SC/F14/J27

Note on sampling and laboratory procedure protocols ofthe genetic work at the Institute of Cetacean Research
Naohisa Kanda, Mutsuo Goto, Hiroyuki Oikawa, and Luis A. Pastene
The Institute of Cetacean Research, Toyomi-cho 4-5, Chuo-ku, Tokyo 104-0055 Japan

Contact email: kanda@cetacean.jp

ABSTRACTThis note presents a summary of sampling and laboratory protocols for the genetic studies under the Objective 3of the JARPAII conducted at the Institute of Cetacean Research.

KEYWORDS: ANTARCTIC, GENETICS, SCIENTIFIC PERMITSINTRODUCTION
Objectives of the JARPAII include the elucidation of temporal and spatial changes in stock structure of Antarctic
minke whale and the description of stock structure of other baleen whale species in the Antarctic feedinggrounds. Genetic analyses utilizing different kinds of genetic markers are one of the important tools to address
the stock structure questions in JARPAII.
In order to minimize errors in genetic data, we were recommended to follow the IWC approved guidelines for
DNA data quality control for genetic studies (IWC, 2009). The purpose of this short note is thus to summarize
the protocols of sampling and laboratory works for the genetic studies at the Institute of Cetacean Research(ICR), on which the population genetic structure studies under the JARPAII are based.
COLLECTION OF GENETIC SAMPLES
Since JARPA started, we have established standard sampling, sample handling, and laboratory practiceprocedures to ensure consistent data generation.
Catches
In JARPAII, this section applies to Antarctic minke and fin whales. Skin tissues are taken as genetic samples
from each individual whale on board by experienced scientists. The sampling involved the use of sterile andsingle-use tools (scalpel and plastic tongs). Two pieces of approximately 5×5×5mm skin tissues are taken andare kept in a 99% ethanol filled tube. Prior to the sampling, all of the tubes are labeled with sample names
following our naming rule for the catches: e.g., 10/11AM001 indicating the austral summer season (=season), thetargeted species (AM= Antarctic minke) and the individual identification number (001). The same codes areapplied to several other biological items and ancillary information so that these codes for the genetic samples
allow for cross referencing with the comprehensive database of JARPAII.
BiopsyBiopsy sampling procedures in JARPAII are similar to those used in the IDCR/SOWER cruises. Biopsy
sampling (skin/blubber) occurs on large vessels using either a crossbow or black powder gun. Photo-id
experiments are conducted in parallel to biopsy sampling. Efforts to avoid re-sampling (two biopsy samples fromthe same individual) are made. A small tissue sample (< 1 gram) is obtained from a free-ranging individual using
a biopsy dart with a stainless tip measuring approximately 4cm in length with an external diameter of 9mm,
which was fitted with a 2.5cm stop to ensure recoil and prevent deeper penetration. Between the sampling
periods, the biopsy tips are thoroughly cleaned and are sterilized with bleach. The obtained biopsy samples aretreated using sterile and single-use tools (scalpel and plastic tongs). Each of the samples is kept in a 99% ethanol
filled tube or in a small zip plastic bag (-20 ºC). Prior to the sampling, all of the tubes are labeled with sample
names following our naming rule for the biopsies: e.g., ‘J03YS2H001’ specifying the research program(J=JARPAII), season (03/04), vessel (YS2= Yushin Maru No. 2), target species (H= humpback whale) andindividual number (001). This code allows cross referencing with ancillary information from the biopsy
sampling experiment.

