Upgrading Citrus Genome Sequence Resources: Providing the Most Complete Tools Necessary for Genome Editing Strategies to Create HLB Resistant Cultivars

Upgrading Citrus Genome Sequence Resources: Providing the Most Complete Tools Necessary for Genome Editing Strategies to Create HLB Resistant Cultivars

Report Date: 05/28/2022
Project: 18-010   Year: 2022
Category: Plant Improvement
Author: Fred Gmitter
Sponsor: Citrus Research and Development Foundation

We are assessing the current inventory of sequence resources we have available, to identify additional needs to successfully complete all 9 genome sequence assemblies to a chromosome scale and with the greatest accuracy and contiguity technically possible. We still are lacking sufficient PacBio long read coverage for Ruby Red grapefruit and Shiikuwasha. New HMW DNA preparations have been made and are pending sequencing. We also need to expand our collection of RNA transcripts, both with Illumina short reads and PacBio long reads, and with broader collections of tissue types to maximize the number of expressed genes we can find for annotation of the assemblies. We have collected samples of tender flush, mature leaves, flowers, young and nearly mature fruit, bark, and for some accessions leaf tissue with and without symptoms of CLas infection and citrus canker. RNA processing and sequencing are pending.Seven of the nine genomes have been assembled using both the PacBio and Hi-C sequencing and assembly using Hi-Rise. Quality control and assessment is underway, to properly phase chromosomes, to identify and anchor unanchored sequence contigs, to find haplotype swaps via 2 different approaches, to define centromeres, and to polish telomeres and resolve highly repetitive sequences in these regions. Phasing of the LB8-9 Sugar Belle® mandarin hybrid genome has been a challenge because of several runs of homozygosity (ROH) throughout the genome; we are trying to address this by using sequence information already available from Dancy tangerine and Duncan grapefruit but using Minneola (the pollen parent) would be better so plans are in place to resequenced it. Comparing the Dovetail assemblies, we found that the highest quality coming from Carrizo citrange, not surprising given that the parents represent the greatest possible genetic diversity.   


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