DNA FINGERPRINTS AS A MEASURE OF GENETIC SIMILARITY IN
THE ENDANGERED SPECIES, ATTWATER'S PRAIRIE CHICKEN
by
MARY MALTBIE, B.A.
A THESIS
IN
ZOOLOGY
Submitted to the Graduate Faculty
of Texas Tech University in
Partial Fulfillment of
the Requirements for
the Degree of
~~STER
OF SCIENCE
Approved
Accepted
December, 1992
ACKNOWLEDGMENTS
I would like to thank my committee chairman, Dr. Robert]. Baker
for all his encouragement, support, and advice since my graduate
studies began at Texas Tech University. I also wish to thank
Dr. Raymond C. jackson and Dr. Nick C. Parker for serving on my
committee and guiding me along in completing my thesis. All the
techniques and ways to analyze DNA fingerprinting data were
taught to me by Jonathan Longmire. The following people have
helped or given advice in the lab work that has gone into this thesis:
Kevin Bowers, Robert D. Bradley, Laura Janecek, Bob Pavelka,
Madison Powell, Andy Simmons and Shelly Witte. Meredith].
Hamilton, Laura Janecek, Jon Longmire, Scott Lutz, Bob Pavelka,
Calvin A. Porter, Madison Powell, and Ron Van Den Bussche have
provided information and good criticism in reviewing my thesis.
Samples of Attwater' s Prairie Chickens and Greater Prairie Chickens
have been kindly provided by Dr. Rodney Honeycutt's Lab and Dr.
Nova Silvy at Texas A&M University. I also wish to thank Steve
Labuda, George Levandoski, and the Gulf Coastal Prairie Association
for support and advice in working with Attwater's Prairie Chickens
and also allowing us to visit the Attwater' s Prairie Chicken Wildlife
Refuge.
I would also really like to thank my parents and my brothers for
supporting me in accomplishing my goals and getting me through
the tough times.
11
Funding for this research project has been supplied by the Office
of Information Transfer, U.S. Fish and Wildlife Service at Fort
Collins, Colorado through the leadership of Dawn P. Jennings and
RichardS. Sojda. Support has also come from Helen Hodges
Educational Scholarships.
iii
TABLE OF CONTENTS
ACKNOWLEDGEMENTS ................................. ii
LIST 0 F TABLES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vi
LIST 0 F FIGURES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .vii
CHAPTER
I. INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
II. MATERIALS AND METHODS ......................... 7
Tissue Samples ............................... 7
DNA Isolation ................................ 8
Selecting the DNA Fingerprint Probe and Enzyme
Combination for Analysis . . . . . . . . . . . . . . . . . . . . 9
Restriction Endonuclease Digestion .............. 10
Southern Blots ............................... 10
Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Scoring of Autoradiograms ..................... 12
III. RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Scoring and Analysis of Au to radiograms .......... 14
DNA Markers ................................ 2 7
IV. DISCUSSION ..................................... 34
Choice of Fingerprint DNA Probes and Restriction
Endonucleases ............................. 34
Frequency of Minisatellite Fragments ............. 34
Polymorphic minisatellite fragments ........... 35
Mean frequency of all bands .................. 36
Index of Similarity and Selection of Individuals for
Captive Breeding . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 7
iv
The Search for Genetic Markers .................. 3 8
Future Goals in Producing a Genetic Profile of
Attwater' s Prairie Chicken ..................... 40
V. RECOMMENDATIONS ...............................42
BIBLIOGRAPHY ....................................... 44
v
LIST OF TABLES
1.
Binary code that was set up for part of one scored
autoradiogram of sixteen individual Attwater's Prairie Chicken
genomic DNA samples digested with Hae III and probed
with p VP01- 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
2.
The Mean Frequency of Polymorphic Fragments is the average
occurance of bands in percent that are different among all
individuals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 2
3.
Index of similarity for sixteen Attwater's Prairie Chicken
genomic DNA samples digested with Hae III and probed with
p VP01- 3 .......................................... 24
4.
Selection of mates for females 2 and 3, Attwater's Prairie
Chicken, based on autoradiogram of sixteen individual
Attwater's Prairie Chicken DNAs digested with Hae III and
probed with pVP01-3 ................................ 25
5.
Most preferred and least preferred mates for two female
Attwater's Prairie Chickens based on analysis of several
autoradiograms of genomic DNA digested with Hae III and
probed with pVP01-3 ................................ 26
Vl
LIST OF FIGURES
1.
An autoradiogram showing the genomic DNA of four individual
Greater Prairie Chickens in groups of four digested with four
different enzymes (Hae III, Hinf I, Pst I, and Sau 3A I) and
probed with pVP01-3 at 4091> strigency .................. 15
2.
Sixteen individual Attwater' s Prairie Chicken genomic DNA
samples digested with Hae III and probed with pVP01-3 at a
40% s trig ency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1 7
3.
Four individual Attwater's Prairie Chicken genomic DNAs and
three individual Greater Prairie Chicken genomic DNAs on the
same autoradiogram, digested with Hae III and probed with
pVP01-3 at a 40% stringency .......................... 19
4. Autoradiogram of sixteen Greater Prairie Chicken genomic
DNAs digested with Hae III and probed with (TC)n ......... 28
5. Sixteen Attwater's Prairie Chicken genomic DNAs digested
with Hae III and probed with (TC)n ..................... 30
6. Eleven samples of genomic DNA from Greater Prairie
Chickens were digested with Hae III and electrophoresed
through a 0.8% agarose gel ............................ 32
Vll
CHAPTER 1
INTRODUCTION
Attwater's Prairie Chicken (Tympanuchus cupido attwateri), a
subspecies of the Greater Prairie Chicken (Typanuchus cupido), was
placed on the Federal Endangered Species List in 1967 (Lawrence
and Silvy, 1980; King, 1979). The number of individuals and the
range of this prairie chicken has been reduced drastically in the last
few decades (Lehmann, 1971). It has been estimated that this
subspecies once inhabited millions of acres of the Gulf Coastal
Prairie of the Southern United States with population size of at least
a million when conditions were right (Lehmann, 1971; King, 1979).
