Proc. R. Soc.

B (2009) 276, 21952208

doi:10.1098/rspb.2008.1930
Published online 18 March 2009

Genetic linkage map of the guppy, Poecilia

reticulata, and quantitative trait loci analysis
of male size and colour variation
Namita Tripathi, Margarete Hoffmann, Eva-Maria Willing, Christa Lanz,
Detlef Weigel and Christine Dreyer*
Department of Molecular Biology, Max Plank Institute for Developmental Biology,
Spemannstrasse 37-39, 72076 Tubingen, Germany
We report construction of a genetic linkage map of the guppy genome using 790 single nucleotide
polymorphism markers, integrated from six mapping crosses. The markers define 23 linkage groups
(LGs), corresponding to the known haploid number of guppy chromosomes. The map, which spans a
genetic length of 899 cM, includes 276 markers linked to expressed genes (expressed sequence tag), which
have been used to derive broad syntenic relationships of guppy LGs with medaka chromosomes. This
combined linkage map should facilitate the advancement of genetic studies for a wide variety of complex
adaptive phenotypes relevant to natural and sexual selection in this species. We have used the linkage data
to predict quantitative trait loci for a set of variable male traits including size and colour pattern.
Contributing loci map to the sex LG for many of these traits.
Keywords: linkage map; single nucleotide polymorphism markers; expressed sequence tag linked;
bacterial artificial chromosome ends; synteny; quantitative trait loci mapping

1. INTRODUCTION
Fishes of the family Poeciliidae have served as model
systems for the study of evolution, ecology, behaviour,
tumour genetics and genomics. Natural populations of
guppies (Poecilia reticulata) have been studied for more
than 80 years ( Winge 1922a), and many of the selective
forces driving phenotypic variation between populations
are well known (Haskins et al. 1961). In addition to a
plethora of ecological and evolutionary studies, guppies
have been studied historically as a model system for the
sex-linked inheritance of a variety of male ornamental
traits important for sexual selection and adaptation in the
natural populations. The evolution of several interesting
behavioural and adaptive strategies of this species in the
wild is specifically driven by a delicate balance of natural
and sexual selective forces. A variety of studies regarding
these aspects of guppy biology have already generated a
basis to establish this species as an evolutionary model
system (Houde & Endler 1990; Magurran & Nowak 1991;
Magurran 2001, 2005; Brooks 2002; Crispo et al. 2006;
Olendorf et al. 2006).
We would like to understand the underlying molecular
basis for the extreme male colour polymorphism and other
variable traits that have been important for evolution and
adaptive success of guppies in their wild habitat (Haskins
et al. 1961; Reznick & Endler 1982; Endler 1991). Genetic
maps are thus needed and will allow the mapping
of loci underlying quantitative variation (quantitative
trait loci (QTL); Lander & Botstein 1989; Leder et al.
2006; Shirak et al. 2006), map-based cloning of genes
* Author for correspondence (christine.dreyer@tuebingen.mpg.de).
Electronic supplementary material is available at http://dx.doi.org/10.
1098/rspb.2008.1930 or via http://rspb.royalsocietypublishing.org.
Received 22 December 2008
Accepted 24 February 2009

defined by mutant or natural alleles (Karsi & Waldbieser

2004; Kazianis et al. 2004b) and comprehensive investigation of genome evolution between related strains and
species (Gates et al. 1999; Naruse et al. 2000; Danzmann
et al. 2005; Stemshorn et al. 2005).
Detailed genetic linkage maps have been developed for
several commercially and scientifically important fishes,
including tilapia, rainbow trout, catfish, Atlantic salmon,
medaka, Arctic char, zebrafish, fugu and platyfish ( Young
et al. 1998; Gates et al. 1999; Naruse et al. 2000; Sakamoto
et al. 2000; Gilbey et al. 2004; Kazianis et al. 2004a;
Woram et al. 2004; Kai et al. 2005; Lee et al. 2005).
The guppy was one of the first vertebrates for which
sex-linked inheritance was demonstrated and the first
linkage map of the guppy was published in 1927, although
it was restricted to some sex-linked loci causing variation
in ornamental traits ( Winge 1927). There is extensive
genetic evidence supporting an XY sex-determination
system in the guppy, suggesting that a limited nonrecombining region of the guppy Y chromosome harbours
the male sex-determining locus in tight genetic linkage to
male-advantageous ornamental genes ( Winge 1922a,b,
1927; Winge & Ditlevsen 1947; Haskins et al. 1970).
A few later studies have also shown genetic mapping of
different colour patterns to the sex chromosomes, using
ornamental guppy strains ( Khoo et al. 1999ac).
Additional partial linkage maps for the guppy genome
have been constructed ( Foo et al. 1995; Khoo et al. 2003)
using randomly amplified polymorphic DNA, amplified
fragment length polymorphism and microsatellite DNA
markers ( Watanabe et al. 2005; Shen et al. 2007), as well
as microsatellite markers from the closely related genus
Xiphophorus (Brummell et al. 2006). However, none of

2195

This journal is q 2009 The Royal Society

2196 N. Tripathi et al.

Genetic map of the guppy

Table 1. Description of mapping crosses. (Only informative SNP markers, with each grandparent homozygous for polymorphic
alleles, are listed. For details, see table 1 in the electronic supplementary material.)
(a) grandparents and number of offspring in mapping populations

cross

populationsa

no. of F2
individuals

additional markersb
no. of informative
markers (both grand- (one grand-parent
parents homozygous) heterozygous)

Cross76
Cross99
Cross150
Cross153
Cross157
Cross158

\Cumana!_QuaII_215-3
\QuaII_203-4!_Cumana
\QuaII_203-4!_Cumana
\QuaII_203-4!_Cumana
\QuaII_215-3!_Cumana
\QuaII_215-3!_Cumana

230
133
99
281
896
354

510
461
504
495
577
583

209
151
123
116
135
167

subpopulationsc

two F1 pairs

five F1 pairs
nine F1 pairs
three F1 pairs

(b) number of informative markers shared between the six mapping crosses
cross
158
157
153

150

99

76

158
157
153
150
99
76

504
377
373

461
363

510

583
501
401
423
396
424

577
415
408
389
416

495
379
366
399

All Quare originated from Quare 6, the numbers indicate laboratory-reared families from this location.
SNP markers for which one of the grandparents was homozygous, while the other grandparent was heterozygous.
F2 offspring from each F1 pair considered as an independent population (to facilitate inclusion of a maximum number of informative markers
including those for which either grandparent of the mapping cross showed heterozygous alleles).
b
c

these previous maps reproduced the correct haploid

chromosome number of the guppy, because of an
insufficient number of markers.
Some teleosts show differences in recombination rates
between the sexes. An androgenetic haploid linkage map
of Danio was estimated at 1010 cM, but a gynogenetic
map at 2583 cM (Singer et al. 2002). By contrast,
Xiphophorus does not show any sex-specific differences in
recombination rates (Kazianis et al. 2004a; Walter et al.
2004). Most previous studies have not revealed consistent
differences in recombination rates between guppy sexes
(Khoo et al. 2003; Watanabe et al. 2004; Brummell et al.
2006), although one report suggested a greater length in
males (Shen et al. 2007). We report a detailed and
complete genetic linkage map for the guppy, with
polymorphic single nucleotide polymorphism (SNP)
markers linked to either expressed protein-coding genes
(expressed sequence tag, EST) or bacterial artificial
chromosome (BAC) genomic clones.
Male guppies exhibit high levels of phenotypic and
genetic variability for several traits such as size, shape
and colour, which have been implicated to influence female
choice, male mating success and their susceptibility to
predation (Reznick & Endler 1982; Houde & Endler 1990;
Endler 1995; Brooks & Endler 2001a; Hughes et al. 2005;
Lindholm et al. 2005). These secondary sexual characters
are important for male fitness. We report predictions of
QTL peaks for selected traits showing variation in the male
progeny in one of our mapping crosses.
2. MATERIAL AND METHODS
(a) BAC library screening
All of the clones were obtained from a guppy BAC library,
prepared from males of the Cumana population, with an
average insert size of 160 kbp (constructed by Bio S&T,
Montreal, Canada).
Proc. R. Soc. B (2009)

