Copyright 0 1990 by the Genetics Society of America
Transposable Element-Induced Response to Artificial Selection in
Drosophila melanogaster: Molecular Analysis of Selection Lines
Anthony E. Shrimpton,' Trudy F. C. Mackay2 and Andrew J. Leigh Brown
Department of Genetics, University of Edinburgh, Edinburgh EH93JN, Scotland
Manuscript receivedJune 2, 1989
Accepted for publication April 19, 1990
ABSTRACT
Artificial selection lines for abdominal bristle scoreof Drosophila melanogaste?; established from PM hybrid dysgenic crosses showed increases in selection response, heritability and phenotypic variance
compared to similar lines started from nondysgenic crosses. To determine whether this increased
genetic variance could be due to enhanced transposition ofP elements following the dysgenic cross,
the cytological locations (sites) of P elements were determinedby in situ hybridization for the whole
genome of samples of 20 individuals from the parentalP strain, 20 individuals from each ofthe eight
dysgenicselectionlines,andtenindividualsfromeachof
theeightnondysgenicselectionlines.
Variation among and within the selection lines and the parental P strain in P element insertion sites
was exceptionallyhigh. A total of 601siteswere identified, but there was no difference in total
number of sites per line, mean number of sites per individual, mean copy number per individual, or
site frequency between dysgenic and nondysgenic selection lines, or between lines selected for high
and low bristle score. Transposition following nondysgenic crosses
may explain additional observations
of accelerated selection responsesin nondysgenic selectionlines. It was not possible to deducewhich,
if any, of the several hundred insertions in the dysgenic selection lines were responsible for their
extreme bristle phenotypes.
T
0 what extent dotransposable elementsgenerate
quantitativegeneticvariation?Approximately
10% of the Drosophila melanogaster genome is composed of dispersed, repetitive, transposable element
(TE)sequences (RUBIN 1983; FINNEGANand FAWCETT 1986), many of which have structural similarity
to the endogenous proviruses of vertebrate retroviruses and to other dispersed repetitive sequences in
taxa ranging from prokaryotes to mammals. T h e distribution of TEs in natural Drosophila populations
has been determined by in situ hybridization of labeled
T E DNA to polytene salivary gland chromosomesand
by restriction fragment analysis of cloned DNA regions. While copy number per individual ofeach
element family is relatively stable, although characteristically different for the various families, their sites
of insertion are highly variable between individuals
from the same population (summarized by MACKAY
1989a). O n average, each site of insertion of an element is represented by a single individual from the
population.
It is clear from this distribution that
mobile elements transpose relatively frequently, andit is known
that T E transposition is mutagenic. Indeed, most
spontaneous major morphological mutations in Dro-
'
'
Present address: MRC Human Genetics Unit, Western General Hospital,
Crewe Road, Edinburgh EH4 ZXU, Scotland.
Presentaddress:Department of Genetics, Box 7614, North Carolina
State University, Raleigh. North Carolina 27695-7614.
Genetic5 125: 805-81 1 (August, 1990)
sophila have been shown to be caused by insertions of
TEs (GREEN 1988). However, T E insertions can have
manifold subtle effects on gene expression (summarized by MACKAY1989a), so it is of interest to inquire
whetherthesemutanteffects
encompass the small
allelic differences assumed to be typical of loci affecting quantitative traits (or polygene loci). In addition,
because loci tagged with transposons in this way can
be readily cloned (BINGHAM,LEVISand RUBIN 1981),
this line of investigation may pave the way for our
understanding of the nature of quantitative genetic
variation at the molecular level.
P transposableelements in D. melanogaster have
been used extensively and successfully totagand
facilitate the cloning ofmany known loci (for areview,
see KIDWELL1986). T h e P family ofelements was
discovered because it mediates the phenomenon of PM hybrid dysgenesis in this species. When males from
strains containing P elements (P strains) are crossed
to females from strains with no P element (M strains),
the F, hybrid offspring are dysgenic: they display a
number of aberrant genetic traits, including a
high
frequencyoflethal
and visible mutations,chromosome rearrangements, male recombination and temperature-sensitive sterility (KIDWELL,KIDWELL and
SVED1977). These traits are presentmuch
at reduced
frequency in the FI progeny of the reciprocal, nondysgenic cross, and have been shown to be associated
with a high frequency of transposition of P elements
804
A. E. Shrimpton, T. F. C. Mackay and A. J. Leigh
Brown
in the germline of F1 dysgenic hybrids (BINGHAM,
KIDWELLand RUBIN 1982).
