Resistance to Downy Mildew in Lettuce 'La Brillante' is Conferred by Dm50 Gene and Multiple QTL.

Many cultivars of lettuce (Lactuca sativa L.) are susceptible to downy mildew, a nearly globally ubiquitous disease caused by Bremia lactucae. We previously determined that Batavia type cultivar 'La Brillante' has a high level of field resistance to the disease in California. Testing of a mapping population developed from a cross between 'Salinas 88' and La Brillante in multiple field and laboratory experiments revealed that at least five loci conferred resistance in La Brillante. The presence of a new dominant resistance gene (designated Dm50) that confers complete resistance to specific isolates was detected in laboratory tests of seedlings inoculated with multiple diverse isolates. Dm50 is located in the major resistance cluster on linkage group 2 that contains at least eight major, dominant Dm genes conferring resistance to downy mildew. However, this Dm gene is ineffective against the isolates of B. lactucae prevalent in the field in California and the Netherlands. A quantitative trait locus (QTL) located at the Dm50 chromosomal region (qDM2.2) was detected, though, when the amount of disease was evaluated a month before plants reached harvest maturity. Four additional QTL for resistance to B. lactucae were identified on linkage groups 4 (qDM4.1 and qDM4.2), 7 (qDM7.1), and 9 (qDM9.2). The largest effect was associated with qDM7.1 (up to 32.9% of the total phenotypic variance) that determined resistance in multiple field experiments. Markers identified in the present study will facilitate introduction of these resistance loci into commercial cultivars of lettuce.

Identification of QTLs conferring resistance to downy mildew in legacy cultivars of lettuce.

Many cultivars of lettuce (Lactuca sativa L.), the most popular leafy vegetable, are susceptible to downy mildew disease caused by Bremia lactucae. Cultivars Iceberg and Grand Rapids that were released in the 18th and 19th centuries, respectively, have high levels of quantitative resistance to downy mildew. We developed a population of recombinant inbred lines (RILs) originating from a cross between these two legacy cultivars, constructed a linkage map, and identified two QTLs for resistance on linkage groups 2 (qDM2.1) and 5 (qDM5.1) that determined resistance under field conditions in California and the Netherlands. The same QTLs determined delayed sporulation at the seedling stage in laboratory experiments. Alleles conferring elevated resistance at both QTLs originate from cultivar Iceberg. An additional QTL on linkage group 9 (qDM9.1) was detected through simultaneous analysis of all experiments with mixed-model approach. Alleles for elevated resistance at this locus originate from cultivar Grand Rapids.

Association mapping and marker-assisted selection of the lettuce dieback resistance gene Tvr1.

BACKGROUND: Lettuce (Lactuca saliva L.) is susceptible to dieback, a soilborne disease caused by two viruses from the family Tombusviridae. Susceptibility to dieback is widespread in romaine and leaf-type lettuce, while modern iceberg cultivars are resistant to this disease. Resistance in iceberg cultivars is conferred by Tvr1 - a single, dominant gene that provides durable resistance. This study describes fine mapping of the resistance gene, analysis of nucleotide polymorphism and linkage disequilibrium in the Tvr1 region, and development of molecular markers for marker-assisted selection. RESULTS: A combination of classical linkage mapping and association mapping allowed us to pinpoint the location of the Tvr1 resistance gene on chromosomal linkage group 2. Nine molecular markers, based on expressed sequence tags (EST), were closely linked to Tvr1 in the mapping population, developed from crosses between resistant (Salinas and Salinas 88) and susceptible (Valmaine) cultivars. Sequencing of these markers from a set of 68 cultivars revealed a relatively high level of nucleotide polymorphism (theta = 6.7 x 10-3) and extensive linkage disequilibrium (r(2) = 0.124 at 8 cM) in this region. However, the extent of linkage disequilibrium was affected by population structure and the values were substantially larger when the analysis was performed only for romaine (r(2) = 0.247) and crisphead (r(2) = 0.345) accessions. The association mapping approach revealed that one of the nine markers (Cntg10192) in the Tvr1 region matched exactly with resistant and susceptible phenotypes when tested on a set of 200 L. sativa accessions from all horticultural types of lettuce. The marker-trait association was also confirmed on two accessions of Lactuca serriola - a wild relative of cultivated lettuce. The combination of three single-nucleotide polymorphisms (SNPs) at the Cntg10192 marker identified four haplotypes. Three of the haplotypes were associated with resistance and one of them was always associated with susceptibility to the disease. CONCLUSION: We have successfully applied high-resolution DNA melting (HRM) analysis to distinguish all four haplotypes of the Cntg10192 marker in a single analysis. Marker-assisted selection for dieback resistance with HRM is now an integral part of our breeding program that is focused on the development of improved lettuce cultivars.

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.

The disease resistance gene Dm3 is infrequent in natural populations of Lactuca serriola due to deletions and frequent gene conversions at the RGC2 locus.

