An abscisic acid-responsive transcriptional regulatory module CsERF110-CsERF53 orchestrates citrus fruit coloration.

Carotenoid biosynthesis is closely associated with abscisic acid (ABA) during the ripening process of non-climacteric fruits, but the regulatory mechanism between ABA signaling and carotenoid metabolism remains largely unclear. Here, we identified two master regulators of ABA-mediated citrus fruit coloration, CsERF110 and CsERF53, which activated the expression of carotenoid metabolism genes (CsGGPPS, CsPSY, CsPDS, CsCRTISO, CsLCYB2, CsLCYE, CsHYD, CsZEP, and CsNCED2) to facilitate carotenoid accumulation. Further investigations showed that CsERF110 not only activated the expression of CsERF53 by binding to its promoter, but also interacted with CsERF53 to form a transcriptional regulatory module CsERF110-CsERF53. Furthermore, we discovered a positive feedback regulation loop between the ABA signal and carotenoid metabolism regulated by the transcriptional regulatory module CsERF110-CsERF53. Our results reveal that the transcriptional regulatory module CsERF110-CsERF53 responded to ABA signaling, thereby orchestrating citrus fruit coloration. Considering the importance of carotenoid content for citrus and many other carotenoid-rich crops, the revelation of molecular mechanisms underlying ABA-mediated carotenoid biosynthesis in plants will facilitate transgenic/gene editing approach development, further contributing to improving the quality of citrus and other carotenoid-rich crops.

Myo-inositol oxygenase CgMIOX3 alleviates S-RNase-induced inhibition of incompatible pollen tubes in pummelo.

Pummelo (Citrus grandis L. Osbeck) exhibits S-RNase-based self-incompatibility (SI), during which S-RNase cytotoxicity inhibits pollen tubes in an S-haplotype specific manner. The entry of S-RNase into self-pollen tubes triggers a series of reactions. However, these reactions are still poorly understood in pummelo. In the present study, we used S-RNases as baits to screen a pummelo pollen cDNA library and characterized a myo-inositol oxygenase (CgMIOX3) that physically interacts with S-RNases. CgMIOX3 is highly expressed in pummelo pollen tubes and its down-regulation leads to a reduction in pollen tube growth. Upon entering pollen tubes, S-RNases increase the expression of CgMIOX3 and enhance its activity by directly binding to it in an S-haplotype-independent manner. CgMIOX3 improves pollen tube growth under oxidative stress through ascorbic acid accumulation and increases the length of self-pollen tubes. Furthermore, over-expression of CgMIOX3 increases the relative length of self-pollen tubes growing in the style of petunia (Petunia hybrida). This study provides intriguing insights into the pumelo SI system, revealing a regulatory mechanism mediated by CgMIOX3 that plays an important role in the resistance of pollen tubes to S-RNase cytotoxicity.

Involvement of CgHSFB1 in the regulation of self-incompatibility in 'Shatian' pummelo.

As self-incompatibility is a major issue in pummelo breeding and production, its mechanism in citrus was analyzed to improve breeding efficiency and reduce production costs. Rutaceae belongs to S-RNase type of gametophytic self-incompatibility. While the function of S-RNase/SLF and the mechanism of self-incompatibility have been studied extensively, the transcriptional regulation of S-RNase has been less studied. We performed transcriptome sequencing with the styles of 'Shatian' pummelo on the day of anthesis and 1-5 days before anthesis, and found that the transcript level of S-RNase gradually decreased with flower development. By analyzing differentially expressed genes and correlation with the expression trend of S-RNase, we identified a candidate gene, CgHSFB1, and utilized biochemical experiments such as yeast one-hybrid assay, electrophoretic mobility shift assay and dual-luciferase assay, as well as transient transformation of citrus calli and Citrus microcarpa and demonstrated that CgHSFB1 could directly bind to the S1-RNase promoter and repress the expression of S1-RNase, which is involved in the pummelo self-incompatibility response. In contrast, CgHSFB1 did not bind to the promoter of S2-RNase, and there was specificity in the regulation of S-RNase.

Genetic Identification of Medicinal Citrus Cultivar 'Local Juhong' Using Molecular Markers and Genomics.