(b) SNP discovery from BACs

Plasmid DNA was isolated from overnight cultures
of random BAC clones with a REAL Prep96 plasmid
kit (Qiagen, Hilden, Germany) and sequenced at both
ends with BIG DYE T ERMINATOR chemistry using the
standard pIndigoBAC-5 vector-specific sequencing primers
(forward 5 0 GGA TGTGCTGCAAGGCGATTAAGTT
GG3 0 , reverse 5 0 CTCGTATGTTGTGTGGAATTGT
GAGC3 0 ) on a 3730xl DNA analyser (Applied Biosystems,
Darmstadt, Germany).
The resulting sequence trace files in ABI format were
processed with pregap4 (STADEN package), using the phred
base-calling algorithm (Ewing et al. 1998). Vector sequence
and low-quality insert sequence were trimmed (Staden 2000).
BAC end sequences were blasted against each other, to
identify high copy number, repeat-rich sequences, which were
excluded from further marker development. PCR primers
were designed from all unique sequences using Primer3
(Rozen & Skaletsky 2000). PCR products were generated
with genomic DNA pooled from six individuals representing
each strain and sequenced. These sequences were
compared and SNP markers were selected that distinguish
the Cumana and Quare strains (see table 1a,b and the
electronic supplementary material, table 1, for details on
the number of informative markers in each mapping cross).
(c) Mapping panel and genotyping
Several reciprocal mapping crosses were set up between the
Cumana and Quare-6 wild populations, which were maintained in the laboratory for few generations. Cumana is
derived from a high-predation stream in Cumana, Venezuela
(Alexander & Breden 2004) and Quare-6 is derived from a
low-predation population in the lower Quare River, Trinidad
(Kelly et al. 1999). F1 individuals from each intercross were
mated in single pairs. The crosses with Quare \ ! Cumana _
were found to be more successful in producing fertile F1

Genetic map of the guppy

pairs, compared with reciprocal crosses. A final set of
six populations with the highest number of F2 offspring
were selected for genotyping. Only one of these, Cross76,
was from a Cumana \ ! Quare _ pair of grandparents.
Quare-6 females used were from two separate families, Quare-6
II-203-4 and Quare-6 II-215-3 maintained in our
laboratory (table 1a).
A total of 2060 individuals from the mapping populations
including the F1 and F2 offspring from the six intercrosses
were genotyped for 224 EST-linked (Dreyer et al. 2007) and
819 BAC-linked markers developed in this work. The
information about the primer sequences and accession
numbers, of all the SNP markers generated and used in this
study, is provided in tables 2 and 3 in the electronic
supplementary material. A fraction of the markers were
linked with coding genes (electronic supplementary material,
table 4; additional information is available at http://guppy.
weigelworld.org/weigeldatabases/). Genomic DNA was
isolated from the tail muscle of each fish using Qiagen
DNeasy96 kit (catalogue no. 69582) according to the
manufacturers instructions. Genotyping was done using the
MassArray MALDI-TOF mass spectrometry assay
(Sequenom, San Diego, CA, USA).
Mapping population was analysed for the marker genotypes and the markers were classified as informative if both
grandparents in a cross were homozygous for alternative
alleles (table 1; see table 1 in the electronic supplementary
material). Between 461 and 583 markers were in this
category, depending on the cross. Among the remaining
markers, those for which either of the grandparents was
heterozygous formed the second category. This comprised
116209 additional markers per cross (table 1a,b).
(d) Generation of the linkage map
Calculation of the linkage groups (LGs), order and distance
between the markers was carried out with the software
JOINMAP4 ( Van Ooijen 2006). Based on the first set of
informative markers, LGs were calculated using the independence logarithm of the odds (LOD) at a minimum of 4.0
and recombination frequencies at a maximum of 0.40 as the
threshold for each cross. The correct haploid number of 23
LGs was obtained for each of the six mapping populations.
The map order of each LG was estimated with the regression
mapping algorithm using Kosambis (1944) mapping function. An initial framework map was generated for each cross.
For comparison of the basic framework map, maximumlikelihood mapping, which uses Haldanes mapping function,
was also used. The marker order was the same with
both mapping functions, but occasionally maximumlikelihood mapping resulted in larger inter-marker distances.
For the final combined map, all map distances were computed
with regression algorithm using Kosambis mapping function.
To integrate the maximum number of additional markers
from the second category into the framework map, the F1
pairs with a large number of F2 offspring from each intercross
were analysed as a separate mapping population. For this, the
genotypes of all markers were used to calculate the linkages
(table 1a), applying the fixed marker order obtained from the
framework map when required ( JOINMAP; Van Ooijen 2006).
This resulted in individual F1 pair-specific maps with
additional markers incorporated into the framework map for
each cross. The multiple maps obtained from each pair of
grandparents were combined to obtain a single map using
the map integration function of JOINMAP4 (Van Ooijen 2006).
Proc. R. Soc. B (2009)

N. Tripathi et al.

2197

This calculated the virtual numbers of recombinant and

non-recombinant gametes estimated in each population for
each pair of loci, which is then averaged across populations to
obtain the mean recombination frequencies and corresponding LOD score values. The integrated maps for each
of the six mapping crosses were studied individually and the
best order of the markers was confirmed for each LG.
Consensus maps from the six grandparent crosses were then
compared and markers whose positions were ambiguous or
that added significantly (more than 20 cM) to the map length
were removed. Each of the 23 LGs from different populations
was then combined, to obtain the final integrated map. For
calculating the map orders, the consensus framework
maps for each LG were given as fixed orders ( JOINMAP;
Van Ooijen 2006).
(e) Phenotype analyses
All mature males were submerged in tricaine as terminal
anaesthetic, and photographed with a digital single lens reflex
camera under incident light, at an average age of 120 days.
Lateral views, each with a coloured scale bar, were used for
the measurements of the colour patterns on male body and
fins, as well as for the size estimates of each male. The
measurements of the phenotypes of all males were performed
using CELL IMAGING software (Olympus Soft Imaging
Solutions GmbH, Munster, Germany). This allows semiautomatic identification of each area of similar pixel density
on the fish, and calculates associated parameters for it. The
parameters measured for each colour pattern were area size,
perimeter length, mean colour intensity, mean hue (red, blue
and green) and position of the centre of mass with respect to a
reference point that was arbitrarily set to the middle of the
eye. The final trait values used for QTL mapping calculations
were direct measurements as well as derived numerical values
from each property of the trait. For example, for the anterior
orange spot, we used its area, mean colour intensity and mean
hue individually as well as the products of these measurements. Here we present the results of the QTL mapping of
area (mm2) of each colour trait. Besides recording all colour
patterns on males, specific length measurements (mm)
between fixed points on each fish were taken. Variation in
trunk length was measured as the gap between fixed points on
the snout and the caudal or dorsal fin; the width was
measured as the height of the anterior and posterior caudal
peduncle and the gap between dorsal fin and gonopodium.
The caudal peduncle was surrounded by fine margins at its
dorsalventral, anteriorposterior boundaries and the total
area of the enclosed surface was estimated. These length
measurements and their ratios were used for QTL mapping of
size and shape variation among males.
(f ) QTL predictions
All estimations of genotypephenotype correlations for
QTL mapping were performed with the MAPQTL5 software
( Van Ooijen 2004). Under the null hypothesis, i.e. when the
segregating QTL has no effect, the KruskalWallis (KW )
statistic is distributed approximately as a chi-square distribution with the number of genotype classes minus 1 as the
degrees of freedom. The power of the test depends on
the degrees of freedom and the number of individuals in the
test. We used a KW score (K
) of 10.0 and a significance level
of 0.005 as the threshold for all our tests. The traits with
significant K
values on any LG were further analysed
using interval mapping (IM) and multiple-QTL model

2198 N. Tripathi et al.

Genetic map of the guppy

Table 2. Marker distributions across linkage groups among six mapping crosses. (no., the number of markers; L, the genetic
length of each linkage group, for every cross.)
Cross76

Cross99

Cross150a

Cross153a

Cross157a

Cross158a

LG

no.