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STORAGE OF GENETIC SAMPLES
Prior to DNA extraction, the individual tissue samples from catches in the ethanol filled tubes are stored at room
temperature. The biopsy samples in the ethanol filled tubes are stored at room temperature and those in the small
plastic bag are stored in a freezer (-20ºC) as frozen tissues.
DNA EXTRACTION
Total DNA of each individual is extracted from 0.05g of the collected tissue using either the standard
phenol/chloroform extractions protocol of Sambrook et al. (1989) or the GENTRA PUREGENE DNA extraction
kit (QIAGEN) following the company’s manual. Extracted DNAs are stored in the TE buffer (10 mM Tris-HCl,
1 mM EDTA, pH 8.0).
SEQUENCING AND GENOTYPING PROCEDURE
At present, two different kinds of genetic markers, mitochondrial DNA (mtDNA) and microsatellite DNA, areused in the ICR.
mtDNA
The first 490 base-pairs at the 5' end of the control region of mtDNA are sequenced for the genetic analysis. Aset of primers for PCR amplification is usually MT4 (Arnason et al. 1993) and Dlp 5R (5'-CCA TCG AGA TGTCTT ATT TAA GGG GAA C-3'). The PCR reactions are carried out in 25 uL volumes containing 10-100ng ofDNA, 2.5 pmole of each primer, 0.5 units of Ex Taq DNA polymerase (Takara), 2mM of each dNTP, and 10x
reaction buffer. After an initial denaturation step at 95° C for 5 minutes, a PCR amplification cycle of 30 seconds
at 94°C followed by 30 seconds at 50°C and 30 seconds at 72°C is repeated 30 times. The amplification iscompleted with a final extension step of 10 minutes at 72°C. Subsequent cycle sequencing reactions areperformed with 100ng each of the PCR products generated in the above amplifications using the BigDye®
Terminator Cycle Sequencing Kit (Applied Biosystems). The primers for the cycle sequencing reactions are thesame as used in the initial PCR amplification listed above. The cycle sequencing of 10 seconds at 96°C followed
by 20 seconds at 56°C and four minutes at 60°C is repeated 25 times. The nucleotide sequence of each cycle
sequencing reaction is determined using Applied Biosystems 3500 Genetic Analyzer (Life Technology) understandard conditions. Both strand samples are sequenced in their entirety for all samples.
Microsatellite DNA
Different sets of the microsatellite DNA loci are used for different species.
Species
Antarctic minke whale

Microsatellite DNA lociAC045, AC082, AC087, AC137, CA234, DlrFCB14, EV1, EV104, GT23,GT129, GT195, GT211

Fin whale

DlrFCB14, EV1, EV14, EV21, EV37, EV94, EV104, GATA28, GATA98,GAT417, GT23, GT195, GT211, GT310, GT575, TAA31

Humpback whale

AC137, CA234, EV1, EV14, EV37, GATA28, GATA53, GATA98, GATA417,
GT23, GT195, GT271, GT310, TAA31

Southern right whale

DlrFCB17, EV1, EV14, EV21, EV37, EV94, GATA28, GT23, GT211, GT310,
TR2F3, TR3G2, TR2G5, TR3F2

All of these loci have been already published:
Bérubé et al. (2005) : AC045, AC082, AC087, AC137, CA234, and GT129
Buchanan et al. (1996): DlrFCB14, DlrFCB17
Valsecchi and Amos (1996): EV1, EV14, EV21, EV37, EV94
Bérubé et al. (2000): GT23, GT195, GT211, GT271, GT310
Palsbøll et al. (1997): GATA28, GATA53, GATA98, GATA417, TAA31
Frasier et al. (2006): TR2F3, TR3G2, TR2G5, TR3F2
Although primer sequences follow those of the original authors, an annealing temperature of each of the loci is
optimized for each of the whale species. Although the IWC guidelines (IWC, 2009) recommended to sequence
the microsatellite loci for marker validation, we have not had the opportunity yet to do so by ourselves. The
published primers were designed from the cetacean species and were checked for the reliability of amplificationand scoring prior to use by the original authors.

