Copyright ? 2009 by the Genetics Society of America
Mapping Loci Associated With Tail Color and Sex Determination
in the Short-Lived Fish Nothobranchius furzeri
Dario Riccardo Valenzano,*,1Jeanette Kirschner,†,1Roarke A. Kamber,* Elisa Zhang,*
David Weber,†Alessandro Cellerino,‡Christoph Englert,§Matthias Platzer,†
Kathrin Reichwald†and Anne Brunet*,2
*Department of Genetics, Stanford University, Stanford, California 94305 and†Department of Genome Analysis,
‡Department of Biology of Aging, and§Department of Molecular Genetics, Leibniz Institute for
Age Research–Fritz Lipmann Institute, 07745 Jena, Germany
Manuscript received August 16, 2009
Accepted for publication September 21, 2009
The African fish Nothobranchius furzeri is the shortest-lived vertebrate species that can reproduce in
captivity, with a median life span of 9–11 weeks for the shortest-lived strain. Natural populations of N.
furzeri display differences in life span, aging biomarkers, behavior, and color, which make N. furzeri a
unique vertebrate system for studying the genetic basis of these traits. We mapped regions of the genome
involved in sex determination and tail color by genotyping microsatellite markers in the F2progeny of a
cross between a short-lived, yellow-tailed strain and a long-lived, red-tailed strain of N. furzeri. We identified
one region linked with the yellow/red tail color that maps close to melanocortin 1 receptor (mc1r), a gene
involved in pigmentation in several vertebrate species. Analysis of the segregation of sex-linked markers
revealed that N. furzeri has a genetic sex determination system with males as the heterogametic sex and
markedly reduced recombination in the male sex-determining region. Our results demonstrate that both
naturally-evolved pigmentation differences and sex determination in N. furzeri are controlled by simple
genetic mechanisms and set the stage for the molecular genetic dissection of factors underlying such
traits. The microsatellite-based linkage map we developed for N. furzeri will also facilitate analysis of the
genetic architecture of traits that characterize this group of vertebrates, including short life span and
adaptation to extreme environmental conditions.
populate ephemeral water pools that often undergo
complete desiccation during the dry season (Terzibasi
et al. 2008; Reichard et al. 2009; Wildekamp 2009).
Nothobranchius species tend to live in extreme habitats
and have evolved unique adaptations to harsh envi-
ronmental conditions, including extremely short life
cycles, resistance to a wide range of temperatures and
water salinity, embryonic development that does not
require the presence of water, and a developmental
diapause that allows embryos to survive for months in
dry conditions (Wourms 1972; Inglima et al. 1981;
Genade et al. 2005).
Nothobranchius furzeri is the shortest-lived species of
the Nothobranchius genus, with an intergeneration
time of 40 days, a median life span of 9–11 weeks, and
a maximum life span of 12–15 weeks for the shortest-
HE Nothobranchius fish species are present in
eastern and southeastern Africa, where they
lived strain GRZ (Valdesalici and Cellerino 2003;
Genade et al. 2005; Valenzano et al. 2006; Terzibasi
et al. 2008, 2009; Hartmann et al. 2009). Natural
populations of N. furzeri can vary substantially in
phenotypic traits. For example, N. furzeri strains derived
from Zimbabwe and northern Mozambique (e.g., GRZ)
exhibit a shorter life span than strains derived from
more humidareasinsouthern Mozambique(e.g.,MZM-
0403) under controlled conditions (Terzibasi et al.
2008). The extremely short life cycle of N. furzeri and
the presence of natural populations with phenotypic
variations make this species a promising model system
for studying aging and adult-specific traits, including
color and behavior.
The color pattern of the adult male tail differs among
N. furzeri strains. GRZ males show a yellow submarginal
band and a black marginal band (yellow morph)
whereas MZM-0403 males display a broad red band
(red morph) in the caudal fin (Figure 1A). This
dichromatism is present in natural populations of
N. furzeri (Terzibasi et al. 2008; Reichard et al. 2009).
Similar color polymorphism among males is also ob-
served in other species of Nothobranchius (Wildekamp
2009) and in other fish species, including guppies and
1These authors contributed equally to this work.
Stanford, CA 94305. E-mail: firstname.lastname@example.org
Genetics 183: 1385–1395 (December 2009)
cichlids (Hughes et al. 1999; Brooks and Endler 2001;
Maan et al. 2004). Differences in male color morphs
within the same species are associated with sexual
preference by females, different recognition by preda-
tors depending on the habitat, and differential suscep-
tibility to pathogens (Price et al. 2008), which could all
influence the evolution of this trait. Despite the wide-
spread variation in Nothobranchius coloration, the
genetic basis of this trait is unknown.
Genetic information on N. furzeri is still limited. The
N. furzeri genome is 1.6–1.9 Gb in size and is character-
ized by a high repeat content (45%) (Reichwald et al.
2009). N. furzeri has 19 chromosomes, but no morpho-
logically discernible sex chromosomes (Reichwald
et al. 2009). The sex determination system in N. furzeri
has not been characterized yet. Sex can be determined
either genetically or environmentally in fish (Volff
2005; Marshall Graves 2008). For example, medaka,
platyfish, guppy, and sticklebacks all have recently
evolved genetic sex-determining systems (Volff and
Schartl 2002; Peichel et al. 2004; Shapiro et al. 2009;
Tripathi et al. 2009a), whereas zebrafish do not have a
clear genetic basis of sex determination (von Hofsten
and Olsson 2005).
Genetic studies in N. furzeri would greatly benefit
However, to date there is no linkage map available for
N. furzeri or for any Nothobranchius species, although
linkage maps have been generated for fish of the same
order, e.g., Poecilia reticulata (guppy) and Xiphophorus
maculatus (platyfish) (Khoo et al. 2003; Walter et al.
2004; Tripathi et al. 2009b), and of the same superor-
der, e.g., Oryzias latipes (medaka) (Wada et al. 1995).
Here, we report a microsatellite-based linkage map for
N. furzeri using a genetic cross between the short-lived
yellow-tailed GRZ strain and the long-lived red-tailed
map the male-specific tail color trait on linkage group
to a region of the medaka genome that contains the
melanocortin 1 receptor (mc1r) gene, which isknown to play
a key role in vertebrate pigmentation. We identified a
sequence polymorphism in mc1r between the two strains
revealed that mc1r is located in close proximity to the
color locus, but that the sequence polymorphism is
probably not causative for the color difference. We also
males as the heterogametic sex. The sex-determining
specific suppression of recombination. Our findings will
be pivotal for the identification of the genetic determi-
nants of color in N. furzeri and for expanding our
knowledge about sex-determination mechanisms in ver-
tebrates. Due to the array of intraspecific phenotypic
differences displayed by the various populations of
N. furzeri, this linkage map will also be a key tool for
mapping phenotypic variation in this short-lived verte-
brate species, including differences in life span.