L (cM)

no.

L (cM)

no.

L (cM)

no.

L (cM)

no.

L (cM)

no.

L (cM)

01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
23

16
23
14
15
8
29
37
28
28
19
20
14
19
15
21
13
26
20
22
10
17
15
23

27.50
28.80
19.90
31.60
20.70
36.00
37.00
29.30
33.80
34.10
25.40
25.80
35.80
27.20
36.00
24.00
32.50
29.00
23.50
30.30
23.00
10.70
28.10

18
27
17
15
14
25
29
19
26
17
20
15
19
14
16
11
24
20
17
11
12
11
18

32.70
29.90
21.90
49.10
23.80
45.70
41.70
28.80
53.50
31.30
38.80
30.20
44.90
24.90
47.70
30.60
39.40
35.80
32.60
37.30
26.70
15.50
28.70

25
29
17
19
10
30
30
28
23
21
15
15
18
20
15
17
24
19
19
14
14
13
16

32.70
43.30
19.30
39.80
17.50
38.70
38.90
37.60
50.80
52.00
33.80
35.40
43.20
36.80
48.20
24.40
34.50
45.00
31.40
31.90
28.30
28.20
35.60

20
31
14
11
15
31
37
26
33
23
25
19
23
19
19
19
30
25
29
18
16
25
20

32.50
29.90
22.20
43.50
29.50
45.40
34.30
32.00
52.00
40.30
32.90
29.80
37.50
33.90
32.80
29.40
27.80
34.80
30.80
30.00
25.40
20.80
27.90

28
35
15
22
18
35
26
30
33
24
28
25
30
31
21
22
32
23
24
21
23
23
30

30.46
40.29
22.69
54.23
30.02
49.60
33.04
33.15
52.75
41.24
40.90
27.55
42.54
31.21
52.85
30.43
34.63
38.16
28.81
32.60
37.69
26.29
28.90

30
41
26
31
21
32
34
34
34
28
24
22
28
30
28
17
30
22
27
18
22
24
23

38.10
41.40
35.80
51.70
24.50
41.90
38.70
31.00
50.80
50.20
38.80
34.10
42.40
32.00
45.40
31.70
30.40
33.90
33.30
31.80
32.50
30.30
33.00

Combined map from individual F1 pair subpopulations for the cross.

(MQM) functions of the MAPQTL5 software. A genomewide permutation test was performed for each trait with 1000
iterations, to establish LOD thresholds at a p-value of 0.05 for
each LG. Following the initial round of IM, markers with the
highest LOD score for a trait were selected as cofactors, and
MQM analysis was performed. The analysis was repeated,
until no further significant peaks were obtained. The
automatic cofactor selection function of MAPQTL5 was
then used to select the significant set of cofactors and MQM
was repeated, until no more peaks with higher significance
were obtained. All peaks with a LOD score above the
calculated threshold for their specific LG were considered
to represent the QTL linked with the trait. The markers
closest to the maximum significant interval in the QTL
likelihood map are listed with their positions, LOD values.
The estimates of the additive and dominant effects and the
estimated mean distributions of the quantitative trait
associated with each genotype, for all significant loci, are
also shown. The estimated mean trait value is received as an
output from the final mapping of the trait through MQM
analysis, and it takes into account the additive and dominance
effects of all the cofactors influencing a trait. For an F2
population, in addition to the three QTL means, the
associated additive and dominance effects for every cofactor
marker are also modelled ( Van Ooijen 2004). Because there
are roughly as many heterozygous as homozygous (Cumana
and Quare) genotypes, the positive and negative additive
effects do not always average to 0, affecting the overall values
around which the three QTL means are estimated and
sometimes giving a negative estimate mean trait value.
A correlation matrix was established to identify the size
and shape traits that co-segregated in a significant manner. To
test whether the size and shape traits covary, Pearson
correlation coefficients were calculated for all traittrait
Proc. R. Soc. B (2009)

comparisons, for the 16 size- and shape-related traits. This

analysis used the trait scores for each individual in the mapping
panel. The correlation matrix retained the magnitude and
direction (positive and negative) of each correlation coefficient
(see table 5 in the electronic supplementary material).

3. RESULTS
(a) A complete genetic map of the guppy
The linkage map obtained from each of the mapping
crosses represents the correct number of 23 haploid
chromosomes for the guppy ( Winge 1922b). The
individual maps were found to be consistent regarding
the marker orders and overall map lengths (table 2), and
were combined to obtain a final consensus linkage map for
the species (figure 1). The sum of intervals between all loci
on the final combined map was 850 cM. Two approaches
were used to estimate map length: first, the genome length
was estimated by adding 2s (s is the average space between
adjacent markers on the linkage map) to the length of each
group to allow for chromosome ends ( Fishman et al.
2001). This yielded a corrected genome length of 896 cM.
By the second approach, the genome length was calculated
by multiplying the length of each LG by (mC1)/(mK1),
where m is the number of markers in each group. The
estimated map length is the sum of the revised length of
all LGs (Chakravarti et al. 1991) and was calculated to be
902 cM by this method. The average of the two corrected
genetic length estimates was 899 cM and includes 790
markers on 23 LGs (table 3).
In addition, the positions of a few markers were
uncertain (from 2 to 7 per LG; see table 6 in the electronic
supplementary material), either because they were

Genetic map of the guppy

LG01
0
0.1
0.5
1.2
1.6
2.4
3.0
4.4
7.6
8.1
8.3
9.2
11.4
13.5
14.1
15.5
16.0
16.1
16.2
16.3
18.5
18.7
19.0
20.0
20.1
21.7
24.0
24.4
24.9
26.7
26.9
27.2
27.3
29.6
30.3
31.9
34.1

LG02
0213
0813
0099
1011
0058
0509
0444
0958
0548
0072
0660
0154
0063
0671
0087
0619
0146
0938
0885
0571
0581
0550
0455
0285
0708
0932
0445
0400
0831
0192
0559
0939
0848
0670
0898
0140
0040

0
0.6
0.8
1.9
2.0
2.1
2.2
2.5
2.7
4.7
5.3
5.7
5.9
6.8
7.2
7.9
8.3
9.1
9.3
10.2
10.6
12.8
13.4
14.0
14.2
14.6
15.2
15.8
16.1
16.2
16.4
17.5
18.0
20.1
20.6
21.4
22.0
22.7
23.2
24.5
26.5
26.6
28.5
28.8
30.2
30.8
32.3
33.1
35.5
37.2
40.6

LG03
0803
0451
0337
0431
0254
0738
0327
0531
0122
0309
0712
0820
0845
0629
0956
0520
0742
0382
0292
0096
0319
0050
0024
0621
0911
0202
1035
0119
0416
0683
1003
0776
1009
0919
0555
0439
0864
0817
0901
0645
0329
0924
0883
0821
0649
0252
1027
0705
0799
0608
0967
0317
0654
0299

0
0.4
1.1
2.0
2.1
2.8
3.1
4.3
5.1
5.2
5.5
7.6
8.4
9.2
9.6
10.0
11.0
11.7
14.0
15.3
15.9
17.9
18.0
19.2
20.1
21.7
22.8
29.1

LG04
0136
0204
0688
0348
0311
0570
0768
0721
0113
0519
0733
0941
0906
0273
0286
0810
0342
0198
0226
0504
0465
1002
0002
0527
0323
0575
0405
0368
0347

