Molecular Epidemiology of Xanthomonas euvesicatoria Strains from the Balkan Peninsula Revealed by a New Multiple-Locus Variable-Number Tandem-Repeat Analysis Scheme

Bacterial spot of pepper and tomato is caused by at least three species of Xanthomonas, among them two pathovars of Xanthomonas euvesicatoria, which are responsible for significant yield losses on all continents. In order to trace back the spread of bacterial spot pathogens within and among countries, we developed the first multilocus variable number of tandem repeat analyses (MLVA) scheme for pepper- and tomato-pathogenic strains of X. euvesicatoria. In this work, we assessed the repeat numbers by DNA sequencing of 16 tandem repeat loci and applied this new tool to analyse a representative set of 88 X. euvesicatoria pepper strains from Bulgaria and North Macedonia. The MLVA-16 scheme resulted in a Hunter–Gaston Discriminatory Index (HGDI) score of 0.944 and allowed to resolve 36 MLVA haplotypes (MTs), thus demonstrating its suitability for high-resolution molecular typing. Strains from the different regions of Bulgaria and North Macedonia were found to be widespread in genetically distant clonal complexes or singletons. Sequence types of the variable number of tandem repeats (VNTR) amplicons revealed cases of size homoplasy and suggested the coexistence of different populations and different introduction events. The large geographical distribution of MTs and the existence of epidemiologically closely related strains in different regions and countries suggest long dispersal of strains on pepper in this area.

Microsatellite DNA markers in Shorea platyclados (Dipterocarpaceae): genetic diversity, size homoplasy and mother trees

Cross-specific amplification of microsatellite loci greatly enhances the effectiveness of this marker system. This shortcut would greatly enhance our examination of the gene flow and population structure of trees in diverse tropical rainforests. To explore the effectiveness and limitations of this approach, we examined allelic diversity at six microsatellite loci, originally developed in a congeneric species, in three populations of Shorea platyclados from Peninsular Malaysia. Fragment sizing was performed by an efficient and sensitive (1 bp resolution) technique using capillary electrophoresis, ethidium bromide detection, and minimal clean-up. Fragment size ranges were conserved between species and null allele frequencies were low. Higher overall levels of genetic diversity were detected in our study. Variation among populations was directly related to geographic distance. Fragment size class distributions suggest that each locus should be studied using different evolutionary models. Direct sequencing of SSR fragments revealed that size differences were due to changes in both the flanking regions and repeat motifs. Several clear examples of size homoplasy were observed, along with the disruption of perfect repeats, suggesting that cross-specific amplification of microsatellite loci requires an additional level of confirmation at the DNA sequence level before the influence of size homoplasy and changes in repeat structure can be assessed. Simulation studies demonstrate that the increasing intensity of timber harvest leads to higher variability in levels of potential heterozygosity and decreasing total number of alleles in the remnant "mother trees" The careful selection of "mother" trees can greatly enhance the future genetic diversity of populations.   

Exon-primed intron-crossing (EPIC) PCR markers ofHelicoverpa armigera(Lepidoptera: Noctuidae)

Applying microsatellite DNA markers in population genetic studies of the pest mothHelicoverpa armigerais subject to numerous technical problems, such as the high frequency of null alleles, occurrence of size homoplasy, presence of multiple copies of flanking sequence in the genome and the lack of PCR amplification robustness between populations. To overcome these difficulties, we developed exon-primed intron-crossing (EPIC) nuclear DNA markers forH. armigerabased on ribosomal protein (Rp) and the Dopa Decarboxylase (DDC) genes and sequenced alleles showing length polymorphisms. Allele length polymorphisms were usually from random indels (insertions or deletions) within introns, although variation of short dinucleotide DNA repeat units was also detected. Mapping crosses demonstrated Mendelian inheritance patterns for these EPIC markers and the absence of both null alleles and allele ‘dropouts’. Three examples of allele size homoplasies due to indels were detected in EPIC markers RpL3, RpS6 and DDC, while sequencing of multiple individuals across 11 randomly selected alleles did not detect indel size homoplasies. The robustness of the EPIC-PCR markers was demonstrated by PCR amplification in the related species,H. zea,H. assultaandH. punctigera.