An early attempt to address the
question of the
extent to which transposable elements can generate
mutations affecting quantitative traits used the P-M
system of hybrid dysgenesis to mobilize P elements by
a dysgenic cross, followed by artificial selection for a
quantitative trait, abdominal bristle count, to bring to
high frequencies any new P-induced mutations affecting this trait (MACKAY1985). This experimentis only
competent to detect new mutations of largeeffect
because of the segregating genetic background resulting from crossing the strains. A control for this segregation was to select also from the progeny of the
reciprocal nondysgenic cross; it was assumed that
these populations would have nearly the same genetic
background as the dysgenically derived selection lines,
and that repression of transposition in the nondysgenic F1hybrids would continue through several generations.
T h e results of this experiment, which was replicated
four times, werestriking.After
tengenerations of
selection the dysgenic lines selected for high and low
bristle score had diverged by 10.1 phenotypic standard deviations, and the nondysgenic selection lines by
only 4.3standarddeviations.
Accompanying this
greater than 2-fold difference in response was a 50%
increment in the realized heritability and a 2.5-fold
increase of the phenotypic variance of bristle score in
the dysgenic selection lines relative to thenondysgenic
selection lines. Since rapid jumpsin response, increase
in heritability and increase in phenotypic variance in
a selection line are all hallmarks of the presence of
genes of large effect increasing in frequency in that
line (FRANKHAMand NURTHEN198 l),it was inferred
that P element transposition in the dysgenic selection
lines caused new mutations affecting abdominal bristle
score.
However, repetitions of this experimental design
using the same P and M strains and selecting for the
same bristle trait (TORKAMANZHEI,MORANand NICHOLAS 1988) and extending the design to another
bristle trait and another system of hybrid dysgenesis
(PIGNATELLIand MACKAY1989) yielded less clear-cut
results. In the experimentof T ~ R K A M A N ~ H EMORAN
I,
and NICHOLAS (1988), which was replicated twice,
only one of thefour selection lines displayed the
features typical of the increase in frequency of a gene
of large effect on the selected trait, andthis was a line
started from a nondysgenic cross. Of the 40 selection
lines initiated by PIGNATELLIand MACKAY (1989)
from dysgenic and nondysgenic crosses, only 14
showed increased responses to selection, of which six
werefrom initial dysgenic crosses andeightfrom
initial nondysgenic crosses. MACKAY (1986,1987)
used a different experimental design and found that
“strain second chromosomes passed through “nondysgenic” crosses accumulated nearly equal amounts
of mutationalvariance as those passed through an
initial dysgenic cross. LAI and MACKAY(1990) demonstrated increased variance for bristle traits on initially isogenic X chromosomes passed through nondysgenic crosses. An interpretation of these results is
that transposition of P elements is capable of causing
variation for quantitativetraits, but that transposition
is not after all repressed in the populations started
from nondysgenic hybrids.
T h e aboveinferenceshavebeen
based solely on
phenotypic observations and analyses of selection responses; there is no direct evidence that the unusual
responses are caused by P element-induced mutations.
Since data on the amount and pattern of P element
transposition in the selection lines are critical to the
evaluation of the hypothesis that P element-induced
mutations were responsiblefor theincreased selection
response, we determined the sites of residence of P
elements by in situ hybridization in the 16 selection
lines of MACKAY(1985) andalso in the parentP strain
used to initiate them. We report here the results of
this investigation.
MATERIALS AND METHODS
Selection lines:The derivation of the selection lines from
crosses between a P strain (Harwich) andan M strain (Canton-S) is described by MACKAY(1985). Briefly, divergent
artificial selectionfor high and low abdominal bristle score
was applied to four replicate F, populations founded from
dysgenic crosses ( M E X Pdd), and four replicate F1populations founded from nondysgenic crosses (Po0 X Mdd). Replicates 1 and 2 in eachcaseusedHarwichandCanton-S
strains that had been maintained as laboratory stocks for
many generations in small bottle cultures (“noninbred”
crosses), and replicates 3 and 4 were of single Harwich and
Canton-S sublines derived by eight generations of full-sib
inbreeding (“inbred”crosses). Selection of the 10 most extreme individuals out of 50 scored of each sex was practiced
for 16 generationsin each of the 16 selection lines. Subsequently, the lines derived from dysgenic crosses were maintained with 70 pairs of parents per generation,with accompanying mild visual selection for the extreme bristle phenotypes. [Whenselection was relaxed, the population means
of the dysgenic selection lines tended to revert toward less
extreme values (MACKAY1985).] The lines derived from
nondysgenic crosses were maintainedwithout selection with
35 pairs of parents per generation.The mean bristle scores
of all selection lines remained near the level they had attained by the last generation of selection. The lines were
maintained in bottle cultures on cornmeal-molasses-agar
medium, at 20 .
