A chromosome-level phased genome enabling allele-level studies in sweet orange: a case study on citrus Huanglongbing tolerance

Abstract

Sweet orange originated from the introgressive hybridizations of pummelo and mandarin resulting in a highly heterozygous genome. How alleles from the two species cooperate in shaping sweet orange phenotypes under distinct circumstances is unknown. Here, we assembled a chromosome-level phased diploid Valencia sweet orange (DVS) genome with over 99.999% base accuracy and 99.2% gene annotation BUSCO completeness. DVS enables allele-level studies for sweet orange and other hybrids between pummelo and mandarin. We first configured an allele-aware transcriptomic profiling pipeline and applied it to 740 sweet orange transcriptomes. On average, 32.5% of genes have a significantly biased allelic expression in the transcriptomes. Different cultivars, transgenic lineages, tissues, development stages, and disease status all impacted allelic expressions and resulted in diversified allelic expression patterns in sweet orange, but particularly citrus Huanglongbing (HLB) shifted the allelic expression of hundreds of genes in leaves and calyx abscission zones. In addition, we detected allelic structural mutations in an HLB-tolerant mutant (T19) and a more sensitive mutant (T78) through long-read sequencing. The irradiation-induced structural mutations mostly involved double-strand breaks, while most spontaneous structural mutations were transposon insertions. In the mutants, most genes with significant allelic expression ratio alterations (≥1.5-fold) were directly affected by those structural mutations. In T19, alleles located at a translocated segment terminal were upregulated, including CsDnaJ, CsHSP17.4B, and CsCEBPZ. Their upregulation is inferred to keep phloem protein homeostasis under the stress from HLB and enable subsequent stress responses observed in T19. DVS will advance allelic level studies in citrus.

Acknowledgements

This work was supported in part by the United States Department of Agriculture National Institute of Food and Agriculture (NIFA) under Grant 2017-70016-26051 to F. L. and F. G., and the U.S. National Science Foundation (NSF) under Grant ABI-1759856 and MTM2-2025541 to F.L. This research was partially supported by grants from the Citrus Research and Development Foundation (CRDF 15-010, CRDF RMC 18-010, and CRDF RMC 18-011), and the New Varieties Development and Management Corporation to F.G.

Author contributions

F.G. and F.L. conceived and designed this project. F.G. and Q.B.Y. observed and assessed the HLB tolerance of the plants. Q.B.Y carried out all the biological experiments. B.W. did all the bioinformatical and statistical analyses. B.W., Q.B.Y., F.G., and F.L. wrote the paper. Z.D and Y.P.D evaluated the plant materials and the data analysis results. All authors have read and approved the final version of this paper.

Data availability

All sequencing data generated in this study (PacBio sequencing, NGS sequencing, and RNA-seq data) have been deposited in the National Center for Biotechnology Information (NCBI) under BioProject ID PRJNA735893. The genome assembly and gene annotation of DVS have been submitted to NCBI under the GenBank assembly accessions GCA_022201045.1 (DVS_A) and GCA_022201065.1 (DVS_B). Data supporting the findings of this work are available within the paper and its Supplementary Information files. The allelic expression patterns of 749 sweet orange RNA-seq data sets (Supplementary File 1) can be accessed from https://doi.org/10.6084/m9.figshare.19424699.v2. The scripts and configuration files used in the assembly processes and the allele-aware RNAseq pipeline are accessible from https://github.com/TheLuoFengLab/DVS-assembly-and-allele-aware-RNAseq-pipeline.git.

Conflict interests

The authors declare no competing interests.

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