In a recent survey, 456 birds, 228 of these birds being males, were
sighted in five counties in south coastal Texas (Steve Labuda, pers.
comm.,1992).
The decrease in population size has resulted from many factors
with habitat destruction being the most significant (Lehmann and
Mauermann, 1963; King, 1979; Lawrence and Silvy, 1980). Since the
mid 1960s, when management programs were initiated for
Attwater's Prairie Chicken (Lawrence and Silvy, 1980 ), the
understanding of the ecological needs of these birds has improved
considerably. As a result of the continued loss of population size,
another management program has been initiated to breed these
birds in captivity. Researchers at Fossil Rim in Glenn Rose, Texas,
have collected some Attwater's Prairie Chicken clutches from wild
1
populations, these eggs were hatched at Fossil Rim to produce a
captive population of sexually mature individuals.
An understanding of the genetic makeup of the potential
breeders is an important part of a successful breeding program.
This allows the managers to make the best possible mate pairings of
birds to produce genetically healthy offspring as well as to maintain
genetic diversity within these and future captive populations. This
thesis is concerned with the molecular techniques and analyses
necessary to give a genetic profile of Attwater's Prairie Chicken and
how such information can be used in setting up a captive breeding
program.
Restriction fragment length polymorphisms (RFLPs) of nuclear
DNA have been used to examine the relationship among taxa of
prairie chickens to determine if each taxon is genetically unique
(Ellsworth, 1991). A problem with the use of RFLPs to determine
relationship is that the amount of variation found between
individuals is low (Jeffreys and Morton, 1987; Meng et al.,1990), so
that the amount of information provided for a breeding program is
minimal. Mitochondrial DNA also has been used to understand the
systematics of the prairie chicken taxa (Ellsworth, 1991). However,
this method is limited because mitochondrial DNA is inherited only
from the mother and relative to the nuclear genome is a small piece
of DNA (Lewin, 1990).
The technique used in this study to assess genetic variation
among Attwater's Prairie Chicken is DNA fingerprinting. DNA
fingerprinting is based upon the variation in the length of small
2
tandemly repeated sequences called Variable Number Tandem
Repeats (VNTR) which are, usually 15 to 60 base pairs long, such
variable repeats are also known as minisatellites (Jeffreys et al.,
1985a). When the genomic DNA of the animal being studied is
digested with restriction enzymes, separated by size using
electrophoresis and probed with one of the minisatellite or
fingerprint probes, a distinct banding pattern can be created for
each individual (Jeffreys and Morton, 1987). Many studies have
been performed to determine the validity of DNA fingerprint data
and most current controversy considers the accuracy of the
technique ( 1 in one thousand versus 1 in one million)
(Budowle,1992; Risch and Devlin, 1992; Sullivan, 1992). Georges
( 1988) has shown that DNA fingerprints produced with human DNA
fingerprinting probes show enough differences that to be "used
efficiently in animal identification, paternity testing, and as a source
of genetic markers for linkage analysis"(p. 127). Gilbert et al.
( 1990) worked on the probability of unrelated humans having the
same DNA fingerprints in their work on cell line individualization.
They calculated that "the chance of two or more common DNA
fingerprints among 1 million distinct individuals is
< 0.001"
(p. 499). Jeffreys et al. (1985a) have shown also the individuality of
DNA fingerprints with his 33.6 and 33.15 probes among related and
unrelated humans. They demonstrated that there is stability in the
DNA fingerprints (Jeffreys et al., 1985a).
The use of DNA fingerprinting among humans has been
extensive. It has been used in forensics (Gill et al., 1985), linkage
3
analysis and human pedigree (Jeffreys et al., 1986; Nakamura et al.
1987) and also as a method of positive identification of individuals
(Jeffreys et al., 1985a;1985b).
As DNA fingerprinting became a valid technique and as other
probes, for the example, M13 sequence from a bateriophage that
requires no competitor DNA to effectively produce fingerprints in
humans and animals (Yassart et al., 1987)were found, the use of
fingerprinting spread into many areas beyond work on humans.
DNA fingerprints have been used for analysis in plants (Dallas,
1988), insects (Blanchetot,1989;1991), fish (Turner, 1990; 1991),
mammals (Jeffreys and Morton, 1987; Hoelzel and Amos, 1988;
Reeve et al., 1990; Gatei, 1991; Gilbert et al., 1991; Packer et al.,
1991; Dolf et al., 1992; Lehman et al., 1992), and birds (Burke and
Bruford, 1987; Wetton et al., 1987; Burke et al., 1989; Hillel, 1989;
Gyllensten et al., 1990; Meng et al., 1990; Rebenold, et al., 1990;
Brock and White, 1991; Haberfeld, 1992; Love and Deininger, 1992;
Triggs et al.,1992; Longmire et al., in press).
The value of DNA fingerprinting to the captive breeding program
has been shown in many studies with captive and also among free
ranging animals. An extensive study of African lions of the
Serengeti (Gilbert et al.,1991) has shown the power of analysis of
DNA fingerprinting in a free ranging population. There has been
some argument as to how well DNA fingerprinting analysis can be
used to distinguish kinship (Lynch, 1988;1990). Gilbert et al.
