A target cultivar-specific identification system based on the chromatographic printed array strip method for eight prominent Japanese citrus cultivars.

Citrus is a major cultivated crop in Japan, and new cultivars are of great interest in the Japanese and global market. Recently, the infringement of breeders' rights to citrus cultivars bred in Japan has become a problem related to the agricultural product export strategy promoted by the Japanese government. Cultivar identification systems using DNA markers are an effective tool for protecting breeders' rights. Here, a novel target cultivar-specific identification system using the chromatographic printed array strip method was developed for eight prominent Japanese citrus cultivars. A polymorphic InDel fragment specific to each cultivar was explored through the screening of published citrus InDel markers and next-generation sequencing of retrotransposon libraries. The cultivar-specific DNA marker set for each cultivar comprised 1-3 polymorphic InDel fragments in combination with a PCR-positive DNA marker for the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit gene. The DNA markers were detected within 3 hours from DNA extraction to the detection by the C-PAS4 membrane stick following multiplex PCR. The developed system is superior as a convenient, rapid, and cost-effective DNA diagnostic method during inspection. The proposed target cultivar-specific identification system is expected to serve as an efficient tool for the injunction of suspicious registered cultivars, contributing to the protection of breeders' rights.

Fast-track breeding system to introduce CTV resistance of trifoliate orange into citrus germplasm, by integrating early flowering transgenic plants with marker-assisted selection.

BACKGROUND: Global warming will expand the range of new and invasive pathogens in orchards, and subsequently increase the risk of disease epidemics and economic losses. The development of new resistant plant varieties can help to reduce the impact of pathogens, however, the breeding speed can be extremely slow, due to the growth rates of the plants, and the availability of resistance genes. Citrus trees are suffering immense damage from serious diseases such as citrus canker (XCC), huanglongbing (HLB), and citrus tristeza virus (CTV). A fast-track breeding system, that aimed at shortening the duration for disease resistance breeding by incorporating the resistance genes from related species to commercial varieties, has been developed using the integration of precocious transgenic trifoliate orange with the overexpression of CiFT and MAS. It was applied here to incorporate CTV resistance of trifoliate orange into citrus germplasm. RESULTS: One generation of backcrossed breeding, that would normally take at least 5 years, was achieved in a single year by fast-track breeding system. Linkage analysis using the corresponding DNA markers revealed that CTV resistance and T-DNA integrated regions were found in different linkage groups, and they were independently segregated in the BC progenies. The CTV resistant null segregants, in which the T-DNA integrated region was removed from their genome, were feasibly obtained by MAS in each generation of the BC progenies, and their CTV resistance was confirmed by immunological analysis. Several BC3 null segregants, whose genetic backgrounds had been substituted into citrus germplasm, except for the haplotype block of CTV resistance, were successfully obtained. CGH and NGS analyses revealed that the T-DNA integrated region was safely segregated out in null segregants. CONCLUSION: Fast-track breeding systems are expected to shorten the required breeding time by more than one-fifth in comparison with conventional cross breeding techniques. Using this system, we obtained BC3-8, whose genetic background was successfully substituted except for the CTV resistance locus, and could be a novel mandarin breeding material. The fast-track breeding system will be useful to introduce important traits from related species to citrus germplasm while also drastically reducing the time required for breeding.

TaqMan-MGB SNP genotyping assay to identify 48 citrus cultivars distributed in the Japanese market.

A citrus cultivar identification system using CAPS marker has been developed on nursery trees, but this needs to be extended to include various product types, such as imported fruits and processed products. Here, we developed a new cultivar identification system using TaqMan-MGB SNP genotyping assay. Eight probe and primer sets were designed to amplify PCR fragments <100 bp to enable the genotyping of fresh and processed fruits in which predicted that insufficient quantities of DNA and residual impurities in the DNA extracts. The TaqMan-MGB SNP genotyping assay was stable and reproducible, and were confirmed to apply various sample sources, including leaves, fresh fruit, juice, canned fruit, and dry fruit. They could provide at least a single differentiating SNP to discriminate any paired combination among 48 citrus cultivars. Minimal marker subsets to identify the target cultivar were listed for each of 18 registered cultivars with valid patent. The allelic SNP genotypes of 48 citrus cultivars, which cover more than 98% of all citrus fruit shipment produced in Japan, is valuable for the referencing information in the DNA-based identification for fresh and processed fruits. This identification system will help protect registered cultivars and facilitate food fraud inspections.

