Genomics for African CattleChallenges 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 amountsof cereals, dairy and meat will be needed by 2050

From 2bn to 3bntonnes cereals each year

From 664m to 1bntonnes 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 sectorand 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 systemsE.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

Interpolation Interpolation

mapmap

38 %38 %

PC1PC1

Interpolation Interpolation

mapmap

38 %38 %

PC2PC2

Interpolation Interpolation

mapmap

9.5 %9.5 %

PC2PC2

Interpolation Interpolation

mapmap

9.5 %9.5 %

PC3PC3

Interpolation Interpolation

mapmap

6.6 %6.6 %

PC3PC3

Interpolation Interpolation

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 – descriptOutbreed – 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 – crosseswith 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)

New technologies

Mapping of genomic and

environmental diversity

Diversity of livestock genotypes

Human selection

Natural selection

Population genetics structure

Genome association study

Livestock genotype tailored

to the local production

environment

Genomic 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 PhD2008-2012

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)

PC

2 (

14

% v

aria

tio

n)

EASZ GENOME ADMIXTURE ANALYSIS STRUCTURE

-23000000

-22500000

-22000000

-21500000

-21000000

-20500000

-20000000

-19500000

0 1 2 3 4 5 6 7 8

Me

an E

st.L

n P

rob

of

dat

a

K

MEAN LnP(D)

Optimal K = 3 - 4

European taurine introgression‘Shared African – European taurine’ background in Jersey

Individual chromosome admixture

East African Shorthorn Zebu Friesian N’dama ShekoJersey Nelore

Jersey

Sheko

‘AT’ background in Sheko or Jersey

AT’ in EASZ

Selection and/or genetic drift

Examining only the non-Europeantaurine introgressedanimals (n = 425)Show an ‘even’zebu-taurineancestry (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%

No

rth

-So

uth

gen

etic

clin

e

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 schemesBreeds used: Holstein, Aryshire, Jersey, Guernsey

Impacts:Offspring born AI : 84,749Bull 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 Europeantaurine 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 Biology13(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

HussainBahbahani, 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 taurineand 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 cattleGenome wide scan analysis

QC: MAF= 0.005, SNP call rate = 0.95, ibs.threshold = 0.95.

45227 Autosomal SNPs

SamplesEASZ: 421 pure calves (18 sublocations)Friesian: 62 animalsJersey: 28 animalsNelore: 21 animalsSheko: 20 animals

AnalysisBetween pair of populationsBetween ‘’group of populationsWithin 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 (Bovinaesubfamily), 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

3Ref: Reference number for previous studies reporting overlapping regions with the identified candidate regions. ** Commercial breeds 4studies. ΔAZ: The average excess/deficiency in Asian zebu ancestry at each SNP calculated by subtracting the average estimated Asian zebu 5ancestry of the SNP from the average estimated Asian zebu ancestry of all SNPs. Bold (deviation by more or less than 1 s.d. from the 6genome-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 inacquired immune response (e.g. IL17D and IRAK1), mRNA processingregulation (e.g. U5 and U6), and cell cycle regulation (HECTD3). Thecandidate region on BTA7, which shows an excess in zebu ancestry,contains genes associated with critical biological pathways suggestedto be under selection in tropical adapted cattle, such as protein foldingand heat shock response (DNAJC7), and male reproduction andfertility (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 Hpmean (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 (Bta5)

- 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, sangaEuropean taurine and Nelore

African zebu + Nelore

East Africa

Nigeria

Asian zebu

Africantaurine

Africanzebu

Asianzebu

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

B3GLTL

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 transfersglucose to O-linked fucosylglycans onthrombospondin type-1 repeats (TSRs) ofseveral proteins. Defects in B3GALTLgene are a cause of Peters-plussyndrome (PPS).

Peters plus syndrome is an recessiveinherited condition that is characterized byeye abnormalities, short stature,developmental delay, an opening in the lip(cleft lip) with or without an opening in theroof of the mouth (cleft palate), anddistinctive 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…..