Methods of molecular identifications and lab. protocols
PCR protocols for amplification of Trichoderma phylogenetic markers
Here is the collection of PCR protocols for the amplification of ITS1 and 2, two fragments of tef1 gene, cal1 and chi18-5 (former ech42) which are used for phylogenetic analyses of Hypocrea/Trichoderma.
use the tef1 map below to locate tef1 primers. Note that the large intron of tef1 gene is the most informative phylogenetic marker for Trichoderma/Hypocrea.You can use either EF1/EF2 primer pair to amplify the 5 prime end or EF1-728F/LLErev primer pair to amplify the 3 prime end. The LLErev is not shown on the figure but it is located in the middle of the last large exon
- use ITS1 and 2 and TrichOKey as the first markerr.
- If TrichOKey identifies some of your strains belonging to Section Trichoderma, sequence tef1as ITS1 and 2 is not polymorphic enough to identify all species within this group. Use TrichoBLAST to identify tef1 sequences. Search in the main ISTH database and in the 'koningii tef1' database which has all sequences published here.
- If you assume that your Hypocrea/Trichoderma strain is new to science, amplyfy rpb2 and cal1 to multiloci phylogeny to test your hypothesis. If it belongs to Section Longibrachiatum or Harzianum-Catoptron Clade, also sequence chit18-5.
DO NOT HESITATE TO CONTACT US FOR QUESTIONS OF HYPOCREA/TRICHODERMA IDENTIFICATION
posted by Irina Druzhinina on 24 Jul, 2009