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Screening Chickens for Endogenous Virus ev 21 Viral Element by the Polymerase Chain Reaction
RESEARCH NOTES Screening Chickens for Endogenous Virus ev21 Viral Element by the Polymerase Chain Reaction MICHELE H. TIXIER-BOICHARD Laboratoire de Gtnitique Factorielle, Institut National de la Recherche Agronomique, 78352 Jouy-en-Josas, Cedex, France BERNHARD F. BENKEL, JIM R. CHAMBERS, and JAN S. GAVORA
ABSTRACT The molecular architecture of the sex-linked late-feathering region of the chicken genome is still poorly defined. Current evidence points to a strong association between the presence of the endogenous viral element evil and the late-feathering phenotype. However, analysis at the molecular level has demonstrated that this is not a simple case of insertional mutagenesis. Instead, the structure of the region of the chicken genome containing the feathering locus is complex and variable between and within lines of chickens. Significant clues to the molecular structure of this genomic region can be obtained by analyzing rare and revertant genotypes. However, searching for rare genotypes can only be carried out effectively using quick screen methodology. This paper describes a quick, polymerase chain reaction-based test for evil that facilitates the search for rare genotypes. (Key words: polymerase chain reaction-based test, endogenous provirus detection, rapid nonradioactive screening, late-feathering mutation, chicken) 1994 Poultry Science 73:1612-1616
between evil and the K allele. Molecular analysis has revealed that the structure of The sex-linked, dominant late- the K-evll genomic region is complex, due feathering mutation (K) used in poultry to the simultaneous presence of a duplibreeding for feather-sexing, is associated cated insertion site that is unoccupied by with the endogenous viral gene, evil, in ei>21 (unoccupied site = US), together with White Leghorn chickens (Bacon et at, the occupied site (OS) in late-feathering 1988). The presence of ev2\ is associated birds (Smith and Levin, 1991). However, with a reduced immune response to other configurations have also been lymphoid leukosis viruses (Smith and reported. Of particular interest are certain Fadly, 1988). This observation corrobo- strains of brown egg layers that carry the rated previous results showing immuno- evil insertion (OS) alone without the US logical tolerance towards lymphoid leuko- region. These birds are phenotypically sis virus in slow-feathering White Leghorn early-feathering (Boulliou et ah, 1992). dams and chicks (Harris et at, 1984). Evidently, the evil insert by itself is not The cloning of a locus-specific probe sufficient to produce late-feathering in corresponding to the evil insertion site, chickens. The fact that chickens can carry ez>21-int (Levin and Smith, 1990), has the evil element yet appear phenotypifacilitated the analysis of the relationship cally early-feathering as shown above means that the frequency of evil in modern chicken lines is not known at present and may be much higher than is Received for publication March 11, 1994. currently appreciated. A rapid test to Accepted for publication May 24, 1994. INTRODUCTION
1612
Downloaded from http://ps.oxfordjournals.org/ at D H Hill Library - Acquis Dept S on March 10, 2015
Centre for Food and Animal Research, Agriculture Canada, Ottawa, Ontario, Canada, K1A 0C6
RESEARCH NOTE
MATERIALS AND METHODS Chickens An Ottawa synthetic broiler dam line (Strain 30, Chambers et al, 1984) segregates for the K mutation (Chambers and Gavora, 1993) and for many ev genes of the Rousassociated virus family (Sabour et al, 1992). A sample of 106 female progeny of Strain 30 was obtained from 58 sires. The frequency of the late-feathering mutation in the contemporary generation was .42 out of a total of 317 female progeny. The chicks were vent-sexed and scored for feathering type on the day of hatch and bled at 12 wk of age. The test included females only because the hemizygosity for Z-linked traits warrants a straightforward relationship between the genotype and the phenotype.
