Characterization of Novel Di-, Tri-, and Tetranucleotide Microsatellite Primers Suitable for Genotyping Various Plant Pathogenic Fungi with Special Emphasis on Fusaria and Mycospherella graminicola

Abstract

The goals of this investigation were to identify and evaluate the use of polymorphic microsatellite marker (PMM) analysis for molecular typing of seventeen plant pathogenic fungi. Primers for di-, tri-, and tetranucleotide loci were designed directly from the recently published genomic sequence of Mycospherlla graminicola and Fusarium graminearum. A total of 20 new microsatellite primers as easy-to-score markers were developed. Microsatellite primer PCR (MP-PCR) yielded highly reproducible and complex genomic fingerprints, with several bands ranging in size from 200 to 3000 bp. Of the 20 primers tested, only (TAGG)4, (TCC)5 and (CA)7T produced a high number of polymorphic bands from either F. graminearum or F. culmorum. (ATG)5 led to successful amplifications in M. graminicola isolates collected from Germany. Percentage of polymorphic bands among Fusarium species ranged from 9 to 100%. Cluster analysis of banding patterns of the isolates corresponded well to the established species delineations based on morphology and other methods of phylogenetic analysis. The current research demonstrates that the newly designed microsatellite primers are reliable, sensitive and technically simple tools for assaying genetic variability in plant pathogenic fungi.

Characterization of Fusarium proliferatum and Fusarium verticillioides based on Species-Specific Gene and Microsatellites Analysis

“…From the results of our study (Figure 1), sizes of PCR fragments amplified from primers (AG)7C and (TCC)5 were as expected ranging from 250-2000 bp and 300-2000 bp, respectively. Meanwhile for primer (TAGG)4, the fragments amplified ranged from 500-1500 bp in range, however, as reported by Bahkali et al (2012) the expected size ranged from 200-2000 bp. For primer (TTTC)4, there are negative results and no amplifications produced even though modifications were made on the annealing temperature and PCR reaction volume.…”

“…For primer (TTTC)4, there are negative results and no amplifications produced even though modifications were made on the annealing temperature and PCR reaction volume. It can be concluded that this primer is not suitable for analysis on F. verticillioides and F. proliferatum and from all the results, only partial agreement can be made with previous study by Bahkali et al (2012).…”

“…Standard PCR master mix for all reactions were each 20 μL comprises 1 PCR buffer, 0.5 μM primer, 0.2 mM of each deoxynucleotide triphosphate (dNTPs), 2.5 mM magnesium chloride (MgCl 2 ), 0.125 U GoTaq polymerase, nuclease free water and 20 ng DNA. The following PCR amplification was followed as: initial denaturation at 94°C for 2 min, 39 cycles of denaturation at 94°C for 1 min, annealing as follow the optimal Tm for each primer (Table 1) for 90 min, extension at 72°C for 2 min and a final extension at 72°C for 6 min (Bahkali et al 2012). …”

“…To validate the polymorphism and genetic diversity among all the Fusarium isolates, 6 established primers were selected from Bahkali et al (2012). Five primers have successfully amplified bands while one primer showed negative result.…”

SSR Markers for Trichoderma virens: Their Evaluation and Application to Identify and Quantify Root-Endophytic Strains

“…This made it possible to search for all scaffolds in one go. Screening the genome for microsatellite motifs was based on published data about existing motifs that were detected in T. virens by ISSR and RAMS analysis [31,32]. Microsatellite loci sufficiently flanked by unique sequences were selected for primer design using the Primer3 software [33] with parameter settings that included the GC-clamp option and an allowance for primer annealing at approximately 60 °C.…”