RAPD-PCR to detect genomic polymorphisms among geographically-dispersed populations of Cephus cinctus

Wheat stem sawfly, Cephus cinctus Norton, is the most destructive chronic insect pest of wheat in the northern Great Plains. However, little is known about the extent and distribution of genetic variability in the species. Over the past 20 years in Montana, C. cinctus has changed from an insect pest exclusive to spring wheat to a pest which causes major economic damage in both spring and winter wheat. Preliminary examination suggests that phenology and maturity of wheat cultivars grown in Montana has not changed enough over this period to account for the difference in insect virulence. Knowledge of the genetic variability within endemic populations of C. cinctus is important for developing management and resistance-breeding strategies. Our objective in this study was to assay the genomic variability within and among geographically-dispersed collections of C. cinctus from the northern U.S. Great Plains using RAPD-PCR markers. Overwintering sawfly larvae were collected from wheat stubble at eight sites in Montana, six sites in North Dakota, and one site in Wyoming. DNA was extracted and evaluated from individual larvae from each collection site. Sixty-two random decamer primers were screened and 20 of them consistently produced well-amplified and reproducible polymorphic bands. The size of amplified DNA fragments produced by these primers ranged from 200-1900 bp, with individual primers generating from two to nine bands. Genetic distances among 186 individuals based on 60 RAPD loci were calculated using similarity index, 1-M (where M is the fraction of matches). Each sawfly individual was a unique RAPD multiband phenotype. Based on UPGMA cluster analysis all Montana sawflies clustered separately from all North Dakota and Wyoming sawflies. Principal coordinate analysis based on the band frequency within each population showed a similar result. Analysis of molecular variance partitioned the RAPD variation into the among- and within-population components. The within-population component accounted for 71.6% of the variation and was significantly different from zero at the 1% probability level. The among-population and among-region components accounted for 6.3% and 22.1% of the total variation, respectively. Subset analyses of MT and ND populations showed that there were significant differences among populations in MT but not in ND. Pairwise tests for the homogeneity of the RAPD variance between populations suggested significant divergences among 81 of the 105 (77%) population pairs including all but three of the MT pairwise comparisons. A dendrogram based on the Euclidean distance among populations showed that all the Montana populations were grouped together, with all North Dakota populations in another group. The high degree of structuring in Montana populations suggests the high degree of reproductive isolation among geographically-separated populations is contributing to development of geographic and/or host races due to adaptation to local environment conditions and/or host differences.