Genomics for African Cattle
Challenges and Opportunities
The East African shorthorn zebu story
Mary Mbole-Kariuki, Gemma Murray, Hussain Bahbahani,
Abdulfatai Tijjani, Olivier Hanotte
ILRI, Addis Ababa, 24 March 2016

Huge increases over the 2005/7 amounts
of cereals, dairy and meat will be needed by 2050
From 2bn to 3bn
tonnes cereals each year
From 664m to 1bn
tonnes dairy each year
From 258m to 460m
tonnes meat each year

High growth systems with externalities is at one end of the rapid growth spectrum where
dynamic markets, IT, investment capital, infrastructure and increasingly skilled human resources
result in fast-changing small-scale livestock systems. NOT OUR CLIENTS
Rapid inclusive growth systems address the need to develop sustainable food systems that
deliver key animal-source nutrients to the poor while facilitating a structural transition in the
livestock sector of developing countries. Mostly the transition is from many smallholders
keeping livestock in low-productive systems to eventually fewer households raising more
productive animals in more efficient, intensive and market-linked systems. Productivity traits -
Adaptive traits.
Fragile growth systems recognize that rapid, market-focused growth will not be the trajectory
for all poor livestock keepers. In areas where productivity is severely limited by remoteness,
harsh climates or environments, or by poor institutions, infrastructure and market access,
much emphasis will be to enhance the important roles of livestock in the resilience of people and
communities to environmental variability.
Productivity traits - Adaptive traits.
Trajectories for the livestock sector
and opportunities for genomics
*Smith, J.W., Tarawali, S., Grace, D. and Sones, K. 2013. Feeding the
world in 2050: Trade‐offs, synergies and tough choices for the
livestock sector. Tropical Grasslands - Forrajes Tropicales 1(2): 125-
136.

The largest dairy herd in the world (Al Karj – Kingdom of Saudi Arabia) ?
67,000 dairy cow heads – 2,5 million liters per day ( 40 l/day/per cow)
Almarai Dairy Farms
Sustainability ….
High growth with externality

Rapid inclusive growth systems
Small-holders farmers from Western Kenya
Milk
East Coast Fever
Milk

Fragile growth systems E.g. Pastoral systems
Maximize Adaptive Diversity

Genomic applied to livestock systems: no one-size-fit
• High growth Classical
Genomics
• Strong growth
• Fragile growth
“Innovatives”
genomics
approaches

© Dan Bradley

Felius
1995

X
X
Hanotte et al. Science 2002
Microsatellites study (15)
Hanotte et al. Mol. Ecol 2000
PC1PC1
InterpolationInterpolation
mapmap
38 %38 %
PC1PC1
InterpolationInterpolation
mapmap
38 %38 %
PC2PC2
InterpolationInterpolation
mapmap
9.5 %9.5 %
PC2PC2
InterpolationInterpolation
mapmap
9.5 %9.5 %
PC3PC3
InterpolationInterpolation
mapmap
6.6 %6.6 %
PC3PC3
InterpolationInterpolation
mapmap
6.6 %6.6 %

To remember…..
From:
Bradley et
al.
1996
Taurine and zebu cattle - common ancestor
mtDNA data: 117,000 - 275,000 BP (Bradley et al., 1996)
Microsatellite data: 610,000 - 850,000 years (MacHugh et al., 1997)
African and European taurine have a common maternal ancestry in the
Near East (Bonfiglio 2012)
First African cattle were of the taurine type
Majority of African cattle are crossbreed zebu x taurine (Hanotte et al.
2000, 2002)
‘All’ African cattle (zebu, taurine, crossbreed) have a taurine mtDNA
(several studies) but both zebu and taurine Y chromosomes are present on the
African continent (Hanotte et al. 2000)
African wild auroch Bos primigenius africanus introgression is a possibility …..
(J.E. Decker et al. 2014)

The East African Shorthon Zebu (EASZ) genomics
story

East African
Shorthorn Zebu
Non – descript
Outbreed – Panmictic (?)
‘Human selection’ (?)
Natural selection (?)
The today genome of the East African Shorthorn
Zebu is a legacy of a thousands of years of admixture,
selection and adaptation, genetic drift……

Funded by the Wellcome Trust 2007 - 2013

IDEAL Project: Understanding diseases burden in tropical
cattle including the effect of co-infections
Recruitment criteria: No F1 – crosses
with European taurine
(farmers’ interviews)
552 calves:
88 (16%) died ECF, heartwater,
helminthosis
243 (44%) with clinical
episodes/illness
221 (40%) healthy survivors
Breeding improvement

