Hi Manee

See whether your primers are also binding to plant rDNA regions or not....you can also get some information on this by checking the specificity of your primer sequences on primer blast site at NCBI website...

bye

Hi!

I guess in your DNA preparation the quantity of yeast rDNA is much lower then the plant one. In this case the primers may tend to anneal on plant DNA rather than yeast DNA. You can also try to optimize your PCR conditions, e.g. increase temperature of primers annealing. If there a some mismatches between primers and plant rDNA you will definitely end up with specific PCR product of yeast rDNA.

hi,

firstly check your primers sequences on primer blast site at NCBI website and do BLAST ... and using primer 3 software. Firstly u should go for in silico analysis then after u may go with ITS 1, 4 n 7 makers.

Thank you very much for your advice.

I'm not sure about what I'm going to say, but you could try a DNA extraction protocol where yeast DNA yield is increased and plant DNA is decreased. After on, using the comments from above, your PCR will be better.

Dear Dr. Farshad Darvishi

According to your recommendation and most paper worked on fungi, ITS primers were used rather than D1/D2.

Please explain why it is, since ITS1 & ITS4 primers seem to be universal for eukaryote same as NL1 & NL4 for D1/D2 region.

Why do not use ITS1F (fungal specific primer) /ITS4??

These primers are commonly used for fungal DNA amplification from plant material (e.g mycorrhizal roots...) or complex matrix such as soil...

Thank you Erica

I suggest using fungi-specific primers for endophytes. I cannot suggest primer pair ITS1-ITS4 as this will most likely amplify plant DNA as well. There is also fungi-specific ITS4-B primer but it has been reported being less efficient.

Also, amplification of ITS+D1 or ITS+D1/D2 is possible.

If the PCR-product length is limiting factor, you can consider amplifying ITS2+D1 using primers for NGS approaches.