Recombination and spontaneous mutation at the major cluster of resistance genes in lettuce (Lactuca sativa)

Two sets of overlapping experiments were conducted to examine recombination and spontaneous mutation events within clusters of resistance genes in lettuce. Multiple generations were screened for recombinants using PCR-based markers flanking Dm3. The Dm3 region is not highly recombinagenic, exhibiting a recombination frequency 18-fold lower than the genome average. Recombinants were identified only rarely within the cluster of Dm3 homologs and no crossovers within genes were detected. Three populations were screened for spontaneous mutations in downy mildew resistance. Sixteen Dm mutants were identified corresponding to spontaneous mutation rates of 10(-3) to 10(-4) per generation for Dm1, Dm3, and Dm7. All mutants carried single locus, recessive mutations at the corresponding Dm locus. Eleven of the 12 Dm3 mutations were associated with large chromosome deletions. When recombination could be analyzed, deletion events were associated with exchange of flanking markers, consistent with unequal crossing over; however, although the number of Dm3 paralogs was changed, no novel chimeric genes were detected. One mutant was the result of a gene conversion event between Dm3 and a closely related homolog, generating a novel chimeric gene. In two families, spontaneous deletions were correlated with elevated levels of recombination. Therefore, the short-term evolution of the major cluster of resistance genes in lettuce involves several genetic mechanisms including unequal crossing over and gene conversion.     

Sources and genetic structure of a cluster of genes for resistance to three pathogens in lettuce

The second largest cluster of resistance genes in lettuce contains at least two downy mildew resistance specificities, Dm5/8 and Dm10, as well as Tu, providing resistance against turnip mosaic virus, and plr, a recessive gene conferring resistance against Plasmopara lactucae-radicis, a root infecting downy mildew. In the present paper four additional genetic markers have been added to this cluster, three RAPD markers and one RFLP marker, CL1795. CL1795 is a member of a multigene family related to triose phosphate isomerase; other members of this family map to the other two major clusters of resistance genes in lettuce. Seven RAPD markers in the region were converted into sequence characterized amplified regions (SCARs) and used in the further analysis of the region and the mapping of Dm10. Three different segregating populations were used to map the four resistance genes relative to molecular markers. There were no significant differences in gene order or rate of recombination between the three crosses. This cluster of resistance genes spans 6.4 cM, with Dm10 1.2 cM from Dm8. Marker analysis of 20 cultivars confirmed multiple origins for Dm5/8 specificity. Two different Lactuca serriola origins for the Du5/8 specificity had previously been described and originally designated as either Dm5 or Dm8. Some ancient cultivars also had the same specificity. Previously, due to lack of recombination in genetic analyses and the same resistance specificities, it was assumed that Dm5 and Dm8 were determined by the same gene. However, molecular marker analysis clearly identified genotypes characteristic of each source. Therefore, Dm5/8 specificity is either ancient and widespread in L. serriola and some L. sativa, or else has arisen on multiple occasions as alleles at the same locus or at linked loci.     

The evolution of disease resistance genes

Several common themes have shaped the evolution of plant disease resistance genes. These include duplication events of progenitor resistance genes and further expansion to create clustered gene families. Variation can arise from both intragenic and intergenic recombination and gene conversion. Recombination has also been implicated in the generation of novel resistance specificities. Resistance gene clusters appear to evolve more rapidly than other regions of the genome. In addition, domains believed to be involved in recognitional specificity, such as the leucine-rich repeat (LRR), are subject to adaptive selection. Transposable elements have been associated with some resistance gene clusters, and may generate further variation at these complexes.     

Convergent evolution of disease resistance genes

The resistance genes Rpg1-b in soybean and RPM1 in Arabidopsis recognize the same bacterial avirulence protein (AvrB). Recent map-based cloning of Rpg1-b has provided the first opportunity to compare functionally analogous R genes in distantly related species. Rpg1-b and RPM1 are not orthologs. Rather, these genes descended from distinct evolutionary lineages in which recognition of AvrB has probably evolved independently. This result, together with new insights into RPM1-mediated recognition of AvrB, provides an exciting opportunity to reconsider classical views on the evolution of pathogen recognition specificity.     

