer, 1989. Absence of endogenous viral genes in an inbred line of Leghorn chicks selected for high egg production and Marek's disease resistance. J. Anim. Breed. Genet. 106:217. Gudkov, A. V., E. Korec, M. V. Chernov, A. T. Tikhonenko, I. B. Obukh, and I. Hlozanek, 1986. Genetic structure of the endogenous proviruses and expression of the gag gene in Brown Leghorn. Folia Biol. (Cracow) 32:65-72. Gudkov, A. V., I. B. Obukh, S. M. Serov, and B. S. Naroditsky, 1981. Variety of endogenous proviruses in the genomes of chickens of different breeds. J. Gen. Virol. 57:85-94. Haywood, W. S., S. B. Braverman, and S. Astrin, 1980. Transcriptional products and DNA structure of endogenous avian proviruses. Cold Spring Harbor Symp. Quant. Biol. 44:1111-1121. Ignjatovic, J., 1986. Replication-competent endoge-
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associated with differences in the traits selected in the meat-type chickens. In some chickens analyzed in the present study, a 9.5-kb Sacl fragment was associated with a 5.2-kb BamHl fragment, which is characteristic of locus evl in the egg strains (Smith, 1986). However, due to comigration and possible polymorphism, it was difficult to ascertain the presence of an evl. Using a probe specific for evl, this locus was detected in White Plymouth Rock chickens at low frequency (Aarts et al, 1991). White Plymouth Rock chickens were a major contributing breed to the dam population in the current study. The results of the present study further demonstrate the complexity of ev loci in meat-type chickens. Several of ev loci present in these chickens create problems in identifying specific ev loci, without which an understanding of their possible association with production traits will be very difficult. Unequivocal identification of individual ev genes and loci, presumably by assays using cloned flanking regions of the loci, will be required for future analyses of phenotypes of the as yet uncharacterized loci in meat chickens and their association with production trait differences. Such research is justified by the implication of ev genes as factors that influence economic traits in White Leghorns.
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SABOUR ET AL. Pym, R.A.E., P. J. NichoUs, E. Thomson, A. Choice, and D. J. Farrell, 1984. Energy and nitrogen metabolism of broilers selected over ten generations for increased growth rate, food consumption and conversion of food to gain. Br. Poult. Sci. 25:529-539. Reed, K. C, and D. A. Mann, 1985. Rapid transfer of DNA from agarose gels to nylon membranes. Nucleic Acids Res. 13:7207-7221. Robinson, H. L., S. M. Astrin, A. M. Senior, and I. H. Salazar, 1981. Host susceptibility to endogenous viruses: defective glycoprotein-expressing proviruses interfere with infections. J. Virol. 40: 745-751. Rovigatti, U. G., and S. M. Astrin, 1983. Avian endogenous viral genes. Curr. Top. Microbiol. Immunol. 103:1-21. SAS Institute, 1985. SAS® User's Guide. Version 5 Edition. SAS Institute Inc., Cary, NC. Smith, E. J., 1986. Endogenous avian leukemia viruses. Pages 101-120 in: Avian Leukosis. G. F. de Boer, ed. Martinus Nijhoff Publishing, Boston, MA. Smith, E. J., and L. B. Crittenden, 1986. Endogenous viral genes in a slow-feathering line of White Leghorn chickens. Avian Pathol. 15:395-406. Smith, E. J., and A. M. Fadly, 1988. Influence of congenital transmission of endogenous virus-21 on the immune response to avian leukosis virus infection and the incidence of tumors in chickens. Poultry Sci. 67:1674-1679. Tereba, A., and S. M. Astrin, 1980. Chromosomal localization of ev-1, a frequently occurring endogenous retrovirus locus in White Leghorn chickens, by in situ hybridization. J. Virol. 35: 888-894. Tereba, A., and S. M. Astrin, 1982. Chromosomal clustering of fine defined endogenous retrovirus loci in White Leghorn chickens. J. Virol. 43: 737-740. Tereba, A., M.M.C. Lai, and K. G. Murti, 1979. Chromosome 1 contains the endogenous RAV-0 retrovirus sequence in chicken cells. Proc. Natl. Acad. Sci. 76:6486-6490.
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nous avian leukosis virus in commercial lines of meat chickens. Avian Dis. 30:264-270. Iraqi, F., M7 Soller, and J. S. Beckmann, 1991. Distribution of endogenous viruses in some commercial chicken layer populations. Poultry Sci. 70:665-679. 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, R. 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. Levin, L, and E. J. Smith, 1990. Molecular analysis of endogenous virus eu21 slow-feathering complex of chickens. 1. Cloning of proviral-cell junction fragment and unoccupied integration site. Poultry Sci. 69:2017-2026. Merritt, E. S., and R. S. Gowe, 1962. Development and genetic properties of a control strain of meat-type fowl. Pages 66-70 in: Proceedings of the 12th World's Poultry Congress, Sydney, Australia. Merritt, E. S., M. Zawalsky, and S. B. Slen, 1966. Direct and correlated responses to selection for 63-day body weight in chickens. Pages 86-91 in: Proceedings of the 13th World's Poultry Congress, Kiev, Russia. Murray, J. C, R. Shiang, L. R. Carlock, M. Smith, and K. H. Buctow, 1987. Rapid RFLP screening procedure identifies new polymorphisms at albumin and alcohol dehydrogenase loci. Hum. Genet. 76:274-277. Plotsky, Y., A. Cahaner, A. Haberfeld, U. Lavi, and J. Hillel, 1990. Analysis of genetic association between DNA fingerprint bands and quantitative traits using DNA mixes. Pages 133-136 in: Proceedings of the 4th World Congress on Genetics Applied to Livestock Production, Volume XIII, Edinburgh, Scotland.
