Unusually High Variance at Pinyon Jay Microsatellite Loci

1
Unusually High Variance at Pinyon Jay
Microsatellite Loci
Holmes, L., Benford, R., and Balda, R.P. Avian
Cognition Laboratory Department of Biological
Sciences Northern Arizona University
Flagstaff, AZ 86011
Abstract
Results
Variance in allelic polymorphism at 8
microsatellite loci is unusually high in pinyon
jays compared to other animal species. At least
three hypotheses could explain this phenomenon.
The first hypothesis suggests that there is
difference in mutation rates among loci. The
second hypothesis suggests that the time that
each locus began to mutate was different, and
older loci had more opportunity to accumulate
diversity than did younger loci. The third
hypothesis suggests that microsatellites linked
to conserved genes are limited in their potential
to diversify. Testing these three hypotheses
requires knowledge of the rate of mutation at
each locus. Therefore, the goals
Pedigree genotyping revealed no evidence of
mutation at any of the 8 microsatellite loci in
any of the 11 pinyon jay parent-offspring triads.
Genotypes of offspring followed predicted
genotypes based on parent alleles. Therefore, in
the 704 opportunities for mutation to have
occurred, it occurred in none.
Discussion
Three hypotheses were proposed to explain the
significant polymorphism of alleles among 8
microsatellite loci in the nuclear genome of the
pinyon jay. To test these hypotheses, information
about mutation rate at each microsatellite locus
was needed. Information on mutation rates was
derived from pedigree genotyping, a process in
which genotypes of parents and known offspring
are evaluated for discrepancies. This technique
was used in 11 parent-offspring triads of pinyon
jays, but it revealed no evidence of mutation at
any of the 8 microsatellite loci considered.
There are several possible reasons that no
mutations were detected. One possibility is that
mutation in the pinyon jay genome no longer
occurs at these 8 loci. Another possibility is
that it occurs at such a slow rate that it is not
detectable with this experimental design. One
potential drawback of this experimental design is
the small sample size. The sample was limited to
11 parent-offspring triads. The number of triads
was low because of difficulties associated with
holding and breeding pinyon jays in captivity. A
sample size of 11 might have been prohibitively
small. Thus, it is possible that mutation at
these loci does occur, but that the consideration
of 704 opportunities for mutation was
insufficient to measure the real mutation rate.
Despite the potential shortcomings of this
investigation, it does yield some positive
outcomes. The pedigree chart generated from the
data collected on allele sizes will be useful in
future studies. The techniques used provide a
model by which we can base further research on
mutation at microsatellite loci in the pinyon jay
genome. They also confirm the reliability and
validity of current genotyping methods, and they
have provided a preliminary data set that can be
expanded in subsequent investigations. This study
has therefore been helpful in gaining a broader
understanding of the techniques and methods of
analysis useful in answering questions about
pinyon jay genetics.
of this investigation are to determine if
mutations are occurring and, if so, at what rate.
Mutations were looked for using DNA from 20
parent-offspring triads in 5 nuclear families of
jays. Pedigree genotyping on each family was
performed, but no mutations were detected.
Failure to detect mutations could be caused by an
indiscernibly low mutation rate at each locus, or
by the limited sample size of the study. To
discriminate between these possibilities, future
research should increase the sample size of
parent-offspring triads and include additional
microsatellite loci.
Figure 2. Pairings in which a mutation did
(right) or did not (left) occur. Each box
represents an individual, and each number
represents an allele.
Introduction
Methods
Pinyon jays (Gymnorhinus cyano- cephalus) have a
high amount of variability in some non-coding
regions of their DNA (Figure 1). Among 8
microsatellite loci, allelic diversity ranges
from 3 to 44 alleles (Busch et al. in
preparation). This variability seems exceptional
compared to other species. In American alligators
(Alligator mississippiensis), microsat- ellite
diversity ranges from 1 to 16 alleles (Glenn et
al. 1996). In fishers (Martes pennanti),
diversity ranges from 2 to 10 alleles (Williams
et al. 2000). And in 10 breeds of domestic dogs
(Canis familiaris), diversity ranges from 3 to 6
alleles (Kim et al. 2001). Why is there such a
high genetic diversity in pinyon jays? There are
at least three possible answers to this
To detect mutations in the pinyon jay genome, DNA
was extracted from 20 wild-caught, captive pinyon
jays. Of the 20 individuals, 9 were paired,
breeding adults (one adult male died during the
study, and his female partner re-paired with a
different male). The 11 additional birds were the
offspring that resulted from these pairings. All
parents and offspring belonged to five nuclear
families. From each individual, a tissue
sample was taken. Tissue samples were digested,
and DNA was extracted using a PureGene DNA
extraction kit (Gentra Systems). DNA was
amplified at 8 microsatellite loci (MJG6, GATA2,
AAAG9, GATA3, GATA1, GATA4, AAAG1, and AAAG5)
using PCR. Amplicon sizes were estimated using
capillary electrophoresis on an Advanced
BioSystems 3100 genetic sequencer.
