Richard W. Michelmore

Development of reliable PCR-based markers linked to downy mildew resistance genes in lettuce

SummarySequence characterized amplified regions (SCARs) were derived from eight random amplified polymorphic DNA (RAPD) markers linked to disease resistance genes in lettuce. SCARs are PCR-based markers that represent single, genetically defined loci that are identified by PCR amplification of genomic DNA with pairs of specific oligonucleotide primers; they may contain high-copy, dispersed genomic sequences within the amplified region. Amplified RAPD products were cloned and sequenced. The sequence was used to design 24-mer oligonucleotide primers for each end. All pairs of SCAR primers resulted in the amplification of single major bands the same size as the RAPD fragment cloned. Polymorphism was either retained as the presence or absence of amplification of the band or appeared as length polymorphisms that converted dominant RAPD loci into codominant SCAR markers. This study provided information on the molecular basis of RAPD markers. The amplified fragment contained no obvious repeated sequences beyond the primer sequence. Five out of eight pairs of SCAR primers amplified an alternate allele from both parents of the mapping population; therefore, the original RAPD polymorphism was likely due to mismatch at the primer sites.

Genome-Wide Analysis of NBS-LRR–Encoding Genes in Arabidopsis

The Arabidopsis genome contains ∼200 genes that encode proteins with similarity to the nucleotide binding site and other domains characteristic of plant resistance proteins. Through a reiterative process of sequence analysis and reannotation, we identified 149 NBS-LRR–encoding genes in the Arabidopsis (ecotype Columbia) genomic sequence. Fifty-six of these genes were corrected from earlier annotations. At least 12 are predicted to be pseudogenes. As described previously, two distinct groups of sequences were identified: those that encoded an N-terminal domain with Toll/Interleukin-1 Receptor homology (TIR-NBS-LRR, or TNL), and those that encoded an N-terminal coiled-coil motif (CC-NBS-LRR, or CNL). The encoded proteins are distinct from the 58 predicted adapter proteins in the previously described TIR-X, TIR-NBS, and CC-NBS groups. Classification based on protein domains, intron positions, sequence conservation, and genome distribution defined four subgroups of CNL proteins, eight subgroups of TNL proteins, and a pair of divergent NL proteins that lack a defined N-terminal motif. CNL proteins generally were encoded in single exons, although two subclasses were identified that contained introns in unique positions. TNL proteins were encoded in modular exons, with conserved intron positions separating distinct protein domains. Conserved motifs were identified in the LRRs of both CNL and TNL proteins. In contrast to CNL proteins, TNL proteins contained large and variable C-terminal domains. The extant distribution and diversity of the NBS-LRR sequences has been generated by extensive duplication and ectopic rearrangements that involved segmental duplications as well as microscale events. The observed diversity of these NBS-LRR proteins indicates the variety of recognition molecules available in an individual genotype to detect diverse biotic challenges.

Plant NBS-LRR proteins: adaptable guards

The majority of disease resistance genes in plants encode nucleotide-binding site leucine-rich repeat (NBS-LRR) proteins. This large family is encoded by hundreds of diverse genes per genome and can be subdivided into the functionally distinct TIR-domain-containing (TNL) and CC-domain-containing (CNL) subfamilies. Their precise role in recognition is unknown; however, they are thought to monitor the status of plant proteins that are targeted by pathogen effectors.

Molecular Basis of Gene-for-Gene Specificity in Bacterial Speck Disease of Tomato

Transient expression of the Pseudomonas syringae avirulence gene avrPto in plant cells resulted in a Pto-dependent necrosis. The AvrPto avirulence protein was observed to interact directly with the Pto resistance protein in the yeast two-hybrid system. Mutations in the Pto and avrPto genes which reduce in vivo activity had parallel effects on association in the two-hybrid assay. These data suggest that during infection the pathogen delivers AvrPto into the plant host cell and that resistance is specified by direct interaction of Pto with AvrPto.

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.

