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Sequence analysis of the Equine ACTN3 Gene in Australian horse breeds

K.C. Thomas, N.A. Hamilton, K.N. North, P.J. Houweling

PII: S0378-1119(14)00035-3DOI: doi: 10.1016/j.gene.2014.01.014Reference: GENE 39384

To appear in: Gene

Accepted date: 6 January 2014

Please cite this article as: Thomas, K.C., Hamilton, N.A., North, K.N., Houweling, P.J.,Sequence analysis of the Equine ACTN3 Gene in Australian horse breeds, Gene (2014),doi: 10.1016/j.gene.2014.01.014

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Sequence analysis of the Equine ACTN3 Gene in Australian horse breeds.

KC Thomas1, NA Hamilton

2, KN North

1,3,4, PJ Houweling

1,3,4

Affiliations:

1. Institute for Neuroscience and Muscle Research, Children’s Hospital at

Westmead, Sydney, NSW, Australia.

2. Faculty of Veterinary Science, University of Sydney, Sydney, NSW, Australia.

3. Murdoch Childrens Research Institute, The Royal Children’s Hospital,

Melbourne, VIC, Australia.

4. Discipline of Paediatrics and Child Health, Faculty of Medicine, University of

Sydney, Sydney, NSW, Australia.

Corresponding Author

Prof Kathryn North

Director, Murdoch Children’s Research Institute (MCRI)

Telephone: +61 3 8341 6226

Fax: +61 3 9348 1391

Email: kathryn.north@mcri.edu.au

Abstract..................................................................................................................... 2

1. Introduction ...................................................................................................... 2

2. Methods: ........................................................................................................... 4

2.1 Animals ...................................................................................................... 4

2.2 DNA Extraction and Whole Genome Amplification ..................................... 5

2.3 Polymerase Chain Reaction (PCR) .............................................................. 5

2.4 Sequencing and Promoter prediction analysis ............................................ 6

3. Results/Discussion ............................................................................................. 8

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Abstract

The sarcomeric α-actinins, encoded by the genes ACTN2 and ACTN3, are

major structural components of the Z-line and have high sequence similarity. α-

Actinin-2 is present in all skeletal muscle fibres, while α-actinin-3 has developed

specialized expression in only type 2 (fast, glycolytic) fibres.

A common single nucleotide polymorphism (SNP) in the human ACTN3 gene

(R577X) has been found to influence muscle performance in elite athletes and the

normal population. For this reason, equine ACTN3 (eACTN3) is considered to be a

possible candidate that may influence horse performance. In this study, the

intron/exon boundaries and entire coding region of eACTN3 has been sequenced in

five Australian horse breeds (Thoroughbred, Arabian, Standardbred, Clydsdale and

Shire) and compared to the eACTN3 GenBank sequence. A total of 34 SNPs were

identified, of which 26 were intronic and eight exonic. All exonic SNPs were

synonymous; however, five intronic SNPs showed significant differences between

breeds. A total of 72 horses were genotyped for a SNP located in the promoter

region of the eACTN3 gene (g. 1104 A>G) which differed significantly between breed

groups. We hypothesize that this polymorphism influences eACTN3 expression and

with further studies may provide a novel marker of horse performance in the future.

Key Words: Horse, polymorphism, ACTN3, α-actinin-3.

1. Introduction

The contractile apparatus of skeletal muscle (the sarcomere) is composed of

highly organized thick (myosin containing) and thin (actin containing) filaments,

anchored to the three-dimensional Z-line. The sarcomeric α-actinins (ACTN2 and

ACTN3) are major components of the Z-line and are highly analogous (81% identical)

to each other (Beggs et al., 1992, Blanchard et al., 1989, MacArthur and North,

2004). Importantly, α-actinin-3 has developed specialized expression in type 2 (fast,

glycolytic) muscle fibres which are required for fast muscle contraction, whereas α-

actinin-2 is present in all skeletal muscle fibres (MacArthur and North, 2004, Mills et

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al., 2001). The α-actinins are thought to perform a static function in skeletal muscle,

maintaining the myofibrillar network as well as coordinating muscle contraction (Lek

et al., 2010, MacArthur and North, 2004).

