Citrus Huanglongbing (HLB) poses the greatest threat to the survival of the Florida and US citrus industry. Research to incorporate HLB resistance/tolerance into citrus has been recommended by the National Research Council as one of the top priority topics for addressing the HLB threat. A number of Poncirus and Citrus cultivars have been found to be tolerant to HLB. Microarray-based profiling of the transcriptomes of two cultivars with HLB tolerance (Poncirus hybrid US-897and rough lemon) and two cultivars without HLB tolerance have identified ~1,200 genes that are differentially expressed in HLB-tolerant cultivars. These genes constitute a valuable pool of potential candidates from which true HLB tolerance genes can be identified. The original project proposal aimed to identify approximately 1,200 potential candidate genes ~differentially expressed in HLB-tolerant Poncirus and rough lemon, conduct massively parallel sequencing of the ~candidate genes, perform genetic association and linkage analysis to find most likely candidate gene(s) for HLB tolerance, and clone these candidates for function validation. We have identified up-regulated genes in rough lemon in response to CLas inoculation. By combining our new gene expression data with 14 other published data sets, using two gene expression meta-analysis tools, we identified 3,122 probe sets differentially expressed between HLB-tolerant and susceptible varieties or in response to CLas inoculation. These probe sets correspond to 2,147 genes in the Clementine genome. To capture the sequence polymorphisms in these candidate genes for association and linkage analysis and to obtain full-length sequences for gene cloning, we sequenced the genomes of more than 40 Poncirus and Citrus accessions from 25-50x coverage and obtained partial genome sequences of 30 Poncirus and Citrus accessions from our collaborators, resulting in >700 Gb of sequence data. We have assembled the sequence reads into contigs and mapped these to the candidate genes for 48 Poncirus and Citrus accessions. To perform association and linkage analysis, in collaboration with USDA colleagues we collected HLB severity and CLas Ct data from more than 70 Poncirus and Citrus hybrids and accessions planted at the Picos Road Farm (USDA, Ft. Pierce). These accessions were replicated in 8 randomized complete blocks. For approximately 60% of the accessions, data were collected for two growing seasons, in addition to recently published information from the same planting. For the remaining 40%, plants were poorly established initially, and we will continue to collect phenotypic data for one additional growing season on HLB severity and CLas Ct value. Gene cloning efforts have focused on genes expressed at significantly higher levels in HLB-tolerant accessions or after CLas inoculation and with known functions in plant defense or immunity. However, some of the selected genes seemed to encode proteins toxic to E. coli or Agrobacterium cells, resulting in failure in gene cloning. To overcome this, gene expression vectors were modified. Two gene constructs have been developed and are being introduced into citrus to produce transgenic plants. To speed up the cloning of additional genes at full length, we have begun to complete a high-quality reference genome sequence of Poncirus, which currently does not exist. Based on these results, we have published two refereed papers and several abstracts. HLB-tolerant citrus so engineered are expected to be deregulated at much quicker speeds and less expense and be better accepted by consumers. Combining tolerance genes from Poncirus and rough lemon in the same citrus cultivars may result in augmented tolerance, even resistance. The primers and new DNA markers derived from HLB tolerance candidate genes also can be used to screen existing rootstock and scion breeding populations to facilitate development of non-engineered HLB-tolerant/resistant citrus scion or rootstock cultivars by conventional breeding.