All selection lines were assessed for sites of insertion of P
element sequences between generations 25 and 40. The
combination of the different populationsizes of the dysgenic
and nondysgenic selection lines and the variable times at
which the lines were sampled gives rise to variation in
expected average inbreeding coefficients among the lines.
However, thisis not great since most inbreeding for all lines
occurred during the 16 generations of selection when the
lines were maintained with ten pairs of parents per generaO
805
P Elements and Selection Response
TABLE 1
TABLE 2
Total number of sites per line
Number of sites per individual
Selection Line
Selection line
Dysgenic ( M E
x Pdd)
Sites
Replicate
1
A
A
High
Low
A
N
208
130
125
71
142
74
134
76
N
131
61
195
128
166
96
178
112
A
N
161
98
89
52
87
46
108
62
N
154
94
154
86
134
72
144
85
2
3
Nondysgenic
(PPP X Mdd)
High
Low
High
4
Sites
All (A) sites and sites not present in the sample from Harwich
(N) are tabulated for the selection lines of MACKAY
( 1 985). Replicates 1 and 2 correspond to selection lines started from noninbred
P and M strains, and replicates 3 and 4 to selection lines started
from inbred P and M strains.
tion. If the effective population size is estimated byNe =
0.7N (CROWand MORTON 1955), then the average inbreeding coefficient at (316, Fls, is 0.44, where F, = 1 - (1 - 1/
(2NJ)’ (FALCONER
1981). After 25 generations the average
inbreeding coefficient is expected to have risen to 0.46 in
the dysgenic linesand 0.48 in the nondysgenic lines, and by
40 generations to 0.5 in the dysgenic lines and 0.56 in the
nondysgenic lines.
In situ hybridization: In situ hybridizations were perMONTGOMERYand
formed as described by SHRIMPTON,
LANGLEY (1986) andLEIGHBROWNand Moss (1987) on
third instar larvae raised at 18 O on a glucose-yeast medium
(10% glucose, 10% dried, killed yeast, 3% agar). Plasmid
pr25.1 (O’HARE andRUBIN 1983) was used throughout as
the P element probe, labeled with biotinylated dUTP (bio1 1-dUTP,BRL) by nick translation. Hybridization was detected using the Vectastain ABC kit (Sera Labs) and visualized with horseradish peroxidase/diaminobenzidine.
Because P elements vary in size from the 2.9-kb complete
et
element to less than 100-bp deletion derivatives (SEARLES
al. 1982), we were concerned that insertions of small deletion derivatives, which nonetheless might be instrumental
in causing mutations, might be missed by the biotin-labeling
technique. A crude positive control for the strength of the
signal was hybridization to the heldup locus at 17C on the X
chromosome, because the probe used contains a complete
P element and 1.8 kb of single-copy heldup DNA. To
determine more precisely the sensitivity of the technique,
three slides from the strain containing the white locus mutant, d’,were prepared as described above. This mutation
is due toan insertion of approximately 500 bp of P element
DNA in the white gene at 3C (K. O’HARE,personal communication), and was detected in two of three slides. T o
reduce variation in detection efficiency as much as possible,
preparations with obviously weak signalwere not scored.
Cytological positions (sites) of P element homology were
recordedfortheentire
genomes of 280 individuals; 20
individuals from each of the eight selection lines of MACKAY
( 1 985) founded from initial dysgenic crosses, 10 individuals
from each of the eight selection lines founded from initial
nondysgenic crosses, and 20 individuals from Harwich and
Canton-S, the parent P and M strains used in these crosses.