In humans, a common nonsense polymorphism in the α-actinin-3 gene (ACTN3

R577X) has been identified (North et al., 1999). Homozygosity for the ACTN3 577 X-

allele (577XX) results in complete absence of α-actinin-3 in approximately 16 - 18%

of humans worldwide (Yang et al., 2003). We have previously shown that ACTN3

genotype significantly influences elite athletic performance; elite sprint athletes

have a significantly decreased frequency of α-actinin-3 deficiency compared to

healthy non-athlete controls (Yang et al., 2003). This association has been replicated

in elite athletes from around the world (Niemi and Majamaa, 2005, Papadimitriou et

al., 2008, Ahmetov et al., 2008, Druzhevskaya et al., 2008, Santiago et al., 2008, Roth

et al., 2008, Eynon et al., 2009). In contrast, α-actinin-3 deficiency is associated with

an increased response to resistance training (Clarkson et al., 2005) and a higher

frequency of α-actinin-3 deficient endurance athletes (Eynon et al., 2009, Yang et al.,

2003), suggesting that the loss of α-actinin-3 is beneficial under specialized

circumstances. Similar findings have been demonstrated in non-athletes, with α-

actinin-3 deficiency significantly associated with slower 40m sprint times (Moran et

al., 2007), lower isometric maximal voluntary muscle contractions (Clarkson et al.,

2005, Vincent et al., 2007, Walsh et al., 2008), reduced muscle mass (Delmonico et

al., 2007, Walsh et al., 2008, Zempo et al., 2010) and decreased fast fibre area

(Vincent et al., 2007).

The sarcomeric ACTN genes are highly conserved across species. In the horse,

equine ACTN3 (eACTN3) has been mapped to chromosome 12, is 13.2 kb long and

contains 21 exons with a coding region of 3.47 kb and an open reading frame of

2709 base pairs (bp) (Mata et al., 2012).

The horse has been selected for specific athletic phenotypes/traits including

sprint, endurance, and strength. In 2009, a genome wide study for positive selection

in Thoroughbreds identified the sarcomeric α-actinins (ACTN2 and ACTN3) as

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potential target genes for athletic performance (Gu et al., 2009). Concurrently,

(Schröder et al., 2011) identified ACTN3 among 27 other potential candidate genes

that may provide insight into equine performance. Expanding on the 2009 study, (Gu

et al., 2010) identified four SNPs in intron 7 of eACTN3, which were not found to be

associated with racing performance of elite Thoroughbreds. More recently,

sequence analysis of the entire eACTN3 coding region in four French horse breeds

(French trotter, French Saddlebred, Thoroughbred and Cob Normand) identified

eight SNPs (six coding and two in the 3’ non-coding region) (Mata et al., 2012).

Taken together, each of these studies failed to identify polymorphisms in

eACTN3 that were associated with altered muscle performance in horses. However,

to date, no studies have examined the ACTN3 gene in Australian horse populations,

or in elite endurance (Arabian) or heavy draught breeds. The aim of this study was to

sequence eACTN3 in Australian horses and identify genetic variants that segregate

between breeds selected for specific performance traits.

2. Methods:

2.1 Animals

Five horse breeds (Thoroughbred, Arabian, Standardbred, Shire and Clydesdale)

from the Australian horse population were selected for this study. Four distinct

groups were established to assess genetic differences between their proven abilities.

Group (1) Sprint: Thoroughbreds (n = 39) were selected as proven performers over

short (1000 – 3200 m) distances which are completed at a fast gallop, with the

Australian horse racing industry preferentially selecting for shorter/faster race

events. Group (2) Endurance: the Arabian horse breed (n = 13) was chosen as proven

long distance performers. All Arabian horses genotyped in this study were successful

performers over 80 – 160 km events. Typically Arabian horses are leaner than

Thoroughbreds and races are completed at a fast trot rather than a gallop. Group (3)

Pace: the Standardbred (n = 13), which undertake harness races and perform over

shorter distances (1609 – 2650 m) at a pace (move both legs on the same side

together, rather than a gallop) while pulling a cart and driver were selected as an

intermediary performance breed. Finally, Group (4) Strength: consisting of Shire (n =

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1) and Clydesdales (n = 6) which are considered to be traditional heavy horses that

excel at slow powerful work.

