PertanikaJ. Trop. Agric. Sci. 28(1): 79 - 85 (2005) ISS : 1511-3701© Universiti Putra Malaysia Press

Isolation of DNA Microsatellite Markers in theGreen-lipped Mussel, Perna viridis

JULY ONG CHIN CHIN, ITAN SOON GVAN,2KHATIJAH YUSOFF & JYAP CHEE KONG

lDepartment of Biology Faculty of Science,Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia

2Faculty of Biotechnology and Biomolecular Sciences,Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia

E-mail: ongchinchin@yahoo.com

Keywords: Perna viridis, green-lipped mussel, microsatellite, RAMs

ABSTRAK

Sejumlah 21 mikrosatelit DNA telah dipencilkan daripada Perna viridis dengan menggunakan teknik rantaiantindak balas '5' anchored polymerase '. Primer-primer telah direka untuk tujuh lokus mikrosatelit dan amplifikasiPCR bagi ketujuh-tujuh lokus mikrosatelit ini menunjukkan bahawa lima adalah polimorfik dengan jumlah alelper lokus menjulat daripada 2 hingga 4. Ini merupakan penanda set mikrosatelit pertama yang telahdibangunkan untuk spesies ini. Penanda-penanda ini amat berguna sebagai alat untuk kajian terperinci latarbelakang genetik P. viridis di negara kita memandangkan spesies ini dikultur sebagai sumber protein murah bagimanusia dan telah dikenal pasti sebagai agen biomonitor yang berpotensi untuk pencemaran logam berat diPantai Barat Semenanjung Malaysia.

ABSTRACT

A total of 21 DNA microsatellites were isolated from Perna viridis lJy using a 5' anchored polymerase chainreaction technique. Primers were designed for seven microsatellite loci and the peR amplifications of these sevenmicrosatellite loci showed that five were polymorphic with the number ofalleles per locus rangingfrom 2 to 4. Theseare the first set of microsatellite markers developed for this species. These markers are useful as tools for moredetailed studies of the genetic backgrounds of the green-lipped musse~ P. viridis, in our country as this speciesis being cultured as a cheap source of protein for human consumption and has been identified as a potentialbiomonitoring agent for heavy metal pollution in the west coast of Peninsular Malaysia.

INTRODUCTION

The green-lipped mussel, Perna viridis, is widelydistributed in the Indo-Pacific region (Siddall1980). This mussel is important ecologicallybecause of its widespread distribution andbiological filtration activity, and also economicallybecause of its value as a cheap source of animalprotein for human consumption. It was onceregarded as a nuisance by oyster farmers in thePhilippines and today it is being extensivelycultured in many Asian countries includingMalaysia (Rosell 1991; Monirith et al. 2003).Recently, this species has been used as abiomonitor for a wide range of contaminantssuch as metals, organochlorines, polycyclicaromatic hydrocarbons and organotins throughout

the Indo-Pacific region and has considerablepotential as a pollution monitoring agentthroughout its geographical range ( icholsonand Lam 2005).

In Malaysia, P. viridis is widely distributedalong the west coast of Peninsular Malaysia andto a lesser extent, in certain parts of Sabah andthe east coast of Peninsular Malaysia. It is a localseafood delicacy and is one of the few localspecies that is successfully cultured in the Straitsof Malacca. Furthermore, its sessile lifestyle andwidespread distribution in the Straits hasprompted its use as a bioindicator of heavymetal contamination in the Straits of Malacca;which is one of the busiest shipping lanes in theworld (Ismail et at. 2002). Therefore, information

ULY 0 G CHIN CHIN, TAN SOO GUAN, KHATIJAH YUSOFF & YAP CHEE KO G

on the population genetic structure of P. viridisalong the Straits of Malacca is undoubtedly veryimportant since different species may accumulatemetals at different rates (Gyllensten and Ryman1985). Besides, this information can be appliedto improve the broodstocks of this commerciallyimportant seafood delicacy and hence increasethe productivity of mussel farms.

