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vested from the plant and planted in the
greenhouse. Seven plants were obtained
and selfed.
Selfed seeds from the greenhouse-grown
plants were planted in the field in 1993 at
the rate of one seed per 30 cm. Stands
were not thinned after planting. Approximately 1 month after panicle emergence,
plants were rated for panicle characteristics.
Pollen was collected from one vigorous
plant with a mutant phenotype during the
1993 evaluation. A sample of the pollen
was used to pollinate the cytoplasmic
male-sterile pearl millet, Tift 23DA*. The
rest of the pollen was dried and stored
(Hanna et al. 1983), and used to pollinate
the Tift 23DA, X mutant selection hybrids
to produce testcross progeny. Selfed seed
of the mutant selection from 1993 were
grown in the greenhouse to determine if
the trait was expressed in greenhousegrown plants.
Testcross progeny and remnant F, seed
of Tift 23DA, X the 1993 mutant selection
were grown in the field in 1994 as described above. Panicle characteristics of
plants were evaluated approximately 1
month after panicle emergence.
Observed ratios of normal/mutant
plants in the F2 and testcross progenies
were tested to Mendelian ratios with the
X2 test. F2 progenies were evaluated with a
heterogeneity x2 test to determine if segregation was consistent among families.
Results and Discussion
Progeny derived from open-pollinated
seed from the original mutant had normal
panicle characteristics. This indicated
that the trait was either recessive, environmentally sensitive to expression, or
not heritable.
Because pearl millet is cross-pollinated,
the plants derived from selfing the putative open-pollinated progenies in the
greenhouse were assumed to be F2 progeny derived from random crosses between
the mutant phenotype and the surrounding normal plants. In the field in 1993, segregation of panicle phenotype fit a 3:1 normal/mutant ratio in six of the seven F2 families (Table 1). The heterogeneity x2 value
was significant, indicating that not all families segregated similarly. Family 4 affected
the heterogeneity x2 value, but not the
pooled x2 value. When data from family 4
was eliminated, the heterogeneity x2 was
not significant (.25 < P < .10). Modifying
genes that may have been derived from
the unidentified random pollen source
Table 1. Segregation of a bomeotic panicle mutation in progeny of pearl millet crosses
Number of plants with
panicle phenotype
Generation"
F2
Family 1
Family 2
Family 3
Family 4
Family 5
Family 6
Family 7
Pooled
Heterogeneity
Testcross
Mutant
Ex
ted
ratio
39
582
12
17
72
22
61
17
14
215
83
89
Normal
61
30
209
35
152
56
X2
P
3:1
3:1
3:1
3:1
3:1
3:1
3:1
3:1
2.854
3.128
0.058
5.620
1.504
0.114
0.057
1.639
13.335
.10 -.05
.10 -.05
.90 -.75
.025-.01*
.25 -.10
.75 -.50
.90 -.75
.10 -.25
.05 -.025*
1:1
0.209
.50 -.75
" F2 families obtained by selfing plants grown from open-pollinated seed taken from original mutant observed in the
field. Testcross progeny obtained from the cross (Tift 23DA, X mutant selection) X mutant selection.
' Significant.
may have affected expression of the trait
in this family.
Selfed progeny of the vigorous mutant
selected in 1993 expressed the mutant
phenotype in the greenhouse in the winter
greenhouse cycle of 1993-1994, indicating
that the expression of the trait was not
sensitive to environmental conditions.
Panicle characteristics segregated in a
1:1 normal/mutant phenotypic ratio in the
testcross progeny grown in the field in
1994 (Table 1). All F, plants of Tift 23DA4
X the 1993 mutant selection had normal
panicles.
It can be concluded from the segregation ratios that the mutant phenotype is
controlled by a recessive allele at a single
locus. The designation of phm, for phylloid homeotic mutant, is proposed for this
allele.
The mutant phenotype is more strongly
expressed on the apical portion of the
panicle. Generally, the imperfect male floret is normally formed, whereas the primary perfect floret is transformed into
vegetative leaves with a meristematic region. If environmental conditions are favorable for further development, small
poorly developed plants are formed. This
atypical development can be classified as
a phylloid homeotic mutant as defined by
Acquaah et al. (1992).