Mikan Genome Database (MiGD): integrated database of genome annotation, genomic diversity, and CAPS marker information for mandarin molecular breeding.

Citrus species are some of the most valuable and widely consumed fruits globally. The genome sequences of representative citrus (e.g., Citrus clementina, C. sinensis, C. grandis) species have been released but the research base for mandarin molecular breeding is still poor. We assembled the genomes of Citrus unshiu and Poncirus trifoliata, two important species for citrus industry in Japan, using hybrid de novo assembly of Illumina and PacBio sequence data, and developed the Mikan Genome Database (MiGD). The assembled genome sizes of C. unshiu and P. trifoliata are 346 and 292 Mb, respectively, similar to those of citrus species in public databases; they are predicted to possess 41,489 and 34,333 protein-coding genes in their draft genome sequences, with 9,642 and 8,377 specific genes when compared to C. clementina, respectively. MiGD is an integrated database of genome annotation, genetic diversity, and Cleaved Amplified Polymorphic Sequence (CAPS) marker information, with these contents being mutually linked by genes. MiGD facilitates access to genome sequences of interest from previously reported linkage maps through CAPS markers and obtains polymorphism information through the multiple genome browser TASUKE. The genomic resources in MiGD (https://mikan.dna.affrc.go.jp) could provide valuable information for mandarin molecular breeding in Japan.

MITE insertion-dependent expression of CitRKD1 with a RWP-RK domain regulates somatic embryogenesis in citrus nucellar tissues.

BACKGROUND: Somatic embryogenesis in nucellar tissues is widely recognized to induce polyembryony in major citrus varieties such as sweet oranges, satsuma mandarins and lemons. This capability for apomixis is attractive in agricultural production systems using hybrid seeds, and many studies have been performed to elucidate the molecular mechanisms of various types of apomixis. To identify the gene responsible for somatic embryogenesis in citrus, a custom oligo-DNA microarray including predicted genes in the citrus polyembryonic locus was used to compare the expression profiles in reproductive tissues between monoembryonic and polyembryonic varieties. The full length of CitRKD1, which was identified as a candidate gene responsible for citrus somatic embryogenesis, was isolated from satsuma mandarin and its molecular function was investigated using transgenic 'Hamlin' sweet orange by antisense-overexpression. RESULTS: The candidate gene CitRKD1, predominantly transcribed in reproductive tissues of polyembryonic varieties, is a member of the plant RWP-RK domain-containing protein. CitRKD1 of satsuma mandarin comprised two alleles (CitRKD1-mg1 and CitRKD1-mg2) at the polyembryonic locus controlling embryonic type (mono/polyembryony) that were structurally divided into two types with or without a miniature inverted-repeat transposable element (MITE)-like insertion in the upstream region. CitRKD1-mg2 with the MITE insertion was the predominant transcript in flowers and young fruits where somatic embryogenesis of nucellar cells occurred. Loss of CitRKD1 function by antisense-overexpression abolished somatic embryogenesis in transgenic sweet orange and the transgenic T1 plants were confirmed to derive from zygotic embryos produced by self-pollination by DNA diagnosis. Genotyping PCR analysis of 95 citrus traditional and breeding varieties revealed that the CitRKD1 allele with the MITE insertion (polyembryonic allele) was dominant and major citrus varieties with the polyembryonic allele produced polyembryonic seeds. CONCLUSION: CitRKD1 at the polyembryonic locus plays a principal role in regulating citrus somatic embryogenesis. CitRKD1 comprised multiple alleles that were divided into two types, polyembryonic alleles with a MITE insertion in the upstream region and monoembryonic alleles without it. CitRKD1 was transcribed in reproductive tissues of polyembryonic varieties with the polyembryonic allele. The MITE insertion in the upstream region of CitRKD1 might be involved in regulating the transcription of CitRKD1.

