A comparison of UP‐PCR and RAPD markers to study genetic diversity of Fusicladium effusum (G. Winter), cause of pecan scab

Abstract

Summary Fusicladium effusum infects pecan causing yield loss, but no information is available on the genetic diversity of F. effusum. Randomly amplified polymorphic DNAs (RAPDs) and universally primed polymerase chain reaction (UP‐PCR) were compared to detect polymorphisms on a group of 20 isolates of F. effusum from 11 geographical locations in the southeastern USA. Two tests (run 1 and 2) of both the RAPD and UP‐PCRs were conducted to assess the repeatability of the methods, and the markers scored on agarose gels. In addition, the UP‐PCR markers from run 1 were scored using an automated capillary system. Both RAPDs and UP‐PCR markers detected a high level of polymorphism among the scored markers (92 and 91% of RAPD markers, and 86 and 87% of manually scored UP‐PCR markers in run 1 and 2 were polymorphic, respectively; 93% of UP‐PCR markers were polymorphic when scored using the automated system). Unweighted paired group method of arithmetic averages (UPGMA) analysis showed both RAPDs and UP‐PCR markers individually identified each isolate, producing three groupings, but only the groupings based on run 1 and 2 of the UP‐PCR contained the same isolates. Bootstrap analysis based on the Dice coefficient produced phenograms from the UP‐PCR data with weak to moderate node support (≥54) for the primary branch, but no support for the RAPDs data (≤34). A Mantel test of runs 1 and 2 using RAPDs or UP‐PCR showed good agreement (r = 0.8761 and 0.8289, p < 0.0001), but poor agreement between RAPDs and UP‐PCR. UP‐PCR results based on the interisolate Dice coefficients showed a weak to strong association with distance. Based on these results, both RAPDs and UP‐PCR markers were capable of demonstrating polymorphisms and identifying relationships among isolates of F. effusum; however, UP‐PCR markers appear to be more reliable.

Genetic Diversity and Population Structure of Fusicladium effusum on Pecan in the United States

“…The process of isolation for F. effusum. has been previously described (6). Briefly, conidia of F. effusum were scraped from single lesions on the sampled leaf using a scalpel, and a dilute spore solution was prepared in sterile distilled water.…”

“…Once obtained, the DNA was quantified using a Nanodrop spectrophotometer (Nanodrop Products, Wilmington, DE) and stored in TE buffer at -20°C. The UP-PCR primers (obtained from Eurofins MWG Operon, Huntsville, AL) bad been previously tested and found to he reliable and repeatahle (6). UP-PCR primers produce dominant markers (9), and have been previously used on pathogens related to F. efftisum (44).…”

“…Fragment sizes were esfimated based on tbeir migration relative to a known standard (50 to 10,000 bp Ladder; Bionexus, Inc., Oakland, CA). Any amplicon tbat was sufficiently pronounced to be readily visible was scored as present (6).…”

“…Apart from a comparison of two dominant marker methods (6), there is no information available on tbe population genetics of F effusum (although limited molecular genetic data has been used to aid classification of this fungus; 3,54). Population genetics and genetic diversity have been studied for several related species.…”

“…(6) recently compared RAPDs and universally primed polymerase chain reaction (UP-PCR) as dominant marker systems to investigate the molecular genetic diversity of F. effusum. Both systems have the advantages of being inexpensive and relatively straightforward, but the UP-PCR system was found to be more reliable.…”