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PCR amplifications are performed in 15ml reaction mixtures containing 10-100ng of DNA, 5 pmole of eachprimer, 0.625 units of Ex Taq DNA polymerase (Takara), and 2mM of each dNTP, and 10x reaction buffer
containing 20mM MgCl2 (Takara). The amplified products, each with an internal size standard(GENESCAN400HD, Life Technology), are run on a 6% polyacrylamide denaturating gel (Long Ranger) using
BaseStation100 DNA fragment analyzer (BioRad). Although the amplified alleles are visualized and are size
determined using the computer software CARTOGRAPHER specifically designed for the BaseStation, allelicsizes for the data base are determined manually in relation to the internal size standard and DNA of known sizes
from the 'control' individual that is rerun on each gel.
Sex determination
For the biopsy samples, the slightly modified method of Abe et al. (2001) is used, which involves
co-amplification of SRY locus on the Y chromosome and a microsatellite locus (usually GT23). With thiscombination of loci, males show the amplified products of both SRY and microsatellite loci, while females show
only the product of the microsatellite locus. Molecular sex determination is not performed for the catches
because the sexes of the whales were determined on board by the experienced scientists by observing thereproductive organs.
CONTROL RELIABILITY
For both mtDNA and microsatellites analyses, no attempt has been made to estimate statistical error rates.
Although the IWC guidelines recommend scoring by two or more people for double-checking, scores are in
general conducted by a single person.
mtDNA
For each individual sample, both forward and reverse sequences are typed for error check. After the first run, allof the ambiguous sequences, singletons, and sequences with the unusual number of transversion, are repeated forconfirmation.
Microsatellites
Although typing procedure is repeated only in the case of samples not providing satisfactory results in the firstattempt, some individuals, chosen arbitrarily, from the samples are genotyped more than twice for doublechecking. In some cases, side by side running to check genotypes is conducted when there is some uncertainty inthe pattern found. For data consistency, a computer program MICRO-CHECKER (van Oosterhout et al., 2004)is used to check for null alleles and reading/typing errors. A computer program GENEPOP (Rousset, 2008) isused to test deviation from Hardy-Weinberg genotypic expectations and, if needed, linkage disequilibrium.
Identity check, such as duplicate sampling in the biopsy samples, is conducted using a computer program
CERVUS (Marshall et al., 1998). Re-sampled animals are not used in the statistical analyses. Input data files forthe computer programs are in general made first in the GENEPOP format, and are converted to the otherrequired formats using the computer software, such as through the file conversion option in GENEPOP.
DATA BASE STRUCTURE
Sequence and microsatellite genotype data are stored in Excel files by markers and species.
REFERENCES
Abe, H., Goto, M., Pastene, L.A., Dewa, K. and Naito, E. 2001. Practical use of multiplex fluorescent PCR for
cetacean sex identification. Mar. Mammal Sci. 17: 657-664.
Arnason, U., A. Gullberg, and B. Widegren. 1993. Cetacean mitochondrial DNA control region: sequences of allextant baleen whales and two sperm whale species. J. Mol. Biol Evol. 70: 960-970.
Bérubé, M., Jørgensen, H., Mcewing, R., and Palsbøll, P.J. 2000. Polymorphic di-nucleotide microsatellite lociisolated from the humpback whale, Megaptera novaeanglliae. Mol. Ecol. 9: 2181-2183.
Bérubé, M., Rew, M.B., Skaug, H., Jørgensen, H., Robbins, J., Best, P., Sears, R., and Palsbøll, P.J. 2005.
Polymorphic microsatellite loci isolated from humpback whale, Megaptera novaeanglliae and fin whale,balaenoptera physalus. Cons. Genet. 6: 631-636.
Buchanan, F.C., Friesen, M.K., Littlejohn, R.P., and Clayton, J.A. 1996. Microsatellites from beluga whale
Delphinapterus leucas. Mol. Ecol. 5: 571-575.
Frasier, T.R., Rastogi, T., Brown, M.W., Hamilton, P.K., Kraus, S.D., and White, B.N. 2006. Characterization oftetranucleotide microsatellite loci and development and validation of multiplex reactions for the study ofright whale species (genus Eubalaena). Mol Ecol. Notes 6: 1025-1029.
International Whaling Commission. 2009. Report of the Scientific Committee. Annex I. Report of the working
group on stock definition. Appendix 2. Guidelines for DNA data quality control for genetic studies relevant
to IWC management advice. J. Cetacean Res. Manage. (Suppl.) 11:252-256.

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Marshall, T.C., Slate, J., Kruuk, L., and Pemberton, J.M. 1998. Statistical confidence for likelihood-based
paternity inference in natural populations. Mol. Ecol. 7: 639-655.
Palsbøll, P.J., Bérubé, M., Larsen, A.H., Jørgensen, H. 1997. Primers for the amplification of tri-and tetramer
microsatellite loci in baleen whales. Mol. Ecol. 6:893-895.
Rousset, F. 2008. Genepop'007: a complete re-implementation of the genepop software for Windows and Linux.
Mol. Ecol. Resources 8:103-106.
Sambrook, J., Fritsch, E.F., and Maniatis, T. 1989. Molecular cloning: A laboratory manual. 2nd Ed., ColdSpring Harbor, NY: Cold Spring Harbor Laboratory.
Valsecchi, E., and Amos, W. 1996. Microsatellite markers for the study of cetacean populations. Mol. Ecol. 5:151-156.
van Oosterhout, C., Hutchinson, W.F., Wills, D.P., and Shipley, P. 2004. MICRO-CHECKER: software foridentifying and correcting genotyping errors in microsatellite data. Mol. Ecol. Notes 4:535-538.

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