EASZ Genomics – data
548 calves + (reference breeds)
Full genome sequencing : 10 calves (5 dead/
5 alive) + (reference breed UMD 3.1)
Mapping of
genomic and
environmental
diversity
Diversity of livestock genotypes
Human selection
Natural selection
Vr.1
114 calves + (reference breeds)
Exomes: 10 animals ECF targeted
(resistance – susceptible)

18
Breeds – population others
Illumina BovineSNP50 chip
Holstein = 63, Jersey = 28, N’Dama = 25, Sheko = 20
Nelore = 21
__________
Illumina BovineHD Genotyping BeadChip
Holstein = 59, Jersey = 32, N’Dama = 24, Nelore = 34,
Gir = 28, Sheko = 18
Nigerian zebu: A.Gudali = 23, Azwaka = 1, Bunaji = 23,
Red bororo = 22, S. Gudali = 21, Wadara
= 2, Yakanaji = 12
Ugandan zebu: Ankole = 25, Karamojong = 16,
Nanda = 23, Serere = 12

The East African
Shorthorn Zebu
(EASZ) story ……..
The genetic make-up
Coat patterns
Mary Ndila PhD
2008-2012
Mbole-Kariuki MN et al.(2014). Genome-wide analysis reveals the ancient and recent admixture history
of East African Shorthorn Zebu (EASZ) from Western Kenya. Heredity 113, 207-305.

EASZ GENOME ‘ADMIXTURE’ ANALYSIS: PCA
EASZ
Sheko
N’dama
Nelore
Jersey
Holstein
PC 1 (65% variation)
PC2(14%variation)

EASZ GENOME ADMIXTURE ANALYSIS STRUCTURE
-23000000
-22500000
-22000000
-21500000
-21000000
-20500000
-20000000
-19500000
0 1 2 3 4 5 6 7 8
MeanEst.LnProbofdata
K
MEAN L
Optimal K = 3 - 4
European taurine introgression
‘Shared African – European taurine’ background in Jersey

Individual chromosome admixture
East African Shorthorn Zebu Friesian N’dama Sheko
Jersey Nelore

Jersey
Sheko
‘AT’ background in Sheko or Jersey
AT’ in EASZ
Selection and/or genetic drift
Examining only the non-
European
taurine introgressed
animals (n = 425)
Show an ‘even’
zebu-taurine
ancestry (0.84/0.16),
s.d. 0.009) with
significant difference
in ‘African’ taurine and
zebu background across
chromosomes (P <
0.0001).
Strong correlation
betweeb EASZ and
Sheko but not between
EASZ and Jersey

Key points
European taurine background:
More evenly distributed across chromosomes
Unevenly distributed across animals
Recent introgression
African taurine - zebu background:
Unevenly distributed across chromosomes
Evenly distributed across animals
Ancient admixture which might have been selected…
M.N. Mbole-Kariuki et al. (2014) Genome-wide analysis reveals the ancient and recent
admixture history East African Shorthorn Zebu (EASZ) from western Kenya. Heredity.

“Substantial”; 2-3 ET generations ago
“Moderate” 6 or more ET generations ago
“Pure” 6 or more ET generations ago
European taurine
introgression

Origin of the European taurine introgression
‘’substantial’’ > 12.5%
Livestock markets

Moderate 4-5 generations 1.56% >x ≤ 12.5%
North-Southgeneticcline
Breeding programmes
• Rural Development Project
(1979-1989) - Kitinda dairy
Bungoma
• Kenyan Dairy project ~1980s
• Kenya – Finland Livestock
• Development Programme
(1991-2003)
Services offered:
AI upon request, in-calf
heifers, bull schemes
Breeds used: Holstein,
Aryshire, Jersey, Guernsey
Impacts:
Offspring born AI :
84,749
Bull schemes: 138,904
Origin of the European taurine introgression
‘’moderate’ , 1.56% < x < 12.5%
r= 0.82 P < 0.0005

The East African
Shorthorn Zebu
(EASZ) story ……..
The effect of European
taurine introgression
Coat patterns
Gemma Murray
MSc 2013
Murray GG et al. (2013). Genetic susceptibility to infectious disease in East African Shorthorn Zebu: a
genome-wide analysis of the effect of heterozygosity and exotic introgression. BMC Evolutionary Biology
13(1), 246 http://www.biomedcentral.com/1471-2148/13/246

Negative effect of European taurine introgression ?
Proportion of ET
background similar
across chromosomes
Apparently no difference in proportion dead whether or not you are
introgressed…..
Origin
r2
P-values
African taurine 0.0089 0.380
European
taurine 0.0074 0.426
Asian zebu 0.0104 0.344
No significant relationships between
age of dead and proportion of ET
background

But the reality is more complex ………….
Murray et al. 2013

Negative associations
between
heterozygosity and
both death and illness
as a result of
infectious diseases
Positive association
between European
taurine introgression
and episodes of
clinical illness.

