Location: National Clonal Germplasm Repository for Citrus
Project Number: 2036-21000-012-007-R
Project Type: Reimbursable Cooperative Agreement
Start Date: Oct 1, 2023
End Date: Sep 14, 2028
Objective:
Hybrids have been generated by crossing citrus cultivars with HLB-resistant/tolerant Australian limes to incorporate and introgress crucial resistance traits into cultivated citrus. Promising individuals of the F1 generation were backcrossed with citrus varieties to generate advanced hybrids with predominantly citrus-like fruit quality. Greenhouse evaluations against two isolates of CLas in Riverside-contained research facilities have shown HLB resistance in some hybrids. Here, the evaluation of 24 additional, selected accessions for HLB resistance in real-world situations by conducting field trials in FL and TX under severe disease pressure is proposed. Fruit traits of four selected hybrids have been evaluated in Riverside and various taste panels conducted. Some F1 hybrids selected for the study have lemon-like fruit quality traits, and if resistant, these may be useful for commercial cultivation in HLB-affected areas. A subset of hybrids (eight) with excellent HLB resistance but no fruit evaluation data as of now are included because of their value for the identification of resistance genes. A combination of resistance studies and molecular analysis will help in understanding the basis of resistance in these hybrids. The molecular analysis will be facilitated by previously generated, phased, genomic sequences of Australian citrus relatives and a few mandarins used in breeding. Horticultural evaluations will be conducted in CA, FL, and TX. Objectives are 1. Evaluation of 24 novel citrus scion hybrids in FL, TX, and CA for HLB resistance/tolerance. Obj. 2: Analysis of resistance genes and targeted gene expression in 24 hybrids through PacBio DNA sequencing and single-cell sequencing. Obj. 3: Metabolomic analysis and assessment of fruit quality to identify hybrids with potential for cultivar development. Obj. 4: Extension and outreach to share information with stakeholders.
Approach:
Objective 1: Twenty-four selected F1 and back-cross hybrids will be tested in TX and FL to evaluate HLB resistance to different pathogen strains since broad-spectrum resistance to CLas determines the suitability of hybrids as resistant varieties. Evaluation of the type of resistance and identifying durable resistance against several pathogen isolates is deemed essential. Horticultural evaluations will be done in all three states. The field trees will also be evaluated for susceptibility to other citrus pathogens using the pathogen testing arrays developed previously.
Objective 2: PacBio long-read sequencing of the 24 selected hybrids will be done. Phased genomic sequences of the parental accessions determined in a previous project will be used as a reference. Identification of genomic fragments associated with resistance traits in various groups of hybrids (with different parentages) will be useful for understanding the resistance traits in the breeding population. Single-cell sequencing will be carried out in this study to understand gene expression in different tissues. This novel approach will allow detailed, cellular-level resolution of gene expression changes within the phloem where the pathogen resides. These advanced tools will further the understanding of the mechanism of HLB resistance.
Objective 3: The fruit quality of hybrids generated in the breeding program will determine their usefulness as new varieties. Due to linkage drag, undesirable horticultural traits can be associated with resistance-associated gene fragments. Taste panels will be conducted to document the organoleptic properties of fruits, determine the acceptability of the novel hybrids, and conduct detailed metabolomic studies to understand the composition of the fruit juice in the selected hybrids compared with standard mandarin, grapefruit, and lemon selections. This approach will identify the most promising hybrids from the breeding population.
Objective 4: The research data generated will be presented at scientific and grower meetings. The novel fruits produced by the hybrids will be shared with the industry to obtain feedback and improve strategies for the development of useful hybrids in the breeding program. The extension is an integral part of this project.
