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Resistance Gene Candidate2 (RGC2) genes belong to a large, highly duplicated family of nucleotide binding site-leucine rich repeat (NBS-LRR) encoding disease resistance genes located at a single locus in lettuce (Lactuca sativa). To investigate the genetic events occurring during the evolution of this locus, approximately 1.5- to 2-kb 3' fragments of 126 RGC2 genes from seven genotypes were sequenced from three species of Lactuca, and 107 additional RGC2 sequences were obtained from 40 wild accessions of Lactuca spp. The copy number of RGC2 genes varied from 12 to 32 per genome in the seven genotypes studied extensively. LRR number varied from 40 to 47; most of this variation had resulted from 13 events duplicating two to five LRRs because of unequal crossing-over within or between RGC2 genes at one of two recombination hot spots. Two types of RGC2 genes (Type I and Type II) were initially distinguished based on the pattern of sequence identities between their 3' regions. The existence of two types of RGC2 genes was further supported by intron similarities, the frequency of sequence exchange, and their prevalence in natural populations. Type I genes are extensive chimeras caused by frequent sequence exchanges. Frequent sequence exchanges between Type I genes homogenized intron sequences, but not coding sequences, and obscured allelic/orthologous relationships. Sequencing of Type I genes from additional wild accessions confirmed the high frequency of sequence exchange and the presence of numerous chimeric RGC2 genes in nature. Unlike Type I genes, Type II genes exhibited infrequent sequence exchange between paralogous sequences. Type II genes from different genotype/species within the genus Lactuca showed obvious allelic/orthologous relationships. Trans-specific polymorphism was observed for different groups of orthologs, suggesting balancing selection. Unequal crossover, insertion/deletion, and point mutation events were distributed unequally through the gene. Different evolutionary forces have impacted different parts of the LRR.

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

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

Genetic structure and diversity of natural populations of prickly lettuce (Lactuca serriola) were studied using AFLP markers and then compared with the diversity of the RGC2 disease resistance gene cluster. Screening of 696 accessions from 41 populations using 319 AFLP markers showed that eastern Turkish and Armenian populations were the most diverse populations and might be located in the origin and center of diversity of L. serriola. Screening 709 accessions using the microsatellite MSATE6 that is located in the coding region of most RGC2 homologs detected 366 different haplotypes. Again, the eastern Turkish and Armenian populations had the highest diversities at the RGC2 cluster. The diversities at the RGC2 cluster in different populations were significantly correlated with their genomewide diversities. There was significant variation of copy number of RGC2 homologs in different populations, ranging from 12 to 22 copies per genome. The nucleotide diversities of two conserved lineages (type II) of RGC2 genes (K and L) were not correlated with diversities calculated using the MSATE6 or AFLP data. We hypothesize that the high genomewide diversity and diversity of the RGC2 cluster in eastern Turkish and Armenian populations resulted from high abiotic and biotic stresses in the regions of origin of L. serriola.

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 sativaL. and three wild Lactuca spp., L. serriolaL., L. saligna and L. virosaL. 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.