Molecular Ecology (2009) 18, 1801–1813 doi: 10.1111/j.1365-294X.2009.04141.

Detection of selection signatures within candidate regions

Blackwell Publishing Ltd

underlying trypanotolerance in outbred cattle populations

G . - K . D AY O ,*† S . T H E V E N O N ,‡ D . B E RT H I E R ,‡ K . M O A Z A M I - G O U D A R Z I ,§ C . D E N I S ,§
G . C U N Y ,* A . E G G E N § and M . G A U T I E R §
*IRD (Institut de Recherche pour le Développement), Unité Mixte de Recherche Trypanosomes, TA A-17/A Campus international
de Baillarguet, 34398 Montpellier cedex 5, France, †CIRDES (Centre International de Recherche-Développement sur l’Elevage en
zone Subhumide), Unité de Recherche sur les bases Biologiques de la lutte intégrée BP454 Bobo Dioulasso, Burkina Faso, ‡CIRAD
(Centre de coopération Internationale en Recherche Agronomique pour le Développement), Unité Mixte de Recherche 17 Trypanosomes,
F-34398, Montpellier, France, §INRA (Institut National de la Recherche Agronomique), Unité de Recherche 339 Laboratoire de
Génétique Biochimique et de Cytogénétique, 78350 Jouy-en-Josas, France

Abstract
Breeding indigenous African taurine cattle tolerant to trypanosomosis is a straightforward
approach to control costs generated by this disease. A recent study identified quantitative
trait loci (QTL) underlying trypanotolerance traits in experimental crosses between tolerant
N’Dama and susceptible Boran zebu cattle. As trypanotolerance is thought to result from
local adaptation of indigenous cattle breeds, we propose an alternative and complementary
approach to study the genetic architecture of this trait, based on the identification of selec-
tion signatures within QTL or candidate genes. A panel of 92 microsatellite markers was
genotyped on 509 cattle belonging to four West African trypanotolerant taurine breeds and
10 trypanosusceptible European or African cattle breeds. Some of these markers were
located within previously identified QTL regions or candidate genes, while others were
chosen in regions assumed to be neutral. A detailed analysis of the genetic structure of
these different breeds was carried out to confirm a priori grouping of populations based on
previous data. Tests based on the comparison of the observed heterozygosities and variances
in microsatellite allelic size among trypanotolerant and trypanosusceptible breeds led to
the identification of two significantly less variable microsatellite markers. BM4440, one of
these two outlier loci, is located within the confidence interval of a previously described
QTL underlying a trypanotolerance-related trait.
Detection of selection signatures appears to be a straightforward approach for unravelling
the molecular determinism of trypanosomosis pathogenesis. We expect that a whole genome
approach will help confirm these results and achieve a higher resolving power.
Keywords: African cattle, Bos indicus, Bos taurus, European cattle, selection signatures,
trypanotolerance

Received 24 October 2008; revision received 16 December 2008; accepted 21 January 2009

later into the African continent, via the Horn of Africa

Introduction
Previous studies on African bovine populations have
shown that the humpless taurine populations (Bos taurus)
are the original indigenous cattle of Africa while the
humped zebu populations (Bos indicus) were brought only
Correspondence: Mathieu Gautier, INRA, UR339 Laboratoire de
Génétique Biochimique et de Cytogénétique, 78350 Jouy-en-Josas.
Fax: +33 1 34 65 24 78; E-mail: Mathieu.Gautier@jouy.inra.fr
(Epstein 1971; Loftus et al. 1994; Bradley et al. 1996; MacHugh
et al. 1997; Hanotte et al. 2002). Indeed, it is generally
believed that taurine cattle penetrated West African forests
around 4000 years before present (bp; Freeman et al.
2004; MacDonald & Hutton MacDonald 2000; Marshall &
Hildebrand 2002) while zebu populations arrived since
1300 bp–1000 bp (Epstein 1971; MacHugh et al. 1997;
MacDonald & Hutton MacDonald 2000). Hence, the earlier
arrival of taurine breeds in West African savannah and

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1802 G . - K . D AY O E T A L .
forests may explain their better adaptation to subtropical
regions infested by tsetse flies and their tolerance to
trypanosomosis (trypanotolerance). In contrast, humped
zebu populations started to inhabit these regions only
with the development of veterinary prophylaxis and the
destruction of the tsetse habitat through widespread
deforestation (Lhoste 1991). Trypanotolerance is presumed
to have arisen through natural selection acting on cattle
exposed to trypanosome infection. Trypanotolerance is a
heritable trait associated with ability to control parasitaemia
and anaemia, a major clinical sign during disease develo-
pment (Murray et al. 1984). Unravelling the molecular
mechanisms underlying trypanotolerance is a major issue,
first to develop new control methods against the disease,
and second to elucidate the evolutionary mechanisms
involved in the adaptation of populations to environmental
constraints.
It has been shown that immune molecules such as
cytokines are involved in either trypanosome or trypano-
somosis control (Olsson et al. 1991; Bakhiet et al. 1996;
Taylor et al. 1996a, b). For instance, in humans, genetic
polymorphisms located in IL-6, IL-10, IL-1α and tumour
necrosis factor-alpha genes are associated with an increased
risk (IL-1α and TNFα) or a decreased risk (IL-6 and IL-10)
of developing human African trypanosomiasis (Courtin
et al. 2006, 2007, 2008). However, in cattle, only a limited
number of similar studies exist.
Hanotte et al. (2003) published the results of a quantitative
trait loci (QTL) mapping experiment involving an experi-
mental F2 cross between N’Dama (trypanotolerant) and
Boran zebu (trypanosusceptible) subjected to experimental
infection. They have identified QTL on 18 different bovine
autosomes and underlying 16 trypanotolerance related
traits (Hanotte et al. 2003). Although this pioneering
work significantly improved our knowledge on the genetic
determinism of trypanotolerance, these results need to be
extended in at least two directions. First, QTL intervals
are still too large to undertake an efficient candidate gene
approach. Second, QTL need to be validated in outbred
populations subjected to natural infection. Considering the
recent settlement of cattle populations in regions with high
trypanosomosis pressure and their natural adaptation,
favourable allelic variants might have left distinctive
signatures of selection in their vicinity via the so-called
hitchhiking phenomenon (Schlötterer 2003). Such selection
signatures are characterized by a lower variability on an
extended region compared to that expected under neutrality.
Although both the impact and extent of such a phenom-
enon remain to be evaluated in the genome of adapted cattle
populations, searching for such signatures could represent
a promising approach for validating and refining the
localization of previously identified QTL.
In this study, our aim was to search for selection sig-
natures, using the lnRH and lnRV tests (Schlötterer 2002;
Kauer et al. 2003; Schlötterer & Dieringer 2005), in 14 cattle
populations exposed to different parasitic pressures and
displaying different and already known tolerance statuses,
i.e. four West African trypanotolerant taurine breeds and
10 trypanosusceptible European or African cattle breeds.
We focused on four QTL regions previously identified
as underlying cattle trypanotolerance traits. In addition,
microsatellites close to nine candidate genes (eight inter-
leukin genes and the IFNG gene) located elsewhere in
the genome were also genotyped. Finally, other selected
markers expected to be located in neutral regions with
respect to trypanotolerance were also used. A detailed
analysis of the population structure was carried out in
order to confirm the different groups of populations to
be compared. Overall, the density of markers in the QTL
regions included in this study is expected to be sufficient to
identify selection signatures with moderate power.
Materials and methods
Animal material
Five hundred and nine individuals belonging to 14 different
bovine breeds originating from Africa and Europe were
included in the study (21–50 individuals per breed, for
further details see Table 1). African taurine populations
have been living for hundreds of generations under
trypanosomosis selective pressure. The African zebu
terminology is somewhat improper. Indeed, as shown by
several studies, there is a decline in the proportion of Zebu
genome introgression from East to West in Africa, and
some indicine alleles also segregate in taurine populations
(MacHugh et al. 1997; Hanotte et al. 2000, 2002; Freeman
et al. 2004; Ibeagha-Awemu et al. 2004; Freeman et al. 2006).
Hence, in general African ‘zebu’ cattle are trypanosusce-
ptible while hybrid cattle show intermediate phenotypes.
European cattle, which have not been exposed to any
trypanosome pressure during their history, are (expected
to be) trypanosusceptible (Murray et al. 1984; Mattioli et al.
1998; D’Ieteren & Kimani 2006).
All individuals were chosen to be unrelated to each
other (as far as available information allowed). Genomic
DNA samples are part of the INRA-CIRDES DNA bank and
originate from previous published studies (see Table 1)
and (Souvenir Zafindrajaona & Lauvergne 1993; Moazami-
Goudarzi et al. 1997, 2001; Souvenir Zafindrajaona et al.
1999; Beja-Pereira et al. 2003; Thevenon et al. 2007).
Genotyping data
Ninety-two microsatellite markers were genotyped among
which 52 were selected using available genetic map
information (Ihara et al. 2004) so that they extensively cover
trypanotolerance QTLs (Hanotte et al. 2003), 27 belong to
© 2009 Blackwell Publishing Ltd
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Table 1 Origin and sample size of cattle breeds included in the study

