J. gen. ViroL 0976), 3z, 441-453
44I
Printed in Great Britain
Further Studies in Genetic Resistance o f F o w l to
R S V ( R A V 0): Evidence for Interaction between Independently
Segregating Turnout Virus b and Turnout Virus e Genes
By P. K. P A N I
Houghton Poultry Research Station, Houghton,
Huntingdon, Cambs., P E I 7 2 D A
(Accepted 26 April I976 )
SUMMARY
The segregation of resistant and susceptible phenotypes in response to infection
by RSV(RAV 2), RSV(RAV 50) and RSV(RAV o), of avian RNA tumour
virus subgroups B, D and E, respectively, was analysed in several test-crosses
using chickens from the RPRL line 7-2, HPRS-synthetic line E and the Reaseheath line C. The results were fully consistent with our view reported previously
that the genes at the tve and tvb loci segregate independently and recombine
under the Mendelian second law of independent assortment. The dominant
susceptibility e~ gene is expressed phenotypically when associated with the
dominant susceptibility b~ gene, but its expression is suppressed when associated
with two doses of the recessive resistance b ~ gene. Genetic causes such as lack
of penetration, recessive epistasis, and/or complementary interaction between
the tvb and tve genes have been discussed to account for the modified phenotypic
expression of b~b~e~e~ cells, i.e. resistance to subgroup E virus.
Also, as reported previously, it was observed in this study that the tvb genes
control the cellular response to subgroup D virus.
INTRODUCTION
Two autosomal loci, inhibitore (P) and tve (tumour virus e), with one pair of alleles at
each locus I e, i t and e~, er, respectively, control the genetic resistance of fowl to RSV(RAV o)
of subgroup E (Payne, Pani & Weiss, i97i; Pani & Payne, I973; Pani, I974). Tile dominant
I e gene masks the expression of the dominant susceptibility e~ gene, for which reason
chick embryos of IePeSe~ genotype, for example, are resistant to infection with subgroup
E virus, even though they carry the e~ susceptibility genes. Because of this epistatic gene
action the segregation of four subclass phenotypes in the ratio of 9: 3 : 3 : I in the F~ population
is modified to two recognizable classes, i.e. resistant and susceptible, in the ratio of I3:3
(see Pani & Payne, I973, for further details).
It has been reported that chick embryo cells resistant to subgroup B virus (C/B phenotype) are also resistant to subgroup E virus (Weiss, I97I; Crittenden, Wendel & Motta,
I973; Pani, I974), whereas those which are susceptible can be either resistant or susceptible
to subgroup E virus (Payne et aL I97I; Crittenden et aL I973; Pani, I974).
In other avian species, such as quail and pheasant, the B-E relationship does not hold
because quail and pheasant cells of C/B phenotype are susceptible to subgroup E virus
(Tooze, I973). Recently we have demonstrated that chicken × quail hybrid cells of C/B
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P.K. PANI
phenotype that carry one-half of the chicken genome and one-half of the quail genome
are also susceptible to subgroup E virus (Pani, 1975b).
It seems therefore that, in the chicken, the concept of resistance of C/B cells to subgroup E virus is largely based on the b'~b r genotype regardless of the genes at the tve
locus. Whereas the resistance of C/B cells of b~b~e~e r genotype to subgroup E virus can be
envisaged, that of C/B cells of brbre~e * genotype cannot readily be explained. Also, because
the I e gene is probably widespread in different strains of fowl (Pani & Payne, 1973;
Crittenden et al. 1973; Pani, 1974) study of the effect of the b r gene on the expression of
the e8 gene is not straightforward.
Nevertheless, by careful planning of crosses between chicken lines of known genotype
that lack the I e gene but which carry the tvb and tve genes, the relationship between
B-resistance and E-susceptibility can be ascertained, and this study was pursued with
this objective.
In this paper we report strong evidence in support of our earlier view that the tvb and
tve are two independent loci (Payne et aL 1971; Pani, 1974) and that the e* gene is
expressed in the presence of the b8 gene in the genotype but its expression is suppressed
when associated with two (homozygous) doses of the b ~ gene, possibly because of recessive
epistasis or other gene interaction. The hypothesis proposed recently by Crittenden &
Motta (1975) that the tvb genes control E-resistance and that therefore the assumption
of an independent tve locus as originally proposed by Payne et al. (1971) is unnecessary, has
been tested and rejected.
C h i c k e n lines
METHODS
L i n e E . This synthetic line was developed from the cross between the two highly
inbred Reaseheath lines I and C, of FI*e~e ~ and i*i*e~'e ~ genotypes respectively (Payne e t al.
I97I), and is maintained as a closed flock at Houghton Poultry Research Station (HPRS).
