Molecular Markers Used to Analyze Species

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Molecular markers used to analyze species-specific status in

abalones with ambiguous morphology.ABSTRACT Pacificabalone abalone (bl`n), popular name in the UnitedStates for a univalve gastropod mollusk of the genusHaliotis, members of whichare also called ear shells, or sea ears, as their shape resembles the human ear...... Click the link for more information. (Haliotis discus hannai) and Californianred abalone (Haliotis rufescens) are easily distinguished by their shell andepipodial characteristics. A few individuals of H. rufescens were discovered tohave epipodial coloration similar to that of H. discus hannai suggesting that theymay have been hybrids of the two species, because hybridizationhybridization /hybridization/ (hi?brid-i-zashun)1. crossbreeding; the act or process of producing hybrids.

2. molecular hybridization

3...... Click the link for more information. of these species has been informallyreported from local hatcheries. The present study uses molecular genetic methodsto determine whether the rarely colored variants represented hybrids between thetwo species. Two hundredRAPD RAPD Randomly Amplified Polymorphic DNARAPD relative afferent pupillary defect (ophthalmology; aka Marcus-GunnPupil) primers were tested with PCRPCR polymerase chain reaction.

PCRabbr.

polymerase chain reaction

Polymerase chain reaction (PCR)..... Click the link for more information. on two DNA DNA: see nucleic acid.

DNAordeoxyribonucleic acid

One of two types of nucleic acid (the other is RNA); a complex organiccompound found in all living cells and many viruses. It is the chemical substance

of genes. pools of eight individuals of each species. Primers that showed differentamplification profiles between species were tested on 27 randomly sampledindividuals of each species to check the existence of polymorphisms. Two primersdifferentiate both species. Primer 356 amplified an approximately 450 bp and 460

bp specifics-DNA fragments in H. discus hannai that were not present in redabalone, whereas primer 368 amplified a 750 bp, 850 bp, 860 bp, and 1,190 bpspecific-DNA fragments in H. rufescens that were not present in H. discus hannai.The amplification pattern of the DNA of individuals with ambiguous morphologyfor both primers was characteristic of red abalone. The results strongly suggestedthat the specimens with unusual epipodial coloration were not hybrids, but rather,

phenotypic variants of H. rufescens. Future studies should focus on the cause of

the variation of epipodium color in H. rufescens, which could be either genetic
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polymorphism polymorphism, of minerals, property of crystallizing in two ormore distinct forms. Calcium carbonate is dimorphous (two forms), crystallizing

as calcite or aragonite. Titanium dioxide is trimorphous; its three forms arebrookite, anatase (or octahedrite), and rutile. or phenotypic plasticity.

KEY WORDS: abalone, hybridization, Haliotis rufescens, Haliotis discus hannai,DNA testing DNA testingAnalysis of DNA (the genetic component of cells) in order to determine changesin genes that may indicate a specific disorder.

Mentioned in: Acoustic Neuroma, Retinoblastoma, Von Willebrand Disease ,

RAPD, culture, Chile

INTRODUCTION

Abalones aregastropod gastropod, member of the class Gastropoda, the largestand most successful class of mollusks (phylum Mollusca), containing over 35,000living species and 15,000 fossil forms. molluscs belonging to the genus Haliotis(Mollusca: Gastropoda). These molluscs have a dorsally flattened bowl-shapedshell that protects most of the body, and a large ventral foot only slightly smallerthan the shell. A major feature of the exterior morphology is the epipodium, amantle extension around the edge of the foot. The epipodium varies in itsstructural detail, coloration, and texture among species and is one way todistinguish between the different species of abalone (Anderson 2003).

Two species of abalone have been introduced to Chile for culture purposes, thePacific abalone Haliotis discus hannai Ino 1953 and the Californian red abalone,H. rufescens Swainson 1822. These two species are easily differentiated based ontheir shell and epipodium morphology. The shell of the Pacific abalone isnormally green and variably rugose rugose orrugousadj.Having many wrinkles or creases; ridged or wrinkled.

rugose

marked by ridges; wrinkled. with brown and white epipodia. In contrast, the shellof the California abalone cultured in Chile has a finely lined shell with variablecoverage of red and turquoise color and black, smooth epipodia.