Molecular diversity at the major cluster of disease resistance genes in cultivated and wild Lactuca spp.

Diversity was analyzed in wild and cultivated Lactuca germplasm using molecular markers derived from resistance genes of the NBS-LRR type. Three molecular markers, one microsatellite marker and two SCAR markers that amplified LRR-encoding regions, were developed from sequences of resistance gene homologs at the main resistance gene cluster in lettuce. Variation for these markers were assessed in germplasm including accessions of cultivated lettuce, Lactuca sativa L. and three wild Lactuca spp., L. serriola L., L. saligna and L. virosa L. Diversity was also studied within and between natural populations of L. serriola from Israel and California; the former is close to the center of diversity for Lactuca spp. while the latter is an area of more recent colonization. Large numbers of haplotypes were detected indicating the presence of numerous resistance genes in wild species. The diversity in haplotypes provided evidence for gene duplication and unequal crossing-over during the evolution of this cluster of resistance genes. However, there was no evidence for duplications and deletions within the LRR-encoding regions studied. The three markers were highly correlated with resistance phenotypes in L. sativa. They were able to discriminate between accessions that had previously been shown to be resistant to all known isolates of Bremia lactucae. Therefore, these markers will be highly informative for the establishment of core collections and marker-aided selection. A hierarchical analysis of the population structure of L. serriola showed that countries, as well as locations, were significantly differentiated. These differences may reflect local founder effects and/or divergent selection. Received: 7 March 1999 / Accepted: 25 March 1999     

Receptor-like genes in the major resistance locus of lettuce are subject to divergent selection.

Disease resistance genes in plants are often found in complex multigene families. The largest known cluster of disease resistance specificities in lettuce contains the RGC2 family of genes. We compared the sequences of nine full-length genomic copies of RGC2 representing the diversity in the cluster to determine the structure of genes within this family and to examine the evolution of its members. The transcribed regions range from at least 7.0 to 13.1 kb, and the cDNAs contain deduced open reading frames of approximately 5. 5 kb. The predicted RGC2 proteins contain a nucleotide binding site and irregular leucine-rich repeats (LRRs) that are characteristic of resistance genes cloned from other species. Unique features of the RGC2 gene products include a bipartite LRR region with >40 repeats. At least eight members of this family are transcribed. The level of sequence diversity between family members varied in different regions of the gene. The ratio of nonsynonymous (Ka) to synonymous (Ks) nucleotide substitutions was lowest in the region encoding the nucleotide binding site, which is the presumed effector domain of the protein. The LRR-encoding region showed an alternating pattern of conservation and hypervariability. This alternating pattern of variation was also found in all comparisons within families of resistance genes cloned from other species. The Ka /Ks ratios indicate that diversifying selection has resulted in increased variation at these codons. The patterns of variation support the predicted structure of LRR regions with solvent-exposed hypervariable residues that are potentially involved in binding pathogen-derived ligands.     

The major resistance gene cluster in lettuce is highly duplicated and spans several megabases.

At least 10 Dm genes conferring resistance to the oomycete downy mildew fungus Bremia lactucae map to the major resistance cluster in lettuce. We investigated the structure of this cluster in the lettuce cultivar Diana, which contains Dm3. A deletion breakpoint map of the chromosomal region flanking Dm3 was saturated with a variety of molecular markers. Several of these markers are components of a family of resistance gene candidates (RGC2) that encode a nucleotide binding site and a leucine-rich repeat region. These motifs are characteristic of plant disease resistance genes. Bacterial artificial chromosome clones were identified by using duplicated restriction fragment length polymorphism markers from the region, including the nucleotide binding site-encoding region of RGC2. Twenty-two distinct members of the RGC2 family were characterized from the bacterial artificial chromosomes; at least two additional family members exist. The RGC2 family is highly divergent; the nucleotide identity was as low as 53% between the most distantly related copies. These RGC2 genes span at least 3.5 Mb. Eighteen members were mapped on the deletion breakpoint map. A comparison between the phylogenetic and physical relationships of these sequences demonstrated that closely related copies are physically separated from one another and indicated that complex rearrangements have shaped this region. Analysis of low-copy genomic sequences detected no genes, including RGC2, in the Dm3 region, other than sequences related to retrotransposons and transposable elements. The related but divergent family of RGC2 genes may act as a resource for the generation of new resistance phenotypes through infrequent recombination or unequal crossing over.     