Trypanotolerance Sample
Continent Breed Code (a priori status) Sampling area (reference) size

European taurine Aubrac AUB No France (Beja-Pereira et al. 2003) 42

Charolaise CHA No France (Moazami-Goudarzi et al. 1997) 45
Montbeliard MON No France (Moazami-Goudarzi et al. 1997) 21
Normande NOR No France (Moazami-Goudarzi et al. 1997) 30
African taurine Baoule BAO Yes Burkina Faso (Souvenir Zafindrajaona et al. 1999) 30
Lagune LAG Yes Benin (Moazami-Goudarzi et al. 2001) 37
N’Dama NDA Yes Burkina Faso (Souvenir-Zafindrajaona et al. 1999) 30
Somba SOM Yes Benin/Togo (Moazami-Goudarzi et al. 2001) 40
African hybrid Borgou BOR Variable Benin (Moazami-Goudarzi et al. 2001) 50
Kuri KUR Variable Chad (Souvenir-Zafindrajaona et al. 1999) 39
West African zebu Zebu peul* ZPB Variable Burkina Faso (Thevenon et al. 2007) 47
Zebu Choa† ZCH No Chad (Souvenir-Zafindrajaona et al. 1999)
Zebu Bororo ZBO No Chad (Souvenir-Zafindrajaona et al. 1999) 25
East African zebu Zebu Madagascar ZMA No Madagascar (Souvenir Zafindrajaona & Lauvergne 1993) 38

*This population is described in Thevenon et al. (2007).

†zebu Choa, zebu Arab.

the Food and Agriculture Organization panel recommended
for livestock biodiversity studies and 13 are new markers
(except for that in gene IL4) developed from sequence
information from the latest bovine genome assembly
(ftp://ftp.hgsc.bcm.tmc.edu/pub/data/Btaurus/fasta/
Btau20070913-freeze/) in the vicinity of some candidate
genes: eight interleukins (IL1A, IL1B, IL4, IL6, IL10, IL12A,
IL12B, IL18), Interferon Gamma (IFNG) and transmembrane
chemokine receptor (CXCR4). Based on intermediate
results (see below), this latter gene could be a candidate
and four microsatellites (named CXCR4_1, CXCR4_2,
CXCR4_3, CXCR4_4) were detected around this gene. As
shown in Table 2, most of the markers (77 out of 92) come
from the Ihara et al. (2004) bovine linkage map and map to
the bovine genome assembly (88 out of 92). In addition,
these 92 markers are assigned to 23 different autosomes
(from 1 to 20 markers per chromosome and 4 on average).
For markers mapping to the same chromosome, the
average intermarker spacing is 7.27 centimorgans (cm)
(0.31 to 54.1 cm) on the genetic maps (N = 56 markers) and
8.97 Mb (0.048 Mb to 72 Mb) on the bovine genome
assembly (N = 65 markers). Thus, linkage disequilibrium
is expected to be negligible for most marker pairs as reported
in previous studies in the same or similar populations
(Gautier et al. 2007; Thevenon et al. 2007). In addition, based
on the QTL results obtained by Hanotte et al. (2003), 37 of
the 92 markers (40%) are located within large QTL
confidence intervals (> 20 cm), and thus, most of them
might not be associated to putative causal variant(s)
responsible for the observed QTL effect. Assuming that a
selection signature associated with a QTL could cover
0.5–1 cm and using the marker density described above,
it should be possible to detect a selection signature with
moderate power. Thus, we do not expect that more than
one marker per region will show evidence of a signature,
and we do not expect to identify a signature in each QTL
region. Characteristics of the markers considered in this
study are summarized in Table 2.
For some populations/markers, data were available
from previous studies (Moazami-Goudarzi et al. 2001;
European Cattle Genetic Diversity Consortium 2006;
Thevenon et al. 2007) representing about 20% of the data
set considered. For the remainder, new genotyping data
were produced according to standard protocols described
elsewhere (Gautier et al. 2006; Thevenon et al. 2007).
Genetic diversity and tests for Hardy–Weinberg
equilibrium
For each population, allele numbers, allele frequencies,
expected heterozygosities and observed heterozygosities
were estimated using Genetix 4.03 (http://www.genetix.
University-montp2.fr). Measures of allelic richness for
each locus were corrected for sample size variation using
the rarefaction method implemented in fstat software
(Goudet 2001).
Departure from Hardy–Weinberg equilibrium over
all the loci was evaluated using Fisher’s exact test as imple-
mented in GenePop 3.4c (Raymond & Rousset 1995) and
the permutations method (Genetix 4.03). The inbreeding
coefficient measured as the deficit in heterozygotes
(Wright’s FIS) and the standardized variance between sites
measured as the relative excess in heterozygotes found by
pooling different subpopulations (FST, global and pairwise)

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1804 G . - K . D AY O E T A L .

