Is there any free software for in silico functional inference based on ITS (fungi) sequences?

Dear researchers

I hope you are doing well. We got an invitation from Elsevier for the contribution in their upcoming book entitled "Natural Remedies and Drug Discovery: Nanotechnology and In Silico Tools". if you wish to join us we would like to include you as an author. I also like to inform you that there are not any publication charges. If you are ready to join us as an author then please let us know as soon as possible.

Thank you.

"Title of the chapter: Biogenically efficient production and characterization of silver nanoparticles using the marine fungus Hamigera terricola along with their antimicrobial and antioxidative efficacy"

Vegetative compatibility assay in fungi is a time consuming process for huge numbers of isolates, so is there any alternative molecular method for these assays? I think the best method may be use of hybridization with probe. What is your opinions? 

there are already existing some markers like 18S (ITS region), 28S ect. but i want to know that how we could develop now such markers (step by step). is this type of study only related to in silico analysis or require some in vivo experiments?

Thanks in advance for your precious time and lending me your expertise in this regard. 

I was assigned to do PCR on fungi with three different molecular markers mentioned above. The time recorded to complete the PCR work was 1hr &30 min for SSR, 2hrs & 45 min for RAPDs, and 3hrs & 50 min for UP-PCR for the same sample size and materials.

In our preliminary study we confirmed the presence of Fungal DNA contaminated with host plant DNA using ITS 1 and ITS 4 universal primers for fungi. Since presence of endophytic fungi has been reported in many plants and animals systems and has been exploited for the improvement of functional traits of host organism, sincere efforts have been made to separate endophytic and host plant DNA not taken so far. Zhang et al., reported the PCR amplification of numerous types of fungal ribosomal RNA gene (rDNA) sequences from (apparently healthy) leaves of eight different bamboo rDNA amplicons were only obtained when leaves were successively treated with 95% ethanol and 5% NaOCl, reimmersed in 95% ethanol, and blooted dry prior to DNA extraction.

Although epiphyllous mycelia and spores can thus effectively be removed through surface sterilization, contamination by endophytic fungi (which are present in a wide variety of plant without causing obvious symptoms) is almost unavoidable, and many compromise studies using PCR-generated molecular markers. Saar et al., recommended that plant genomic DNA should be test amplified with universal primers specific for the internal transcribed spacer 2 (ITS2) region of rDNA, whenever fungal contamination is suspected. Hence, I would like to get your generous comments on above subject and please provide efficient protocols if possible!

Use of auxotrophic mutants lines for transformation of fungi is a traditional practise but it does not enable future discrimination between the wild type and transgenic line. Could basta gene serve to enable selection of the transgenic line from the wild type or from the back mutants of the transgenic line?

Is there any free software anyone could recommend for genotype x environment interaction analysis, Quantitative Trait Loci (QTL) analysis?