In the last few years, microsatellites havebecome one of the most popular molecularmarkers used in various fields of study. Beingcodominant, PCR-based, highly polymorphic andeasy to score contribute to the popularity ofmicrosatellites as a marker of choice. One of themajor drawbacks of this DNA marker is that itneeds to be isolated de novo from species thatare being examined for the first time (Zane et al.2002). Although microsatellite isolation is atedious, expensive and laborious procedure, thisdoes not seem to be a strong deterrent factor.The conven tional method of isolatingmicrosatellites is like searching for a needle in ahaystack. It involves the creation of a size­fragmented genomic library, cloning of bandsand subsequently screening thousands of coloniesfor the presence of microsatellite repeats throughhybridization. The success rate usually rangesfrom 12% to less than 0.04% (Zane et al. 2002).Here we report on the development ofmicrosatellites from P. viridis using a modificationtechnique of the 5' anchored PCR technique ofFisher et al. (1996).

MATERIALS AND METHODS

Sample Collection

Mussels were collected from five differentlocations along the west coast of PeninsularMalaysia. The five collection sites were Tanjung

Rhu (Pulau Langkawi, Kedah), Bagan Tiang(Perak), Pulau Ketam (Selangor), Muar (Johor)and Kampung Pasir Puteh (Johor) with a samplesize of 20 individuals per location. The adductormuscle was excised from the mussel and kept at-80Q C prior to DNA analysis.

DNA Extraction

DNA was extracted from approximately 20-30mg of the adductor muscle by using a CTABbased protocol described by Winnepennincks etal. (1993) with minor modifications.

5' Anchored PCR Amplification

Two degenerate RAMs primers, \]1 and BP2,were used to produce a genomic library enrichedfor microsatellites (Table 1).

A polymerase chain reaction (PCR) wascarried out in a total volume of 10 J1L containing25 ng of genomic D A, IX PCR buffer, 0.2 mMeach of dATP, dGTP, dCTP and dTTP, 15 pmolof either primer \]1 or BP2, 2mM MgCI

2, and

1.5 U of Tag DNA polymerase (Promega, USA).Amplifications were performed in a PeltierThermal Cycler PTC-220 (MJ Research, USA)with an initial 3 min of predenaturation at 96°C,followed by 40 cycles of denaturation at 96°C for15 seconds, an appropriate annealingtemperature for 15 seconds (as shown in Table1) and extension at 72°C for 30 seconds. A finalextension step at 72QC for 7 min was includedfor the attachment of a dATP at the 3' terminalfor the cloning reaction. The PCR product wasrun on a 2% agarose gel and visualised byethidium bromide staining to confirm thepresence of bands. Mter performing the PCRreaction, the resultant products were then readyfor cloning.

TABLE 1Degenerate RAMs primers used to produce a genomic library enriched for microsatellites

and the annealing temperatures for the PCR amplifications

Primer

VJ1BP2

Sequence 5' to 3'

NNN NNN NKK VRV RV (CT) 10

NNN NNK KYW (BD)sB(CA)10

Annealing temperature (QC)

5655

Note: K = G/T, N = A/C/G/T, V = G/C/A, R = G/A, Y = T/C, B = C/G/T, D =A/G/T (IUB code)

80 PERTANlKAJ. TROP. AGRIC. SCI. VOL. 28 NO.1, 2005

ISOlATION OF DNA MICROSATELLITE MARKERS IN THE GREEN-LIPPED MUSSEL, PERNA VIRIDIS

Cloning of the 5' Anchored PCR Products into pCR2.1-TOPO Vector

The PCR products were then cloned into theTapa TA cloning vector according to themanufacturer's instructions (Tapa TA Kit,Invitrogen). Five recombinant clones from eachprimer were randomly selected for plasmidextraction.

Plasmid Extraction

The plasmid extractions were performedaccording to the protocol of Sambrook et aL(1989) and the clones were sequenced by usingthe BigDye Terminator Cycle Sequencing ReadyReaction Kit (Applied biosystems, USA) on theABI PRISM 377 DNA sequencer at Institute ofBiosciences, Universiti Putra Malaysia.