No other comparable homeotic mutant
of pearl millet has been described previously. The present, apparently unique
homeotic mutant will be useful as an easily identifiable marker in linkage studies,
and may be of value in studies of floral
morphogenesis. Pollen can be collected
easily from the homozygous recessive
phenotype, and thus it can be readily used
as the pollen parent in crosses. Because
of its erratic floral development within the
panicle, it is difficult to use as a female
parent in crosses. Seed of the homozygous
recessive mutant will be maintained by
the author, and limited quantities are
available upon request.
From the U.S. Department of Agriculture, Agricultural
Research Service, University of Georgia Coastal Plain
Experiment Station, Tifton, GA 31793. This article is the
result of a cooperative investigation of USDA-ARS and
the University of Georgia, College of Agricultural and
Environmental Sciences, Agricultural Experiment Stations. I thank A. Hornbuckle for his technical assistance.
The Journal of Heredity 1996:87(1)
References
Acquaah G, Saunders JW, and Ewart LC, 1992. Homeotic floral mutations. Plant Breed Rev 9:63-99.
Hanna WW, Wells HD, Burton GW, and Monson WG,
1983. Long-term pollen storage of pearl millet. Crop Sci
23:174-175.
Koduru PRK and Rao MK, 1983. Genetics of qualitative
traits and linkage studies in pearl millet. Z Pflanzenzuchtg 90:1-22.
Kumar KA and Andrews DJ, 1993. Genetics of qualitative traits in pearl millet: a review. Crop Sci 33:1-20.
Received December 12, 1994
Accepted July 7, 1995
Corresponding Editor: Prem Jauhar
Linkage Analysis of
Endogenous Viral Element 1,
Blue Eggshell, and Pea
Comb Loci in Chickens
J. R. Bartlett, C. P. Jones, and
E. J. Smith
Linkage analyses were conducted among
endogenous viral element 1 (ev1), pea
comb (P), and blue eggshell (O) loci,
mapped to the short arm of chromosome
1 in chickens. The F, males were pro-
Brief Communications 6 7
Figure 1. Analysis of evi amplification products for birds produced from a test cross of F, to Rhode Island Red hens. Lane Mb_ is a 100-bp ladder. Homozygous evi* or
evl~ have either a 300- or 510-bp DNA fragment. Heterozygous evi birds have both fragments.
duced from a cross between pea comb
Araucana males negative for ev1 and
known to be homozygous at the blue eggshell locus, and single comb White Leghorn females positive for ev1 and laying
white shelled eggs. Recombination data
were based on birds from a test cross of
three F, birds, each mated to 10 single
combed, brown eggshell laying Rhode Island Red (RIR) hens, negative for ev1 integration. Birds were screened for ev1 by
DNA amplification using the polymerase
chain reaction (PCR). Analyses of 207
progeny segregating for linkage between
ev1 and P showed an estimated average
map distance of 4.0 cM. Linkage analyses
based on 44 progeny resulted in estimates
of map distances of 1.8 and 4.1 cM between ev1 and O and P and O loci, respectively. Based on these estimates, the
order of these three loci on the short arm
of chromosome 1 can be inferred to be
centromere, P, ev1, and O.
As researchers move to develop a consensus chicken genetic map, the integration
of molecular and classical maps provides
a unique and difficult challenge. This difficulty is primarily due to the use of different reference populations on which the
classical and molecular maps are based.
An integrated linkage map provides obvious advantages. These advantages could
include the use of morphological, rather
6 8 Trie Journal of Heredity 1996:87(1)
than molecular markers, in marker-assisted selection programs if both are linked to
genes with significant effect on a trait of
economic importance in poultry.
Endogenous viral elements, with sequences homologous to avian leukosis virus, have been reported to be stably integrated in many chicken populations,
with a Mendelian mode of inheritance
from one generation to the next (Smith
1986; Tereba et al. 1979). Earlier reports of
work done in White Leghorns, for which
most studies of evi genes have been conducted, suggested a random distribution
of endogenous viral elements in the chicken genome (Crittenden 1991). More recent
work, however, indicates a clustering of ev
genes on chromosome 1 (Burke et al.
1994).
There are conflicting reports of the
chromosomal position of the endogenous
viral element characterized by Astrin
(1978) as evi. Based on in situ hybridization, Tereba and Astrin (1980) localized
evi to the long arm of chromosome 1.