2.2 SNP discovery cohort

The eACTN3 gene was initially sequenced in a total of 15 horses consisting of a

sub cohort of Thoroughbred (n = 5), Arabian (n = 5), Standardbred (n = 3) and

Clydesdales (n = 2). Following the identification of a significant SNP in the 5’UTR, the

remaining individuals (n = 57) were sequenced for this region and further analyses

performed.

2.3 DNA Extraction and Whole Genome Amplification

DNA was extracted from fresh blood as previously described (Montgomery and

Sise, 1990) or from frozen blood samples using a QIAamp® DNA Blood Mini Kit

(Qiagen) as per the manufacturer’s instructions.

Whole Genome Amplification (WGA) was required to ensure sufficient amounts

of DNA were available for each animal to complete the study. The Illustra GenomiPhi

V2 DNA Amplification Kit (GE Healthcare) was used to amplify extracted genomic

DNA. A minimum of 10ng of genomic DNA for each individual was used to ensure

accurate amplification. The quality of the WGA product was evaluated on a 1%

agarose gel. The samples were stored at -20˚C and diluted for all subsequent

polymerase chain reactions (PCR).

2.4 Polymerase Chain Reaction (PCR)

Polymerase Chain Reaction (PCR) was used to amplify each region of the eACTN3

gene prior to Sanger sequencing. Primers were designed to amplify all 21 exons

using Primer-BLAST (Rozen and Skaletsky, 1999) based on the available eACTN3 gene

sequence (GenBank accession: HQ005425) (Table 1). Specificity of primers to the

eACTN3 gene was tested using nucleotide BLAST (BLASTn) (Altschul et al., 1990). PCR

was performed using AmpliTaq Gold DNA Polymerase (Invitrogen). The number of

cycles, MgCl₂ concentration (mM), annealing temperature (Tm) and primers used

were dependent on the primer optimization (Table 1). Each PCR was performed in

20μL reaction with 2μL of WGA DNA used as a template. PCR was performed using a

Veriti 96 well PCR thermal cycler (Applied Biosystems). All samples were subjected to

a 5 min 95˚C polymerase activation step, followed by 35-40 cycles of denaturing

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(95˚C for 15 seconds), annealing (60 - 65˚C for 15 seconds) and extension (72˚C for 1

minute). After thermal cycling, samples were subjected to a final extension (72˚C for

10 minutes). PCR product size (539 - 696bp) and quality were assessed using 2%

agarose gel electrophoresis with ethidium bromide and a size standard DNA ladder

(Hyperladder I, Bioline).

2.5 Sequencing and Promoter prediction analysis

PCR products were purified using SAP (Shrimp Alkaline Phosphatase in buffer;

Promega) and Exonuclease I (New England Biolabs) prior to Sanger sequencing at the

Australian Genome Research Facility (AGRF, Westmead, Australia). Sequences were

aligned to the eACTN3 gene (GenBank accession no. HQ005425) and SNPs identified

based on differences between the template and individuals DNA using Sequencher

5.1 (GeneCodes). All primer and SNP reference positions have been determined

using the available eACTN3 GenBank entries (Genomic - accession no. HQ005425

and amino acid - accession no. NP_001157341.1).

Exonic SNPs were compared with eACTN3 mRNA (GenBank accession no.

NM_001163869.1) and protein (GenBank accession no. BAD18923.1) sequences to

identify the location and infer possible functional changes.

ACTN3 gene sequences were obtained for the cow, dog, pig, orangutan,

macaque, human and chimpanzee using Ensembl (Birney et al., 2004) and aligned

using BLASTn (Altschul et al., 1990) and ClustalW (Larkin et al., 2007) to infer the

level of gene homology. In silico analysis of the eACTN3 promoter region was

performed using the online prediction package, RegRNA (Bengert and Dandekar,

2004) http://regrna.mbc.nctu.edu.tw/html/about.html).

2.6 Statistical analyses

Allele and genotype frequencies, along with observed and expected

heterozygosity and Hardy-Weinberg Equilibrium (HWE) were calculated using

Haploview 4.2 software (Barrett et al., 2005). A two-sided Fisher’s Exact Test was

performed using the statistical analysis package, R (Ihaka and Gentleman, 1996) to

assess for significant differences in allele and genotype frequency between the

breed groups.

