The water had a salinity of 3.5 psu and the temperature during measurements was either 24 or 27◦C, which was taken into account for calibration. We used Clark-type oxygen microsensors , and calibrated before and after measurements in air-saturated and anaerobic 3.5% NaCl. The micro-profiling apparatus and software Sensor Trace Suite was also provided from Unisense. Starting at the water surface, microprofiles through individual aggregates were measured in the light, and light-dark shifts. Time-lapse lightdark-shift recordings included sensor tips placed directly above the aggregate ; and inserting the tip into the central core of the aggregate. Recordings of the oxygen signal were taken every second. Light sources were 65 W halogen lamps and experiments were conducted with either 170 µmol photons m−2 s −1 , or 320 µmol photons m−2 s −1 . Dark conditions were realized by switching off the lamps, removing them from the table, and carefully placing a carton box over the entire profiling setup to avoid residual light from the room. Background light intensities under the box were < 1 µmol m−2 s −1 . Theoretical limits of oxygen and DIC flux and whole aggregate O2 flux calculations were calculated from depth concentration profiles according to Ploug et al. , with a diffusion coefficient for O2 in 3.5% saline water of 2.175 × 10−5 cm2 s −1 at 24◦C and 2.3535 × 10−5 cm2 s −1 at 27◦C. Inside the aggregate,square flower bucket the apparent diffusivity of O2 was assumed to be 0.95 . Carbon fixation was estimated based on a photosynthetic quotient of 1.2 .

The diffusion of oxygen in agar was not found to be different than in water over a wide range of salinities . Fusarium oxysporum, a widespread soil-borne pathogen, causes vascular wilt disease in several economically important plants , in addition to the broad spectrum human disease known as ‘fusariosis’ . F. oxysporum is one of the most destructive plant-pathogenic fungi worldwide, with a long and storied history of outbreaks and epidemics that have caused signifcant production losses and disrupted food and fber production . One of the earliest reports of the disease arose from outbreaks on banana in the late 1800s that progressively annihilated the widely grown susceptible cultivar ‘Gros Michel’, forced the abandonment of export plantations, and caused a gradual, albeit inexorable shift in production from susceptible ‘Gros Michel’ to resistant ‘Cavendish’ cultivars . Similar production shifts have unfolded over the last century in tomato , cotton , and other economically important plants , and more recently strawberry . The discovery of sources of resistance and development and deployment of resistant cultivars has been critical for limiting disease losses and sustaining agricultural production in strawberry and other host plants affected by the pathogen . Fusarium wilt of strawberry is caused by F. oxysporum f. sp. fragariae , one of more than 100 documented host-specific pathogens , many of which have been widely disseminated . Although the strawberry-specific Fof has been reported in many countries, the disease has been most widely reported and studied in Japan, South Korea, Australia, and California, between which virulent strains have been disseminated . Fusarium wilt was first reported on strawberry in Australia in the 1960s , and was not reported on strawberry in California until the mid-2000s . The disease has been aggressively spreading and poses a serious threat to production in California .

Fusarium wilt has not yet become a serious threat to production everywhere strawberries are grown; however, there is a significant risk of virulent strains being disseminated through global trade, and the ever present danger of the evolution and emergence of virulent races of the pathogen that defeat known resistance genes . One of the motivations for the present study was to prepare for that inevitability by delving more deeply into the genetics of resistance and developing the resources and knowledge needed to accelerate the development of Fusarium wilt resistant cultivars through marker-assisted selection . To that end, we initiated studies in 2015 to identify sources of resistance to California isolates of the pathogen and shed light on the genetics of resistance to Fusarium wilt in strawberry . The prevalence, diversity, strength of resistance, and genetic mechanisms underlying resistance to Fusarium wilt were unknown when those studies were initiated . Significant insights into the Fragaria-Fusarium pathosystem have since emerged. Pincot et al. identified multiple sources of resistance to Fusarium wilt in a closed breeding population developed at the University of California, Davis . The isolate they used was subsequently classified as Fof race 1 . From the resistance phenotypes of plants artificially inoculated with AMP132, they observed a nearly bimodal distribution of resistant and susceptible individuals in a genome-wide association study of the California population, observed near-Mendelian distributions for resistance phenotypes in segregating populations, and showed that resistance to AMP132 was conferred by a single dominant gene in the California population. The resistant allele had a low frequency and was only homozygous in 3% of the resistant individuals in the California population .