Pooled semen from four to six I line sires was used to inseminate about ten to twelve C line
dams for the production of the F1 progeny. The F2 progeny were produced by mating of
the F 1 sires and dams selected at random. The F2 parents were selected on the basis of
the performance of their embryos in response to infection with RSV(RAV o) of subgroup
E. It is known from the two loci hypothesis of Payne e t aL (1971) that chick embryos
that lack the I * gene, i.e. when they are of either i*iee.~es or i*iee*e ~ genotype, are susceptible
to subgroup E virus. Thus, on the basis of E-susceptibility of the Fa progeny, the genotypes
of their F2 parents can be predicted. The F2 individuals, as parents, were therefore progenytested to ascertain their putative genotypes. Those sire-dam families (F2) which had
produced at least 7o ~ E-susceptible embryos were selected as the potential producers of
the Fz generation.
The criterion of E-susceptibility on a family basis was chosen to eliminate the F2 parents
that carried the I t gene, because in the F2 families segregating for the I e gene the proportion
of E-susceptibility was not expected to be as high as 7° ~ . The maximum susceptibility
of 5o ~ is expected only in the mating type, Iei ~ × i*i ~, one or both parents being homozygous
for the e' gene. But the proportion of 7o ~ or more E-susceptibility can occur in the
F~ families where the parents are of genotypes i*i*e'e ~ or i~iee~e ~. The F8 and F4 parents
selected at random and mated as sibs were the progeny and grand-progeny, respectively
of the selected F2 parents. The average cumulative inbreeding coefficient ( f ) of the F~
generation progeny was estimated to be o'45 on the basis of the standard formula used
for the calculation of f i n small inbred populations (Lush, 1948; Falconer, 196o).
Line E is assumed to lack the P gene (see later for more evidence) but is heterogeneous
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for the e~ gene. This line also carries the bsbs genotype because it was developed from the
cross between the two highly inbred Reaseheath lines, I and C, each of the bsb~ genotype
(Payne & Biggs, I966; Pani, I975a).
Line 7-2. Detailed information about this inbred line has been given elsewhere (Pani,
I975a). The line was obtained from the Regional Poultry Research Laboratory (RPRL),
East Lansing, U.S.A., is maintained as a closed flock at HPRS, and is of the C/ABD
phenotype (resistant to virus subgroups A, B and D; Pani, I975a).
Mating design. Eight test-crosses (TC) were made using line E, line 7-2 and Reaseheath
line C, as follows:
Test-cross (~ × 9)
TCI : Line 7-z × line C
TCz : Line 7-2 × TCI
TC3: Line C x TCI
TC4: Line E × Line C
TC5: Line 7-2 × line E
TC6: TC5 x line E
TC7: line 7-2 x TC5
TC8 : TC5 × TC5
Line 7-2 dams could not be used in reciprocal crosses of the appropriate test-crosses
because of mating restrictions imposed by disease control measures. Six to seven dams in
each of four test-crosses, TCI, TC2, TC 5 and TC7, were inseminated with pooled semen
from line 7-2 sires. In TC3, semen from line C sires was used to inseminate eight TCI
dams. In TC4, six E line sires selected at random were naturally mated to C line dams
(4~:I~). The TC6 and TC8 dams, 6 to 8 per cross, were artificially inseminated with
semen from the appropriate single sires. Eggs were pedigreed to dams as necessary.
Virus strains. Single stocks of RSV(RAV o) of subgroup E (Payne et al. 197I; Pani,
I974), RSV(RAV 2) and RSV(RAV 5o) of subgroups B and D respectively (Pani, I975a),
were stored at - 7 0 °C and used at appropriate dilutions. The diluent used was phosphate
buffered saline (PBS) containing 5 ~ calf serum.
Chick embryo fibroblast (CEF) cultures and challenge with viruses. Eleven-day-old
embryos from eight test-crosses and three lines, 7-2, C and E, were individually cultured
according to the procedure of Temin & Rubin (I958). An appropriate number of plates
of secondary monolayer cultures were prepared for challenge with the viruses. Cultures
of embryos of HPRS-Brown leghorn (BrL) of C[E phenotype (cells resistant to subgroup
E but susceptible to virus subgroups B and D) and of Japanese quail of Q]BD phenotype
(cells resistant to virus subgroups B and D) were used as controls to check the titres of
the virus strains. All plates including controls were infected with at least IO3.3 f.f.u, of
B and D subgroup viruses. For infection of plates with subgroup E virus, cultures were
treated with polybrene (2o #g/ml for I h), challenged with a dose containing IO2's f.f.u, of
RSV(RAV o) and overlayed with nutrient agar containing I ~ dimethylsulphoxide
(DMSO) and I ~ inactivated chick serum. The overlay agar medium foi the B- and D-plates
lacked DMSO and chick serum, and cultures not pre-treated with polybrene for challenge
with B and D subgroup viruses. RSV induced foci were counted IO days later.
Statistical analysis of the results. On the basis of the Mendelian hypothesis appropriate
to the particular test-cross, the agreement between the observed and expected segregation
ratios of resistant and susceptible phenotypes in response to each virus strain were tested
by use of X2 analysis (see later for further details).
RESULTS
The distribution patterns of loglo transformed focus counts (count + I) of lines and
crosses in response to the three virus subgroups are presented in Table I.