More recently, observations by Bitgood
and Crittenden reported in Bitgood (1993)
suggested that evi is located on the short
arm of chromosome 1 and is linked to the
pea comb gene (P) locus at a map distance
of <10 cM. Additionally, a recent report
presented by Burke et al. (1994) supports
the observation that evi is mapped to the
short arm of chromosome 1. The objective
of the present work was to evaluate linkage between evi and two other loci: O, the
gene for blue eggshell, and P, previously
reported to be linked and mapped to the
short arm of chromosome 1 (Bitgood et
al. 1980). This work provides an opportunity to further integrate the classical and
molecular linkage maps of the chicken.
Materials and Methods
Animals and Mating Scheme
The Araucona (AR), Rhode Island Red
(RIR), and White Leghorn (WL) populations from which birds in the present studies were produced had been maintained
for several generations as true breeding
stock. Two WL males of genotype, based
on evi, O, and P loci, evl+-o+-p+/evl+-o+-
p+, were each mated to AR hens that were
pea comb, laid blue shell eggs and were
negative for evi integration and of genotype evl~-O-P/evl'-O-P. Three pea comb F,
males from this mating, heterozygous at
the evi (Figure 1), O, and P loci, were each
mated to 10 RIR females that were single
comb, laid brown shelled eggs, negative
for evi, with a genotype of evl~-o+-p*l
evl'o+-p*. Linkage analysis among the
loci was based on data from both male
and female progeny for P and evi, and for
females only for P and 0, and 0 and evi.
Figure 2. evi amplified PCR products analyzed on a 1.2% agarose gel and stained in ethidium bromide. Lane (Ln) B is control reaction without template. Lanes with AR,
RIR, and WL prefix are amplified products from Araucona, Rhode Island Red and White Leghorn birds, respectively. Lane F,<3 is amplification product from a bird produced
by mating Ln WL, birds to birds in Ln AR2. Homozygous evi* or evl~ have either a 300- or 510-bp DNA fragment. Heterozygous evi birds have both fragments. Ln Mbp is a
100-bp DNA ladder.
Molecular Analysis
DNA isolation. Blood collected in 0.5 M
EDTA by brachial venipuncture was stored
in 50-JJLI aliquots at -20°C until used. High
molecular weight DNA was isolated from
each 50-n-l aliquot by a modification of
standard protocols (Sambrook et al. 1989).
Briefly, 600 |il of lysis buffer (10 mM TrisCl, pH 8.0, 100 mM NaCl, 0.5% SDS, and 50
(jig/ml proteinase K) was added to each
sample. Samples were then incubated for
6-12 h with gentle shaking at 55°C. Phenolchloroform extraction and ethanol precipitation were by conventional methods
(Sambrook et al. 1989).
Genotyping of birds for EVI. Birds were
genotyped for evi by the polymerase
chain reaction (PCR) using a modification
(Smith et al. 1993) of the approach of Benkel et al. (1992). Briefly, the PCR amplification reaction was in a final volume of 50
\xi, containing 200 ng of genomic DNA as
template, 200 \JM of each deoxyribonucleotide triphosphate, 2 mM MgCl2, and the
primers PR-A, PR-B, and PR-C described by
Benkel et al. (1992), each to a final concentration of 370, 330, and 462 ng, respectively, and 2.5 units of Taq Polymerase
(Promega, Madison, WI). The reaction
mixture was then overlaid with mineral
oil. Cycling parameters for PCR in a PTC100 (MJ Research, Watertown, MA) were
as follows: initial denaturation at 95°C for
5 min, followed by 30 cycles of 1 min each,
at 94°C, 3 min at 49°C, and 3 min at 72°C
for denaturation, annealing, and exten-
Table 1. x ! analysis of segregation at the evi, O, and P loci
Phenotype
F°
No"
Endogenous viral element 1
Blue eggshell
Pea comb
Comb type and evi
Egg color and evi
Comb type and egg color
0.5
0.5
0.5
0.25
0.25
0.25
207
44
207
207
44
44
Probability
0.40
3.28
4.06
179.06
43.81
42.90
.10
.10
.10
.001
.001
.001
• Expected frequency of progeny within each phenotype from the test cross of F, males mated to Rhode Island Red
females.
* Total number of progeny analyzed by the polymerase chain reaction for each phenotype.
sion, respectively. A final extension for 5
min was at 72°C. Twenty-five microliters of
each amplification product was analyzed
on a 1.2% agarose gel and stained with
ethidium bromide. DNA from birds known
to be positive for evi were included in the
PCR analysis as a standard (a gift from Dr.
