The Development of a Fluorescent Microsatellite Marker Assay for the Pitaya Canker Pathogen (Neoscytalidium dimidiatum)

Abstract

Pitaya canker, caused by Neoscytalidium dimidiatum, is a destructive disease that significantly threatens the safety of the pitaya industry. The authors of previous studies have mainly focused on its biological characteristics and chemical control. However, there are no molecular markers available thus far that can be used for the population genetics study of this pathogen. In the present study, a draft genome of N. dimidiatum with a total length of 41.46 MB was assembled in which 9863 coding genes were predicted and annotated. In particular, the microsatellite sequences in the draft genome were investigated. To improve the successful screening rate of potentially polymorphic microsatellite makers, another five N. dimidiatum isolates were resequenced and assembled. A total of eight pairs of polymorphic microsatellite primers were screened out based on the polymorphic microsatellite loci after investigating the sequencing and resequencing assemblies of the six isolates. A total of thirteen representative isolates sampled from different pitaya plantations were genotyped in order to validate the polymorphism of the resulting eight markers. The results indicated that these markers were able to distinguish the isolates well. Lastly, a neighbor-joining tree of 35 isolates, sampled from different pitaya plantations located in different regions, was constructed according to the genotypes of the eight molecular markers. The developed tree indicated that these molecular markers had sufficient genotyping capabilities for our test panel of isolates. In summary, we developed a set of polymorphic microsatellite markers in the following study that can effectively genotype and distinguish N. dimidiatum isolates and be utilized in the population genetics study of N. dimidiatum.