Conservation Genetics Resources
Online Resource 1
Development of 20 TaqMan assays differentiating the endangered shortnose and Lost River suckers
M. S. Hoy and C. O. Ostberg
U.S. Geological Survey, Western Fisheries Research Center, 6505 NE 65th Street, Seattle, WA 98115
Corresponding author: Carl O. Ostberg, costberg@usgs.gov
Methods
Samples
Shortnose sucker (SNS) (Chasmistes brevirostris) and Lost River sucker (LRS) (Deltistes luxatus) fin tissues
were collected from adults (2004 – 2006, 2009, and 2011) in Upper Klamath Lake, southern Oregon, and
Clear Lake, northern California, and whole young-of-the-year (YOY) LRS and SNS collected from Upper
Klamath Lake (2004 – 2011). Adults were identified to species by snout and lip morphology. Young-ofthe-year were identified to species by vertebral counts, with the exception that gill raker counts were
incorporated for individuals having 44 vertebrae or less. Young-of-the-year SNS and LRS are primarily
differentiated by their number of vertebrae; SNS have 41 – 44 and LRS have 44 – 48 (Markle et
al. 2005). Gill raker number is used to assist species identification for YOY having 44 vertebrae,
although gill raker number overlaps between LRS and SNS. In addition, SNS and Klamath
largescale sucker (Catostomus snyderi) have similar numbers of vertebrae and their
differentiation relies on gill rakers and other meristic characters. Species identification was
performed by field biologists at U.S. Geological Survey (Western Fisheries Research Center, Klamath Falls
Field Station) and Bureau of Reclamation (Klamath Basin Area Office). Herein, we designate the species
identifications by field biologists as the a priori assignment. Genomic DNA was extracted from fin tissues
using the UltraClean Tissue and Cells DNA Isolation Kit (MO BIO Laboratories).
Marker discovery using locus-specific sequencing
Two marker discovery approaches were applied to identify species-diagnostic candidate SNPs; locusspecific sequencing and restriction-site-associated DNA (RAD) sequencing (Baird et al. 2008). For locusspecific sequencing, PCR primers were generated from SNS nuclear DNA (nDNA) sequence data
available in Genbank and were used to amplify and sequence 15 YOY LRS and SNS each. Sequences were
edited and aligned using SEQUENCHER v 4.10.1 (Gene Codes Corporation). One PCR primer set designed
from accession number AY351369.1 (AL176; F:5’-TCATAATAACCATCACATCAC-3’; R:5’AGTCCCATTGGCGAGAG-3’) amplified a 435 base pair product that contained a species-diagnostic
candidate SNP. PCR chemistry for AL176 amplification consisted of 10 ng genomic DNA, 10x Reaction
Buffer (Bulldog Bio), 500 µM dNTP Mix (Bioline), 0.5 µM of each primer, and 1.0 units BioReady rTaq
(Bulldog Bio). Cycling conditions consisted of initial denature at 94oC for 5 minutes, followed by 35 cycles
at 94oC for 30 seconds, anneal at 52oC for 15 seconds, and extension at 72oC for 25 seconds, followed by
a final extension at 72oC for 5 minutes. A restriction enzyme assay was designed for the AL176 candidate
SNP using TasI (MBI Fermentas) and was applied to 66 adults from each species. The TasI-digested
AL176 amplicon produced two restriction fragment length polymorphism patterns: one common to SNS
(21, 26, and 388 base pairs) and a second common to LRS (21, 26, 58, and 330 base pairs).
We also amplified and sequenced 771 base pair of the mitochondrial DNA (mtDNA) Cytochrome
B (CytB) gene in 15 YOY LRS and SNS each, using primers L14725 and H15573 (Meyer 1993). PCR
chemistry consisted of 10 ng genomic DNA, 10x Reaction Buffer (Bulldog Bio), 500 µM dNTP Mix
(Bioline), 0.5 µM of each primer, and 1.0 units BioReady rTaq (Bulldog Bio). Cycling conditions consisted
of initial denature at 94oC for 5 minutes, followed by 35 cycles at 94oC for 30 seconds, anneal at 56oC for
1 minute, and extension at 72oC for 1 minute, followed by a final extension at 72oC for 5 minutes. The
CytB amplicons were sequenced, edited and aligned using SEQUENCHER v 4.10.1 (Gene Codes
Corporation), and yielded two haplotypes: one common to SNS and an alternate common to LRS. A
restriction enzyme assay that differentiated the two CytB haplotypes was designed using BamHI (MBI
Fermentas), and this restriction enzyme assay was applied to 66 adults from each species. The BamHIdigested CytB amplicon produced two restriction fragment length polymorphism patterns: one common
to SNS (102,159, and 510 base pairs) and a second common to LRS (159 and 612 base pairs).