Each preparation was scored twice initially, by assigning
elements to bands on photocopies of LEFEVRE’S
(1 976)pho-
Dysgenic ( M E
Replicate
X
Pdd)
Low
Nondysgenic (PPPX Mdd)
High
Low
1
A
N
66.05 (7.69) 59.95 (2.78) 70.40 (3.60) 53.30 (5.46)
35.85 (4.90) 30.00 (2.60) 29.00 (2.1 1 ) 21.10 (4.91)
2
A
N
64.85 (6.29) 50.90 (5.08) 62.80 (6.00) 73.40 (2.63)
22.95 (4.26) 31.50 (4.17) 29.40 (4.97) 39.30 (3.16)
3
A
N
58.85 (5.47) 39.85 (3.79) 50.00 (4.76) 43.00 (3.62)
30.80 (3.78) 16.80 (2.33) 18.60 (2.72) 20.40 (3.34)
4
A
N
57.15 (5.98) 62.05 (5.68) 55.60 (8.06) 74.20 (6.73)
24.90 (3.82) 28.90 (3.88) 21.30 (4.67) 38.10 (4.17)
Mean (2 standard deviation) numbers of sites per individual are
tabulated separately for all (A) sites, and sites not present in the
sample from Harwich (N), for the selectionlines of MACKAY( 1 985).
Replicates 1 and 2 correspond to selection lines started from noninbred P and M strains, and replicates 3 and 4 to selection lines
started from inbred P and M strains.
tographic map of the salivary chromosomes. The sites were
designated by number and letter, and further resolved into
proximal, distal, and (occasionally) mid as necessary. When
ambiguities arose the slides were rescored.
RESULTS
The objectiveofthis
study was to determine
whether the twofold difference in response to selection for abdominal bristle number among selection
lines started from dysgenic crosses,compared to selection lines started from nondysgenic crosses (MACKAY
1985), could be associatedwith differences in the
distribution of P element insertions. The data on the
P element insertion sites in samples of individuals
from
the 16 selection lines and the parental P strain were
summarized at variouslevels: the total number of
occupiedsites per line, the mean and variance of
number ofsites per diploid individual, meancopy
number per individual, and site frequency. These
statistics were computed both for all identified sites,
and for those sitesnot present in the sample from the
parent Harwich strain. The latter “new” siteswere
considered separately since any new P element insertions affecting the selected quantitative trait will fall
in this category; therefore differences in the distribution of new sites between dysgenic and nondysgenic
selection lines, and between high and low directions
of selection, might aid
in pinpointing transposition
events associated with selection response.
Total number of sites: All Canton-S slideshad
hybridization at position 17C only, confirming the
absence ofP elements from this stock.The individuals
from Harwich and the selection lines all had multiple
P element insertions. The basic data (not given) are
of the cytological locations of the sites occupied in
each individual. Given that a site is identified as occupied if it is found in at least one of the 260 individ-
A. E. Shrimpton, T. F. C. Mackay a n d A. J. Leigh Brown
806
:,”
TABLE 3
Analysis of variance of number of P element sites per
individual from the selection lines of MACKAY(1985)
45 -.
40.-
All sites
Source of variation
Cross
Selection
Cross: Selection
Keplicate (Cross* Selection)
Chromosome
Cross * Chromosome
Selection :Chromosome
Cross * Selection :
Chromosome
Replicate*Chromosome
(Cross and Selection)
d.f.
MS
New sites
F
MS
1
82.9 0.31”‘
3.2
1 149.0 0.55”’ 24.8
1 270.7 1.00”’ 134.4
12 27 1 .i
146.5
4 1894.7 22.61*100.9
94.6
4
1.13”’ 28.9
645.1
4 7.69*
113.2
4
46.4 0.55”’ 25.5
F
0.02”’
0.17”‘
0.92
2.02”s
0.58“’
2.26”‘
0.51”5
f
b
E
J
35“
30.-
25.20“
15.-
10
5
”
”
07
0
5
83.8
20
NUMBER OF INDIVIDUALS
FIGURE1.-Occupancy
48
15
10
profile, Harwich.
50.0
ns = not significant; P > 0.05. * P < 0,0001.
The variance i n number of sites per individual was partitioned
for all sites and new sites separately as described in the text.
Significance of the first three sources of variance was tested using
the Replicilte (Cross*Selection)mean square as the error term, and
significanceof the sources of variation associated with chromosome
arms tested using the Keplicate:Chromosome (Cross*Selection)
mean square as the error term.
uals in the total sample, 60 1 separateP element insertion sites were identified. The sites were scattered
apparently uniformly over the cytological map, which
contains 600 major and minor band designations at
the level of resolution of in situ preparations (the 20
major bands on each of the five long chromosome
arms are each subdivided into 6 minor bands). Of
these sites, 148 were occupied in the sample taken
from theHarwich population; the remaining 453 sites
were new. Seventy percent of the new sites were
present in more than one selection line, with an average of three lines in which each new site occurred.