MATERIALS AND METHODS
Fish housing and husbandry: Fish were grown at 25? in a
centralized filtration water system at a density of two fish per
gallon tank. Fish were fed freshly hatched Artemia brine
shrimp until 3 weeks of age and then dried Chironomid
in the system. Tanks were inspected daily, and freshly laid
embryos were collected and stored in dry peat moss until they
were ready to hatch, as indicated by spontaneous twitching
inside the eggshell. Once ready to hatch, embryos were
immersed in Yamamoto embryo solution (17 mm NaCl,
2.7 mm KCl, 2.5 mm CaCl2, 0.02 mm NaHCO3, pH 7.3)
(Rembold et al. 2006). Fry were placed in 0.2-gallon tanks at
the density of five fry per tank.
Cross between two strains of N. furzeri: One male from the
GRZ strain was crossed with one female from the MZM-0403
strain (cross 1). From the F1 generation (54 fish), nine
independent ‘‘families’’ (eight tanks with a spawning pair in
each tank and one tank with three males and four females
spawning together) were formed, and fertilized eggs were col-
lected. A total of 413 F2individuals were produced. An in-
MZM-0403 and one female GRZ (cross 2). Two F1individuals
from cross 2 produced a total of 34 F2individuals.
Color phenotyping: Fish were removed from tanks at death,
rinsed in tap water, and stored in 100% ethanol. Tail color
(yellow vs. red) was scored in all 203 F2adult males and all
23 F1males from cross 1 by visual inspection, immediately
preceding submersion in ethanol. N. furzeri male fish are
extremely colorful, and thus male color at death was clearly
visible. F1and F2fish with yellow tails overlaid with red spots
were scored as ‘‘yellow.’’
Sex phenotyping: Sex was assessed at death on the basis of
that had not yet reached sexual maturity at death were scored
Microsatellite identification by hybridization: Genomic
DNA was isolated from a 7-week-old MZM-0403 male and
digested with RsaI. Fragments (1000–1650 bp) were cloned
into the pCR-Blunt-TOPO vector (Invitrogen) and trans-
formed into chemically competent Escherichia coli. Transform-
ants were selected for the presence of CA/GT microsatellites
using a [32P]dCTP end-labeled (CA)15hybridization probe
(Elim Biopharmeceuticals) following a described method
(Peichel et al. 2001). Of the 773 positive clones analyzed,
318 contained microsatellites and 311 met the following
criteria: (i) at least 15 repeat units and (ii) at least 100 bp of
sequence flanking the microsatellite. To amplify these micro-
satellite repeats by PCR, primers that have melting temper-
atures between 57?–63? and that amplify fragments between
150 and 400 bp were designed using Primer3 (http:/ /frodo.
wi.mit.edu/). A 59 M13 sequence (59-TGTAAAACGACGGC
CAGT-39) was added to all forward primers to enable fluores-
cent labeling of fragments during PCR (Schuelke 2000).
Microsatellite identification by whole-genome sample
sequencing: Whole-genome sample sequencing of a male
specimen of the GRZ strain resulted in 5540 sequences
in silico analyses using Sputnik (http:/ /cbi.labri.fr/outils/
Pise/sputnik.html), 289 microsatellites that met the following
criteria were identified: (i) one microsatellite per sequence,
(ii) at least 20 repeat units for dinucleotide repeats, (iii) at
least 100 bp of microsatellite flanking sequence, and (iv)
1386D. R. Valenzano et al.
perfect repeats. Primers were designed using the GAP4
(Reichwald et al. 2009). One hundred and thirty-nine
microsatellites were experimentally validated by PCR and
subsequent genotyping using ABI3730xl analyzers and Gene-
Mapper software v4 (Applied Biosystems).
Genotyping: A total of 244 pairs of primers were used to
amplify microsatellites from the grandparents and 246 F2
offspring (160 males and 86 females) of cross 1. One hundred
and fifty-two microsatellite markers were informative for the
grandparents, 148 of which were used to genotype F2
individuals. Of the 246 individuals genotyped to build the
map, 234 were genotyped at all the markers whereas 12 were
genotyped only at the 121 markers corresponding to the four
terminal markers of each LG and to the singletons. PCRs were
performed in 5 ml in 384-well plates with 0.2 units Taq DNA
polymerase, 13 PCR buffer (50 mm KCl, 100 mm Tris–HCl,
pH 9.0, 0.1% Triton-X), 0.5 ng/ml DNA, 0.25 mm dNTP
(Invitrogen), 800 nm FAM56-labeled M13 primer, 10–20 nm
M13-forward primer, and 400–800 nm reverse primer. Samples
were heated to 94? for 5 min, followed by 30 cycles of 30 sec at
94?, 45sec at 56?, and 45sec at 72?and8 cycles of 30 sec at94?,
45 sec at 53?, and 45 sec at 72?. Amplicons were denatured by
incubation in denaturation solution (1:1.15 Hi-Di Formamide
(Applied Biosystems) and 1:300 GeneScan-500 LIZ Size Stan-
dard (Applied Biosystems)) at 95? for 5 min and electrophor-
esed on an ABI 3730 capillary sequencer. Chromatographs
were analyzed manually using PeakScanner software v1.0
Linkage map generation and map length calculation:
and Voorrips 2001). The Kosambi mapping function was
used to convert recombination frequencies (REC ¼ 0.4) to
centimorgans (cM). The map was also calculated using
Haldane’s function to account for double crossing-overs,
which gave similar results. The assignment of markers to LG
was carried out with a LOD score threshold of 4 and a
maximum linkage distance of 25 cM. The calculation of phase
cannot be exploited in this particular case, since parental (F1)
genotypes were tractable for only 96 of 246 F2individuals.
Raw data are available for download (supporting information,
The map length was computed by adding 2 3 s to each LG
length in centimorgans, where s is the average intermaker
distance, to account for chromosome ends, as described
(Tripathi et al. 2009b). This measure was averaged with the
measure obtained by multiplying each LG’s length in centi-
morgans by (m 1 1)/(m ? 1), where m is the number of
markers in each LG, as described (Tripathi et al. 2009b).
