Search

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.

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.

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

We have categorized the dataset from content and emission of terpene volatiles of peel and juice in both Navelina and Pineapple sweet orange cultivars in which D-limonene was either up- (S), down-regulated (AS) or non-altered (EV; control) (“Impact of D-limonene synthase up- or down-regulation on sweet orange fruit and juice odor perception”(A. Rodríguez, J.E. Peris, A. Redondo, T. Shimada, E. Costell, I. Carbonell, C. Rojas, L. Peña, (2016)) [1]). Data from volatile identification and quantification by HS-SPME and GC–MS were classified by Principal Component Analysis (PCA) individually or as chemical groups. AS juice was characterized by the higher influence of the oxygen fraction, and S juice by the major influence of ethyl esters. S juices emitted less linalool compared to AS and EV juices.

To enrich carotenoids, especially β-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 β-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 β-cryptoxanthin and violaxanthin, in juice sac tissues. Moreover, the pyramiding of these alleles also increased the β-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.

Salicylic acid and jasmonic acid are two important plant hormones that trigger the plant defense responses and regulate the accumulation of bioactive compounds in plants. In the present study, the effects of salicylic acid (SA) and methyl jasmonate (MeJA) on flavonoid and carotenoid accumulation were investigated in the juice sacs of Satsuma mandarin in vitro. The results showed that SA treatment was effective to enhance the contents of eriocitrin, narirutin, poncirin, and &beta, cryptoxanthin in the juice sacs (p &lt, 0.05). In contrast, the MeJA treatment inhibited flavonoid and carotenoid accumulation in the juice sacs (p &lt, 0.05). Gene expression results showed that the changes of flavonoid and carotenoid contents in the SA and MeJA treatments were highly regulated at the transcriptional level. In addition, a transcriptional factor CitWRKY70 was identified in the microarray analysis, which was induced by the SA treatment, while suppressed by the MeJA treatment. In the SA and MeJA treatments, the change in the expression of CitWRKY70 was consistent with that of flavonoid and carotenoid biosynthetic key genes. These results indicated that CitWRKY70 might be involved in the regulation of flavonoid and carotenoid accumulation in response to SA and MeJA treatments in the juice sacs of citrus fruit.

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.

Terpene volatiles play an important role in the interactions between specialized pathogens and fruits. Citrus black spot (CBS), caused by the fungus Phyllosticta citricarpa, is associated with crop losses in different citrus-growing areas worldwide. The pathogen may infect the fruit for 20-24 weeks after petal fall, but the typical hard spot symptoms appear when the fruit have almost reached maturity, caused by fungal colonization and the induction of cell lysis around essential oil cavities. d-Limonene represents approximately 95% of the total oil gland content in mature orange fruit. Herein, we investigated whether orange fruit with reduced d-limonene content in peel oil glands via an antisense (AS) approach may affect fruit interaction with P. citricarpa relative to empty vector (EV) controls. AS fruit showed enhanced resistance to the fungus relative to EV fruit. Because of the reduced d-limonene content, an over-accumulation of linalool and other monoterpene alcohols was found in AS relative to EV fruit. A global gene expression analysis at 2 h and 8 days after inoculation with P. citricarpa revealed the activation of defence responses in AS fruit via the up-regulation of different pathogenesis-related (PR) protein genes, probably as a result of enhanced constitutive accumulation of linalool and other alcohols. When assayed in vitro and in vivo, monoterpene alcohols at the concentrations present in AS fruit showed strong antifungal activity. We show here that terpene engineering in fruit peels could be a promising method for the development of new strategies to obtain resistance to fruit diseases.

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.

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, β-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 β,β-branch to β,e-branch of the carotenoid biosynthetic pathway.

