GENETIC RELATIONSHIPS BETWEEN HORSE BREEDS BASED ON

MICROSATELLITE DATA: APPLICATIONS FOR LIVESTOCK CONSERVATION

A.M.P. Behara’, D.T. Collins, E.G. Cothran3 and J.P. Gibson1

1 Centre for Genetic Improvement of Livestock, University of Guelph, Guelph, Canada

2 Mann Equitest Inc., Guelph, Canada
3 Department of Veterinary Science, University of Kentucky, Lexington, 40546-0099, USA.

SUMMARY
Data on 11 microsatellite loci were analysed in 903 horses of 11 different breeds to determine the
genetic relationships between breeds and heterozygosity in endangered vs. common breeds.
Neighbor-Joining trees constructed using three different distance measures produced slightly
different topologies. Preliminary analysis of the effect of sample size on clustering patterns
suggests that the resolution of more highly isolated breeds and very closely related breeds is less
dependant on distance measure or sample size, and that sample sizes as small as 30 animals are
sufficient to place isolated breeds over 98% of the time.
Keywords: Equus caballus, conservation, inbreeding, microsatellites, phylogeny

INTRODUCTION
The conservation of genetic variation found in rare breeds of livestock is a growing worldwide
concern due to the increasing risk of loss of these breeds. Breeds of horses which have suffered
a substantial decrease in population size may harbour elevated levels of inbreeding which may
result in inbreeding depression and increase the breed’s risk of extinction (Hedrick 1994).

Microsatellites were chosen to analyse ten popular North American breeds and one breed listed as
endangered (le Cheval Canadian) for levels of inbreeding and’degree of genetic isolation, due to
their high degree of variability resulting from a high mutation rate (eg. Bowcock et al. 1994) and
their ease of amplification by PCR. Their use in equine studies is further facilitated through the
existence of an efficient, highly multiplexed panel developed for parentage testing by Mann
Equitest (Colling and Kelly 1996).

Three genetic distances were compared in this study for their applicability in establishing the
present genetic relationships among livestock breeds, many of which have been incompletely
isolated for short periods of time. Distances were chosen on the basis of simulations which have
demonstrated their superiority to more complex distance measures for closely related populations
(Goldstein et al. 1995; Takezaki and Nei 1996). Suitable sample sizes for the resolution of
phylogenetic relationships have been suggested by Takezaki and Nei (1996), but their relevance
when determining present genetic relationships for conservation purposes is uncertain. We tested
this by taking different sized subsamples from the complete data set and comparing the number of
times the same nodes occurred in 100 random samples.
MATERIALS AND METHODS
Sampling. Breeds: Eleven breeds were included in this analysis: Appaloosa (APP, n=65);
Arabian (ARA, n=93); American Saddlebred (ASB, n=55); Belgian (BEL, n=54); Canadian (CAN,
n=33); Miniature Horse (MIN, n=126); Morgan (MOR, n=98); Quarter Horse (QHH, n=95);
Standardbred (STB, n=98); Thoroughbred (TBD, n=94); and Tennessee Walking Horse (TWH,
n=92).
Loci: Genotype data used in this study were generated by Mann Equitest for parentage testing
purposes. Thirteen microsatellite loci (AHT4, AHTS, ASB2, HMSl, HMS2, HMS3, HMS6,
HMS7, HTG4, HTG6, HTG7, HTGlO, VHL20) were amplified in two multiplex reactions and
co-loaded for detection on an ABI 377 DNA Sequencer. All loci except for ASB2 and HMS2 were
included in analyses.
Analysis. Trees and distance matrices were generated using the PHYLIP program
(Felsenstein 1988), with the exception of D,, which was generated using Microsat (Goldstein et
al. 1995). Trees were plotted with TreeView (Page 1996).
Genetic Distance and Relationship. Matrices of genetic distance between 11 populations were
computed for the following distance measures: Bowcock et al.3 (1994) proportion of shared alleles
transformed by -ln(PS) (D&; Cavalli-Sforza and Edwards’ (1967) chord distance (I&); and Nei’s
(1972) standard genetic distance (DNs). Phylogenies of relationships were constructed Tom these
distance matrices by the Neighbor-Joining (NJ) algorithm(Saitou and Nei 1987).
Subsampling. Neighbor-Joining trees based on chord distance matrices were generated for 100
random subsamples of lo,20 and 30 animals from each breed. A consensus tree of all 100 trees
was generated and the number of times each node occurs in the subsamples is given.

