LETTUCE BREEDING. MARCH 2015 Richard W. Michelmore, María José Truco, Oswaldo Ochoa, Pauline Sanders, Cayla Tsuchida, Lorena Parra and German Sandoya Objectives: 1) To produce advanced crisphead, romaine, green and red leaf and butterhead breeding lines which have resistance to multiple diseases, superior appearance and quality, high yielding ability, uniform maturity, and are slow bolting. 2) To determine the genetic inheritance of agriculturally important traits, particularly disease resistance. 3) To identify new genes for disease resistance in wild germplasm and incorporate them into advanced breeding lines. 4) To identify molecular markers linked to disease resistance genes to accelerate breeding. Procedures and Results: Lettuce Downy Mildew: Bremia lactucae, the causal pathogen of LDM, is highly variable and continuously evolving requiring regular monitoring of isolates. One-hundred-eighty-five isolates were sampled and characterized during 2014. The data is available at http://bremia.ucdavis.edu. Nearly half of the isolates had novel virulence phenotypes (46%); 29% were Pathotype CAVII, 9% were Pathotype CAIX and CAV, 6% were Pathotype CAVIII, and 2% were Pathotype CAVI. Cv. Balesta was resistant to 99 % of the isolates, followed by Dm17, Bellisimo, RYZ-2164, Dm38, and cv. RYZ-910457. Eighteen isolates were mating type B1. Of these, 50% had the same novel avirulence phenotype (Avr2, 7, 16, 17, 18, 36, 37, 38, d and e). Crosses between B1 and B2 isolates from the same field resulted in progeny with a wide range of novel phenotypes. The progeny from this and another cross are also segregating for metalaxyl resistance; if this resistance has a simple molecular basis, it may lead to a rapid PCR-based assay for metalaxyl resistance allowing decisions on whether to spray. We continue backcross programs to introgress resistance from new sources into iceberg, romaine, and leafy types. Genes from different sources are being introgressed into each type. We are genetically characterizing 13 putative new sources of resistance to LDM from wild donors that are resistant to the most isolates currently detected in California. We have sequenced 35 advanced breeding lines carrying these resistances. Comparing these sequences with the sequence of cultivar Salinas (L. sativa reference genome) allows the detection of putative genome regions associated with the introgression of the resistance (Figure 1). Additionally, we are sequencing 45 lettuce cultivars carrying known Dm genes to characterize them at the sequence level. This will allow the diagnosis of which Dm genes have been introgressed and are present in different cultivars. Detailed information of two Major Resistance Clusters (MRCs) is available at Christopoulou et al. (2015) MPMI http://dx.doi.org/10.1094. Figure 1. SNP density in two advanced breeding lines relative to the lettuce reference genome of cv. Salinas. A: Chromosome 1 for release UC02-105. The polymorphic introgressed segment corresponds to MRC 1. B: Chromosome 2 for release UC02-101. The polymorphic introgressed segment corresponds to MRC 2. Verticillium Wilt: Identification of resistance to wilt caused by Verticillium dahliae is one of our top priorities. We developed methods to screen germplasm for resistance in the greenhouse. So far over 400 germplasm accessions have been evaluated. Some lines had resistance to race 1; in collaboration with Ryan Hayes, we identified a major locus for this resistance that co-segregated with a gene similar to the Ve gene for resistance to Verticillium in tomato. Molecular markers are now available for this locus. None of the accessions screened are fully resistant to race 2. Seven lines from Armenia showed some resistant to Vdl 17 (race 2) with no seed transmission. Because development of disease symptoms is influenced by the transition to flowering, scoring for resistance is difficult if the population segregates for bolting. We therefore generated a RIL population by crossing the resistant L. serriola Arm09-170 with PI251246, a L. sativa oil type that bolts similarly to L. serriola and is highly susceptible to Vdl 17. We have tested 96 of the 240 RILs in 2014. We are currently genetically characterizing these 96 RIL lines to be able to conduct QTL analysis for resistance. This population is being retested for resistance to V. dahliae race 2 at USDA Salinas. This will allow us to compare the resistance of the RILs under different greenhouse conditions. In addition, we re-screened 14 accessions of new germplasm from Russia that had shown potential in earlier tests; however, none of the lines showed resistance in the repeated test. We increased seed of new germplasm from Georgia, Kirghizstan and Uzbekistan that we received last year and will test these lines for resistance this year. Fusarium wilt: In collaboration with Tom Gordon, we previously analyzed the progeny of a cross between cvs. Valmaine and Salinas for resistance to Fusarium wilt in infected field plots at UC Davis. Cv. Valmaine showed higher levels of resistance than cv. Salinas. QTL analysis revealed three loci conferring resistance to Fusarium, two resistant alleles originating from Valmaine and one from Salinas. We have analyzed five additional populations: Salinas x Green Towers, Lolla Rosa x Salinas, Lolla Rosa x Green Towers, Red Tide x Lolla Rosa x Red Tide and Red Tide x Salinas to study the genetic basis of resistance in these lines. None of the ~ 100 F2 individuals tested in the field from Lolla Rosa x Green Towers showed symptoms of disease indicating that resistance in these lines is allelic. Segregation on F3 families of crosses between Lolla Rosa and Salinas and between Salinas and Green Towers indicated one or two genes were responsible for the differences in resistance. We are constructing genetic maps in these two populations to genetically characterize the resistance further. 112 F2 individuals from the cross Lolla Rosa x Red Tide have been characterized with molecular markers to construct a genetic map with 132 markers. QTL analysis of the resistance on F3 families yielded QTLs for resistance on LG1, 4 and 8. These QTLs mapped in different genomic regions from the ones observed in the Valmaine x Salinas population indicating that several regions of the genome can contribute to resistance and it may be possible to pyramid genes to provide high levels of resistance. Last summer, we conducted field selections at the Fusarium infected field in UC Davis on lines deriving from the above mapping populations. We selected ~ 20 lines previously characterized as resistant from each population. However, timing was not optimal and some lines particularly iceberg types failed to set seed. Selection will be repeated this year. Releases: Further crosses and selections were made to generate lines with multiple disease resistances. Seed is available for the latest releases that are resistant to the most common but not all isolates of downy mildew.