To explore the transcription factors associated with carotenoid metabolism in citrus fruit, one transcription factor (CubHLH1) was selected through microarray screening in Satsuma mandarin (Citrus unshiu Marc.) fruit, which was treated with exogenous ethylene or gibberellin (GA), accelerating or retarding carotenoid accumulation in peel, respectively. The amino acid sequence of CubHLH1 has homology to Arabidopsis activation-tagged bri1 suppressor 1 (ATBS1) interacting factor (AIF), which is functionally characterized as a negative regulator of the brassinolide (BR) signalling pathway. Yeast two-hybrid analysis revealed that protein for CubHLH1 could interact with Arabidopsis and tomato ATBS1. Overexpression of CubHLH1 caused a dwarf phenotype in transgenic tomato (Solanum lycopersicum L.), suggesting that CubHLH1 has a similar function to Arabidopsis AIF. In the transgenic tomato fruit at ripening stage, the lycopene content was reduced along with the changes in carotenoid biosynthetic gene expression. The abscisic acid (ABA) content of all the transgenic tomato fruit was higher than that of the wild type. These results implied that CubHLH1 is considered to have a similar function to Arabidopsis AIFs and might be directly involved in carotenoid metabolism in mature citrus fruit.

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').

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. Electronic supplementary material The online version of this article (10.1186/s12870-018-1369-3) contains supplementary material, which is available to authorized users.

Flavonoid biosynthesis in grape ( Vitis spp.) berry skin is affected by environmental factors such as light andtemperature. However, the components of the light signaling and low-temperature-induced abscisic acid(ABA) signaling networks related to flavonoid accumulation in grape berry skin have not been fullyelucidated, and how environmental conditions affect these components remains unclear. To clarify the detailsof the possible light- and ABA-related signal transduction networks, we developed a grape oligo-DNAmicroarray (38,549 independent probes) using the publicly available genomic sequence of grape, andperformed comprehensive transcriptome analysis using detached ‘Pione’ grape (Vitis labruscana × V. vinifera)berries cultured under different light and temperature conditions. Using the microarray data, we explored thelight-inducible and low-temperature-inducible genes in post-veraison grape berries. We identified 40 light-inducible genes, 55 low-temperature-inducible genes, and 34 genes induced by light plus low temperature.Among these, we selected elongated hypocotyl 5 (HY5), open stomata 1 (OST1 ), and enhanced response to ABA 1(ERA1) as candidate light-inducible, low-temperature-inducible, and light- plus low-temperature-induciblegenes, respectively. We investigated their detailed expression characteristics in grape accessions by means ofquantitative real-time polymerase chain reaction analyses, and hypothesized that HY5, OST1 , and ERA1 mightbe involved in flavonoid biosynthesis via light signaling and low-temperature signaling networks. We alsoestablished an extensive catalog of gene expression patterns to support future investigations of other candidategenes that respond to light and temperature in grape berry skin.Key Words: abscisic acid, anthocyanins, environmental factors, flavonoids, transcriptome analysis.IntroductionAnthocyanins and flavonols, which are two classes offlavonoids, are secondary plant metabolites that accu-mulate in various plant organs. The skin color of grape(black, red, or white) is mainly determined by the con-tent and composition of anthocyanins, and flavonolscontribute to wine color through co-pigmentation withanthocyanins ( Baranac et al., 1997 ). Coloration of grapeberry skin is an important determinant of consumerpreference and marketability. However, in regions witha warm climate, decreased grape quality, such as poor

Terpene volatiles play an important role in the interactions between specialized pathogens and fruits. Citrus black spot (CBS), caused by the fungus Phyllosticta citricarpa, is associated with crop losses in different citrus‐growing areas worldwide. The pathogen may infect the fruit for 20–24 weeks after petal fall, but the typical hard spot symptoms appear when the fruit have almost reached maturity, caused by fungal colonization and the induction of cell lysis around essential oil cavities. d‐Limonene represents approximately 95% of the total oil gland content in mature orange fruit. Herein, we investigated whether orange fruit with reduced d‐limonene content in peel oil glands via an antisense (AS) approach may affect fruit interaction with P. citricarpa relative to empty vector (EV) controls. AS fruit showed enhanced resistance to the fungus relative to EV fruit. Because of the reduced d‐limonene content, an over‐accumulation of linalool and other monoterpene alcohols was found in AS relative to EV fruit. A global gene expression analysis at 2 h and 8 days after inoculation with P. citricarpa revealed the activation of defence responses in AS fruit via the up‐regulation of different pathogenesis‐related (PR) protein genes, probably as a result of enhanced constitutive accumulation of linalool and other alcohols. When assayed in vitro and in vivo, monoterpene alcohols at the concentrations present in AS fruit showed strong antifungal activity. We show here that terpene engineering in fruit peels could be a promising method for the development of new strategies to obtain resistance to fruit diseases.

