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 generated raw sequence data for Valencia orange (S, sensitive), Ruby Red grapefruit (S), Clementine mandarin (S), LB8-9 Sugar Belle® mandarin hybrid (T, tolerant), and Lisbon lemon (T) and preliminary assemblies and analyses were carried out. Because of reduced sequencing costs, we were able to enter additional important genomes into the pipeline beyond those originally proposed, including Carrizo citrange, sour orange, and Shekwasha (an important breeding parent for HLB tolerance); these also have now been sequenced and preliminarily assembled. Original plans for transcriptome sequencing, necessary to annotate the genome assembles produced, were to rely on Illumina short reads; but we decided to also include long PacBio Sequel II3 reads to capture full length transcripts as well. The first transcriptome data for two target genomes, were found to be inadequate. So, we identified a new vendor for this service. New samples have been collected for RNA preparation.We have focused on the first two genomes for which we had PacBio long read assemblies coupled with Hi-C sequencing using Hi-Rise software for the best quality chromosome scale assemblies. For these two citrus types, we also have access to a collection of resequenced genomes of related mutants and closely related accessions which is enabling us to explore additional potential HLB tolerant or resistant rootstocks. Because these assemblies have much improved contiguity (i.e., completeness), we have been able to better characterize the MITE sequence diversity (MITEs are a type of mobile DNA that inserts into different locations and contributes to genetic and phenotypic diversity) at the locus that controls nucellar embryony, a very important and widespread trait in commercial citrus cultivars. And, as these two genomes contain contributions for several biological citrus species, we can now begin to look at species specific genes. The new Hi-C assemblies have now been completed by Dovetail Genomics, and they have been transferred to us. Next, we can begin the long process of analyzing the assembly output for possible Type 2 errors, attempting to anchor several still unanchored sequence contigs, etc. to polish and produce the most complete and accurate assemblies.   


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