Resistance genes can exhibit heterogeneous patterns of variation. However, there are few data on their frequency and variation in natural populations. We analysed the frequency and variation of the resistance gene Dm3, which confers resistance to Bremia lactucae (downy mildew) in 1033 accessions of Lactuca serriola (prickly lettuce) from 49 natural populations. Inoculations with an isolate of Bremia lactucae carrying avirulence gene Avr3 indicated that the frequency of Dm3 in natural populations of L. serriola was very low. Molecular analysis demonstrated that Dm3 was present in only one of the 1033 wild accessions analysed. The sequence of the 5' region of Dm3 was either highly conserved among accessions, or absent. In contrast, frequent chimeras were detected in the 3' leucine-rich repeat-encoding region. Therefore low frequency of the Dm3 specificity in natural populations was due to either the recent evolution of Dm3 specificity, or deletions of the whole gene as well as variation in 3' region caused by frequent gene conversions. This is the most extensive analysis of the prevalence of a known disease resistance gene to date, and indicates that the total number of resistance genes in a species may be very high. This has implications for the scales of germplasm conservation and exploitation of sources of resistance.

Insensitivity to the Fungicide Fosetyl-Aluminum in California Isolates of the Lettuce Downy Mildew Pathogen, Bremia lactucae.

Lettuce downy mildew, caused by Bremia lactucae, is the most important foliar disease of lettuce in California. In recent years, there were apparent failures of fungicides containing fosetyl-aluminum (Aliette) to control downy mildew in commercial lettuce fields in California. Consequently, we characterized 134 isolates collected over 2 years from throughout the coastal growing areas of California for insensitivity to the fungicides fosetyl-aluminum and maneb, pathotype, and mating type. Tests using seedlings in controlled growth room conditions demonstrated the widespread occurrence of insensitivity to fosetyl-aluminum in California populations of B. lactucae. Fifty percent of the isolates assayed sporulated profusely in the presence of fosetyl-aluminum applied at rates twice the normal field dosage, and an additional 40% showed moderate sporulation at this rate. Fosetyl-aluminum-insensitive isolates were detected from all regions sampled. Insensitivity was also observed in multiple pathotypes. Insensitivity was not complete, however, because quantitative analysis of the number of lesions on older plants revealed that applications of fosetyl-aluminum could reduce the levels of disease by 50%. Therefore, while fosetyl-aluminum may have utility under low disease pressure in the field, other control measures are required to provide control under conditions favorable to the disease.

SNP-based codominant markers for a recessive gene conferring resistance to corky root rot (Rhizomonas suberifaciens) in lettuce (Lactuca sativa).

The analysis of F2 progeny and derived F3 families of Lactuca sativa segregating for resistance to corky root rot caused by Rhizomonas suberifaciens permitted the identification of restriction fragment length polymorphism (RFLP) and single nucleotide polymorphism (SNP) markers linked to the recessive resistance gene cor. PCR-based markers were identified by bulked segregant analysis (BSA). Allele-specific primers were generally designed with the 3 terminal base coinciding with an SNP, matching one of the alleles and mismatching the other, and with an additional subterminal 3 base mismatching both alleles. Codominant, robust, and inexpensive molecular markers were obtained that used standardized PCR conditions. Some of the markers could be analyzed in multiple Lactuca mapping populations that did not segregate for disease resistance allowing the cor locus to be located on several maps. The consistent low density of markers around cor in these maps suggests that cor may be in an area with an elevated rate of recombination. Evaluation of these markers in a large sample of cultivars and landraces identified pairs of flanking polymorphic markers that can be used for marker-assisted selection of corky root resistance.

Dm3 is one member of a large constitutively expressed family of nucleotide binding site-leucine-rich repeat encoding genes.

The major cluster of resistance genes in lettuce cv. Diana contains approximately 32 nucleotide binding site-leucine-rich repeat encoding genes. Previous molecular dissection of this complex region had identified a large gene, RGC2B, as a candidate for encoding the downy mildew resistance gene, Dm3. This article describes genetic and transgenic complementation data that demonstrated RGC2B is necessary and sufficient to confer resistance with Dm3 specificity. Ethylmethanesulphonate was used to induce mutations to downy mildew susceptibility in cv. Diana (Dm1, Dm3, Dm7, and Dm8). Nineteen families were identified with a complete loss of resistance in one of the four resistance specificities. Sequencing revealed a variety of point mutations in RGC2B in the six dm3 mutants. Losses of resistance were due to single changes in amino acid sequence or a change in an intron splice site. These mutations did not cluster in any particular region of RGC2B. A full-length genomic copy of RGC2B was isolated from a lambdaphage library and introduced into two genotypes of lettuce. Transgenics expressing RGC2B exhibited resistance to all isolates expressing Avr3 from a wide range of geographical origins. In a wildtype Dm3-expressing genotype, many of the RGC2 family members are expressed at low levels throughout the plant.