in a nut shell
In general, the following order of steps may be suggested for those researchers who study Hypocrea/Trichoderma biodiversity or perform a global screening for industrially important Trichoderma strains.
1) Prepare monospore (single spore) cultures for every strain to be identified.
This step is of crucial importance since several Trichoderma spp. may occupy one ecological niche. Moreover, it is well known that due to homoplasy of morphological characters it is often impossible to discriminate species.
2) Extract genomic DNA from young cultures preferably before sporulation begins
We suggest to cultivate Trichoderma strains on agar plates covered by cellophane no longer than 2 - 4 days in order to control possible cross contamination by other Trichoderma cultures.
It is also advisable to check DNA quality on agarose gels. If possible, measure DNA concentration
3) make PCR amplification and sequencing of the following marker(s):
|Case||Phylogenetic marker (locus)||Tools to be used|
Large scale biodiversity assays with a big number of Trichoderma strains isolated from several locations with certain gradient of conditions;
Identification of individual strains
Species identity control for industrial isolates
Screening for certain Hypocrea/Trichoderma species distinguished by ITS1 and 2
ITS1 and 2
internal transcribed spacers�1�and�2�of rRNA gene cluster
a universal fungal BarCode marker
DNA oligonucleotide BarCode program
Identification of strains which were not identified by TrichOKEY
Identification of species from Trichoderma Section Trichoderma
Detection of new species
translation elongation factor 1-alpha encoding gene
TrichoMARK is highly recommended as an important tool for pre-BLAST sequence diagnosis. The program retrieves the most diagnostic fragments for the subsequent similarity search what significantly increases the accuracy of the similarity search in TrichoBLAST or NCBI BLAST
TrichoBLAST a multiloci local alignment each tool against verified database of Hypocrea/Trichoderma sequences
it includes three phylogenetic loci within tef1 gene (see here for details), ITS1 and 2 and rpb2
|Detection of a new species||
endochitinase 18 - 5 (former ech42)
Detect next neighbours using TrichoBLAST (Note: cal1 and chi18-5 are not yet included)
Retrieve sequences of corresponding sequences using ISTH Multiloci Database of Phylogenetic Markers
This database contains only verified type sequences of all molecularly characterized species of Hypocrea/Trichoderma. Database may be searched by species or locus name, sequences may be retrieved in the multiple FASTA format suitable for subsequent alignment and phylogenetic analysis