I am assuming you are trying to develop markers for both plant and endophyte. Extract the DNA from plant tissue which also has endophyte fungus. Use specific restriction enzymes which can cut in different places for both types of DNA and construct specific primers using the adapters specific to the restriction enzymes cut differently the two genomic DNA. PCR using the primers to do the sequencing and develop markers. It is a pretty long process involving lots of digestion and sequencing. Happy to discuss more if you are interested.

@Rajasekaran Palanisamy

Actually we are trying to develop markers only for host plant not for the endophyte. Our interest to recover host plant DNA which contaminated by endophytic DNA for that we require a protocol of DNA isolation. I will be happy if you could kindly help me in this regard.

What kind of host plant we are talking about?

I dont think the endophyte genome will interfere much in the way of gettin information from molecular markers if you choose good informative markers (SSR or SNPs). When you are using markers developed for your host plant then there is minimum chance of getting other genomes flanked.If you are in doubt you can try control plant or tissue which you think that endophyte free along with your host plants. Having said that how much concentration you get out of endophyte hyphae matters in this case. One can find out taking section of the tissue and estimate the incidence of the mycelium in the tissue you are using for DNA extraction.If you are still convinced that you are amplifying fungal DNA in your PCR you can use fungal specific universal primers like ITS to confirm the inteference and work it from there. Or isolate the RNA and using two species specific primers (for host plant and endophyte) and do QPCR to see how much interference you are dealing with. If you are desperate then you can try using cesium hloride/bisbenzimide density gradient centrifugation to separate the fungal DNA from the host DNA. OR suspending of cell contents after grinding the tissues in buffer containing different concentrations of protease and EDTA to avoid Endophyte DNA.

As I mentioned before choosing specific SSR or SNPs markers may not pose this problem. Hope this may be of some help.

The host plant is Tea (Camellia sinensis)

We are trying initial screening with RAPD primers for generating trait specific markers in F1 progenies. We tried both visibly healthy tissue and in vitro grown plant tissue, confirming presence of endophytes using ITS universal primers for both bacteria and fungi. Fungal ITS1 and ITS4 primers showed positive and bacterial primers showed negative.

I hope that, Cesium chloride-bisbenzimide gradients for separation of endophytic and plant DNA, or using different concentration of protease and EDTA methods will promote endophyte free DNA. Can you please send me a protocol that can help me out of this problem?

Dear Edwin Raj,

I suggest you to read the following article and hope that it will help you.

http://www.fs.fed.us/rm/pubs/rmrs_p052/rmrs_p052_087_090.pdf

Regards,

As the previous answer to your question started out, it is not clear what your aim is. Just biochemically separating the DNA, I guess this is not possible (e.g. like a bacterial plasmid from bacterila genome based on diffeent physical properies). However, if you first of all want to know whether and how strongly your plant/ animal DNA is contaminated, you already described the methods, if you develop this e.g. in a qPCR, you even quantify the relative conatmination i different samples.

However, when I first read your question I had the impression that you want to "sort out" the DNA from different sources not physically, but rather decide which sequence is coming from which contributor. As far as I know, the bioinformaticians dealing with whole genome sequencing are confromted with this problem all day, so they have developed algorithms based on different sequence peculiarities etc. to align and sort the DNA sequences obtained. So, you might want to contact thoese people (if this is the aim of your work, otherwise, you may want to specify your question including the experiment you are aiming at).

Good luck,

Tony Schaeffner

Sorry, I only had looked at the top answer not realizing that there was also an intense discussion with newer entries below, where also the aims of the study are better defined.

Tony

Isolate Endophyte and plant DNA separately and find markers is the only way. Few papers also available on that area.

Please do qPCR to be sure of contamination . Else, specific primers are known to genrate DNA of your interest. As it is without growing in water agar or defined medium endophytic fungi would be just negligible to give you any significant amount of DNA unless you amplify.

Hi,

since some of you suggested gradients or even different protease treatment, I am wondering about the physical basis. Is fungal DNA so much denser or lighter due to a considerable difference in GC content, otherwise there would be no difference at all for (in fact) fragments of eukarytic DNA.

Tony

@Anton Schäffner

CsCl–bisbezimide gradient centrifugation is a method to separate phytoplasma from host plant DNA. Bisbenzimide forms a complex with A + T-rich DNA thereby lowering its relative density. During centrifugation the A + T-rich phytoplasma DNA is spatially separated from the less A + T-rich host plant DNA. The difference in buoyant density between phytoplasma DNA and plant DNA varies according to the host–pathogen combination. The phytoplasma DNA forms a distinct band above the host plant DNA and can be collected. Depending on the phytoplasma titer and the scale of extraction, highly purified DNA is obtained in sufficient quantities for the construction of a genomic library, a sequencing project or hybridization studies.

Based on above mentioned abstract (taken from

http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-62703-089-2_32)

I hope that they have done A+T and Bisbenzimide complex for separating DNA from host DNA. But it applicable only if the less A+T-rich host plant DNA. The universal methodology should be developed for the DNA isolation since researcher around world developing trait specific markers using random primers for initial screening in the various crops. But i should be applicable to every researcher as like CTAB, Doyel and Doyel, Murrey and Thomsom etc.,

Dear Golam Dastogeer

I hope that your information useful in charaterisation and identification of unculturable endophytic fungi. However we need DNA isolation protocol as mentioned in the following hyperlink http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-62703-089-2_32

My suggestion for example identify gene or bio marker of a host which is responsible for metabolite production in plant annotate that with fungal DNA which will help to identify the plant DNA

This might be helpful

Genomic DNA Extraction and Barcoding of Endophytic Fungi