NEWS NO.6

Isolation of 43 Microsatellite Loci from Paphiopedilum rothschildianum,
An Endangered Species of Slipper Orchid.
Rodrigues K.F., Kumar S. V.

Biotechnology Research Institute, Universiti Malaysia Sabah, Locked Bag 2073, Kota Kinabalu, 88999, Sabah, Malaysia.

E-mail: ccegoa@yahoo.com

INTRODUCTION

Paphiopedilum rothschildianum is one of the endangered species of orchids associated with specific microhabitats within Sabah. The genomic characterization and subsequent assessment of inter specific genetic variability of Paphiopedilum rothschildianum will provide a valid basis for the determination of those genetic factors which are significant for the survival of a species classified as endangered.
Microsatellites are significant molecular markers in the context of population genetic studies as they can be employed to determine interspecific and intraspecific genetic variability. This variability can then be utilized to characterize the genetic resilience of a particular species when subjected to environmental stresses.
Leaf tissue was sampled randomly from mature Paphiopedilum rothschildianum plants at the in-situ conservation site managed by the Sabah Parks Authority at Kinabalu National Park, Sabah. DNA was extracted, purified and amplified using the 5ﾕ anchored Polymerase Chain Reaction with a degenerate primer incorporating a dinucleotide repeat motif.
The PCR products were cloned into a pCR 2.1 TOPO TA cloning vector and subsequently chemically transformed in E. Coli competent cells . A total of 155 positive clones were obtained of which 19 were sequenced. The sequence data yielded 43 unique microsatellite loci.
Investigations are now being directed towards obtaining a multiplicity of unique sequences incorporating microsatellite loci with di-, tri- and tetranucleotide repeat motifs. This sequence data will subsequently be utilized to design specific primers which will be employed for genetic profiling of the slipper orchid.

METHODOLOGY

DNA Extraction

5' Anchored PCR

Cloning using TOPO TA Cloning Kit

Plasmid Extraction

DNA Sequencing

RESULTS & DISCUSSION

DNA Extraction

M 　1 　 2 3 4 　 5 　 6 　 7 　 8 　 9 10 11

M: l Hind III DNA Marker; Lanes 1 ﾐ 11: DNA Extract
( 0.8% Agarose Gel: TBE: 75 Volts: 60 Mins )
DNA was extracted using a modified protocol based on Dellaporta

5ﾕ- Anchored PCR Amplification

1 　 　　 2 　　 M 　　　 3 　　　 4

　　　　　　200bp

M: 100 Bp. Marker; Lanes 1,2: Mg++ (2.5 mM); Lanes 3, 4: Mg++ (1.5 mM)
( 1.2% Agarose Gel: TBE: 75 Volts: 60 Mins )

5ﾕ Anchored PCR amplification can be used to successfully amplify regions of DNA which contain microsatellite Loci.

Plasmid RE Digestion

M 　 1 　 2 　 3 　4 　5 　 6 　 7 　　M 　 8 　 9 　 10 　 11

M: 100 bp DNA Ladder; Lanes 1 - 11: Plamid s digested with EcoRI (Promega)
( 1.0 % Agarose Gel: TBE: 75 Volts: 60 Mins )

Microsatellite Locus

Microsatellite locus showing 5ﾕ and 3ﾕ terminal repeats.

Microsatellite Loci

Clone 5ﾕ Terminal Repeat Internal repeat(s) 3ﾕ Terminal Repeat

Clone

5' Terminal Repeat

Internal repeat(s)

3' Terminal Repeat

PRA04

(AC) 10

(AT) 7

(GT) 13

PRA06

(AC) 11

--

--

PRA08

(AC) 10

(GT) 4

( GT) 11

PRA09

(AC) 11

(GT) 6

(GT) 10

PRA11

(AC) 10

--

--

PRA12

(AC)10

(AC) 4

(GT) 11

PRA17

(AC) 11

--

--

PRC03

(AC) 10

(GA) 8

(GT) 10

PRC09

(AC) 11

--

(GT) 11

PRC10

(AC) 11

--

(GT) 13

PRC11

(AC) 10

(AT) 5

(GT) 10

PRC12

(AC)12

(GT) 6

(GT) 10

PRC13

(AC) 10

--

--

PRC14

(AC) 12

--

(GT) 10

PRC15

(AC) 10

(CT) 7

(GT) 10

PRC 16

(AC) 10

--

(GT) 10

PRC18

(AC) 12

--

(GT) 10

PRC 19

(AC) 13

(AT) 5

(GT) 10

PRC 20

(AC) 10

--

(GT) 11

43 Microsatellite Loci were characterized for the first time in Paphiopedilum rothschildianum, these comprise 34 terminal repeat sequences located at the 5ﾕ and 3ﾕ end and 9 internal repeat sequences.

CONCLUSION
The 5ﾕ-Anchored PCR Method has proven to be an efficient, rapid and cost-effective method in isolating and characterizing microsatellite loci in genomes where no prior DNA sequence data is available. A total of 43 microsatellite loci were isolated and characterized.
REFERENCES
1. Dellaporta S. L., Wood J., Hicks, J. B., 1983. A Plant DNA Minipreparation: Version II. Plant Molecular Biology Reporter 1: 19 - 21.
2. Fisher, P. J. , Gardener, R.C., Richardson, T.E., 1996. Single locus microsatellites isolated using 5ﾕ Anchored PCR. Nucleic Acid Research. 24: 4369 - 4371
3. Kumar, S. V., Tan, S. G.,Quah, S. C., Yusoff, K. 2002. Isolation and characterization of seven tetranucleotide microsatellite loci in mungbean, Vigna radiata. Molecular Ecology Notes 2: 293 -295.
4. Sambrook, J., Fritsch, E. F. and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, CSH, NY.
This project is funded by the Malaysian Government IRPA Grant No. 01-02-10-0054-EA0052
The authors are grateful to the Sabah Parks Authority for providing specimens of Paphiopedilum rothschildianum from their in-situ conservation facilities at Poring and Kinabalu.

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