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P.K. P A N I
Table I. Loglo transformed focus counts o f lines and test-crosses showing
distribution patterns in response to virus subgroups B, D and E
Log~o ( m e a n c o u n t + i)
Line and
cross
o'oo-43'40
o'4I-0'80
TC2
TC7
TC8
i6
39
24
5
---
TC2
TC7
TC8
19
38
24
2
I
--
Line E
TC4
TC5
TC6
TC7
TC8
8
13
9
8
50
37
4
t3
-IO
t
t
0.8I-I'2O
I'2I-I'6O
I'61-2"00
2.oi-2.40
> 2.41
13
12
33
9
38
II
---
I4
7
25
4
37
24
-7
--
25
18
2
9
5
i2
2I
I
3
2
3
I5
I
I
5
3
4
8
Subgroup B virus
--3
4
I
7
Subgroup D virus
--2
8
-3
Subgroup E virus
2
3
-3
I4
I
IO
27
4
I4
13
4
Virus subgroups B and D
On the basis of the bimodal patterns of distribution of focus counts of the TC2, TC7
and TC8 populations in response to the B and D virus subgroups, embryos which had
5 loci or less ( 4 o.81og) were considered to be resistant and those which had more than
5 foci were considered to be susceptible.
Virus subgroup E
The focus count distribution of line E and test-crosses was also bimodal (Table I).
On the basis of the distribution pattern in line E, embryos with a count of 14 foci (~< 1.2 log)
were designated as resistant and those with a count greater than 14 foci were designated
as susceptible. These criteria were used to recognize the resistant (R) and susceptible (S)
phenotypes in the lines and test-crosses. Because line E was the reference line in this
study, the criteria used to identify the R and S phenotypes in the lines and test-crosses
were considered to be unbiased within the limits of experimental errors.
The distributions of resistant and susceptible phenotypes within lines and test-crosses
in response to viruses o f subgroups B, D and E are presented in Table 2.
Estimation o f the frequencies o f the e"~and e ~ genes in line E
Because the F 5 embryos in line E were inbred, Wright's formula (Li, 1968) was used to
estimate the frequency of the e r gene, as follows:
Proportion of the R phenotype = q 2 ( t - f ) +fq, where q is the frequency of the e ~ gene
and f the average inbreeding coefficient of the F 5 embryo population. The proportion of
the R phenotype in the F 5 population was about o.20 (Table 2) and the average f was
estimated to be o'45. Thus the estimate of the e r gene frequency using the above formula
was 0"32, and therefore the frequency of the e s gene was calculated as o.68.
For the estimation of the frequencies of the e s and e ~" genes in the F 5 generation we
assumed that the genotypic frequencies at the tve locus are at equilibrium because of
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Table 2. Segregation o f resistant and susceptible phenotypes within lines
and test-crosses in response to viruses o f subgroups B, D and E
Virus subgroup
c
Chicken line
and test-cross
(c~x ~)
Line E x line E
Line C × line C
Line 7-z x line 7-2
Line 7-2 x line C (TCI)
Line 7-2 x TCI ( T C 2 )
Line CxTCI
(TC3)
Line E × line C (TC4)
Line 7-2 × line E (TC5)
TC5 × line E (TC6)
Line 7-2 x T C 5 ( T C 7 )
TC5 × TC5 (TC8)
B
D
E
c - - ~ - - ~
r-----~-----~
c- ~ ,
Susceptible Resistant Susceptible Resistant Susceptible Resistant
7x
5
o
6
26
o
o
8
o
2I
7I
5
o
6
26
o
o
8
o
2I
57
o
o
o
0
I4
5
8
6
47
I2
0
12
0
0
12
76
23
49
51
54
o
o
o
39
24
76
23
49
5I
54
o
o
o
39
24
47
I4
28
25
39
29
9
2I
65
39
random mating of the Fa and Fa parents and because line E lacks the 12 gene (see Methods).
I f it is assumed that line E lacks the I ~ gene but is heterogeneous for the e~ gene, the
proportion of E-susceptibility in TC 4 (line E x line C) should not exceed that of line E,
and is limited to the frequency of the e~ gene because line C is of iei"e~e~ genotype (Payne
et al. I971 ; Pani, I974). It can be seen in Table 2 that E-susceptibility in TC4 was about
0.62, which is not different from the frequency of the es gene in line E, indicating that
line E lacked the 12 gene.
Assessment o f the putative genotype o f line 7-2 in response to R S V ( R A V o)
The segregation patterns of the R and S phenotypes in response to RSV(RAV o) of
line 7-2 and of its test-crosses with line E are shown in Table 2. Line C is known to be of
iq~e~e " genotype (Payne et al. I97I; Pani, ~974). Preliminary assay of RSV(RAV o) on
the chorioallantoic membrane (CAM) of 2o embryos of line 7-2 indicated that the line
is resistant to this virus, and this was confirmed by CEF assay (Table 2). None of the
65 embryos tested in TCI, TC2 and TC3 (Table 2), in which line 7-2 was crossed with
line C, was observed to be susceptible to RSV(RAV o). On the other hand, in test-crosses
TC5 and TC6, in which line 7-2 was crossed with line E, segregation of S and R phenotypes occurred.