Crude DNA extracts for the PCR assay were prepared according to the Protocol B of Higuchi (1989) as described in Benkel et al (1992). Reference DNA samples were a gift from A. Boulliou (Centre National de la Recherche Scientifique, Unite de Recherche Assoctee 256, Rennes I, 35042 Rennes Cedex, France) and represented 1) an earlyfeathering layer with OS only; 2) a revertant early-feathering female broiler with US only; and 3) a revertant early-feathering female broiler with OS only. The genotypes had been characterized by a Southern procedure with several enzymes and the probe evll-mi (Boulliou et al, 1992). Polymerase Chain Reaction Amplification Oligonucleotides were synthesized on an Applied Biosystems synthetizer Model 381A11.1 Sequences for the upstream flanking primer PR-UP (5'-GTGGGAATGGTACTACAGAGAAGG-3'), the downstream flanking region primer PR-DWN (5'CATTTCAAGCAAGGGACTGGC-3'), and the oligomer specific for the retroviral long terminal repeat Pl-IN (5'-ACCTGAATGAAGCTGAAGGCTTC-3') were chosen based on the results of Levin and Smith (1991). Amplifications were carried out in 50-/tL reactions in a Perkin Elmer Cetus2 thermal cycler. A modified "touchdown" procedure consisting in two cycles at 96 C for 1 min, 66 C for 1 min, and 72 C for 2 min; two cycles at 96 C for 1 min, 63 C for 1 min, and 72 C for 2 min; 28 cycles at 96 C for 1 min, 57 C for 1 min, and 72 C for 2 min; followed by a 10-min extension at 72 C, was used to minimize secondary priming (Don et al, 1991; Benkel and Smith, 1993). Reaction mixtures were overlaid with sterile mineral oil. Amplified products were separated on a 2% agarose gel containing ethidium bromide. An amplified product of 390 bp indicated the presence of a viral insert at the ez>21 locus, (OS), whereas an amplified product of 515 bp was expected with a US (Figure 1). RESULTS AND DISCUSSION
1
Applied Biosystems Canada Inc., Mississauga, ON, Canada, L5N 2M2. 2 Perkin Elmer (Canada) Ltd., Rexdale, ON, Canada, M9W 1A4.
Figure 2 shows the results of the evil PCR assay applied to a number of test samples. As expected, standard earlyfeathering females produced a single am-
Downloaded from http://ps.oxfordjournals.org/ at D H Hill Library - Acquis Dept S on March 10, 2015
identify eu21 would help to address this question. One interpretation of the available evidence on the association between e»21 and late-feathering would be that the eu21 insertion site and the feathering gene are closely linked but physically separate genomic regions, which would make possible the segregation between alleles at the K locus and the evil integration site. The search for recombinants at the K-ev21 locus requires the analysis of large numbers of chickens due to the close linkage between the two loci. Such an extensive screening operation would be both expensive and time-consuming to perform by Southern blotting. Polymerase chain reaction (PCR)-based diagnostics (Mullis and Faloona, 1987) are becoming increasingly popular as an alternative to Southern analysis due to their speed and sensitivity. In this report, we describe a PCR-based method for the rapid identification of the US and OS components of the K-evll locus, following an approach developed previously for the evl locus (Benkel et al, 1992).
1613
1614
TIXIER-BOICHARD ET AL. 515
bp
PR-UP
ev21 PR-DWN PR-UP
P1-1N
c PR-DWN 390 bp H
FIGURE 1. Schematic representation of the insertion site for the endogenous virus element ev21 provirus, showing the annealing sites for the primers (short arrows) used in the Polymerase Chain Reaction (PCR), and the sizes of the DNA fragments resulting from amplification of a chromosome lacking ro21 (upper line) and containing the proviral insert (lower line). Hatched and solid boxes represent the upstream and downstream flanking regions of the element, respectively. The open box represents the ev21 provirus. The amplification products of the interactions with either the upstream P1-1N site in the element or PR-UP, in combination with PRDWN, are over 7.0 kbp in length and are not detected under standard PCR conditions.
