Homologues of a single resistance-gene cluster in potato confer resistance to distinct pathogens: a virus and a nematode.

The isolation of the nematode-resistance gene Gpa2 in potato is described, and it is demonstrated that highly homologous resistance genes of a single resistance-gene cluster can confer resistance to distinct pathogen species. Molecular analysis of the Gpa2 locus resulted in the identification of an R-gene cluster of four highly homologous genes in a region of approximately 115 kb. At least two of these genes are active: one corresponds to the previously isolated Rx1 gene that confers resistance to potato virus X, while the other corresponds to the Gpa2 gene that confers resistance to the potato cyst nematode Globodera pallida. The proteins encoded by the Gpa2 and the Rx1 genes share an overall homology of over 88% (amino-acid identity) and belong to the leucine-zipper, nucleotide-binding site, leucine-rich repeat (LZ-NBS-LRR)-containing class of plant resistance genes. From the sequence conservation between Gpa2 and Rx1 it is clear that there is a direct evolutionary relationship between the two proteins. Sequence diversity is concentrated in the LRR region and in the C-terminus. The putative effector domains are more conserved suggesting that, at least in this case, nematode and virus resistance cascades could share common components. These findings underline the potential of protein breeding for engineering new resistance specificities against plant pathogens in vitro.

Inheritance of resistance to beet necrotic yellow vein virus in Beta vulgaris conferred by a second gene for resistance.

Rhizomania is a serious disease of sugar beet, caused by beet necrotic yellow vein virus (BNYVV). The disease can only be controlled by the use of resistant cultivars. The accession Holly contains a single dominant gene for resistance, called Rz. The identification of a locus for resistance that differs from Rz would provide possibilities to produce cultivars with multiple resistance to BNYVV. Inheritance of resistance to BNYVV was studied by screening progenies of crosses between resistant plants of the accessions Beta vulgaris subsp. maritima WB42 and B. vulgaris subsp. vulgaris Holly-1-4 or R104. Observed and expected segregation ratios were compared to elucidate whether the resistance genes in the three accessions are alleles or situated on different loci. STS markers, linked to the genes for resistance, were used to study the segregation in more detail. The results demonstrated that the genes for resistance to BNYVV inHolly-1-4 and WB42 are closely linked. The gene for resistance in R104 is at the same locus as in Holly-1-4, and also closely linked to the gene in WB42. As the Holly resistance gene has been named Rz, the name Rz2 is proposed to refer to the resistance gene in WB42. Consequently, the gene Rz should be referred to as Rz1.

Positional cloning of a gene for nematode resistance in sugar beet.

The Hs1(pro-1) locus confers resistance to the beet cyst nematode (Heterodera schachtii Schmidt), a major pest in the cultivation of sugar beet (Beta vulgaris L.). The Hs1(pro-1) gene was cloned with the use of genome-specific satellite markers and chromosomal break-point analysis. Expression of the corresponding complementary DNA in a susceptible sugar beet conferred resistance to infection with the beet cyst nematode. The native Hs1(pro-1) gene, expressed in roots, encodes a 282-amino acid protein with imperfect leucine-rich repeats and a putative membrane-spanning segment, features similar to those of disease resistance genes previously cloned from higher plants.

Identification and mapping of random amplified polymorphic DNA (RAPD) markers linked to resistance against beet necrotic yellow vein virus (BNYVV) in Beta accessions.

Molecular markers linked to resistance genes are useful to facilitate the introgression of one or more of these genes in breeding materials. Following the approach of bulked segregant analysis, RAPD markers linked to resistance genes against beet necrotic yellow vein virus were identified in the four Beta accessions Holly-1-4, R104, R128 and WB42. Two primers were found which generate RAPD markers tightly linked to resistance in segregating families of Holly-1-4, R104 and R128, indicating that the resistance genes in these accessions might be situated at the same locus. Other, specific, primers were identified which generate RAPD markers linked to resistance in each of these accessions. Short-range maps were established around the resistance locus in these accessions. For WB42, RAPD markers were only identified at a relatively large distance from the resistance gene. Conversion of three RAPD primers of Holly-1-4, R104 and R128 into STS primers resulted in STS markers which can be readily used for marker-assisted selection in breeding programmes.