In other words………
In the EASZ we do find evidences of association
between inbreeding (Ho) and death and/or illness
(clinical episodes)
Introgression with European taurine breeds increase
vulnerability to infectious diseases (outbreeding
depression)
Genetic basis to resistance to infectious
diseases
Better breeding practices
Murray, G.G.R. et al. (2013). Genetic susceptibility to infectious disease in East African
Shorthorn Zebu: a genome-wide analysis of the effect of heterozygosity and exotic
introgression. BMC Evol. Biol. 13, 246.

Crossbreeding
Red: unselected
Purple: selected

Identifying fixed (or nearly) fixed
signature selection could provide insight
into the key genome requirements for
survival as a crossbreed in the African
environments and therefore entry
points to inform crossbreeding
programs with exotic for productivity
improvement…

The East African
Shorthorn Zebu
(EASZ) story ……..
Signature of selection
Coat patterns
Hussain
Bahbahani, PhD
(2012-2015)
H. Bahbahani et al. (2015). Signatures of positive selection in East African Shorthorn Zebu: A
genome-wide SNP analysis. Scientific Reports http://www.nature.com/articles/srep11729

Guadeloupe: Creole admixed ‘’breed’’
European taurine, African taurine
and Asian zebu
2011

Questions …….
- Can we detect signature of selection in the EASZ ?
- How unique are they (EASZ, crossbreed, African) ?
- What is their biological significances (selection
pressures) ?
- What is/are the genetic controls behind these
signatures ?

Signature of selection in East African Shorthorn Zebu cattle
Genome wide scan analysis
QC: MAF= 0.005, SNP call rate = 0.95,
ibs.threshold = 0.95.
45227 Autosomal SNPs
Samples
EASZ: 421 pure calves (18
sublocations)
Friesian: 62 animals
Jersey: 28 animals
Nelore: 21 animals
Sheko: 20 animals
Analysis
Between pair of populations
Between ‘’group of populations
Within population
Allele frequencies / Haplotype
segregation
Extended Haplotype Homozygosity
(EHH)
Identify region with unusually long range
of haplotype homo. and high pop. frequency
|iHs| ‘’integrated Haplotype Score’’
(Voight et al. 2006)
Comparison of EHH between derived and
ancestral alleles
Rbs Across populations (Tang et al. 2007)
Fst analysis: Inter-population Wright’s Fst
(sliding windows 10 SNP, 5 SNP overlaps)
Ancestral states: Bovinae subfamily data
(Decker et al. 2009) applied the
BovineSNP50 13 of the 70 species (Bovinae
subfamily), Most common allele inferred as
ancestral.

Manhattan plots of the pairwise genome-wide autosomal Fst analyses
(A) EASZ with European taurine (Holstein-Friesian, Jersey), (B) EASZ with
African taurine (N’Dama), and (C) EASZ with Asian zebu (Nellore). The significant
thresholds (dashed line) are set at the top 0.2% of the FST distribution.

Genome-wide Rsb analyses: 50K SNP chip
41
EASZ versus Hol,Jer,NDM,NEL
Candidate selected region (green highlight) defined if at least 2 SNPs separated
by less than 700 kb interval passing a threshold of –log10(P-value) = 4
But genome coverage for informative markers ~ 50% Ndila et al . (2014)
9 candidates regions

Following and two extended haplotype homozygosity-based
(iHS and Rsb) analyses 24 candidate genome regions within
14 autosomes and the X chromosome were revealed, in which
18 and 4 were previously identified in tropical-adapted and
commercial breeds, respectively. These regions overlap with
340 bovine QTL (productivity traits). They include 409
annotated genes, in which 37 were considered as candidates.
These genes are involved in various biological pathways (e.g.
immunity, reproduction, development and heat tolerance)
Bahbahani et al. (2015)
Signature of selection in East African Sorthorn Zebu
cattle genome wide scan analysis: Summary of the 50 KB
SNPS chips analysis