The absence of segregation of the S phenotype in TCI, TC2 and TC3 suggested that
line 7-2 could be one of the three genotypes, Iq2eSe s, IeFe*e ~ and i~i~e~er, that code for
resistance to subgroup E virus based on the two loci hypothesis (Payne et al. T97r; Pani,
I974)- I f line 7-2 is assumed to be one of the two genotypes that carry the I t gene all
embryo cultures in TC5 and about 9o ~ of the embryo cultures in TC6 should have been
of the R phenotype because the I" gene contributed by line 7-2 should have masked the
phenotypic expression of the es gene contributed by line E. However, about 62 ~ of embryos
in each test-cross were of the S phenotype, suggesting that line 7-2 does not carry the
I e gene and therefore does not have either of the first two genotypes. The occurrence of
only the R phenotype in TCI, TCz and TC3, and the segregation of the S and R phenotypes in TC5 and TC6, were fully compatible with the assumption that line 7-2 is of the
third genotype, i~i~erer.
29
VIR 32
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v . K . PANI
Table 3. The X ~ analysis for the segregation results of TC4, TC5 and TC6 in response to
infection with subgroup E virus based on the iqee~e~ genotype o f line C, line 7-2 and the
TC6 sire
Source
d.f.
SS
MS
P
Xz heterogeneity
X~ deviation
Total:
2
I
3
0'53
3"47
4"oo
0"27
3'47
0"40-0"30
o'95-o'9o
Assessment o f the putative genotype of TC6 sire
In TC6 (line 7-2 × line E) an F1 sire was backcrossed with line E dams. The F1 sire was
the progeny of the line E dam 21 that had progeny segregating for susceptibility to
RSV(RAV o). The segregation of 18 susceptible and 2I resistant phenotypes in TC6 was
an excellent fit for the assumption of i"i~e~e~ genotype for the F1 sire (X2 value=2.66,
P > 0"05).
Test for the reliability of the estimates o f frequencies of the
e s and e ~ genes in line E
Because the estimated frequencies of the e~ and e" genes of line E were used later for
calculating the expected genotypic frequencies in TC7 and TC8, it was necessary to assess
the reliability of these estimates.
Line C is known to be of iei~e~e~ genotype and line 7-2 and the sire of TC6 were
deduced to be of i~i'e~e~ genotype in this study. Since the estimated frequencies of the
e s and e ~ genes in line E were o.68 and o'32, respectively and because TC4, TC5 and TC6
were test-crossed with line E (Table 2), the proportions of the S and R phenotypes in
each test-cross should conform with that of the e~ and e ~ genes, respectively. This hypothesis was tested by use of the X2 heterogeneity test shown in Table 3. It is seen that
neither the X2 heterogeneity nor the X2 deviation component of the analysis was significant
(P > o'o5), indicating that the estimated frequencies of the e ~ and er genes were reliable.
Association between responses o f embryo cultures to infection
by virus subgroups B, D and E
The association between responses to infection by the three virus subgroups can be
analysed in three steps: (I) Association between B and D subgroup responses, (2) association
between B and E subgroup responses and (3) association between D and E subgroup
responses. Previously we reported that the B and D responses are under the control o f
the tvb genes (Pani, I975a). Under the assumption that the B - D association is confirmed
in this study (see later) the D - E association cannot be other than identical to the B - E
association.
Association between B and D subgroup responses
For the genes at the tvb, tve and inhibitor e (I e) loci the complete description o f the
genotype of line E is bSbSeS-i~i~. The dash (-) in the genotype indicates that the e s and
e r genes segregate in this line. Similarly the genotypic descriptions of line 7-2 and line C
are brb~ere~iei~ and b~b~e"e~i~i", respectively. Because the i s gene has been fixed in these
three lines, contributing no genotypic variation at the inhibitor e locus, the i*i ~ genotype
will be deleted later in the text.
It is seen in Table z that embryo cultures resistant to subgroup B virus were also
resistant to subgroup D virus. Again, as expected in accordance with the tvb genotypes
of line 7-2, line C and line E, the segregations of B-resistant and B-susceptible phenotypes
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agreed completely with the D-resistant and D-susceptible phenotypes, respectively, in all
test-crosses (Table 2). This confirms the pleiotropic action of the tvb genes in the control
of response to subgroup B and D viruses (Pani, I975a ).
Association between B and E subgroup responses
The association between the B and E subgroup responses can readily be understood
from the segregation results of TCI-TC6 shown in Table 2. It is seen that the phenotype
resistant to subgroup B virus was always found to be resistant to subgroup E virus (TC2).
On the other hand, the phenotype susceptible to subgroup B virus was observed to be
either susceptible or resistant to subgroup E virus (TCI, TC3, TC4, TC5 and TC6). This
type of B-E relationship which was reported previously by us (Payne et al. I97~; Pani,
I974), was confirmed in this study. The observations that B-resistant phenotype was not
found to be E-susceptible in any one of the six test-crosses (TCI-TC6) was expected
under the assumption of independent action of the tvb and tve genes. On the basis of
genotypes of line 7-2, line E and line C, the combination of b~b ~ genotype with es- genotype
was not possible in TCI-TC6 under the random segregation of the b~ and e'~ genes (the
dash (-) in the genotype can be replaced by either es or e~ gene without any change in the
phenotypic status in response to subgroup E virus) and hence the phenotype resistant to
subgroup B virus but susceptible to subgroup E virus (C/B segregant) did not occur in
these test-crosses. But in TC7 and TC8, combination between b"b ~ and es- genotype was
theoretically possible and therefore the occurrence of the C/B phenotypic segregant
was expected. The absence of the C/B phenotype in these two test-crosses was revealed
by further analysis of the segregation results (see below).