Eugene Smith, Avian Disease and Oncology Lab, East Lansing, Ml).
Statistical Analysis
Independence of segregation and assortment at the evi, P, and O loci was tested
by x2 (Green 1963). Linkage analysis was
by the direct method based on the frequency of recombinant gametes as a measure of the distance between any of two
loci (Ott 1991). Standard errors were estimated according to Green (1963) for a
double back-cross in coupling.
Results and Discussion
Results of the PCR analysis of the backcross population indicated segregation at
the evi locus (Figure 1) as expected from
matings involving the heterozygous F,
males (Figure 2) and homozygous evi'
RIR hens. Birds homozygous for either
evi* or evi' produced on amplification by
Brief Communications 6 9
Table 2. Analysis of recombination data for
linkage between evl and P
Sire
4692
4691
4202
Total
79
76
52
207
N."
cM
2
3
3
8
2.5
3.9
5.7
3.8
±
±
±
±
0.06
0.06
0.07
0.04
" Where Nv is the total number of birds analyzed lor evl
by the polymerase chain reaction within comb type.
» Where N, is the number of recombinants for evl and P.
PCR a fragment of size 300 or 510 bp as
reported by Benkel et al. (1992) for evl.
The co-dominance at this locus resulted in
two amplified products, 300 and 510 bp,
for birds heterozygous for evl.
Segregation was normal at each of the
evl, 0, and P loci. However, there was no
random assortment among the three
genes as determined by a x2 test of independence, suggesting linkage (Table 1).
The deviation from the expected assortment was highly significant (P = .001).
Linkage analysis between evl and P, evl
and 0, and O and P for each F, male are
presented in Tables 2, 3, and 4, respectively. Estimates of map distance between P
and evl varied between males from 2.5 to
5.7 cM with an average of 4.0 cM (Table
2). Map distance between evl and O
ranged from 0.0 to 5.5 cM with an average
of 1.8 cM (Table 3). These results indicate
that evl is on the short arm of chromosome 1 and linked to the P and 0 loci. This
is contrary to the report by Tereba and
Astrin (1980) that evl is localized to the
long arm of chromosome 1, but consistent
with previous reports by Burke et al.
(1994) and Levin et al. (1994).
Based on the recombination data from
the crosses in the current work, the average map distance between P and O loci
was 4.1 cM. This is consistent with previous reports suggesting linkage between
these two loci. Bitgood et al. (1983) reported a map distance of 4.28 cM between
these two loci. The classic work by Bruckner and Hutt (1939) tested only 35 birds
and reported a map distance of 6 cM, and
Table 3. Analysis of recombination data for
linkage between evl and O
Sire
4692
4691
4202
Total
17
9
18
44
N,"
cM
0
0
1
1
0
5.5 ± 0.12
2.3 ± 0.08
0
" Where Na is the total number of birds analyzed for evl
by the polymerase chain reaction within eggshell color
type.
* Where A', is the number of recombinants for evl and 0.
7 0 The Journal of Heredity 1996:87(1)
Table 4. Analysis of recombination data for
linkage between 0 and P
Crittenden LB, 1991. Retrovfral elements in the genome
of the chicken: implications for poultry genetics and
breeding. Crit Rev Poult Biol 3:73-109.
Sire
Green MC, 1963. Methods for testing linkage. In: Methodology in mammalian genetics (Burdette WJ, ed). San
Francisco: Holden-Day.
4692
4691
4202
Total
cM
17
9
18
44
1
0
1
2
6.7 ± 0.12
0.0
5.5 ± 0.12
4.5 ± 0.08
" Where A^ is the total number of birds analyzed for
evl by the polymerase chain reaction within eggshell
color type.
" Where N, is the number of recombinants for 0 and P.
Crawford (1986) calculated this distance
to be 2.4 cM. Based on these results, the
order of the three loci on chromosome 1
is determined to be centromere, P, evl,
and O. The order of centromere, P and 0
is consistent with that reported by Bitgood (1985). However, due to the relatively small number of hens included in the
analysis, the map distance and order of 0
and evl may need to be confirmed.