LG05

0191

3.1

0441

16.3
16.6
20.2
25.0
25.6
26.0
26.1
26.3
28.4
28.8
30.2
30.6
31.9

0018
0325
0965
0296
0711
0259
0890
0238
1006
0847
0973
0601
0468
0411
0658
1031
0528
0394
0852
0013
0709
0895
0469
0306
0214
0134
0951
0354

34.2
34.8
34.9
35.7
36.4
36.5
37.8
41.3
43.8
46.4
52.4
52.7

0
1.7
2.1
2.2
3.3
4.8
5.0
6.5
6.9
7.1
8.6
10.0
10.4
10.6
10.7
11.6
11.7
13.1
14.3
17.4
17.7
18.4
18.5
22.2
25.9
30.3

N. Tripathi et al.

LG06
0318
0858
0464
0447
0549
1004
0701
0125
0417
0482
0648
0745
0276
0415
0100
0028
0159
0507
0737
0224
0729
0808
0503
0374
0249
0751
0679

0
1.0
1.5
2.8
4.6
6.3
7.3
7.4
7.6
11.3
12.7
13.9
14.6
14.7
15.1
16.9
17.4
17.7
18.7
19.9
20.5
20.8
21.0
21.4
22.4
22.5
22.7
26.1
27.1
27.3
27.4
29.3
29.8
30.3
31.9
33.1
33.6
40.9
48.3
48.8

2199

LG07
0065
0196
0943
0232
0180
0693
0195
0923
0686
0169
0485
1043
0874
0270
0816
0702
0992
1023
0641
0362
1026
0092
0127
0287
0366
0783
0114
0785
0373
0331
0420
0944
0933
0917
0626
0492
0681
0355
0631
0577
0767
0034
0422

0
1.5
1.9
2.1
2.2
2.3
2.4
2.5
3.1
3.7
3.9
4.4
5.0
6.8
7.0
9.6
10.3
10.5
10.6
11.1
12.1
13.1
13.5
14.3
14.5
14.6
16.1
18.4
19.5
19.9
21.6
21.9
23.4
26.0
26.3
26.8
27.9
28.0
28.3
28.5
29.7
31.2
34.7
35.8
39.8

0827
0290
0607
0533
1021
0595
0556
0937
0537
0188
0413
0971
0105
0171
0075
0936
0728
0854
0222
0432
0130
0077
0086
0622
0190
0562
0867
0010
0743
0161
0969
1040
0141
0795
0393
0609
0375
0489
0047
0976
0792
0377
0655
0789
0530
0839
0053
1007
0304

Figure 1. The genetic linkage map of the guppy genome. The numbers to the right on each bar are the markers and the numbers
on the left indicate the genetic distance in centimorgans (cM). The numbering of LGs is based on homology with medaka
chromosomes. The markers showing no recombination in between them on an LG are shown at the same locus. The putative
location of the sex-determining locus (Sex) is shown at the distal end of LG12 (sex chromosome).

successfully typed in only a small number of individuals, or

because they were informative in only a few individuals.
A few markers were excluded from the final map
because they would have added significantly to the
map distances. These markers mapped terminally on
the LG, with abnormally large distances (more than 20 cM)
to the nearest markers, rendering their positions doubtful.
Examples include marker 0484 on LG01, marker 0003 on
LG16, marker 0145 on LG03 and marker 0046 on LG20
(see table 6 in the electronic supplementary material).
The largest gap on the framework map is 13.2 cM, on
LG04. The number of mapped loci ranges from 23 to
54 per LG, and the average length of an LG is 39 cM,
ranging from 29 to 58 cM, with an average of one marker
per 1.1 cM. Based on the predicted genome size of
Proc. R. Soc. B (2009)

700 Mbp (Khoo et al. 2003), we expect the average intermarker distance to be approximately 1 Mbp (table 3).
(b) Distribution of crossovers
Cytogenetic studies have established the acrocentric
nature and similar size of all 23 pairs of guppy
chromosomes ( Nanda et al. 1993; Traut & Winking
2001). With 23 haploid chromosome arms, the predicted
genome size should be in the range of 1150 cM (50!23)
under the complete interference model ( Villena &
Sapienza 2001; Guyomard et al. 2006; Moen et al.
2008). Our estimates of the total genome length are
slightly lower than this. We estimated the average
proportions of F2 offspring inheriting recombinant
gametes, from meiosis in both F1 parents. The mean

2200 N. Tripathi et al.

LG08
0
0.1
0.6
1.7
4.5
4.6
4.7
4.8
5.3
6.4
6.5
10.6
10.9
13.5
14.1
15.2
15.3
16.0
17.2
20.5
20.6
21.5
22.6
22.7
22.9
23.4
23.9
26.8
27.1
27.6
28.0
28.9
33.1

LG09
0352
0643
1018
0074
0085
0396
0150
0999
0183
0554
0807
0623
0975
0102
0064
0022
1032
0640
0131
0639
0160
0201
1014
0341
0121
0830
0774
0539
0312
0695
0383
0771
0753
0790
0208
0910
0207

0
1.9
2.5
3.0
3.5
4.0
8.6
9.1
9.5
11.5
11.6
11.7
12.6
12.7
13.5
14.6
16.0

0589
0748
0041
0724
0110
0434
1008
0842
0467
0587
0948
0866
0949
0725
0083
0233
0812
0955
0567
0066
0663
0610
0984
0993
0726
0888
0811

0
0.1
0.2
0.9
1.2
1.7
2.2
2.8
3.5
5.0
5.3
5.5
5.9
7.3
10.2
10.4
10.6
13.0
14.2
15.7
16.8
17.2
17.8
18.7
19.2
19.3
19.4
20.6
21.5
22.4
22.5
24.6
26.5
27.7
29.7
31.2
32.6

16.2
16.3
16.9
17.0
17.7
17.9
18.2
18.3
21.0
21.6
22.1
22.3
22.9
24.0
24.3
25.1
29.0
33.4
38.6
48.1
50.3

LG16
0
2.5
3.3
4.2
4.4
6.0
6.9
9.8
12.3
12.5
12.7
13.8
14.9
15.0
15.7
16.4
17.4
17.5
18.3
18.9
19.2
21.6
21.7
24.4
25.2
30.5

Genetic map of the guppy

LG10
0281
0314
0138
0079
0668
0625
0463
0558
0957
0647
0345
0682
0038
0142
0435
0300
0471
0731
0793
0837
1020
0777
0715
0108
0020
0069
1042
0859
0218
0005
0117
0841
0351
0945
0262
0462
0579
0128
0572
0395
0182

0
2.4
2.5
2.6
2.7
3.5
4.3
4.8
5.2
5.6
8.0
11.6
12.1
12.3
12.6
15.7
16.5
16.6
17.5
18.2
18.4
19.3
19.5
19.6
21.4
21.5
22.1
23.4
26.1
28.4
34.3
36.5
39.9
42.2

0
0.8
1.4
2.7
3.1
6.5
7.8
11.8
16.0
17.3
19.9
20.6
21.1
21.4
21.6
21.9
23.5
23.9
24.8
25.1
25.4
26.0
27.2
30.2
30.8
31.0
32.3
34.1
40.0

LG12
0321
0305
0089
0710
0277
0786
0532
0060
0757
0541
0840
0605
0147
0889
0877
0616
0217
0042
1019
0486
1022
0878
0440
0818
0031
1028
0642
0653
0665
0004
0152

LG13
0429
0517
0398
0442
0009
0987
0380
0155
0030
0061
0247
0244
0568
0231
0230
0148
0248
0666
0090
0210
0245
0423
0691
0032
0228
0490
1025
0315
0073
0229
Sex

0
0.1
0.3
2.1
4.8
5.7
8.1
8.5
9.5
11.1
12.0
13.2
14.1
14.9
15.3
15.7
15.8
16.3
18.0
18.9
19.9
21.3
21.8
22.8
23.1
25.0
25.4
25.6
26.8
27.2