After a long juvenile period, citrus trees undergo seasonal flowering cycles. Under natural conditions, citrus flowering is regulated mainly by low ambient temperatures around 15-20 °C and water deficit stress. Recent studies have revealed that fluctuations in the expression of citrus homologs of FLOWERING LOCUS T (FT, encoding a flowering integrator) are correlated with their presumed role as flower-promoting signals. Previous ectopic expression analyses have demonstrated the flower-promoting function of citrus FT homologs. In this study, we examined whether abscisic acid (ABA) affects the expression of FT homologs and the flowering induced by low ambient temperatures. Application of exogenous ABA to potted Satsuma mandarin (Citrus unshiu Marc.) trees resulted in transient accumulation of citrus FT homolog transcripts. The promoter of one citrus FT homolog, CiFT3, was active in transgenic A. thaliana (Arabidopsis thaliana) and responded to exogenous and endogenous ABA. CiFT3 is preferentially expressed in shoots, and its expression was affected by flower-inductive treatments. Endogenous ABA accumulated in mandarin shoots during the floral induction period at 15 °C and under field conditions. The accumulation of ABA was correlated with the accumulation of FT homolog transcripts and flowering intensity. It was consistent with changes in the expression of genes related to ABA metabolism. The abundance of carotenoid precursors that serve as substrates for ABA biosynthesis decreased in leaves during the accumulation of ABA. Our data indicate that ABA and carotenoid precursors in leaves influence the flowering of mandarin trees induced by low temperature.

[EN] Citrus fruits are characterized by a complex mixture of volatiles making up their characteristic aromas, being the D-limonene the most abundant one. However, its role on citrus fruit and juice odor is controversial. Transgenic oranges engineered for alterations in the presence or concentration of few related chemical groups enable asking precise questions about their contribution to overall odor, either positive or negative, as perceived by the human nose. Here, either down- or up-regulation of a D-limonene synthase allowed us to infer that a decrease of as much as 51 times in D-limonene and an increase of as much as 3.2 times in linalool in juice were neutral for odor perception while an increase of only 3 times in ethyl esters stimulated the preference of 66% of the judges. The ability to address these questions presents exciting opportunities to understand the basic principles of selection of food. (C) 2016 Elsevier Ltd. All rights reserved., We are grateful to Drs. Lorenzo Zacarias and M. Jesus Rodrigo (IATA-CSIC) for the GC-MS facilities and support. We would like to acknowledge also to Drs. Berta Alquezar and Elsa Pons for their critical review of the manuscript and to all the panelists that participated in the sensory panel. This research is being funded in part by Fundo de Defesa da Citricultura (Fundecitrus).

We developed 708 gene-based markers for citrus genome analysis. Sequence-tagged site (STS) primers were designed that were located in conserved exon regions and whose PCR products spanned genomic introns. Of these, 79.7 % comprised cleaved amplified polymorphic sequence markers. The gene-based markers and their annotation and position on Clementine scaffolds ver. 1.0 permitted comparison of the genetic map and the Clementine genome sequence. The 708 gene-based markers were used to construct a genetic map using the 87 progenies (AG population) from the cross between ‘Okitsu 46 gou’ (‘Sweet Spring’ (‘Ueda unshiu’ (Citrus unshiu) × Hassaku (Citrus hassaku Hort. ex Tanaka)) × ‘Trovita’ orange) × ‘Kankitsu Chukanbohon Nou 5 gou’ (‘Lee’ (Citrus clementina × tangelo) × Citrus kinokuni). The markers were integrated using common STSs on different phase maps in cross-pollination mode. The integrated map (AGI map) comprised 706 loci, including two morphological traits, and spanned 990.9 centimorgans (cM) with an average marker distance of 1.40 cM. These markers formed nine linkage groups (LGs) (corresponding to citrus physical chromosomes): LG-01 to LG-09 corresponded to Scaffold_01, Scaffold_07, Scaffold_09, Scaffold_06, Scaffold_03, Scaffold_02, Scaffold_04, Scaffold_08, and Scaffold_05, respectively. LG-08 and LG-09 contained morphological traits controlling embryo color and seedlessness. Eighty-eight loci comprised three or more alleles on the AGI map; 36.4 % of them were related to transcription factors and DNA-binding proteins. The 708 gene-based markers and the AGI map are valuable for integrating various citrus genetic maps, alignment of genomic sequences, chromosome assignment, and understanding the diversity of citrus germplasms.