From the Department of Agricultural Sciences, Tuskegee University, Tuskegee, AL 36088. We are grateful to
Drs. T. F. Savage, Oregon State University, J. Bitgood,
University of Wisconsin, and M. Egnin and S. Nahashon
of Tuskegee University for suggestions and review of
the manuscript, and to Paul Drummond for technical
assistance. This work was funded in part by the U.S.
Agency for International Development grant number
PCE-5053-G-OO4017-00 and NSF HRD 94-50385.
The Journal of Heredity 1996:87(1)
References
Astrin S, 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.
Benkel BF, Mucha J, and Gavora JS, 1992. A new diagnostic method for the detection of endogenous Rousassociated virus-type provirus in chickens. Poult Sci
71:1520-1526.
Bitgood JJ, 1985. Locating pea comb and blue egg in
relation to the centromere of chromosome 1 in the
chicken. Poult Sci 64:1411-1414.
Bitgood JJ, 1993. The genetic map of the chicken and
availability of genetically diverse stocks. In: Manipulation of the chicken genome (Etches RJ and Verrinder
Gibbins AM, eds). Boca Raton, Florida: CRC Press; 6179.
Bitgood JJ, Otis JS, and Shoffner RN, 1983. Refined linkage value for pea comb and blue egg: lack of effect of
pea comb, blue egg and naked neck on age at first egg
in the domestic fowl. Poult Sci 62:235-238.
Bitgood JJ, Shoffner RN, Otis JS, and Briles WE, 1980.
Mapping of the genes for pea comb, blue egg, barring,
silver, and blood groups A, E, H, and P in the domestic
fowl. Poult Sci 59:1686-1693.
Bruckner JH and Hutt FB, 1939. Linkage of pea comb
and blue egg in the fowl. Science 90:88-89.
Burke D, Ponce de Leon FA, and Smith EJ, 1994. Simultaneous assignment of eu/,3,6 and 12 loci in White Leghorn chickens. Poult Sci 73:4.
Crawford RD, 1986. Linkage between pea comb and
melanotic plumage loci in chickens. Poult Sci 65:1859—
1862.
Crittenden LB, 1981. Exogenous and endogenous leukosis virus genes—a review. Avian Pathol 10:101-112.
Levin I, Santangelo L, Cheng H, Crittenden LB, and
Dodgson JB, 1994. An autosomal genetic linkage map
of the chicken. J Hered 85:79-85.
Ott J, 1991. Analysis of human genetic linkage. Baltimore: Johns Hopkins University Press.
Sambrook J, Fritsch EF, and Maniatis T, 1989. Molecular
cloning: a laboratory manual, 2nd ed. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.
Smith EJ, 1986. Endogenous avian leukemia viruses. In:
Avian leukosis (De Boer GF, ed). Boston: Martinus Nijhoff; 101-120.
Smith EJ, Bartlett JR, and Scott TR, 1993. Molecular
analysis of chicken lines divergently selected for aflatoxin B, resistance. Poult Sci 72:79.
Tereba A and Astrin SM, 1980. Chromosomal localization of evl, a frequently occurring endogenous retrovirus locus in White Leghorn chicken by in situ hybridization. J Virol 35:888-894.
Tereba A, Lai MMC, and Murti KG, 1979. Chromosome
1 contains the endogenous RAV-O retrovirus sequence
in chicken cells. Proc Natl Acad Sci USA 76:6486-6490.
Received December 29, 1994
Accepted July 7, 1995
Corresponding Editor: Lyman Crittenden
Genetic Regulation of Border
Zone Formation in Female
Mastomys (Praomys coucha)
Adrenal Cortex
S. Tanaka, M. Nozaki-Ukai, J.
Kitoh, and A. Matsuzawa
The unique border zone between the zona
fasciculata and z. reticularis of the female
adrenal cortex is formed in the wild-colored inbred mastomys (Praomys coucha)
strain, MWC, but never in the chamois-colored inbred strain, MCC. This clear strainspecific trait was genetically analyzed using F,, F2, and backcross progenies produced between MWC and MCC. Reciprocal crosses gave no significant
differences in the phenotypic ratio of F, or
F2 progeny. Border zone formation was detected in 0% of F, females, 25.8% of F2
females, 0% of backcross females between F, and MCC, and 47.7% of backcross females between F, and MWC. From
these results, it was concluded that border
zone formation in the female MWC adrenal
is regulated by a single autosomal recessive gene and this gene was named bzf
(border zone formation).
A specific border zone between the zona
fasciculata and z. reticularis of the female