Molecular Ecology Notes (2005)

5

, 231–233 doi: 10.1111/j.1471-8286.2005.00905.x

© 2005 Blackwell Publishing Ltd

Blackwell Publishing, Ltd.

PRIMER NOTE

Isolation and characterization of polymorphic microsatellites in

Labeo rohita

and their cross-species amplification in related species

P . DAS, A. BARAT, P . K . MEHER, P . P . RAY and D. MAJUMDAR

Fish Genetics and Biotechnology Division, Central Institute of Freshwater Aquaculture, Kausalyaganga, Bhubaneswar — 751002, Orissa, India

Abstract

The major Indian carps namely rohu (

Labeo rohita

), catla (

Catla catla

), mrigal (

Cirrhinusmrigala

) and calbasu (

Labeo calbasu

) are important freshwater species of the Indian sub-continent constituting over 65% of the fish produce. In the present study, isolation of 12microsatellite loci from rohu has been reported. Cross-species amplification in relatedcarps and their implication in population genetic studies as well as selective breedingprogram were discussed.

Keywords

:

cross species amplification,

Labeo rohita

, microsatellite, population genetics

Received 28 September 2004; revision accepted 24 November 2004

Rohu (

Labeo rohita

) is one of the major cultivable carpspecies native to India. Most of the seed produced in thisspecies comes from hatchery breeding which resulted ininbreeding in most of the hatcheries (Eknath & Doyle1990). For genetic upgradation and management of stocksof

L. rohita

, information about its genetic background isessential. Though DNA markers are known as a powerfultool for genetic analysis (Avise 1994), appropriate DNAmarkers like polymorphic microsatellite loci are lackingin this species. This is the first report of isolation andcharacterization of microsatellite loci from the rohugenome.

Microsatellite loci are now commonly used as geneticmarkers for population genetic studies to resolve the phylo-genetic relationships between different populations, toclassify individuals by relatedness and to determine geneticvariation within populations. In the present investigation,we report 12 microsatellite loci in rohu. These markershave a great potential in terms of studying genetic variationwithin and between populations, selective breeding pro-grams as well as gene mapping in fish species.

Isolation of microsatellite loci from the rohu genomehas been performed based on the protocols of Strassmann

et al

. (1996) with some modification. Genomic DNA isolatedfrom fin tissues of a male individual following standardprocedure was subjected to cleavage with the restrictionenzyme

Sau

3AI (New England Biolabs). Digestion prod-ucts were separated on 0.7% agarose. DNA fragments cor-responding to 0.3 to 0.4 kb size range was eluted from thegel (QIAquick Gel Extraction Kit, QIAGEN) for ligationinto

Bam

HI site of the dephosphorylated pUC18 cloningvector. DH5 alpha Competent Cells were transformedwith the ligation product and grown on agar plates. About3000 recombinants were screened through colony hybridi-zation. Colonies were transferred on to nylon membrane(Hybond N+, Amersham) and hybridized with labelleddinucleotide probes (CT)

15

and (GT)

15

, using DIG Oligo-nucleotide Tailing Kit (Roche Applied Sciences). This resultedin 25 positive clones, which after reconfirmation throughdot blot gave rise to 17 clones which contain repeat sequences.The inserts were sequenced on ABI Prism 377, version 3.0DNA sequencer by cycle sequencing (ABI Prism BigDye Kit)using M13 and T7 universal sequencing primers. Sequenceanalysis and designing of 17 pairs of primers were doneusing

lasergene

version 5.0 (DNASTAR) and sequenceswere deposited in EMBL.

Correspondence: P. Das, Fax

:

+6742465407; E-mail: pdas77@hotmail.com

232

P R I M E R N O T E

© 2005 Blackwell Publishing Ltd,

Molecular Ecology

Notes

, 5, 231–233

To examine microsatellite variability, genomic DNA of18 unrelated farm individuals of rohu was prepared usingphenol–chloroform extraction method (Sambrook

et al

.1989). Polymerase chain reaction (PCR) was set in 10-

µ

Lreaction volumes containing 1

×

Taq

DNA polymerase reac-tion buffer [10 m

m

Tris-HCl (pH 8.0), 50 m

m

KCl, 1.5 m

m

MgCl

2

and 0.01% gelatin]; 5 pmol of each primer; 100

µ

m

ofdNTPs; 0.25 units of T

aq

DNA polymerase (Bangalore Genei);and 20 ng of genomic DNA templates. Amplification wasperformed in a GeneAmp 9700 thermocycler (AppliedBiosystems), programmed for initial denaturation of4 min at 94

°

C followed by 35 cycles of 45 s at 94

°

C, 1 min30 s at annealing temperature (Table 1) and 2 min at 72

°

Cwith a final extension of 7 min at 72

°

C. Amplified productswere dried on a vacuum concentrator (DNA plus, Heto),mixed with 5

µ

L of formamide loading dye, heat denaturedat 94

°

C for 5 min and then separated on 6% denaturingpolyacrylamide gel with 7.5

m

urea and 1

×

TBE buffer.

φ

X174 DNA-

Hae

III digest (Finnzymes) was used as a sizemarker for the microsatellite alleles. Detection of allele wasdone by silver staining (Riesner

et al

. 1989).Out of the 17 loci, 12 were amplified and were found to

be polymorphic in

L. rohita

. The number of alleles at thepolymorphic loci showed a low but distinct allelic variationranging from two to seven alleles (Table 1). Allele numbers,expected and observed heterozygosities were measuredusing

gda

, version 1.1 (Lewis & Zaykin 1997). Expectedheterozygosity (

H

E

) for the 12 loci varied between 0.39 and0.74 whereas observed heterozygosity (

H

O

) ranged from0.27 to 0.88 (Table 1). Observed heterozygosities at all theloci except Lr1, Lr6 and Lr24 were as per Hardy–Weinbergequilibrium expectations. The deficit was observed in thethree loci mentioned and this may be attributed to thesmall number of samples studied.