Allelic diversity of phytoene synthase gene influences the transcription level in citrus fruit among a citrus F1 hybrid population.

Phytoene synthase (PSY) is one of the key regulatory enzyme on the biosynthesis and accumulation of carotenoid in citrus fruits. The transcriptional diversity of PSY is mainly attributed to the structural variation in promoter region among PSY alleles. In aim to clarify how this transcriptional diversity is regulated among them, PSY alleles responsible for carotenoid biosynthesis in the fruits are characterized and their promoter sequences were compared. Based on gene structure and expression pattern of PSY homologues on the clementine mandarin genome sequence, PSY alleles responsible for carotenoid biosynthesis are derived from a single locus in the scaffold 6. AG mapping population possessed four PSY alleles derived from parent lines of A255 and G434, and their F1 individuals with PSY-g2 allele tended to have low transcription level. From sequence comparison of their promoter regions, the cis-motif alternation from MYBPZM to RAV1AAT might be a candidate to influence the transcription level. Among the ancestral pedigree varieties of AG mapping population, the transcription level of PSY correlated with genotypes of MYBPZM and RAV1AAT motifs in the promoter region of PSY alleles, so that homozygous genotype of MYBPZM showed higher transcription level while heterozygous genotype of MYBPZM and RAV1AAT showed lower transcription level.

Citrus breeding, genetics and genomics in Japan.

Citrus is one of the most cultivated fruits in the world, and satsuma mandarin (Citrus unshiu Marc.) is a major cultivated citrus in Japan. Many excellent cultivars derived from satsuma mandarin have been released through the improvement of mandarins using a conventional breeding method. The citrus breeding program is a lengthy process owing to the long juvenility, and it is predicted that marker-assisted selection (MAS) will overcome the obstacle and improve the efficiency of conventional breeding methods. To promote citrus molecular breeding in Japan, a genetic mapping was initiated in 1987, and the experimental tools and resources necessary for citrus functional genomics have been developed in relation to the physiological analysis of satsuma mandarin. In this paper, we review the progress of citrus breeding and genome researches in Japan and report the studies on genetic mapping, expression sequence tag cataloguing, and molecular characterization of breeding characteristics, mainly in terms of the metabolism of bio-functional substances as well as factors relating to, for example, fruit quality, disease resistance, polyembryony, and flowering.

Parental diagnosis of satsuma mandarin (Citrus unshiu Marc.) revealed by nuclear and cytoplasmic markers.

Satsuma mandarins (Citrus unshiu Marc.) are the predominant cultivated citrus variety in Japan. Clarification of its origin would prove valuable for citrus taxonomy and mandarin breeding programs; however, current information is limited. We applied genome-wide genotyping using a 384 citrus single nucleotide polymorphism (SNP) array and MARCO computer software to investigate the satsuma mandarin parentage. Genotyping data from 206 validated SNPs were obtained to evaluate 67 citrus varieties and lines. A total of five parent-offspring relationships were newly found by MARCO based on the 206 SNP genotypes, indicating that 'Kishuu mikan' type mandarins (Citrus kinokuni hort. ex Tanaka accession 'Kishuu mikan' and 'Nanfengmiju') and 'Kunenbo' type mandarins (Citrus nobilis Lour. var. kunip Tanaka accession 'Kunenbo' and 'Bendiguangju') are possible parents of the satsuma mandarin. Moreover, cleaved amplified polymorphic sequences analysis showed that the genotypes of four regions in chloroplast DNA of 'Kishuu mikan' type mandarins were identical to that of the satsuma mandarin. Considering the historical background, satsuma mandarins may therefore derive from an occasional cross between a 'Kishuu mikan' type mandarin seed parent (derivative or synonym of 'Nanfengmiju') and a 'Kunenbo' type mandarin pollen parent (derivative or synonym of 'Bendiguangju').