Structure and evolution of plant disease resistance genes

This article reviews recent advances that shed light on plant disease resistance genes, beginning with a brief overview of their structure, followed by their genomic organization and evolution. Plant disease resistance genes have been exhaustively investigated in terms of their structural organization, sequence evolution and genome distribution. There are probably hundreds of NBS-LRR sequences and other types of R-gene-like sequences within a typical plant genome. Recent studies revealed positive selection and selective maintenance of variation in plant resistance and defence-related genes. Plant resistance genes are highly polymorphic and have diverse recognition specificities. R-genes occur as members of clustered gene families that have evolved through duplication and diversification. These genes appear to evolve more rapidly than other regions of the genome, and domains such as the leucine-rich repeat, are subject to adaptive selection     

Multiple genome comparison within a bacterial species reveals a unit of evolution spanning two adjacent genes in a tandem paralog cluster

It has been assumed that an open reading frame (ORF) represents a unit of gene evolution as well as a unit of gene expression and function. In the present work, we report a case in which a unit comprising the 3' region of an ORF linked to a downstream intergenic region that is in turn linked to the 5' region of a downstream ORF has been conserved, and has served as the unit of gene evolution. The genes are tandem paralogous genes from the bacterium Staphylococcus aureus, for which more than ten entire genomes have been sequenced. We compared these multiple genome sequences at a locus for the lpl (lipoprotein-like) cluster (encoding lipoprotein homologs presumably related to their host interaction) in the genomic island termed nuSaalpha. A highly conserved nucleotide sequence found within every lpl ORF is likely to provide a site for homologous recombination. Comparison of phylogenies of the 5'-variable region and the 3'-variable region within the same ORF revealed significant incongruence. In contrast, pairs of the 3'-variable region of an ORF and the 5'-variable region of the next downstream ORF gave more congruent phylogenies, with distinct groups of conserved pairs. The intergenic region seemed to have coevolved with the flanking variable regions. Multiple recombination events at the central conserved region appear to have caused various types of rearrangements among strains, shuffling the two variable regions in one ORF, but maintaining a conserved unit comprising the 3'-variable region, the intergenic region, and the 5'-variable region spanning adjacent ORFs. This result has strong impact on our understanding of gene evolution because most gene lineages underwent tandem duplication and then diversified. This work also illustrates the use of multiple genome sequences for high-resolution evolutionary analysis within the same species.     

Bisexual branching processes in a genetic context: rates of growth for Y-linked genes

A multitype bisexual branching process is considered to model the behaviour of a Y-linked gene with two genotypes in a two-sex population. It is assumed perfect fidelity mating with preference of females for the males carrying certain allele of the gene. Under these assumptions, we study the rate of growth of each genotype on the event of non-extinction. The rate of growth of a genotype may depend on whether the other survives or becomes extinct and, in general, both genotype frequencies grow at different rates. We also investigate conditions for the simultaneous explosion of both genotypes to have positive or zero probability.     

Roles of selection intensity, major genes, and minor genes in evolution of insecticide resistance

A prominent hypothesis about insecticide resistance is that genes of major effect play a key role in field-evolved resistance because the intensity of selection is extremely high in the field. A corollary hypothesis is that the lower intensity of selection in laboratory selection experiments favors polygenic control of insecticide resistance. Contrary to these hypotheses, a literature review revealed that the intensity of selection for insecticide resistance in the field varies widely and overlaps broadly with selection intensities in the laboratory. Also contrary to these hypotheses, results from simulations of population genetic models suggest that selection intensities typical of laboratory selection experiments favor resistance that is conferred by major genes. Major genes dominated responses to selection for resistance across a wide range of simulated selection intensities, with and without fitness costs and refuges. The simulation results also suggest that the intensity of selection, rather than the number of loci conferring resistance, is central in determining rates of resistance evolution and effectiveness of refuges.     