Primer Design

Primer pairs flanking each of the microsatelliteregions were designed by using a free onlineprimer designing software, PRIMER 3 (Rozenand Skaletsky 1997) provided at http:/ /www.genome.wi.mit.edu/genome_software/other/ primer3.htrnl.

Amplifying Microsatellites

The primer pairs so designed were then used toscreen for polymorphisms in P. viridis. PCRamplifications were performed in a 10 J1L volumecontaining 25 ng of genomic D A, IX PCRbuffer, 0.2 mM each of dNTPs, 0.15,uM of eachreverse and forward primers, 1 - 3 mM MgCI2,

and 0.5 - 1 U of Taq DNA polymerase (Promega,USA). The PCR amplifications were performedin a Peltier Thermal Cycler PTC-220 (M]Research, USA) with the following temperatures:a predenaturation for 3 min at 95°C, followed by35 cycles of 30 seconds denaturation at 94°C, 30seconds annealing at appropriate temperatures(as shown in Table 2), 30 seconds extension at68°C and concluded with a 5 min final extensionat 68°C. The PCR products were run on a 4%(w/v) horizontal MetaPhor gel (BMA, USA) andvisualized over UV after ethidium bromidestaining. A 20 bp DNA ladder (BioWhittakerMolecular Applications) was used as themolecular weight standard. Twenty samples fromeach of the following P. viridis populations:Tanjung Rhu, Bagan Tiang, Pulau Ketam, Muarand Kampung Pasir Puteh were typed for eachof the five microsatellite loci. The population

data were analysed by using the POPGENE(version 1.32) computer software (Yeh & Boyle,1997). An Unweighted Pair-Group Method withArithmetic Mean (UPGMA) dendrogram wasconstructed based on Nei's (1978) geneticdistance estimates by using the TSYS software(Rohf 1990).

RESULTS AND DISCCUSION

5' anchored PCR is a technique that involves theuse of arbitrary primers, which are specificallydesigned to incorporate an anchor and amicrosatellite repeat motif to produce a library,which is highly enriched for microsatellites. Thistechnique offers a number of advantages. Oneof the advantages is that the amplification ofgenomic DNA yields PCR products that containa microsatellite region at both the 5' and the 3'terminal ends. This means every band sequencedwould theoretically contain at least twomicrosatellites. Another advantage of thistechnique is its ability to target a specific repeatmotif that is required, which means we canproduce a library that is highly enriched for thespecific repeat motif that we want (Fisher et al.1996).

In this study, a total of ten clones (five fromeach of the degenerate RAMs primer, VJl andBP2) were randomly selected and sent forsequencing. The ten clones sequenced revealeda total of 21 microsatellite regions. Of the 21microsatellite regions, ten were perfectmicrosatellites while eight were imperfect orinterrupted microsatellite and the rest werecompound microsatellites. Five of the cloneshad one or more additional microsatellites inthe internal sequence. The presence of additionalinternal microsatellites was an unexpected bonus,implying that there may be clustering ofmicrosatellites in some genomic regions (Fisheret al. 1996).

Primer pairs were designed for sevenmicrosatellite loci. These are the first set ofmicrosatellite markers that have been identifiedand characterized in P. viridis. Out of theseseven loci, five were polymorphic with thenumber of alleles per locus ranging from 2 to 4with an average of 3.2. The observedheterozygosity ranged from 0.0104-0.3776 (Table2) and was similar with those reported usingallozyme studies (Yap et al. 2002).