Citrus fruits contain significant amounts of various carotenoids and some of them are known to benefit human health. Approximately 115 different carotenoids have been reported in citrus fruits, and the color of the fruit and peel are caused by carotenoid accumulation (Stewart and Wheaton, 1973). The carotenoid content and composition in

Apomixis is a common reproduction system in the genus Citrus. To investigate the molecular mechanism of apomictic embryogenesis in Citrus, genes expressed specifically in an apomictic genotype were isolated by PCR-selected suppression subtractive hybridization with total RNAs obtained from the ovule at anthesis. Several genes showing conspicuously different expressions between polyembryonic (apomictic) and monoembryonic (nonapomictic) genotypes were selected, and their expression profiles during ovule development were analyzed in detail. This analysis identified two apomictic and three nonapomictic genotype-specific genes. Among the latter, msg-2 was highly expressed in the late stage of somatic embryogenesis. Specific expression during ovule development in monoembryonic cultivars and in the late stage of somatic embryogenesis indicated that msg-2 is not expressed in the initiation stage of polyembryogenesis and somatic embryogenesis, suggesting its role in suppressing initial cell formation of somatic embryos. The full-length complementary DNA of msg-2 contained small open reading frames in its sequence but showed no homology to functionally known genes in the public databases. As sequences similar to msg-2 were frequently found among Citrus expressed sequence tags, msg-2 may be associated with polyembryogenesis and somatic embryogenesis in a Citrus-specific manner.

In order to clarify whether high linalool content in citrus leaves alone induces strong field resistance to citrus canker caused by Xanthomonas citri subsp. citri (Xcc), and to assess whether this trait can be transferred to a citrus type highly sensitive to the bacterium, transgenic 'Hamlin' sweet orange (Citrus sinensis L. Osbeck) plants over-expressing a linalool synthase gene (CuSTS3-1) were generated. Transgenic lines (LIL) with the highest linalool content showed strong resistance to citrus canker when spray inoculated with the bacterium. In LIL plants inoculated by wounding (multiple-needle inoculation), the linalool level was correlated with the repression of the bacterial titer and up-regulation of defense-related genes. The exogenous application of salicylic acid, methyl jasmonate or linalool triggered responses similar to those constitutively induced in LIL plants. The linalool content in Ponkan mandarin leaves was significantly higher than that of leaves from six other representative citrus genotypes with different susceptibilities to Xcc. We propose that linalool-mediated resistance might be unique to citrus tissues accumulating large amounts of volatile organic compounds in oil cells. Linalool might act not only as a direct antibacterial agent, but also as a signal molecule involved in triggering a non-host resistance response against Xcc.

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’).

CuSTS1 cDNA clone was isolated from Satsuma mandarin ( Citrus unshiu Marc.) and functionally characterized. Genomic clone corresponding to CuSTS1 consists of 7 exons and 6 introns which is typical structure of angiosperm sesquiterpene synthase genes. Their predicted proteins possess a general feature of plant sesquiterpene synthases and RR motif and DDXXD motifs were conserved. Phylogenetic tree analysis showed that the molecular evolutions of citrus germacrene synthase might occur according to enzymatic function before the divergence of Citrus species, in contrast to citrus monoterpene synthase genes. Functionally analysis indicated that CuSTS1 protein produces mainly germacrene A from farnesyl diphosphate (FPP) in accompany with side product. In Satsuma mandarin, transcription of CuSTS1 were comparatively abundant in flowers and young fruit at 60 days after flowering (DAF), and peel at 120 DAF. The transcription was decreasing toward fruit maturing. In young fruit at 120 DAF, CuSTS1 is induced by methyl jasmonic acid, salicylic acid and ethylene treatments, suggesting it would be involved in plant defense response.