Global eQTL Mapping Reveals the Complex Genetic Architecture of Transcript-Level Variation in Arabidopsis

The genetic architecture of transcript-level variation is largely unknown. The genetic determinants of transcript-level variation were characterized in a recombinant inbred line (RIL) population (n = 211) of Arabidopsis thaliana using whole-genome microarray analysis and expression quantitative trait loci (eQTL) mapping of transcript levels as expression traits (e-traits). Genetic control of transcription was highly complex: one-third of the quantitatively controlled transcripts/e-traits were regulated by cis-eQTL, and many trans-eQTL mapped to hotspots that regulated hundreds to thousands of e-traits. Several thousand eQTL of large phenotypic effect were detected, but almost all (93%) of the 36,871 eQTL were associated with small phenotypic effects (R2 < 0.3). Many transcripts/e-traits were controlled by multiple eQTL with opposite allelic effects and exhibited higher heritability in the RILs than their parents, suggesting nonadditive genetic variation. To our knowledge, this is the first large-scale global eQTL study in a relatively large plant mapping population. It reveals that the genetic control of transcript level is highly variable and multifaceted and that this complexity may be a general characteristic of eukaryotes.

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 ~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.

Multiple genetic processes result in heterogeneous rates of evolution within the major cluster disease resistance genes in lettuce.

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, ∼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.

Resistance Gene Candidates Identified by PCR with Degenerate Oligonucleotide Primers Map to Clusters of Resistance Genes in Lettuce

The recent cloning of genes for resistance against diverse pathogens from a variety of plants has revealed that many share conserved sequence motifs. This provides the possibility of isolating numerous additional resistance genes by polymerase chain reaction (PCR) with degenerate oligonucleotide primers. We amplified resistance gene candidates (RGCs) from lettuce with multiple combinations of primers with low degeneracy designed from motifs in the nucleotide binding sites (NBSs) of RPS2 of Arabidopsis thaliana and N of tobacco. Genomic DNA, cDNA, and bacterial artificial chromosome (BAC) clones were successfully used as templates. Four families of sequences were identified that had the same similarity to each other as to resistance genes from other species. The relationship of the amplified products to resistance genes was evaluated by several sequence and genetic criteria. The amplified products contained open reading frames with additional sequences characteristic of NBSs. Hybridization of RGCs to genomic DNA and to BAC clones revealed large numbers of related sequences. Genetic analysis demonstrated the existence of clustered multigene families for each of the four RGC sequences. This parallels classical genetic data on clustering of disease resistance genes. Two of the four families mapped to known clusters of resistance genes; these two families were therefore studied in greater detail. Additional evidence that these RGCs could be resistance genes was gained by the identification of leucine-rich repeat (LRR) regions in sequences adjoining the NBS similar to those in RPM1 and RPS2 of A. thaliana. Fluorescent in situ hybridization confirmed the clustered genomic distribution of these sequences. The use of PCR with degenerate oligonucleotide primers is therefore an efficient method to identify numerous RGCs in plants.

Multiple Paleopolyploidizations during the Evolution of the Compositae Reveal Parallel Patterns of Duplicate Gene Retention after Millions of Years

Of the approximately 250,000 species of flowering plants, nearly one in ten are members of the Compositae (Asteraceae), a diverse family found in almost every habitat on all continents except Antarctica. With an origin in the mid Eocene, the Compositae is also a relatively young family with remarkable diversifications during the last 40 My. Previous cytologic and systematic investigations suggested that paleopolyploidy may have occurred in at least one Compositae lineage, but a recent analysis of genomic data was equivocal. We tested for evidence of paleopolyploidy in the evolutionary history of the family using recently available expressed sequence tag (EST) data from the Compositae Genome Project. Combined with data available on GenBank, we analyzed nearly 1 million ESTs from 18 species representing seven genera and four tribes. Our analyses revealed at least three ancient whole-genome duplications in the Compositae-a paleopolyploidization shared by all analyzed taxa and placed near the origin of the family just prior to the rapid radiation of its tribes and independent genome duplications near the base of the tribes Mutisieae and Heliantheae. These results are consistent with previous research implicating paleopolyploidy in the evolution and diversification of the Heliantheae. Further, we observed parallel retention of duplicate genes from the basal Compositae genome duplication across all tribes, despite divergence times of 33-38 My among these lineages. This pattern of retention was also repeated for the paleologs from the Heliantheae duplication. Intriguingly, the categories of genes retained in duplicate were substantially different from those in Arabidopsis. In particular, we found that genes annotated to structural components or cellular organization Gene Ontology categories were significantly enriched among paleologs, whereas genes associated with transcription and other regulatory functions were significantly underrepresented. Our results suggest that paleopolyploidy can yield strikingly consistent signatures of gene retention in plant genomes despite extensive lineage radiations and recurrent genome duplications but that these patterns vary substantially among higher taxonomic categories.