plified band at about 515 bp, whereas normal late-feathering chickens gave composite patterns consisting of the 515-bp US-specific band plus the shorter 390-bp band that is diagnostic of the OS. Of the early-feathering females analyzed previously by Boulliou et ah (1992), one of the revertant female broilers and the brown egg layer, shown in Lanes 2 and 3 respectively, yielded only the 390-bp fragment, which is specific for the presence of evil. A second revertant female produced only the 515-bp fragment, shown in Lane 4, that is diagnostic of the US. Results obtained previously with Southern blotting on the reference samples (Boulliou et ah, 1992) showed a perfect association between the amplified product of 515 bp and the 3.5-kbp EcoRI restriction fragment that is diagnostic of US, as well as the amplified product of 390 bp and the EcoRI restriction fragment of 2.5 kbp, which corresponds to the 3' flanking junction fragment described by Smith and Levin (1991). Overall, among the 106 females of Strain 30 that were studied using the PCR
-
ev 21 ev 21
3
4
FIGURE 2. Gel analysis of amplification products for the endogenous virus element evl\ assay. Females carrying the occupied site (OS) only show a band of 390 bp; chickens with the unoccupied site (US) only produce a band of 515 bp; females with both the OS and the US show a composite pattern consisting of both bands. Samples are as follows: Lanes 1 and 6, normal late-feathering broiler females containing US in addition to OS; lane 2, revertant early-feathering broiler female carrying OS only; lane 3, early-feathering layer female with the OS only; lane 4, revertant early-feathering broiler female with the US only; lane 5, normal early-feathering broiler female containing the US only. The molecular weight standard represents a Haelll digest of 0X174 DNA.
Downloaded from http://ps.oxfordjournals.org/ at D H Hill Library - Acquis Dept S on March 10, 2015
ev21
P1-1N
1615
RESEARCH NOTE TABLE 1. Distribution of molecular types according to the feathering phenotype in 106 female progeny Feathering phenotype Molecular type
Early (k) Late (K)
Unoccupied site (US) only Occupied site (OS) only US + OS
57 3 0
1 0 45
ACKNOWLEDGMENTS
This work started during a sabbatical leave of M. H. Tixier-Boichard at the Centre for Food and Animal Research which was supported by a NATO postdoctoral fellowship.
REFERENCES Bacon, L. D., E. J. Smith, L. B. Crittenden, and G. B. Havenstein, 1988. Association of the slowfeathering (K) and an endogenous viral («>21) gene of the Z chromosome of chickens. Poultry Sci. 67:191-197. Benkel, B. F., J. Mucha, and J. S. Gavora, 1992. A new diagnostic method for the detection of endogenous Rous-associated virus-type provirus in chickens. Poultry Sci. 71:1520-1526. Benkel, B. F., and E. J. Smith, 1993. Research note: A rapid method for the detection of the Rous-
Downloaded from http://ps.oxfordjournals.org/ at D H Hill Library - Acquis Dept S on March 10, 2015
approach, there was a good agreement between the late-feathering phenotype (K) and the presence of the evil element (OS; evll+), as well as the early-feathering phenotype (k) and the unoccupied site, (US; evil-) (see Table 1). Four exceptions to the correlation were found. The three early-feathering females carrying the OS only were found in unrelated families and were probably the result of a type of reversion that cannot easily be detected in a segregating line. The number of revertants (3 out of 49) appears to be too high to have occurred in a single generation and probably resulted from an accumulation of reversion events in previous generations. This suggests that the revertants are stable and not adversely affected by the presence of evil. An alternative explanation would be that these birds are recombinants between the two closely linked loci; i.e., the evil integration site and the feathering locus. Based on this hypothesis, recombinants should also have been found that were late-feathering (K) but lacked the evil insert (US), unless such chickens have a higher mortality rate. In fact, one was found with these characteristics. Unfortunately, this bird was not reproduced and its phenotype could not be inspected further. The possibility that an error was made in recording its phenotype on the day of hatch cannot be ruled out. It should be pointed out, however, that apparent recombinants between the feathering locus and the evil integration site have been reported previously; i.e., late-feathering (K) chickens carrying the US only (Smith and Levin, 1991) and early-feathering (k) females carrying both the US and the OS (Boulliou et al, 1992). To our knowledge,
none of these individuals has been studied further. Obviously, chickens carrying a late-feathering allele at the K locus in combination with an unoccupied evil insertion site would be of prime commercial interest. The relationship between the evil insertion and the K phenotype requires further clarification. The technique described in this communication is relatively simple and rapid. It combines a quick DNA extraction method with the speed and sensitivity of PCR analysis. Such a test is ideal in the search for rare genotypes and could be used commercially to identify late-feathering (K) individuals carrying the US only and establish a line where the late-feathering phenotype would not be associated with tolerance to exogenous avian leukosis virus infection. Due to sex-linkage of the feathering gene, the test described in this report is fully informative when applied to females, although it cannot distinguish heterozygous from homozygous late-feathering males. A separate PCR assay has recently been proposed that would distinguish between the two variants of the US found in White Leghorn chickens, and facilitate the identification of heterozygous males (Iraqi and Smith, 1993). In addition, other PCR procedures could be designed to study the 5' flanking region of the viral insertion and provide a more complete picture of this genomic region.