We developed a 384 multiplexed SNP array, named CitSGA-1, for the genotyping of Citrus cultivars, and evaluated the performance and reliability of the genotyping. SNPs were surveyed by direct sequence comparison of the sequence tagged site (STS) fragment amplified from genomic DNA of cultivars representing the genetic diversity of citrus breeding in Japan. Among 1497 SNPs candidates, 384 SNPs for a high-throughput genotyping array were selected based on physical parameters of Illumina’s bead array criteria. The assay using CitSGA-1 was applied to a hybrid population of 88 progeny and 103 citrus accessions for breeding in Japan, which resulted in 73,726 SNP calls. A total of 351 SNPs (91 %) could call different genotypes among the DNA samples, resulting in a success rate for the assay comparable to previously reported rates for other plant species. To confirm the reliability of SNP genotype calls, parentage analysis was applied, and it indicated that the number of reliable SNPs and corresponding STSs were 276 and 213, respectively. The multiplexed SNP genotyping array reported here will be useful for the efficient construction of linkage map, for the detection of markers for marker-assisted breeding, and for the identification of cultivars.

Citrus tristeza virus (CTV) is an acute pathogen that causes serious damage to the citrus industry. Poncirus trifoliata (L.) Raf., a sexually compatible species with Citrus, has resistance against a broad range of CTV strains. Breeding programs have been conducted to introduce the CTV resistance gene from P.trifoliata to Citrus, but no commercial cultivar has yet been developed. In this study, we developed four selection markers linked to CTV resistance to enable marker assisted selection to efficiently introduce CTV resistance into Citrus. The four CTV resistance-linked markers were composed of a co-dominant single nucleotide polymorphism (SNP) marker and three dominant sequence tagged site (STS) markers. All four markers were fitted to the progeny and were linked to CTV resistance, with only 2.8% exceptions. We also developed 46 P.trifoliata allele identification markers from alleles of 35 Citrus species. Among the 46 markers, nine were located in linkage group 2, on which the CTV resistance locus is located. We located the other 31 markers on the rest of the linkage groups so that these markers could be used to distinguish P.trifoliata genome regions remaining in the hybrid progenies. The set of PCR primers developed in this study will be useful for marker assisted backcrossing to introduce the P.trifoliata CTV resistance gene into Citrus.

To understand the role of the MIKC-type dormancy-associated MADS-box (DAM) genes in the regulation of endodormancy in japanese pear (Pyrus pyrifolia), we isolated two DAM genes from ‘Kosui’ and characterized their expression throughout the seasonal endodormancy phases in ‘Kosui’, as well as in TP-85–119 taiwanese pear (P. pyrifolia), which is a less dormant type. Several copies of the corresponding DAM genes are present in the P. pyrifolia genome. Rapid amplification of cDNA ends enabled the isolation of two full-length cDNAs, designated as PpMADS13–1 and PpMADS13–2, with complete open reading frames encoding 227 and 234 amino acids, respectively. Multialignment of the two ‘Kosui’ and the database DAM genes (based on the deduced amino acid sequences) showed that PpMADS13–1 and PpMADS13–2 were highly identical to the Rosaceae DAM genes and encoded the conserved domains characteristic of other MIKC-type MADS-box genes. The phylogenetic relationships showed that PpMADS13–1 and PpMADS13–2 were more closely related to the Prunus DAM, though they formed a unique subclade. The specific expression analysis of PpMADS13–1 and PpMADS13–2 by real-time polymerase chain reaction showed that both DAM genes are gradually down-regulated concomitant with endodormancy breaking. PpMADS13–1 and PpMADS13–2 showed similar fluctuations in expression patterns, although PpMADS13–2 was more highly expressed relative to PpMADS13–1. The expression of PpMADS13–1 and PpMADS13–2 in the less dormant taiwanese pear, TP-85–119, was quite low (nearly zero level), which is consistent with a down-regulated pattern of expression of the DAM genes in japanese pear, peach (Prunus persica), and japanese apricot (Prunus mume). Differential genomic DNA methylation patterns detected in PpMADS13–1 and PpMADS13–2 were not concomitant with seasonal endodormancy transition phases, suggesting that DNA methylation in these loci under investigation may not be linked to endodormancy progression in ‘Kosui’.