RESULTS AND DISCUSSION

Levels of expected vs. observed heterozygosity. The average level of heterozygosity (computed
from allele frequencies) for the 11 loci was 0.69, with a range of 0.65 to 0.74 (data not shown).
In general, observed levels of heterozygosity were similar to expected levels. Exceptions were the
Arabian and Morgan breeds (F,,=O.l 1 and 0.07) where observed heterozygosity was notably lower
than expected. This could be an indication of line breeding or inbreeding within these breeds.

Relationships of breeds and comparison of distance measures. Figure 1 depicts the NJ trees
generated for the three distance measures. Topologies differ somewhat with respect to clustering
arrangements. Breeds most highly diverged from the other breeds (BEL, MIN, CAN) remain
isolated in all three trees and closely related breeds (STB and TBD) cluster together in all three
trees. The other breeds show different clustering arrangements in different trees, leaving these
relationships unresolved. The consistent clusters all fit expectations based on historical
relationships and type, and are consistent with results from blood typing (Cothran, personal
communication).

Simulations have shown DPS to be superior to hs and DcH in resolving phylogenetic relationships
between closely related breeds (Goldstein et al. 1995; Takezaki and Nei 1996). The chord distance
can be expected to perform better than Nei’s standard distance in livestock situations, as unlike
Nei’s distance, it does not assume a constant population size, which is unlikely to have been
 BEL BEL BEL

MOR

ARA L - MOR

QHH
ASB - APP

STB
TBD

ARA

APP - QHH

QHH WH
o-1 0 nq

Figure 1. Comparison of distance measures: NJ trees created from DPS, D,, and D,, matrices.
maintained in most horse breeds. The DPS tree fits expectations based on presumed breed
relationships quite well: The Canadian is believed to have influenced both the Morgan and the
Saddlebred (Hendricks 1995), which cluster with the Canadian in this tree; Tennessee Walking
Horses cluster with Thoroughbreds and Standardbreds, both of which breeds are closely related
and are known to have contributed to the Tennessee Walking Horse.

As the degree of genetic isolation and genetic relationship between related breeds is of greater
importance than phylogenetic relationships when considering conservation priorities and strategies,
we looked at distances between the breeds without using cluster analysis on the assumption that
average genetic distance (which is closely related to the additive genetic relationship) from other
breeds should tell us something about degree of genetic separation or isolation. This approach
appears useful at first glance, in that Belgians and Miniatures exhibit the highest average distances
from the remaining breeds. However, the minimum genetic distance of almost all breeds is with
either the Appaloosa or Quarter Horse. Relationships inferred from clustering methods agree better
with historical accounts of breed origins and phenotypic similarities between breeds.

Although trees generated based on the Neighbor-Joining algorithm account for different
evolutionary rates in different lineages, thereby accounting for varying breed sizes and bottlenecks,
they do not allow for formation of breeds through crossbreeding and subsequent migration. Thus,
when addressing present genetic relationships between breeds, simpler clustering methods such as
unweighted pair group method with arithmetic mean (UPGMA) (Sneath and Sokal 1973) or
principle components analysis may be superior and will be investigated in future studies.

Number of animals and loci to be sampled. Figure 2 depicts the consensus trees generated for
preliminary analysis of random subsampling within breeds. Numbers at nodes represent the
number of times each branch in the consensus tree occurred in the 100 subsamples. For a sample
size of 30 animals, the consensus tree is almost identical in topology to the tree resulting from use
of the full sample. Although the topologies change with sampling of fewer animals, however the
branches pertaining to the more distinct breeds (BEL, MIN, CAN) as well as to very closely related
breeds (STB and TBD) are more robust to changes in sample size, suggesting that it may be
possible to pinpoint more highly isolated breeds through the use of as few as 30 animals. Nodes
joining the same branches in the three trees decline in occurrence with declining sample size.

ri 34
a- MOR
- C A N
a7
CAN

l-i
la- ASB
- 6 5 -55
MOR
ASB
1-APp 5+ APP APP
f-l -47
TBD

r
- 6 a
-88-88 STB

- TBD

-ARA
10

Figure 2. Effect of sample size on branching order: NJ trees based on D,, for 100 subsamples of
lo,20 and 30 animals.
Future studies will incorporate additional loci and subsampling of these loci to empirically
determine the number of loci best suited to resolving genetic relationships. Preliminary analysis
‘of data incorporating an additional 2 loci (for a total of 13 loci) on a smaller subset of the animals
results in an almost identical topology to that of 11 loci with more animals. The inclusion of two
additional loci also does not appear to alter heterozygosity estimates substantially and does not
significantly alter F,, estimates in any.of the breeds.

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