Ten individuals obtained from the Center for Abalone Production of theUniversidad Catolica del Norte (CAP-UCN) in 2004 had the typical shell andepipodial morphology of H. rufescens, but the coloration of the epipodia weresimilar to that of H. discus hannai. Two possible hypotheses were considered: (1)hybridization had occurred between Pacific and Californian abalone and (2)

phenotypic variation within H. rufescens caused the unusual coloration of the
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epipodia. The latter might be explained by environmental adaptation and/orgenetic polymorphism.

Several authors have reported the existence of hybrids between abalone species(Owen et al. 1971, Leighton & Lewis 1982, Brown 1995, Freeman 2001, Zhao etal. 2004, Ibarra et al. 2005), and hybridization between H. rufescens and H. discushannai has been informally noted by commercial abalone growers in Chile (A.Zuniga, pers. comm.). On the other hand, morphological variation produced bygenetic factors has been described for variousbivalve bivalve, aquatic mollusk ofthe class Pelecypoda ("hatchet-foot") or Bivalvia, with a laterally compressed

body and a shell consisting of two valves, or movable pieces, hinged by an elasticligament. molluscs, particularly with regard to shell color (Chanley 1961, Innes& Haley 1977, Mitton 1977, Newkirk 1980, Adamkewicz & Castagna 1988,

Wada & Komaru 1990, WinklerWinkler may refer to: Winkler, Manitoba, a Canadian city

Winkler(novel), by Giles Coren

Winkler (crater), a crater on the Moon

Winkler (surname), people with the surname WinklerorWinckler

See also

et al. 2001), abalones (Kobayashi et al. 2004) and other gastropods (Boulding &Hay 1993, Rolan et al. 2004). Morphological variability produced by phenotypic

plasticity has also been reported for gastropod shells (Cole 1975, Wullschleger &Jokela 2002, Veliz et al. 2001, Smith & Ruiz 2004). However, we have found no

published information on the variability of epipodial coloration within abalonespecies.

The use of molecular markers has proved to be a useful tool for complextaxonomic identification where morphological characteristics are ambiguous orcryptic (e.g., Douek et al. 2002, Westheide et al. 2003, Miura et al. 2005, Park etal. 2005). The randomly amplified polymorphic DNA-polymerase chain reaction(RAPD-PCR) technique is a relatively simple and cheap method capable ofdifferentiating taxa taxa: see taxon. without the need to know their genomes

(Welsh & McClelland 1990, Williams et al. 1990). RAPDs are dominant markersthat result from the use of short (10 bases long) primers (syntheticoligonucleotides) of random sequence that can amplify multiple segments ofgenomic DNA by PCR. The number of segments depends on the number of sitesof the genome recognized by a particular primer. The main reason for the successof RAPD analysis is the gain of a large number of genetic markers that requiresmall amounts of DNA without the need of a molecular characterization of thegenome of the taxa under study. Species in which such markers were used includeoyster genera Crassostrea, Saccostrea, and Striostrea (Klinbunga et al. 2000),amphipods (Gammarus: Costa et al. 2004) and the tropical abalones Haliotisasinina, H. ovina and H. varia varia
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n.A miscellany, especially of literary works.

[Latin, from neuter pl. ofvarius, various.] (Klinbunga et al. 2004).

The goal of the present study was to obtain species-specific RAPD molecularmarkers that could distinguish between H. discus hannai and H. rufescens andverify if individuals with ambiguous morphology found at CAP-UCN representhybrids orintraspecific intraspecific also intraspeciesadj.Arising or occurring within a species: intraspecific competition. phenotypicvariability.

