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

Authors: WU, BO - Clemson University; YU, QIBIN - University Of Florida; DENG, ZHANAO - University Of Florida; Duan, Ping; LUO, FENG - Clemson University; GMITTER, FRED - University Of Florida

Submitted to: Horticulture Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/18/2022
Publication Date: 11/3/2022
Citation: Wu, B., Yu, Q., Deng, Z., Duan, Y., Luo, F., Gmitter, F. 2022. A chromosome-level phased genome enabling allele-level studies in sweet orange: a case study on citrus Huanglongbing tolerance. Horticulture Research. 10(1): Article uhac247. https://doi.org/10.1093/hr/uhac247.
DOI: https://doi.org/10.1093/hr/uhac247

Interpretive Summary: Sweet orange (Citrus sinensis, SWO) has more than 2,300 years of recorded domestication history, and most of its cultivars could date back to a single plant through somatic mutations. The primitive Citrus species contributing to the hybridizations generally diverged between 3 and 8 million years ago and vary substantially in genomes and phenotypes. Interspecific and introgression hybrids in citrus are phenotypically distinct from their parents. How the allelic genes from their parental pummelo and mandarin cooperate in shaping the phenotypes of SWO, and how differently they react in SWO under different environmental conditions remain largely unknown. In this study, we assembled a chromosome-level phased diploid Valencia SWO (DVS) genome with significantly improved K-Mer completeness, base accuracy, and gene annotation completeness compared to previous published a di-haploid sweet orange genome. In addition, by taking advantage of our nearly complete phased DVS genome, we further identified the target genes at the allelic level, and revealed the heat shock protein (HSP)-related molecular mechanism underlying the budsport selection of SWO with high HLB tolerance. Altogether, our complete sweet orange genome via phase assembly provides new insights into HLB tolerance, and will advance allelic level studies in other citrus species and relatives.

Technical Abstract: Sweet orange (SWO) originated from introgressive hybridization of pummelo and mandarin resulting in a highly heterozygous genome. How their alleles cooperate in shaping SWO phenotypes, and how differently they react under different conditions remain unknown. Here, we assembled the first chromosome-level phased diploid Valencia SWO (DVS) genome. DVS harbors high level intra-genomic variances and enables allelic level studies. Reanalyzing published datasets with DVS as the reference, we found a wide existence of allelic expression patterns associated with tissues, development stages, SWO cultivars, and distinct transgenic lineages. We also found that citrus Huanglongbing (HLB) shifted the allelic expression patterns in SWO leaves and calyx abscission zones from the healthy status. With long-read sequencing and RNAseq, we detected allelic structural mutations and corresponding allelic expression alterations in HLB-tolerant mutant T19, a more susceptible mutant T78, and an ordinary Valencia orange. Most structural mutations induced by irradiation involved double-strand breaks, while the majority of natural structural mutations were transposon insertions. We found that 84.0% of genes with significant allelic expression ratio alterations (=1.5 fold) had one allele directly affected by a structural mutation. In T19, alleles located at one terminal of a translocated segment are upregulated, including three associated with the regulation of heat shock protein genes (HSPs). Over half HSPs, significantly overrepresented with small HSPs, were upregulated in T19. Therefore, increased levels of HSPs and particularly small HSPs may directly affect T19’s HLB tolerance by reducing phloem necrosis/plugging and promoting phloem homeostasis. More importantly, DVS will advance allelic level studies in citrus.