0
0.8
1.0
1.2
1.3
1.4
3.9
5.1
6.5
6.8
9.8
10.3
10.4
10.9
11.9
12.0
12.1
14.1
15.0
17.4
18.3
20.1
20.3
20.6
23.6
24.4
24.8
25.1
25.6
26.9
27.9
29.0
29.6
38.8

LG17
0106
0615
0880
0470
0017
0782
0565
0120
0039
0126
0266
1037
0662
0884
0253
0672
0772
0076
0758
0661
1016
0274
0359
0036
0333
0674
0268
0794
0153
0651
0236
0057
0696
0825
0634
0897
0667

LG11
0557
0477
0590
0011
0324
0896
0474
0566
0988
0346
0027
0700
0084
0454
0026
0170
0044
0021
0552
0770
0124
0367
0052
0372
0740
0513
0834
1033
0995
0521
0873
0070
0339
0133

LG18
0
6.4
6.6
6.8
8.3
9.2
9.7
10.4
10.6
10.8
15.0
15.1
15.2
16.7
17.0
18.3
18.5
18.7
20.3
21.5
21.8
22.7
24.0
26.0
30.3
30.9
31.1
32.5
36.2

0336
0940
0480
0025
0139
0449
0981
0773
0437
0714
0157
0428
0189
0900
0611
0363
0928
0582
0814
0511
1038
0379
0593
0828
0637
0481
0970
0815
1015
0406

LG19
0
2.1
2.6
2.7
4.6
4.9
5.3
6.8
8.4
8.7
8.8
9.0
9.1
9.4
9.6
12.0
15.4
20.5
21.0
21.1
21.6
21.8
21.9
22.7
23.7
27.2
27.3
28.8
29.2

LG20
0894
0997
0545
0491
0162
0144
0293
0260
0167
0407
0734
0893
0185
0869
0576
0690
0991
0054
0088
0118
0515
0694
0497
0553
0959
0741
0508
0107
0588
0968
0123

0
0.7
2.0
4.1
4.6
5.3
9.5
10.4
11.4
11.9
13.1
16.1
19.6
19.9
20.6
20.8
21.0
21.5
23.5
25.7
28.0
30.0
32.5

0569
0280
0699
0452
0913
0909
0275
0633
0401
0755
0926
0101
0892
0051
0677
0903
0418
0506
0334
0563
0326
0881
0335

LG14
0761
0644
0855
0718
1024
0857
0433
0353
0638
0664
0016
0223
1001
0356
0376
0412
0876
0912
0143
0781
0392
0800
0283
0680
0094
0798
0826
0998
0082
0364
0780
0585
0488
0430
0754
0656
0205

LG21
0
6.4
6.8
7.5
9.6
10.0
10.4
10.9
11.5
11.6
11.9
12.0
12.7
14.4
14.8
15.3
17.4
19.3
23.2
23.3
29.5
32.2
32.3
34.5
36.5
36.7

LG15
0925
0344
0752
0240
0019
0055
0179
0172
0186
0963
0242
0635
0836
0289
0381
0602
0294
0211
0796
0397
0151
0574
0279
0861
0596
0510
0174
0961
1017
0221
0872
0282
0538
0494
0427
0628

0
0.6
0.7
0.8
1.2
3.0
5.5
7.4
8.9
9.2
9.4
9.5
11.0
12.3
12.8
13.3
14.0
14.7
15.9
17.0
18.0
18.1
18.8
19.5
23.6
23.8
24.4
27.7
30.7
39.1
43.3
46.0
54.2
55.1

0964
0824
0080
1030
0762
0360
0129
0832
0426
0340
0860
0476
0624
0856
0349
0560
0578
0219
0915
0868
0301
1012
0410
0819
0804
0822
0985
0586
0158
0849
0316

0
0.2
0.3
1.0
1.4
1.7
1.8
2.0
3.2
4.6
5.1
5.2
5.4
5.9
6.0
6.5
7.4
7.7
9.0
9.8
10.7
11.3
13.3
16.5
17.2
17.6
18.8
24.6
25.0
25.3
25.6
27.7
28.5
29.5
29.7

0
1.6
2.0
2.2
3.8
4.1
4.6
5.6
6.6
8.1
9.0
9.4
13.2
13.4
14.5
14.9
15.5
16.0
17.1
17.9
18.9
21.4
22.2
22.5
23.3
24.1
24.4
25.6
28.4
28.7
30.6
30.7
31.4
32.5
33.0

LG22
0704
0749
0320
0727
0419
0386
0165
0775
0620
0675
0239
0234
0678
0920
0687
0632
0446
0498
0950
0659
0178
0597
0962
0095
0295
0343
0173
0899

0
2.6
5.1
6.2
6.8
8.3
10.0
11.1
11.6
12.1
12.7
12.9
14.6
14.7
15.2
15.7
16.0
16.4
17.1
17.4
19.2
19.7
20.0
20.3
20.4
21.6
22.1
23.4
26.2
27.1

1000
0863
0438
0613
0135
0750
0902
0068
0905
0328
0255
0916
0784
0722
0779
0614
0692
0271
0914
0298
0524
0685
0278
0308
0946
1010
0989
0525
0990
0093
0267
0483
0071
0684

LG23
0209
0466
0862
0978
0599
0237
0843
0835
0307
0163
0443
0838
0350
0879
0657
0947
0697
0646
0330
0216
0787
1039
0627
0461
0977
0473
0716
0475
0387
0797
0561
0425
0805
0006
0604

Figure 1. (Continued.)

frequency of non-recombinant and single recombinant

gametes, across all LGs, was 44 per cent. The frequency of
F2 offspring inheriting two recombined gametes (one from
each F1 parent) for a chromosome was 10 per cent.
Moreover, the distribution of the estimated proportion of
Proc. R. Soc. B (2009)

individuals with two recombined gametes was roughly

correlated with the genetic size of the LG. A very
low proportion of F2 individuals (0.43%) inherited
recombined chromosomes with more than two
crossover events (table 4). These results demonstrate

Genetic map of the guppy

Table 3. Guppy genetic map overview.
(a) summary of the integrated genetic map of the guppy
no. of individuals
2060
no. of linkage groups
23
no. of markers on the map
790
marker distribution
34 markers/ LG
total map length
899 cMa
average length /LG
39 cM
average marker density
1 marker/1.1 cM
estimated physical spacing
1.19 cM MbpK1
range of marker distribution
2354
range of length /LG
2958 (cM)
(b) length and marker distribution on linkage groups
linkage group

no. of markers

length (cM)

LG01
LG02
LG03
LG04
LG05
LG06
LG07
LG08
LG09
LG10
LG11
LG12
LG13
LG14
LG15
LG16
LG17
LG18
LG19
LG20
LG21
LG22
LG23
total

37
54
29
30
27
43
49
37
41
34
31
29
37
36
34
27
37
30
31
23
28
31
35
790

36.04
42.34
31.12
55.57
32.41
50.97
41.64
34.98
52.57
44.47
42.29
29.14
40.93
34.95
57.82
32.72
34.47
38.47
31.21
35.00
39.01
29.01
31.60
899a

The total map length is the derived estimate, incorporating the

correction for chromosome ends.

crossover interference and comply with the predictions based on the known acrocentric nature of guppy
chromosomes.
(c) Identification of the sex LG
LG12 was identified as the sex LG in the guppy, since the
markers showed clear sex-linked segregation and recombination between X- and Y-linked alleles was suppressed
(Tripathi et al. in press). The sex-determining locus (Sex)
mapped genetically to the distal end of LG12 in all crosses
(figure 1), marker 0229 being mapped closest to this locus;
although the gap between marker 0229 and Sex could not
be predicted with certainty. This gap is expected to
include the differentiated region of the Y chromosome
and has not been taken into account in genome length
calculations, due to its uncertain length. None of the
known molecular markers on LG12 are present
exclusively on the Y chromosome, although X- and
Y-linked alleles for all markers on this LG show distinct
segregation, due to reduced recombination. The putative
Y-specific region at the distal end of this chromosome is
also predicted to contain a few colour pattern loci in tight
Proc. R. Soc. B (2009)

N. Tripathi et al.

2201

Table 4. Distribution of the predicted number of recombination events per linkage group in the combined mapping
population. (The values represent the percentage of F2
individuals showing null, 1, 2 or more recombination (rec.)
events for the respective linkage groups. The results obtained
from the six mapping crosses have been averaged.)
LG

no rec.