The citrus cultivar 'Local Juhong', which has historically been used as a traditional Chinese medicinal material, originated in Yuanjiang County, Hunan Province.Its parental type and genetic background are indistinct as of yet. Morphological observation shows that 'Local Juhong' has a slight oblateness in fruit shape, a relatively smooth pericarp, a fine and slightly raised oil vacuole, and an inward concave at the blossom end. The tree form and fruit and leaf morphology of 'Local Juhong' are similar to those of 'Huangpi' sour orange. To reveal the genetic background of 'Local Juhong', 21 citrus accessions were evaluated using nuclear and chloroplast SSR markers and whole-genome SNP information. 'Local Juhong' was grouped with mandarins and sub-grouped with 'Miyagawa Wase' and 'Yanxi Wanlu' in a nuclear SSR analysis, which indicated that its pollen parent might be mandarins. It was closely clustered with orange and pummelo in the chloroplast SSR analysis. The genomic sequence similarity rate of 'Local Juhong' with mandarin and pummelo heterozygosity was 70.88%; the main part was the heterozygosity, except for the unknown (19.66%), mandarin (8.73%), and pummelo (3.9%) parts. Thus, 'Local Juhong' may be an F1 hybrid with pummelo as the female parent and mandarin as the male parent, sharing sisterhood with 'Huangpi' sour orange.

Adventitious embryonic causal gene FhRWP regulates multiple developmental phenotypes in citrus reproduction.

Citrus is a model plant for studying adventitious embryos, a form of asexual reproduction controlled by a single dominant gene, RWP. This gene has been identified as the causal gene for nucellar embryogenesis, but its function has not yet been fully understood. In this study, we used the fast-growing Fortunella hindsii as a system to explore chromatin accessibility during the nucellar embryony initiation, emphasizing elevated chromatin accessibility in polyembryonic (PO) genotypes compared to monoembryonic ones (MO). Notably, a higher level of accessible chromatin was observed in one allele of the promoter region of FhRWP, consistent with increased expression of the allele carrying the causal structural variant. By independently performing RNAi and gene editing experiments on PO genotypes, we found the downregulation of FhRWP expression could reduce the number of nucellar embryos, while its knockout resulted in abnormal axillary bud development. In overexpression experiments, FhRWP was identified as having the unique capability of inducing the embryogenic callus formation in MO stem segments, possibly through the regulation of the WUS-CLV signaling network and the ABA and cytokinin pathway, marking the inaugural demonstration of FhRWP's potential to reignite somatic cells' embryogenic fate. This study reveals the pleiotropic function of RWP in citrus and constructs a regulatory network during adventitious embryo formation, providing a new tool for bioengineering applications in plant regeneration.

An S-locus F-box protein as pollen S determinant targets non-self S-RNase underlying self-incompatibility in Citrus.

Self-incompatibility (SI) is a crucial mechanism that prevents self-fertilization and inbreeding in flowering plants. Citrus exhibits SI regulated by a polymorphic S-locus containing an S-RNase gene and multiple S-locus F-box (SLF) genes. It has been documented that S-RNase functions as the pistil S determinant, but there is no direct evidence that the SLF genes closely linked with S-RNase function as pollen S determinants in Citrus. This study assembled the genomes of two pummelo (Citrus grandis) plants, obtained three novel complete and well-annotated S-haplotypes, and isolated 36 SLF or SLF-like alleles on the S-loci. Phylogenetic analysis of 138 SLFs revealed that the SLF genes were classified into 12 types, including six types with divergent or missing alleles. Furthermore, transformation experiments verified that the conserved S6-SLF7a protein can lead to the transition of SI to self-compatibility by recognizing non-self S8-RNase in 'Mini-Citrus' plants (S7S8 and S8S29, Fortunella hindsii), a model plant for citrus gene function studies. In vitro assays demonstrated interactions between SLFs of different S haplotypes and the Skp1-Cullin1-F-box subunit CgSSK1 protein. This study provides direct evidence that SLF controls the pollen function in Citrus, demonstrating its role in the 'non-self recognition' SI system.

Molecular regulation of oil gland development and biosynthesis of essential oils in Citrus spp.

Secretory structures in terrestrial plants serve as reservoirs for a variety of secondary metabolites. Among these, the secretory cavity of the Rutaceae family is notable for containing essential oils with a wide range of applications. However, the molecular basis underlying secretory cavity development is unknown. Here, we reveal a molecular framework for Citrus oil gland formation. Using genetic mapping and genome editing, we demonstrated that this process requires LATE MERISTEM IDENTITY1 (LMI1), a key regulator of leaf serration. A conserved GCC box element of the LMI1 promoter recruits DORNROSCHEN-like (DRNL) for transcriptional activation. This DRNL-LMI1 cascade triggers MYC5 activation, facilitating the development of oil glands and the biosynthesis of essential oils. Our findings spotlight cis-regulatory divergence within leaf shape genes, propelling novel functional tissue formation.