( 1991) collected detailed information on lions for more than 25
years and therefore could compare the known lineages with the
4
conclusions drawn from data from DNA fingerprinting. Their study
did show a relationship among kinship and fingerprint band
sharing. They concluded, however, that it is important to
appreciate the amount of variance in DNA fingerprint data and
where possible to calibrate with known genealogy (Gilbert et al.,
1991).
In a study on whooping cranes in a captive breeding program at
Patuxent Wildlife Research Center, Longmire et al. (in press) was
able to determine the paternity of the cranes using fingerprint
analysis. This was important since the female cranes were
artificially inseminated with sperm from several male cranes to
improve the chance of fertilization. DNA fingerprinting of the
chicks was not only able to give the paternity of each crane but was
also used to determine the level of genetic variation among
whooping cranes and to compare this variation to that characteristic
of other (free living) birds (Burke and Bruford, 1987; Longmire et
al., 1991). Another part of this study that was not included in the
paper is that a pairwise comparison was done for all cranes so that
an index of relatedness could be set up in a matrix (Longmire, pers.
comm.,1992). This matrix would allow managers of the whooping
crane breeding program to match the most genetically different
individuals for breeding to decrease the possibility of inbreeding
(O'Brien et al., 1985;1986; Wildt, 1987; Reeve et al., 1990) and
increase the probablity of maintaining genetic diversity.
In this study, I used DNA fingerprints of Attwater's Prairie
Chicken to estimate the amount of genetic variation through band
5
sharing and to estimate inbreeding among these chickens, especially
since these data can be compared to similar studies done on other
birds and those of endangered species. Second, I generated a
pairwise comparison of Attwater' s Prairie Chicken individuals and
set up an index of similarity matrix so that breeding pairs could be
matched to maintain genetic variation among the chickens. Finally,
I compared DNA fingerprints of Attwater's Prairie Chicken and the
Greater Prairie Chicken to document the level of genetic uniqueness
between these two taxa.
6
CHAPTER II
MATERIALS AND METHODS
Tissues Samples
Attwater's Prairie Chickens used in this study were not part of
the breeding program at Fossil Rim, but were individuals that had
either been bled and released back into their range or were
individuals that had died and been preserved. The 16 blood and
tissue samples from Attwater's Prairie Chicken used in this study
were supplied by personnel at Texas A&M University. Upon arrival
samples identified by a Texas A&M University number (in
parentheses) were given a Texas Tech number, Tk numbers as
follows: Tk 27462 (Ak 8971), Tk 27989 (1946), Tk 27991 (1948),
Tk 27992 (1947), Tk 30986 (Alumin. 108 R40), Tk 30981 (yellow
#29 Alum.#109), Tk 27457 (Ak 8970), Tk 27953 ( 12808),Tk 27990
(1925), Tk 30984 {#1 female), Tk 27993 (1924), Tk 30987 (yellow
#17 Alum.#110), Tk 27465 (Ak 8975), Tk 30983 (red 29 silver 104
APC NWR), Tk 30982 (yellow 11 Alum. 120), and Tk 30985 (female
Aluminum 101). We also received blood and tissue samples of
Greater Prairie Chicken from Texas A&M for this study. Those used
in this analysis are the following: Tk 27467 (8968), Tk 27469
(8961), Tk 27464 (8947), Tk 27466 (8948), Tk 27470 (Ak
8965),Tk 27468 (8964), Tk 27459 (8965), Tk 27456 (8966), Tk
27461(8962),Tk 27463 (8967), Tk 27454 (8949), Tk 27460 (8945),
and Tk 27455 (8946).
7
DNA Isolation
About 0.25 - 0.50 ml of freshly collected blood was placed in 5
ml of lysis buffer, (0.1 M Tris-HCl pH 8.0, 0.1 M ethylenediaminetetraacetic acid (EDTA), 0.01 M NaCl, 0.5% (w/v) sodium dodecyl
sulfate(SDS) and shaken (Longmire et al., 1988). Blood was stored
safely in this lysis buffer at room temperature and shipped to us to
proceed with the DNA isolation. Most of the Greater Prairie Chicken
samples and a few Attwater's Prairie Chicken samples were
maintained as frozen tissue. The DNA isolation technique was
modified from Longmire et al. (1991). DNA was isolated after 1/2 to
1/3 grams of tissue had been macerated and mixed with 5 ml of
lysis buffer. Proteinase K of a stock solution ( 10 mg/ml), 250 111 was
added to each sample and incubated at 3 7°C for 24 hours on a tube
rotator. An equal volume of phenol was added to digested samples
(preheated to 50°C) to remove proteins and the solution was rotated
for 30 minutes at room temperature. Samples were then
centrifuged for 5 minutes at 2000 rpm to separate the phases. The
aqueous phase was removed and placed in dialysis tubing
(Spectra/Par, flat width 25 mm and molecular weight cut off: 1214,000) and dialyzed for 48 hours against 3 changes of 1 X TEat
4°C. All tissue samples had to be purified further due to the
presence of RNA. This was done by adding pre boiled RNase to each
sample to give a final concentration of l0011g per ml. Samples were
incubated for 3 hours at 3 7°C; then SDS was added to denature the
enzyme at a concentration of 0.5% of the final volume. This step
was then followed by another phenol extraction and centrifugation
8
before samples were dialyzed. DNA concentration was determined
by UV- spectrophotometry. The concentration of DNA in all samples
was adjusted to 300J.Jg /ml by dilution or precipitation and
resuspension with the appropriate volume of 1 X TE. DNA was
precipitated by adding 0.1 sample volume of 3 M sodium acetate
and 2.5 to 3 times sample volume cold (0°C) 200 proof ethanol.