MATERIALS AND METHODS

A total of 27 individuals of each species (H. discus hannai and H. rufescens) wereobtained for this study from the CAP-UCN. About 2 [cm.sup.3] of muscle tissuedissected from the foot of each sacrificed specimen was kept in a cryogenic tubeat -20[degrees]C until DNA was extracted. The 10 DNA samples of the abaloneswith ambiguous morphology were obtained from the same tank of H. rufescenshatchery hatchery

a commercial establishment dedicated to the hatching of bird eggs to provide day

old chicks and poults to the poultry industry.

hatchery liquidthe contents of unfertilized eggs. Used in petfood manufacture. broodstock (fromCAP-UCN) and were given to the Laboratory of Molecular Diversity when theywere already dead.

DNA extraction was carried out using a QIAamp Mini Kit (Qiagen Inc.). DNAextractions were verified using the PCR reaction with the HCO-LCO universal

primers (Folmer et al. 1994) for the Cytochrome Oxidase cytochrome oxidasen.

An oxidizing enzyme containing iron and a porphyrin, found in mitochondria andimportant in cell respiration as an agent of electron transfer from certaincytochrome molecules to oxygen molecules. I mitochondrialmitochondrial

pertaining to mitochondria.

mitochondrial RNAsa unique set of tRNAs, mRNAs, rRNAs, transcribed from mitochondrial DNA bya mitochondrial-specific RNA polymerase, that account for about 4% of the totalcell RNA that gene. The presence and intensity of amplification (visualized in anagarose agarose

more highly purified form of agar with similar uses to agar and widely used in the
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separation of nucleic acid fragments. electrophoresis) was used as DNA qualitycriterion. Clear and intense amplicons (bands) in the gel were considered to reflect

that the DNA used was of high quality and quantity. The extracted DNA wasstored at -20[degrees]C until further use.

To analyze a large number of individuals in each PCR reaction, two pools of DNAwere prepared for each species, each pool made up of equal aliquots of DNA fromeight different individuals of the species. Consequently, with two PCR reactions

per species, 16 individuals of each species would be screened. Each DNA poolwas assayed using 200 RAPD primers (RAPD primers 300-500, University ofBritish ColumbiaLocationsVancouverThe Vancouver campus is located at Point Grey, a twenty-minute drive from

downtown Vancouver. It is near several beaches and has views of the North Shoremountains. The 7. ) in independent reactions (each primer and DNA sample in anindependent reaction). The PCR reactions were carried out in a final volume of 10uL, which contained 1X PCR buffer (200 mM Tris-HCL, pH 8.4, 500 mMKCLKCL - Kyoto Common Lisp ), 1.5 mM Mg[Cl.sub.2] 0.1 mM of each dNTP, 0.2[micro]M of each RAPD primer, 1.45 U TaqDNA polymerase DNA

polymerase /DNA polymerase/ (pah-limer-as) any of various enzymescatalyzing the template-directed incorporation of deoxyribonucleotides into aDNA chain, particularly one using a DNA template...... Click the link for more information. (Invitrogen) and approximately 5 ng/[micro]L of genomic DNA. In all the PCRs a negative and positive control were

included, to verify the absence of contamination in the samples and the success ofPCRs respectively.

Amplification was carried out in a PTC (PTC, Needham, MA, www.ptc.com)Long a world leader in mechanical computer-aided design, manufacturing andengineering software, PTC, through acquisitions and reorganization, hastransformed itself into a leading provider of Internet-based B2B solutions fordiscrete manufacturers. 100-MJ Research thermal cycler, with an amplification

program starting with 2 min at 94[degrees]C, followed by 40 cycles consisting of1 min at 94[degrees]C, 1 min at 42[degrees]C, and 2 min at 72[degrees]C,followed by a final extension of 5 min at 72[degrees]C. The PCR products(amplicons) were separated in a 6% denaturing Polyacrylamide gel

polyacrylamide geln.A hydrated polymer consisting of a long chain of amide groups, used as a mediumfor substances that undergo gel electrophoresis. containing 19:1 acrylamideacrylamide /acrylamide/ (ah-krilah-mid) a vinyl monomer used in the

production of polymers with many industrial and research uses; the monomericform is a neurotoxin. : bis-acrylamide and 5 M urea. Electrophoresis wasconducted using a SequiGen sequencing gel electrophoresis system (BIO-RADlaboratories, Hercules, CA). Runs were performed at 6(>100 Watts and55[degrees]C for 7-24 h depending on the sizes of the amplicons. Amplicons werevisualized on the gel using the Promega (Madison, WI) silver-staining protocol