1 rec.

2 rec.

O2 rec.

LG01
LG02
LG03
LG04
LG05
LG06
LG07
LG08
LG09
LG10
LG11
LG12
LG13
LG14
LG15
LG16
LG17
LG18
LG19
LG20
LG21
LG22
LG23
average

47.33
40.80
62.23
31.39
44.01
34.21
42.61
43.35
32.13
38.59
44.90
49.76
35.83
49.73
37.07
47.79
43.92
42.18
48.74
41.58
47.72
56.65
47.77
43.93

43.74
45.60
34.66
43.62
43.72
45.72
46.26
46.89
45.99
47.23
45.10
47.23
46.17
42.70
42.94
42.31
46.74
44.71
45.97
46.00
41.84
38.98
44.81
44.30

7.96
10.44
2.72
21.20
9.70
18.22
9.57
7.91
19.64
12.52
9.03
2.43
14.81
6.89
16.50
8.20
8.22
12.14
3.69
10.19
9.47
3.59
5.87
10.04

0.24
0.79
0.10
0.95
0.64
0.46
0.39
0.46
0.56
0.41
0.24
0.15
0.80
0.17
0.87
0.42
0.28
0.24
0.40
0.56
0.24
0.19
0.39
0.43

linkage with the master sex-determining locus ( Winge &

Ditlevsen 1947; Lindholm & Breden 2002).
(d) Synteny with medaka
Our markers included 332 markers linked to coding genes
with significant similarity to sequences in the medaka
genome (as determined by BLASTN, e-value%10K5,
identity R80%). Of these, 276 (83%) could be assigned to
specific LGs on the guppy map. For each guppy LG, we
found between 2 and 16 markers that belong to a single
medaka chromosome (table 5; see table 4 in the electronic
supplementary material). We can thus infer orthologous
chromosomes in these two species, which have been
separated for approximately 100 million years ( Volff 2005;
Volff et al. 2007). We used this information to name guppy
LGs corresponding to the medaka chromosome with the
maximum number of shared markers (table 5; see table 4
in the electronic supplementary material). Since medaka
(24 chromosomes) has one chromosome more than the
guppy, LG02 of the guppy corresponds to chromosomes
2 and 21 of the medaka, while LG21 of the guppy
corresponds to chromosome 24 of the medaka. Despite
ample evidence for collinear blocks, the orthology between
medaka chromosomes and guppy LGs is not absolute,
since, on average, approximately a quarter of gene-linked
markers from a given LG in the guppy mapped to a
different medaka chromosome (table 5; see table 4 in the
electronic supplementary material).
(e) QTL predictions
We mapped QTL influencing a selected set of size, shape
and colour traits, segregating in one of the mapping

2202 N. Tripathi et al.

Genetic map of the guppy

Table 5. Synteny between medaka chromosomes and guppy linkage groups (LGs). (Distribution of the markers linked with
coding genes found common between guppy linkage groups and medaka chromosomes. The numbers in italics along the
diagonal indicate the number of markers showing major syntenic relationships between guppy LGs and corresponding medaka
chromosomes, and comprise 190 EST-linked markers.)
medaka chromosomes
guppy
LG
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
23

01

02

03

04

05

06

07

14

08

09

10

11

12

13

14

15

1
1

1
2
1
1

7
1

1
2
16

21

1
1

12
1

1
2

23

1
1

24

1
1

1
1
1

1
1

11

1
1

1
1

10
1

22

13

20

12
1

19

5
1

18

5
8

17

16

1
1

9
1

1
6

6
9

1
1
1

1
1

2
1

1
1

1
1

1
11

1
1
1
2

1
1

4
1

1
1

crosses (Cross157, Quare \ ! Cumana _). The nonparametric KW test showed a clear correlation between
quantitative trait and markers on specific LGs. Traits with
a high KW significance were mapped independently using
MQM mapping. MQM mapping resulted in the detection
of loci influencing the traits on some additional LGs and
further strengthened the confidence of the QTL prediction (table 7 in the electronic supplementary material).
This resulted in selection as cofactors, of a subset of
markers from each contributing LG, showing the highest
correlations with the trait (see table 8 in the electronic
supplementary material).
Discrete genetic polymorphism for male size is
characteristic of some species in the family Poeciliidae
(Kallman 1989). Guppies are sexually dimorphic for adult
size and males are highly variable in size at maturity
(Reznick & Endler 1982). Size variation in male guppies is
a quantitative trait, approximately normally distributed
within populations and characterized by high heritability
(Reznick et al. 1997; Nakajima & Taniguchi 2002; Hughes
et al. 2005). While the females continue to grow
throughout their adult lives, males reach their maximum
body size at approximately three months of age, when the
growth rate sharply decreases. We used the genetic map to
predict QTL for various parameters related to the size and
shape of males. We directly measured the distances
between fixed landmarks on each adult male and used
these as quantitative traits (figure 2a). We used the derived
ratios of specific length measurements and caudal
peduncle area, to map QTL, which reflect the relative
proportions and geometry of the body and are indicative
of shape variations.
Proc. R. Soc. B (2009)

5
10
5

For all of the length measurements and the caudal

peduncle area, there was a major effect QTL in the
proximal region of LG12. This peak accounted for a total
of 2030% variation in the length traits, with additional
contributions from QTL on three to five other LGs.
In combination, all the significant peaks could explain
3545% of the total variation for the length traits, while
peaks on five different LGs, including LG12, could explain
43 per cent variance in caudal peduncle area. QTL on
LG12, 09, 02, 07, 18 and 04 seemed to contribute
predominantly to most of the size-related traits (see table 8a
in the electronic supplementary material).
The derived quantitative traits reflecting the relative
proportions and aspect ratios of the fish were used to map
QTL affecting body shape. Ratios of various length
measurements and the area of the caudal peduncle were
calculated, to represent components of the overall
geometry of the fish. QTL for these ratios, indicative of
shape variation, also mapped to the proximal region of
LG12 for many traits, confirming the presence of at least
one major locus influencing size and shape in the pseudoautosomal region of the sex chromosome (see table 8b in
the electronic supplementary material). This QTL
explained 521% of the total variance for different ratios.
QTL on LG01, 02, 04, 07, 08, 09, 11, 12, 18 and 22
seemed to contribute predominantly to most of the shaperelated traits. In combination, all the significant peaks
could explain 1749% of the total variation in different
shape-related traits (see table 8b in the electronic
supplementary material).
A correlation matrix generated to analyse the correlation coefficients between all the size and shape-related

N. Tripathi et al.

Genetic map of the guppy

(a)

length: 9.51 mm
length: 16.01 mm

area: 1.03 mm2

area: 0.20 mm2
area: 1.27 mm2
area: 0.14 mm2
area: 0.48 mm2 length: 22.98 mm
area: 0.39 mm
area: 0.97 mm2 area: 0.18 mm2
area: 153 mm2
area: 4.11 mm2
area: 0.53 mm2

L2
L1

(b)
L3

3 2

length: 2.86 mm

1
8

area: 2.23 mm2

12

L5

area:2 0.25 mm2

area: 1.22 mm
area: 0.09 mm2

area: 1.40 mm2

area: 17.09 mm2

L4

length: 3.47 mm

(c) (i)

area: 1.30 mm2

area: 0.03 mm2

area: 0.13 mm2

CPA

3 2

2203

9 10 11

(ii)

1
8
7

12

(iii)
5

12

10 11

3 2

(iv)

