9698

• UCREFRP Catalog Number: 9698
• Author: Borley, K., and M.M. White.
• Title: Mitochondrial DNA variation in the endangered Colorado pikeminnow
• USFW Year: 2006.
• USFW - Doc Type: a comparison among hatchery stocks and historic specimens.
• Copyright Material: YES

[Article]

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DNA VARIATION IN COLORADO PIKEMINNOW

917

cted to
intains
}reen-
e three
n 1890
edin2
leek in

these populations may have existed historically but has
been masked by stocking of hatchery fish and
anthropogenic barriers to movement. They recom-
mended maintaining three hatchery stocks (Yanlpa,
Green, and Colorado rivers), and they suggested that
the Green and Colorado rivers should be considered
separate MUs (Morizot et al. 2002).
In this paper, we compare mitochondrial DNA
(mtDNA) sequence variation within and anlong the
three stocks maintained at DNFHTC. For comparison,
we also surveyed mtDNA variation in museum
specimens collected from the wild before initiation of
the stocking progranl. Our goal was to assist in
determining whether the three hatchery stocks should
be maintained separately.

;e's (USFWS)
nology Center
leveloped the
Yanlpa River
;ollected from
81 year-class
tablished from
orado rivers in
lorado DX-Fo;
:cted from the
(Willianlson et
. Willianlson,

Methods
Tissue samples.-Tissue or DNA sanlples were
obtained from 74YC (N = 8), 81YC (N = 12), and
91 YC (N = 10) from DNFHTC (Table 1). Tissues were
obtained from museum specimens collected from the
Green (N = 5), San Juan (N = 2), lower Colorado (N =
2), and upper Colorado (N = 2) rivers before (1890-
1974) or just after (1976) establishment of hatchery
stocks (Figure I; Table 2).
Extraction, amplification, and sequencing of
DNA.- The DNA was extracted from hatchery-derived
tissues with a DNeasy tissue isolation kit (Qiagen, Inc.,
Valencia, California). The DNA was extracted from
museum sanlples with the DNeasy kit following the
protocol in Chase et al. (1998). A 1,009-base Rair (bp)
mtDNA fragment consisting of portions of theND-4 and
ND-4L NADH dehydrogenase subunits was anlplified
with the primers NAP2 (Hogan et al. 1997) and ArgBL
(Bielawski and Gold 2002) for 81YC and 91YC.
Sanlples from 74YC were anlplified with two sets of
primers with overlapping polymerase chain reaction
(PCR) products (Table 3). The ArgBL and ND4LH
primers anlplified a 513-bp fragment. The primer pair
ND4LB (Bielawski and Gold 2002) and NAP2
anlplified a 695-bp fragment. Museum sanlples were
anlplified with NAn and ND4LB. All PCR reactions
were in 20-IlL volumes (IX NH4 reaction buffer
[Bioline USA, Inc., Randolph, Massachusetts], 3 roM
MgC~, 0.2 mM each deoxynucleotide triphosphate
[dNTP], 0.5 ~lM each primer, 2.5 units Biolase DNA
polymerase [Bioline USA], and genomic DNA).
Thermal cycling was performed at 950C for 30 s, 450C
for 30 s, and 720C for 30 s for 25-40 cycles. Sequencing
was accomplished with an ABI BigDye terminator cycle
sequencing kit (PerkinElmer, Inc., Wellesley, Massa-
chusetts). For sequencing, half-volume reactions (10:2
J.LL BigDye terminator ready reaction. mix, 2 ilL
sequencing buffer, 0.025 pg/J.LL of primer, and approx-

~ been used to
980 and 1984,
ividuals from
:olorado River
1998) reported
IW for reestab-
:ocked through
74YC brood-
o hatchery fish
ere introduced
ad 1998. Since
YC have been
Uver and an
I the Gunnison
nmunication).
Jared allozyme
:luded that the
;imilar to wild
rivers. Morizot
nine variation
ild-caught fish.
ow constitute a
~rgence anlong

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TABLE I.-Sample numbers, year-class designations, PIT
tag numbers, and haplotypes for Colorado pikeminnow
obtained from the Dexter National Fish Hatchery and
Technology Center, New Mexico.
Sample number Year class PIT -tag number Haplotype
400 74 7F7E353264 A
403 74 7F7E340704 A
406 74 7F7E390B38 A
410 74 7F7E2D3113 A
414 74 7F7E35236F A,
419 74 7F7E362314 A
421 74 7F7E2D7CIO A
429 74 7F7EID2F3E A
E6679 81 7F7FIE6679 B
E7 A02 81 7F7F1E7A02 A
E7A1F 81 7F7F1E7A1F A
E7 A22 81 7F7F1E7A22 A
E7647 81 7F7F1E7647 A
FOF52 81 7F7F1FOF52 A
F0535 81 7F7F1F0535 A
F1E06 81 7F7FIF1E06 A
F1F24 81 7F7F1F1F24 A
FI022 81 7F7F1FI022 A
FllOF 81 7F7F1FllOF B
FI666 81 7F7F1FI666 A
91-1 91 7F7BIA5F22 A
91-2 91 7F7D14OBIF A
91-3 91 7F7D131617 A
91-4 91 7F7B145416 A
91-5 91 7F7B3C4657 A
91-6 91 7F7B012EOE A
91-7 .91 7F7BI24773 A
91-8 91 7F7D136A57 A
91-9 91 7F7D487571 A
91-10 91 7F7B 180029 A

imately 75 ng of DNA) were run in a thermal profile of
25 cycles of 960C for 10 s, 500C for 5 s, and 6QoC for 4
min. The reactions were resolved with an ABI PRISM
310 genetic analyzer (PerkinElmer). Chromas (C.
McCarthy, Griffith University, Southport, Australia)
and Clustal X (Jeanmougin et al. 1998) software
programs were used to edit and align sequences.

Results
A total of 872 bp, which consisted of 288 bp of the
ND-4L gene and 591 bp of the ND-4 gene (the genes
overlap by 7 bp), was obtained for the hatchery
sanlples. The sequences were submitted to GenBank
(DQ248317 and DQ248318). Two haplotypes (A and
B) were present in the hatchery stocks (Table 1). All
individuals sequenced from 74YC and 91 YC possessed
haplotype A. The 81 YC sanlple consisted of 10
individuals with haplotype A and 2 individuals with
haplotype B. Haplotype B differed from haplotype A
by a single substitution in the ND-4 coding region (C to
G at base position 866). A total of 612 bases spanning
the -ND-4L (30 bases) and ND-4 (592 bases) regions
were obtained from the museum sanlples. All museum
sanlples had haplotype A.