Cloning and development of pathotype-specific SCAR marker associated with Sclerospora graminicola isolates from pearl millet

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doi:10.1071/AP08098

Cloning and development of pathotype-specific SCAR marker associated with Sclerospora graminicola isolates from pearl millet

J. Sudisha ^A, S. Ananda Kumar ^B, S. R. Niranjana ^A, N. P. Shetty ^C, H. Shekar Shetty ^A ^D

^A Downy Mildew Research Laboratory, Department of Studies in Applied Botany, Seed Pathology and Biotechnology, University of Mysore, Manasagangotri, Mysore 570 006, Karnataka, India.
^B Microbiology and Cell Biology Division, Indian Institute of Science (IISc), Bangalore 560 012, Karnataka, India.
^C Department of Plant Biology, Royal Veterinary and Agricultural University, Thorvaldsensvej 40, 1871 Frederiksberg C, Copenhagen, Denmark.
^D Corresponding author. Email: hss_uom@hotmail.com; downymildew@hotmail.com





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Australasian Plant Pathology 38(3) 216–221 http://dx.doi.org/10.1071/AP08098
Submitted: 31 July 2008 Accepted: 14 November 2008 Published online: 3 April 2009

Abstract

Downy mildew pathogen of pearl millet in India is associated with the spread of the highly virulent Sclerospora graminicola pathotype-1. Twenty-seven S. graminicola isolates were screened using 20 inter simple sequence repeats (ISSR). Dinucleotide repeat primer [17898A-(CA)₆ AC] amplified a ~600 bp fragment specific to five isolates of pathotype-1 (Sg 048, Sg 153, Sg 212, DM-11 and DM-90). The ISSR fragment linked with pathotype-1 was cloned successfully and sequenced. To convert ISSR fragments into pathotype-specific sequence characterised amplified region (SCAR) markers, PCR primers were designed using a sequence of the cloned DNA fragment. PCR amplification using SCAR primer pair (UOM3-Sg-Path1-F/R) amplified a single 284 bp band only in isolates of S. graminicola pathotype-1. This SCAR primer pair did not amplify the 284 bp product from the other five S. graminicola pathotypes or a negative control, which demonstrates primer specificity for pathotype-1. The SCAR primer pair (UOM3-Sg-Path1-F/R) obtained in this study will provide a valuable tool for rapid identification and specific detection of S. graminicola pathotype-1.

Keywords: PCR, Pennisetum glaucum, SCAR markers.