Table 1: Candidate regions for signature of positive selection in EASZ (50K SNPs results)1
2
3
Ref: Reference number for previous studies reporting overlapping regions with the identified candidate regions. ** Commercial breeds4
studies. ΔAZ: The average excess/deficiency in Asian zebu ancestry at each SNP calculated by subtracting the average estimated Asian zebu5
ancestry of the SNP from the average estimated Asian zebu ancestry of all SNPs. Bold (deviation by more or less than 1 s.d. from the6
genome-wide mean ΔAZ).7
8
BTA
Position of most significant
SNPs (bp)
Candidate region intervals (bp) Candidate genes Test Ref
Median
ΔAZ
3 101,942,771 101,442,771 - 102,442,771
TMEM53
C1orf228
RNF220
Rsb
16
19
-0.132
7 52,419,683 52,224,595 - 52,720,797 UBE2D2 Fst 19 0.07
12 27,181,474 26,681,474 - 27,681,474 _ Rsb 19 -0.188
12 35,740,174 35,240,174 - 36,240,174 EFHA1 Rsb -0.084
X 9,201,028 8,582,093 - 9,248,137 bta-mir-2483 Fst -0.113
X 40,738,704 39,942,044 - 43,999,854 Metazoa_SRP Fst 46 -0.05
The five regions with zebu ancestry deficiency carry genes involved in
acquired immune response (e.g. IL17D and IRAK1), mRNA processing
regulation (e.g. U5 and U6), and cell cycle regulation (HECTD3). The
candidate region on BTA7, which shows an excess in zebu ancestry,
contains genes associated with critical biological pathways suggested
to be under selection in tropical adapted cattle, such as protein folding
and heat shock response (DNAJC7), and male reproduction and
fertility (SPATA24).
Regions with excess or deficiency in zebu ancestry*
*LAMP analysis: Sankararaman et al. (2008). Estimating local ancestry in admixed populations. Am. J. Hum. Genet. 82, 290-303(2008).

But 50k SNPs chips only provide
partial genome coverage (~ 50%
Ndila et al. 2014)

Genome-wide Rsb analysis - HD SNP chip
45
Rsb (EASZ vs
Hol,Jer,NDM,
NEL, Gir)
-log10 (one-
sided upper tail
p-values)
Candidate selected regions (green highlight) defined if at least 5 adjacent SNPs
not separated by more than 600 kb pass a threshold of –log10(P-value) = 4
23 candidates regions

46
Genome-wide analysis - pooled heterozygosity depression
Hp
- EASZ genome mapped against
UMD 3.1 reference sequence
and SNPs identified
- EASZ autosomes divided into
100 Kb windows (increment 10
Kb)
- Pooled heterozygosity Hp index
was calculated for each window
to calculate the autosomal Hp
mean (Rubin et al., 2010)
- The Hp values were Z-
transformed, and Z-score ≤ - 4
(threshold)

EASZ genome (autosomes)
164 significant selective sweep regions
0
2
4
6
8
10
12
14
16
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
Bovine autosomes
No. of sweep regions
With genes = 130
No genes (gene deserts islands) = 19
No genes but flanked (within 100 kb)
with at least one gene = 15

130 regions = 513 genes
Functional terms enriched using “Functional Annotation Clustering” tool in DAVID:
– Cell-cell adhesion (Enrichment score*= 4.46)
– JAK-STAT signalling pathway and hormone stimuli response (EC = 1.6)
– Regulation of development and cell differentiation (ES = 1.4)
– Immune response regulation (ES = 1.3)
*The overall score for the group based on the EASE score of each term member, modified Fisher Exact test P-value, for
gene-enrichment analysis. The higher the more enriched group.
Immunological-related
genes
- interleukin 17
receptor E-like (Bta
5)
- Leukotriene A-4
hydrolase (Bta 5)
- C-C chemokine
receptor type 7
precursor (Bta 19)
- Interleukin 2
receptor subunit
alpha (Bta 13)
Reproduction and
development-related
genes
- RXFP2 (Bta 12)
- zona pellucida-
binding protein 2
precursor (Bta 19)
- stAR-related lipid
transfer protein 3
(Bta 19)
Thermotolerance-
related gene
- Heat shock protein
beta-9 (Bta 19)
Feeding behaviour-
related gene
- Orexin- A (Bta 19)

Questions …….
- Can we detect signature of selection in the EASZ ? ✔
- How unique are they (EASZ, crossbreed, African) ? ✔
- What is their biological significances (selection
pressures) ? ✔
- What is/are the genetic controls behind these
signatures?

