A rapid and versatile combined DNA/RNA extraction protocol and its application to the analysis of a novel DNA marker set polymorphic between Arabidopsis thaliana ecotypes Col-0 and Landsberg erecta.
BACKGROUND
Many established PCR-based approaches in plant molecular biology rely on lengthy and expensive methods for isolation of nucleic acids. Although several rapid DNA isolation protocols are available, they have not been tested for simultaneous RNA isolation for RT-PCR applications. In addition, traditional map-based cloning technologies often use ill-proportioned marker regions even when working with the model plant Arabidopsis thaliana, where the availability of the full genome sequence can now be exploited for the creation of a high-density marker systems.


RESULTS
We designed a high-density polymorphic marker set between two frequently used ecotypes. This new polymorphic marker set allows size separation of PCR products on agarose gels and provides an initial resolution of 10 cM in linkage mapping experiments, facilitated by a rapid plant nucleic acid extraction protocol using minimal amounts of A. thaliana tissue. Using this extraction protocol, we have also characterized segregating T-DNA insertion mutations. In addition, we have shown that our rapid nucleic acid extraction protocol can also be used for monitoring transcript levels by RT-PCR amplification. Finally we have demonstrated that our nucleic acid isolation method is also suitable for other plant species, such as tobacco and barley.


CONCLUSIONS
To facilitate high-throughput linkage mapping and other genomic applications, our nucleic acid isolation protocol yields sufficient quality of DNA and RNA templates for PCR and RT-PCR reactions, respectively. This new technique requires considerably less time compared to other purification methods, and in combination with a new polymorphic PCR marker set dramatically reduces the workload required for linkage mapping of mutations in A. thaliana utilizing crosses between Col-0 and Landsberg erecta (Ler) ecotypes.
Berendzen
Kenneth
K
Max-Planck-Institute for Plant Breeding Research, Department of Developmental Biology, Carl-von-Linné Weg 10, D-50829 Köln, Germany.
Searle
Iain
I
Max-Planck-Institute for Plant Breeding Research, Department of Developmental Biology, Carl-von-Linné Weg 10, D-50829 Köln, Germany.
Ravenscroft
Dean
D
Max-Planck-Institute for Plant Breeding Research, Department of Developmental Biology, Carl-von-Linné Weg 10, D-50829 Köln, Germany.
Koncz
Csaba
C
Max-Planck-Institute for Plant Breeding Research, Department of Developmental Biology, Carl-von-Linné Weg 10, D-50829 Köln, Germany.
Batschauer
Alfred
A
Philipps-Universität, Biology-Plant Physiology/Photobiology, Karl-von-Frisch-Str. 8, D-35032 Marburg, Germany.
Coupland
George
G
Max-Planck-Institute for Plant Breeding Research, Department of Developmental Biology, Carl-von-Linné Weg 10, D-50829 Köln, Germany.
Somssich
Imre E
IE
Max-Planck-Institute for Plant Breeding Research, Department of Plant-Microbe Interactions, Carl-von-Linné Weg 10, D-50829 Köln, Germany.
Ülker
Bekir
B
Max-Planck-Institute for Plant Breeding Research, Department of Plant-Microbe Interactions, Carl-von-Linné Weg 10, D-50829 Köln, Germany.