Table 2 Details for the different markers considered in the study. Markers displaying highly significant (P < 0.0001) departure from HWE
are in italics

Position Position Within

Markers Note BTA (cM)* (Mb)† P value‡ QTL CI§ Forward/reverse primers (5′–3′)

IL12AMS New 1 109.37 0.2945 GCAGCATTCCAAAATGAAACA/TCCAGATTTCTAGCTTCTCCTGA

BM1824 FAO 1 122.4 134.01 0.4972 GAGCAAGGTGTTTTTCCAATC/CATTCTCCAACTGCTTCCTTG
BM2113 FAO 2 115.4 129.68 0.5432 GCTGCCTTCTACCAAATACCC/CTTCCTGAGAGAAGCAACACC
TGLA61 2 23.11 18.81 0.0031 GGCAATCTGGATACCTGACATA/CCTTGAAATAGTAAAAAGACCATAT
DIK1081 2 27.29 23.98 0.9164 CCCACCTACTCCTTGTCAGAG/ACTTTCTCACCCTCAGGATCG
DIK4334 2 34.65 30.89 0.0045 CCCTCCTCTGACTCATTTCCT/GTCAACTGACCGAAGCCCTA
BM3010 2 38.90 35.02 0.9399 TCATCTTTGTCAAGACCTGGC/AGTGGGAGAGGGCTTTGG
DIK5022 2 42.29 40.48 0.5071 Yes CCCAATGTAGCCTTCAAACC/GGAAGGACTGGTGTCCTGAG
DIK2853 2 45.32 46.90 0.5852 Yes GGACAAAAACATGCTCTTCCTT/GGTCACGGAGAGTCCAACAT
DIK1140 2 46.26 47.95 0.1649 Yes GCATGTTGGTTTCAGGTGTAC/GTGGGGGTGCTGTGGAAAGT
DIK2496 2 49.57 50.84 0.9836 Yes GGAGGAATTGGCTATGGTCTC/CGACTTCCGTCTGTCTCACA
DIK2729 2 50.88 53.98 0.1512 Yes CTCCCACTGTGGTTCTGACA/GTCCTGGGCCTGGTAAAATA
DIK4025 2 56.91 59.14 0.5990 Yes AGCTCCACATTTTCAGTGATTG/CTGTAGCCCTCCAGGAACAC
DIK4673 2 59.32 61.67 0.8065 Yes GTACTTGGGGAAGCCCTCTC/ACTGCGCTTGAGGGAAAATA
BM4440 2 60.26 62.33 0.6832 Yes CCCTGGCATTCAACAAGTGT/CACCCTGTTAGGAATCACTGG
CXCR4_1 New 2 62.69 0.0007 Yes TGGCTATGATTGAGCAACAAGA/TCTACTGCCCTGTCAGCATCT
CXCR4_2 New 2 62.74 0.2386 Yes GGAACCAGATGAACTCAGCAC/AGAGCTTAGCACGCTTGTGTC
CXCR4_3 New 2 62.84 0.0331 Yes AGTAGGAAATGGCAACCCACT/CTAGGCTCAGTGCCACATTTC
CXCR4_4 New 2 63.01 0.4199 Yes CCCTGCAATCTTGAACCTACA/TGCATCCATCTGTTTTTGTCC
DIK2719 2 62.01 65.33 0.0467 TGATCCCATTTGAGACAGCA/CCTTACTGTCCCCACTCTGC
DIK4972 2 69.77 48.66 0.0253 GCCTCCCAGAATCCTGACTA/GAATTTCCTGGCAGTTCCTG
DIK4208 2 79.61 92.16 0.5261 GTCCGTGTTGCTGCAAATAG/AGCAGCACTAGTTGCCAAGG
INRA023 FAO 3 33.57 0.0458 GAGTAGAGCTACAAGATAAACTTC/TAACTACAGGGTGTTAGATGAACTC
IL6MS New 4 30.28 0.1510 TCCTTTTCATATTGCTTTGAAGG/GGGAGGGCTGATTTCATAGAT
MNB-42 4 77.58 79.78 0.3676 CTTTAGCTGTTCAGCAGATGG/TGATTCTCAGCTCTTTCTAGCC
DIK4042 4 90.45 96.96 0.1263 Yes GCATCTTCCCGATGTCTGAG/CTCTGAAGCCCAGTTGGAAG
MS2079 4 93.06 99.90 < 0.0001 Yes GCTAGGTGATGCACTTTATACACTT/CATACATAACTGAGTGACTTAGCAT
DIK4259 4 94.11 100.88 0.0517 Yes CATCTCCACTTCACGCTTTG/CATGACTCAGTTCACGCACA
DIK4236 4 95.17 102.21 0.7469 Yes CCACCTCTGCAGTCAGAAAT/CTGGAAGACAGCTAGCACCA
DIK2646 4 96.40 104.98 0.4228 Yes GGTCGACCAGATGAAGAAGG/AATTCCATGGGCAGAGGAC
DIK2579 4 100.9 106.01 0.9910 Yes CACTGGCATCGTCTTGATTG/TCTGTACCTTGGCCCCTTCT
DIK089 4 104.5 110.99 0.3222 Yes CTCAATCCTTCCTATAATATAG/TAGAGGATTGAAGAGAAAAC
DIK4404 4 108.9 0.2006 Yes CAGGGATTGAACCCACTTTG/ATGGCCATTCGTGATAAAAA
MGTG4B 4 112.8 117.29 0.2523 Yes GAGCAGCTTCTTTCTTTCTCATCTT/GCTCTTGGAAGCTTATTGTATAAAG
IFNGMS New 5 47.29 0.2374 AGAATGGACACTTCAGGTTGG/GCCCCCAAAATGTTCTGTACT
ETH10 FAO 5 71.76 58.07 0.0401 GTTCAGGACTGGCCCTGCTAACA/CCTCCAGCCCACTTTCTCTTCTC
ETH152 FAO 5 121.75 0.3024 TACTCGTAGGGCAGGCTGCCTG/GAGACCTCAGGGTTGGTGATCAG
IL4MS 7 30.50 22.11 0.0059 GTGCTGGACATCTGCAAGTG/ACATTCAGGTCTGTGATCCATG
DIK2819 7 47.91 29.84 0.7359 TTACTTTTCGTGGGCCAGAG/GGAACTGTGCCACATAGCAA
DIK4606 7 55.29 37.04 0.4434 TCTTGGAAAGGGGAAAAAGC/TGCTTCATAGCACTTATCTCTTCA
DIK4739 7 58.55 39.59 0.6872 Yes TGAAACACTCTAAAAGGGTATGC/CAGCCTGTGGGAGAAGACAT
DIK2689 7 59.30 40.55 0.0564 Yes GAGCACCTTCCACACACAAA/AAGAAAAGCTGATCCCATGC
DIK4145 7 62.25 43.88 0.0205 Yes TGGATGCTTGGATTGACTGA/TCCACAAACAGGCACACAC
DIK4563 7 63.46 48.03 0.2384 Yes TGAAAGAGAGCTGCCTCCTG/GGGTGAGGAACTGATCCAGA
DIK2256 7 67.73 51.55 0.1359 Yes CAGCACACAAACTAAGGCACA/GATAGGAAAGGAGGGGAAGG
CSSM057 7 66.92 55.33 0.9166 Yes TGTGGTGTTTAACCCTTGTAATCT/GTCGCTGGATAAACAATTTAAAGT
BMS904 7 70.32 57.33 0.5357 Yes GTCCTTTGTTCGTTCTAGCAGG/TGTGATATCCACTTTCCCCG
DIK4135 7 71.73 58.89 0.5752 Yes TCTGCAATGATTTATATGGGAAT/CTACTCTCCAGCTGCTGTGC
DIK050 7 72.05 59.01 0.1570 Yes CAGACATCCACAAGGAAAC/AACCAAGATGGGGAAGTACA
DIK2407 7 73.74 60.37 0.3555 AATTGATTCGGCACACACAC/CTGCTGTACTGGGTGGAAGG
MNB-15 7 72.89 61.35 0.0070 CCATTTAAACAGAGCTCAGTCAC/CTACTTCCTGAGAGTCCAGCAC
DIK4386 7 75.50 62.37 0.7898 TCCAGAAGAGAGATGATGATGG/AAGGCAATGGTTTGTCCTTG
DIK2915 7 76.22 63.90 0.5057 TCTCACCCTCACATGGTTCA/GTGGAGCCAAGGTGAAAGAA
INRABERN192 7 82.48 69.76 0.6590 AGACCTTTACAGCCACCTCTTC/GTCCCAGAAACTGACCATTTTA
IL12BMS New 7 73.25 0.8871 CAAGTTGGGCTTCCTTTTACC/GGACCTCACAGTGACAGCATT

