Lehrstuhl für Pflanzenzüchtung

Cloning of a beet cyst nematode resistance gene from the wild beet Patellifolia procumbens

h_schachtii_sugar_beet_female.jpg

 

Background

The beet cyst nematode (BCN, Heterodera schachtii Schmidt) is a severe pest in sugar beet (Beta vulgaris L.). It has a broad host range including many species from different plant families such as Amaranthaceae and Brassicaceae. After finishing its life cycle in the root, a cyst is formed with several hundred eggs which can survive in the soil for up to ten years.

Resistance to BCN has not been observed in B. vulgaris. The only sources of resistance available for beet breeding are the wild species Patellifolia procumbens and related species P. patellaris. However, gene transfer by crossing and recombination is hampered by strong crossing barriers.

During >20 years of selection, resistant beet lines have been established carrying translocations from the wild beet P. procumbens. The translocation lines A906001 and TR363 carry a fragment of P. procumbens chromosome 1 at the end of chromosome 9. Both lines exhibit complete resistance to the BCN. The Hs1pro-1 gene had been cloned from the translocation line A906001 (Cai et al. 1997). This gene gives complete resistance in hairy roots after transformation into susceptible sugar beet. However, only partial resistance was found in whole plants transformed with this gene. Moreover, the sister line TR363 does not carry the Hs1pro-1 . Thus, it can be followed that a 2nd gene named Hs1-2 is located in the vicinity of Hs1pro-1 .

Objectives

We assume that the Hs1-2 nematode resistance gene is located within the region which both translocations share in common. The objectives of this work are to completely sequence the translocation region of A906001, to clone the Hs1-2 gene from its position on the translocation region, and to analyze its function after transformation into beet hairy roots and Arabidopsis thaliana.

Results

Second and third generation physical maps covering the translocation A906001 have been established (Schulte et al., 2006; Capistrano 2010). Because the whole translocation is excluded from recombination, seeds were 400 Gy gamma irradiated to produce breakages within the translocation and thus narrow down to the resistance gene. The mutants were screened with translocation specific markers for identification of lines with smaller translocations.

As further extension of the BAC-based physical map by chromosome walking is a tedious work, the translocation lines TR520 and TR363 were sequenced by a WGS approach using an Illumina HiSeq2000 sequencer. As a result, 477 kbp of new sequences were identified anchored to the physical map. Thus, the total sequence of the physical map adds up to 1,509 kbp. In addition, 13 new super scaffolds were assembled that integrate scaffolds and contigs of P. procumbens and the translocations TR520 and TR363 that are not anchored to the physical map. As a major result, the new sequences identified (716 kbp) increase the size of the TR520 translocation to 2,226 kbp, corresponding to ca. 150% of the previously estimated value (1,500 kbp). It was demonstrated that the translocation is interrupted by large sequences from the B. vulgaris genome. This renders the identification of the translocation breakpoint(s) less important, as there is no single start or end of the translocation.

Further analyzes of the translocation specific sequences resulted in the prediction of 320 ORFs. We selected eleven ORFs as candidate sequences according to their position on the physical map and to their homology to known genes from other species. One ORF shows overall sequence similarity to an A. thaliana resistance gene. This ORF is presently investigated in detail.

Project team

Prof. Dr. Christian Jung
Dr. Gina Capistrano-Goßmann

Scientific Partners

Prof. Andre Franke, Dr. Georg Hemmrich-Stanisak, Dr. Markus Schilhabel, Institut für Klinische Molekularbiologie (IKMB)

Publications

Jäger, S. Hybrid Assembly of Whole Genome Shotgun Sequences of Two Sugar Beet (Beta vulgaris L.) Translocation Lines Carrying the Beet Cyst Nematode Resistance Gene Hs1-2 and Functional Analysis of Candidate Genes, Doctoral Thesis, Christian-Albrechts University of Kiel, (2013).

Capistrano, G.G.G. 2011. A candidate sequence for the nematode resistance gene Hs1-2 in sugar beet. 2. Quedlinburger Pflanzenzüchtungstage und 15. Kurt von Rümker-Vorträge. March 28-29, 2011, Quedlinburg, Germany.