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Table 1: PCR primer specifications

Exon Name Position Primer Sequence (5'-3') MgCl2

(mM)

Tm

(˚C)

Size

(bp)

1 A35'F 894 - 912 GCTTTCCCAAGGTCACACA 1 62 605

A3I1R 1479 - 1498 AGGCTCCCAGTCCTCTCTTC

2, 3 A3I1F 2227 - 2246 GACCCCATTTCTCCTCTTCC 2 60 719

A3I3R 2927 - 2946 GTCGGCACTTTGGGTACATT

4, 5 A3I3F 3067 - 3086 TGGCTGACAAGTGGTGTAGG 1 65 696

A3I5R 3742 - 3762 CCCTCCTCATGGATTCTCTCT

6, 7 A3I5F 3864 - 3883 AGGGGAGGTGAAGGAATTGT 1 60 611

A3I7R 4456 - 4474 GCAGGGGGATGAGAGAGAG

8 A3I7F 4744 - 4763 TGGAGTCATGGGAGGGTTAG 2 60 593

A3I8R 5316 - 5336 GAGGGGAGCACATCTTTTAGG

9 A3E8F 5065 - 5082 CCAAAGCCCGATGAGAAG 1 65 615

A3I9R 5316 - 5336 GGTGAAGATGGCAGGAGAAG

10 A3I9F 5787 - 5806 GCTCTGTGCCGACCTCTACT 1 60 630

A3E11 6396 - 6416 CCTCATAGCCCTTCTCCACTT

11 A3E10F 6091 - 6110 TGGAGGACTTTCGGGACTAC 2 60 508

A3I11R 6581 - 6598 GCCCTGACCTGTGCATTT

12 A3I11F 7447 - 7465 GGTAGACTGGGCAGGTGTG 2 60 520

A3I12R 7949 - 7966 CCGGTGGGGAAGAGTATG

13, 14 A3I12F 8140 - 8159 TTGAGGGATTGGAGTTGAGG 2 60 629

A3I14R 8749 - 8768 CACAGGAGTGGCTGATAGGA

15 A3I14F 8901 - 8920 CTGTTGCCTGTGGGAAGTGT 2 65 546

A3I15R 9427 - 9446 CATCTGTGAGAGGGGGTCAG

16, 17 A3I15F 9443 - 9464 GATGAGGAAACGAGCCTAGAGA 2 65 687

A3I17R 10092 - 10111 CCTAGAGCAGACGTGCTGTG

18, 19 A3I17F 10190 - 10209 ACCAGGCCAGGATTGGCCGT 2 65 853

A3I19R 11021 - 11042 GCCCCCTGTGGCTCATTGGTTC

20, 21 A3I19F 11228 - 11246 ATGGTGTCTGCCGTCTGTC 2 65 539

A3’R 11747 - 11766 AGCACTTGGCTGGTCATTCT

Optimal PCR conditions for each primer pair, including the exon it contains, position

(Genomic - accession no. HQ005425), forward and reverse primer sequences, MgCl2

concentration, annealing temperature (Tm) and PCR product size.

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3. Results/Discussion

Using the publicly available eACTN3 sequence obtained from the horse

genome project we established 14 primer pairs (Table 1) to sequence the eACTN3

gene in the genomic DNA of five Australian horse breeds.

Recently the genetic diversity of modern horses has been examined in

greater detail. Using genome-wide SNP analyses, the within- and among- breed

diversity has been assessed (Petersen et al., 2013). Using this data we established

four groups (Sprint, Endurance, Pace and Strength) based on the genomic

information and proven athletic performance of the five selected horse breeds.

WGA genomic DNA from 15 horses (Thoroughbred (n = 5); Arabian (n = 5),

Standardbred (n = 3) and Clydesdale (n =2)) was initially used to sequence the entire

eACTN3 coding region and surrounding intron/exon boundaries for SNP discovery.

This equated to approximately 7,546 base pairs (bp) of ACTN3 sequence per

individual. Due to the conserved nature of the ACTN3 gene, low genetic variability

was expected within these populations. In the domestic horse population SNPs are

estimated to be present every 644-891 bp (Orlando et al., 2013), which corresponds

to 8 - 12 SNPs within the eACTN3 gene sequence obtained from this analysis. A total

of 34 SNPs were identified, of which six were exonic and 28 were non-coding (27

base changes and 1 single base insertion/deletion) (Table 2). All exonic SNPs

identified in this study were synonymous and either corresponded to previously

characterized polymorphisms, as outlined in Table 2, or were novel polymorphisms

in this population.