Electrophoretic data were analyzed (Figure 3)
using Advanced BioSystems Genemapper software (v.
4.0). Raw electropherogram data were normalized
on known alleles present in the progenitor
population. The progenitor population in this
study consisted of 652 wild pinyon jays living in
7 flocks east of Flagstaff, Arizona. Individuals
in the progenitor population were genotyped
genotypes were reported in prior studies (Nunes
et al. 2005, Benford et al. 2006, Busch et al. in
preparation). Known alleles in the population
were used as standards to which allelic data in
this study were normalized. Normalization
involved shifting data from a measured value to
the reported value of the known allele. In all
cases, the shift was less than one base pair in
distance. If an allele was more than one base
pair dissimilar from a known allele, it was
discarded from this analysis, because in these
cases the validity of the raw data was
questionable. Alleles were evaluated for
evidence of mutation using pedigree genotyping
(Yue et al. 2000). Pedigree genotyping involves
comparing parent and offspring alleles and
looking for insertions or deletions of di-, tri-,
or tetra-nucleotide repeats (Figure 2). If
evidence of insertions or deletions is found, the
exact nature of the mutation can be determined by
sequencing discrepant genes. When mutations
are identified, mutation rate can be determined
using the formula R N/T, where R mutation
rate, N number of mutant alleles detected, and
T total number of transferred alleles in one
generation at all loci considered (Yue et al.
2000).
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Figure 1. Variance in allele polymorphism among
microsatellite loci. Mean 20.8 S.E.M. 15.5.
question. One hypothesis is that a difference in
mutation rate among loci exists. Differences in
mutation rates among microsatellite loci have
been reported in other species. In fruit flies
(Drosophola melanogaster), trinucleotide repeats
mutate at a rate 6.4 times slower than
dinucleotide repeats (Schug et al. 1998). In
domestic swine (Sus scrofa), the mutation rate
varies between 1.72 x 10-4 and 4.08 x 10-5
mutations per locus per generation (Yue,
Beeckmann, and Geldermann 2000). And in superb
fairy-wrens (Malurus cyaneus), dinucleotide
repeats mutate at a rate 1.27 times slower than
tetranucleotide repeats (Beck et al.
2002). Different mutation rates among loci could
account for different amounts of diversity.
However, other hypotheses could also explain this
phenomenon. A second hypothesis is that the time
that each locus began to mutate is different,
with older mutations having more opportunity to
accumulate diversity than younger mutations.
Said differently, the duration of time that a
locus mutates could determine the amount of
diversity found at that allele (Slatkin 1994).
A third hypothesis is that microsatellites that
are linked to conserved genes might be limited in
their potential to become diverse. This occurs
in Norwegian rats (Rattus norvegicus), where
levels of microsatellite polymorphism are known
to be linked to selection on coding genes (Kohn
et al. 2002). This also happens in silkworms
(Bombyx mori), where linkage affects polymorphism
at 8 microsatellite loci (Prasad et al. 2004).
However, in humans (Homo sapiens), microsatellite
polymorphism seems unrelated to linkage with any
coding genes (Payseur and Nachman 1999). If the
first (mutation rate) hypothesis were correct
and one measured mutation rates at all 8 loci in
the pinyon jay genome, then one would expect the
mutation rate to be different at each locus, with
faster mutation rates at more diverse loci. If
the second (mutation time) hypothesis were
correct and one determined the time at which each
of the 8 loci started to mutate, one would expect
that the more diverse alleles be the oldest ones.
If the third (linkage) hypothesis were correct
and one sequenced the pinyon jay genome, then one
would expect less diverse loci to be located in
genomic regions that are more closely associated
with conserved genes, and more diverse loci to be
located in regions that are less closely
associated with conserved genes. Testing the
mutation time hypothesis requires that specific
mutations be identified, and that mutation rates
at each locus are known (Fisher et al. 2000).
Testing the linkage hypothesis requires more
knowledge about the behavior of mutations at each
locus. Therefore, the primary goals of this
research are to determine if mutations are
occurring at the 8 known microsatellite loci in
the pinyon jay genome, and, if mutations are
detected, to determine the mutation rate at each
mutating locus. When these goals are
accomplished, the times when each locus began to
mutate could be approximated, and the possibility
that mutations accumulate in non-conserved
regions of the genome could be investigated.
Acknowledgements
We thank the National Science Foundations
Research Experience for Undergraduates program
(Summer 2007) and Stephen Shuster. Additional
thanks go to Paul Keim, James Schupp, Ben Leadem,
Christine Clark Friedman, Melanie Hadlock, and
other helpful and skilled people in NAUs Keim
Genetics Lab and EnGGen Facilities. Finally, we
wish to thank Brandon Waddle, Christian Nunes,
Michael Barber, and Erin Strasser from the Avian
Cognition Lab, whose dedication and support made
this research possible.
Figure 3. Sample electropherogram. Green, black,
and blue peaks are created by fluorescently
labeled alleles as they pass through a laser.