Designing a SCAR molecular marker for monitoring Trichoderma cf. harzianum in experimental communities

Abstract

Several species of the fungal genus Trichoderma establish biological interactions with various micro-and macro-organisms. Some of these interactions are relevant in ecological terms and in biotechnological applications, such as biocontrol, where Trichoderma could be considered as an invasive species that colonizes a recipient community. The success of this invasion depends on multiple factors, which can be assayed using experimental communities as study models. Therefore, the aim of this work is to develop a species-specific sequence-characterized amplified region (SCAR) marker to monitor the colonization and growth of T. cf. harzianum when it invades experimental communities. For this study, 16 randomly amplified polymorphic DNA (RAPD) primers of 10-mer were used to generate polymorphic patterns, one of which generated a band present only in strains of T. cf. harzianum. This band was cloned, sequenced, and five primers of 20-23 mer were designed. Primer pairs 2F2/2R2 and 2F2/2R3 successfully and specifically amplified fragments of 278 and 448 bp from the T. cf. harzianum BpT10a strain DNA, respectively. Both primer pairs were also tested against the DNA from 14 strains of T. cf. harzianum and several strains of different fungal genera as specificity controls. Only the DNA from the strains of T. cf. harzianum was successfully amplified. Moreover, primer pair 2F2/2R2 was assessed by quantitative real-time polymerase chain reaction (PCR) using fungal DNA mixtures and DNA extracted from fungal experimental communities as templates. T. cf. harzianum was detectable even when as few as 100 copies of the SCAR marker were available or even when its population represented only 0.1% of the whole community.

Design of Bacterial Strain-Specific qPCR Assays Using NGS Data and Publicly Available Resources and Its Application to Track Biocontrol Strains

“…In the course of this research, closer reference genomes have been released, so using them would most likely reduce the number of unmapped reads and therefore the number of output primer pairs. The high number of potential markers obtained with the proposed workflow contrasts with the few -in the order of tens -markers obtained usually identified in RAPD or AFLP experiments (Hermosa et al, 2001;Dauch et al, 2003;Felici et al, 2008;Xiang et al, 2010;Perez et al, 2014). The workflow offers a number of tuning alternatives that would yield different, yet valid, output.…”

“…PCR is highly specific so long as the primers anneal only to DNA sequences that are specific of the intended target strain. These DNA sequences are most often identified by DNA fingerprinting techniques such as RAPD (Random Amplified Polymorphic DNA) or AFLP (Amplification Fragment Length Polymorphism) followed by sequence characterization (Hermosa et al, 2001;Dauch et al, 2003;Felici et al, 2008;Xiang et al, 2010;Perez et al, 2014). Combining DNA fingerprinting and real-time quantitative PCR (qPCR) provides a powerful tool to identify and quantify bacteria at strain level, and this has made it a widespread method to monitor bacterial strains when dilution-plate counting is not feasible.…”

“…Despite of the abundant literature reporting bacterial strain monitoring methods by PCR using sequence-characterized DNA fragments obtained by RAPD, AFLP, and other DNA fingerprinting methods, these procedures are laborious and time-and resource-consuming and usually produce very few useful marker sequences, which may become helpless as new strains of closely related taxa are discovered and characterized (Hermosa et al, 2001;Dauch et al, 2003;Felici et al, 2008;Xiang et al, 2010;Perez et al, 2014). Next-generation sequencing (NGS) technologies offer the possibility to obtain DNA sequence information from bacterial strains of interest in a faster and more cost-effective manner as compared to the aforementioned methods (Marx, 2016).…”

Short Communication: Rapid and accurate genetic authentication of Penthorum chinense by improved RAPD-derived species-specific SCAR markers

“…In our lab, we have successfully characterized a number of plant or fungal species by developing SCAR markers based on RAPD amplicons; namely Dimocarpus longan , Acorus species (Ryuk et al 2014), Lonicera japonica (Yang et al 2014), Trichoderma cf. harzianum (Pérez et al 2014), Cordyceps sinensis (Lam et al 2015), Litchi chinensis , Angelica sinensis , and Ganoderma lucidum (Khan et al 2016).…”

Genetic authentication of Eclipta prostrate (Asteraceae) from Penthorum chinense (Penthoraceae) by Sequence Characterized Amplified Region (SCAR) markers

“…Our lab and other groups, have successfully characterized a number of plant or fungal species by developing SCAR markers based on RAPD amplicons, such as, Dimocarpus longan (Yang, Fu, Khan, Zeng, & Fu, 2013), Acorus species (Ryuk, Kim, Lee, & Ko, 2014), Lonicera japonica (Yang et al, 2014), Trichoderma cf. harzianum (Pérez, Verdejo, Gondim-Porto, Orlando, & Carú, 2014), Cordyceps sinensis (Lam et al, 2015), Litchi chinensis (Cheng et al, 2015), Angelica sinensis (Zhang et al, 2015), Gardenia jasminoides Ellis var. grandiflora Nakai (Mei et al, 2015b), Ganoderma Lucidum (Khan et al, 2016), Penthorum chinense Pursh (Mei et al, 2017b), as well as developing diagnostic SCAR markers from female breast carcinomas (Fu et al, 2017).…”