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Interspecific hybridization is a common and effective strategy for producing disease resilient citrus cultivars, including those with tolerance to Huanglongbing (HLB) disease. Several HLB-tolerant cultivars have been developed through hybridization of mandarins (Citrus reticulata) with their wild relativePoncirus trifoliata. One such cultivar, ‘US-897’, exhibits robust tolerance to the bacteria causing HLB disease,Candidatus Liberibacter asiaticus(CLas). To explore the genetic architecture of the early transcriptional response toCandidatusLiberibacter asiaticus (CLas) infection in ‘US-897’, we performed transcriptomic analysis of the hybrid and its parents, ‘Cleopatra’ (C. reticulata) and ‘Flying Dragon’ (P. trifoliata). A haplotype-resolved genome for ‘US-897’ was generated using PacBio HiFi sequencing reads to support quantification of the expression of both theCitrus and Poncirusalleles. By profiling gene expression in this parent-offspring trio, we were able to determine the mode of inheritance for genes differentially expressed between parents (‘Cleopatra’ and ‘Flying Dragon’) and their interspecific hybrid (‘US-897’), with the majority genes exhibiting non-additive patterns of gene expression inheritance. Additionally, analysis of allele-specific expression in the hybrid ‘US-897’ revealed the contribution of cis- versus trans-acting regulatory variants on genes with additive and non-additive modes of inheritance. A strong correlation between differential expression between parents and allele-specific expression in ‘US-897’ suggests that cis-regulatory variation is a significant source of expression divergence between species. Finally, genes responsive to infection withCLas were identified to explore how gene regulation associated with tolerance to HLB was rewired betweenCitrusand its relativePoncirus. 

Abstract BackgroundGenetic and epigenetic perturbation of cis-regulatory sequences can shift patterns of gene expression and result in novel phenotypes. Phased genome assemblies now enable the local dissection of linkages between cis-regulatory sequences, including their epigenetic state, and allele-specific gene expression to further characterize gene regulation and resulting phenotypes in heterozygous genomes. ResultsWe assembled a locally phased genome for a mandarin hybrid named ‘Fairchild’ to explore the molecular signatures of allele-specific gene expression. With local genome phasing, genes with allele-specific expression were paired with haplotype-specific chromatin states, including levels of chromatin accessibility, histone modifications, and DNA methylation. We found that 30% of variation in allele-specific expression could be attributed to haplotype associated factors, with allelic levels of chromatin accessibility and three histone modifications in gene bodies having the most influence. Structural variants in promoter regions were also associated with allele-specific expression, including specific enrichments of hAT and MULE-MuDR DNA transposon sequences. Integration of haplotype-resolved genetic and epigenetic landscapes with high-throughput phenotypic analysis of fruit traits in a panel of 154 accessions with mandarin and pummelo ancestry revealed that trait-associated variants were enriched in regions of open chromatin. Mining of trait-associated variants uncovered a Gypsy retrotransposon insertion in a gene that regulates potassium transport and may contribute to the reduction in fruit size that is observed in mandarins. Conclusions​​Using a locally phased assembly of a heterozygous cultivar of citrus, we dissected the interplay between genetic variants and molecular phenotypes to reveal cis-regulatory sequences with potential functional effects on phenotypes relevant for genetic improvement.