Molecular characterization of the beet cyst nematode (Heterodera schachtii) resistance locus Hs1

The white beet cyst nematode (BCN), Heterodera schachtii Schm. is a serious pest in sugar beet ( B. vulgaris L.) cultivation and is widely distributed throughout most of the beet-growing areas in the world (Cooke 1987). The economical losses due to infestation with the nematode are considerable (approximately 1200 dutch guilders or $ 600 per ha. at a rate of 25 % -30 % loss) and can mainly be ascribed to the intensive growing of sugar beet and other crops like oilseed rape which allow the nematode to multiply. The damage consists of wilting and a loss in root yield and sugar content (Mesken & Lekkerkerker 1988). Due to the lack of paying non-host crops to widen the rotation scheme, control of the beet cyst nematode population relies heavily on the use of nematicides. An alternative way, in which control might be achieved, is the use of resistant varieties. However, breeding for nematode resistance in sugar beet is extremely difficult and time consuming and did not yet result in stable nematode resistant material. Nevertheless, from crosses between sugar beet and BCN resistant wild beets three types of BCN resistant plants were obtained: monosomic additions, monosomic fragment additions and diploid introgressions. The BCN resistance of the monosomic additions (2n = 18 + 1) and the monosomic fragment additions (2n = 18 + f) is highly unstable whereas the resistance of the diploids appears to be more stable but also does not reach an acceptable level of stability (Lange et al. 1990; Van Geyt et al. 1990). Because of the difficulty to obtain stable resistant sugar beet varieties by traditional breeding, a program was started in april 1988 which aims at the isolation of BCN resistance gene(s) from wild beets of the section Procumbentes. The ultimate goals of this project are the transfer of the isolated resistance gene(s) to sugar beet to obtain stable resistant varieties and to elucidate the mode of action of the BCN resistance gene. Although, several groups are working on the isolation of genes conferring resistance to plant-parasitic nematodes, no such gene is isolated yet.

This thesis describes work aimed at the isolation of the BCN-resistance genes Hs1 ^pat-1 and Hs1 ^pro-1 via 'positional cloning' (Wicking and Williamsom 1991). 'Positional cloning' is a strategy for isolating genes which are only defined by their phenotype, a condition that holds for the BCN-resistance genes. For positional cloning the gene of interest is localized on the genome with respect to molecular markers. Next, flanking markers can be identified and used for the onset and termination of a chromosomal walk, which is the identification of a continuous set (contig) of overlapping DNA clones that connect the two flanking markers. A Yeast Artificial Chromosome (YAC) library (Burke et al. 1987, Ward & Jen 1990) that contains large cloned DNA-fragments of several hundred kilobases can aid the spanning of large chromosomal distances between the markers. Furthermore, the separation and manipulation of large chromosome fragments by Pulsed Field Gel Electrophoresis can be employed for the construction of a long-range physical map of the region. Finally, the essential chromosomal region, cloned in one or several contiguous YACs and subcloned in cosmids, is analyzed for the presence of candidate genes which are then screened for a functional BCN resistance gene.

Chapter 1 of this thesis describes morphological and genetic features of the plant-BCN interaction. This information is important for the ultimate development of nematode resistant plants. Furthermore, the positional cloning strategy for isolating genes is described in detail and the state of art for the identification and cloning of various nematodes resistance genes is given.

Chapter 2 describes the isolation of molecular markers linked to the BCN resistance locus, Hs1, which is the first prerequisite for positional cloning of the gene(s). The plant material which was used consisted of sugar beets with an introgressed wild beet chromosome fragment containing the resistance gene(s). Since the resistance in this material segregates in a nonMendelian way, a deletion mapping strategy was employed to order the markers with respect to the resistance locus.

Chapter 3 describes the characterization of a marker which is highly repetitive in wild beets and closely linked to Hs1 ^pat-1 and Hs1 ^pro-1 . The long-range physical organization of the repeat is studied by employing the PFGE-technology.

Chapter 4 describes the construction of a YAC-library from BCN-resistant sugar beet (AN5-203b) containing an additional fragment of a wild beet chromosome. This library was screened with a marker localized near the Hs1 ^pat-1  gene to provide a starting point for the assembly of a YAC- contig spanning the resistance locus.

Chapter 5 describes the isolation, characterization and deletion mapping of additional PCR-based RAPD markers.

In Chapter 6 physical distances around three markers linked to Hs1 ^pat-1 are determined which has resulted in a first generation physical map of the resistance locus.

Chapter 7 is a general discussion of the research which will be necessary for the ultimate positional cloning of the resistance genes. Furthermore, different strategies for the engineering of nematode resistant plants are discussed.