1. Develop new rootstocks that impart HLB-tolerance to scion cultivars. Seeds were extracted from 27 candidate rootstock seed source trees, including several UFRs and other experimental rootstocks, that were fund to be free of seed transmissible pathogens by DPI testing. We are authorized to distribute seeds to licensed nurseries for the UFRs, and 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 updated and added new data to existing rootstock trial files and added new files to our website (https://crec.ifas.ufl.edu/citrus-research/rootstock-trials/), currently there is information from 24 locations. We planted seed from 2020 diploid and tetraploid rootstock crosses into calcareous, high pH soil inoculated with two species of Phytophthora, the first step of the `gauntlet’ screening. We stepped up rooted cuttings from more than 50 gauntlet rootstock candidates to citripots in preparation for grafting of replicate trees as necessary to meet the new CRDF guidelines for Stage 1 rootstock evaluations (approximately 500 liners). 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 37 interploid crosses made using selected HLB tolerant plants in 2020 was completed, including 10 crosses for red grapefruit improvement, 10 for sweet orange improvement, and 10 for mandarin improvement. Shoots have been generated from many of the excised embryos, and flow cytometry was used to verify triploidy among some of the hybrid populations. Micrografting of the earliest to germinate normal shoots has begun and will continue through the next quarter. Seeds harvested from 2 diploid crosses made for sweet orange improvement were planted and have germinated. 3. Screen our ever-growing germplasm collection for more tolerant types and evaluate fruit quality of candidate selections. Evaluations of existing breeding populations in the field were somewhat restricted by UF COVID travel regulations, but we nonetheless went through our materials and made selections for further evaluations and testing. Four new HLB tolerant red grapefruit hybrids, one sweet-orange like hybrid, and three seedless easy to peel mandarins were selected and submitted to the DPI Parent Tree Program for cleanup and production of certified budwood for future trials. We have continued monitoring 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, by collecting detailed HLB phenotypic data, including Ct values and other tree health measures as described previously. The frequency of CaLas-negative trees remains unexpectedly high.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 were prepared, each individual was genotyped using the citrus Axiom SNP array, and GWAS analysis is proceeding. This work will validate previously identified, or identify new genomic regions, associated with HLB tolerance or sensitivity. 5. Other related activities. We have continued testing the alternating temperature chamber regime to produce CaLas- and CTV-free lines of promising new scion and rootstock candidates for propagation and to hasten their use in advanced field trials. Five Vernia seedling selections, one grapefruit hybrid, and ten rootstocks were treated, and all were PCR negative, and will now be tested for CTV.