Taqman assays were designed for the AL176 SNP (designated as Dlux176) and a SNP in CytB
(designated as Dlux434) using Primer Express v3.0 (Applied Biosystems) (See Table 1 in main article).
For each assay, we designated the allele most common to a priori identified SNS and LRS as ‘A’ and ‘B’,
respectively.
Marker discovery using RAD sequencing
For RAD sequencing, we selected 24 SNS, consisting of 12 adults from Upper Klamath Lake and 12 from
Clear Lake, and 24 LRS, consisting of 13 adults and 4 YOY from Upper Klamath Lake and 7 adults from
Clear Lake. A RAD library was generated for each individual using 500 ng of SbfI digested genomic DNA
following the methods described by Etter et al. (2011). Two pooled libraries of bar-coded fragments,
each containing 12 LRS and 12 SNS, were sequenced by University of Oregon on an Illumina Hi Seq 2000
instrument. We analyzed single-reads and applied several modules from STACKS v. 0.99992 (Catchen et
al. 2011) to sort, filter, and align sequence reads into RAD loci. The application process-radtags was used
to filter read quality and remove sequences without intact barcodes. Reads with quality score < 33 were
removed. Next, we applied ustacks to build stacks of RAD loci for each individual, using a minimum
coverage depth of 5 total reads per individual, and enabled the removal and deleveraging algorithms to
filter out highly repetitive loci. The application cstacks was used to form a catalog of RAD loci common
to all members by merging loci across individuals. We allowed three nucleotide mismatches between
loci for the between-individual distance parameter. Last, sstacks was used to match RAD loci from
individuals against the catalog formed in cstacks.
Species-diagnostic candidate SNPs were identified within the catalogue by first applying scripts
that removed monomorphic loci and loci containing two or more nonconsecutive SNPs
(https://github.com/marshalhoy/RAD-suckers). We then removed loci having > 20 % heterozygosity
across all individuals. Next, individuals having > 10 % heterozygosity over all loci were removed from the
catalogue. This step was performed to remove potential SNS-LRS hybrids from the catalogue. Finally, we
applied a script to retain loci fixed for alternate alleles between SNS and LRS. The average read coverage
for species-diagnostic candidates was 64 reads/SNP for SNS and 56 reads/SNP for LRS.
We designed 28 TaqMan assays using the first 75 bases of each RAD locus to avoid sequencing
errors that may be incorporated toward the 3’ terminus of single reads. TaqMan sssays were designed
using Primer Express v3.0 (Applied Biosystems) and assays were run on a viiA7 Real-Time PCR System
(Applied Biosystems). Eighteen of the 28 Taqman assays produced high quality amplification products
(See Table 1 in main article). Validation of these 18 assays against the individuals included in the final
catalogue indicated that 99.2 % of genotypes were congruent between RAD sequences and TaqMan
assays. For each assay, we designated the allele most common to a priori identified SNS and LRS as ‘A’
and ‘B’, respectively.
Evaluation of TaqMan assays
TaqMan assays were evaluated in 160 YOY identified a priori as LRS (N = 79) and SNS (N = 81) from
Upper Klamath Lake. These YOY were not used in the RAD library construction and consisted of
individuals pooled across years (2004 – 2010). Allele frequencies and observed and expected
heterozygosity were calculated for the a priori identified species, and multilocus FST was estimated
between SNS and LRS using GENEPOP 4.2 (Raymond and Rousset 1995). We tested each locus for
conformation to Hardy-Weinberg equilibrium and tested all permutations of nuclear locus pairs for
genotypic linkage disequilibrium using GENEPOP 4.2. Significance levels were adjusted using the false
discovery rate (FDR) procedure (Benjamini and Yekutieli 2001). Test results were considered significant
when P exceeded the FDR adjusted critical value (α) for each series of simultaneous tests.
References
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