(v)

4 3

12

10 11

(vi)

1
8

11
1

12

10

5 4

3
7

10

11

1
8

7
12
6

10 11
9

12
6

10 11

Figure 2. Variable quantitative traits segregating in Cross157 F2 males. (a) The length measurements between fixed points on
each fish, used for QTL predictions for the size and shape variation (table 7a,b in the electronic supplementary material). L1,
snout to hind fin; L2, snout to dorsal fin; L3, posterior caudal peduncle height; L4, anterior caudal peduncle height; L5,
gonopodium to dorsal fin; CParea, caudal peduncle area. (b) The polymorphic colour traits used for QTL predictions (table 7c
in the electronic supplementary material). The numbers mark following traits: (1) dorsal fin black; (2) dorsal fin orange; (3)
central blue white spot; (4) anterior main black stripe; (5) anterior orange spot; (6) black spot by gonopodium; (7) central orange
spot; (8) posterior main black stripe; (9) posterior ventral black stripe; (10) posterior orange spot; (11) hind fin lower orange;
(12) hind fin caudal black spot. (c) The segregation of colour pattern variation among males of Cross157. (i)(vi) Arrows show
the extreme variation for different traits among six representative F2 males.

traits indicated that all the size traits and the area of the
caudal peduncle showed very high correlations (8097%)
among themselves, and with selected shape traits (see
table 5 in the electronic supplementary material). Since
most of these traits also show a major effect QTL on LG
12, this locus could be responsible for the coregulation of
the size and shape variation.
Highly polymorphic colour patterns of guppy males
have been long known to play important direct and
indirect roles in various aspects of their biology, influencing sexual selection through female preference (Hughes
et al. 1999; Brooks & Endler 2001a,b; Eakley & Houde
2004; Miller & Brooks 2005) and natural selection due to
a variable susceptibility to predation (Endler 1991; Karim
et al. 2007; Reznick et al. 2008). Guppy males exhibit
diverse phenotypic patterns, including variation in colour,
number, shape, size and position of spots, and this
polymorphism is known to have a substantial genetic
component ( Winge 1922a; Winge & Ditlevsen 1947;
Haskins et al. 1961; Brooks & Endler 2001a; Lindholm
et al. 2004). We have mapped some of the colour traits
prevalent in two geographically distant populations
(Quare and Cumana), segregating in Cross157. The
selected traits that have been used for QTL mapping
include all prominent orange or black spots and stripes as
well as an iridescent patch on the body of the fish. These
patterns exhibit visible polymorphism, segregating among
F2 males of this cross (figure 2b), and show distinct
patterns of expression having dominant, suppressive,
mutually exclusive or co-dominant effects over other traits
Proc. R. Soc. B (2009)

( Tripathi et al. 2008). These colour patterns are

potentially important for the fitness and adaptive success
of the parental populations in the wild. We detect
significant QTL peaks on various LGs underlying some
of these colour traits. The results of the MQM mapping
for the area of the colour traits are shown (see table 8c in
the electronic supplementary material).
Estimated mean values of the quantitative traits
( Van Ooijen 2004) associated with homozygous and
heterozygous genotypes, at all significantly contributing
loci, indicated obvious genetic polymorphisms linked
with the segregating traits, in the two parental populations. For many of the size, shape (figure 3a) and
colour (figure 3b) traits, a clear correlation with either
Cumana or Quare alleles could be detected. For several
traits, the alleles showed obvious additive or dominant
effects. For instance, the lengths L2, L3 and L4 appear
to be positively correlated with the presence of Quare
alleles on LG12 and intermediate values in heterozygotes
suggest additive effects of both alleles. The ratio of L1
over L2 maps to a QTL on LG04, but for this trait the
Cumana dominates the Quare allele (figure 3a). Also,
with respect to the orange area on the dorsal fin, an
allele on LG16 of Cumana is dominant over the
corresponding Quare allele. The total percentage of
variance explained (PVE) by all significantly contributing QTL for a trait, ranged between 9 and 32 per cent
for the colour traits.
Furthermore, fine mapping of the loci underlying the
quantitative traits, broadly mapped in this study, will

2204 N. Tripathi et al.

Genetic map of the guppy

(a)
snout to hind fin (L1)

PVE = 43.90

L1 : L2

20.00
18.00
16.00
14.00
12.00
10.00
8.00
6.00
4.00
2.00

1.80

1.50

PVE = 33.20

20.00

1.70

15.00

1.65

LG09

LG02

LG07

LG18

snout to dorsal fin (L2)

1.60

10.00

1.55

5.00

mean

PVE = 45.20

0
LG04

LG11

LG09

LG08

L1 : L5

LG18

mean

PVE = 16.70

LG12

LG02

LG07

LG18

LG09

posterior CP height (L3)

mean

PVE = 38.50

LG12

LG22

LG01

L2 : L5

LG09

LG12

LG02

LG04

LG09

LG11

LG05

mean

PVE = 42.5

anterior CP height (L4)

3.50
3.40
3.30
3.20
3.10
3.00
2.90
2.80
2.70
2.60
2.50

PVE = 17.30

LG09

LG02

LG07

LG18

gonopodium to dorsal fin ( L5)

LG04

LG12

LG22

LG09

L4 : L3

mean

PVE = 35.30

4.50
4.00
3.50
3.00
2.50
2.00
1.50
1.00
0.50
0

LG22

LG02

mean

PVE = 32.90

LG12

LG09

LG22

LG03

L2/L5 : CParea

mean

PVE = 49.00

0.80
0.70
0.60
0.50
0.40
0.30
0.20
0.10
0
LG01

mean

PVE = 19.40

LG09

LG12

LG18

LG22

L1/L4 : L3

LG07

mean

PVE = 35.30

16.00
15.80
15.60
15.40
15.20
15.00
14.80
14.60
14.40
14.20
14.00

1.11
1.10
1.09
1.08
1.07
1.06
1.05
1.04
LG12

LG09

L1 : CParea

mean

2.75
2.70
2.65
2.60
2.55
2.50
2.45
2.40
2.35
2.30

3.20
3.10
3.00
2.90
2.80
2.70
2.60
2.50
2.40

LG12

1.06
1.04
1.02
1.00
0.98
0.96
0.94
0.92
0.90
0.88
0.86

4.70
4.60
4.50
4.40
4.30
4.20
4.10
4.00
3.90

10.80
10.60
10.40
10.20
10.00
9.80
9.60
9.40
9.20
9.00
8.80
8.60

PVE = 32.80

25.00

1.75

LG12

CParea : L4/L3

LG11

LG12

LG06

CParea : L4

LG09

mean

LG12

LG02

LG11

LG04

LG08

LG18

mean

PVE = 23.20

5.70
5.60
5.50
5.40
5.30
5.20
5.10
5.00
LG12

LG22

LG09

caudal peduncle area (area)

LG02

mean

PVE = 42.80

LG12

CParea : L3

LG09

mean

PVE = 45.20

8.00
7.00
6.00
5.00
4.00
3.00
2.00
1.00
0

25.00
20.00
15.00
10.00
5.00
0
LG12

LG09

LG07

LG18

LG02

mean

LG12 LG09 LG11 LG07 LG04 LG21 LG13 mean

Figure 3. Segregation of colour pattern, size and shape QTL in adult male guppies. The estimated mean values of the
distribution of quantitative trait associated with the paternal (Cumana), maternal (Quare) and heterozygous genotypes
among F2 males of Cross157 are plotted for each contributing linkage group that has a significant QTL (see 2 for
explanation of estimated mean trait values). Mean refers to the average value of the trait when all loci significantly linked
with the trait have homozygous Cumana, or, heterozygous or homozygous Quare alleles. The total percentage of variance
explained (PVE) by all significantly contributing QTL for each trait is shown. The linkage groups for each trait are shown
in descending order of the PVE. Only the loci closest to the significant maxima in the QTL likelihood map, with a LOD
higher than the estimated threshold based on the permutation test for respective linkage groups and explaining at least
2.5% of the total variance, are shown here. (a) Segregation of size (mm) and shape (ratio) QTL in Cross157 males.
(b) Segregation of colour pattern QTL in Cross157 males, using the area (mm2) of each component as trait keys. Red,
Cumana alleles; yellow, heterozygous; green, Quare alleles; x -axis, linkage group; y-axis, estimated mean trait value.