The TC7 was a dihybrid test-cross between the dominant double heterozygous and
double recessive homozygous parents of b*b"e*- and b~b~e~e~ genotypes, respectively. Under
the law of independent assortment of the tvb and tve genes, four distinct subclass phenotypes C/O, C/B, C/E and C/BE were expected to segregate in TC7. The C/O phenotype is
assumed to be susceptible to virus subgroups B and E and the C/BE phenotype to be
resistant to these subgroups. The C/B and C/E phenotypes are regarded as resistant to
the virus subgroups B and E, respectively, and susceptible to the other virus subgroup
under consideration.
Segregation of the four subclass phenotypes should occur in accordance with the
frequencies of the four types of gametes, bse~, b~e~, b*e~ and b~e ", produced by the TC7
dams. Since the frequencies of the b" and es genes in line E were I.o and o.68 respectively,
they should be o'5 and o'34, respectively, in line E x line 7-2 (TC5) population. The four
types of gametes produced by the TC7 dams were therefore expected to be approximately
in the proportion of o.2, o.2, o'3 and o'3 (the estimated proportions being o'~7, o'I7, o'33
and o'33) respectively. Hence the segregation of the four subclass phenotypes C/O, C/B,
C/E and C/BE in TC7 should occur in the ratio of approx. 2: 2: 3 : 3, a modification of the
dihybrid test-cross ratio ~ : l : ~ : I because of the production of gametes in unequal
frequencies.
The segregation of the four subclass phenotypes within dam families of TC7 is presented
in Table 4. The segregation of C/O, C/E and C/BE phenotypes occurred in dam families
that had produced sufficient numbers of progeny, but the C/B phenotype was absent in
all families. By use of the X2 analysis (Mather, I949) shown in Table 5, the observed
segregation ratio was compared with the expected segregation ratio of 2:2:3:3. A significant deviation (X2 value = 26"o9, P < o.o~) caused by the absence of the C/B phenotype
and an excess of the C/BE phenotype was observed. Further analysis of bs: b ~ segregation
z9-e
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v . K . PANI
Table 4. The distribution of four subclass phenotypes within dam families of TC7
in response to infection with subgroup B and E viruses
Subclass phenotype
D a m no.
/O
C/B
C/E
-6
-9
3
9
I
6
5
23
I
I8
II
6
9
io
21
5
I6
I7
I8
2
8
-3
-----
27
32
4
I
--
I82
Total :
C/BE
Total
7
--
--
5
I2
25
--
26
39
90
Table 5. The X2 analysis for the test of random association between the tvb and tve genes
in TC7 on the basis of the segregation of four subclass phenotypes in the ratio of 2:2:3:3
Phenotype
Observed
Expected
deviation
C/O
C/B
C/E
C/BE
25
0
26
39
18"00
18"0o
27'OO
27"oo
2"72
18'00'
0'04
5"33]"
Total :
90
90-00
26"o9*
* P < 0"0I.
P < 005.
%
showed a good agreement with the expected segregation ratio of I : I (•2 value = 1.60, P > o'o5)
whereas the e~:e~ segregation did not conform to the expected 4:6 ratio based on the
frequencies of the e~ and e ~ genes (X2 value=5"22, P > o ' o 5 ) , which raised the question
of why segregation of the e s and e ~ genes did not occur at random.
One possible answer is the view of Crittenden & Motta (1975) , who hypothesized that
the E-response is under the control of multiple alleles at the tvb locus and that therefore
a tve locus, independent of the tvb locus, does not exist. We considered their view but
found that, regardless of the number of alleles chosen at the tvb locus, segregation of three
subclass phenotypes on a dam family basis is not genetically possible (see Discussion).
We believe therefore that at least two pairs of independently segregating genes at the
two loci, tvb and tve, are necessary to code for three or more distinct phenotypes on a
dam family basis. The absence of the C/B phenotypes can be explained under this two
loci hypothesis, if it is assumed that the C/B phenotype is indistinguishable from one of
the four possible phenotypes. For this we assumed that the dominant e s gene is not
expressed while in association with two doses of recessive resistance b ~ gene. This
assumption is based on a well recognized concept of recessive epistasis (Sinnot, Dunn &
Dobzhansky, I95o), which explains that when a trait is under the control of two pairs
of genes, one pair of recessive genes at one locus may mask or modify the phenotypic
expressions of the dominant gene at the other locus.
