ITS1/2 primers for fungal endophytes in trees

Question

[User 4014]

Hi,

I want to look at diversity of fungal endophytes in trees using MiSeq. I tried a couple of primer pairs targeting either ITS1 or ITS2 e.g., ITS1F/ITS2, ITS3_KYO2/ITS4, ITS3_KYO2/ITS4_KYO3 (Toju et al., 2012) and BITSf/B58S3R (Bokulich and Mills, 2013), but none of them returned a satisfied result. All of the primers amplified a relatively large portion of trees ITS1/2.

Do you have any suggestions which primer pair I should try on, or any method I can try to reduce amplification of trees ITS?

Thank you in advance and have a great day!

[emericklarkin]

Hello User 4014,

I am at a similar position to you, when you wrote this post. Did you ever settle on an optimal primer set and if so, would you mind sharing them with me ?

Thanks : )

Answer 1

[User 4014]

Hi there!

I ended up with BITSf/B58S3R, but it doesn't mean the primer pair is good for you. My recommendation is that go to NBCI, download ITS sequence(s) of your tree host(s), align them if needed, blast primers of your interest to the ITS sequence(s), and choose the one with many mismatches to the host ITS as possible. From my experience, some of the primers mentioned above match perfectly with my host species. And despite using the primer pair with 2-3 mismatches, you will still get host ITS to some extent ...

I hope this helps. Good luck!