Cytochrome P450 CitCYP97B modulates carotenoid accumulation diversity by hydroxylating ß-cryptoxanthin in Citrus.

Carotenoids in plant foods provide health benefits by functioning as provitamin A. One of the vital provitamin A carotenoids, ß-cryptoxanthin, is typically plentiful in citrus fruit. However, little is known about the genetic basis of ß-cryptoxanthin accumulation in citrus. Here, we performed a widely targeted metabolomic analysis of 65 major carotenoids and carotenoid derivatives to characterize carotenoid accumulation in Citrus and determine the taxonomic profile of ß-cryptoxanthin. We used data from 81 newly sequenced representative accessions and 69 previously sequenced Citrus cultivars to reveal the genetic basis of ß-cryptoxanthin accumulation through a genome-wide association study. We identified a causal gene, CitCYP97B, which encodes a cytochrome P450 protein whose substrate and metabolic pathways in land plants were undetermined. We subsequently demonstrated that CitCYP97B functions as a novel monooxygenase that specifically hydroxylates the ß-ring of ß-cryptoxanthin in a heterologous expression system. In planta experiments provided further evidence that CitCYP97B negatively regulates ß-cryptoxanthin content. Using the sequenced Citrus accessions, we found that two critical structural cis-element variations contribute to increased expression of CitCYP97B, thereby altering ß-cryptoxanthin accumulation in fruit. Hybridization/introgression appear to have contributed to the prevalence of two cis-element variations in different Citrus types during citrus evolution. Overall, these findings extend our understanding of the regulation and diversity of carotenoid metabolism in fruit crops and provide a genetic target for production of ß-cryptoxanthin-biofortified products.

Transcription factor CrWRKY42 coregulates chlorophyll degradation and carotenoid biosynthesis in citrus.

Chlorophyll degradation and carotenoid biosynthesis, which occur almost simultaneously during fruit ripening, are essential for the coloration and nutritional value of fruits. However, the synergistic regulation of these 2 processes at the transcriptional level remains largely unknown. In this study, we identified a WRKY transcription factor, CrWRKY42, from the transcriptome data of the yellowish bud mutant "Jinlegan" ([Citrus unshiu × C. sinensis] × C. reticulata) tangor and its wild-type "Shiranui" tangor, which was involved in the transcriptional regulation of both chlorophyll degradation and carotenoid biosynthesis pathways. CrWRKY42 directly bound to the promoter of ß-carotene hydroxylase 1 (CrBCH1) and activated its expression. The overexpression and interference of CrWRKY42 in citrus calli demonstrated that CrWRKY42 promoted carotenoid accumulation by inducing the expression of multiple carotenoid biosynthetic genes. Further assays confirmed that CrWRKY42 also directly bound to and activated the promoters of the genes involved in carotenoid biosynthesis, including phytoene desaturase (CrPDS) and lycopene ß-cyclase 2 (CrLCYB2). In addition, CrWRKY42 could bind to the promoters of NONYELLOW COLORING (CrNYC) and STAY-GREEN (CrSGR) and activate their expression, thus promoting chlorophyll degradation. The overexpression and silencing of CrWRKY42 in citrus fruits indicated that CrWRKY42 positively regulated chlorophyll degradation and carotenoid biosynthesis by synergistically activating the expression of genes involved in both pathways. Our data revealed that CrWRKY42 acts as a positive regulator of chlorophyll degradation and carotenoid biosynthesis to alter the conversion of citrus fruit color. Our findings provide insight into the complex transcriptional regulation of chlorophyll and carotenoid metabolism during fruit ripening.

The transcriptional regulatory module CsHB5-CsbZIP44 positively regulates abscisic acid-mediated carotenoid biosynthesis in citrus (Citrus spp.).

Carotenoids contribute to fruit coloration and are valuable sources of provitamin A in the human diet. Abscisic acid (ABA) plays an essential role in fruit coloration during citrus fruit ripening, but little is known about the underlying mechanisms. Here, we identified a novel bZIP transcription activator called CsbZIP44, which serves as a central regulator of ABA-mediated citrus carotenoid biosynthesis. CsbZIP44 directly binds to the promoters of four carotenoid metabolism-related genes (CsDXR, CsGGPPs, CsBCH1 and CsNCED2) and activates their expression. Furthermore, our research indicates that CsHB5, a positive regulator of ABA and carotenoid-driven processes, activates the expression of CsbZIP44 by binding to its promoter. Additionally, CsHB5 interacts with CsbZIP44 to form a transcriptional regulatory module CsHB5-CsbZIP44, which is responsive to ABA induction and promotes carotenoid accumulation in citrus. Interestingly, we also discover a positive feedback regulation loop between the ABA signal and carotenoid biosynthesis mediated by the CsHB5-CsbZIP44 transcriptional regulatory module. Our findings show that CsHB5-CsbZIP44 precisely modulates ABA signal-mediated carotenoid metabolism, providing an effective strategy for quality improvement of citrus fruit and other crops.