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 S E L E C T I O N S I G N A T U R E S I N A F R I C A N C A T T L E 1805

Table 2 Continued

Position Position Within

Markers Note BTA (cM)* (Mb)† P value‡ QTL CI§ Forward/reverse primers (5′–3′)

DIK630 7 90.70 80.98 0.8921 GCCCCTTAAAGGAGTGTTATTT/TGTGACTGAGTCCTCCTACCAA

HEL9 FAO 8 84.84 80.71 0.0003 CCCATTCAGTCTTCAGAGGT/CACATCCATGTTCTCACCAC
ETH225 FAO 9 12.75 10.86 0.3345 GATCACCTTGCCACTATTTCCT/ACATGACAGCCAGCTGCTACT
MM12 FAO 9 82.85 0.0543 CAAGACAGGTGTTTCAATCT/ATCGACTCTGGGGATGATGT
CSRM60 FAO 10 77.82 77.23 0.0066 AAGATGTGATCCAAGAGAGAGGCA/AGGACCAGATCGTGAAAGGCATAG
INRA037 FAO 10 79.01 77.43 0.4283 GATCCTGCTTATATTTAACCAC/AAAATTCCATGGAGAGAGAAAC
ILSTS005 FAO 10 108.0 97.48 0.2067 GGAAGCAATGAAATCTATAGCC/TGTTCTGTGAGTTTGTAAGC
IL1AMS New 11 47.34 0.1589 TTCACTGGCAAAGAGAAAAGC/GGCAGACATTGTGAGGGATAC
IL1BMS New 11 47.43 < 0.0001 GAAATGGAGTAGGAGCCTGGT/CCAAACCTACTATAGTTCCCTCAC
INRA032 FAO 11 68.18 60.79 0.3919 AAACTGTATTCTCTAATAGCTAC/GCAAGACATATCTCCATTCCTTT
HEL13 FAO 11 122.4 102.48 0.0444 TAAGGACTTGAGATAAGGAG/CCATCTACCTCCATCTTAAC
INRA005 FAO 12 86.85 79.26 0.4016 CAATCTGCATGAAGTATAAATAT/CTTCAGGCATACCCTACACC
DIK2309 13 74.37 63.54 0.0049 Yes GAGTGGAAAACCGACACCAT/GAGTTGGACACGACTGAGCA
DIK2890 13 75.11 64.37 0.0186 Yes ATTTGTGTGTGGCGTGTTTG/TGGTGGAGAGACACAAGTGG
DIK5305 13 76.51 66.47 0.9471 Yes GGAATCCACACTTCCACTGC/TGTGCTAATCGCTAGGTCCA
DIK4350 13 77.10 66.94 0.0494 Yes TGGCACATGGGATCTTATTTC/GACATTGCACTTTCGGTGTG
BL1071 13 80.98 72.92 0.0021 Yes AGAAGGACAGAGACCACAGGC/TTGAGGTGAAGAGGTCCACC
DIK2867 13 82.12 73.02 < 0.0001 GGCTGTTGGCAAAGGAAATA/ATCTGTCTCCTCCAGGCTCA
DIK537 13 85.90 75.18 0.3728 TCCACCAGACTCACATGTTCA/TGTTCATCTAGGGGGCTCTG
DIK4871 13 87.00 76.93 0.1615 CCCATGCTCTTAGCCTCTCA/GGGGTCAAGAATGACTCCAA
DIK5250 13 91.38 78.50 0.8968 TCTGCAAAAGGACCAGATCA/TCTCCTCACACGTCTGCATC
DIK2117 13 94.47 80.12 0.2880 AGGGGTCCTGGAATCTGAG/TCTGCCGCCAGTTGTGAC
CSSM66 FAO 14 5.130 4.68 0.0429 ACACAAATCCTTTCTGCCAGCTGA/AATTTAATGCACTGAGGAGCTTGG
HEL1 FAO 15 37.96 34.63 0.2124 CAACAGCTATTTAACAAGGA/AGGCTACAGTCCATGGGATT
SPS115 FAO 15 22.09 22.06 0.0245 AAAGTGACACAACAGCTTCTCCAG/AACGAGTGTCCTAGTTTGGCTGTG
IL18MS New 15 22.98 0.5565 TTTCCCTTCTTACGTTTCTCCA/AAAAGGCCCTGTTTCTGACA
IL10MS New 16 4.45 0.6438 Yes CTGGAACAGCATCCATCTCAT/GGTAGAAGAAGGGAACCAACG
INRA035 FAO 16 72.00 < 0.0001 ATCCTTTGCAGCCTCCACATTG/TTGTGCTTTATGACACTATCCG
ETH185 FAO 17 54.71 44.75 < 0.0001 TGCATGGACAGAGCAGCCTGGC/GCACCCCAACGAAAGCTCCCAG
INRA063 FAO 18 47.95 41.02 0.4343 ATTTGCACAAGCTAAATCTAACC/AAACCACAGAAATGCTTGGAAG
ETH3 FAO 19 90.04 56.63 0.0222 GAACCTGCCTCTCCTGCATTGG/ACTCTGCCTGTGGCCAAGTAGG
TGLA126 FAO 20 31.87 22.54 0.5630 Yes CTAATTTAGAATGAGAGAGGCTTCT/TTGGTCTCTATTCTCTGAATATTCC
HEL5 FAO 21 13.52 8.60 0.0409 GCAGGATCACTTGTTAGGGA/AGACGTTAGTGTACATTAAC
TGLA122 FAO 21 62.69 56.60 0.2723 CCCTCCTCCAGGTAAATCAGC/AATCACATGGCAAATAAGTACATAC
HAUT24 FAO 22 66.10 47.29 0.5254 CTCTCTGCCTTTGTCCCTGT/AATACACTTTAGGAGAAAAATA
BM1818 FAO 23 58.20 37.31 0.0536 AGCTGGGAATATAACCAAAGG/AGTGCTTTCAAGGTCCATGC
HAUT27 FAO 26 35.19 29.27 0.0001 TTTTATGTTCATTTTTTGACTGG/AACTGCTGAAATCTCCATCTTA