Mapping color: Color mapping was computed manually by
scoring the recombination events in all the F2red-tailed fish
(n ¼ 61) of cross 1, genotyped for all 148 microsatellites. Only
red-tailed F2fish were used to map color because the red
phenotype is more reliable than the yellow phenotype, as
yellow-tailed fish can develop red spots with advancing age
(see below). These genotypes were then further included in
the whole-genome map calculation. For each microsatellite
marker, the presence of two, one, or none of the two alleles
inherited from the red-tailed MZM-0403 grandparent was
scored. The LOD score for each marker was calculated ac-
cording to the following formula: LOD ¼ M 3 LOD10(m) 1
(N ? M) 3 LOD10(1 ? m) ? N 3 LOD10(0.5), where M is the
number of recombination events, m is the fraction of re-
combination events over all the alleles (m ¼ M/N), N is twice
the number of genotyped individuals (corresponding to the
total number of alleles genotyped), and LOD10 is the 10-base
logarithm. Considering the color marker position as the
position at which all red individuals have both MZM-0403
alleles, m 3 100 corresponds to the distance in centimorgans
of each marker from the color gene. This analysis was
performed over all the markers in the map.
Synteny analysis: BLASTn searches were performed using
the flanking regions from all 11 microsatellites that cosegre-
gated with tail color as query against the medaka genome
(October 2005 MEDAKA1 assembly) at Ensembl (http:/ /www.
ensembl.org/Oryzias_latipes/Info/Index). The microsatellites
flanking regions having a P value ,10?5, and 100% sequence
identity over .22 bp was considered a significant hit. Search
sensitivity was set to ‘‘no optimization.’’
Cloning of mc1r: Total RNA was isolated from caudal fins of
three male adult individuals (two GRZ and one MZM-0403)
using Trizol (Invitrogen). cDNA was generated using MMLV
reverse transcriptase (Clontech), according to the manufac-
turer’s protocol. A 681-bp fragment of N. furzeri mc1r was
generated by PCR using primers derived from conserved mc1r
regions in medaka, stickleback, Takifugu, and Tetraodon
(forward primer: 59 GAA CCG CAA CCT GCA CTC 39; reverse
primer: 59 GGG TCG ATG AGC GAG TTA CA 39). A 1402-bp
and 39 untranslated regions was amplified by RACE PCR
(Clontech) and subcloned in the pCR 2.1-TOPO cloning
Identification and genotyping of mc1r sequence polymor-
phism: The mc1r 1402-bp region was amplified from the
grandparents of cross 1 (male GRZ and female MZM-0403),
cloned, and entirely sequenced. A sequence polymorphism
the mc1r coding sequence. This polymorphism was genotyped
in 61 F2fish from cross 1. For sequencing, a 312-bp region
flanking nucleotide 67 of mc1r was amplified by PCR using the
following primers (forward primer: 59 GTG GAC CCC TGC
TTT AAT GA 39; reverse primer: 59 TAG TAC ATG GGC GAG
TGC AG 39). The PCR products were purified using a PCR
purification kit (Qiagen) and sequenced using Molecular
Cloning Laboratories (http:/ /www.mclab.com). Sequences
were analyzed using Sequencher 4.7 (Gene Codes).
Mapping sex: The genotypes at 148 microsatellites of 239 F2
individuals from cross 1 were sorted by sex (female, male,
unknown) to isolate the markers carrying a significant sex-
biased allele distribution. To confirm these results, F2individ-
uals from cross 2 (34 individuals) were also genotyped at 9
sex-linked microsatellites identified in cross 1 and sorted by
sex (female, male, unknown). The presence of potentially sup-
pressed recombination in the male vs. female sex-determining
region was determined by scoring recombination events in
two specific F1families of cross 1 (family 3 and family 7), in
which the two parental pairs were both heterozygous at all the
sex-linked loci. The sex-specific recombination events were
determined by assessing the sex-specific allele distribution in
the offspring of these two F1families (22 F2individuals for
family 3 and 46 F2individuals for family 7—68 F2individuals
total) at the sex-linked markers that were heterozygous for
both F1parents and that had more than two alleles.
Microsatellite-based linkage map for N. furzeri: To
develop genetic markers for linkage mapping in N.
furzeri, we identified microsatellites using two strategies
of large-scale genomic library screening and sequenc-
from MZM-0403, a wild-derived strain of N. furzeri with a
broad red marginal band in the caudal fin (‘‘red
morph’’), by using a (CA)15probe because CA micro-
Tail Color and Sex Determination in N. furzeri
satellite repeats are frequent in fish genomes. We
sequenced 773 positive clones and identified 318 clones
containing microsatellites, 105 of which gave rise to a
PCR product. In the second approach, we performed
whole-genome sample sequencing of GRZ, which has a
black marginal band and a yellow submarginal band in
the caudal fin (‘‘yellow morph’’) (Reichwald et al.
2009). We identified 289 clones containing microsatel-
lites by in silico analysis using Sputnik (http:/ /cbi.labri.
fr/outils/Pise/sputnik.html),139 of which were experi-
To generate a linkage map, we set up a cross between
one long-lived red-tailed MZM-0403 female and one
short-lived yellow-tailed GRZ male (Figure 1A). We
obtained 54 F1progeny, which were used to form nine
families (Figure 1B). A total of 413 F2progeny from
these F1families developed into adulthood (Figure 1B).
We genotyped 244 microsatellite markers in the grand-
parents and found that 152 (62%) were polymorphic
and thus informative for building a genetic map (Table
S1). All microsatellite markers were homozygous in the
GRZ male grandparent, except those linked with sex
(see below), confirming that GRZ is an inbred strain
(Reichwald et al. 2009). Forty-one percent of the
microsatellite markers (63 of 152) were heterozygous
in the MZM-0403 female grandparent, which is consis-
tent with this strain being recentlyderived from the wild
and propagated in captivity for no more than seven
generations (Terzibasi et al. 2008; Hartmann et al.
To build the linkage map, we genotyped 246 F2fish at
148 of the 152 informative microsatellite markers (see
materials and methods). Significant linkage was
found for 138 (93%) microsatellite markers, and 10
markers were singletons. Six of the 138 linked markers
were excluded from the map calculation because they
gave incomplete genotypes in .50% of the individuals.
The resulting N. furzeri linkage map consists of 25 LGs,
only 19 chromosomes (Reichwald et al. 2009), we
anticipate that some of the linkage groups will coalesce
when additional markers are included. The total calcu-
lated map length is 1012 cM, with an average inter-
marker distance of 5.3 cM. Considering 25 cM as the
maximum intermarker distance, 10 singletons, and 6
more LGs than chromosomes, we estimate that up to
400 cM could still not be accounted for in this map
(25 3 10 1 25 3 6 ¼ 400 cM), corresponding to 28% of
the N. furzeri genome [400/(400 1 1012)]. Thus, we
have generated a first-generation microsatellite-based
linkage map for N. furzeri that can be used to map
phenotypic variation between the populations of this
Mapping tail color on LG V in N. furzeri: A conspic-
uous difference between the GRZ and MZM-0403
strains of N. furzeri is the color and pattern of the caudal
fin in males (Figure 1A, Figure 3A). To examine the
genetic basis of this dichromatism, we scored all males
in the F1and F2generations for a ‘‘yellow’’ vs. a ‘‘red’’
caudal fin. All F1males display a yellow morph, in-
dicating that yellow is dominant over red (Figure 3A).