Purified samples were stored at 4°C.
Selecting the DNA Fingerprint Probe and Enzyme
Combination for Analysis
DNA samples from four individual Greater Prairie Chicken were
digested and electrophoresesd on a 0.8% agarose gel. Each
individual's DNA was set up in four different digestions. The
enzymes ( New England Biolabs, Beverly, MA) used were Pst I, a six
base cutter; Hinf I, a five base cutter; and two four base cutters, .Haf.
III and
.s.a.u 3A I .
The restriction fragments were transferred to a
nylon membrane, probed and stripped repeatedly with different
fingerprint probes. The following were probes tried in this study:
Poly( dA-dC) ·( dG-dT)( supplied by Pharmacia, Piscataway, NJ),
Poly(dA-dG)·(dC-dT)(supplied by Pharmacia), Poly(dG-dC)·(dGdC)(supplied by Pharmacia), pV47(Longmire et al., 1990), and
pVP01-3(Longmire et al., unpubl.). The enzyme and probe
combinations selected for scoring from this were Hae III, probe
pVP01-3, Haf III, and probe (TC)n. The enzyme Hinf I was not used
in scoring autoradiograms since it did not have as much variation as
pVP01-3 but was informative in locating genetic markers.
9
Restriction Endonuclease Digestion
Genomic DNA ( 10 pg) was digested using the suppliers'
recommended buffer and endonuclease concentrations. The
restriction enzymes used in this project were Hae III and Hinf I.
Digestions were carried out for 7 to 16 hours in a 37oe incubator.
This was followed by heat denaturation of the endonucleases at 70°e
for 10 minutes. Digested samples were electrophoresed in a 0.8%
agarose gel (350 ml of lX TAE with 2.6 gms of agarose, 20 em X 25
em) with 1 X TAE (49.3 grams Trizma base, 4.1 grams EDTA-Na2, 9.1
ml HOAc, pH to 8.2) as the running buffer. A maximun of 42 pl was
placed in each well, and gels were run at 40 volts for 48 to 72 hours.
Sou them Blots
The gels were photographed under UV light with a red filter
before Southern blotting. Gels were submerged for 5 minutes in
0.25 M Hel, followed by two 15-minute submersions in 0.4 M
NaOH. Restriction fragments were transferred to a nylon membrane
(Boehringer Mannheim, Indianapolis, IN) using the procedure of
Southern ( 197 5). After transfer the membranes were washed gently
by hand in 2 X sse, followed by one 15 minute wash in a
neutralization solution (0.5 M Tris pH 7.5; 1.5 M Nael). Two ISminute washes in 2 X sse were done at room temperature on an
orbital mixer. Membranes were dried between two clean sheets of
Whatman paper (3 MM chromatography paper) and stored in plastic
coverings until used.
10
Hybridization
Immediately prior to hybridization, membranes were washed in
0.1 X SSe, 0.1% SDS at 60°e for 1 hour on an orbital mixer.
Membranes were then treated with a prehybridization solution at
42 oe for 45 minutes on an orbital mixer. The two prehybridization
solutions used were a 35% blotto (30 ml 20 X sse, 35 ml formamide,
1 ml 0.5 M EDTA, 5 ml 20% SDS, 0.25 grams powdered milk and
brought up to volume 100ml with distilled water) and a higher
stringency solution, 40% blotto, formamide increased by 5 ml. The
40% blotto was used with the fingerprint probe, pVP01-3 to
decrease some of the intense hybridization so scoring would be
possible. DNA probes were labeled with 3 2 P as follows: 1.0 J,Jg
probe DNA, 5 J,Jl of marker probe ( 15 ng of Rae III o X174 DNA plus
25 ng Hind III cut lambda DNA), 5 pl 10 X nick translation buffer
(0.5 M Trisel (pH 7.5), 0.1 M MgS04, 1 mM dithiothreitol, 5 pg/ml
bovine serum albumin), 5 pl of 32 P deTP, 16 pl of distilled water,
5 J,Jl DNase/Polymerase I(Gibco BRL, Grand Island, NY). This
solution was incubated for 45 minutes (except for (Te)n, which was
incubated for 30 minutes) at 15°e. Solutions of 50 pl of 1 X TE and
10 pl 0.5% SDS were then added and run through a G-50 in 1 X TE
sephadex spin column by centrifugation. The solution containing
the labeled probe was treated with 1/9 volume of 1 M NaOH and
then incubated for 10 minutes at 37°e. The probe was added
directly to the prehybridization solution containing the membranes
and allowed to hybridized overnight at 42oe on an orbital mixer.
Following hybridization, membranes were rinsed in 2 X sse at room
11
temperature followed by a 15-minute wash on an orbital mixer at
room temperature. Membranes were then washed for 15 minutes in
1 X SSC, 0.1 % SDS at 50°C. Membranes were dried between two
clean sheets of Whatman paper and put in plastic protectors. The
covering with the radioactive membrane inside was then placed in
cassettes with intensifying screens and film (Kodak XAR-5) and
exposed overnight at- 70°C. Some membranes took longer than one
night exposure since some hybridization were not as strong as
others.