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.

To obtain a peel-specific promoter useful for citrus transgenic research, the 5' flanking region of a citrus d-limonene synthase gene (CitMTSE2), which showed a high degree of expression in the peel of mature fruits, was isolated from the satsuma mandarin (Citrus unshiu Marc.) Bacterial Artificial Chromosome (BAC) library. Promoter activity was characterized by particle bombardment, agroinjection and Arabidopsis transformation analyses. Approximately 5 kbps in the 5' upstream region (PCuMTSE2) contained several cis-motifs relating to guard cell-specific and stress-related genes. PCuMTSE2 fused to the uidA gene was directly incorporated into tissues from citrus and its relatives using particle bombardment. The results showed that PCuMTSE2 conferred β-glucronidase (GUS) activity in fruits rather than in seed and leaf tissues. Agroinjection analysis using citrus fruits showed PCuMTSE2 has promoter activity in peel but not in juice sacs. In a transgenic Arabidopsis-incorporated PCuMTSE2::uidA construct, GUS activity was detected in the joint between the stalk and silique. These results suggest that PCuMTSE2 could be utilized as a promoter regulating the quantitatively preferential expression in peel, and is useful for studies of manipulation by genetic engineering in citrus.

Gibberellin 3 (GA 3 )-responsive genes were investigated with a citrus 22K oligoarray 1 to further the understanding of transcriptional regulation by GA 3 treatment in Satsuma mandarin fruit ( Citrus unshiu Marc.). 213 GA 3 -responsive genes were identified that showed a 3-fold or greater expression change after 72 h GA 3 treatment, compared to expression after 72 h air treatment. GA 3 treatment induced expression of pathogenesis-related (PR) proteins and genes that function in photosynthesis, chloroplast biogenesis, resistance, defense and stress. Also, GA 3 treatment reduced the transcription of several ethylene-inducible genes, such as carotenoid metabolic genes, which are associated with fruit ripening. Contrasting effects between GA 3 and ethylene were observed on photosynthesis and chloroplast biogenesis, chlorophyll metabolism, and carotenoid metabolism, indicating that the endogenous GA 3 level might be important for the endogenous regulation of maturation and senescence in mature citrus fruit. It was also found that the GA response pathway was likely to take part in cross-talk with the pathogen-related pathway in mature citrus fruit.

Polyembryony, in which zygotic and apomictic embryos develop concurrently in a seed, is widespread in Citrus. To obtain further genomic information about the previously characterized map position of the polyembryony locus, the relationship among linkage maps from 3 different hybrid populations was determined using common markers flanking the polyembryony locus. By mapping the polyembryony locus with cleaved amplified polymorphic sequence (CAPS) markers in a F180 × ‘Harumi’ population, the polyembryony locus was detected in a high-density marker region co-segregated to marker Mf0086. By screening a bacterial artificial chromosome (BAC) library with marker Mf0086, two contigs reflecting the polyembryony and monoembryony haplotype were constructed. The single nucleotide polymorphism (SNP) markers detected on these contigs were mapped on the nearest position to the polyembryony locus in linkage group 1 of ‘Miyagawa wase’ and ‘Harumi’. The highly conserved marker lineage in the flanking regions of the polyembryony locus in these polyembryonic cultivars of different parentage suggested that the genomic region of the polyembryony locus might have a common structure in a wide range of Citrus cultivars.