We also observed the progressive appearance of red
spots in the caudal fin of adult F1 male fish with
advancing age (data not shown). In the 203 males of
the F2generation, the yellow/red tail-color trait segre-
gated as 142 yellow (yellow and yellow with red spots)
and 61 red (Figure 3A). The ratio betweenthe two color
morphs is close to 3:1 (x1
suggests Mendelian transmission.
To map the yellow/red tail-color trait, we genotyped
We found a strong linkage signal on LG V (Figure 3B,
Table 1). The peak LOD score corresponds to the
map resolution, the NfuFLI0030 marker is indistin-
guishable from the color locus; i.e., all red males in
the F2generation inherited both alleles from the red
MZM-0403 grandparent. These results indicate that the
yellow/red tail-color trait is determined mainly by one
locus, although we cannot exclude that multiple linked
loci contribute to this color trait or that other minor
unlinked loci may influence the trait.
We sought to identify candidate genes that underlie
the tail-color trait in N. furzeri. Synteny with other spe-
cies can be used to infer the position of genes on a link-
age map of a species without a sequenced genome
(Gross et al. 2008). We performed a BLASTn search in
the 11 microsatellite markers of LG V as queries, since
2¼ 2.76, P ¼ 0.09), which
Figure 1.—Cross between two strains of
N. furzeri that differ in color and life span (A)
Color phenotypes of GRZ and MZM-0403. (B)
A yellow-tailed, short-lived male GRZ and a red-
tailed, long-lived female MZM-0403 were the
founders of cross 1.
1388D. R. Valenzano et al.
to N. furzeri (Reichwald et al. 2009). Markers Nfu-
FLI0122, NfuSU0050, and NfuSU0046 displayed signifi-
muscle actin, orthologous to human ACTC1; P , 10?17),
sox6 (sry box containing gene 6; P , 10?13), and cttn
(cortactin; P , 10?11) on medaka chromosome 3 (Fig-
ure 3B). In addition, NfuFLI0122, NfuSU0050, and
NfuSU0046 are in the same order on N. furzeri LG V as
their respective counterparts on medaka chromosome
3 (Figure 3B). Together, these results suggest that there
is synteny and colinearity between N. furzeri LG V and
medaka chromosome 3.
hair pigmentation in other vertebrate species are pre-
sent in the region that is syntenic to N. furzeri LG V on
medaka chromosome 3 (Figure 3B). The genes that we
specifically examined were the following: melanocortin
1 receptor (Mc1r) and its ligand (Asip) (Rees 2003;
Figure 2.—Microsatellite-based genetic linkage map of N. furzeri. Each linkage group is designated by a Roman numeral and
ordered on the basis of genetic length. The map distance in centimorgans is reported on the left side of each linkage group.
Microsatellite loci identified at Stanford University were labeled as SU followed by a four-digit number based on their order
of identification. Microsatellite loci identified at the Fritz Lipmann Institute were labeled as FLI followed by a four-digit number.
In the text, these markers are termed NfuSU or NfuFLI followed by a four-digit number, with Nfu standing for N. furzeri.
Tail Color and Sex Determination in N. furzeri
Shriver et al. 2003; Hoekstra 2006; Sulem et al. 2008;
LePapeet al.2009),theKitreceptorand itsligand(Kitl)
(Geissler et al. 1988; Miller et al. 2007; Sulem et al.
2007), CBD103 (Candille et al. 2007), MATP/SLC45A2
(Norton et al. 2007), SLC24A4 (Sulem et al. 2007),
SLC24A5 (Lamason et al. 2005), IRF4 (Sulem et al.
2007; Han et al. 2008), TPCN2 (Bonilla et al. 2005;
Sulem et al. 2008), Sly (Alizadeh et al. 2009), oca2
(Shriver et al. 2003; Protas and Patel 2008), and TYR
(Shriver et al. 2003; Sulem et al. 2007). Of all these
genes, only mc1r and slc24a5 were located on the me-
daka chromosome syntenic to N. furzeri LG V. Con-
versely, the annotated genes on medaka chromosome 3
do not include any other genes known to be associated
with color determination, although this does not rule
out their existence. In medaka, mc1r is located 2.6 Mb
from olma1, the marker that is syntenic with Nfu-
FLI0122, whereas slc24a5 is located outside of the me-
daka region that is syntenic to the region linked with
color in N. furzeri (Figure 3B). Together, these results
suggest that mc1r is a better functional candidate for tail
color than slc24a5 in N. furzeri.
To determine if mc1r is the gene underlying male tail
color, we cloned N. furzeri mc1r cDNA and mapped mc1r
on the linkage map. Sequence comparison of GRZ and
MZM-0403, the strains used in our cross, revealed a
single nucleotide polymorphism at position 67 of the
mc1r coding sequence (C in GRZ and G in MZM-0403)
(Figure S1; GenBank GQ463613). This variation is a
nonsynonymous substitution that results in a change
from histidine (GRZ) to aspartic acid (MZM-0403) at
amino acid 23 in the N-terminal region of the Mc1r
protein (Figure S1). Sequencing three additional speci-
mens of each strain confirmed that MZM-0403 individ-
uals were homozygous for G, and GRZ individuals were
homozygous for C at position 67 of the mc1r coding
sequence. The segregation of this mc1r polymorphism
in 61 F2fish allowed us to place mc1r on LG V, the LG
Figure 3.—Genetics of
the tail color in N. furzeri
males. (A) Transmission of
the color trait in the F1
and F2 generations. A x2
test shows that the ratio is
close to 3:1. Yellow tails al-
ways have black vertical
bars. Red tails can be with
(bottom) or without (top)
black vertical bars. (B) LG
V contains the color locus,
which corresponds to mark-
er NfuFLI0030 (FLI0030).
The expected map location
of N. furzeri mc1r is indi-
cated in bold. The distance
in centimorgans is reported
on the left side of LG V.
NfuFLI0008 because there
is stronger linkage among
NfuFLI0122 than between
mc1r and NfuFLI0008 and
because mc1r is 4 cM from
NfuFLI0122 and 2 cM from
NfuFLI0030. Note that the
distances in centimorgans
NfuFLI0008 do not add
up because the distances
between mc1r and each of
these three microsatellite
markers were calculated independently. Dashed lines indicate the synteny of microsatellite markers on N. furzeri LG V and an-
notated genes on medaka chromosome 3. The distance in megabases is reported on the right side of medaka chromosome 3.
