9411

• UCREFRP Catalog Number: 9411
• Author: Williamson, J. H., D. C. Morizot and G. J. Carmichael.
• Title: Biochemical Genetics of Endangered Colorado Pikeminoow from the Green, Yampa, Colorado, and San Juan Rivers.
• USFW Year: 1998.
• USFW - Doc Type: \
• Copyright Material: NO

YOY fish were captured during annual fall monitoring trips in 1990 and 1991, and were collected using
electroshock and small-mesh brail seines.
Biochemical Genetic Analyses.- YOY fish collected in the field were euthanized with an overdose
of MS-222 (tricaine methane sulfonate). Some fish were frozen whole on dry ice and some were frozen
after the head and viscera were removed for uses peripheral to this study (see discussion below). After
transfer to the laboratory and storage at -80 C, necropsy samples of skeletal muscle and fins were obtained
and stored at -80 C. Adult and juvenile Colorado pikeminnow collected in the field were anesthesized with
MS-222 and biopsy samples of skeletal muscle and fin were taken. In order to prevent cross sample
contamination, biopsy samples of skeletal muscle and fin were taken from fish using separate disposable
muscle punches, and minced with sterilized, rinsed, and dried scissors. Biologists wore new disposable
latex gloves for each fish. Separate syringes, needles, and latex gloves were used on individual fish to
collect blood. Biopsy samples of skeletal muscle and fins intended for protein electrophoresis were frozen
on dry ice until transfer to the laboratory and long term storage at -80 C. Muscle samples intended for
DNA analyses were stored in 95% ethanol at ambient temperatures.
In order to determine expression of biochemical genetic loci in various tissues, 34 YOY individuals
produced at the Dexter NFH&TC from Yampa DX-FI(74) broodfish were euthanized with an overdose of
MS-222 and necropsy samples were stored at -80 C. These individuals were sampled in 1991 and bear the
designation of Yampa River DX-FZ(91). Samples of skeletal muscle, liver, brain, eye, caudal fin, pectoral
fin, erythrocytes, blood plasma, and gonads were prepared and subjected to vertical starch gel
electrophoresis and histochemical visualization using the methods of Morizot and Schmidt (1990).The
products of > 89 loci were resolved and assessed for electrophoretic polymorphism(Table 1).
The number of loci is somewhat uncertain because several of the loci included have not been
studied extensively in cyprinids. Gene and protein nomenclature follows that proposed by Shaklee et al.
(1990). Isozymes (numerically) and allozymes (alphabetically in lower case letters) were designated in
order of increasing anodal mobility. Data analyses were performed using the BIOSYS- I program of
Swofford and Selander (1981), with the data entered as individual genotypes at each locus. These
analyses, however, were performed solely on a subset of loci as only polymorphic loci and several
monomorphic loci were analyzed on the majority of fish sampled.
Nuclear DNA Analyses.- A polymerise chain reaction (PCR)-based analysis of potential
Mendelian polymorphisms from anonymous single-copy nuclear DNA was conducted on blood samples
taken from Colorado pikeminnow from the Dexter NFH&TC (Yampa River DX-FI(74) and
Colorado/Green DX-F1(81)), the Green River(Desolation Canyon), Yampa River, and 10 wild fish from the
Colorado River. Blood samples were kept in DNA-stabilizing EDTA buffer (0.25 mL blood and 7.5 mL
DNA lysis and preservation buffer in 15 mL tubes) at ambient temperatures. This approach examined fish
for restriction fragment-length polymorphisms using primers constructed from clones isolated from a
bovine nuclear DNA library. Primer pairs complementary to two single copy nuclear genes, gastrin and
thyroxine were designed and provided by J. N. Derr (Texas A&M University, College Station, TX). These
sequences are highly conserved in human, rodent, and bovine DNA, and the primers are complementary to
the conserved regions (exons) which flank regions that are potentially more polymorphic (introns).
Total DNA was extracted from 200-600 uL of blood diluted in lysis buffer (Longmire et al.
1988). Lysates were digested for 2 hours at 55 C in 500 ug/ml proteinase K. Two 10 minute extractions
with phenol:chloroform:isoamyl alcohol were followed by two 5 minute extractions with chloroform and
final precipitation with two volumes of ethanol. DNA was spooled, dissolved in 1 mM Tris, 0. 1 mM
EDTA (pH 8.0), and stored at -80 C.
PCR reactions contained 50 mM KCI, 10 mM Tris-HCI, pH 8.3,1.5 mM MgCl, and 0.2 mM
each of the four dinucleotide triphosphates, primers at 0.33 mM, and 2.5 units of Taq polymerase in a 50
uL volume overlaid with mineral oil. Reactions were run in a Coy DNA thermal cycler for 30 cycles with
a 48 C annealing cycle (1 min.), a 72 C extension cycle (1 min.), and a 93 C denaturing cycle (0.5 min.). A
single I min. denaturation at 95 C preceded the 30 cycles, and the final cycle was extended for 10 min. at