1616
TTXIER-BOICHARD ET AL. Iraqi, F., and E. J. Smith, 1993. Determination of the zygosity of ev21-K in late-feathering (LF) roosters using the polymerase chain reaction. Poultry Sci. 72(Suppl. l):ll.(Abstr.) Levin, I., and E. J. Smith, 1990. Molecular analysis of endogenous virus eo21 slow-feathering complex of chickens. 1. Cloning of proviral-cell junction fragment and unoccupied integrate site. Poultry Sci. 69:2017-2026. Levin, I., and E. J. Smith, 1991. Association of a chicken repetitive element with the endogenous virus-21 slow-feathering locus. Poultry Sci. 70: 1948-1956. Mullis, K. B., and F. A. Faloona, 1987. Specific synthesis of DNA in vitro via a polymerasecatalyzed chain reaction. Methods Enzymol. 155:335-350. 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., 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. Smith, E. J., and I. Levin, 1991. Application of a locusspecific DNA hybridization probe in the analysis of the slow-feathering endogenous virus complex of chickens. Poultry Sci. 70:1957-1964.
Downloaded from http://ps.oxfordjournals.org/ at D H Hill Library - Acquis Dept S on March 10, 2015
associated endogenous solitary long terminal repeat,
ABSTRACT The molecular architecture of the sex-linked late-feathering region of the chicken genome is still poorly defined. Current evidence points to a strong association between the presence of the endogenous viral element evil and the late-feathering phenotype. However, analysis at the molecular level has demonstrated that this is not a simple case of insertional mutagenesis. Instead, the structure of the region of the chicken genome containing the feathering locus is complex and variable between and within lines of chickens. Significant clues to the molecular structure of this genomic region can be obtained by analyzing rare and revertant genotypes. However, searching for rare genotypes can only be carried out effectively using quick screen methodology. This paper describes a quick, polymerase chain reaction-based test for evil that facilitates the search for rare genotypes. (Key words: polymerase chain reaction-based test, endogenous provirus detection, rapid nonradioactive screening, late-feathering mutation, chicken) 1994 Poultry Science 73:1612-1616
between evil and the K allele. Molecular analysis has revealed that the structure of The sex-linked, dominant late- the K-evll genomic region is complex, due feathering mutation (K) used in poultry to the simultaneous presence of a duplibreeding for feather-sexing, is associated cated insertion site that is unoccupied by with the endogenous viral gene, evil, in ei>21 (unoccupied site = US), together with White Leghorn chickens (Bacon et at, the occupied site (OS) in late-feathering 1988). The presence of ev2\ is associated birds (Smith and Levin, 1991). However, with a reduced immune response to other configurations have also been lymphoid leukosis viruses (Smith and reported. Of particular interest are certain Fadly, 1988). This observation corrobo- strains of brown egg layers that carry the rated previous results showing immuno- evil insertion (OS) alone without the US logical tolerance towards lymphoid leuko- region. These birds are phenotypically sis virus in slow-feathering White Leghorn early-feathering (Boulliou et ah, 1992). dams and chicks (Harris et at, 1984). Evidently, the evil insert by itself is not The cloning of a locus-specific