(C) LOD score plot of the color locus region. The LOD score is undetermined at marker NfuFLI0030 (FLI0030) due to lack
of recombination between this marker and the color locus. Therefore the peak at NfuFLI0030 is represented by two short hor-
izontal lines. slc24a5 was not directly mapped and therefore is represented by a horizontal line between marker NfuSU0050
(SU0050) and NfuSU0046 (SU0046).
1390 D. R. Valenzano et al.
that contains the color locus (Figure 3B). However,
there was one recombinant red-tailed F2fish with a GC
genotype, as well as three recombinant F2yellow-tailed
and these genotypes were independently confirmed.
These results indicate that mc1r is closely linked to the
color locus, but 1.7 cM away from it. The marker to
which mc1r is most closely linked is NfuFLI0030 (Figure
3B). The distances between mc1r and NfuFLI0030
(2 cM) and between mc1r and the color locus (1.7 cM)
are not the same, because the former was calculated in
both male and female F2individuals whereas the latter
was calculated only in red F2males. The distance be-
tween mc1r and the color locus indicates that the Mc1r
amino acid difference is unlikely to be causal for the
yellow/red color determination in N. furzeri, although
we cannot rule out the involvement of cis-acting ele-
ments for the mc1r gene in the determination of color.
An alternative possibility is that another gene in close
proximity to mc1r is involved in color determination in
A sex determination system on linkage group XIII in
N. furzeri: Sex is another phenotype that differs between
the two grandparents of our cross. To establish the
sex determination system in N. furzeri, we counted the
number of males and females in the F1and F2gener-
4A), consistent with a genetic sex determination system.
2¼ 0.184, P ¼ 0.668; F2: x1
2¼1.64, P ¼ 0.2) (Figure
or in females. We identified nine microsatellite markers
these makers (NfuSU0004, NfuSU0007, NfuSU0010,
NfuSU0090, and NfuFLI0091) show a significant male-
specific allelic bias in that most of the F2males carry a
combination of a fixed, male-specific allele and a non-
fixed, non-sexually-biased allele (Table 2). Importantly,
the male-biased markers are the only ones for which the
male grandparent (GRZ) is heterozygous. These results
strongly suggest that male is the heterogametic sex in
N. furzeri and that the sex determination system is XY/
XX. To analyze whether the male-specific allelic bias is
shared by different N. furzeri strains, we genotyped the
sex-linked markers in the F2progeny (20 males and
14 females) of a reciprocal cross of a male MZM-0403
and a female GRZ (cross 2) and found a male-allelic bias
at the same loci (Table 2). Together, these results indi-
cate the presence in N. furzeri of a conserved haplotype
for the male sex chromosome with marked sex-linkage
Chromosomal regions carrying a sex-determining
locus are characterized by suppressed meiotic recombi-
nation (Charlesworth 2004; Marshall Graves
2008). To test whether recombination is suppressed at
the sex-linked markers in N. furzeri, we independently
calculated male and female meiotic recombination
frequencies in 68 F2offspring from two F1families of
our first cross (cross 1). We performed this analysis in F2
individuals instead of F1individuals because the P0
F1females were heterozygous at the sex-linked loci,
except at marker NfuSU0004, which was therefore
excluded from the analysis. In F2progeny from each
ofthe twoF1families, there wasno recombination event
for markers NfuSU0090, NfuSU0007, NfuSU0010, and
NfuFLI0091 in males, whereas females displayed a total
of six recombination events, which account for a map
distance of 2 cM for family 3 and 9 cM for family 7 at
markers NfuSU0007 and NfuSU0090, respectively (Fig-
ure 4B). This result indicates that recombination is
largely suppressed in males in the sex-linked region of
LG XIII and further supports the conclusion that male
is the heterogametic sex in N. furzeri.
To determine if the sex-linked markers in N. furzeri
have similarities with sex-determining regions in other
fish species, we searched for synteny between the nine
microsatellite markers on LG XIII on N. furzeri and
medaka chromosomes. Marker NfuSU0015 on LG XIII
shows significant homology with a region on medaka
chromosome 16 (BLASTn P ¼ 1.5 3 10?7), although
there is no annotated gene in this region. Interestingly,
chromosome 16 corresponds to the sex chromosome in
one medaka species(Oryzias javanicus) (Takehana et al.
2008), raising the interesting possibility that sex de-
termination in N. furzeri and O. javanicus might have
evolved from a common system. However, N. furzeri LG
XIII is not syntenic with the sex-determining chromo-
Genotype frequencies of microsatellite markers on the
color locus containing LG V
Genotype frequencies for 11 markers and for mc1r on LG V
in 61 F2red-tailed males. The number of red individuals is
presented. Genotype frequencies are presented as percen-
tages in parentheses. Only complete genotypes with both al-
leles present were reported; therefore, percentage values do
not add up to 100.
aMarker corresponding to the LOD peak.
Tail Color and Sex Determination in N. furzeri
some in another species of medaka (Oryzias latipes)
(Matsuda et al. 2002; Kasahara et al. 2007) and with
the sex-determining LGs in two stickleback species
(Peichel et al. 2004; Ross and Peichel 2008; Shapiro
et al. 2009), suggesting that the sex-determination
system of N. furzeri probably arose independently of
Linkage map in N. furzeri: Our first-generation
microsatellite-based linkage map consists of 25 LGs.
The number of LGs is higher than the number of
chromosomes visible in a metaphase spread (19)
(Reichwald et al. 2009). First-generation linkage maps
LGs tend to collapse if more markers are added
(Ohtsuka et al. 1999; Naruse et al. 2000). Thus, it is
likely that the current LG number will eventually
collapse to 19 if additional markers/meioses are added.
Our analyses also show that the phylogenetic relation-
ship between N. furzeri and medaka allows the use of
sequence similarity and synteny to predict the location
of specific genes on the N. furzeri map. The synteny
between medaka and N. furzeri is likely to be high, given
that medaka and the more distant stickleback genomes
500.html). A high level of synteny will be particularly
useful in identifying candidate genes underlying spe-
cific traits, as was recently described for the cave fish
Astyanax mexicanus (Gross et al. 2008, 2009).