Cross-species amplification with primers from the 12loci was examined in 10 species (one individual each) ofthe family Cyprinidae. Each primer pair amplified targetlocus in one or more species (Table 2). These results are con-sistent with the systematic relationship of rohu with otherspecies of same genus i.e. higher amplification success inmore closely related species. The present set of microsatel-lites will be useful for assessing genetic diversity at species,population and individual level of the species examinedhere.

Table 1 Microsatellite loci identified from the genome of Labeo rohita with GenBank Accession no., primer sequence, annealing temperatureand expected fragment size

LocusRepeat sequence

GenBank Accession no. Primer sequence (5′–3′)

Allele Size (bp) HO HE Ta (°C)

No. of alleles

Lr1 (TG)14 AJ507518 F-GACCCTTAACCCTTGACCTT 171 0.437 0.514 58 2R-TGGGATAATGCAGGGAAAAC

Lr3 (TG)19 AJ507520 F-ATCTGGCTGCCTATTCACC 152 0.444 0.390 58 5R-CATCGGCGACTGCACTGGA

Lr6 (TG)16 AJ507522 F-TATCCTGGCTGAAAACTTTG 160 0.294 0.620 56 3R-CCCCTACAGGAACAACCAT

Lr10 (CA)13 AJ507523 F-GATCTTCAGCGCCAGCGTG 250 0.833 0.650 60 4R-GAGGACCTGCCCAGCATG

Lr12 (CA)13 AJ507524 F-CACCGCTGCTGTCCATCA 161 0.882 0.668 58 4R-AGGTCGGCCAGATACACG

Lr14a (CA)24 AJ831434 F-GATCAACTGCTGCCACAC 183 0.733 0.544 58 3R-GGTTTGTTGTTCCCATGTG

Lr14b (CA)12 AJ831434 F-TCACATGGGAACAACAAACC 172 0.882 0.755 58 5R-CCGCCGCTTACCCATCAC

Lr20 (CA)9 AJ831435 F-GCCCGCTGCCGTCTGAC 144 0.833 0.553 58 4R-CAATAACTCAGCATGTGGAG

Lr21 (CA)11 AJ831436 F-GATCAGAGGGTCAATGTGG 148 0.777 0.674 58 6R-CAGCAGAGTACTATGGAAGA

Lr23 (TG)12 AJ831437 F-GGAAATTACAAATGTGCTGATG 186 0.588 0.506 60 3R-AGCGCTGCTGTCACTTCTGT

Lr24 (TG)17 AJ831438 F-CAAGGCCAAAAGTGTCCAT 170 0.444 0.742 56 7R-AGGAAATTGGTAAAGTGTTTC

Lr26 (TG)8 AJ831439 F-CCAGGGAGCTGCTAAGAAT 151 0.777 0.488 56 2R-AGCGCTTCATGCAGTCTAC

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© 2005 Blackwell Publishing Ltd,

Molecular Ecology Notes

, 5, 231–233

Acknowledgements

We gratefully acknowledge the financial help by the Departmentof Biotechnology, Government of India, New Delhi. Sincerethanks are due to Dr (Mrs) S. Nayak, RPRC, Bhubaneswar for hervaluable input during genomic library screening. We are thankfulto the Director of our institute for continuous encouragement andsupport for the work.

References

Avise JC (1994) Molecular markers.

Natural History and Evolution

.Chapman & Hall, New York.

Eknath AE, Doyle RW (1990) Effective population size and rate ofinbreeding in aquaculture of Indian major carps.

Aquaculture

,

85

, 293–305.

Lewis PO, Zaykin D (1997)

GENETIC

DATA ANALYSIS: Computer Pro-gram for the Analysis of Allelic Data, version 1.0. Free programdistributed by the authors over the internet from the gda homepage at http://www.chee.unm.edu/gda/

Riesner DG, Steger RZ, Owens RA et al. (1989) Temperature-gradient gel electrophoresis of nucleic acids: analysis of confor-mational transition, sequence variations and protein nucleicacid interactions. Electrophoresis, 10, 377–389.

Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: ALaboratory Manual, 2nd edn. Cold Spring Harbor LaboratoryPress, New York.

Strassmann JE, Solis CR, Peters JM, Queller DC (1996) Strategiesfor finding and using highly polymorphic DNA microsatelliteloci for studies of genetic relatedness and pedigrees. In: Molecu-lar Zoology: Advances, Strategies and Protocols (eds Ferraris JD,Palumbi SR), pp. 163–180. Wiley-Liss, Inc.

Table 2 Cross species amplification of Labeo rohita microsatellite loci in different Labeo species and other unrelated carp species

Species Lr1 Lr3 Lr6 Lr10 Lr12 Lr14a Lr14b Lr20 Lr21 Lr23 Lr24 Lr26

Labeo bata – + – + + – + – + – + +Labeo calbasu + + – + + + + – + + + +Labeo dyocheilus + + + + + + + – + – + +Labeo fimbriatus + + + + + + + – + + + +Labeo gonius + + + + + + + + + – + +Catla catla + – – + + – + – + – – –Cirrhinus mrigala + + + + + – – + + + + –Hypophthalmicthys molitrix + + + + + – + – – – – –Ctenopharyngodon idella + + – + + – + + – – + +Cyprinus carpio – + – + – – – – – – – –

+, specific amplification; –, no amplification.