*linkage map (Ihara et al. 2004).

†latest bovine genome assembly Btau4.1.
‡global Fisher’s exact test across all populations.
§confidence interval from Supplementary Table 5 in Hanotte et al. (2003). See this reference for details about the trait(s) affected by the
corresponding QTL.
FAO, Food and Agriculture Organization.

were calculated by the Weir & Cockerham (1984) method

as implemented in Genetix 4.03. Significance levels were
determined using the permutations method (104 permuta-
tions), which consists in comparing the observed F-statistics
values in the sample to the distributions obtained with
permutations under the null hypothesis. The significance
of FIS was tested by randomizing alleles between individuals
within populations while the significance of FST was tested
by randomizing individuals among populations.
Population structure
The Bayesian clustering method, as implemented by the
Structure 2.2 program (Pritchard et al. 2000), was used to
assess the level of population structure. As genotyping
information for the putative parental populations was not
available, we hypothesized k unknown parental populations
(k ranging from 1 to 14). When working with complex data
sets, Garnier et al. (2004) suggested choosing the k-value

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1806 G . - K . D AY O E T A L .

that maximizes the gain of information such as [Ln P(D)k – Table 3 Observed and expected heterozygosity (HO and HE) and
Ln P(D)k–1] where Ln P(D) represents the posterior prob- allelic richness in the 14 different cattle breeds. Standard deviations
ability of the data. Structure 2.2 was run five times with a (SD) of the different measures are given in parenthesis. Population
abbreviations are as in Table 1
burn-in period of 5.104 iterations followed by 105 iterations
for each k-value. Populations HO (SD) HE (SD) Allelic richness (SD)
Nei’s genetic distances (Nei 1978) between the different
pairs of populations were also estimated using the phylip AUB 0.59 (0.17) 0.62 (0.17) 3.8 (1.2)
3.2 package (Felsenstein 1989). The resulting distances BAO 0.52 (0.22) 0.52 (0.21) 3.3 (1.1)
matrix was used for the construction of dendograms accord- BOR 0.70 (0.15) 0.72 (0.14) 4.7 (1.2)
ing to the neighbour-joining (NJ) algorithm (Saitou & Nei CHA 0.63 (0.18) 0.64 (0.17) 3.9 (1.1)
KUR 0.68 (0.17) 0.71 (0.15) 4.6 (1.2)
1987) implemented in the phylip package (Felsenstein
LAG 0.45 (0.22) 0.48 (0.22) 2.8 (0.9)
1989). The reliability of each node was estimated from 10 000 MON 0.61 (0.20) 0.61 (0.18) 3.7 (1.2)
random bootstrap resamplings of the data. NDA 0.54 (0.22) 0.57 (0.21) 3.7 (1.2)
NOR 0.62 (0.22) 0.61 (0.20) 3.7 (1.3)
SOM 0.54 (0.21) 0.57 (0.20) 3.6 (1.1)
Selective sweep tests: ln RV and ln RH statistics ZPB 0.68 (0.16) 0.71 (0.15) 4.7 (1.2)
The relative variance in microsatellite repeat number for ZCH 0.70 (0.17) 0.72 (0.15) 4.8 (1.3)
ZBO 0.70 (0.18) 0.72 (0.15) 4.7 (1.3)
the compared populations (ln RV) and the relative expected
ZMA 0.57 (0.18) 0.60 (0.15) 3.5 (0.9)
heterozygosity for the compared populations (ln RH)
statistics (Schlötterer 2002; Kauer et al. 2003; Schlötterer &
Dieringer 2005) were calculated for each marker and for
different groups of populations using Microsatellite Analyser displayed highly significant departure from HWE
(msa) software (Dieringer & Schlötterer 2003). (P < 0.0001 using the global Fisher’s exact test). In addition,
Under the null hypothesis of neutrality, lnRV and lnRH these markers exhibited high FIS values (mean FIS ≥ 0.1 and
are expected to follow a Gaussian distribution (Schlötterer FIS ≥ 0.2 in most of the populations). Although departure
2002). Assuming most loci in the study are neutral, the from HWE might result from selection, it is most likely that
moments of this distribution could reasonably be estimated either technical problems or null alleles with these five
empirically from the data. After normalization, P values markers in several populations explain this result and they
were estimated for each locus (for a given group of popu- were therefore excluded from subsequent analyses (note
lations in the study) to identify outlier loci (at 5% and 1% that no clear difference in the overall results was observed
thresholds). whether they were included or not).
Using both statistics lowers the rate of false positives
by a factor of 3 (Schlötterer & Dieringer 2005). For each
comparison, we checked for the normality of ln RV and
Genetic variability and population structure
ln RH values using the Kolmogorov–Smirnov test. Information on the number of alleles/locus and number of
alleles/locus/population, expected heterozygosities (HE)
and observed heterozygosities (HO) are summarized in the
Annotation of the region of interest
Support Information Table S1. As shown in Table 3, gene
Because of the current lack of annotation information on diversity (HE) varied from 0.48 (LAG) to 0.72 (BOR, ZCH
the bovine genome assembly, we used the hg18 human and ZBO). The overall diversity (HO and allelic richness) is
genome sequence assembly available from the UCSC higher in African zebu and crossbred populations than in
genome browser (http://genome.ucsc.edu/) to search for the African and European taurine populations.
genes located in chromosome regions of interest (surround- Among all the populations, the overall value for FST
ing outlier loci). Each bovine outlier locus was anchored was 0.14 ± 0.0017 while the average FST among zebuine
directly to the human genome after extracting a surrounding populations (ZBO, ZCH, ZMA and ZPB) was 0.06 ± 0.0014
bovine sequence of several thousand kilobases using the and decreased to 0.03 ± 0.0053 when ZMA was removed.
online blast alignment tool. Among African taurine populations (BAO, LAG, NDA and
SOM) the average FST was 0.08 ± 0.0017 and 0.04 ± 0.0015
when LAG was removed and it was 0.065 ± 0.0013 among
Results
European breeds.
With the Bayesian clustering method (Pritchard et al.
Hardy–Weinberg equilibrium (HWE) analysis
2000), the most consistent gain in information was obtained
As shown in Table 2, of the 92 microsatellite markers used, with a number of clusters, k, between 4 and 7. Figure 1
five (ETH185, INRA35, MS2079, IL1BMS and DIK2867) shows the population clustering obtained with different