Under an assumption of recessive epistasis segregation of three subclass phenotypes in
a true dihybrid test-cross should occur in the ratio of 2:1:2. In this study, however, the
segregation of C/O, C/E and C/BE phenotypes should be in the ratio of 2:3:5. The X2
analysis based on this ratio is presented in Table 6 and strongly upheld the 3-subclass
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Table 6. The X~ analysis for the segregation of three subclass phenotypes
in TC7 based on the 2 : 3 : 5 ratio
~2
Phenotype
Observed
Expected
deviation
C/O
25
i8-oo
2-7z NS*
C/E
26
27-00
0"04 N S
C/BE
39
45"oo
0"8o N S
Total :
90
90.00
3"56 INS
* NS, not significant at 0'o5 level.
Table 7- The distribution of the three subclass phenotypes within dam families
of TC8 in response to infection with subgroup B and E viruses
Subclass phenotype
A
D a m no.
C/O
C/E
C/BE
Total
I69
170
I7I
I72
I73
I74
5
7
2
7
12
6
2
3
-6
-4
3
6
2
4
4
5
Io
I6
4
X7
I6
I5
Total:
39
15
24
78
phenotypic segregation, because none of the observed frequencies of the three subclass
phenotypes differed significantly ( P > o ' o 5 ) from the expected frequencies. We concluded
therefore that the expression of the e ~ gene is modified or masked in genotypes that carry
two doses of recessive resistance b " gene.
Additional support in favour of the hypothesis of recessive epistasis was obtained from
the segregation results of TC8. The TC8 was an F~, a cross between line 7-2 x line E F1
sire and dams of TC5. The segregation of three subclass phenotypes, C/O, C/E and C/BE
occurred in four of the six dam families (Table 7), but the C/B phenotype was absent in
all dam families. These results were consistent with the assumption of the hypothesis of
recessive epistasis.
For the statistical analysis of the segregation results of TC8 it was necessary to ascertain
the complete genotype of the F1 sire. At the tvb locus ali F1 parents (sire and dams) were
expected to be of b~br genotype. But the genotypic status at the tve locus was uncertain
because two genotypes, eSe~ and e"e", were possible. The genotype of the F~ sire was
known on the basis of the segregation of susceptible and resistant phenotypes in the cross
between this sire and line C dams. Since line C dams are of e~e~ genotype, segregation of
at least one susceptible phenotype in response to subgroup E virus is adequate to reveal
the eSer genotype of the F1 sire. Using the CAM assay method we observed segregation
of four susceptible to six resistant phenotypes, which thus favoured the eSe~ genotype.
Hence the complete genotype of the F1 sire was bsb~e~e".
In a true F2, the segregation of C/O, C/E and C/BE phenotypes should occur in the
ratio of 9:3:4. As TC8 was not a true F2 because of production of gametes (b~e~, b~e~,
bSe~ and b~e~) in unequal frequencies by the dams, the segregation ratio of the three subclass phenotypes should be calculated according to the zygotic frequencies expected on
the basis of frequencies of b ~ and e~ genes in the sire and dams. The frequencies of the
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450
P.K.
PANI
Table 8. Compar&on of the observed and expected frequencies of
the segregation of three subclass phenotypes in TC8
Phenotype
Expected
frequency*
Expected
no.
Observed
no.
X2
deviation
C/O
C/E
C/BE
0'5025
0"2475
o'25oo
39"195
19'3o5
19"5oo
39
15
24
o'ooo9t
o-96oot
I'O385t
Total:
1.oooo
78"ooo
78
1.9994t
* Expected frequencies for the 3-subclass phenotypes were calculated assuming that in TC8 the sire
was a double heterozygote of b*bre*e ~ genotype and that the dams of varying genotypes were a random
sample from the TC5 population in which the frequencies of the bs and e ~genes were 0"5 and 0'34 respectively.
"i" P > 0"05.
Table 9. The X~ analysis for testing heterogeneity among dam families for the segregation
of resistant and susceptible phenotypes in TC8 in response to infection with subgroup B and
E viruses
Virus of subgroup
E
_
_
z
~
_
z
Dam no. Susceptible Resistant
169
I7O
171
172
173
I74
Total:
7
1o
3
6
2
2
I3
12
IO
4
4
5
54
24
* P < 0"05.
X2(3:1)
Susceptible Resistant
o'14
1"33
--
5
7
X2(2:I)
5
9
I '3I
3"9I*
2
2
0"0I
0"OO
O'56
7
I2
6
I0
4
9
5"I3"
0"46
4"94*
2"04 NS
39
39
15"75"~
t P < o.oi.
- -
X~(I:I)
o.oo
0"25
- -
0"53
4"00*
0"60
5'38 NS$
~: NS, not significant.
b s and e* genes in the F1 dams were 0"5 and 0"34, respectively, and those in the F1 sire
were 0"5 and o'5, respectively.
Thus the segregation of C/O, C/E and C/BE phenotypes in TC8 was expected to be in
the ratio of approx. 8 : 4: 4, and the X2 analysis based on this ratio is presented in Table 8.
It is seen that the deviations of the observed numbers of each phenotype subclass from the
expected numbers were not significant ( P > o'o5), which adds further evidence in support
of the hypothesis of recessive epistasis.