Proc. R. Soc. B (2009)

N. Tripathi et al.

Genetic map of the guppy

(b)

PVE = 22.90

dorsal fin black

1.40
1.20
1.00
0.80
0.60
0.40
0.20
0
0.20

LG12

LG01

LG04

LG02

dorsal fin orange

LG09

mean

PVE = 26.80

1.40
1.20
1.00
0.80
0.60
0.40
0.20
0
0.20
0.40
0.60
LG16

LG01

LG06

LG04

LG02

central blue white spot

LG07

mean

PVE = 26.50

1.00
0.80
0.60
0.40
0.20
0
0.20

LG12

LG02

LG23

LG12

LG17

anterior main black stripe

LG21

mean

PVE = 09.40

central orange spot

1.00
0.90
0.80
0.70
0.60
0.50
0.40
0.30
0.20
0.10
0

0.80

0.80

0.60

0.60

0.40

0.40

0.20

0.20

0.20

anterior orange spot

PVE = 23.00

2.50
2.00
1.50
1.00
0.50
0

LG12

LG07

LG06

LG20

black spot by gonopodium

LG09

mean

PVE = 19.10

2.50
2.00
1.50
1.00
0.50
0

LG12

LG04

LG16

LG07

LG01

mean

LG20

LG22

LG13

LG22

LG04

LG13

LG02

LG16

LG08

LG12

LG23

LG10

LG20

LG01

LG18

LG18

LG16

LG04

LG12

LG15

mean

PVE = 23.30

LG12

LG03

LG06

hind fin caudal black spot

3.50
3.00
2.50
2.00
1.50
1.00
0.50
0

mean

PVE = 35.90

LG09

LG04

mean

PVE = 17.70

hind fin lower orange

1.40
1.20
1.00
0.80
0.60
0.40
0.20
0

mean

PVE = 21.60

posterior orange spot

1.00

mean

LG05

posterior ventral black stripe

0.90
0.80
0.70
0.60
0.50
0.40
0.30
0.20
0.10
0

1.00

LG19

PVE = 15.30

posterior main black stripe

4.50
4.00
3.50
3.00
2.50
2.00
1.50
1.00
0.50
0

1.20

LG08

LG08

2205

LG02

LG02

mean

PVE = 32.10

LG21

LG06

LG15

mean

Figure 3. (Continued.)

require more individuals segregating for the phenotypes

and a higher marker density.

4. DISCUSSION
We have reported here a dense genetic linkage map of the
guppy, which provides a basis for further genetic and
molecular studies of sex linkage of male-advantageous
genes, as well as natural variation of quantitative adaptive
traits. The synteny information facilitates selection of
candidate genes during fine mapping of QTL, since the
guppy genome has not yet been sequenced.
Based on crosses involving ornamental guppy strains,
quite variable lengths of the genetic map had been
estimated (Khoo et al. 2003; Watanabe et al. 2005;
Proc. R. Soc. B (2009)

Shen et al. 2007). Our genetic map has a length of

899 cM, which is shorter compared with that of the
medaka at 1354 cM ( Naruse et al. 2000), or even the
closely related Xiphophorus at 2486 cM (Kazianis et al.
2004a). Both species have 24 chromosomes compared
with a haploid set of 23 in guppies and marginally higher
estimated genomic DNA content (Cimino 1974;
Lamatsch et al. 2000). Owing to the absence of an ESTlinked map for the platyfish genome, we could not
compare the synteny between guppy and platyfish
genomes. In a comparative study with common microsatellite markers, the guppy was also found to have lower
rates of recombination than Xiphophorus, which is in the
same family as the guppy (Brummell et al. 2006). An
explanation for the relatively short genetic length of the

2206 N. Tripathi et al.

Genetic map of the guppy

guppy map could be sequence inversions between the

Cumana and Quare populations, due to significant
divergence (Alexander & Breden 2004), which might
result in reduced recombination frequency.
We have tested microsatellite markers from the sex
chromosomes of platyfish, in addition to a set of selected
candidate genes from the sex chromosome, but did not
find any evidence of its sex linkage in the guppy (data not
shown). Although from the present data, it seems likely
that the sex-determining loci in platyfish and guppy are
not homologous, we cannot prove it until we have
additional markers from the diverged gonosomal region
of guppy sex chromosomes, and more gene-linked
markers from the sex chromosome of platyfish.
The differentiated region of the sex chromosome
is likely to consist of expanded heterochromatin-rich
sequences. Hence, due to the uncertain gap between
the last mapped markers and the Sex locus, we have not
included it in genome length estimates. Several additional
markers that were placed with confidence on an LG,
but could not be assigned to their accurate position (see
table 6 in the electronic supplementary material), may add
to the total genetic length when correctly positioned on
the map. Therefore, the present calculated length could be
a slight underestimate.
The multiple QTL mapped in this work affect traits
that contribute to phenotypic variation of males in the two
mapping populations. Several QTL were located on
LG12, indicating that loci responsible for visible polymorphism in size, shape and colour patterns are enriched
on the sex chromosome. This is in agreement with the
previous knowledge of physical linkage of major colour
pattern loci to sex chromosomes in the guppy ( Winge
1927; Winge & Ditlevsen 1947; Haskins et al. 1970;
Brooks & Endler 2001a; Lindholm & Breden 2002). We
do not believe that the enrichment for QTL reflects
exceptional gene density on the sex chromosome, based
on the extensive conserved synteny of the major portion of
the guppy sex chromosome with an autosome of medaka.
While we do not yet have any markers from the
Y-specific segment, due to the suppressed recombination
between X and Y chromosomes, the reduced recombination facilitates the identification of QTL mapping to the
sex LG. Unfortunately, for the same reason, it is difficult
to resolve the precise locations of the sex-linked QTL.
Based on the visual analysis of their segregation, we can
predict certain Cumana-derived alleles affecting quantitative colour traits (1, 2, 3 and 9 in figure 2b) to be in
tight linkage with the Sex locus. Not all of these mapped to
the expected region in the present QTL analysis, due
to the absence of Y-linked markers.
The multiple genes detected for most of the traits
mapped in this study probably reflect the complex nature
of the traits, and indicate that their final expression
may require a range of biological pathways and involve
products of many genes. Furthermore, there will undoubtedly be additional loci, as a single mapping cross does not
generally segregate all of the quantitative loci affecting any
given trait (Mackay 1996).
The MQM mapping results for selected shape traits
and for areas of multiple colour traits suggest that multiple
QTL of minor effect (lower LOD scores) contribute to
Proc. R. Soc. B (2009)

each colour trait, while fewer large effect QTL with higher
LOD scores may explain the selected shape traits.
In summary, the guppy genetic map will allow efficient
use of the extensive genomic information available for
several reference teleost fishes. In the long term, it will help
to further our understanding of the genetic basis of
adaptive evolution in this species and to explore the
processes governing the evolution of genomes.
All experiments were performed in agreement with German
regulations for keeping lower vertebrates in the laboratory
(Regierungsprasidium Tubingen).
We wish to thank Felix Breden and David Reznick for
providing guppies, Jakob Hodyl, Bartosz Pieczara and
Gertrud Scheer for their help with photography and image
analysis, and Norman Warthmann for help with bioinformatics tools. Supported by a Gottfried Wilhelm Leibniz
Award ( DFG) to D.W. and the Max Planck Society.
Sequence data from this paper have been deposited with
GenBank Data Libraries under accession numbers
FH888484FH893712.

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