Selection of monosomic addition plants in offspring families using repetitive DNA probes in Beta L.

The distribution of two repetitive DNA probes Sat-121 and PB6-4, specific for the section Procumbentes of the genus Beta, was tested in 16 B. patellaris monosomic addition families using a dot-blot hybridization procedure. All monosomic additions were accurately distinguished from diploid sib plants with both DNA probes. The probe PB6-4, with the strongest signal after hybridization, was selected for rapid screening of an extensive number of putative monosomic additions in B. patellaris or B. procumbens addition families using a squash-blot hybridization procedure. The probe PB6-4 detected 118 monosomic additions in 640 plants (18.4%) in eight different B. procumbens addition families. The addition family with chromosome 4 of B. procumbens was semi-lethal and could not be tested. The distribution of PB6-4 in B. patellaris addition families was confirmed in 63 addition families using the squash-blot procedure. In 4580 plants of these addition families, 628 individual monosomic additions (13.7%) were found. The relationship of the morphological characteristics of monosomic addition plants to the results of the squash-blot hybridization (plants with signal) using probe PB6-4 is quite rigorous but not complete. The correlation between plants with a signal and chromosome number (2n=19) is complete. These results indicate that sequences present on PB6-4 are probably present on all chromosomes of B. patellaris and B. procumbens. The possibility of utilizing the sequence information of Sat-121 for a PCR-based assay to screen for putative monosomic addition plants was also investigated as an alternative to chromosome counting. The DNA-amplification profiles using the primers REP and REP.INV clearly distinguished monosomic addition plants from their diploid sibs.

Isolation and characterization of RAPD-based markers linked to the beet cyst nematode resistance locus (Hs1 (pat-1)) on chromosome 1 of B. patellaris.

A beet cyst nematode (BCN)-resistant telosomic addition of B. patellaris chromosome 1 in B. vulgaris was used to isolate 6 RAPD markers linked to the BCN resistance locus Hs1 (pat-1). Southern analysis showed that the analyzed RAPD products contain either low-, middle or high-repetitive DNA. The relative positions of the random amplified polymorphic DNA (RAPD) markers and of the restriction fragment length polymorphism (RFLP) loci corresponding to the low-repetitive RAPD products were determined by deletion mapping using a panel of seven nematode-resistant B. patellaris chromosome-1 fragment additions. One RAPD marker, OPB11800, was found to be present in two copies on the long arm telosome of B. patellaris chromosome 1. These copies are closely linked to the BCN resistance gene and flank the gene on both sides. On the basis of the nucleotide sequence of OPB11800, sequence-tagged site (STS) primers were developed that amplify specific fragments derived from the two OPB11800 loci. These STS markers can be used in the map-based cloning of the BCN gene, as they define start and finishing points of a chromosomal walk towards the Hs1 (pat-1) locus. Two copies of the middle-repetitive OPX21100 marker were mapped in the same interval of the deletion mapping panel as the resistance gene locus and thereby belong to the nearest markers as yet found for the BCN gene in B. patellaris.

Construction of a YAC library from a Beta vulgaris fragment addition and isolation of a major satellite DNA cluster linked to the beet cyst nematode resistance locus Hs1 (pat-1.).

A YAC library was constructed from the Beta vulgaris fragment addition AN5-203b. This monosomic fragment addition harbors an approximate 12-Mbp fragment of B.patellaris chromosome 1 accomodating the Hs1 (pat-1) conferring resistance to the beet cyst nematode (Heterodera schachtii). The YAC library consists of 20,000 YAC clones having an average size of 140 kb. Screening with organelle-specific probes showed that 12% of the clones contain chloroplast DNA while only 0.2% of the clones hybridizes with a mitochondrial specific probe. On the basis of a sugar beet haploid genome size of 750 Mbp this library represents 3.3 haploid genome equivalents. The addition fragment present in AN5-203b harbors a major satellite DNA cluster that is tightly linked to the Hs1 (pat-1) locus. The cluster is located on a single 250-kb EcoRI restriction fragment and consists of an estimated 700-800 copies of a 159-bp core sequence, most of which are arranged in tandem. Using this core sequence as a probe, we were able to isolate 1 YAC clone from the library that contains the entire 250-kb satellite DNA cluster.