Color determination in N. furzeri: Our linkage map
allowed us to identify a locus linked with male yellow/
red tail color on LG V in N. furzeri. Synteny analysis
between N. furzeri and medaka revealed a potential
candidate gene for color, mc1r (Rees 2003; Hoekstra
2006). The Mc1r protein is a G-protein-coupled re-
Figure 4.—Sex determination in N.
furzeri (A) The proportion of males
and females in the F1(23 males and
26 females) and F2 (203 males and
178 females) generations. The x2test
shows that the ratio is 1:1 (F1 x1
0.184, P ¼ 0.668; F2 x1
0.2). The individuals that could not
be phenotyped as males or females (un-
determined) are not reported; there-
fore the proportion of males and
females does not add up to 1. (B) Re-
combination scores for males and fe-
males at LG XIII for two F1families
(family 3 ¼ 22 individuals; family 7 ¼
46 individuals). In both families, F2
males do not show recombination at
the four sex-linked markers NfuSU0010
NfuSU0007 (SU0007), and NfuSU0090
(SU0090). In contrast, F2females show
2¼1.64, P ¼
recombination at markers NfuSU0007 (SU0007) and NfuSU0090 (SU0090). Note that F2females show recombination at different
markers in family 3 and family 7, which is likely due to the low number of individuals that were genotyped. This figure displays only
the markers on LG XIII that were heterozygous in the F1parents of family 3 and family 7 and therefore allowed the analysis of male
vs. female meioses. The partial representation of LG XIII is depicted by dotted lines. The map distance in centimorgans is re-
ported on the left of the LGs.
Genotype frequencies of sex-linked markers on the
sex-determining region on LG XIII
m1 allele (%) m2 allele (%) f allele (%)
Genotype frequencies of sex-linked markers on LG XIII in
160 males and 86 females (cross 1) and in 20 males and 14
females (cross 2). m1 and m2 are alleles derived from the
male grandparent. The m1 allele is exclusively present in F2
males. The f allele is derived from the female grandparent.
Cross 1: GRZ male crossed with MZM-0403 female. Cross 2:
MZM-0403 male crossed with GRZ female.
1392D. R. Valenzano et al.
ceptor for the Agouti ligand Asip. In mice, mutations in
either Mc1r or Asip result in changes in the pattern of
melanogenesis and in coat color (Jackson 1993) and
affect the relative amounts of eumelanin and pheome-
lanin in mammalian melanocytes (Andersson 2003).
In humans, MC1R mutations are associated with red
hair and fair skin (Valverde et al. 1995; Mundy 2009)
and with the presence of freckles and skin sensitivity to
sun (Sulem et al. 2007). However, while mc1r is impor-
tant for the brown pigmentation in different popula-
tions of the cave fish A. mexicanus (Gross et al. 2009),
whether mc1r plays any role in yellow/red pigmentation
in fish is unknown. In fact, fish pigmentation is funda-
mentally different from that of mammals because it
depends on several cell types, including xantophores
and erythrophores, which can synthesize yellow pig-
ments de novo (Parichy 2003; Braasch et al. 2007;
Protas and Patel 2008).
The red and yellow grandparents of our cross have a
nonsynonymous variation in the coding sequence of
mc1r, leading to a change in amino acid in the Mc1r
protein from an aspartic acid in the red strain to a
histidine in the yellow strain. However, this nonsynon-
ymouschange inMc1rdoesnot map exactlyat the color
locus, but 1.7 cM from it, likely ruling out its direct
functional implication in male coloration. This amino
acid change isinthe extracellularN-terminaldomain of
Mc1r, a region poorly conserved between different
teleost species and not essential for ligand binding
(Selz et al. 2007). This region does not contain any of
the known Mc1r mutations that have previously been
found to be causative for color differences in other
species (Selz et al. 2007). Thus, at this point, we
consider unlikely an implication of mc1r in fish yel-
low/red color determination, although we cannot
exclude a potential involvement of mc1r via cis-regula-
tion. It is more likely that another gene located in the
same region is responsible for color determination in
N. furzeri. Fine mapping will reveal whether color de-
termination in N. furzeri is mediated by mc1r or by
Mapping the color locus will provide important cues
In the genus Nothobranchius, coloration is specific to
males and is likely shaped by female mate selection. For
example, in Nothobranchius guentheri, females prefer
conspecific males on the basis of color (Haas 1976).
More intensely colored males are preferred over less
intensely colored ones (Haas 1976). However, a bright
coloration also renders males more conspicuous to
predators and likely comes with a survival cost, in line
with the observation that, in the wild, the sex ratio for
this species is biased toward females, which are not
between the fitness advantage due to mate preference
and the fitness costs due to predation may underlie the
rate of evolution of male coloration in this species.
When we identify a gene or regulatory region underly-
ing the difference in color in N. furzeri, it will be
interesting to test whether natural populations of N.
furzeri, as well as different species of the genus Notho-
branchius, harbor polymorphisms in this region.
Sex determination in N. furzeri: Sex can be de-
termined by mechanisms that are genetic, environmen-
tal, or a combination of both (Volff 2005; Marshall
Graves 2008). Environmental factors that control sex
determination in fish species include water tempera-
ture, density, and social interactions. Genetic control of
sex determination is governed by the presence of sex
chromosomes (visible sex chromosomes or heteromor-
phic chromosomes) that can be present either in males
(XY) or in females (ZW). We found that N. furzeri has a
genetic sex-determination system, with males as the
heterogametic sex, indicative of an XY/XX system. The
male sex-determining region in N. furzeri harbors a
nonrecombining region, similar to that in medaka,
guppies, platyfish, and sticklebacks (Kondo et al. 2001;
Matsuda et al. 2002; Nanda et al. 2002; Volff and
Schartl 2002; Peichel et al. 2004; Schultheis et al.
2006; Ross and Peichel 2008; Shapiro et al. 2009;
Tripathi et al. 2009a). In line with sticklebacks and
et al. 2009), the sex linkage group in N. furzeri does show
major differences in recombination rates when com-
puted independently for males and females, and N.
furzeri males consistently share a sex haplotype. Synteny
analysis between N. furzeri sex-linked markers and the
known medaka and stickleback sex chromosomes re-
vealed that the sex-linked LG in N. furzeri (LG XIII) is
syntenic with the O. javanicus sex chromosome (chro-
the sex-determining regions in these two different
species were derived from the same ancestral chromo-
some or arose independently from one another.
Color and sex determination in N. furzeri: Genes
underlying sexually attractive traits, such as bright
coloration, are often located on sex chromosomes
(Lindholm and Breden 2002). In guppies, many of
the male color traits (dorsal fin black, central blue
white spot, anterior orange spot, etc.) map to the sex
chromosome, although other color traits map to auto-
somes (Lindholm and Breden 2002; Tripathi et al.
2008, 2009b). Similarly, in platyfish (Xiphophorus mac-
ulatus), a number of color traits (iris color, body colors,
fin color) map to sex chromosomes, while others (black
comets on the fin) map to autosomes (Basolo 2006).
Having color and sex linked may help maintain sexual
dimorphism and provide an evolutionary advantage. In
N. furzeri, malecolordoesnot mapto the LGcontaining
the sex-determining region, at least in our first-genera-
tion linkage map. This could be due to the incomplete-
ness of the map: it is conceivable that LG Vand LG XIII
would mergeifadditionalmarkers/specimensare used.