The exonic SNPs were assigned to mRNA conserved domains; E1 (g. 2742

C>T) and E2 (g. 4264 G>T) are located within the calponin homology domain, E3 (g.

6206 G>A) and E4 (g. 8427 A>G) within the spectrin repeats and E5 (g. 11304 C>T)

and E6 (g. 11517 A>G) within the two EF-hands (Marchler-Bauer et al., 2011).

Of the non-coding SNPs, two (E7 g. 11649 T>C & E8 g. 11730 T>C) occurred

after the stop codon and therefore the effect on the amino acid could not be

evaluated. In silico microRNA analyses performed by Mata et al., (2012) using

targetscan.org did not identify any predicted microRNA sites. Replication of this

analysis using MicroRNA.org (Betel et al., 2008), also failed to identify any novel

microRNA sites in this analysis. As suggested by Mata et al., (2012) no significant

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effects on gene/protein function are expected from these changes. Although an

underlying role in gene transcription may yet be identified.

Previous studies focused on cDNA sequence analysis and thus could not

assess possible intronic/splice site polymorphisms. The non-coding nature of the

remaining SNPs makes inferring functional implications difficult. However, five

intronic SNPs showed significantly different frequencies between the breed groups

(Table 3) and a novel SNP located in the 5’UTR (g. 1104 A>G) showed the most

significant difference between the selected breeds.

A total of 39 Thoroughbreds, 13 Arabians, 13 Standardbreds, 6 Clydesdales

and 1 Shire horse have now been genotyped for the 5’UTR g. 1104 A>G

polymorphism. Allele and genotype frequency analyses showed significant

segregation between individuals when separated into their proven athletic tasks

(Sprint, Endurance, Pace and Strength) (Table 4); with an overrepresentation of the

AA genotype in the Clydesdales and Shire horses (AA>86%; AG>14%; GG>0%), and

Standardbreds (AA>54%; AG>31%; GG>15%) compared to both Thoroughbred

(AA>0%; AG>33%; GG>67%) and Arabian (AA>23%; AG>31%; GG>46%) horse breeds

(Figure 1).

The location of the g. 1104 A>G SNP within the promoter region (5’UTR),

33bp from the transcription start site, may result in altered gene expression.

However, the ACTN3 promoter is yet to be fully characterized. In silico analysis of the

eACTN3 5’UTR identified a putative exon enhancer or riboswitch at position 550 to

557 when the A allele (5’ GCCGACG 3’) is present. This predicted enhancer is lost

following conversion to the G allele (5’ GCCGGCG 3’). The presence of this predicted

binding domain suggests that this region may be involved in controlling eACTN3 gene

expression. No additional regulatory sites were predicted within the region of this

polymorphism.

The sequence surrounding this SNP shows a high degree of genetic

conservation across several species (human, horse, primate, bovine and canine)

(Supplementary Figure 1), suggesting that this region is functionally important. We

hypothesize that replacing the 1104 G allele may result in altered eACTN3 gene

expression that could contribute to the muscle performance differences between

these horse breeds. No Thoroughbreds were homozygous AA at position 1104 which

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suggests that the A-allele could be detrimental in sprint performance. However,

segregation between the five horse breeds and targeted selection for specific

performance traits (Sprint, Endurance and Strength) may also explain the genotype

frequency difference observed in this publication.

All five breeds studied in this publication have been maintained under strict

stud conditions and as closed population for as long as 222 years in the case of the

Thoroughbred (Stud Book started in 1791). While the Standardbred (1871),

Shire/Clydesdales (1877 and 1879, respectively) and Arabian (1908) studs range from

142 to 105 years old (Petersen et al., 2013). There is a clear difference in the

proportion of the g. 1104 A>G SNP - with absence of the A allele in the Australian

Thoroughbred population and G allele in the Clydesdale and Shire population. While

the Thoroughbred has been maintained as a closed population for the longest period

of time the level of individual inbreeding (f) has recently been determined across

these populations, with the Clydesdales and Shire horses showing the greatest

(0.261 and 0.187, respectively), followed by the Thoroughbred (0.134), Standardbred

(0.130) and finally Arabian (0.060) showing the lowest level of individual inbreeding

(Petersen et al., 2013). The common hallmarks of inbreeding and selection (i.e.

fixation of the rare allele) are difficult to separate without functional analyses. It is

therefore important to note that even though the g. 1104 A>G allele frequency is

significantly different between the populations examined; it is currently not clear

weather this is a result of inbreeding/random genetic drift or positive selection.