Genome-wide investigation on the genetic variations of rice disease resistance genes

Exploitation of plant disease resistance (R) gene in breeding programs has been proven to be the most efficient strategy for coping with the threat of pathogens. An understanding of R-gene variation is the basis for this strategy. Here we report a genome-wide investigation on the variation of NBS-LRR-encoding genes, the common type of R genes, between two sequenced rice genomes, Oryza sativa L. var. Nipponbare and 93–11. We show that the allelic nucleotide diversity in 65.0% of 397 least-divergent pairs is not high (0.344% on average), while the remaining 35% display a greater diversity (5.4% on average). The majority of conserved R genes is single-copy and/or located as a singleton. The clustered, particularly the complex-clustered, R-genes contribute greatly to the rich genetic variation. Surprisingly only 11.2% of R-genes have remarkably high ratios of non-synonymous to synonymous rates, which is much less than the 17.4% observed between Arabidopsis genomes. Noticeable “artificially selective sweeping” could be detected in a large proportion of the conserved R-genes, a scenario described in the “arms race” co-evolutionary model. Based on our study, a variation pattern of R-genes is proposed and confirmed by the analysis of R-genes from other rice lines, indicating that the observed variation pattern may be common in all rice lines.Electronic Supplementary Material Supplementary material is available for this article at     

The genomic architecture of disease resistance in lettuce

Genbank and The Compositae Genome Project database, containing over 42,000 lettuce unigenes from Lactuca sativa cv. Salinas and L. serriola accession UC96US23 were mined to identify 702 candidate genes involved in pathogen recognition (RGCs), resistance signal transduction, defense responses, and disease susceptibility. In addition, to identify sequences representing additional sub-families of nucleotide binding site (NBS)-leucine-rich repeat encoding genes; the major classes of resistance genes (R-genes), NBS-encoding sequences were amplified by PCR using degenerate oligonucleotides designed to NBS sub-families specific to the subclass Asteridae, which includes the Compositae family. These products were cloned and sequenced resulting in 18 novel NBS sequences from cv. Salinas and 15 novel NBS sequences from UC96US23. Using a variety of marker technologies, 294 of the 735 candidate disease resistance genes were mapped in our primary mapping population, which consisted of 119 F₇ recombinant inbred lines derived from an interspecific cross between cv. Salinas and UC96US23. Using markers shared across multiple genetic maps, 36 resistance phenotypic loci, including two new loci for resistance to downy mildew and two quantitative trait loci for resistance to anthracnose were positioned onto the reference map to provide a global view of the genomic architecture of disease resistance in lettuce and to identify candidate genes for resistance phenotypes. The majority but not all of the resistance phenotypes were genetically associated with RGCs. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. An erratum to this article can be found at     

Variation in seedling susceptibility to Bremia lactucae (lettuce downy mildew) in the absence of effective major resistance genes

In the absence of effective major genes the importance of interactions between cultivar genotype, isolate genotype and environment was investigated in experiments using seedlings of eight lettuce cultivars inoculated with three isolates of Bremia lactucae and grown under a number of different environmental conditions. The outcome of the cultivar-isolate association was measured using four criteria and the data were examined by analysis of variance and correlation. The relative susceptibility of cultivars was generally independent of environment and there was no evidence that isolates were adapted to particular cultivars. Although significant cultivar X isolate interactions were found in individual experiments they were not consistent between experiments, even where these were conducted under apparently identical conditions. Variation resulted from either cultivar X environment or isolate X environment interaction with the environment always the dominant variable.     

Structure, function and evolution of plant disease resistance genes

Gene-for-gene plant disease resistance involves two basic processes: perception of pathogen attack, followed by responses to limit disease. Perception involves receptors with high degrees of specificity for pathogen strains, which are encoded by disease resistance genes. Large repertoires of distantly related resistance (R) genes with diverse recognitional specificities are found within a single plant species. The generation of R-gene polymorphism involves gene duplication, followed by DNA-sequence divergence by point mutation, and by deletion and duplication of intragenic DNA repeats encoding blocks of leucine-rich elements. Recombination between related genes reassorts this variation to further diversify gene sequences. Pathogen pressure selects functional resistance specificities and results in the maintenance of R-gene diversity. Recent genome-sequence data reveal that the NBS-LRR (i.e. nucleotide-binding site-leucine-rich repeat) class of R genes represents as much as 1% of the Arabidopsis genome. Experimental data have shown that the LRR has a role in determination of specificity. Mutation experiments, in which R-gene signaling has been dissociated from specificity in constitutive signal mutants, have provided the potential for non-specific resistance to be expressed from pathogen-infection-induced promoters in transgenic plants.     