Long-range organization of a satellite DNA family flanking the beet cyst nematode resistance locus (Hs1) on chromosome-1 of B. patellaris and B. procumbens.

New members of a satellite DNA family (Sat 121), specific for wild beets of the section Procumbentes of the genus Beta, were isolated. Sequence analysis showed that the members of Sat-121 fall into two distinct classes. The organization of Sat-121 in the vicinity of a beet cyst nematode (Heterodera schachtii Schm.) resistance locus (Hs1) in B. patellaris and B. procumbens was investigated by pulsed-field gel electrophoresis (PFGE) using DNA from a series of resistant monosomic fragment additions, each containing an extra chromosome fragment of B. patellaris chromosome-1 (pat-1) in B. vulgaris. In this way several clusters of Sat-121 flanking the Hs1 (pat-1) locus were identified. In nematode resistant diploid introgressions (2n=18), which contain small segments of B. procumbens chromosome-1 (pro-1) in B. vulgaris, only two major Sat-121 clusters were detected near the Hs1 (pro-1) locus.

Isolation of molecular markers for tomato (L. esculentum) using random amplified polymorphic DNA (RAPD).

A new DNA polymorphism assay was developed in 1990 that is based on the amplification by the polymerase chain reaction (PCR) of random DNA segments, using single primers of arbitrary nucleotide sequence. The amplified DNA fragments, referred to as RAPD markers, were shown to be highly useful in the construction of genetic maps ("RAPD mapping"). We have now adapted the RAPD assay to tomato. Using a set of 11 oligonucleotide decamer primers, each primer directed the amplification of a genome-specific "fingerprint" of DNA fragments. The potential of the original RAPD assay to generate polymorphic DNA markers with a given set of primers was further increased by combining two primers in a single PCR. By comparing "fingerprints" of L. esculentum, L. pennellii, and the L. esculentum chromosome 6 substitution line LA1641, which carries chromosome 6 from L. pennellii, three chromosome 6-specific RAPD markers could be directly identified among the set of amplified DNA fragments. Their chromosomal position on the classical genetic map of tomato was subsequently established by restriction fragment length polymorphism (RFLP) linkage analysis. One of the RAPD markers was found to be tightly linked to the nematode resistance gene Mi.

RFLP markers linked to the root knot nematode resistance gene Mi in tomato.

The Mi gene originating from the wild tomato species Lycopersicon peruvianum confers resistance to all major root knot nematodes (Meloidogyne spp.). This single dominant gene is located on chromosome 6 and is very closely linked to the acid phosphatase-1 (Aps-1) locus. Resistance to nematodes has been introgressed into various cultivars of the cultivated tomato (L. esculentum), in many cultivars along with the linked L. peruvianum Aps-1 (1) allele. By using a pair of nearly isogenic lines differing in a small chromosomal region containing the Mi and Aps-1 loci, we have identified two RFLP markers, GP79 and H6A2c2, which are located in the introgressed L. peruvianum region. Analysis of a test panel of 51 L. esculentum genotypes of various origins indicated that GP79 is very tightly linked to the Mi gene and allows both homozygous and heterozygous nematode-resistant genotypes to be distinguished from susceptible genotypes, irrespective of their Aps-1 alleles. Marker H6A2c2 is linked to the Aps-1 locus and is capable of discriminating between the L. peruvianum Aps-1 (1) allele and the L. esculentum Aps-1 (3) and Aps-1 (+) alleles. In combination, these RFLP markers may provide a powerful tool in breeding tomatoes for nematode resistance.