Terpene down-regulation triggers defense responses in transgenic orange leading to resistance against fungal pathogens.

Terpenoid volatiles are isoprene compounds that are emitted by plants to communicate with the environment. In addition to their function in repelling herbivores and attracting carnivorous predators in green tissues, the presumed primary function of terpenoid volatiles released from mature fruits is the attraction of seed-dispersing animals. Mature oranges (Citrus sinensis) primarily accumulate terpenes in peel oil glands, with d-limonene accounting for approximately 97% of the total volatile terpenes. In a previous report, we showed that down-regulation of a d-limonene synthase gene alters monoterpene levels in orange antisense (AS) fruits, leading to resistance against Penicillium digitatum infection. A global gene expression analysis of AS versus empty vector (EV) transgenic fruits revealed that the down-regulation of d-limonene up-regulated genes involved in the innate immune response. Basal levels of jasmonic acid were substantially higher in the EV compared with AS oranges. Upon fungal challenge, salicylic acid levels were triggered in EV samples, while jasmonic acid metabolism and signaling were drastically increased in AS orange peels. In nature, d-limonene levels increase in orange fruit once the seeds are fully viable. The inverse correlation between the increase in d-limonene content and the decrease in the defense response suggests that d-limonene promotes infection by microorganisms that are likely involved in facilitating access to the pulp for seed-dispersing frugivores.

Corrigendum: Allelic haplotype combinations at the MS-P1 region, including P-class pentatricopeptide repeat family genes, influence wide phenotypic variation in pollen grain number through a cytoplasmic male sterility model in citrus.

[This corrects the article DOI: 10.3389/fpls.2023.1163358.].

Propagation path of a flowering cherry (Cerasus × yedoensis) cultivar 'Somei-Yoshino' traced by somatic mutations.

In the long history of human relations with flowering cherry trees in Japan, 'Somei-Yoshino' occupies an exceptional position among a variety of flowering trees: it is a self-incompatible interspecific hybrid but has been enthusiastically planted by grafting throughout Japan, due most likely to its flamboyant appearance upon full bloom. Thus, 'Somei-Yoshino' gives us a rare opportunity to trace and investigate the occurrence and distribution of somatic mutations within a single plant species through analysis of the genomes of the clonally propagated trees grown under a variety of geographical and artificial environments. In the studies presented here, a total of 46 samples of 'Somei-Yoshino' trees were collected and their genomes were analysed. We identified 684 single nucleotide mutations, of which 71 were present in more than two samples. Clustering analysis of the mutations indicated that the 46 samples were classified into eight groups, four of which included 36 of the 46 samples analysed. Interestingly, all the four tree samples collected in Ueno Park of Tokyo were members of the four groups mentioned above. Based on comparative analysis of their mutations, one of the four trees growing in Ueno Park was concluded to be the closest to the original ancestor. We propose that somatic mutations may be used as tracers to establish the ancestral relationship amongst clonally propagated individuals.

Increased levels of IAA are required for system 2 ethylene synthesis causing fruit softening in peach (Prunus persica L. Batsch).

The fruit of melting-flesh peach (Prunus persica L. Batsch) cultivars produce high levels of ethylene caused by high expression of PpACS1 (an isogene of 1-aminocyclopropane-1-carboxylic acid synthase), resulting in rapid fruit softening at the late-ripening stage. In contrast, the fruit of stony hard peach cultivars do not soften and produce little ethylene due to low expression of PpACS1. To elucidate the mechanism for suppressing PpACS1 expression in stony hard peaches, a microarray analysis was performed. Several genes that displayed similar expression patterns as PpACS1 were identified and shown to be indole-3-acetic acid (IAA)-inducible genes (Aux/IAA, SAUR). That is, expression of IAA-inducible genes increased at the late-ripening stage in melting flesh peaches; however, these transcripts were low in mature fruit of stony hard peaches. The IAA concentration increased suddenly just before harvest time in melting flesh peaches exactly coinciding with system 2 ethylene production. In contrast, the IAA concentration did not increase in stony hard peaches. Application of 1-naphthalene acetic acid, a synthetic auxin, to stony hard peaches induced a high level of PpACS1 expression, a large amount of ethylene production and softening. Application of an anti-auxin, α-(phenylethyl-2-one)-IAA, to melting flesh peaches reduced levels of PpACS1 expression and ethylene production. These observations indicate that suppression of PpACS1 expression at the late-ripening stage of stony hard peach may result from a low level of IAA and that a high concentration of IAA is required to generate a large amount of system 2 ethylene in peaches.

