Hi there,

since I've received several requests for the HAT-RAPD protocol I will just post it here.

We first published the use of the protocol (without the fancy name :-) ) in  Eimert et al. (2003). Since that journal does not allow the distribution of copies I attach here some later papers describing it.

Basically, the trick is in using decamer primers with higher GC content (70-90%) and or di- or trinucleotide microsatellite-like composition. That gives them kind of inter-simple sequence repeat (ISSR)-anchored primer (Zietkiewicz et al. 1994) or SPAR (Gupta et al. 1994) like properties and allows for the higher annealing temperatures (we usually use TA around 45-50°C). We bought the primer sets 1,4 and 8 from the University of British Columbia for the above mentioned reasons. They don’t sell them anymore, but I attach the primer list here so you can order them from your local suppliers. I also attach a short list of primers we tested successfully for several species (Asparagus, Heuchera, Euonymus, Malus, Phalenopsis, Prunus, Ribes, Vitis, Zinnia) and which turned out to work in most of those reasonably well (some better in one species then in the other). You might want to start with those amplifying well in several species.

You will probably have to optimize the Primer/Mg/TA combination for your species of interest. Using thermocyclers with programmable ramp times, we get very reliable results over different labs and operators (repeated checks after several years give the same patterns). One thing you still have to keep tightly controlled is the enzyme – using different brands of (Taq-) polymerase may result in changes in fingerprint patterns (mostly in intensity, but still … - better not to change suppliers during a running experiment).

Good luck to all of you and feel free to ask any questions :-) !

Klaus

Helpful (hopefully :-) literature:

Eimert et al. (2003) J Agric Sci 141:73–78

Zietkiewicz et al. (1994) Genomics 20:176–183

Gupta et al. (1994) Theor Appl Genet 89:998–1006

And attached:

Ruangsuttapha et al. (2007) Gen Res Crop Evol 54:1565–1572

Eimert et al. (2012) Plant Syst Evol 298 609–618

You may use molecular markers MASAP/SSAP or SNP to verify its stability. 

Hi Paromik,

I have no experience with SPAR, but if you don't have the infrastructure for SSR you could always use a HAT-RAPD protocol (more reliable than RAPD) or ISSR - they only require a cycler and agarose electrophoresis. If you'd like I can send you our HAT-RAPD protocol.

Good luck,

Klaus

Dear Klaus

Please send me the protocol. It will be of immense help.

Regards

Hi Klaus Elimert

Could you please send me your HAT-RAPD protocol ,too.

Thanks

Dear Klaus

Could you also please send me the protocol. Thank you.

Regards

Hi Klaus Elimert

Could you please send me your HAT-RAPD protocol ,too.

Thanks

You can check it through ISSR analysis. This article may help you

Ghosh, S., Majumdar, S., Sarkar, D., & Datta, K. An efficient adventitious shoot regeneration system for potato (Solanum tuberosum L.) using leaf discs. Journal of Plant Biochemistry and Biotechnology, 1-7

Hi there,

since I've received several requests for the HAT-RAPD protocol I will just post it here.

We first published the use of the protocol (without the fancy name :-) ) in  Eimert et al. (2003). Since that journal does not allow the distribution of copies I attach here some later papers describing it.

Basically, the trick is in using decamer primers with higher GC content (70-90%) and or di- or trinucleotide microsatellite-like composition. That gives them kind of inter-simple sequence repeat (ISSR)-anchored primer (Zietkiewicz et al. 1994) or SPAR (Gupta et al. 1994) like properties and allows for the higher annealing temperatures (we usually use TA around 45-50°C). We bought the primer sets 1,4 and 8 from the University of British Columbia for the above mentioned reasons. They don’t sell them anymore, but I attach the primer list here so you can order them from your local suppliers. I also attach a short list of primers we tested successfully for several species (Asparagus, Heuchera, Euonymus, Malus, Phalenopsis, Prunus, Ribes, Vitis, Zinnia) and which turned out to work in most of those reasonably well (some better in one species then in the other). You might want to start with those amplifying well in several species.

You will probably have to optimize the Primer/Mg/TA combination for your species of interest. Using thermocyclers with programmable ramp times, we get very reliable results over different labs and operators (repeated checks after several years give the same patterns). One thing you still have to keep tightly controlled is the enzyme – using different brands of (Taq-) polymerase may result in changes in fingerprint patterns (mostly in intensity, but still … - better not to change suppliers during a running experiment).

Good luck to all of you and feel free to ask any questions :-) !

Klaus

Helpful (hopefully :-) literature:

Eimert et al. (2003) J Agric Sci 141:73–78

Zietkiewicz et al. (1994) Genomics 20:176–183

Gupta et al. (1994) Theor Appl Genet 89:998–1006

And attached:

Ruangsuttapha et al. (2007) Gen Res Crop Evol 54:1565–1572

Eimert et al. (2012) Plant Syst Evol 298 609–618