Apparent site identity across lines may be genuine, or
a consequence of the coarse resolution of the in situ
technique. Assuming 120 resolvable bands per chromosome arm, and that all chromosomearms have
roughly the same DNA content as the X chromosome
(3 X lo4 kb) (LEFEVRE1976), each band contains an
average 250-kb DNA.
The distribution of the total number of sites per
selection line is given in Table 1. There was little
difference in average number of sites per line if the
lines were grouped according to whether they were
founded fromdysgenic or nondysgenic crosses (152.1
and 136.6 total sites, and 90.0 and 77.8
new sites,
respectively, averaged over both directions of selection); or if they were grouped according to direction
of selection (147.9 and 140.9 total sites, and 83.9 and
84.0 newsitesin
the high and low selection lines,
respectively, averaged over dysgenic and nondysgenic
crosses). The average total number of sites per chromosome arm per selection line (data not given) was
similar for all major arms (all sites: X , 32.8; 2L, 22.6;
2R, 29.1; 3L, 28.1; 3R, 30.9; new sites: X , 16.0; 2L,
15.3; 2R, 18.6, 3L, 15.4; 3R, 17.9).
Number of sites per individual: The mean and
variance of the total number of sites per individuaI in
each selection line are given in Table 2. The total
number of P element sites per individual averaged
over all 16 selection lines was 58.90, very similar to
the Harwich strainaverage of 58.25. On average,
27.43 new sites were present per individual per selection line. The variance among individuals in number
of sites was in all cases high; 50.72 for the Harwich
strain, and 29.76 for all sites and 14.80 for new sites
averaged over all selection lines.
There was little pattern to the distribution of average number of sites per individual when the selection
lines were grouped into dysgenic (all sites, 57.46; new
sites, 27.71) and nondysgenic (all sites, 60.34; new
sites 27.15) categories, or high (all sites, 60.71; new
sites 26.60) and low (all sites, 57.08; new sites 28.26)
directions of selection. There was a tendency for an
uneven distribution of number of sites per individual
among the chromosome arms (data not shown), with
a trend for an
increased number of sites per individual
on the X and a decreased number on 2L when all P
element sites were considered. The number of sites
per individual was distributed amongthe chromosome
arms of the Harwich strain as X , 16.65; 2L, 5.80; 2R,
11.55; 3L, 11.90; 3R, 12.30; 4,0.05. Thisdistribution
was similar for thetotal numberof sites averaged over
all selection lines: X , 15.91; 2L, 7.88; 2R, 10.47; 3L,
11.45; 3R, 13.00; 4 , 0.19. The number of new sites
per individual per selection line was more evenly
distributed over the major chromosome arms
( X , 5.2 1;
2L, 4.64; 2R, 6.03; 3L, 5.12; 3R, 6.31; 4 , 0.12).
Analysis of variance of number of sites per individual confirmed the general trends found by inspection
of means. Variance was partitioned, separately for all
sites and new sites, among crosses (dysgenic or nondysgenic), directions of selection (high or low), cross
by direction of selection interaction, replicates within
cross by direction of selection, majorchromosome
arms, cross by chromosome arm interaction, direction
807
P Elements and Selection Response
60
b
30 .
25
_I
55.
50.
45.
40 35.
a
b
7
35.
30.
30 -
.
60 55 50.
4540-
2520 15105-
n,
5
0 55y
55
0 601
I
0
10
5
10
6o 1
50
45
m 40
I35
3 30
= ::
15
10
5
0
0
10
NUMBER OF INDIVIDUALS
O
FIGURE2,”Dysgenic high lines. Occupancy profiles, selection
lines. Filled bars are for all P element sites, open bars for new sites
only. Pmels a and b correspond to the “noninbred” and panels c
and d to the “inbred”replicates of MACKAY(1985).
10
NUMBER OF I N D I V I D U ~ L S
FIGURE
4.-Nondysgenic
complete description.)
high lines. (See legend to Figure 2 for
55.
50 -
b
45.
40.
1
35.
w
o
l5
‘0
5
10
15
20
0
5
10
d
d
45-
-
m 40-
z
50
20
15
55 1
551
50 -
35
30
0
10
O
15
20
0
5
10
UMBER OF INDIVIDUALS
FIGURE3,”Dysgenic low lines.(See
complete description.)
