Genome sequencing and CRISPR/Cas9 gene editing of an early flowering Mini‐Citrus (Fortunella hindsii)

Abstract

Summary Hongkong kumquat (Fortunella hindsii) is a wild citrus species characterized by dwarf plant height and early flowering. Here, we identified the monoembryonic F. hindsii (designated as ‘Mini‐Citrus’) for the first time and constructed its selfing lines. This germplasm constitutes an ideal model for the genetic and functional genomics studies of citrus, which have been severely hindered by the long juvenility and inherent apomixes of citrus. F. hindsii showed a very short juvenile period (~8 months) and stable monoembryonic phenotype under cultivation. We report the first de novo assembled 373.6 Mb genome sequences (Contig‐N50 2.2 Mb and Scaffold‐N50 5.2 Mb) for F. hindsii. In total, 32 257 protein‐coding genes were annotated, 96.9% of which had homologues in other eight Citrinae species. The phylogenomic analysis revealed a close relationship of F. hindsii with cultivated citrus varieties, especially with mandarin. Furthermore, the CRISPR/Cas9 system was demonstrated to be an efficient strategy to generate target mutagenesis on F. hindsii. The modifications of target genes in the CRISPR‐modified F. hindsii were predominantly 1‐bp insertions or small deletions. This genetic transformation system based on F. hindsii could shorten the whole process from explant to T1 mutant to about 15 months. Overall, due to its short juvenility, monoembryony, close genetic background to cultivated citrus and applicability of CRISPR, F. hindsii shows unprecedented potentials to be used as a model species for citrus research.

Characterization of the complete chloroplast genome of the Hongkong kumquat (Fortunella hindsii Swingle)

“…Hongkong kumquat (Fortunella hindsii Swingle), belonging to the subfamily Aurantioideae of the Rutaceae, is a wild citrus species widely distributed in low-altitude mountainous areas of Fujian, Jiangxi, Guangdong, Guangxi and some other southern provinces of China. It is native to South China, people there usually apply its fruits as condiments, use its dried roots and leaves as Chinese folk-medicine, and made the F. hindsii plant into miniascape (Zhu et al 2019). Moreover, due to its high callus induction ratio from F. hindsii explants and short juvenility characteristics, it was recommended to be used as model species for citrus studies (Zhang et al 2009;Zhu et al 2019).…”

“…It is native to South China, people there usually apply its fruits as condiments, use its dried roots and leaves as Chinese folk-medicine, and made the F. hindsii plant into miniascape (Zhu et al 2019). Moreover, due to its high callus induction ratio from F. hindsii explants and short juvenility characteristics, it was recommended to be used as model species for citrus studies (Zhang et al 2009;Zhu et al 2019). In this study, we obtained the complete chloroplast genome of F. hindsii and explored its phylogenetic relationship with several citrus species, which would provide valuable genetic information for the plants of Rutaceae and can be subsequently used for researches of citrus species.…”

Development of Improved Fruit, Vegetable, and Ornamental Crops Using the CRISPR/Cas9 Genome Editing Technique

“…When the Cas9 gene was driven by the Arabidopsis YAO gene promoter instead of the commonly used cauliflower mosaic virus 35S promoter, the mutation efficiency increased and in several instances was reported to be 100% [63]. Mutations in the PDS gene introduced by CRISPR/Cas9 have also been shown to confer an albino phenotype in apple [64,65], grapes [66] kiwifruit [67], pear [64], and kumquat [68].…”

Genome-wide analysis of the citrus B3 superfamily and their association with somatic embryogenesis

“…3B), indicating its potential association with callus initiation, because empirically, EC can only be induced from the seeds of the polyembryonic citrus genotypes. With the availability of the citrus genome sequences [43][44][45][46][47], two orthologs of CsARF19, MSYJ162170.1 (amino acids sequence identity of 99.36%) and…”

“…Finally, a self-blast of these protein sequences was performed to remove the redundancy whereas the alternative splice variants were not considered. A total of seven draft genomes of citrus species, including Mangshan mandarin, Clementine mandarin, atalantia, Ichang papeda, kumquat, trifoliate orange (unpublished data) and citron, were used to search the orthologs of CsARF19 [44][45][46][47].…”