The segregation of susceptible and resistant phenotypes in response to subgroup E virus
was analysed further by use of the X2 heterogeneity test to ascertain the presence or absence
of heterogeneity among dam families of TC8. If the e * gene is assumed to be expressed
while in association with either the b s or b ~ gene, segregation of susceptible and resistant
phenotypes in TC8 on a dam family basis should occur in the ratio of approx. 2: I (3 : I
when frequencies of the e"~and e" genes are equal). But according to the assumption o f
recessive epistasis the segregation ratio should be modified to approx. I : I . It is seen in
Table 9 that the X~ heterogeneity value (5"38) was not significant ( P > o ' o 5 ) under the
assumption of recessive epistasis even though one exceptional dam contributed a value
of 4"o0 to the total X2 value. In contrast, on the assumption that the e ~ gene is fully
expressed when associated with either the b ~ or b ~"gene the X2 analysis showed significant
heterogeneity among dam families (Table 9). It is also seen in Table 9 that on a dam family
basis the segregation of susceptible and resistant phenotypes in response to subgroup B
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Genetic resistance to R S V ( R A V
o)
45I
virus was in the ratio of 3 : I (Xz heterogeneity = 2.o4, P > 0"05), as expected, and this occurred
regardless of the segregation of phenotypes at the tve locus. Hence, this analysis provided
more evidence in favour of the hypothesis of recessive epistasis.
DISCUSSION
The two most important conclusions of this study are: (I) that the tvb and tve are two
independent loci with at least one pair of alleles at each locus, which is consistent with
the view reported previously (Payne et al. I97I; Pani, I974). The random recombination
of genes at the two loci occurs according to Mendel's second law of independent assortment;
(2) that the dominant susceptibility e~ gene at the tve locus is fully expressed while in
association with the dominant susceptibility b ~ gene of the tvb locus, but its expression is
suppressed when associated with two doses of the recessive resistance b r gene in the
genotype. Either lack of penetrance of the e~ gene in the b~b~e~- genotype or interaction
between the tvb and tve genes (see later) could be responsible for the modified expression
of the b~'b~e~- genotype, i.e. the C[E phenotypic response to subgroup E virus challenge.
Our results do not support the view of Crittenden et al. (i973) and of Crittenden &
Motta (I975) that cellular resistance to infection by subgroup B virus is under the control
of multiple alleles at the tvb locus. According to them the tvb locus carries at least three
susceptibility alleles, namely b ~, b ~ and b ~3, of which the first two code for susceptibility to
subgroup B and E viruses, although they differ in another respect, whereas the third codes for
susceptibility to subgroup B virus but resistance to subgroup E virus. On this basis the e ~
and er alleles at the proposed tve locus are comparable with the b '2 and b ~3 alleles, respectively, because of the assumption of functional similarity between the twopairs of alleles.
It seems therefore that Crittenden and his associates have assumed the non-existence of
the tve locus by postulating multiple alleles at the tvb locus, whose functions are similar
to those of the tve alleles. We believe, however, that the tvb and tve loci exist independently
of each other, because evidence is clear-cut for the reasons presented in this study. The
patterns of phenotypic segregation predicted by (I) multiple allelism at the tvb locus
(hypothesis I, Crittenden and his associates) and (2) the two loci hypothesis proposed
in this study (hypothesis 2, Table IO), can be compared with those observed on a dam
family and on a population basis.
If we consider the segregation results of each test-cross (TCI-TC6) on a population
basis, it is apparent that the results can be explained by either hypothesis. This is because
of the assumption of functional similarity between the allelic pair, b s2, b ~3of hypothesis
and the allelic pair, e', e ~ of hypothesis 2. The crucial point of contradiction between the
two hypotheses therefore lies with the phenotypic segregation pattern on a sire-dam
family basis. The segregation of phenotypes on a family basis can be analysed in TC7 and
TC8 according to the conditions assumed by the two hypotheses.
In TC7, according to hypothesis I, heterozygous dams of possible genotypes b~2b~ and
b*~b~, assuming segregation of b ~ and b s3 alleles in line E, were mated with b~b " sires
(line 7-2). Hence the maximum number of phenotypes expected to segregate on a dam
family basis was two, the pattern being C/O, C/BE if the darn were b~2b~ (pattern I, Table IO)
and C/E, C/BE, if the dam were b~Sb~ (pattern II, Table io). But the segregation of three
subclass phenotypes, C/O, C/E and C/BE (pattern III, Table IO) on a family basis is
genetically impossible regardless of the number of alleles at the tvb locus. In TC8, however,
mating between parents of genotypes b~2b~ and b~3b~ (on the assumption of hypothesis ~)
was possible and segregation of three subclass phenotypes in pattern III could occur.