15
20
legend to Figure 2 for
of selection by chromosome arm interaction, cross by
direction of selection by chromosome arm interaction,
and replicate by chromosome within cross by direction
of selection (Table 3). All sources of variance were
treated as fixed effects, and no distinction was made
between the replicates arising from “noninbred” and
“inbred” crosses of Harwich and Canton-S (MACKAY
1985). The only significant sources of variation were
among major chromosome arms and direction of se-
5
10
0
5
10
NUMBER OF INDIVIDUALS
FIGURE
5.-Nondysgenic
complete description.)
low lines. (See legend to Figure 2 for
lection by chromosome arm interaction for total number of sites per individual. The former arises because
of the relatively large number of sites on the X and
the small number on2L overall, and the latterbecause
the average totalnumber of sites per individual on 2R
is greater for the low lines (12.2) than the high lines
(8.7), and the average number on 3R is greater for
the high lines (15.8) than the low lines (10.2)
Occupancy frequency:Summarizing the P element
insertion sites in terms of total numbers of sites per
A. E. Shrimpton, T. F. C . Mackay and A. J. Leigh Brown
808
TABLE 4
Mean copy number
Selection line
Dysgenic ( M EX
P66)
Reolicate
Sites
Nondysgenic (PPP
X
M66)
Hieh
Low
Hieh
Low
1
A
N
44.02
23.21
41.94
19.63
55.29
20.74
39.11
12.63
2
A
N
51.41
16.22
34.29
21.55
44.06
18.71
53.58
26.47
3
A
N
44.56
21.63
32.28
12.79
41.64
13.64
30.28
13.67
4
A
N
42.74
15.91
44.40
18.34
41.16
13.13
57.45
28.27
Mean copy number per individual, corrected for heterozygosity
as described in the text, is tabulated separately for all (A) sites, and
sites not present in the sample from Harwich (N), for the selection
lines of MACKAY(1 985). Replicates 1 and 2 correspond to selection
lines started from noninbred P and M strains, and replicates 3 and
4 to selection lines started from inbred P and M strains.
line or per individual takes no account of the frequency with which individual sites are present. Each
site identified in a line canbepresent
in 1 to N
individuals, where N is thenumber of individuals
scored. The occupancy profiles of Figures 1-5 depict
the number of insertion sites present in 1 , 2 . . . N
individuals for the parental Harwich population and
each selection line. Generally the most common categories of sites were those observed in only one individual and those observed in all individuals of the
sample, giving roughly U-shaped occupancy distributions.
For the Harwich population, 31 % of the sites were
present in only one individual. The great variation in
site frequency in this population demonstrates the P
element transposition rate within this strong P strain
is appreciable. This poses problems forinferring
transposition events in the selection lines using the
Harwich pattern as a reference (see DISCUSSION).
Copy number: In situ hybridization cannot differentiate between homozygous or heterozygous P element insertions at a site. However, the average P
element copy number per genome can be estimated
by assuming all sites are independent and P element
presence or absence at each site segregates in HardyWeinberg proportions, with presence dominant. The
average P element copy number perindividual is then:
where ni is the number of element sites seen in
exactly i of the N individuals sampled per line. Mean
copy numbers areshown for theselection line in Table
4. The overall average copy number of the selection
lines was 43.64, very close to the estimate of 44.16
for the Harwich population. There was little difference in P element copy number between dysgenic (all
sites, 41.96; new sites, 18.66) and nondysgenic (all
sites, 45.32; new sites, 18.41) selection lines, or high
(all sites, 45.61 new sites, 17.90) and low(all sites,
4 1.67;new sites, 19.17) directions of selection. As for
the total number of sites per individual, the distribution of copy number by chromosome arm (data not
given) showed atendencyforan
increase incopy
number on the X and a decrease on 2L for Harwich
( X , 14.63; ZL, 4.17; 2R, 8.38; 3L, 8.59; 3R, 8.36) and
for all sites averaged over the selection lines ( X , 12.90;
ZL, 5.28; ZR,7.02; 3L, 8.60; 3R, 9.73). Copy number
of new sites averaged over all selection lines was more
similar among major chromosome arms ( X , 3.58; ZL,
2.99; ZR,3.93; 3L, 3.53; 3R, 4.43).
Site frequency: T h e average frequency of sites in
each line can be estimated by dividing the average
copy number by the total number of sites per line.
