ARS | Publication request: Comparison of whole-genome amplifications for microsatellite genotyping of Rotylenchulus reniformis

Submitted to: Electronic Journal of Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 30, 2011
Publication Date: May 15, 2011
Citation: Arias, R.S., Stetina, S.R., Scheffler, B.E. 2011. Comparison of whole-genome amplifications for microsatellite genotyping of Rotylenchulus reniformis. Electronic Journal of Biotechnology. DOI:10.2225/vol14-issue3-fulltext-13.

Interpretive Summary: In order to develop resistant cultivars of important crops susceptible to reniform nematode, it is important to know the genetic diversity of this plant pathogen. Since this organism is microscopic in size, it would take thousands of them to obtain enough DNA for testing, for example, 100 molecular markers. There is technology available to solve this problem by increasing the amount of DNA from small samples, and it has been tested in other biological systems, but not on reniform nematode. We assessed three commercial kits to multiply the amount of DNA, and then used the DNA to test 96 molecular markers to compare their efficacy in detecting the same regions in the genomes. The results of the most successful technique will be used in a broad screening of reniform nematode in the Cotton-BeltWide Area to determine the degree of genetic diversity of this pathogen.

Technical Abstract: Whole-genome amplification (WGA) of one and five Rotylenchulus reniformis (reniform nematode) individuals was successfully performed on disrupted tissue using three commercial kits. The DNA resulting from the WGA ranged from 0.5 to 8 µg and was used without further purification to test 96 microsatellite markers previously developed for the reniform nematode. The results were compared to those from fingerprinting the original population (MSRR03). Out of 96 markers tested, 71 had amplicons in the MSRR03 population, and subsets of those 71 markers amplified 86-93% of the alleles found on MSRR03 when using WGA of single nematodes as a template, and 87-88% of the alleles found on MSRR03 when using WGA of five nematodes as template. Our results indicate that single reniform nematodes can be used in WGA and direct testing with microsatellites giving consistent results when compared to the original population.