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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. [ABSTRACT FROM AUTHOR]

Background: In citrus, genetic improvement via biotechnology is hindered by the obstacle of in vitro regeneration via somatic embryogenesis (SE). Although a few B3 transcription factors are reported to regulate embryogenesis, little is known about the B3 superfamily in citrus, and which members might be involved in SE. Results: Genome-wide sequence analysis identified 72 (CsB3) and 69 (CgB3) putative B3 superfamily members in the genomes of sweet orange (Citrus sinensis, polyembryonic) and pummelo (C. grandis, monoembryonic), respectively. Genome duplication analysis indicated that segmental and tandem duplication events contributed to the expansion of the B3 superfamily in citrus, and that the B3 superfamily evolved under the effect of purifying selection. Phylogenetic relationships were well supported by conserved gene structure and motifs outside the B3 domain, which allowed possible functions to be inferred by comparison with homologous genes from Arabidopsis. Expression analysis identified 23 B3 superfamily members that were expressed during SE in citrus and 17 that may play functional roles at late SE stages. Eight B3 genes were identified that were specific to the genome of polyembryonic sweet orange compared to monoembryonic pummelo. Of these eight B3 genes, CsARF19 was found to be specifically expressed at higher levels in embryogenic callus (EC), implying its possible involvement in EC initiation. Conclusions: This study provides a genome-wide analysis of the citrus B3 superfamily, including its genome organization, evolutionary features and expression profiles, and identifies specific family members that may be associated with SE. [ABSTRACT FROM AUTHOR]

Somatic embryogenesis ( SE) is a process of somatic cells becoming dedifferentiated and generating embryos. SE has been widely used in biotechnology as a powerful way of regeneration and a model system for studying plant embryogenesis, but the controlling mechanisms of SE are far from clear. Here, we show the genomewide profiles of mi RNAs/si RNAs and their target genes in nonembryogenic and embryogenic tissues of 'Valencia' sweet orange. By high-throughput sequencing ( HTS) of small RNAs and RNA degradome tags, we identified 50 known and 45 novel mi RNAs, 130 miniature inverted-repeat transposable elements ( MITEs) derived, 94 other and 235 phased small interfering RNAs (si RNAs), as well as 203 target genes. The majority of the abundantly expressed mi RNAs/si RNAs exhibit lower expression levels in embryogenic callus ( EC) or during SE process than in nonembryogenic callus ( NEC), which is supposed to derepress the target genes that are involved in development and stress response, thus to activate the biological processes required for cell differentiation. However, the conserved csi-miR156a/b, miR164b and 171c directed suppression of specific transcription factors ( TFs) are supposed to inactivate the postembryonic growth thus to maintain normal SE. In this study, mi RNA- and si RNA-mediated silencing of target genes was found under sophisticated regulation in citrus SE system; the enhancement effect of specific conserved mi RNAs on SE was discussed, providing new clues for future investigation of mechanisms that control SE. [ABSTRACT FROM AUTHOR]

miRNAs have recently been reported to modulate somatic embryogenesis (SE), a key pathway of plant regeneration in vitro. For expression level detection and subsequent function dissection of miRNAs in certain biological processes, qRT-PCR is one of the most effective and sensitive techniques, for which suitable reference gene selection is a prerequisite. In this study, three miRNAs and eight non-coding RNAs (ncRNA) were selected as reference candidates, and their expression stability was inspected in developing citrus SE tissues cultured at 20, 25, and 30 °C. Stability of the eight non-miRNA ncRNAs was further validated in five adult tissues without temperature treatment. The best single reference gene for SE tissues was snoR14 or snoRD25, while for the adult tissues the best one was U4; although they were not as stable as the optimal multiple references snoR14 + U6 for SE tissues and snoR14 + U5 for adult tissues. For expression normalization of less abundant miRNAs in SE tissues, miR3954 was assessed as a viable reference. Single reference gene snoR14 outperformed multiple references for the overall SE and adult tissues. As one of the pioneer systematic studies on reference gene identification for plant miRNA normalization, this study benefits future exploration on miRNA function in citrus and provides valuable information for similar studies in other higher plants. Key message Three miRNAs and eight non-coding RNAs were tested as reference candidates on developing citrus SE tissues. Best single references snoR14 or snoRD25 and optimal multiple references snoR14 + U6, snoR14 + U5 were identified. [ABSTRACT FROM AUTHOR]

Apomixis, is an asexual mode of seed formation resulting in genetically identical or clonal seed with a maternal genotype. Apomixis has not been reported in seed crops where its flexible application in plant breeding could accelerate delivery of new varieties. By contrast, a sporophytic form of apomixis termed nucellar or adventitious embryony is common in the Rutaceae containing Citrus crop species. Here, multiple embryos develop from the maternal, somatic, nucellar cells of the ovule. They are incorporated into the enlarging embryo sac containing the sexually derived zygotic embryo and endosperm, which are products of double fertilization. Recent research has provided insights to the molecular basis for nucellar embryony. Here, we review the current understanding of the initiation, genetic basis and evolution of nucellar embryony in Citrus , and discuss prospects for future study and breeding applications of Citrus sporophytic apomixis. [ABSTRACT FROM AUTHOR]