Transposable elements cause the loss of self-incompatibility in citrus.

Self-incompatibility (SI) is a widespread prezygotic mechanism for flowering plants to avoid inbreeding depression and promote genetic diversity. Citrus has an S-RNase-based SI system, which was frequently lost during evolution. We previously identified a single nucleotide mutation in Sm-RNase, which is responsible for the loss of SI in mandarin and its hybrids. However, little is known about other mechanisms responsible for conversion of SI to self-compatibility (SC) and we identify a completely different mechanism widely utilized by citrus. Here, we found a 786-bp miniature inverted-repeat transposable element (MITE) insertion in the promoter region of the FhiS2-RNase in Fortunella hindsii Swingle (a model plant for citrus gene function), which does not contain the Sm-RNase allele but are still SC. We demonstrate that this MITE plays a pivotal role in the loss of SI in citrus, providing evidence that this MITE insertion prevents expression of the S-RNase; moreover, transgenic experiments show that deletion of this 786-bp MITE insertion recovers the expression of FhiS2-RNase and restores SI. This study identifies the first evidence for a role for MITEs at the S-locus affecting the SI phenotype. A family-wide survey of the S-locus revealed that MITE insertions occur frequently adjacent to S-RNase alleles in different citrus genera, but only certain MITEs appear to be responsible for the loss of SI. Our study provides evidence that insertion of MITEs into a promoter region can alter a breeding strategy and suggests that this phenomenon may be broadly responsible for SC in species with the S-RNase system.

Transcription factor CsTT8 promotes fruit coloration by positively regulating the methylerythritol 4-phosphate pathway and carotenoid biosynthesis pathway in citrus (Citrus spp.).

Carotenoids directly influence citrus fruit color and nutritional value, which is critical to consumer acceptance. Elucidating the potential molecular mechanism underlying carotenoid metabolism is of great importance for improving fruit quality. Despite the well-established carotenoid biosynthetic pathways, the molecular regulatory mechanism underlying carotenoid metabolism remains poorly understood. Our previous studies have reported that the Myc-type basic helix-loop-helix (bHLH) transcription factor (TF) regulates citrus proanthocyanidin biosynthesis. Transgenic analyses further showed that overexpression of CsTT8 could significantly promote carotenoid accumulation in transgenic citrus calli, but its regulatory mechanism is still unclear. In the present study, we found that overexpression of CsTT8 enhances carotenoid content in citrus fruit and calli by increasing the expression of CsDXR, CsHDS, CsHDR, CsPDS, CsLCYE, CsZEP, and CsNCED2, which was accompanied by changes in the contents of abscisic acid and gibberellin. The in vitro and in vivo assays indicated that CsTT8 directly bound to the promoters of CsDXR, CsHDS, and CsHDR, the key metabolic enzymes of the methylerythritol 4-phosphate (MEP) pathway, thus providing precursors for carotenoid biosynthesis and transcriptionally activating the expression of these three genes. In addition, CsTT8 activated the promoters of four key carotenoid biosynthesis pathway genes, CsPDS, CsLCYE, CsZEP, and CsNCED2, directly promoting carotenoid biosynthesis. This study reveals a novel network of carotenoid metabolism regulated by CsTT8. Our findings will contribute to manipulating carotenoid metabolic engineering to improve the quality of citrus fruit and other crops.

Determination of Self-(In)compatibility and Inter-(In)compatibility Relationships in Citrus Using Manual Pollination, Microscopy, and S-Genotype Analyses.