(following manufacturer's instructions) and a 200 base pair (bp) DNA ladder as a
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marker to estimate amplicon size. The primers that produced good amplification(clearly defined bands that differentiated between the two abalone species) were

used in the next amplification step.

We screened the primers that simultaneously showed different and consistentamplification results between species. The potentially useful primers were used toamplify by PCR the DNA of each of the 27 individuals of H. discus hannai and H.rufescens (i.e., primers were used to open the DNA pools). At this stage, primerswere considered as informative and reliable for identification and differentiation

between H. discus hannai and H. rufescens when they produced a specificamplicon in all (27) individuals of one species and none from the other species.These markers were then used to identify the specific status of the 10 abalone withambiguous morphology found at the CAP-UCN.

RESULTS

Of the 200 primers assayed with the pools of DNA from both species, 40 werepotentially useful for differentiating between the two abalone species. Four of the40 primers found produced amplicons exclusive for H. discus hannai and 31 forH. rufescens; whereas five primers simultaneously produced different sizeamplicons unique for each species. The five primers that simultaneously producedamplicons of different sizes and four other primers that produced good bandintensities, difference in molecular weight between amplicons of the two species,and repeatability among assays were chosen and tested in 27 individuals of each

species.

All primers that produced amplicons of different sizes in both species showedintraspecific polymorphisms when they were tested with the 27 individuals ofeach abalone species, disallowing unequivocal differentiation between the twospecies. However, two primers produced specific amplicons on the 27 individuals;

primer 356 (5'GCG GCG Genetics Computer GroupGCG GlucagonGCG Good Corporate GovernanceGCG Global Consumer GroupGCG Global Church of GodGCG Generalized Conjugate GradientGCG Global Change GameGCG Geological Curators' GroupGCG Giant-Cell Granuloma GCC GCC: see Gulf Cooperation Council.

(compiler, programming) GCC - The GNU Compiler Collection, which currentlycontains front ends for C, C++, Objective-C, Fortran, Java, and Ada, as well aslibraries for these languages (libstdc++, libgcj, etc). CTCCTC - Cornell TheoryCenter T 3') for H. discus hannai and primer 368 (5' ACT TGT TGT TargetTGT Ticket Granting Ticket (Windows 2000 Kerberos security)TGT Target Corp (stock symbol)TGT Turbine Gas Temperature

TGT TDRSS Ground Terminal
http://acronyms.thefreedictionary.com/GCGhttp://acronyms.thefreedictionary.com/GCGhttp://encyclopedia2.thefreedictionary.com/GCChttp://encyclopedia2.thefreedictionary.com/CTChttp://acronyms.thefreedictionary.com/TGThttp://acronyms.thefreedictionary.com/GCGhttp://encyclopedia2.thefreedictionary.com/GCChttp://encyclopedia2.thefreedictionary.com/CTChttp://acronyms.thefreedictionary.com/TGT


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TGT Tank Gunnery TrainerTGT Target Tracker GCG G 3') for H. rufescens.