probe sufficient to produce late-feathering in corresponding to the evil insertion site, chickens. The fact that chickens can carry ez>21-int (Levin and Smith, 1990), has the evil element yet appear phenotypifacilitated the analysis of the relationship cally early-feathering as shown above means that the frequency of evil in modern chicken lines is not known at present and may be much higher than is Received for publication March 11, 1994. currently appreciated. A rapid test to Accepted for publication May 24, 1994. INTRODUCTION
1612
Downloaded from http://ps.oxfordjournals.org/ at D H Hill Library - Acquis Dept S on March 10, 2015
Centre for Food and Animal Research, Agriculture Canada, Ottawa, Ontario, Canada, K1A 0C6
RESEARCH NOTE
MATERIALS AND METHODS Chickens An Ottawa synthetic broiler dam line (Strain 30, Chambers et al, 1984) segregates for the K mutation (Chambers and Gavora, 1993) and for many ev genes of the Rousassociated virus family (Sabour et al, 1992). A sample of 106 female progeny of Strain 30 was obtained from 58 sires. The frequency of the late-feathering mutation in the contemporary generation was .42 out of a total of 317 female progeny. The chicks were vent-sexed and scored for feathering type on the day of hatch and bled at 12 wk of age. The test included females only because the hemizygosity for Z-linked traits warrants a straightforward relationship between the genotype and the phenotype.
Crude DNA extracts for the PCR assay were prepared according to the Protocol B of Higuchi (1989) as described in Benkel et al (1992). Reference DNA samples were a gift from A. Boulliou (Centre National de la Recherche Scientifique, Unite de Recherche Assoctee 256, Rennes I, 35042 Rennes Cedex, France) and represented 1) an earlyfeathering layer with OS only; 2) a revertant early-feathering female broiler with US only; and 3) a revertant early-feathering female broiler with OS only. The genotypes had been characterized by a Southern procedure with several enzymes and the probe evll-mi (Boulliou et al, 1992). Polymerase Chain Reaction Amplification Oligonucleotides were synthesized on an Applied Biosystems synthetizer Model 381A11.1 Sequences for the upstream flanking primer PR-UP (5'-GTGGGAATGGTACTACAGAGAAGG-3'), the downstream flanking region primer PR-DWN (5'CATTTCAAGCAAGGGACTGGC-3'), and the oligomer specific for the retroviral long terminal repeat Pl-IN (5'-ACCTGAATGAAGCTGAAGGCTTC-3') were chosen based on the results of Levin and Smith (1991). Amplifications were carried out in 50-/tL reactions in a Perkin Elmer Cetus2 thermal cycler. A modified "touchdown" procedure consisting in two cycles at 96 C for 1 min, 66 C for 1 min, and 72 C for 2 min; two cycles at 96 C for 1 min, 63 C for 1 min, and 72 C for 2 min; 28 cycles at 96 C for 1 min, 57 C for 1 min, and 72 C for 2 min; followed by a 10-min extension at 72 C, was used to minimize secondary priming (Don et al, 1991; Benkel and Smith, 1993). Reaction mixtures were overlaid with sterile mineral oil. Amplified products were separated on a 2% agarose gel containing ethidium bromide. An amplified product of 390 bp indicated the presence of a viral insert at the ez>21 locus, (OS), whereas an amplified product of 515 bp was expected with a US (Figure 1). RESULTS AND DISCUSSION
1
Applied Biosystems Canada Inc., Mississauga, ON, Canada, L5N 2M2. 2 Perkin Elmer (Canada) Ltd., Rexdale, ON, Canada, M9W 1A4.