Allelic haplotype combinations at the MS-P1 region, including P-class pentatricopeptide repeat family genes, influence wide phenotypic variation in pollen grain number through a cytoplasmic male sterility model in citrus.

In citrus breeding programs, male sterility is an important trait for developing seedless varieties. Sterility associated with the male sterile cytoplasm of Kishu mandarin (Kishu-cytoplasm) has been proposed to fit the cytoplasmic male sterility (CMS) model. However, it remains undetermined whether CMS in citrus is controlled by interactions between sterile cytoplasm and nuclear restorer-of-fertility (Rf) genes. Accordingly, mechanisms underlying the control of the wide phenotypic variation in pollen number for breeding germplasm should be elucidated. This study aimed to identify complete linkage DNA markers responsible for male sterility at the MS-P1 region based on fine mapping. Two P-class pentatricopeptide repeat (PPR) family genes were identified as candidates for Rf based on predicted mitochondrial localization and higher expression in a male fertile variety/selected strain than in a male sterile variety. Eleven haplotypes (HT1-HT11) at the MS-P1 region were defined based on genotyping of DNA markers. Association analysis of diplotypes at the MS-P1 region and the number of pollen grains per anther (NPG) in breeding germplasms harboring Kishu-cytoplasm revealed that the diplotypes in this region influenced NPG. Among these haplotypes, HT1 is a non-functional restorer-of-fertility (rf) haplotype; HT2, a less-functional Rf; HT3-HT5 are semi-functional Rfs; and HT6 and HT7 are functional Rfs. However, the rare haplotypes HT8-HT11 could not be characterized. Therefore, P-class PPR family genes in the MS-P1 region may constitute the nuclear Rf genes within the CMS model, and a combination of the seven haplotypes could contribute to phenotypic variation in the NPG of breeding germplasms. These findings reveal the genomic mechanisms of CMS in citrus and will contribute to seedless citrus breeding programs by selecting candidate seedless seedlings using the DNA markers at the MS-P1 region.

Terpene down-regulation in orange reveals the role of fruit aromas in mediating interactions with insect herbivores and pathogens.

Plants use volatile terpene compounds as odor cues for communicating with the environment. Fleshy fruits are particularly rich in volatiles that deter herbivores and attract seed dispersal agents. We have investigated how terpenes in citrus fruit peels affect the interaction between the plant, insects, and microorganisms. Because limonene represents up to 97% of the total volatiles in orange (Citrus sinensis) fruit peel, we chose to down-regulate the expression of a limonene synthase gene in orange plants by introducing an antisense construct of this gene. Transgenic fruits showed reduced accumulation of limonene in the peel. When these fruits were challenged with either the fungus Penicillium digitatum or with the bacterium Xanthomonas citri subsp. citri, they showed marked resistance against these pathogens that were unable to infect the peel tissues. Moreover, males of the citrus pest medfly (Ceratitis capitata) were less attracted to low limonene-expressing fruits than to control fruits. These results indicate that limonene accumulation in the peel of citrus fruit appears to be involved in the successful trophic interaction between fruits, insects, and microorganisms. Terpene down-regulation might be a strategy to generate broad-spectrum resistance against pests and pathogens in fleshy fruits from economically important crops. In addition, terpene engineering may be important for studying the basic ecological interactions between fruits, herbivores, and pathogens.

Characterization of a Caffeic Acid 8-O-Methyltransferase from Citrus and Its Function in Nobiletin Biosynthesis.