The PCR banding pattern (fingerprint) obtained with primer 368 shows anamplicon of approximately 700 bp that is present in most of the individuals of H.discus hannai, and in some of H. rufescens (Fig. 1, band A). Even though it wasnot a specific amplicon for either of the two species, it was much more frequentand intense in H. discus hannai. With this same primer, four anaplicons specificfor H. rufescens were found. These were of approximately 750 bp, 850 bp, 860

bp, and 1,190 bp, which are present in all individuals of H. rufescens, and in noneof H. discus hannai (Fig. 1, bands B-E), therefore making unequivocalidentification of the two species possible using primer 368. The banding patternsobtained from assaying the DNAs of the 10 individuals with ambiguous

morphology with primer 368 showed the amplicons of approximately 750 bp and850 bp unique to H rufescens, in some of the 10 individuals. The 750 bp ampliconis only present in two specimens with ambiguous morphology and the 850 bpamplicon in three individuals (Fig. 1).

The banding pattern produced with primer 356 shows an amplicon ofapproximately 990 bp present in the 27 individuals of H. rufescens and in nine ofH. discus hannai (Fig. 2, band A). This amplicon, therefore, was not specific foreither of the two species, although it was more frequent and intense in H.rufescens. The amplicons of approximately 450 bp and 460 bp, in contrast, were

present in all the individuals of H. discus hannai and none of the H. rufescens

(Fig. 2, bands B and C). Consequently, these two amplicons allow unequivocalspecies identification. The RAPD banding pattern of the 10 individuals withambiguous morphology with primer 356 did not show discriminatory ampliconsunique to H. discus hannai, whereas all the individuals with ambiguousmorphology showed the 990 bp amplicon, frequent and more intense in H.rufescens.

[FIGURE 1 OMITTED]

DISCUSSION

Our results strongly suggest that the 10 specimens with ambiguous morphologyfound at the CAP-UCN correspond to H. rufescens and that there is phenotypicvariability within this species. The banding patterns from the RAPD-PCRs carriedout on the DNA of these specimens show greater similarities with the banding

patterns of H. rufescens. Some of the DNA of the individuals with ambiguousmorphology show amplicons unique to H. rufescens (750 bp and 850 bp with

primer 368), whereas the amplicons unique to H. discus hannai (450 bp and 460bp with primer 356) were not detected in any of them. The specific amplicons ofboth species would be expected to appear in the banding pattern of the individualswith ambiguous morphology if the specimens in question had been hybrids.Although the fingerprint is not always consistent, the whole fingerprint of 10specimens with ambiguous morphology is visibly more consistent with the

fingerprint of H. rufescens than H. discuss hannai (Fig. 1 and Fig. 2). The


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inconsistency in the fingerprint of the specimens with ambiguous morphologycould be caused by the inadequate maintenance of tissue samples that produces

poor and degraded DNA. These results emphasize the importance of goodmaintenance of tissues that will be used for molecular work. Unfortunately we didnot have more or fresh tissues of specimens with ambiguous morphology to

perform the study.

[FIGURE 2 OMITTED]

The results show that RAPD-PCR is a simple and efficient method to identify anddifferentiate between H. discus hannai and H. rufescens. The banding patternsobtained from some RAPDPCR show specific amplicons for moleculardifferentiation.

The RAPD markers tested herein, allowed us to reject the hypothesis that the 10individuals with ambiguous morphology were hybrids between the two species ofabalone. The banding patterns produced with the DNA of the 10 individuals withambiguous morphology did not simultaneously carry the amplicons obtained with

primers 368 and 356, for H. rufescens and H. discus hannai respectively. Othermolecular genetic tools may allow better discrimination of hybrids and could beused to corroborateTo support or enhance the believability of a fact or assertionby the presentation of additional information that confirms the truthfulness of theitem.

The testimony of a witness is corroborated if subsequent evidence, such as acoroner's report or the testimony of other our findings, including microsatellites(Ibarra et al. 2005), sequencing (Spies et al. 2006) and allozymes (Brown 1995,Ibarra et al. 2005).