© 2009 Blackwell Publishing Ltd

 S E L E C T I O N S I G N A T U R E S I N A F R I C A N C A T T L E 1807
Fig. 1 Population Bayesian clustering obtained with different k-values.
k-values. Of particular interest are the low values of k,
which confirm prior grouping of populations based on
geographical and historical data. With k = 3, four main
groups are distinguished: European taurine, EUT (AUB,
CHA, MON, NOR), West African taurine (BAO, LAG,
NDA, SOM), West African hybrid breeds, WAH (BOR,
KUR) and African zebu (ZCH, ZBO, ZPB and ZMA).
Subsequent increases in the prior number of cluster leads
to the separation of ZMA, zebu Madagascar (k = 4) from
the West African zebu (WAZ) group and LAG (k = 5) from
the other West African taurine (WAT) breeds. The hybrid
status (African taurine and zebu) of KUR and BOR appears
clearly at such levels of clustering. These different results
are remarkably robust in different marker sets; in particular,
we obtained almost identical results when removing
(non-neutral) outliers loci as identified using FST based
tests (not shown). The neighbour-joining tree obtained
with Nei genetic distances confirms this Bayesian clustering
(data not shown).
Identification of selection signatures
Based on the genetic structure analysis presented above
(Fig. 1) and the history known for the different populations,
© 2009 Blackwell Publishing Ltd
we can define five different groups of populations for the
detection of selection signatures based on microsatellite
diversity: (i) West African taurine (WAT) comprising BAO,
NDA and SOM (LAG was not included in this group
because of its lower heterozygosity which leads to an
inflated FST value in African taurine populations and was
isolated from other groups at a low clustering level in the
structure analysis); (ii) European taurine (EUT) composed
of four populations: AUB, CHA, MON and NOR; (iii) West
African zebu (WAZ) comprising three populations: ZPB,
ZCH and ZBO; (iv) West African hybrids (WAH) composed
of two populations: BOR and KUR and (v) Zebu Madagascar
(ZMA). Each group was subsequently considered as a
single population for the lnRV and lnRH tests. Focusing on
selection signatures related to trypanotolerance, three
main comparisons were made using WAT as a reference
trypanotolerant group and WAZ, ZMA and EUT as
trypanosusceptible groups. The results are shown in Fig. 2.
Out of the eight tests performed for the WAT trypano-
tolerant group (four group comparisons and two tests
per comparison: ln RV and ln RH), eight and seven were
significant (P < 0.01) for markers DIK5250 and BM4440,
respectively (the remaining test for the latter being very
close to the 1% threshold with P < 0.013). As expected from
1808 G . - K . D AY O E T A L .
these results, BM4440 and DIK5250 showed markedly
reduced variability in the populations of the WAT group
(Table 4). In addition, for each marker, the same allele (101
and 221 for BM4440 and DIK5250, respectively) is fixed
or nearly fixed (frequency always > 0.95%) in all three
populations belonging to the WAT group.
Three other markers (DIK2579, IL6MS and MM12) were
significant (P < 0.01) for only one of the eight tests involving
ZMA and EUT vs. WAT, which gives far less support for
their being related to the trypanotolerant trait. When each
of the other four groups was taken as reference (data not
shown), MM12 was significantly outlying (P < 0.01) in the
four possible comparisons in which ZMA is involved,
while two and three tests out of the possible eight were
significant (P < 0.01) for IL6MS and/or DIK2579, respectively,
for the four comparisons in which the EUT group is involved.
These markers are likely to show selection signatures asso-
ciated with some adaptive processes in ZMA (for MM12)
and EUT (for IL6MS and DIK2579).
Discussion
Population structure
Results from both Nei’s distance-based analysis and
Bayesian analysis tally well with the grouping of populations
according to geographical and historical data when the
Fig. 2 Joint distribution of the ln RH and
ln RV test statistics for the four different
comparisons between trypanotolerant popu-
lation group (WAT) and each trypano-
susceptible cattle group (EUT, WAZ, WAH
and ZMA). Assuming each test statistics
is normally distributed, the dashed lines
indicate the 1% empirical threshold. Names
are indicated for the outlier loci (which
displayed at least one test statistic above the
1% significant threshold).
number of clusters is k = 3. When considering k = 4 (among
the most likely) with the Structure software (Pritchard et al.
2000), a clear separation is observed between European
and African populations. A gradient of admixture among
African taurine populations (with almost pure taurine
populations represented by NDA or BAO) and zebuine
populations (represented by ZCH and ZBO) is observed in
West African populations. Of particular interest is the kuri
breed (KUR) in which animals are humpless and thus were
first classified as taurine. Moreover, they were shown to
carry a Bos taurus submetacentric Y chromosome (Souvenir
Zafindrajaona et al. 1999). In our study, KUR was found to
be closer to other West African zebu cattle (Fig. 1) with
a higher zebuine proportion (88% of West African zebu
proportion for k = 4). Similar results have already been
reported (Souvenir Zafindrajaona et al. 1999; Freeman et al.
2004) and among the different cattle breeds considered,
zebuine and hybrid populations displayed greater genetic
diversity. These results tally with previous reports (Freeman
et al. 2004), indicating that hybridization between African
taurine and African indicine cattle increased diversity by
combining alleles from two distinct lineages as exemplified
in our study in BOR populations. A hybrid position, as in
BOR population, was also observed with the KUR breed
and this result is similar to that observed by Freeman et al.
(2004). Conversely, the lowest heterozygosity was observed
in African taurine populations, especially in the LAG, as
© 2009 Blackwell Publishing Ltd
 S E L E C T I O N S I G N A T U R E S I N A F R I C A N C A T T L E 1809