Alternatively, color and sex-determining regions may
Tail Color and Sex Determination in N. furzeri
segregate independently in N. furzeri, perhaps because
the evolutionary advantage of linking male tail color
and sex has not emerged yet in this species.
Life span, color, and sex determination in N. furzeri:
A major advantage of developing N. furzeri as a model
system is its short life span and the presence of natural
populations with differences in mean and maximal life
loci (QTL) for longevity because a large number of F2
fish died prematurely because of the unexpected
presence of the parasite Glugea sp. in the fish housing
suggest that there is no simple link between the yellow/
red color and longevity or between life span and sex
(data not shown). Additional crosses performed in
controlled environmental conditions will be needed
for the mapping of QTL for longevity in N. furzeri.
Concluding remarks: Our study reports the genera-
tion of the first linkage microsatellite-based map for the
short-lived fish N. furzeri. This genetic tool allowed us to
identify a single locus linked with color determination
in males, as well as to reveal the sex-determination
system of this species. This map will also be of key
importance in determining the genetic architecture of
other traits that characterize this unique group of
organisms, particularly differences in life span.
We thank David Kingsley and Greg Barsh for intellectual input on
the project. We thank Steve Arnott from the Kingsley lab for guidance
Michael Shapiro for helpful suggestions throughout this project. We
thank Sabrina Fullhart for her help with fish husbandry and fish
pictures. We thank Tom Hofmann for expert technical assistance. We
are grateful to the Kingsley lab for the use of the ABI sequencer. We
thank members of the Brunet lab, as well as Steve Arnott, Greg Barsh,
David Kingsley, Craig Miller, Katie Peichel, Dmitri Petrov, and Michael
Shapiro for critically reading the manuscript. We thank Martin
Reichard for communication of his findings on N. furzeri color in
the wild. This work was supported by an R21 grant from National
fellowship from the Stanford Center on Longevity (D.R.V.), and by a
pact for research and innovation of the Joint Science Conference of
the federal and La ¨nder governments from the Leibniz-Gemeinschaft,
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JoinMap 3.0, Software for
Sex determination and sex
Zebrafish sex determina-
Communicating editor: T. R. Magnuson
Tail Color and Sex Determination in N. furzeri
Supporting Information Supporting Information
Mapping Loci Associated with Tail Color and Sex Determination
in the Short-Lived Fish Nothobranchius furzeri
Dario Ricardo Valenzano, Jeanette Kirschner, Roarke A. Kamber, Elisa Zhang,
David Weber, Alessandro Cellerino, Christoph Englert, Matthias Platzer,
Kathrin Reichwald and Anne Brunet
Copyright © 2009 by the Genetics Society of America
D. R. Valenzano et al. 2 SI
N. furzeri mc1r coding sequence
TAC AGT CCG CTG GGG GAC TAC
TAC AGT CCG CTG GGG
5’ UTR3’ UTR
FIGURE S1.—N. furzeri mc1r sequence polymorphism and resulting amino acid change in Mc1r protein in
MZM-0403 and GRZ. UTR: untranslated region.
D. R. Valenzano et al. 3 SI
Informative 152 microsatellite markers used to generate N. furzeri linkage map.
repeat Forward primer Reverse primer
NfuSU0001 CA GGGTGTATGGCAGCAAGATT TGCACTGCAAATCTGAAGGA 211-247
NfuSU0002 CA CACAAGCAAGGTGAAGTCCA GCGACACTCAAGCCTCCTAC 250-258
NfuSU0003 CA CTCCTTTGTCTGGCTTCTGG TGAAGCGGTTTGAAGTTTTG 250-267
NfuSU0004 GAA AATGAAAAGCATCACTGCACA TTTTGTGAAGTGCCTCGTGA 195-207
NfuSU0005 CA AAGGTTCAACCACTCCATCG AGGACAGAAGGCTGCATGTT 208-295
NfuSU0006 CA CTGACGTGCTGCGTCTCTAC GGCATTTTGCCACACATAAA 186-215
NfuSU0007 CA GGCTAAGCCTTGCTGACAGA CAGGGAGCTGAAAACCTCAG 166-214
NfuSU0008 CA GCAAACTCGCTAGCTTGGAT CACCGGCATGATCAGTGTTA 293-301
NfuSU0009 CA TTGGACATGTGGACGTGACT GAAACCTTCAGCATTTGAGCA 217-262
NfuSU0010 CA CGCAGTCTGATCAAATCGTGT TGTTTGAAGGTTCACATTCATTATC 220-272
NfuSU0011 CA AGGAATCAGGCTCAGAGCAA AGCTGAGGATGGTGTTTTGG 215-257
NfuSU0012 CA CCCACAGCGGATTAACAGAG GTTATTGACCAAGGCGAGGA 211-267
NfuSU0013 CA GGGGAGTGCTGTAGGACAAC ACCTGCATTTGCCAGGTTA 606-655
