ABSTRACT

The objective of the work was to tag a locus for white rust resistance from Finnish spring turnip rape material. The ultimate goal is to design molecular markers to be used in marker-assisted selection. In order to find RAPD markers linked to white rust resistance gene(s), an F₂ mapping population was raised from the cross between one resistant and one susceptible individual from the breeding material of Boreal Plant Breeding. The white rust reaction against races 2 and 7 was scored in 20 seedlings from each self-pollinated F₂ individual. The amount of resistant plants among these F₃ families varied from 0 to 67%. Two bulks with nine individuals were constructed from the F₂ progeny: the susceptible bulk (individuals with 0% resistant offspring) and the resistant bulk (individuals with the highest amount of resistant offspring). So far, 409 RAPD primers have been tested for polymorphism between the bulks but none of them produced suitable markers. Therefore, an alternative strategy, based on the existence of conserved structures among several resistance genes, was chosen. Two different PCR primer pairs were designed to amplify resistance gene analogs from the resistant parent. The PCR products of ca. 500 bp in size were cloned and several clones are currently being sequenced to find the putative white rust resistance gene among them.

INTRODUCTION

White rust, caused by Albugo candida (Pers.) Kuntze, is an economically important disease of many crucifers. Yield losses due to white rust on turnip rape (B. rapa) can range from 30 to 60% in heavily infected fields (Bernier 1972). A. candida is classified into ten races based on their specificity to different crucifer species (Pound and Williams 1963, Hill et al. 1988). The races of economic importance in western Canada are race 2 on Brassica juncea and race 7 on B. rapa (Petrie 1988). However, races do not exhibit an absolute adaptation to a particular host species, but can also infect heterologous hosts, especially those sharing a common genome. The development of white rust resistant cultivars is a primary goal in breeding programmes of B. juncea and B. rapa in western Canada. In Finland, white rust is not an economically important disease, but spring turnip rape cultivars of Boreal Plant Breeding aimed at Canadian markets have to be resistant. The genetic control of resistance to A. candida in B. rapa seems to be governed by both major and minor genes (Delwiche and Williams 1974, Edwards and Williams 1978, Kole et al. 1996).

Sequence comparisons between resistance genes against different pathogens (viruses, bacteria, fungi) have revealed structural similarities. Consequently, resistance genes can be classified into two different classes (Staskawicz et al. 1995): leucine-rich repeat (LRR) genes with or without a nucleotide-binding site (NBS), and genes with a serine/threonine kinase domain. The majority of resistance genes have an NBS and LRR, e.g. virus resistance gene N from tobacco (Whitham et al. 1996) and bacteria resistance gene RPS2 from Arabidopsis (Bent et al. 1994). The overall sequences among members of NBS-LRR class are highly divergent, but short peptide sequences are well conserved. The conserved domains offer opportunities to reach other disease resistance sequences (resistance gene analogs, RGAs) via PCR. Primers based on the conserved sequences often amplify a collection of PCR products of the anticipated size. Therefore, only sequencing of several clones from the ligated and cloned PCR fragment reveals the different resistance gene analogs.

The aim of the current study was to tag the locus for resistance to A. candida from Finnish spring turnip rape (B. rapa ssp. oleifera) material to develop molecular markers to be used in marker-assisted selection. Two strategies were adopted, bulked segregant analysis using RAPD markers and the isolation of resistance gene analogs via PCR based on structurally conserved motifs.

MATERIALS AND METHODS

An F₂ mapping population of 99 individuals was derived by self-pollinating one resistant F₁ offspring from the cross between a susceptible and a white rust resistant individual from the spring turnip rape material of Boreal Plant Breeding. The DNAs were extracted by the Dellaporta method (Dellaporta et al. 1983). The F₂ individuals were evaluated for resistance by examining the disease reaction of 20 seedlings from each self-pollinated individual. The cotyledons were inoculated 8 days after sowing with white rust strains 2 and 7. Plant reaction to the disease was recorded 10-14 days after inoculation. Plants were classified either resistant or susceptible.

RAPD primers (from Operon Technologies or self-synthesised) were tested for polymorphism between two bulks of nine individuals from the F₂ progeny: the susceptible bulk (individuals with susceptible offspring only) and the resistant bulk (individuals with the highest amount of resistant offspring). The amplification was carried out for 2 cycles of 30 s at 94°C, 30 s at 32°C and 1 min at 72°C and 33-43 cycles of 5 s at 94°C, 30 s at 34°C and 1 min at 72°C. The programme included an initial denaturation step of 3 min 30 s at 94°C and a final extension step of 6 min at 72°C. Primers generating polymorphic markers between the bulks were further used to amplify DNA from individual plants of each bulk.

The DNA regions used for the amplification of resistance gene analogs included a P loop motif (amino acid sequence GGVGKTT) of NBS and a second region (amino acid sequence GLPLAL) approximately 160 amino acids downstream. Two different primer pairs were used. The first one was designed to contain directly the DNA sequence of RPS2 (primers: GGT GGG GTT GGG AAG ACA ACG and CAA CGC TAG TGG CAA TCC). The second pair contained degenerate primers to the same region (primers: GGI GGI GTI GGI AAI ACI AC and IAG IGC IAG IGG IAG ICC. The PCR programme was as follows: 35 cycles of 30 s at 94°C, 30 s at annealing temperature and 1 min at 72°C preceded by an initial denaturation step and followed by a final extension step. The annealing temperature for the first primer pair was 64°C and for the second 52°C. The PCR fragments of the expected size (ca. 500 bp) were excised from the agarose gel, purified, ligated and cloned. Several clones from each transformation were picked randomly and sequenced with ALF DNA Sequencer or ALFexpress (Pharmacia).

RESULTS AND DISCUSSION

Twenty-three F₂ individuals were classified susceptible (0% resistant offspring). Instead, none of the F₂ plants was 100% resistant, but the amount of resistant offspring varied from 5 to 67%. There was a great deal of variation in sporulation intensity and rapidity among individual plants. This suggests that although one single major gene may control the resistance, other loci are involved in the control of sporulation.

So far, 409 RAPD primers have been tested for polymorphism between the bulks. Fifty-one primers produced polymorphic markers and they were tested on individual plants of the bulks, but either the markers did not amplify reproducibly or did not seem to be (tightly) linked to resistance.

The first primer pair designed for the amplification of RGAs produced one PCR fragment of the expected size from the resistant parent. Seventy-four clones from the transformation of the PCR product were sequenced. Most of the clones were similar, excluding some which contained 1-3 base substitutions. These substitutions most probably do not represent real RGAs, but are mistakes made by taq polymerase during amplification. Thus, with the first primer pair, we were only able to amplify the B. rapa gene corresponding to the RPS2 of Arabidopsis. The 506 bp sequence of B. rapa contained 62 base substitutions compared to the Arabidopsis sequence leading to 24 changes in the amino acid composition.

The degenerate primers amplified four different fragments, one of which corresponds to the 500bp fragment. The fragment(s) will be cloned and several clones sequenced to find the putative white rust resistance gene. The results will be discussed further in the poster.

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