Nobiletin (3',4',5,6,7,8-hexamethoxyflavone) is a polymethoxylated flavonoid specifically accumulated in citrus fruit with numerous beneficial effects to human health. In this study, a novel O-methyltransferase (CitOMT2) was isolated from three citrus varieties, Ponkan mandarin (Citrus reticulata Blanco), Nou 6 ("King mandarin" × "Mukaku-kishu"), and Satsuma mandarin (Citrus unshiu Marc.), and its functions were characterized in vitro. The gene expression results showed that CitOMT2 was highly expressed in the two nobiletin abundant varieties of Ponkan mandarin and Nou 6. However, the expression level of CitOMT2 was low in the flavedo of Satsuma mandarin, in which only a small amount of nobiletin was accumulated. Functional analysis suggested that CitOMT2 was a caffeic acid 8-O-methyltransferase, and it catalyzed the O-methylation of 7,8-dihydroxyflavone at 8-OH. As the methylation of flavone at 8-OH was required for nobiletin biosynthesis, the results presented in this study suggested that CitOMT2 was a key gene regulating nobiletin accumulation in citrus fruit.

Biological and molecular characterization of linalool-mediated field resistance against Xanthomonas citri subsp. citri in citrus trees.

The biological and molecular traits of the Ponkan mandarin (Citrus reticulata Blanco) were characterized in an investigation of the mechanisms of field resistance against citrus canker disease caused by the bacterial pathogen, Xanthomonas citri subsp. citri (Xcc). Various conventional citrus varieties that show diverse responses to Xcc were investigated, and the temporal changes in Xcc titer in response to linalool concentrations among the varieties revealed differences in Xcc proliferation trends in the inoculated leaves of the immune, field-resistant and susceptible varieties. In addition, increased linalool accumulation was inversely related to Xcc titers in the field-resistant varieties, which is likely caused by host--pathogen interactions. Quantitative trait locus (QTL) analysis using the F1 population of the resistant Ponkan mandarin and susceptible 'Harehime' ('E-647' × 'Miyagawa-wase') cultivar revealed that linalool accumulation and Xcc susceptibility QTLs overlapped. These results provide novel insights into the molecular mechanisms of linalool-mediated field resistance to Xcc, and suggest that high linalool concentrations in leaves has an antibacterial effect and becomes a candidate-biomarker target for citrus breeding to produce seedlings with linalool-mediated field resistance against Xcc.

Blue LED light induces regreening in the flavedo of Valencia orange in vitro.

In the present study, the effects of blue LED light on the regreening of citrus fruit were investigated in an in vitro system of Valencia orange flavedos. The results showed that blue LED light irradiation induced regreening in the flavedos. After four-week culture in vitro, the flavedos exhibited obviously green color in the blue LED light treatment, while the flavedos in the control were still in orange color. During the regreening process, the blue LED light treatment induced chlorophyll accumulation, and substantially altered the carotenoid composition in the flavedos. Compared with the control, the content of 9-cis-violaxanthin was decreased, while the contents of lutein, ß-carotene, and all-trans-violaxanthin were increased by blue LED light. In addition, gene expression results showed that the up-regulation of CitLCYe and down-regulation of CitLCYb2 by blue LED light led to a shift from ß,ß-branch to ß,ε-branch of the carotenoid biosynthetic pathway.

Allelic composition of carotenoid metabolic genes in 13 founders influences carotenoid composition in juice sac tissues of fruits among Japanese citrus breeding population.