Variability in the size and morphology of gastropod shells can be influenced bothgenetically and environmentally (Boulding & Hay 1993, Trussell & Etter 2001,Jordanes et al. 2003, Rolan et al. 2004). Phenotypic plasticity represents the

potential of an organism to alter its phenotype caused by environmental stimuli(Levins 1968, Adler & Harvell 1990). Genetic polymorphism is caused by the

presence of two or more alleles at a given locus and depending on thealleleallele (ll`): see genetics.

allele

Any one of two or more alternative forms of a gene that may occur alternatively ata given site on a chromosome. combination, can be expressed as different

phenotypes. Because the 10 abalone individuals showing ambiguous morphologywere obtained from a single breeding population of H. rufescens (even thoughtheirparentage PARENTAGE. Kindred. Vide 2 Bouv. Inst. n. 1955; Branch;Line. is unknown) and cultured under the same hatchery conditions (i.e., samefood and environmental conditions) it seems improbable that the morphologicalvariability found was a manifestation of phenotypic plasticity, and is more likelyascribed to genetic polymorphism. The present study represents the first published

report on intraspecific variability in the color of the epipodium of H. rufescens (or
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any other abalone species). Other experiments therefore could be carried out inwhich crosses are made between abalones to confirm the hypothesis that the color

variation represents intraspecific polymorphism (if more individuals with the traitare found). The crosses would allow verification of the stability of a characterthroughout the ontogenetic ontogeneticadj.Of or relating to ontogeny. development and possible patterns of charactersegregation. Additionally, molecular markers associated with the trait could bedeveloped.

LITERATURE CITED

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bay scallop scallop or pecten, marine bivalve mollusk. Like its close relative theoyster, the scallop has no siphons, the mantle being completely open, but it differsfrom other mollusks in that both mantle edges have a row of steely blue "eyes"and Argopeeten irradians. J. Hered. 79:14-17.

Adler, F. & C. D. Harvell. 1990. Inducible defences, phenotypic variability andbiotic biotic /biotic/ (bi-otik)1.pertaining to life or living matter.

2.pertaining to the biota.

bioticadj.1. Relating to life or living organisms. environments. Trends Ecol. Evol. 5:407-410.

Anderson, G. 2003. Marine Science. Chapter 6.2.1 at http://www.biosbcc.net/ocean/marinesci/06future/abintro.htm

Brown, L. D. 1995. Genetic evidence for hybridization between Haliotis rubra andH. laevigata. Mar. Biol. 123:89-93.

Boulding, E. G. & T. K. Hay. 1993. Quantitative genetics of shell form of anintertidal intertidaladj.Of or being the region between the high tide mark and the low tide mark.

in snail: constrains on short-term response to selection. Evol. 47:576-592.

Chanley, P. E. 1961. Inheritance of shell markings and growth in the hard clamVenus mercenaria. Proc. Natl. Shellf. Assoc. 50:163-169.

Cole, T. J. 1975. Inheritance of juvenile shell colour of the oyster drill, Urosalpixcinerea. Nature 257:794-795.
http://medical-dictionary.thefreedictionary.com/Ontogenetichttp://encyclopedia2.thefreedictionary.com/scallophttp://medical-dictionary.thefreedictionary.com/Biotichttp://www.thefreedictionary.com/intertidalhttp://medical-dictionary.thefreedictionary.com/Ontogenetichttp://encyclopedia2.thefreedictionary.com/scallophttp://medical-dictionary.thefreedictionary.com/Biotichttp://www.thefreedictionary.com/intertidal


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Costa, F. O., M. R. Cunha, T. Neuparth, C. W. Theodorakis, M. H. Costa & L. R.

Shugart. 2004. Application of RAPDDNA fingerprinting DNA fingerprinting orDNA profiling, any of several similar techniques for analyzing and comparingDNA from separate sources, used especially in law enforcement to identifysuspects from hair, blood, semen, or other biological materials found at intaxonomic identification of amphipods: a case-study with Gammarus species(Crustacea: Amphipoda). J. Mar. Biol. Assoc. UK. 84:171-178.