The intensive selection pressure, based on performance and body type,

which has been placed on these breeds may have resulted in the altered allele

frequency observed in this publication – either by chance (due to random genetic

drift) or as a result of a functional impact of this SNP on muscle performance.

Additional studies are required to identify functional changes in eACTN3 expression

as a result of the g. 1104 A>G SNP or any of the other non-coding SNPs identified in

this publication. These novel SNPs may provide a useful marker of muscle

performance in the future while improving our understanding of the functional

domains in the promoter and non-coding regions of ACTN3.

List of Figures/Tables:

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Figure 1: A) Allele and B) Genotype frequency for the eACTN3 5’UTR g. 1104 A>G

SNP in Sprint (Thoroughbred n = 39), Endurance (Arabian n = 13), Pace

(Standardbred n = 13) and Strength (Clydesdale (n = 6 and Shire n = 1) horses.

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Strength/power

A

G

23%

77%

A

BStrength/power

Clydesdale/Shiren = 7

14%

86%

AA

AG

GG

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Table 2: Coding and non-coding SNPs identified in the eACTN3 gene of

Thoroughbred, Arabian and Standardbred, Clydesdale and Shire horses.

Location SNP I.D. EquCab 2.0

Position

Reference

Position*

SNP Amino Acid

Position2

Substitution

Type

SNP previously

reported

5'UTR I1 26511704 1104 A/G

Intron 2 I2 26515793 2593 C/G Equcab 2.0

(Wade et al., 2009)

(rs68947239)

I3 26515795 2595 C/T Equcab 2.0

(Wade et al., 2009)

(rs68947240)

I4 26515807 2607 T/C

I5 26515885 2685 A/T Equcab 2.0

(Wade et al., 2009)

(rs68947241)

Exon 3 E1 26515942 2742 C/T 105 Synonymous Equcab 2.0

(Wade et al., 2009,

Mata et al., 2012)

(rs68947242)

Intron 3 I6 26516020 2820 G/C (Wade et al., 2009)

(rs68947243)

I7 26516067 2867 C/T

I8 26516386.1 3186.1 T Insert

I9 26516432 3232 G/C

Intron 5 I10 26517248 4048 C/T

Exon 7 E2 26517464 4264 G/T 226 Synonymous

Intron 7 I11 26518079 4879 C/T (Gu et al., 2010)

I12 26518108 4908 G/A

I13 26518110 4910 T/C

I14 26518227 5027 G/A (Gu et al., 2010)

I15 26518241 5041 C/T

Exon 10 E3 26519406 6206 G/A 366 Synonymous

Intron 10 I16 26519479 6279 G/C

Intron 11 I17 26519722 6522 C/T

Intron 13 I18 26521510 8310

A/G

Exon 13 E4 26521627 8427 A/G 506 Synonymous

Intron 16 I19 26522964 9764 G/A

I20 26522973 9773 G/A

I21 26522983 9783 G/A

I22 26522989 9789 G/A

I23 26523003 9803 A/G

Intron 17 I24 26523230 10030 C/T

I25 26523237 10037 G/T

I26 26523267 10067 G/C

Exon 21 E5 26524504 11304 C/T 814 Synonymous (Mata et al., 2012)

E6 26524717 11517 A/G 858 Synonymous (Mata et al., 2012)

3’ UTR E7 26524895 11649 T/C 917 (Mata et al., 2012)

E8 26524930 11730 T/C 929 (Mata et al., 2012)

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Intronic and exonic SNP information in the eACTN3 gene including location,

nucleotide position (EquCab 2.0 and reference position) and nucleotide substitution.

1Reference position refers to the position in GenBank (accession no. HQ005425).