Scoring of Autoradiograms
There were two ways to score autoradiograms. One method was
to just score polymorphic fragments and to calculate their
frequency among a bird taxon. The polymorphic fragment
frequency was caculated as the total number of individuals with a
specific band (not shared among all individuals) divided by the total
number of individuals. This frequency was the average of all
polymorphic bands scored on the autoradiogram could then be
compared to other frequencies for different species of birds in work
done by other investigators using DNA fingerprinting (Burke and
Bruford, 1987; Longmire et al., in press). The second approach was
to score all readable bands even if they were shared among all
individuals. This allows calculation of an index of similarity among
all individuals in a pairwise comparison using the same DNA
fingerprinting probe and digested with the same enzyme. The
formula used to calculate the amount of band sharing in a pairwise
12
comparison was the total number of bands shared by two prairie
chickens in the comparison divided by their total number bands
(Wetton et al., 1987). To keep consistency in scoring, standard
markers were run at both ends and the middle of the gel, and
sometimes samples from individual birds were duplicated at
opposite ends of the gel so that the chance of error could be
reduced.
13
CHAPTER III
RESULTS
Scoring and Analysis of Autoradiograms
Figure 1 is an autoradiogram photograph of four individual
Greater Prairie Chicken genomic DNAs digested with different
enzymes and probed with pVP01-3. From this autoradiogram, an
enzyme and probe were selected for DNA fingerprinting of all other
Attwater' s Prairie Chicken DNA samples. Figures 2 and 3
autoradiograms were used in this analysis. Figure 2 is of 16
individual Attwater's Prairie Chicken DNA samples digested with Rae
III and probed with pVP01-3, and Figure 3 displays the DNA
fingerprints of four individual Attwater's Prairie Chicken DNA
samples and three individual Greater Prairie Chicken DNA samples
digested and probed with the same enzyme and probe used in
Figure 2.
Scoring of these autoradiograms was based on the absence or
presence of a band compared to the banding pattern of other
individual birds. A binary code table was set up for each
autoradiogram (Table 1). A "1" represent the presence of a band
and a "0" represents the absence of a band. From this binary table,
several different analyses could be performed.
One analysis that has also been examined in several other papers
(Burke and Bruford, 1987; Longmire et al., 1991; in press) is the
frequency of polymorphic fragments. In this analysis, bands are
scored only if they are not shared among all individuals. Table 2
14
15
Figure 1. An autoradiogram showing the genomic DNA of four
individual Greater Prairie Chickens in groups of four digested
with four different enzymes (liae III, Hinf t £s.LI, and .s.au 3A I)
and probed with pVP01-3 at 40% strigency.
16
17
Figure 2. Sixteen individual Attwater's Prairie Chicken genomic
DNA samples digested with Hae III and probed with pVP01-3
at a 40% strigency.
1 2
3 4
5
6
1
8 9 19 11 12 13 14 15 16 1
18
19
Figure 3. Four individual Attwater's Prairie Chicken genomic
DNAs and three individual Greater Prairie Chicken genomic
DNAs on the same autoradiogram, digested with Hae III and
probed with pVP01-3 at a 40% strigency.
1 2
3
4
56 7
124 53 6 7
20
Table 1 : Binary code that was set up for part of one scored
autoradiogram of sixteen individual Attwater's Prairie
Chicken genomic DNA samples digested with H.ae III and
probed with pVP01-3. A "1" means that a band was
present and a "0" means the absence of a band.
BANDS SCORED
BIRDS
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
ABCDEFGHI
0
1
0
0
0
0
0
0
0
0
0
1
0
0
0
0
1
0
1
0
1
0
0
0
0
0
0
0
0
0
0
0
1
0
1
1
1
1
0
0
0
0
1
0
0
0
0
0
0
0
0
1
1
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
1
0
0
1
0
1
1
1
0
1
1
1
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
1
1
0
0
1
0
0
0
0
0
0
0
21
0
0
1
0
0
1
1
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
KLMN
0
1
1
1
1
1
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
1
1
0
0
0
0
1
1
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
0
0
0
0
Table 2: The Mean Frequency of Polymorphic Fragments is the
average occurance of bands in percent that are different
among all individuals. This frequency has been used
as an indication of how genetic diversity is among the
population being studied.
TAXA
NUMBER OF
INDNIDUALS
FREQUENCIES
Attwater' s Prairie
Chicken
16
0.33
Greater Prairie
Chicken
13
0.27
Peregrine Falcon
110
0.12a
Whooping Crane
42
0.42b
Sparrows
13
0.28C
a Longmire et al., 1991.
b Longmire et al., in press.
c Burke and Bruford, 1987.
22
shows the Mean Frequency of Polymorphic Fragments for Attwater's
shows the Mean Frequency of Polymorphic Fragments for Attwater's
Prairie Chicken, Greater Prairie Chicken, and other taxa of birds that
have been analyzed in similar studies.
The greatest amount of variation in DNA fingerprinting for
Attwater's Prairie Chicken was found following digestion of DNA
with H.a.e III and using probe pVP01-3 to create an index of
similarity (Table 3). This index was produced from the
autoradiogram of 16 Attwater's Prairie Chickens which contained 14
males and 2 females. This was based on pairwise comparisons
among all individuals (Table 3). From this index, a table was
created that lists the most and least preferred mates for each of the
two females represented in Table 4. To select the most preferred
mates, the males on the index with the lowest percentage of bands
shared with each female bird are listed. On selection of the least
preferred mates, the males on the index with the highest percentage
of bands shared to each female bird are lis ted.
An index of similarity was also created for autoradiograms of
these sixteen Attwater's Prairie Chicken DNAs digested with Hae lll
and probed with (TC)n to compare it to the index of pVP01-3. A
direct comparison cannot be made between pVP01-3 and (TC)n
since they are different types of probes. However, certain males
Attwater's Prairie Chicken showed up repeatedly as most preferred
or least preferred for each female bird when a tally was taken of all
indices of similarity produced from autoradiograms of llaf III
digestion and probe pVP01-3 and probe (TC)n (Table 5).