The results support that different selection pressures (e.g.
environmental constraints, human selection, genome
admixture constrains) have shaped the genome of EASZ.
These candidate regions represent genome landmarks to be
maintained in breeding programs aiming to improve
sustainable livestock productivity in the tropics.
The challenge is now to select for these regions in
crossbreeding improvement programs with exotic breeds
‘’genomic selection’’ approach adapted to the small-holder
farmer
Or more targeted approach……
Signature of positive selection in East African Shorthorn
Zebu

52
Illumina BovineHD Genotyping BeadChip
Holstein = 59, Jersey = 32, N’Dama = 24, Nelore = 34,
Gir = 28, Sheko = 18
Nigerian zebu: A.Gudali = 23, Azwaka = 1, Bunaji = 23,
Red bororo = 22, S. Gudali = 21, Wadara
= 2, Yakanaji = 12
Ugandan zebu: Ankole = 25, Karamojong = 16,
Nanda = 23, Serere = 12
Confirming and narrowing down the candidate
selected sweep regions (HD SNPS data)

Selective sweep across breeds approach
From:
Bradley et
al.
1996
HD: 280,613 SNPs
African zebu, taurine, sanga
European taurine and Nelore
African zebu + Nelore
East Africa
Nigeria
Asian zebu
African
taurine
African
zebu
Asian
zebu
Aldufatai Tijjani
EASZ

Rsb Analysis HD data
54
Rsb (EASZ vs
Hol,Jer,NDM,NEL,
Gir)
-log10 (one-sided
upper tail p-
values)
Rsb (Ugandan
zebu vs
Hol,Jer,NDM,NEL,
Gir)
-log10 (one-sided
upper tail p-
values)
Rsb (Nigerian
zebu vs
Hol,Jer,NDM,NEL,
Gir)
-log10 (one-sided
upper tail p-
values)

• Yellow : A candidate peak significant in only EASZ Rsb analysis
• Blue : A candidate peak significant in East (EAZS and Uganda)
African breeds
• Red : A candidate peak significant in East (EAZS and Uganda)
and Nigeria African breeds
Rsb analysis Bta 05

Confirming Rsb Analysis HD data*
56
- EASZ: 23 regions (13 autosomes)
- EASZ and Uganda: 12 regions (10 autosomes)
- EASZ, Uganda and Nigeria: 4 regions (Bta 5, 12, 19)
*Candidate selected sweep regions defined if at least 5 adjacent SNPs not
separated by more than 600 kb pass a threshold of –log10(P-value) = 4

57
EASZ Rsb
~ 50 kb
Legend
~ 300 kb
~ 10 kb
~ 215 kb
Narrowing down the selective sweep regions
Uganda Rsb
Nigeria Rsb
no annotated genes
Bta05
Bta12
Bta19

~ 300 kb
~ 10 kb
Bta05 HD candidate selective sweeps
No genes
6 genes
MSRB3*: - Detected as candidate sweep region in Brahman cattle (Ramey
et al. 2013)
- Related ear morphology and floppiness (Boyko et al 2010,
Vaysse et al. 2011)
Man1** : Critical role in heart development (Ishimura et 2008)
*Methionine sulfoxide reductase B3, **Inner Nuclear Membrane Protein 1

59
EASZ Rsb
~ 50 kb
Legend
Narrowing down the selective sweep regions
Uganda Rsb
Nigeria Rsb
Bta12
B3GLT
L
RXFP2
EHHS plot against position in bp of the most significant SNP at Bta12
(African crossbreeds) – 50 K
HOL, JER, NDM, NEL, Gir

60
Bta12: ~ 50 kb (29,671,166 – 29, 721,207 bp)
B3GALTL is a beta-1,3-
glucosyltransferase that transfers
glucose to O-linked fucosylglycans on
thrombospondin type-1 repeats (TSRs) of
several proteins. Defects in B3GALTL
gene are a cause of Peters-plus
syndrome (PPS).
Peters plus syndrome is an recessive
inherited condition that is characterized by
eye abnormalities, short stature,
developmental delay, an opening in the lip
(cleft lip) with or without an opening in the
roof of the mouth (cleft palate), and
distinctive facial features.
RXFP2: Relaxin/insulin-like family peptide
receptor 2 male fertility, control of the
transabdominal stage of early development
testicular descent and cryptorchidism.
Physiological adaptation (endothermic
mammals)).
Adaptation to HOT CLIMATE (male
reproductive function) ?
★Also involved in horn development and
survival in sheep

Ongoing…..
Abdulfatai Tijjani PhD study…..