1. Develop new rootstocks that impart HLB-tolerance to scion cultivars. We have had 27 candidate rootstock seed trees tested by DPI. All have received a clean bill of health; no seed transmissible pathogens were found. We have harvested fruit from these selections, and seed extractions are in progress. Once completed, we are authorized to distribute seeds to collaborative nurseries and other organizations within Florida, in the US, and globally, to establish advanced trials to compare our best rootstock selections with industry standards. We continuously update and add new data to existing and new rootstock trial files to our website (https://crec.ifas.ufl.edu/citrus-research/rootstock-trials/), currently there is information from 24 locations. 2. Develop new, HLB-tolerant scion cultivars from sweet orange germplasm, as well as other important fruit types such as grapefruit, mandarins, and acid fruit. Embryo rescue from 2019 crosses yielded shoots from >1750 germinating embryos coming from 36 interploid crosses (including 11 targeting sweet orange-like hybrids, and 8 targeting grapefruit improvement), Micrografting to rootstocks has been completed, and nearly all have been transferred to the greenhouses to grow off. Embryo rescue from 37 interploid crosses made using selected HLB tolerant plants in 2020 is nearly completed, including 10 crosses for red grapefruit improvement, 10 for sweet orange improvement, and 10 for mandarin improvement. In addition, 2 diploid crosses were also made for sweet orange improvement. Populations of protoclones from EV1 and 2, were produced to seek more robust early maturing Valencia types; these trees have been prepared for field planting. Cybridization experiments were conducted to combine Meiwa kumquat cytoplasm with OLL and EV sweet oranges, and red grapefruit clone N11-7, to attempt improvements in citrus canker resistance; plant regeneration is well underway, and we expect micrografting and molecular characterization to proceed in early 2021. Three HLB tolerant red grapefruit hybrids and two mandarins were submitted to the DPI Parent Tree Program for cleanup and the production of certified budwood for future trials.3. Screen our ever-growing germplasm collection for more tolerant types and evaluate fruit quality of candidate selections. We used new approaches to quantify tree responses to HLB, in addition to the previously used subjective approaches; we measured photosynthetic parameters and leaf canopy indexes, to produce repeatable and reliable quantitative data in support of further genetic analyses of tolerant types. Objective quantitative data of tree responses provides more reliable information that improves the precision with which we can associate genome regions with tolerance or sensitivity; see Obj 4. We also have been collecting detailed HLB phenotypic data, including Ct values and other tree health measures as described above, from a unique hybrid family of more than 400 individuals (with many of these planted as 3 tree replicates) from the cross of Clementine mandarin with a wild species reported by numerous sources to be nearly resistant to CaLas attacks.4. Conduct studies to unravel host responses to CLas and select targets for genetic manipulations leading to consumer-friendly new scion and rootstock cultivars. We selected ~ 450 mandarin hybrids for GWAS studies, using the data referred to in Obj 3 above. DNA samples from these trees were prepared, and each individual was genotyped using the citrus Axiom SNP array. GWAS analysis is proceeding. This work will validate previously identified, or identify new genomic regions, associated with HLB tolerance or sensitivity. Further, we have contributed budwood of 54 UF-CREC selections to a top-working trial of approximately 200 selections in total, that is a collaboration with USDA-ARS Ft. Pierce and supported by industry. These have been topworked in a commercial grove and are now growing; this smaller but more diverse population will be used for GWAS in the future. We assisted with the collection of DNA samples to fingerprint the plantings to be assured that the trees are properly labeled and coincide with the original clones and budwood sources at DPI. Samples have also been prepared for genotyping all individuals using the GBS approach, to be fed into GWAS analysis in the future.