Capistrano, G., Jäger, S.C., Harloff, H., Jung, C. Irradiation mutant mapping of wild beet translocation lines carrying resistance genes against the beet cyst nematode, GPZ AG Pflanzenzüchtung October 26-28, 2010, Gießen, Germany.

Capistrano, G., Jäger, S.C., Harloff, H., Jung, C. Irradiation mutant mapping of wild beet translocation lines carrying resistance genes against the beet cyst nematode, ICPMB3 September 5-9, 2010, Beijing, China.

Capistrano, G.G.G. 2010. A candidate sequence for the nematode resistance gene Hs1-2 in sugar beet. PhD thesis, Plant Breeding Institute, University of Kiel, Kiel, Germany.

Capistrano, G.G.G, Harloff, H.-J., Jäger, S.C., Cai, D., Jung, C. A second generation physical map around the nematode resistance gene Hs1-1 from the wild beet Beta procumbens. Plant & Animal Genomes XVII Conference January 10-14, 2009, San Diego, USA.

Capistrano, G. G. G., Jäger, S. C., Schulte, D., Harloff, H.-J., Kopisch-Obuch, F. J., Cai, D., and Jung, C. Cloning of the Hs1-1 gene for nematode resistance from the wild beet Beta procumbens. Proceedings of the second COST 872 Annual Meeting 26th - 29th May 2008, Postojna, Slovenia.

Jäger, S.C., Capistrano, G.G.G., Harloff, H.-J., Jung, C. γ-Irradiation of wild beet translocation lines and monosomic addition lines in sugar beet carrying nematode resistance genes. GPZ-Tagung gemeinsam mit der Gesellschaft für Pflanzenbauwissenschaften e.V. 30th Sept. until 2nd. Oct. 2008, Göttingen, Germany.

Jäger, S.C., Capistrano, G.G.G., Harloff, H.-J., Jung, C. γ-Irradiation of wild beet translocation lines and monosomic addition lines in sugar beet carrying nematode resistance genes. Proceeding of the International Symposium in Induced Mutations in Plants 12th until 15th August 2008, Vienna, Austria.

Jäger, S.C. 2007. Physische Kartierung der Wildrüben-Translokation auf Chromosom 9 der Zuckerrübe. Master Thesis, Plant Breeding Institute, University of Kiel, Kiel, Germany.

Capistrano, G.G.G. Translocation of nematode resistance from wild beet into sugar beet. Tagung der Gesellschaft für Pflanzenzüchtung e.V. AG Cytogenetik und Chromosomenanalyse (GPZ). 5-6.7.2007, Gatersleben, Germany.

Capistrano, G.G.G., Schulte, D., Longjiang, F., Cai, D., Jung, C. Determination of Hs1-1 candidates from draft sequences of a wild beet (Beta procumbens) translocation at the end of chromosome 9 of sugar beet. Proceedings of the Plant Genetics Conference 20th-23th September, 2006, Kiel, Germany.

Schulte, D., Cai, D., Kleine, M., Fan, L., Wang, S., Jung, C. 2006. A complete physical map of a wild beet (Beta procumbens) translocation in sugar beet. Molecular and General Genomics 275: 504-511.

Kleine, M., Voss, H., Cai, D., Jung, C. 1998. Evaluation of nematode resistant sugar beet (Beta vulgaris L.) lines by molecular analysis. Theor.Appl.Genet 97: 896-904.

Cai, D., Kleine, M., Kiffle, S., Harloff, H.-J., Sandal, N.N., Marcker, K.A., Klein-Lankhorst, R.M., Salentjin, E.M.J., Stiekema, W.J., Wyss, U., Grundler, F.M.W., Jung, C. 1997. Positional Cloning of a Gene for Nematode Resistance in Sugar Beet. Science 275.5301: 832-34.

Heller, R., Schondelmaier, J., Steinrücken, G., Jung, C. 1996. Genetic localization of four genes for nematode (Heterodera schachtii Sch.) resistance in sugar beet (Beta vulgaris L.). Theor.Appl.Genet, 92: 991-997.

Jung, C., Wricke, G. 1987. Selection of diploid nematode-resistant sugar beet from monosomic addition lines. Plant Breeding, 98: 205-214.