Cluster analysis revealed two major groups.Tanjung Rhu, Pulau Ketam, Bagan Tiang and

PERTANlKAJ. TROP. AGRIC. SCI. VOL. 28 O. 1, 2005 81

(XlN:l

"Ct'l

~~

':-

~~

f;~oVl

parN:l(Xl

z9­N:looC,J1

TABLE 2Microsatellite variation in five populations of Perna viridis

Locus Repeat motif Primer sequence (5'-3') T. eC) Allele size No. of Ho HE GenBank Accession no.range (bp) alleles

\]1-18-1 (CT)n F:GTAGCGGCTCTCTCTCTCT 55 260-290 4 0.3776 0.7105 AY850126R:GCGTGACACTCIIIIICTTT

\]1-12-2 (AG)n(GACA)N' (AG)N F:ATAGGATAGAGTCACGTTAG 40 200-210 2 0.1579 0.1462 AY850124R: TAAGACCTCTCTCTCTCTC

\]1-23-2 (GAAA)N' (AG)n F:CAGGACTCCCGCTGGGTAAA 44 205-220 2 0.0104 0.0104 AY850125R:TCCACTGGCCGGCTCTCT

BP2-49-1 (CA)n' (CAAC)N F: GGTACTTTTCTCACITCACA 44 165-230 4 0.2418 0.2541 AY850129R:GGAGTGAACCTCITCGAC

BP2-49-2 (TG)N F: GTTAAACAACCAACCAACG 44 180-260 4 0.2366 0.2471 AY850129R:GTCIIIIIGTCATTGCACAC

Repeat motif: N, pure; n, interrupted. T. = annealing temperature, Ho = observed heterozygosity, HE = expected heterozygosity

t:~ozon::r:Z(')

.~

~Vloo

.~@

<-=:~~Vlo~fI:"

~ggJ

~Zo

ISOlATIO OF DNA MICROSATELLITE MARKERS IN THE GREEN-LIPPED MUSSEL, PERNA VIRIDIS

Muar were clustered together while KarnpungPasir Puteh was clustered by itself (Fig. 3). TheKampung Pasir Puteh population was collectedfrom a highly polluted environment and thisfinding is in line with the results obtained byYap et al. (2004) based on codominant allozymemarkers but contradicted those of Chua et al.(2003) which were based on the dominant RAPDand RAMs markers. No diagnostic alleles thatcould differentiate between samples frompolluted and non-polluted environments werefound in this study.

This is the first report that microsatellitemarkers were isolated and characterized for P.viridis. Seven microsatellite primer pairs weredesigned based on the P. viridis DNA sequencesobtained from a modified 5' anchored PCRtechnique (Fisher et at. 1996). This study iscurrently ongoing and the primers describedwill be used for more detailed studies on thepopulation genetic structure of the green-lippedmussel, P. viridis from along the west coast ofPeninsular Malaysia.

M 2 3 4 5 6

280 bp

260 bp

Fig. I: MicrosateUite profile of samples from the Bagan Tiang population generated IYy primer pairsVJI-I8-I. Lane M: 20 bp marker; lanes 1-6: microsateUite profiles of samples from Bagan Tiang

1 2 3 4 5 6 7 8 M

260 bp

240 bp

220 bp

Fig. 2: Microsatellite profile of samples from the Muar population generated IYy primer pairs BP2-49-1.Lane M: 20 bp marker; lanes 1-8: microsatellite profiles of samples from Muar

PERTANIKAJ. TROP. AGRIC. SCI. VOL. 28 NO.1, 2005 83

ULY ONG CHIN CHIN, TAN SOON GUAN, KHATIJAH YUSOFF & YAP CHEE KONG

ITanjung Rhu

I Pulau Ketam

Bagan TiangII Muar

Kampung Pasir Puteh

---1I------+------lI----I-----i~0.048 0.040 0.032 0.024 0.016 0.008 0.000

Fig. 3: UPGMA dendrogram of genetic relationships among five populationsof Perna viridis based on Nei's (1978) genetic distance

ACKNOWLEDGEMENT

This work was supported by a IRPA grant 09-02­04-EA001: Development and application of DNA

- microsatellite markers for the biodiversitycharacterization of the green-lipped mussel, Pernaviridis from the Ministry of Science, Technologyand Innovation Malaysia headed by Prof. Dr.Tan Soon Guan. The authors wish to thank Mr.Hoh Boon Peng and Dr. Vijay Kumar forproviding their degenerate RAMs primers in thisstudy.

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(Received: 25 February 2005)(Accepted: 29 August 2005)

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