Table 4 Allele frequency distribution for BM4440 and DIK5250

Markers Alleles BAO NDA SOM LAG BOR KUR ZPB ZCH ZBO ZMA AUB CHA MON NOR

BM4440 101 0.983 1.000 0.950 0.446 0.573 0.679 0.298 0.457 0.420 0.516 0.750 0.591 0.857 0.776
103 0.017 0 0.050 0.554 0.083 0.064 0.202 0.100 0.140 0.109 0.012 0.114 0.024 0.017
105 0 0 0 0 0 0 0 0 0 0.078 0.012 0.023 0 0
108 0 0 0 0 0 0.038 0.011 0 0 0 0.036 0 0 0
110 0 0 0 0 0.052 0.051 0.064 0.114 0.100 0.031 0.107 0.023 0.024 0.034
112 0 0 0 0 0.010 0.051 0 0.014 0 0 0.036 0.136 0.071 0
114 0 0 0 0 0.021 0.013 0.074 0.071 0.020 0 0 0.011 0 0
116 0 0 0 0 0 0 0 0 0 0 0 0 0.024 0
119 0 0 0 0 0 0 0 0 0 0.172 0 0.011 0 0
121 0 0 0 0 0.052 0.064 0.287 0.143 0.100 0.078 0.012 0 0 0
123 0 0 0 0 0.188 0.038 0.032 0.057 0.080 0.016 0.024 0.023 0 0.155
125 0 0 0 0 0.021 0 0.032 0.043 0.140 0 0 0.034 0 0
127 0 0 0 0 0 0 0 0 0 0 0.012 0.023 0 0
129 0 0 0 0 0 0 0 0 0 0 0 0.011 0 0.017
DIK5250 213 0 0 0 0 0.020 0.053 0.043 0.043 0.063 0.361 0 0.011 0 0
219 0 0 0 0 0 0 0 0 0 0 0.202 0.078 0.024 0.034
221 1.000 0.983 0.988 1.000 0.790 0.618 0.681 0.543 0.563 0.417 0.679 0.767 0.905 0.793
223 0 0 0 0 0 0.013 0.011 0.029 0.021 0 0 0 0 0
225 0 0 0 0 0.010 0.013 0 0.043 0.063 0.083 0 0 0 0
227 0 0 0 0 0 0 0 0.014 0.021 0 0 0 0 0
229 0 0.017 0.013 0 0.060 0.066 0.053 0.071 0.063 0.042 0.012 0.022 0 0
231 0 0 0 0 0.010 0.079 0.011 0.043 0.021 0.069 0 0 0 0
233 0 0 0 0 0.110 0.145 0.202 0.200 0.125 0.028 0 0 0 0
235 0 0 0 0 0 0 0 0 0 0 0.107 0.122 0.071 0.172
237 0 0 0 0 0 0 0 0 0.021 0 0 0 0 0
239 0 0 0 0 0 0.013 0 0.014 0.042 0 0 0 0 0

West African taurine: BAO, NDA, SOM and LAG.

West African hybrids: BOR and KUR.
West African zebu: ZBO, ZCH and ZPB.
East African zebu: ZMA.
European taurine: AUB, CHA, MON and NOR.

already reported (Freeman et al. 2004) while average FST
values remained relatively high within this group. This
can be explained by a combination of a relatively small
effective population size and a small migration rate, linked
to a sedentary breeding system. Moreover, during the
migration of cattle from a common centre of origin, genetic
diversity is expected to decline. Hanotte et al. (2002)
showed that a consistent pattern of reduction in diversity
is related to the African colonization route taken by cattle.
Furthermore, it can be presumed that very high selective
pressures, such as diseases and especially trypanosomosis,
decimating a large part of a cattle population, might reduce
the global effective population size. Because of these
specificities, we did not include this breed in subsequent
selective sweep detection analyses, although it might be
classified as trypanotolerant. African zebuine populations
are characterized by smaller FST values and higher genetic
diversities related to a larger effective population size
and a higher migration rate in an usually transhumant
breeding system. Even with a large number of clusters
(k = 14) used to run Structure (Pritchard et al. 2000), African
zebu breeds (ZCH, ZBO and ZPB) remained close to each
other, showing the strong unity of that group. These
populations are herded in a pastoralist breeding system,
which might explain their close genetic relationship.
Identification of selection signatures
Analysis of population structure allowed us to compare
accurately defined groups for the detection of selection
signatures based on microsatellite variability analysis.
The main advantage of the lnRH and lnRV tests is that both
are pairwise comparison tests between two populations,
enabling to rule out differences in absolute levels of variability
between trypanotolerant and trypanosusceptible populations
that could be linked to demographic events. Furthermore,
the combination of these two statistics is powerful to detect
recent and strong selective sweeps. However, their power is
reduced for older selective events (Schlötterer 2002; Schlötterer
& Dieringer 2005).

