MSA - Cell and molecular biology/Physiology & Genetics
Santana, Quentin C. , Coetzee, Martin P. A. , Steenkamp, Emma T. , Wingfield, Michael J. , Wingfield, Brenda D .
Microsatellite discovery in Fusarium circinatum using ISSR-PCR and deep sequencing.
Molecular markers are essential for robust population and biogeography studies of biota. A number of marker types are available, of which microsatellites were shown to be most informative. Microsatellites or simple sequence repeats (SSR) represent polymorphic loci consisting of repeating units of 1 to 6 nucleotide motifs. The use of microsatellites is, however, often hampered by the lack of available primers that targets these regions and absence of polymorphisms among individuals. Consequently new microsatellite regions must be searched for to obtain informative markers and primers. Finding microsatellites regions usually involves some form of enrichment method followed by cloning and sequencing; making it time consuming and labour intensive. The aim of this study was to develop a rapid protocol using the 454 GS FLX genome sequencing platform for identifying microsatellite regions and designing PCR primers for their amplification and scoring. We used genomic DNA sourced from two isolates of the pine pathogen Fusarium circinatum. Microsatellite enrichment was performed using Inter-SSR-PCR, after which the DNA was sequenced. For each isolate, 1.6 to 1.7 megabases of DNA sequences were generated and a total of 382 microsatellite sequences were identified. These microsatellites were represented by repeat classes similar to those found in the sequenced genome of F. verticillioides, showing that the technique does not favour a specific class of microsatellite. For F. circinatum, a total of 29 primer pairs were developed, of which 13 were found to be polymorphic among individuals of this species. Our approach for microsatellite discovery is therefore substantially faster and more robust than other methods which have been reported previously.
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1 - University of Pretoria, Genetics, Lynnwood Road, Hillcrest, Pretoria, Gauteng, 0002, South Africa
2 - Univeristy of Pretoria, Genetics, Lynnwood Road, Hillcrest, Pretoria, Gauteng, 0002, South Africa
3 - University of Pretoria, Microbiology and Plant Pathology, Lynnwood Road, Hillcrest, Pretoria, Gauteng, 0002, South Africa
4 - University of Pretoia, Genetics, Lynnwood Road, Hillcrest, Pretoria, Gauteng, 0002, South Africa
Presentation Type: Oral Paper:Papers for Topics
Location: White Pine/Cliff Lodge - Level C
Date: Monday, July 27th, 2009
Time: 11:15 AM