Citrus uses S-RNase-based self-incompatibility to reject self-pollen and, therefore, requires nearby pollinizer trees for successful pollination and fertilization. However, identifying suitable varieties to serve as pollinizers is a time-consuming process. To solve this problem, we have developed a rapid method for identifying pollination-compatible citrus cultivars that utilizes agarose gel electrophoresis and aniline blue staining. Pollen compatibility is determined based on the identification of S genotypes by extracting total DNA and performing PCR-based genotyping assays with specific primers. Additionally, styles are collected 3-4 days after manual pollination, and aniline blue staining is performed. Finally, the growth status of the pollen tubes is observed with a fluorescence microscope. Pollen compatibility and incompatibility can be established by observing whether the pollen tube growth is normal or suppressed, respectively. Due to its simplicity and cost-effectiveness, this method is an effective tool for determining the pollen compatibility and incompatibility of different citrus varieties to establish incompatibility groups and incompatibility relationships between different cultivars. This method provides information essential for the successful selection of suitable pollinizer trees and, thus, facilitates the establishment of new orchards and the selection of appropriate parents for breeding programs.

Genomic conservation of crop wild relatives: A case study of citrus.

Conservation of crop wild relatives is critical for plant breeding and food security. The lack of clarity on the genetic factors that lead to endangered status or extinction create difficulties when attempting to develop concrete recommendations for conserving a citrus wild relative: the wild relatives of crops. Here, we evaluate the conservation of wild kumquat (Fortunella hindsii) using genomic, geographical, environmental, and phenotypic data, and forward simulations. Genome resequencing data from 73 accessions from the Fortunella genus were combined to investigate population structure, demography, inbreeding, introgression, and genetic load. Population structure was correlated with reproductive type (i.e., sexual and apomictic) and with a significant differentiation within the sexually reproducing population. The effective population size for one of the sexually reproducing subpopulations has recently declined to ~1,000, resulting in high levels of inbreeding. In particular, we found that 58% of the ecological niche overlapped between wild and cultivated populations and that there was extensive introgression into wild samples from cultivated populations. Interestingly, the introgression pattern and accumulation of genetic load may be influenced by the type of reproduction. In wild apomictic samples, the introgressed regions were primarily heterozygous, and genome-wide deleterious variants were hidden in the heterozygous state. In contrast, wild sexually reproducing samples carried a higher recessive deleterious burden. Furthermore, we also found that sexually reproducing samples were self-incompatible, which prevented the reduction of genetic diversity by selfing. Our population genomic analyses provide specific recommendations for distinct reproductive types and monitoring during conservation. This study highlights the genomic landscape of a wild relative of citrus and provides recommendations for the conservation of crop wild relatives.

Transcription factor CsMADS3 coordinately regulates chlorophyll and carotenoid pools in Citrus hesperidium.

Citrus, 1 of the largest fruit crops with global economic and nutritional importance, contains fruit known as hesperidium with unique morphological types. Citrus fruit ripening is accompanied by chlorophyll degradation and carotenoid biosynthesis, which are indispensably linked to color formation and the external appearance of citrus fruits. However, the transcriptional coordination of these metabolites during citrus fruit ripening remains unknown. Here, we identified the MADS-box transcription factor CsMADS3 in Citrus hesperidium that coordinates chlorophyll and carotenoid pools during fruit ripening. CsMADS3 is a nucleus-localized transcriptional activator, and its expression is induced during fruit development and coloration. Overexpression of CsMADS3 in citrus calli, tomato (Solanum lycopersicum), and citrus fruits enhanced carotenoid biosynthesis and upregulated carotenogenic genes while accelerating chlorophyll degradation and upregulating chlorophyll degradation genes. Conversely, the interference of CsMADS3 expression in citrus calli and fruits inhibited carotenoid biosynthesis and chlorophyll degradation and downregulated the transcription of related genes. Further assays confirmed that CsMADS3 directly binds and activates the promoters of phytoene synthase 1 (CsPSY1) and chromoplast-specific lycopene ß-cyclase (CsLCYb2), 2 key genes in the carotenoid biosynthetic pathway, and STAY-GREEN (CsSGR), a critical chlorophyll degradation gene, which explained the expression alterations of CsPSY1, CsLCYb2, and CsSGR in the above transgenic lines. These findings reveal the transcriptional coordination of chlorophyll and carotenoid pools in the unique hesperidium of Citrus and may contribute to citrus crop improvement.

A study on alterations in functional activity in migraineurs during the interictal period.