Figure 2 shows the results of the evil PCR assay applied to a number of test samples. As expected, standard earlyfeathering females produced a single am-
Downloaded from http://ps.oxfordjournals.org/ at D H Hill Library - Acquis Dept S on March 10, 2015
identify eu21 would help to address this question. One interpretation of the available evidence on the association between e»21 and late-feathering would be that the eu21 insertion site and the feathering gene are closely linked but physically separate genomic regions, which would make possible the segregation between alleles at the K locus and the evil integration site. The search for recombinants at the K-ev21 locus requires the analysis of large numbers of chickens due to the close linkage between the two loci. Such an extensive screening operation would be both expensive and time-consuming to perform by Southern blotting. Polymerase chain reaction (PCR)-based diagnostics (Mullis and Faloona, 1987) are becoming increasingly popular as an alternative to Southern analysis due to their speed and sensitivity. In this report, we describe a PCR-based method for the rapid identification of the US and OS components of the K-evll locus, following an approach developed previously for the evl locus (Benkel et al, 1992).
1613
1614
TIXIER-BOICHARD ET AL. 515
bp
PR-UP
ev21 PR-DWN PR-UP
P1-1N
c PR-DWN 390 bp H
FIGURE 1. Schematic representation of the insertion site for the endogenous virus element ev21 provirus, showing the annealing sites for the primers (short arrows) used in the Polymerase Chain Reaction (PCR), and the sizes of the DNA fragments resulting from amplification of a chromosome lacking ro21 (upper line) and containing the proviral insert (lower line). Hatched and solid boxes represent the upstream and downstream flanking regions of the element, respectively. The open box represents the ev21 provirus. The amplification products of the interactions with either the upstream P1-1N site in the element or PR-UP, in combination with PRDWN, are over 7.0 kbp in length and are not detected under standard PCR conditions.
plified band at about 515 bp, whereas normal late-feathering chickens gave composite patterns consisting of the 515-bp US-specific band plus the shorter 390-bp band that is diagnostic of the OS. Of the early-feathering females analyzed previously by Boulliou et ah (1992), one of the revertant female broilers and the brown egg layer, shown in Lanes 2 and 3 respectively, yielded only the 390-bp fragment, which is specific for the presence of evil. A second revertant female produced only the 515-bp fragment, shown in Lane 4, that is diagnostic of the US. Results obtained previously with Southern blotting on the reference samples (Boulliou et ah, 1992) showed a perfect association between the amplified product of 515 bp and the 3.5-kbp EcoRI restriction fragment that is diagnostic of US, as well as the amplified product of 390 bp and the EcoRI restriction fragment of 2.5 kbp, which corresponds to the 3' flanking junction fragment described by Smith and Levin (1991). Overall, among the 106 females of Strain 30 that were studied using the PCR
-
ev 21 ev 21
3
4
FIGURE 2. Gel analysis of amplification products for the endogenous virus element evl\ assay. Females carrying the occupied site (OS) only show a band of 390 bp; chickens with the unoccupied site (US) only produce a band of 515 bp; females with both the OS and the US show a composite pattern consisting of both bands. Samples are as follows: Lanes 1 and 6, normal late-feathering broiler females containing US in addition to OS; lane 2, revertant early-feathering broiler female carrying OS only; lane 3, early-feathering layer female with the OS only; lane 4, revertant early-feathering broiler female with the US only; lane 5, normal early-feathering broiler female containing the US only. The molecular weight standard represents a Haelll digest of 0X174 DNA.
Downloaded from http://ps.oxfordjournals.org/ at D H Hill Library - Acquis Dept S on March 10, 2015
ev21
P1-1N
1615
RESEARCH NOTE TABLE 1. Distribution of molecular types according to the feathering phenotype in 106 female progeny Feathering phenotype Molecular type
Early (k) Late (K)
Unoccupied site (US) only Occupied site (OS) only US + OS
57 3 0
1 0 45
ACKNOWLEDGMENTS
This work started during a sabbatical leave of M. H. Tixier-Boichard at the Centre for Food and Animal Research which was supported by a NATO postdoctoral fellowship.