NfuSU0014 CA CATCAATGATCCCTGGCTCT GATGAAGGCTTTCCGTTCAC 214-222
NfuSU0015 CA CTGAAGGGACCTCAGCACTC CGAGTGAAACTGCTCACCTCT 214-245
NfuSU0016 CA CATGGCTAAACCGTGATGAA GAAGGACGCCAGCTATGAAG 209-240
NfuSU0017 CA CCTCTTCTCCCAACGAACAC TGCATCAGCTTCATTTCTGG 240-261
NfuSU0018 CA GTGGTTTTGGCTGTTCTCGT GAAGAGGAACAAAGGAGGGTTT 274-338
NfuSU0019 CA TTCAAGCAAATGCAGGACAG GCAGGCATGCAATCTTACAG 173-224
NfuSU0020 CA GCCTTCTCCTGTGCACTTTC GCTTCTCCTCTTTTGCGAGA 571-642
NfuSU0021 CA GGGTTAGCACATGGTCGAGT CAACGACTTTGGAGTTGCATT 181-210
NfuSU0022 CA AACACAGCTCTCGTAAGGAGGTA TTCAGACTTGTCTTACTACCATGTTT 198-238
NfuSU0023 CA TGCATTAAAACGCGTTAATCTT CGTTTTCTCCTGTCTTCTGTCC 203-245
NfuSU0024 CA TTCAGCAGCTGTTCACCATC ATGAATTGCATGTTCCCACA 172-226
NfuSU0025 CA TGAATGCCACCTTCTCTCCT CGTCCTGAGCAGAGTTTGAA 183-214
NfuSU0026 CA GCCAGTAATCGAAAGGTTGC AGCTCCTTTCAGCGTCAGAG 261-309
NfuSU0027 CA TCCAGCTGAATCGGTAATGA AAACTCGAGGGTGCAATCTG 164-226
NfuSU0028 CA GCCAGGAAGCAATAAAACCA CAGAACTGATAGCAACGTGAGAA 174-194
NfuSU0031 CA CTGAACAAGCTCCCCAATGT CATGTTTTATGCCCTGCATTT 121-219
NfuSU0032 CA GCCCCACACATCACTTTTCT CCAGAGCCACAAAGACACAA 226-268
NfuSU0033 CA ATTAGGACCGGGTTTTGGTC CCTTTTTCCTCTTGCTTTCG 211-235
NfuSU0034 CA CATTCCTTTGGATGCCATTT CAGGTGCAGAGCTGTCTGAG 310-320
NfuSU0035 CA GCTGCGGTTATTCCTCTGTC ATGAAACTGGCCACACCAAT 240-264
NfuSU0036 CA-GA TGCTTTGTGTGAGCATGTGA CTGGACAGCACTGGGAAGTT 259-298
NfuSU0038 CA TCTCCGTGGTCAAGTCACAG TCCCAAGCAGATCTGGAAAG 118-171
NfuSU0039 CA ATGGCATTTTTCATGGAAGC CAGATGGGTCAACAGGCTTT 202-210
D. R. Valenzano et al. 4 SI Download full-text
NfuSU0040 CA TAAGCACAATCCGCCTCTTT GCCCCTTCAACTCATGTCAC 171-203
NfuSU0042 CA CACACAGAAATGAGGGCAAA GCTGGTATTGCAACAGGACA 222-229
NfuSU0043 CA GATGGCACACACACACAACA GCCTGAGTCTGGTTTGCATT 218-226
NfuSU0044 CA TGTGGCAGCTTTAATGAGGA AACCCAGCTCTGACACTTGTT 608-651
NfuSU0045 CA TGTCTGGATGTGGATGGAGA TACTGGACATGCTGGTCTGG 208-216
NfuSU0046 CA GCCAAACTTAAATAATAAAACTAGGG GTCATCACGTTCACGCATTT 210-226
NfuSU0048 CA TTCTGTTGGTTGCAGAGACG AGATCCACCTGTGCCGTTAC 441-444
NfuSU0049 CA CTGGACAAAGTGCCAATCAC CTCCCACAGTCCCAAAACAT 196-197
NfuSU0050 CA CCAGAATGAACAATACTCAGATCAA GCAGCTTAGTTTAATGATATCACAATG 252-295
NfuSU0052 CA GGGCCAAATTCAAACACATT AGGGGGCTTGTTGATTTTGT 226-236
NfuSU0053 CA GCCGGCTGAAATTACACCTA TGACAGCAAAACCAGGCTAA 256-264
NfuSU0054 CA CAAGACTTTACAGTGTGTGCTTTTG GTCGGACATTACCCCTGCTA 157-174
NfuSU0055 CA GCAGTCATAAGCAGTCTTTTGG TCTTTCCCCCAAAATTCAAA 172-192
NfuSU0056 CA GCAAGGCCTGATGTTGATCT TATGTCAGCAACCCTGGTAGG 160-188
NfuSU0057 CA GCAGGATCGCTCATTACCAT TCCAAGGAAGCGTATTTTCA 337-383
NfuSU0058 CA CTTTTCCCCTCCTTCCTCTG ACTAGCGGCTTGTTTCTCCA 208-255
NfuSU0059 CA GCTGCAACGCAATAGTTTCA AAAGGCAGAGTTGGTCGAAA 220-230
NfuSU0060 CA CTAGCCACTCCCCTGGTTTA CCGTCACGATGTGCTGATAC 216-248
NfuSU0061 CA TGAAGGGAAAGCAGGTGACT CCTCAGGTCTGGCATTCATT 242-255
NfuSU0062 CA GGGCTTTTAATTCCCCTCAT TTCAACCAATTACATCAGTTTGTG 248-258
NfuSU0063 CA TGTCTCCAGATGCAGAGGAA TGCATCATTAGACAGCATAACG 253-263
NfuSU0064 CA CACACTGATGAATCGCATCC CAGCCCAAAATAACCCTCAA 242-257
NfuSU0065 CA ATAACCTAGGCCAGGGAGGA TGTCATGTGCAGACACATCC 176-187
NfuSU0066 CA GCCAGTAATCGAAAGGTTGC AGTTTGTCCCAGGAAGCGTA 447-495
NfuSU0067 A CATGTCCAGCCTCAGAGTGA AGGATACGGACCCTCGAAGA 208-211
NfuSU0068 CA TCACTGGGGATGGAGAAGAC GCTGCTAAATTCCTGCATCA 224-261
NfuSU0069 A AGTGGGGAAAGGGGTAACAG GCAAAACAGAAATATATGAAAAACCA 252-254
NfuSU0070 CA TTGTCCCCTACCAACGCTAA AAACCGTGCGATTTGCTTTA 380-419
NfuSU0071 CA CATAAACCCCACGCTGAGTC CAGAGAAACTTTTGCTGCACAC 254-258
NfuSU0072 CA CTGGTTCAGAGTCCGAGAGC CATGGAGAATGCAGAGTTTCC 210-237
NfuSU0073 CA GCCACTGAATTCCTCAGCTC CCAGCAAAGGCTAGCTTGAA 242-274
NfuSU0074 CA AAATGTGACGCCAAACCTTC CCAACATAGCATCACGGTTG 189-194
NfuSU0075 TAA TTGCTACAAGGCAACAGCTC AAGCAAATATGATTTACCTACAAGAAA 265-388
NfuSU0076 CA CAGCAAGAAAGTTAATTCTGACGA AGGCGCTTTGCAATACAGTT 202-220
NfuSU0077 CA GTTCGGGTGAGAGAGCAGAC CAGTTATAGCTCCGCCCATT 193-269
NfuSU0078 CA GCAGCAATAAGGAATCATCCA TGGTGATGACCGATCACAGT 188-201
NfuSU0079 CA TTGCTTCCAAATCCTAAATCC CACATTGATGTGACGTTTGTGA 296-306
NfuSU0080 CA ATCCTTTCCTCCCTCTCTGC AAAGCAGACCTTGGTGTCACT 175-239
NfuSU0082 CA ACAAATGCACAGCAGCTCAC CCATTTGTTGCCTGAACTGA 258-262
NfuSU0083 CA GGATATGTCACGTGGGGAAC CCGTGGTGCTGAAACAAGAT 221-230
NfuSU0084 CA AACAGCATCACACCAACCAA GTCAGCATCGACAGCAGTGT 245-257