23
Table 3 : Index of similarity for sixteen Attwater's Prairie
Chicken genomic DNA samples digested with liaf III
and probed with pVP01-3. The numbers 1-16 on the lefthand border and the top border represent the birds
in the analysis. Number 2 and 3 are the females while
the rest are males. The numbers are based on the
percentage of bands shared between any two individuals
being compared.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
1 2 3
** 38 44
** 31
**
4
30
15
48
**
5
39
10
54
37
**
7
14
26
21
33
so 26
** 11
**
6
27
0
27
26
8
23
28
23
22
14
8
25
**
9
23
28
23
22
14
8
25
**
**
10
38
10
31
22
30
so
26
7
7
**
11
20
14
20
29
43
20
12
0
0
14
**
12
38
13
38
53
52
30
24
35
35
26
32
**
13
39
8
26
38
40
48
7
18
18
40
32
14
34
28
29
44
28
24
44
53
53
41
9
so 40
** 24
**
**All bands scored were shared between both individuals
24
15
27
25
27
39
25
20
37
36
36
so
11
46
21
67
**
16
22
10
30
36
19
24
33
33
33
48
0
38
23
60
72
**
Table 4 : Selection of mates for females 2 and 3, Attwater' s Prairie
Chicken, based on autoradiogram of sixteen individual
Attwater's Prairie Chicken DNAs digested with Haf III and
probed with pVP01-3. The numbers in parenthesis
represent the percentage of bands sharing between that
male and female.
MOST PREFERRED
LEAST PREFERRED
MATE
MATE
2
male 6 (0)
male 13 (8)
male 8 (28)
male 9 (28)
male 14 (28)
3
male 11 (20)
male 7 (21)
male 5 (54)
male 4 (48)
FEMALE
25
Table 5: Most preferred and least preferred mates for two female
Attwater' s Prairie Chickens based on analysis of several
autoradiograms of genomic DNA digested with Ha.e III and
probed with pVP01-3.a The most preferred mates were
those male birds with the lowest percentage of band
sharing to the selected female and the least preferred
were the males with the highest percentage of band
sharing. The number in parenthesis is the number of
times it showed up in the column after tallying the data.
MOST PREFERRED
MALE
LEAST PREFERRED
MALE
FEMALE
2
Tk 27453 (3)
Tk 30983 (3)
Tk 30982 (0)
Tk 27453 (1)
Tk 30983 (0)
Tk 30982 (3)
3
Tk 27453 (3)
Tk 27457 (3)
Tk 30984 (0)
Tk 27453 (1)
Tk 27457 (1)
Tk 30984 (3)
a Autoradiograms used for this analysis were the following: 1
membrane of sixteen Attwater's Prairie Chicken genomic DNAs
digested with Hae III and probed with pVP01-3, 1 membrane of
eleven Attwater's Prairie Chicken genomic DNAs digested with Hae
III and probed with pVP01-3, and 2 membranes of the sixteen
Attwater's Prairie Chicken genomic DNAs digested with Hill! III and
probed with (TC)n.
26
DNA Markers
Many different types of probes were used to determine the best
one for our analysis. Simple sequences have been used successfully
by many investigators (Tautz, 1989; Kashi et al., 1990; Ellegren et
al., 1992a; 1992b) to show parentage, genetic variation, and DNA
markers. The use of dinucleotide and trinucleotide DNA probes led
to the discovery of a sex marker for Attwater' s and Greater Prairie
Chicken. Sex markers have been identified in other taxa of birds
(Kodoma et al., 1987; Saitoh et al., 1989; 1991; Longmire et al.,
1991). In birds, the sex marker will appear in the female birds since
they are the heterogametic sex. The sex marker was seen with the
probes of (TC)n and (TCC)n after the Attwater's Prairie Chickens
and Greater Prairie Chicken DNAs were digested with either H.ae III
or Hinf I (Figures 4 and 5 ).
An ethidium band in the gel where the digested genomic DNA of
the Prairie Chickens was electrophoresed could also be used to
distinguish females from males when digested with either Haf III or
Hint I (Figure 6).
27
28
Figure 4. Autoradiogram of sixteen Greater Prairie Chicken
genomic DNAs digested with Hae III and probed with (TC)n.
The intense, high molecular band appearing after
hybridization, identified the females of the group correctly.
Scoring of lanes
F F M F F
F
1234567
29
MMMFM
8 9 10 1112
FMFF
1314 1516
30
Figure 5. Sixteen Attwater' s Prairie Chicken genomic DNAs
digested with H..gg III and probed with (TC)n. Number 2 and
3 represent the females.
1 2
3
4
5
6
7
8 9 19 11 12 13 14 15 16 1
31
32
Figure 6. Eleven samples of genomic DNA from Greater
Prairie Chickens were digested with Hill! III and electrophoresed
through a 0.8% agarose gel. An ethidium band can be seen
distinguishing females.
marker
33
CHAPTER IV
DISCUSSION
Choice of Fin~erprint DNA Probes and
Restriction Endonucleases
The critieria used to select a DNA fingerprint probe included ( 1)
variabilty among individual birds, (2) clarity of banding patterns
and (3) a restriction endonuclease that does not digested within the
variable number tandem repeat monomer. Using these criteria, the
most acceptable DNA fingerprint probe for prairie chicken analysis
was pVP01-3 (Longmire et al., unpubl.) and the restriction
endonuclease was Hili! III. Other probes and enzymes evaluated
include: probes (TC)n, (GT)n, (GC)n, pV47(Longmire et al., 1990),
pVP01-3 (Longmire et al., unpubl.) and enzymes Rae III, Hinf I, S.al.l
3A I, and £s.t I. The combination of (TC)n and H.af III also were
scored, but its index of similarity was not as valuable as pVP01-3 in
setting up Table 5 on mate selection due to (TC)n linkage to the
female sex chromosome and the high amount of band sharing
among all individuals.