These estimates are given for each selection line in
Table 5. The frequency ofall sites averagedover
selection lines was 0.31, compared to 0.30 for Harwich. The frequency of new sites averaged over selection lines was somewhat lower, 0.23. There was little
difference in average site frequency between dysgenic
(all sites, 0.29; new sites, 0.22) and nondysgenic (all
sites, 0.34; new sites, 0.24) selection lines, or between
high (all sites, 0.32; new sites, 0.22) and low (all sites,
0.30; new sites, 0.23) directions of selection. T h e
average site frequency was greater for theX chromosomes of the Harwich sample ( X , 0.44; ZL,0.22; ZR,
0.34; 3L, 0.24; 3R, 0.25) andfor all sites of the
selection lines ( X , 0.42; ZL, 0.25; ZR,0.24; 3L, 0.32;
3R, 0.32). T h e higher frequency for sites on the X
chromosome might be expected
because there is more
inbreeding for the X than the autosomes. This trend
was notapparent when new sites were considered
separately ( X , 0.25; ZL, 0.20; ZR,0.21; 3L, 0.24; 3R,
0.25).
Chromosome rearrangements: P element mobilization commonly causes chromosome
rearrangements, the breakpoints of which are usually the sites
of residence of P elements. For example,ENGELSand
PRESTON(1984) found a frequencyof X chromosome
rearrangements of 10% in the germlines of dysgenic
males. We did
not
observe many rearrangements within the selection lines; it is likely that any
rearrangements that did occurwere subsequently lost
through deleterious effects on fitness. Six inversions
were observed, allin selection lines resultingfrom
dysgenic crosses. One inversion, In(2R) 45C-52C was
observed in 17 of the 20 slides scored in one of the
lines selected for low bristle score; in two individuals
it was homozygous. The other five inversions were all
present as heterozygotes at low frequency. Four of
these were found in a single line selected for high
and P Elements
TABLE 5
Mean site frequency
Selection line
Dysgenic (MY?
X
PSt3)
High Sites
Replicate
Nondysgenic (PY?
X
MSS)
Low
High
Low
1
A
N
0.212
0.179
0.336
0.277
0.389
0.280
0.292
0.166
2
A
N
0.392
0.266
0.176
0.168
0.265
0.195
0.301
0.236
3
A
N
0.277
0.221
0.363
0.246
0.479
0.297
0.280
0.221
4
A
0.278
0.169
0.288
0.213
0.307
0.182
0.399
0.333
N
Mean site frequency, corrected for heterozygosity as described
in the text, is tabulated separately for all (A) sites, and sites not
present in the sample from Harwich (N), for the selection lines of
MACKAY(1985). Replicates 1 and 2 correspond to selection lines
started from noninbred P and M strains, and replicates 3 and 4 to
selection lines started from inbred P and M strains.
bristlescore: In(1)7D-l4Dand In(3L)70D-80 were
present in only one individual, and In(2L)35B-39F
and In(3R)89E-98B were each observed twice in the
sample of 20 individuals. In(2R)55E-57A was found
in one individual from one of the lines selected for
low bristle score.
One of the ten slides scored from a nondysgenic
line selected for high bristle score was heterozygous
for a three-point rearrangement in which bands 94D96C of one chromatid had been inserted into 86C.
Examination of a further35 slides from this line
revealed one other example of this rearrangement
and two examples of 94D-96C deficiency heterozygotes.
DISCUSSION
Because P elements are known tobe mobilized
following dysgenic crosses, and P element insertions
can cause visible mutations, it was thought that differences in patterns of P element insertion between the
dysgenic and nondysgenic selection lines of MACKay
(1985) might be informative in identifying loci that
had contributed to the enhanced selection response
of the dysgenic lines. There were apriori several
reasons why this approach might have little power to
detect transposition events associated with mutations
in genes affecting bristle number. For example, excisions of P elements commonly occur following dysgenic crosses (reviewed by ENCELS1989), and these
events for the most part would not be detectable by
in situanalysis of P element sites. Precise excisions of
elements residing near bristle polygenes in the Harwich population and subsequent loss of the site in a
selection line could not be distinguished from loss of
the site by fixation of the M strain homologoussite by
Selection Response
809
drift. Similarly, bristle mutations caused by imprecise
excisions removing the resident P element and some
portion of the neighboring locus, and imprecise excisions resulting in internal deletions which place the
resident P element below the resolution of the in situ
technique, would be missed. Furthermore, new P
insertions can be identifiedonly by comparison to the
occupied sites in the parent P strain used in the crosses
to initiate the selection lines. T o pinpoint unambiguously new insertions potentially affecting bristle loci
therefore requireshomozygosity of the parent P strain
with respect to P elementinsertion sites. We also
recognized the danger thatmany transposition events
may have occurred in each line, in which case narrowing the numberof potential candidates forloci affecting the selected trait would have to rely on frequency
arguments. That is, selection should have brought to
high frequency those insertion mutations affecting
the
trait in the direction of selection. This argument is
fraught with difficulty, as drift is also expectedto
bring to high frequency insertions not affecting
bristle
score, and if the mutations are of the sort with deleterious effects on fitness as homozygotes, but with
heterozygous effects on the selected traits (loci with
such properties are often found segregating in Drosophila selection lines) (reviewed by MACKAY1989b)
the frequency of the insert responsible would be in
any case intermediate.