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452
P . K . PANI
Table io. C o m p a r i s o n o f the p a t t e r n s o f segregation o f p h e n o t y p e s in T C 7 in response to
virus subgroups B a n d E on the basis o f the two hypotheses outlined in t e x t
Putative genotype of
the TC7 dams
Hypothesis I : bS2b~*
Putative
genotype of
TC7 sire
b~b~
c"
Genotype
bS2br
brbr
bS3b~
b~b~
b~Sb~
Hypothesis 2: bSb~eSer
Segregation of possible genotypes and
phenotypes within a dam family
brb~e~er
b"b~e"e~"
b"b~e~e~
b~b~e~e~
b~b~e~e~j
Phenotype
C/O
C/BE
C/E
C/BE
C/O
C/E
Pattern
I
II
III
C/BE
* The TC7 dams could also carry bSlbr genotype, but the phenotypic segregation on a dam family basis
is expected to be in accordance with the pattern I (see text) because the b~lb~ and b~2b~ genotypes give
indistinguishable phenotypic segregation patterns.
However, the proportion of dam families that showed the 3-subclass segregation was
higher than that expected in accordance with the frequencies of b 8~ and b ~z alleles in
TCS. Thus the segregation results of TC7 compared with that of TC8 not only support
hypothesis 2, but also provide a means of differentiating between the two hypotheses.
The other possible explanation of the 3-subclass phenotypic segregation in TC7 and
TC8 is that there is linkage between the tvb and tve loci, with unequal crossing over
between them. Because we used a total of I68 embryos in TC7 and TC8, of which 4~ were
of C/E cross-over phenotype, the absence of the C/B reciprocal cross-over phenotype
could not have happened by chance. We believe, therefore, that linkage with unequal
crossing over is not an explanation o f the 3-subclass phenotypic segregation either on
a family or on a population basis.
One additional point worth mentioning is that Crittenden & Motta 0975) believe their
line 7-2 to be of b~bre~eS genotype, and they have explained their results on this basis,
whereas we have shown in this study that our line 7-2 is of brb~e"e ~" genotype. Because
HPRS-line 7-2 was derived from RPRL-line 7-2 and has since been maintained at
Houghton Poultry Research Station, as a closed flock, we find no reason to expect the two
lines to differ in genotype unless there has been a mutation at the tve locus in one of
the lines, which is very unlikely.
However, the segregation results of TC7 and TC8 are inconsistent with a eSe "~genotype
for line 7-2, but not with the e~'e~ genotype. Because the b ~3allele of the tvb locus is identical
in function with the e r allele of the tve locus in respect of E-susceptibility, the alternative
assumption of b~3b~3 genotype for line 7-2 on the basis of the single locus hypothesis
(Crittenden & Motta, I975) is another possible explanation for the segregation results
of TC7 and TC8. However, line 7-2 is already known to be of b~'b~ genotype (Crittenden
et al. 2973; Pani, I975a). We believe therefore that the tve locus exists independently
in line 7-2, and is detectable from segregation results in an appropriate test-cross. This
was possible in line 7-2 x line E (TC5) where the segregation of E-susceptible phenotype
occurred in-accordance with the frequency of the e ~gene in line E (see Results and Table 2).
Two mechanisms, at least, may be suggested to explain the interaction between the
tvb and tve genes in respect of cellular response to infection by subgroup E virus:
M e c h a n i s m I. It is an established concept that chick cells susceptible to R N A tumour
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Genetic resistance to R S V ( R A V o)
453
viruses of different subgroups carry susceptibility genes that code for subgroup-specific
virus receptors o n the cell m e m b r a n e surface, possibly at different sites. Thus cells of
b~-e ~-, b~-ere r a n d b'bre~e ~ genotypes in response to s u b g r o u p B a n d E viruses should be
o f C/O, C/E a n d C/BE phenotypes, respectively, in agreement with the results of this
study. But the dual resistance of b~bre'~e~ cells to subgroups B a n d E, as observed in this
study and as reported by others, deviates from the concept in respect o f phenotypic
expression to s u b g r o u p E virus. It is possible that the e s gene is n o t p e n e t r a n t when
associated with two doses o f the resistance b" gene, a n d this could be explained by blocking
of the e~-coded receptor b y a substance u n d e r the control o f b~b~ genotype. This b l o c k i n g
o f the e~-coded receptor w o u l d be analogous to the m e c h a n i s m postulated for the inhibitory
effect o f the I" gene over the e ~ gene (Payne et al. I971 ; Pani & Payne, 1973).
Mechanism 2. U n d e r this m e c h a n i s m it is assumed that the E-receptor is coded for by
a c o m p l e m e n t a r y action of the b ~ a n d e s genes, whereas the B-receptor is coded for by the
b s gene alone. T h u s the cells o f genotypes that lack either e ~ or b ~ or b o t h genes, for
example b~b'ere ~, b"bre'e ~ a n d b~b~e"e~ respectively, are resistant to s u b g r o u p E virus because
o f the absence of the E-receptor o n the cell surface, a n d those that carry b o t h b ~ a n d
e ~ genes are expected to code for the E-receptor a n d to be susceptible to this virus.
W e prefer the second mechanism, which is based o n a well recognized genetic hypothesis,
to explain the inheritance of three distinct phenotypes u n d e r the control o f two pairs o f
genes, for instance, coat colour in rodents (see S i n n o t et al. 195o).
I a m grateful to D r L. N. Payne for constructive suggestions for the i m p r o v e m e n t of
the m a n u s c r i p t a n d wish to t h a n k M r s D. H u g g i n s for expert technical assistance.
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