Douek, J., I. Barki, D. Gateno & B. Rinkevich. 2002. Possible cryptic speciationspeciation

Formation of new and distinct species, whereby a single evolutionary line splits

into two or more genetically independent ones. One of the fundamental processesof evolution, speciation may occur in many ways. within the sea anemone Actiniaequina complex detected by AFLP markers. Zool. J. Lin. Soc. 136:315-320.

Folmer, O., M. Black, R. Hoeh, R. A. Lutz & R. Vrijenhoek. 1994. DNA primersfor amplification of mitochondrial cytochrome oxidase subunit I from diversemetazoan metazoan

member of the zoological division of Metazoa. invertebrates. Mol. Mar. Biol.Biot. 3:294-299.

Freeman, K. A. 2001. Aquaculture aquaculture, the raising and harvesting offresh- and saltwater plants and animals. The most economically important form ofaquaculture is fish farming, an industry that accounts for an ever increasing shareof world fisheries production. and related biological attributes of abalone speciesin Australia- a review. Fish. Res. Rep. West. Aust. 128:1-48.

Ibarra, A. M., N. K. Hernandez-Ibarra, P. Cruz, R. Perez-Enriquez, S. Avila & J.L. Ramirez. 2005. Genetic certification of presumed hybrids of blue and redabalone (Haliotis fulgens Philippi and H. rufescens Swainson). Aquac. Res.36:1356-1368.

Innes, D. J. & L. E. Haley. 1977. Inheritance of a shell-color polymorphism in themussel mussel, edible freshwater or marine bivalve mollusk. Mussels are able tomove slowly by means of the muscular foot. They feed and breathe by filteringwater through extensible tubes called siphons; a large mussel filters 10 gal (38liters) of water per day. . J. Hered. 68:203-204.

Jordanes, K., P. Van Riel rieln.See Table at currency.

[Origin unknown.]
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Noun1.riel - the basic unit of money in Cambodia; equal to 100 sen & T.Backeljau. 2003. Molecular and morphological discrimination between the

pulmonate pulmonateadj.1. Having lungs or lunglike organs.

2. Of or belonging to the Pulmonata, a subclass of gastropods including terrestrialsnails and slugs and certain freshwater snails that are capable of breathing airthrough land snail Zonitoides nitidus and Z. excavatus. J. Moll. Stud. 69: 295-300.

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Kobayashi, T., I. Kawahara, O. Hasekura & A. Kijima. 2004. Genetic control ofbluish bluish also blueishadj.Somewhat blue.

blu ishness n. shell color variation in the Pacific abalone, Haliotis discus hannai.J. Shellfish Res. 23:1153-1156.

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Newkirk, G. F. 1980. Genetics of the shell color in Mytilus edulis L. and theassociation of growth rate with shell color. J. Exp. Mar. Biol. Ecol. 47:89-94.

Owen, B., J. H. McLean & R. J. Meyer. 1971. Hybridization in the Eastern Pacific

abalone (Haliotis). Sci. Bull. Nat. Hist. Mus. Los Ang. City. 9:1-37.
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Park, K., J. K. Park, J. Lee & K. S. Choi. 2005. Use of molecular markers for

species identification of Korean Perkinsus sp isolated from Manila clamsRuditapes philippinarum. Dis. Aquat. Org. 66: 255-263.

Rolan, E., J. Guerra-Varela, I. Colson, R. N. Hughes & E. Rolan-Alvarez. 2004.Morphological and genetic analysis of twosympatric sympatricadj.EcologyOccupying the same or overlapping geographic areas without interbreeding. Usedof populations of closely related species. morphs of the dogwhelk Nucella lapilluslapillusn.pl.lapilliA small, solidified fragment of lava.

[Latin, diminutive oflapis,stone. (Gastropoda: Muricidae) from Galicia(northwestern Spain). J. Moll. Stud. 70:179-185.

Smith, N. F. & G. M. Ruiz. 2004. Phenotypic plasticity in the life history of themangrove mangrove, large tropical evergreen tree, genusRhizophora, that growson muddy tidal flats and along protected ocean shorelines. Mangroves are mostabundant in tropical Asia, Africa, and the islands of the SW Pacific. snailCerithidea scalariformis. Mar. Ecol. Prog. Ser. 284:195-209.

