|
|
|
|
|
1. Development of DNA markers for the detection and quantification of pathogens and mycorrhizal fungi in cooperation with partners A1/C4 and B7, respectively
|
Calibration curve for the quantification of Phytophthora citricola using real-time PCR (A) and detection of the pathogen in beech seedlings at the indicated time points after infection (B). (References 1,2,4)
|
Figure A
Figure B
|
2. Development of 7 codominant DNA markers for Norway spruce
|
Survey of newly developed EST markers (C) and verification of codominant inheritance (D) of alleles I and O for marker CAD by comparing PCR products from a diploid bud sample D with corresponding haploid megagametophytes H. (References 5,6)
|
| Marker loci |
Putative function |
| PA0002 |
A-like cyclin |
| PA0034 |
non-identified gene |
| PA0038 |
halotolerance protein |
| PA0043 |
high molecular heat-shock protein |
| PA0055 |
ATP synthase beta-chain |
| PA0066 |
60S ribosomal protein L13-2 |
| CAD |
cinnamyl alcohol dehydrogenase |
Figure C
Figure D
|
3. Studying genetic variation in tree populations using DNA markers and isoenzyme gene markers
|
(E) Minimum, maximum and mean values of genetic variation parameters obtained for the Kranzberg test population (n=110 spruce trees) using six DNA markers (EST) and 18 isoenzyme gene markers (Iso). (F) Genetic distances for the subset of 10 spruce trees localized at the ozone fumigation facility in the Kranzberg forest in comparison with a subset of 100 surrounding trees. Calculations for alleles and genotypes as well as the gene pool and the haplotypes were done using 6 new nuclear EST markers, 18 isoenzyme gene markers and 3 chloroplast markers.(References 3,5,6)
|
Figure E
Figure F
|
Conclusions
|
Species-specific DNA markers ensure an accurate and efficient detection of pathogens and mycorrhizal fungi via PCR in forest trees as indicated by Figure s A and B. Real-time PCR offers new opportunities for quantification of microorganisms especially for large sample sizes.
The newly developed multiallelic DNA markers of spruce (Figure s C and D), targeting to stressdefense genes, became powerful tools for the genetic characterization of population material (Figure s E and F). They are showing a trend to more even frequency distribution and larger intrapopulational variation than isoenzyme gene markers. These data demonstrate that the developed markers are indicative in the monitoring of genetic response of individual trees to environmental stress. DNA markers will be designed for European beech during the second period of the SFB project.
|
References:
[1] Bahnweg, G., Schubert, R., Kehr, R.D., Müller-Starck, G., Heller, W., Langebartels, C. and H. Sandermann Jr.: Controlled inoculation of Norway spruce (Picea abies) with Sirococcus conigenus: PCR-based quantification of the pathogen in host tissue and infection-related increase of phenolic metabolites. Trees: Structure & Function 14: 435-441, 2000.
[2] Böhm, J., Hahn, A., Schubert, R., Bahnweg, G., Adler, N., Nechwatal, J., Oehlmann, R. and W. Oßwald: Real-time quantitative PCR: DNA determination in isolated spores of the mycorrhizal fungus Glomus mosseae and monitoring of Phytophthora infestans and Phytophthora citricola in their respective host plants. Journal of Phytopathology 147: 409-416, 1999.
[3] Riegel, R., Schubert, R. and G. Müller-Starck: Validation of newly-developed codominant DNA-markers for studying intrapopulational genetic variation in Norway spruce [Picea abies (L.) Karst.]. In: S. Espinel, E. Ritter (Eds.) Proceedings of Application of Biotechnology to Forest Genetics, Biofor 99, 22-25 September, Vitoria-Gasteiz, Spain, 1999, pp. 151-154.
[4] Schubert, R., Bahnweg, G., Nechwatal, J., Jung, T., Cooke, D. E. L., Duncan, J. M., Müller-Starck, G., Langebartels, C., Sandermann Jr., H. and W. Oßwald: Detection and quantification of Phytophthora species which are associated with root-rot diseases in European deciduous forests by species-specific polymerase chain reaction. European Journal of Forest Pathology 29: 169-188, 1999.
[5] Schubert, R., Müller-Starck, G. and R. Riegel: Development of EST-PCR markers and monitoring their intrapopulational genetic variation in Picea abies (L.) Karst.. Theoretical and Applied Genetics 103, 1223-1231(2001).
[6] Schubert, R.and G. Müller-Starck: Development and application of molecular markers in conifers. In: S.M. Jain, D.S. Brar, B.S. Ahloowalia (Eds.) Molecular techniques in crop improvement. Kluwer academic publishers, Dordrecht, The Netherlands, 2001 (in press).
|
|
|
|