2

Amino acid positions refer to the location in NCBI Reference Sequence

NP_001157341.1.

Table 3: SNP Analysis –Comparisons between each breed show five statistically

significant SNPs across the eACTN3 gene.

Table showing the observed and expected heterozygosity (Hetero.), Hardy-Weinberg

equilibrium (HWE) P-value, minor allele frequency (freq.) and the Fisher’s Exact test

when comparing the frequencies between breed groups for each SNP. 1 Analyses

performed using the SNP discovery cohort consisting of Thoroughbred (n = 5),

Arabian (n = 5), Standardbred (n = 3) and Clydesdales (n = 2). 2 Analysis of the full

cohort (n = 72) was carried out for the 5’UTR g. 1104 G>A polymorphism. 3

Values

obtained in Haploview 4.2 analysis. 4

Values obtained in R using a Fisher’s Exact Test.

* Significantly different frequencies then expected (HWE P<0.05 and Fisher’s Exact

Test P<0.05).

Table 4: The 5’UTR g. 1104 G>A SNP is not out of Hardy-Weinberg equilibrium

when analysed within each breed. Thoroughbred (n = 39), Arabian (n = 13),

Standardbred (n = 13) and Clydesdale (n = 6) / Shire (n = 1) horses.

SNP

I.D.

Position Allele Number

of

horses

Observed

Hetero.3

Expected

Hetero.3

HWE

P-

value3

Minor

Allele

Freq3

Fisher’s

P-

value4

I1 1104 A:G

151

722

0.333

0.319

0.491

0.472

0.3867

0.0104

0.433

0.382

0.035*

2.09e-

10

I8 3186.1 T

insert

15 0.000 0.426 0.0009* 0.308 0.003*

I18 8310 A:G 15 0.182 0.463 0.1176 0.364 0.003*

I23 9803 A:G 15 0.133 0.498 0.0100* 0.467 0.037*

I24 10030 C:T 15 0.000 0.231 0.0077* 0.133 0.023*

Breed Degrees

of

freedo

m

Allel

e

Numbe

r of

horses

Mino

r

Allele

Freq 1

Observe

d

Hetero.1

Expecte

d

Hetero.1

HWE

P-

value1

Thoroughbred 1 G:A 39 0.167 0.333 0.278 0.603

5

Arabian 1 G:A 13 0.385 0.308 0.473 0.401

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Analyses performed within breeds using Haploview 4.2.

Table 5: 5’UTR g. 1104 A>G allele frequency comparisons between Sprint

(Thoroughbred n = 39), Endurance (Arabian n = 13), Pace (Standardbred n = 13) and

Strength (Clydesdale and Shire n = 7) horse populations.

Allele Frequency

Comparison

Degrees of

freedom

Chi-Squared P-value Fisher’s Exact P-value

Thoroughbred vs. Arabian 1 0.04074 0.0291

Thoroughbred vs.

Standardbred

1 1.386e-6

1.338e-6

Thoroughbred vs.

Clydesdale/Shire

1 3.642e-8

5.525 e-8

Arabian vs. Standardbred 1 0.05151 0.002844

Arabian vs. Clydesdale/Shire 1 2.844 e-3

9.431e-4

Clydesdale/Shire vs.

Standardbred

1 0.1902 0.1243

Values obtained in R using a Fisher’s Exact Test. Significantly different frequencies

than expected are in bold (HWE P<0.05 and Fisher’s Exact Test P<0.05).

Acknowledgements:

This project was funded in part by the Australian Research Council (ARC) grant

DP120100754.

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List of Abbreviations: ACTN3 – alpha-actinin-3 eACTN3 – equine alpha-actinin-3 SNP – single nucleotide polymorphism R – arginine X – stop codon UTR – untranslated region ng – nanograms PCR – polymerase chain reaction mM – milli molar TM – annealing temperature bp – base pair SAP – shrimp alkaline phosphatase HWE- Hardy-Weinberg equilibrium Hetero – heterozygosity

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Highlights

• The alpha-actinin-3 gene represent a candidate for performance in the horse • Thoroughbred, Arabian, Standardbred and Clydesdale/Shire horses perform

differently • Equine ACTN3 was sequence in all five breeds • A SNP in the 5’UTR segregates between these breeds