REFERENCES Bacon, L. D., E. J. Smith, L. B. Crittenden, and G. B. Havenstein, 1988. Association of the slowfeathering (K) and an endogenous viral («>21) gene of the Z chromosome of chickens. Poultry Sci. 67:191-197. Benkel, B. F., J. Mucha, and J. S. Gavora, 1992. A new diagnostic method for the detection of endogenous Rous-associated virus-type provirus in chickens. Poultry Sci. 71:1520-1526. Benkel, B. F., and E. J. Smith, 1993. Research note: A rapid method for the detection of the Rous-
Downloaded from http://ps.oxfordjournals.org/ at D H Hill Library - Acquis Dept S on March 10, 2015
approach, there was a good agreement between the late-feathering phenotype (K) and the presence of the evil element (OS; evll+), as well as the early-feathering phenotype (k) and the unoccupied site, (US; evil-) (see Table 1). Four exceptions to the correlation were found. The three early-feathering females carrying the OS only were found in unrelated families and were probably the result of a type of reversion that cannot easily be detected in a segregating line. The number of revertants (3 out of 49) appears to be too high to have occurred in a single generation and probably resulted from an accumulation of reversion events in previous generations. This suggests that the revertants are stable and not adversely affected by the presence of evil. An alternative explanation would be that these birds are recombinants between the two closely linked loci; i.e., the evil integration site and the feathering locus. Based on this hypothesis, recombinants should also have been found that were late-feathering (K) but lacked the evil insert (US), unless such chickens have a higher mortality rate. In fact, one was found with these characteristics. Unfortunately, this bird was not reproduced and its phenotype could not be inspected further. The possibility that an error was made in recording its phenotype on the day of hatch cannot be ruled out. It should be pointed out, however, that apparent recombinants between the feathering locus and the evil integration site have been reported previously; i.e., late-feathering (K) chickens carrying the US only (Smith and Levin, 1991) and early-feathering (k) females carrying both the US and the OS (Boulliou et al, 1992). To our knowledge,
none of these individuals has been studied further. Obviously, chickens carrying a late-feathering allele at the K locus in combination with an unoccupied evil insertion site would be of prime commercial interest. The relationship between the evil insertion and the K phenotype requires further clarification. The technique described in this communication is relatively simple and rapid. It combines a quick DNA extraction method with the speed and sensitivity of PCR analysis. Such a test is ideal in the search for rare genotypes and could be used commercially to identify late-feathering (K) individuals carrying the US only and establish a line where the late-feathering phenotype would not be associated with tolerance to exogenous avian leukosis virus infection. Due to sex-linkage of the feathering gene, the test described in this report is fully informative when applied to females, although it cannot distinguish heterozygous from homozygous late-feathering males. A separate PCR assay has recently been proposed that would distinguish between the two variants of the US found in White Leghorn chickens, and facilitate the identification of heterozygous males (Iraqi and Smith, 1993). In addition, other PCR procedures could be designed to study the 5' flanking region of the viral insertion and provide a more complete picture of this genomic region.
1616
TTXIER-BOICHARD ET AL. Iraqi, F., and E. J. Smith, 1993. Determination of the zygosity of ev21-K in late-feathering (LF) roosters using the polymerase chain reaction. Poultry Sci. 72(Suppl. l):ll.(Abstr.) Levin, I., and E. J. Smith, 1990. Molecular analysis of endogenous virus eo21 slow-feathering complex of chickens. 1. Cloning of proviral-cell junction fragment and unoccupied integrate site. Poultry Sci. 69:2017-2026. Levin, I., and E. J. Smith, 1991. Association of a chicken repetitive element with the endogenous virus-21 slow-feathering locus. Poultry Sci. 70: 1948-1956. Mullis, K. B., and F. A. Faloona, 1987. Specific synthesis of DNA in vitro via a polymerasecatalyzed chain reaction. Methods Enzymol. 155:335-350. 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., 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. Smith, E. J., and I. Levin, 1991. Application of a locusspecific DNA hybridization probe in the analysis of the slow-feathering endogenous virus complex of chickens. Poultry Sci. 70:1957-1964.
Downloaded from http://ps.oxfordjournals.org/ at D H Hill Library - Acquis Dept S on March 10, 2015
associated endogenous solitary long terminal repeat,