A Citrus FT/TFL1 homologue (CuMFT1) was isolated from Satsuma mandarin (Citrus unshiu Marc.), and a high degree of expression was detected in mature seeds, indicating that its promoter is expected to induce the expression of a target gene in the late embryonic stage of Citrus seed. To obtain a seed-specific promoter useful for Citrus transgenic research, the 5′ upstream region of CuMFT1 was isolated from the BAC library, and its promoter activity was characterized using particle bombardment and transgenic Arabidopsis. The 2.4 kbp in the 5′ upstream region (CuMFT1 promoter) contained RY (CATGCAT), E-box (CANNTG), and distant B-box (GCCACTTGTC) cis-elements, which have been reported to promote seed-specific gene expression in plants. The CuMFT1 promoter fused to the uidA gene was directly incorporated into tissues from Citrus and its relatives using particle bombardment. The results showed that the CuMFT1 promoter conferred high β-glucronidase (GUS) activity in seed. The CuMFT1 promoter-GUS fusion construct was also incorporated into Arabidopsis, and transgenic Arabidopsis was evaluated by histochemical staining and fluorometric GUS analysis. The experiments revealed that the CuMFT1 promoter conferred quantitatively preferential expression in Arabidopsis seeds; thus, it was suggested that the cis-elements in the CuMFT1 promoter required for expression in Citrus seed were functionally conserved in the heterologous Arabidopsis plant. The CuMFT1 promoter could be utilized as a promoter regulating the quantitatively preferential expression in seed, and is useful for studies of seed development and manipulation by genetic engineering in Citrus and Arabidopsis.

A comprehensive transcriptome analysis using a citrus 22K oligoarray was performed to identify ethylene-responsive genes and gain an understanding of the transcriptional regulation by ethylene in mandarin fruit (Citrus unshiu Marc.). In the 72 h after ethylene treatment, 1493 genes were identified as ethylene-responsive with more than 3-fold expression change, and an interesting aspect of gene regulation by ethylene was observed, namely, that more than half of the ethylene-responsive genes were repressed. This aspect might suggest that ethylene demotes numerous biological processes and plays an important role in fruit ripening and senescence. Ethylene repressed the transcription of most genes involved in photosynthesis, chloroplast biogenesis, and sugar metabolism, while it induced the transcription of several genes related to resistance, defense, stress, amino acid synthesis, protein degradation, and secondary metabolism. In carotenoid metabolism, the sensitivity and responsive patterns to exogenous ethylene were significantly different among carotenoid biosynthesis genes. Ethylene might cause a change of their transcriptional balance and influence carotenoid composition of fruits. Besides, most of ethylene biosynthesis genes and its signal transduction components did not show any significant expression change (

The genomic region of a type 3 metallothionein (MT3)-like gene ( CitMT45 ) with extensive expression in citrus fruit was isolated from Citrus unshiu Marc. and its promoter activity was analyzed using particle bombardment. A genomic library was screened with CitMT45 cDNA as a probe, and approximately 9.5 kb genomic fragments were obtained. The nucleotide sequence showed that the genomic CitMT45 consists of 3 exons and 2 introns, similar to other plant MT-like genes. Interestingly, the proximal 5′ flanking region had no canonical TATA-box or several cis -regulatory elements of G-box, MRE and ERE motifs common to plant MT-like genes. Lack of the MRE motif in the promoter region is in agreement with no response of CitMT45 to metal treatment, suggesting that CitMT45 might play other biological roles during fruit development rather than detoxification. Two promoters of proximal 190 bp region (PMT45C) and 4.2 kb full region (PMT45L) were fused to the uidA gene and directly incorporated into Citrus tissues using particle bombardment. The PMT45C promoter is sufficient to direct GUS expression in all tested organs and the upstream region from this proximal 190 bp sequence should have cis -elements that act as enhancers in citrus juice sacs. Moreover, a fluorometric GUS analysis of transgenic Arabidopsis indicated that the PMT45L promoter conferred quantitatively preferential expression in siliques. These results might indicate that the cis -elements required for preferential expression in citrus fruits are functionally conserved in heterologous Arabidopsis plants. Thus, it was demonstrated that the PMT45L promoter could be used for manipulation of fruit quality by genetic engineering in Citrus and Arabidopsis .

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.