To enrich carotenoids, especially ß-cryptoxanthin, in juice sac tissues of fruits via molecular breeding in citrus, allele mining was utilized to dissect allelic variation of carotenoid metabolic genes and identify an optimum allele on the target loci characterized by expression quantitative trait (eQTL) analysis. SNPs of target carotenoid metabolic genes in 13 founders of the Japanese citrus breeding population were explored using the SureSelect target enrichment method. An independent allele was determined based on the presence or absence of reliable SNPs, using trio analysis to confirm inheritability between parent and offspring. Among the 13 founders, there were 7 PSY alleles, 7 HYb alleles, 11 ZEP alleles, 5 NCED alleles, and 4 alleles for the eQTL that control the transcription levels of PDS and ZDS among the ancestral species, indicating that some founders acquired those alleles from them. The carotenoid composition data of 263 breeding pedigrees in juice sac tissues revealed that the phenotypic variance of carotenoid composition was similar to that in the 13 founders, whereas the mean of total carotenoid content increased. This increase in total carotenoid content correlated with the increase in either or both ß-cryptoxanthin and violaxanthin in juice sac tissues. Bayesian statistical analysis between allelic composition of target genes and carotenoid composition in 263 breeding pedigrees indicated that PSY-a and ZEP-e alleles at PSY and ZEP loci had strong positive effects on increasing the total carotenoid content, including ß-cryptoxanthin and violaxanthin, in juice sac tissues. Moreover, the pyramiding of these alleles also increased the ß-cryptoxanthin content. Interestingly, the offset interaction between the alleles with increasing and decreasing effects on carotenoid content and the epistatic interaction among carotenoid metabolic genes were observed and these interactions complexed carotenoid profiles in breeding population. These results revealed that allele composition would highly influence the carotenoid composition in citrus fruits. The allelic genotype information for the examined carotenoid metabolic genes in major citrus varieties and the trio-tagged SNPs to discriminate the optimum alleles (PSY-a and ZEP-e) from the rest would promise citrus breeders carotenoid enrichment in fruit via molecular breeding.

Transcriptional changes in CiFT-introduced transgenic trifoliate orange (Poncirus trifoliata L. Raf.).

Ectopic expression of a Flowering Locus T (CiFT) from Citrus confers an early flowering phenotype on trifoliate orange (Poncirus trifoliata L. Raf.). To profile transcriptional effects of CiFT, mRNA extracted from the shoots of the transgenic trifoliate orange was subjected to genetic analysis using a 22-K oligo DNA microarray. Microarray results and reverse transcription polymerase chain reaction analysis indicated that genes relating to transcription, cell wall modification and defense responses were expressed at higher levels in the transgenic shoots than in the control wild-type shoots. Of the genes showing ectopic mRNA accumulation, two SEPALLATA (SEP) and one FRUITFULL (FUL) homologues (CuSEP1, CuSEP3 or CuFUL) were introduced to Arabidopsis thaliana. Constitutive expression of each gene caused early flowering in transgenic Arabidopsis, suggesting that these genes could function as regulators of flowering time.

Differences in seasonal expression of flowering genes between deciduous trifoliate orange and evergreen Satsuma mandarin.

To determine differences in seasonal flowering between evergreen and deciduous woody perennials, endogenous expression of flowering-related genes was investigated in Satsuma mandarin (Citrus unshiu Marc.) and its close relative, trifoliate orange (Poncirus trifoliata (L.) Raf.), which are evergreen and deciduous, respectively, and show different seasonal flowering characteristics. In Satsuma mandarin, in which floral induction is triggered by low temperatures during fall and winter, mRNA levels of the citrus FLOWERING LOCUS T homologue CiFT increased during fall and winter, corresponding to the floral induction period, and mRNA levels of citrus LEAFY and SEPALLATA homologues (CsLFY and CuSEP) increased during early spring just before blooming. Citrus APETALA1 and FRUITFULL homologues (CsAP1 and CuFUL) did not show a significant association with seasonal flowering. In trifoliate orange, in which floral induction and flower bud development occur during early summer as in many deciduous trees, expression of CiFT, CsLFY, CsAP1, CuSEPs and CuFUL increased during early summer, corresponding to the period of floral induction and flower bud development. The CuSEPs expression peaked again during early spring just before blooming. In both species, the citrus TERMINAL FLOWER1 homologue (CsTFL), which acts as a floral repressor, showed low transcript levels during the period of floral induction and flower bud development. Thus, despite the difference in flowering season, in both species transcriptional changes in CiFT, CsLFY, CsTFL and CuSEPs were correlated with seasonal flowering. In contrast, the correspondence between CsAP1 and CuFUL expression and seasonal flowering differed between the species.