Accelerating invasion rates result from the evolution of density-dependent dispersal

Evolutionary processes play an important role in shaping the dynamics of range expansions, and selection on dispersal propensity has been demonstrated to accelerate rates of advance. Previous theory has considered only the evolution of unconditional dispersal rates, but dispersal is often more complex. For example, many species emigrate in response to crowding. Here, we use an individual-based model to investigate the evolution of density dependent dispersal into empty habitat, such as during an invasion. The landscape is represented as a lattice and dispersal between populations follows a stepping-stone pattern. Individuals carry three ‘genes’ that determine their dispersal strategy when experiencing different population densities. For a stationary range we obtain results consistent with previous theoretical studies: few individuals emigrate from patches that are below equilibrium density. However, during the range expansion of a previously stationary population, we observe evolution towards dispersal strategies where considerable emigration occurs well below equilibrium density. This is true even for moderate costs to dispersal, and always results in accelerating rates of range expansion. Importantly, the evolution we observe at an expanding front depends upon fitness integrated over several generations and cannot be predicted by a consideration of lifetime reproductive success alone. We argue that a better understanding of the role of density dependent dispersal, and its evolution, in driving population dynamics is required especially within the context of range expansions.     

Organization,expression and evolution of a disease resistance gene cluster in soybean

PCR amplification was previously used to identify a cluster of resistance gene analogues (RGAs) on soybean linkage group J. Resistance to powdery mildew (Rmd-c), Phytophthora stem and root rot (Rps2), and an ineffective nodulation gene (Rj2) map within this cluster. BAC fingerprinting and RGA-specific primers were used to develop a contig of BAC clones spanning this region in cultivar "Williams 82" [rps2, Rmd (adult onset), rj2]. Two cDNAs with homology to the TIR/NBD/LRR family of R-genes have also been mapped to opposite ends of a BAC in the contig Gm_Isb001_091F11 (BAC 91F11). Sequence analyses of BAC 91F11 identified 16 different resistance-like gene (RLG) sequences with homology to the TIR/NBD/LRR family of disease resistance genes. Four of these RLGs represent two potentially novel classes of disease resistance genes: TIR/NBD domains fused inframe to a putative defense-related protein (NtPRp27-like) and TIR domains fused inframe to soybean calmodulin Ca(2+)-binding domains. RT-PCR analyses using gene-specific primers allowed us to monitor the expression of individual genes in different tissues and developmental stages. Three genes appeared to be constitutively expressed, while three were differentially expressed. Analyses of the R-genes within this BAC suggest that R-gene evolution in soybean is a complex and dynamic process.     

Induced deletions within a cluster of resistance genes in the mec region of the chromosome of Staphylococcus aureus

Variants of a methicillin-resistant Staphylococcus aureus showing loss of or reduced resistance to the antibiotic were isolated at frequencies of 0.1-100% from cultures which had been starved, grown at elevated temperature, or given small doses of UV radiation. Three types of variant were identified on the basis of population distribution of resistance to the antibiotic, and field-inversion gel electrophoresis of digests of the chromosome cut with the rare-cutting restriction endonuclease SmaI. Type I variants are methicillin-sensitive and have a deletion in the mec region of the chromosome. Type II variants have reduced methicillin resistance and rearranged DNA elsewhere in the chromosome. Type II variants show reduced methicillin resistance and no detectable change in the chromosome. Type I deletions were mapped using cloned fragments from the mec region. In 13 of the 16 independently isolated deletion mutants, one of the deletion endpoints appears to correlate with the positions of insertion sequences or transposons found in this region of the staphylococcal chromosome.