Spies, I. B., S. Gaichas, D. E. StevensonD. E. Stevenson (1892-1973) - marriedname Dorothy Emily Peploe - was a Scottish author of light romantic novels. Herfather was the lighthouse engineer David Alan Stevenson and she was distantlyrelated to the writer Robert Louis Stevenson. , J. W. Orr & M. F. Canino. 2006.DNA-based identification of Alaska skates (Amblyraja, Bathyraja and Raja:Rajidae) using cytochrome c oxidase The enzyme cytochrome c oxidase orComplex IV (PDB 2OCC, EC 1.9.3.1) is a large transmembrane protein complexfound in bacteria and the mitochondrion. FunctionIt is the last protein in the electron transport chain...... Click the link for more information. submit 1 (coI) variation. J. Fish Biol.69:283-292.

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Wada, K. T. & A. Komaru. 1990. Inheritance of white coloration of theprismatic prismatic alsoprismaticaladj.

1. Of, relating to, resembling, or being a prism.
http://www.thefreedictionary.com/sympatrichttp://www.thefreedictionary.com/sympatrichttp://www.thefreedictionary.com/lapillushttp://encyclopedia2.thefreedictionary.com/mangrovehttp://encyclopedia.thefreedictionary.com/D.+E.+Stevensonhttp://encyclopedia.thefreedictionary.com/Cytochrome+c+oxidasehttp://www.thefreedictionary.com/prismatichttp://www.thefreedictionary.com/sympatrichttp://www.thefreedictionary.com/lapillushttp://encyclopedia2.thefreedictionary.com/mangrovehttp://encyclopedia.thefreedictionary.com/D.+E.+Stevensonhttp://encyclopedia.thefreedictionary.com/Cytochrome+c+oxidasehttp://www.thefreedictionary.com/prismatic


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2. Formed by refraction of light through a prism. Used of a spectrum of light.

3. Brilliantly colored; iridescent. layer of shells in the Japanese pearl oysterPincada fucata martensii and its importance in the pearl culture industry. NipponSuisan Gakkaishi 56:1787-1790.

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Any of about 5,400 species of marine worms of the annelid class Polychaeta,having a segmented body with many setae (bristles) on each segment. Species,often brightly coloured, range from less than 1 in. (2.5 cm) to about 10 ft (3 m)long. species: An approach to a molecular taxononly. Helg. Mar. Res. 57:1-6.

Williams, J. G. K., A. R. Kubelik, K. J. Livak, J. A. Rafalski & S. V. Tingey.1990.DNA polymorphism DNA polymorphismn.

A condition in which one of two different but normal nucleotide sequences canexist at a particular site in a DNA molecule. amplified by arbitrary primer areuseful as genetic markers. Nucleic Acids Res. 18:6531-6535.

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S. A. MARIN, (1) P. A. HAYE, (1,2) * S. MARCHANT (1) AND F. M.WINKLER (1,2)

(1) Departamento de Biologia Marina, Facultad de Ciencias del Mar, UniversidadCatolica del Norte; (2) Centro de Estudios Avanzados en Zonas Aridas (CEAZA),Larrondo 1281. Coquimbo, Chile

* Corresponding author. E-mail: [email protected]
http://medical-dictionary.thefreedictionary.com/Nucleic+acidshttp://encyclopedia2.thefreedictionary.com/Polychaetehttp://medical-dictionary.thefreedictionary.com/DNA+polymorphismhttp://medical-dictionary.thefreedictionary.com/DNA+polymorphismhttp://medical-dictionary.thefreedictionary.com/Nucleic+acidshttp://encyclopedia2.thefreedictionary.com/Polychaetehttp://medical-dictionary.thefreedictionary.com/DNA+polymorphism

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Molecular Markers Used to Analyze Species