Migraine is a recurrent disease in which the cumulative effect of repeated pain attacks over a long period of time causes changes in brain function. Although there are some studies focusing on the interictal period of migraine, the reproducibility of these results is poor. Therefore, we intend to use a data-driven functional connectivity (FC) approach to probe the alterations in cerebral functional activity during the interictal period, as well as underlying no-task mechanisms of inducing headache attack in migraine patients. In the current research, 24 episodic migraine patients and 23 healthy controls (HCs) were recruited. By analyzing the magnitude of regional homogeneity (ReHo) and low-frequency fractional fluctuation (fALFF), We identified alterations in spontaneous brain activity in migraineurs, including the bilateral middle frontal gyrus, left postcentral, and right lingual gyrus. Thereafter such abnormalities were selected as seeds (ROIs) for FC analysis to further explore the underlying changes between ROIs and the whole brain areas. Compared with HCs, FC between the right middle frontal gyrus with the left precuneus cortex, and bilateral thalamus were enhanced in migraineurs. In addition, increased FC has been showed between the left postcentral gyrus with the bilateral thalamus. Furthermore, negative correlation existed between fALFF values of the left middle frontal gyrus and the pain intensity of migraine attacks (r = -0.4578, p = 0.0245). In summary, abnormal FC between the bilateral thalamus and right middle frontal gyrus, or the left retrocentral gyrus may occur between attacks in migraineurs, which may be the basis for sensory integration and pain regulation dysfunction. Thus, this could become a promising biomarker for the early diagnosis and evaluation of migraine in the interictal period, and provide a novel view for further investigation of the pathogenesis and etiology of recurrent migraine.

Jasmonate activates a CsMPK6-CsMYC2 module that regulates the expression of ß-citraurin biosynthetic genes and fruit coloration in orange (Citrus sinensis).

Carotenoids are natural pigments that influence the color of citrus fruit. The red-colored carotenoid ß-citraurin is responsible for the peel color in "Newhall" orange (Citrus sinensis). Although jasmonates are known to regulate the biosynthesis and accumulation of carotenoids, their effects on ß-citraurin biosynthesis in citrus fruit remain unclear. Here, we determined that treatment with methyl jasmonate (MeJA) significantly promotes fruit coloration and ß-citraurin production in "Newhall" orange. A MeJA treatment induced the expression of CsMYC2, which encodes a transcription factor that serves as a master regulator of jasmonate responses. CsMYC2 bound the promoter of the gene that encodes carotenoid cleavage dioxygenase 4b (CsCCD4b), the key gene for ß-citraurin biosynthesis, and the promoters of genes that encode phytoene synthase (CsPSY), lycopene ß-cyclase (CsLCYb), and ß-carotene hydroxylase (CsBCH) and induced their expression. In addition, CsMYC2 promoted CsMPK6 expression. Notably, we found that CsMPK6 interacted with CsMYC2 and that this interaction decreased the stability and DNA-binding activity of CsMYC2. Thus, we conclude that negative feedback regulation attenuates JA signaling during the jasmonate-induced coloration of citrus fruit. Together, our findings indicate that jasmonates induce ß-citraurin biosynthesis in citrus by activating a CsMPK6-CsMYC2 cascade, thereby affecting fruit coloration.

Structural variation and parallel evolution of apomixis in citrus during domestication and diversification.

Apomixis, or asexual seed formation, is prevalent in Citrinae via a mechanism termed nucellar or adventitious embryony. Here, multiple embryos of a maternal genotype form directly from nucellar cells in the ovule and can outcompete the developing zygotic embryo as they utilize the sexually derived endosperm for growth. Whilst nucellar embryony enables the propagation of clonal plants of maternal genetic constitution, it is also a barrier to effective breeding through hybridization. To address the genetics and evolution of apomixis in Citrinae, a chromosome-level genome of the Hongkong kumquat (Fortunella hindsii) was assembled following a genome-wide variation map including structural variants (SVs) based on 234 Citrinae accessions. This map revealed that hybrid citrus cultivars shelter genome-wide deleterious mutations and SVs into heterozygous states free from recessive selection, which may explain the capability of nucellar embryony in most cultivars during Citrinae diversification. Analyses revealed that parallel evolution may explain the repeated origin of apomixis in different genera of Citrinae. Within Fortunella, we found that apomixis of some varieties originated via introgression. In apomictic Fortunella, the locus associated with apomixis contains the FhRWP gene, encoding an RWP-RK domain-containing protein previously shown to be required for nucellar embryogenesis in Citrus. We found the heterozygous SV in the FhRWP and CitRWP promoters from apomictic Citrus and Fortunella, due to either two or three miniature inverted transposon element (MITE) insertions. A transcription factor, FhARID, encoding an AT-rich interaction domain-containing protein binds to the MITEs in the promoter of apomictic varieties, which facilitates induction of nucellar embryogenesis. This study provides evolutionary genomic and molecular insights into apomixis in Citrinae and has potential ramifications for citrus breeding.