© 2009 Blackwell Publishing Ltd

1810 G . - K . D AY O E T A L .
BM4440 locus on BTA02 and DIK5250 locus on BTA13
were systematically outlier loci in comparisons involving
the only trypanotolerant group of West African taurine
breeds (WAT). Given that microsatellite markers themselves
are not likely to be the target of selection, these results
suggest that BM4440 and DIK5250 markers may be linked
to a hypothetical gene that has been swept into the African
taurine population. Moreover, the two loci displayed lower
variability in the African taurine populations than in the
others (European taurine and indicine populations) as
shown in Table 4. The distribution of allele frequencies of
BM4440 and DIK5250 loci in the West African taurine
populations is skewed in comparison to the European
taurine and indicine populations, probably highlighting a
recent selective sweep, as reported by Wiehe (1998) and
Schlötterer (2002). Furthermore, allelic richness of these
loci is reduced in African taurine cattle compared to other
populations. Allelic richness is influenced by selection
given its correlation with expected heterozygosity (Reed
et al. 2007). Interestingly, within the WAT group, the loss of
variability is explained within each of the breeds by the
near fixation (frequency always above 0.95) of the same
allele, 101 and 221 for BM4440 and DIK5250, respectively.
In LAG, also considered trypanotolerant but not included
in the WAT group for reasons explained above, DIK5250 is
also completely fixed for allele 221 while only two alleles
(101 and 103 only one mutation step apart) are segregating
at similar frequencies for BM4440. Assuming these loci
have been hitchhiking, these observations would tally with
rather recent induced selective pressure — relatively to the
(unknown) mutation rate at these two loci and the historical
effective population size.
The introduction of the West African taurine cattle is
known to have occurred in two waves (Epstein 1971). The
first started about 8000 bp and concerned the longhorn
cattle (NDA), which most likely originated from cattle
domesticated in the Fertile Crescent. The second wave
started about 4750–4500 bp and is characterized by the
introduction of shorthorn taurine cattle (BAO, LAG and
SOM in our sample). Some authors have hypothesized the
existence of another domestication centre for shorthorn
taurine cattle in North Africa from which West African
shorthorn cattle might have originated, while other
authors simply consider shorthorn cattle as deriving
from longhorn cattle in the Fertile Crescent (Payne &
Hodges 1997). Thus, assuming that the same causative
variants in the different WAT breeds are under selection
in the vicinity of the observed outlier markers, they might
have either appeared shortly before domestication of
longhorn cattle or been introduced through migration
into each of the population. In the first case, if the selective
pressure is represented by trypanosomosis disease, a
variant conferring resistance might have existed prior to the
selective pressure. However, our data cannot discriminate
between these different hypotheses and further studies are
needed.
Annotation of the regions surrounding BM4440 and
DIK5250
BM4440 locus is within the QTL confidence interval
underlying trypanotolerance traits on BTA02 while DIK5250
locus is located at about 6 cm from a QTL on BTA13, as
previously identified in an experimental cross (Hanotte
et al. 2003). Given the characteristics of the markers chosen
and the size of the QTL confidence intervals in this latter
study, 40% of the markers considered were located within
a QTL identified in the ILRI design (Hanotte et al. 2003).
The probability of observing two randomly chosen markers
within a QTL and assuming they are independent was
equal to 0.16 a priori. More precisely, BM4440 lies within
the confidence interval of the QTL underlying PCVM
(minimum packed red cell volume recorded during the
post-challenged period with Trypanosoma congolense) and
the QTL underlying BWD150 (percentage decrease in body
weight up to day 150 after challenge). DIK5250, on BTA13,
is located about 6 cm from a QTL underlying the DR60–150
trait (number of times an individual is detected to be
infected between days 60–150 after challenge with T.
congolense).
Assuming overall conservation of the region containing
the BM4440 marker between cattle and humans (as con-
firmed by sequence analysis) and after its anchorage on
the human genome, the closest RefSeq gene found in the
region is CXCR4 located approximately 500 kb away. This
gene is the specific receptor of chemokine CXCL12. The
CXCL12/CXCR4 complex is involved in the pathogenesis
of the Chagas disease caused by Trypanosoma cruzi. Thymo-
cytes from mice infected by T. cruzi display a higher
membrane receptor level of CXCR4 concomitantly to the
enhanced chemokine ligand, CXCL12 (Mendes-da-Cruz
et al. 2006), than uninfected mice. A study with knockout
mice revealed that the CXCR4 receptor plays an important
role in the haematopoiesis, development and organization
of immune systems. Indeed, CXCL12 and CXCR4 gene-
deleted mice produce identical lethal phenotypes, charac-
terized by defective myelopoiesis and B-lymphopoiesis,
and abnormal neuronal and cardiovascular development
(Nagasawa et al. 1996, 1998; Ma et al. 1998; Zou et al. 1998;
cited by Burger & Bürckle 2007). Thus, CXCR4 was con-
sidered as a candidate gene for the trypanotolerance-related
trait, and we developed and genotyped four additional
markers (CXCR4_1 to CXCR4_4) around this gene (BTA2:
62 690 000–63 010 000). However, none of these additional
markers displayed significant ln RH and ln RV statistics,
preventing validation of this candidate gene with those
statistic tests. Nevertheless, BM4440 may lie within or close
to a cis-regulating region of the CXCR4 gene. For DIK5250,
© 2009 Blackwell Publishing Ltd
 S E L E C T I O N S I G N A T U R E S I N A F R I C A N C A T T L E 1811
the closest gene (EYA2 for eyes absent 2, homologue)
identified using this same annotation approach was 1 Mb
away. Although this gene was recently shown to be differ-
entially expressed in muscle tissue after infection by an
intracellular parasite of the genus Trichinella (Wu et al.
2008), it was not considered as a clear candidate gene
underlying trypanotolerance.
The best approach would be to genotype additional
microsatellite markers (for instance, between four and
eight every 50 kilobases) around the two outlier loci to
confirm the signal observed. However, since DNA chips
are now available, we plan to use them on the same samples.
The distance at which a hitchhiking signal is observable is
difficult to determine a priori since it depends on several
unknown parameters for our populations such as the
intensity of selection, the demographic process and the age
of the selective process. Statistical simulations based on
the variation of bi-allelic markers showed that it is very
unlikely to detect any genetic hitchhiking at recombination
distances larger than 0.5 cm (~500 kb in the bovine genome),
unless the population divergence time is short and selec-
tion is very strong (De Kovel 2006). A similar study using
multiallelic markers is necessary but we expect that the
higher mutation rate of microsatellites (compared to single
nucleotide polymorphism) might decrease the detectable
signal as suggested by other empirical studies (Kohn et al.
2000). In our study, the closest neighbouring markers were
located 400 kb and 1.5 Mb away from BM4440 and DIK5250,
respectively. Thus, we confirm that at such distances, the
initial signal cannot be observed anymore. The available
microsatellite density in the bovine genome (approximately
one every 50 kb) might be an optimum for validating our
observations. Alternatively, the advent of high-throughput
genotyping techniques based on SNP chips might facilitate
detection of selection signatures using other methodologies.
Conclusion
We were able to identify two significant signals for selection
signatures for markers located within QTL underlying
traits related to trypanotolerance. If confirmed by additional
genotyping of closely linked markers (< 100 kb) or dense
genotyping approaches, this could contribute to a better
understanding of the molecular determinism of trypanotoler-
ance, provided additional functional tests unambiguously
relate the responsible selective pressure to trypanosomosis.
Moreover, the nature of the selective pressure needs to be
confirmed, for instance by carrying out an association
analyses between markers within these candidate regions
and phenotypes related to trypanotolerance collected from
field populations. Even if a large number of genes with a
small effect are expected to be involved, major genes are
likely to be involved in cattle trypanotolerance (Kemp &
Teale 1998; Hanotte et al. 2003). Hence, although hitchhiking
© 2009 Blackwell Publishing Ltd
mapping approaches are less powerful than conventional
QTL studies, their expected mapping resolution is much
higher.
Acknowledgements
The authors thank the farmers and the CIRDES team for their help
in providing the samples from Burkina Faso and INRA-LGBC for
other DNA samples. We thank Hélène Hayes (INRA) and Peter
Biggins (CIRAD) for English revision of the manuscript. We also
thank the INRA Department of Animal Genetics, Laboratoire de
Génétique Biochimique et de Cytogénétique (E.P. Cribiu director),
and Labogena. G. Dayo was supported by a PhD scholarship from
IRD (France). Genotyping were funded by the CORUS program
(Coopération pour la Recherche Universitaire et Scientifique) from
the French Ministry of Foreign Affairs, by the BRG Programme
(Bureau des Ressources Génétiques, France) and by the INRA
Department of Animal Genetics.
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GK Dayo is a doctoral student working on the identification of
loci underlying trypanotolerance in cattle. The research interests
of S. Thevenon, D. Berthin and G. Cuny include the study of the
trypanotolerance in cattle. C. Denis participated to the project as a
laboratory technician. K. Moazami-Goudarzi’s research interests
include livestock biodiversity characterization. A. Eggen has
research interests in cattle structural genomics. M. Gautier is
interested in the genetic mechanisms of adaptation.
Supporting information
Additional Supporting Information may be found in the online
version of this article:
Table S1 Allelic richness, observed and expected heterozygosities
for each locus/breed.
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