Freguency of Minisatellite
Fra~ments
In examining the conservation genetics of various species, two
related scores have been used to indicate similarity. The first
commonly used scored reflects mean frequency of polymorphic
fragments. The second score provides a frequency measurement
using all bands including those that are monomorphic. The
34
frequency of all bands scored provide considerably more
information than does examination of only polymorphic fragment
bands. Unfortunately, the most commonly published values in the
literature are restricted to polymorphic fragments. The use of only
polymorphic fragments becomes a function of sample size because
most bands will eventually vary if enough individuals are examined.
The problem would be further complicated if only individuals of a
single population were sampled. Table 2 compares published
literature.
Polymorphic minisatellite fragments. To get an estimation of the
frequency of polymorphic minisatellite fragments, I used an average
of scores from several autoradiograms of Attwater' s Prairie Chicken
and those of Greater Prairie Chicken calculated separately. The
reason for this was the electrophoresis of each gel, especially the
gels containing samples of Attwater's Prairie Chickens, were run for
different lengths of time so that bands at higher molecular weights
could be separated out and scored properly.
For Attwater's Prairie Chicken, the estimated value was 0.33, and
the estimated value for the Greater Prairie Chicken, was 0.27. These
values can be compared broadly to estimated values of mean
frequencies of other bird species to give a general overview of
genetic variation and the level of inbreeding(Table 2). However,
these values cannot be directly compared because different probes
and enzymes were used to calculate the mean frequency of
polymorphic fragments. The values for Attwater's Prairie Chicken
and Greater Prairie Chicken demonstrated that there was a
35
considerable amount of genetic variation within the animals
analyzed. Although the Attwater's Prairie Chickens' population size
is very low now, this does not seem to have yet affected the level of
genetic variation among the population. This is probably due to the
fact that the Attwater' s Prairie Chicken populations once covered a
widespread area and had a population size in the millions(Lehmann,
1971). This is a unique aspect of the biology of the prairie chicken
as an endangered species. Most endangered species have gone
through severe bottlenecks and have reduced amounts of variation
within their populations. The Attwater's Prairie Chicken is facing a
potentially severe bottleneck, but the variation in existing
individuals is still great and potentially can be maintained for future
populations. Greater genetic variation is an advantage for a captive
breeding program since genetic variation does not have to be
created through selective breeding-- just maintained.
Mean freQuency of all bands. To obtain an overall view of
genetic variation, the mean frequency of polymorphic fragments
should not be the only value examined to generate conclusions
about the genetic profile of Attwater's Prairie Chickens as a
population or even an individual.
When calculating the mean frequency, the exclusion of bands
that were shared among all individuals in a population when
calculating the mean frequency will not give a full profile of the
genetic variation and level of inbreeding. To eliminate this
problem, the mean frequency of all bands scored was calculated for
both groups of prairie chicken. The value for Attwater's Prairie
36
Chicken was 0.35 and the value for Greater Prairie Chicken was
0.33. These values do not show a high level of band sharing and
give confidence to the conclusion that Attwater's Prairie Chicken
still has an adequate level of genetic variation.
Index of Similarity and Selection of Individuals
for Captive Breeding
A pairwise comparison of band sharing was performed on all
autoradiograms containing 16 Attwater's Prairie Chicken (2 females
and 14 males). An index of similarity (Table 3) was produced from
this comparison, giving a percentage of bands shared between each
two individuals compared. Since maintaining genetic variation is a
goal of a captive breeding program, mate selection for the two
females should be for male Attwater's Prairie Chicken with the
lowest percentage of bands shared between them. For example, the
most preferred male for female 2 would be male Attwater's Prairie
Chicken 6 because they shared no common bands. Although in the
case of female 2, probably all the males compared to it would be
good mates since the highest percentage of bands shared was 0.28
which is below the estimated level of the genetic variation found
(0.33). There would have to be a more careful selection of a mate
for female 3 since males, such as 4 and 5 have 0.48 and 0.54 band
sharing, respectively, both of which are higher than the mean
frequency of 0.33 for genetic variation and could lead to a decrease
in genetic variation if mating took place.
Another factor of DNA fingerprinting that should be taken into
consideration is the banding pattern itself. If one individual
37
appears with a fingerprint pattern that no other individual has, it
could be important to the population to maintain these different
DNA fingerprint patterns in the group. DNA fingerprinting could be
used on the genes to identify individuals with rare alleles and make
sure they were included in a captive breeding program in Attwater's
Prairie Chicken.
When analyzing the index of similarity (Table 3), a comparison
of male 8 and 9 revealed identical fingerprints in all cases. The
blood and tissue samples of all the birds sent to us in this study
included a catalog number and sometimes sex identification.
Samples 8 and 9 had two different catalog numbers and were
assumed to be different individuals until their DNA fingerprints
were examined with several different fingerprint probes. Based on
the results given in Table 3, no other conclusion can be made except
that they are the same individual.
The Search for Genetic Markers
Genetic markers are valuable in many biological studies.
Markers have been used as diagnostic signs for certain diseases
(Nakamura et al.,1987) to possibly identify the home location of an
organism (Longmire et al., 1991) or to study the dynamics of a
contact zone between two subspecies (Baker et al., 1989).