From analyses of pedigree records and haplotypes of SNP markers in linkage disequilibrium with the FW1 locus, Pincot et al. predicted that 99% of the resistant individuals in the California population carried FW1. They concluded that the resistant allele had fortuitously survived early breeding bottlenecks and originated in the earliest known ancestors of the California population . Pincot et al. screened two non-California cultivars , both of which were shown to be resistant to race 1 and had SNP marker haplotypes diferent from the FW1 SNP marker haplotype. The only AMP132-resistant cultivar in the California population without the FW1 SNP marker haplotype was the heirloom cultivar ‘Wiltguard’. We speculated that Earliglow, Guardian, and Wiltguard might carry novel R-genes, a hypothesis tested in the present study. To build on earlier findings in the California population and develop a deeper understanding of the genetics of resistance, we screened a diverse collection of elite and exotic germplasm accessions for resistance to race 1 and selected several additional race 1 resistant donors for further study. Here, we show that resistance to race 1 is widespread in elite and exotic germplasm, including geographically diverse ecotypes of the wild octoploid progenitors of strawberry . Plant genes that confer strong race-specific resistance frequently encode proteins with nucleotide-binding leucine-rich repeat domains or surface localized pattern recognition receptors . Several of the previously described Fusarium wilt R-genes encode proteins with NLR and PRR architecture . R-genes that confer resistance to F. oxysporum f. sp. lycopersici in tomato are among the most well studied examples . PRRs are capable of recognizing conserved pathogen features and extracellular effectors, while NLR receptors recognize secreted pathogen effectors inside plant cells, resulting in disease resistance . Although the gene encoded by FW1 has not yet been identified, we posited that FW1 might encode an NLR or PRR immune receptor protein that recognizes an effector protein encoded by Fof race 1 isolates . Because R-genes often have short-lived utility , the continual discovery and deployment of novel R-genes has been critical for keeping pace with the evolution of pathogen races in the gene-for-gene ‘arms race’ . The durability of FW1 and other race-specific R-genes is uncertain , and depends on the speed of emergence of novel Fof races through pathogen mutation . If FW1 encodes an NLR or PRR, a mutation of AvrFW1 could lead to an evasion of host immune perception and regained pathogenicity . Currently, only race 1 isolates of Fof have been found in California,black flower bucket and none cause disease in cultivars carrying the dominant FW1 allele . However, race 2 isolates that cause disease on FW1-carrying cultivars have been observed . The identification of race 2 reinforces the expectation that novel strains of the pathogen could eventually evolve and defeat defeat race 1 R-genes through mutation, loss, or expression polymorphism in AvrFW1. The identification of FW1 and AvrFW1 and advances in the development of genomic resources for Fragaria and Fusarium laid the foundation for the present study. FW1 was originally discovered by GWAS using a diploid reference genome .

The approximate location of FW1 in the octoploid genome was subsequently ascertained by genetic mapping in octoploid segregating populations genotyped with a single nucleotide polymorphsim array designed with probe DNA sequences anchored to a diploid reference genome . The octoploid genome has since been sequenced , thereby opening the way for octoploid genome-informed breeding and genetic studies in strawberry. Those genome assemblies supplied the foundation for several additional technical advances, the most important of which were the genome-wide discovery and physical and genetic mapping of millions of DNA variants in the octoploid genome, the development of 50K and 850K SNP genotyping arrays with probe DNA sequences uniformly distributed and anchored to physical positions throughout the octoploid genome, and telomere-to-telomere resolution of the A, B, C, and D subgenomes of octoploid strawberry . These breakthroughs and resources were critical for the present study, which included: pinpointing the genomic location of the FW1 locus and four newly discovered Fusarium wilt resistance loci ; expanding the database of octoploid germplasm accessions screened for resistance to Fusarium wilt races 1 and 2; identifying SNPs and other DNA variants in linkage disequilibrium with FW1–FW5; and identifying plausible candidate genes for FW1–FW5 through genotype-to-phenotype associations. Finally, we describe high-throughput genotyping assays for SNPs in strong LD with FW1–FW5 to facilitate the development of Fusarium wilt resistant cultivars through MAS.The plant materials for our studies included 309 F. × ananassa, 62 F. chiloensis, and 40 F. virginiana germplasm accessions preserved in the University of California, Davis Strawberry Germplasm Collection or the United States Department of Agriculture, Agricultural Research Service, National Plant Germplasm System , National Clonal Germplasm Repository, Corvallis, Oregon . The original ‘mother’ plants of individuals acquired from the USDA were asexually multiplied in a Winters, CA field nursery and preserved in the UC Davis Strawberry Germplasm Collection throughout the course of our studies . Bare-root plants of every individual were produced by asexual multiplication in high-elevation field nurseries in Dorris, CA from mother plants propagated in low-elevation field nurseries in Winters, CA. The mother plants were planted mid-April and daughter plants were harvested and trimmed in mid-October and stored in plastic bags at 3.5 °C for two to three weeks before pathogen inoculation and planting. The daughter plants for growth chamber and greenhouse experiments were stored at –2.2 °C for 5 to 27 weeks and ultimately thawed and stored at 3.5 °C for one to three days prior to pathogen inoculation and planting. S1 families were developed by self-pollinating three Fusarium wilt race 1 resistant F. × ananassa cultivars identified by Pincot et al. : Guardian , Wiltguard , and Earliglow . An S1 family was developed by self-pollinating a resistant individual we identified in a population developed by crossing the susceptible cultivar Cabrillo with the resistant F. virginiana subsp. glauca ecotype PI612500. An S2 family was developed by self-pollinating 61S016P006, a highly resistant S1 individual identified in our resistance screening study. These individuals were known from genome-wide DNA profiling to be highly heterozygous and predicted a priori to either be heterozygous or homozygous for alleles affecting resistance. We developed interspecific full-sib families by crossing a susceptible F. × ananassa parent with race 1 resistant ecotypes of Fragaria virginiana subsp. virginiana , Fragaria chiloensis subsp. patagonica , and Fragaria virginiana subsp. grayana . These ecotypes were identified in the present study, known to be highly heterozygous from genome-wide DNA profling, and, as before, predicted a priori to either be heterozygous or homozygous for alleles affecting resistance. The parents of these populations were grown in greenhouses at UC Davis. S1 and S2 family seeds were produced by hand pollinating unemasculated flowers of Guardian, Wiltguard, Earliglow, 17C327P010, and 61S016P006. The PI612569 × 12C089P002, 12C089P002 × PI602575, and PI552277 × 12C089P002 full-sib families were produced by emasculating flowers on greenhouse grown plants of the female parent and hand pollinating the emasculated flowers with pollen from male parents. Ripe fruit were harvested and macerated in a pectinase solution to separate achenes from receptacles. Scarified seeds were germinated on moistened blotter paper at room temperature . Seedlings were transplanted to sterilized soil and were greenhouse grown for 9 months in Winters, CA before transplanting to the field, or were grown in a growth chamber for two to four months in Davis, CA before transplanting to the greenhouse.