Regardless of the above caveats concerningthe
possibility of identifying individual loci associated with
selection response from this analysis, we anticipated a
description of the distributions of P element sites and
theirfrequencies in the selection lines of MACKAY
(1985) might shedsome light on why the large difference in selection response between dysgenic and nondysgenic lines observed in this experiment have not
been repeatable(TORKAMANZEHI,
MORANand NICHOLAS 1988; PICNATELLI
and MACKAY1989). Is it because transposition occurs in appreciable amounts in
both dysgenic and nondysgenic crosses after the first
generation, as suggested by PIGNATELLI
and MACKAY
(1989), or must other reasons be sought for the discrepancy?
Variation among the selection lines in P element
insertion sites was exceptionally high. The mean P
element copy number per individual averaged over
all selection lines was 43.6, compared to 44.2 for the
Harwich population. A total of 601 distinguishable P
element sites were identified in all lines. There was
no difference in total number of sites per line, mean
number of sites per individual, mean copy number
per individual, or site frequencybetween dysgenic
and nondysgenic selection lines. Only 148 of the 601
sites identified were found in the sample from Harwich, but similar analyses for the 453 “new”
sites failed
to distinguish dysgenic and nondysgenic selection
810
A. E. Shrimpton, T. F. C. Mackay and A. J. Leigh Brown
lines. However, the Harwich strain was, surprisingly,
highly heterozygous for P element sites. Therefore it
is not possible to ascertain which of these “new” sites
represent transposition events; which are sites that
were present in the Harwich population at the time
the selection lines were started, butwere subsequently
lost from Harwich by drift or excision; and which are
sites present in Harwich but missed by chance in the
sample from it. A considerable fraction of the new
sites may fall in the latter two categories: 70% of new
sites are foundin more than oneselection line, suggestive of common origin; and theprobability of missing
low frequency sites in a sample of 20 individuals is
appreciable (0.14 for a site at frequency 0.05).
Given theseuncertainties,
identifying individual
sites that may be associated with loci affecting bristle
number is ruled out. Neither is it possible to estimate
the number of sites in the nondysgenic selection lines
that representtransposition events. However, the similarity inall regards of P element site distribution
pattern between dysgenic and nondysgenic selection
lines, and the evidence from Harwich that transposition occurs in appreciable amountseven within strong
P strains, lends credenceto theargumentthat
P
element transposition has occurred in the nondysgenic
selection lines. This may account for the discrepancy
between the patterns of selection response observed
by MACKAY(1985) and by TORKAMANZEHI,
MORAN
and NICHOLAS(1988) and PIGNATELLIand MACKAY
(1989), and the accumulation of mutational variance
forquantitativetraits
on M strainchromosomes
passed through bothdysgenic and nondysgenic crosses
(MACKAY1986, 1987; LAI and MACKAY1990). It is
importanttonotethatthe
aspect of the selection
was not repeatable
experiment of MACKAY(1 985) that
was the difference between reciprocal crosses in response. The observation that unusual patterns of response happen following crosses of P and M strains,
interpretable as arising from mutational variance affecting the selected trait, was repeatable.
Further more extensive genetic analysis of the dysgenic selection lines should reveal what factors are
responsible fortheextreme
bristle phenotypes of
these lines. This approach has been successful already
in identifying an allele at the smooth locus in low
selection lines derived fromdysgenic crosses (MACKAY
1985). Subsequent analysis has shown this mutation
to be caused by a full-length P insertion at 56E on2R,
which has enabled us to clone this gene (A. SHRIMPTON, T. MACKAYand A. LEIGH BROWNunpublished
data).
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