Various minisatellite and microsatellite probes were used in this
analysis to search for a marker that could separate Attwater's Prairie
Chicken from Greater Prairie Chicken. Microsatellite probes have
been very informative in other studies (Kashi, 1990; Ellegren, et al.,
38
1991; Epplen et al., 1991; Ellegren et al., 1992a,1992b) and also
have proved useful in the study of Attwater's Prairie Chicken.
The microsatellite probe, (TC)n, was one of the fingerprint
probes that was able to identify a genetic marker. It was not used in
the similarity studies because probing with (TC)n revealed a high
level of band sharing among the 16 Attwater' s Prairie Chickens
(Table 3). The importance of (TC)n was observed when Attwater's
Prairie Chicken and Greater Prairie Chicken DNAs were digested
with either Hae III or Hinf I and then hybridized with (TC)n. This
combination of probe and enzymes revealed a genetic marker for
sex identity. The sex marker was a very intense band of high
molecular weight estimated to be about SO kb in the female prairie
chickens (Figures 4 and 5). The only other probe to show a genetic
marker for sex identity was a trinucleotide, (TCC)n that was not as
intense as (TC)n but was about the same high molecular weight.
Many different probes and enzyme combinations were tried to
find a genetic marker to separate the Attwater' s Prairie Chicken
from the Greater Prairie Chicken. Probes such as (GT)n and pV47,
produced variable DNA fingerprints when hybridized to DNA of
Attwater's Prairie Chicken but were not informative as genetic
markers. When attempts were made to hybridize the microsatellite
probe (GC)n to DNA of Attwater's or Greater Prairie Chicken, no
hybridization was detected. This does not mean that there were no
(GC)n sequences; the sequences could be present in small copy
number and therefore hard to detect. Two autoradiograms
containing Attwater's and Greaters' DNA samples were scored using
39
the same method as used for the autoradiogram with Attwater's
Prairie Chicken. There were 12 individual Greater Prairie Chicken
DNAs and 4 individual Attwater' s Prairie Chicken DNAs and on the
other, 4 Attwater's Prairie Chicken DNAs were compared to 3
Greater Prairie Chicken DNAs. Analysis of these results,
demonstrated that Attwater' s Prairie Chicken was genetically very
similar to Greater Prairie Chicken, but Attwater's demonstrated
more band sharing among themselves than with the Greater Prairie
Chicken.
Future Goals in Producing a Genetic Profile
of Attwater' s Prairie Chicken
Many other uses can be made of DNA fingerprinting in
Attwater's Prairie Chicken. In this analysis, I have examined the
variation in minisatellite and microsatellite sequences to obtain an
overview of genetic variation. What would probably be more
informative is to examine the variation of the genes in the DNA of
Attwater's Prairie Chicken and see if a link can be made between the
amount of variation between the genes and minisatellite and
microsatellite sequences. This project will be made possible since a
genomic library has already been created from Attwater's Prairie
Chicken DNA and is ready to be used.
Dunnington et al. ( 1990) has shown that a DNA fingerprint can
be linked to phenotypic traits in lines of chickens that have been
bred for specific body weights. In a captive breeding program, if
certain diseases or unacceptable physical traits appear, it might be
40
possible for DNA fingerprints to be used as a diagnostic test on the
selection of parents to see if they carry these traits in their genetic
makeup before mating takes place.
Another factor that can be examined is the genetic effect of a
species going into a bottleneck and hopefully coming out of it
successfully. Kuhnlein et al. ( 1990) have created a calibration curve
to assess inbreeding with known strains of domestic chickens
and believe it will be useful in examining other species. This
calibration curve could possibly be used on Attwater's Prairie
Chicken from different locales to determine if and where inbreeding
may be taking place and where management might be needed to
keep different populations of Attwater's Prairie Chicken genetically
healthy.
41
CHAPTER V
RECOMMENDATIONS
Some of the problems encountered during this study, such as the
inability to define the exact locations from which the Attwater's
Prairie Chickens were collected, and duplicate samples taken from
one bird but identified as two birds could easily have been avoided
by adequate planning. However, birds were collected by several
different people over a number of years prior to initiation of this
project and procedures had not been established. Results from the
molecular techniques developed during this study provide a level of
management information for greater than that commonly availiable
using other procedures. These techniques have similar nature to
management and recovery of other endangered species.
Specific recommendations are as follows:
1. Record date and location for each tissue sample (carcass,
feathers, egg shells, blood, etc.) collected.
2. Coordinate all tissue collections through one central point
to avoid duplication and establish reliable records.
3. Ration all tissue samples once collected. For example, if
collecting blood samples for serum analysis do not discard the blood
cells but maintain them for analysis by others.
4. Foster open communication among all participants involved
in research, management, or recovery of endangered species.
Perhaps a regularly distributed newsletter would serve this need.
42
5. Assure that tissue samples collected for DNA analysis are
property taken, handled, and stored. Have protocols available for
anyone that might be involved.
6. Develop DNA libraries for endangered species.
7. Evaluate the ability of at least three restriction
endonucleases to produce polymorphic fragments.
8. Screen polymorphic fragments with a series of probes to
select those yielding the greatest amount of information.
9. Have mate selection for captive brood stock based on
maintaining the greatest blend of genetic diversity.
10. Avoid paired matings of individuals with genetic diversity
less than the average found within the population.
11. For Attwater's Prairie Chicken collect egg shells from birds
nesting in the wild, extract residual DNA and screen for genetic
diversity.
12. Develop specific molecular probes for Attwater's Prairie
Chicken based on the DNA libraries.
43
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