[Show abstract][Hide abstract] ABSTRACT: The establishment of a method for citrus cultivar identification is required to protect domestic citrus producers and breeders’ rights. In order to establish a scientific method for citrus cultivar identification with marked reliability, it is necessary to develop appropriate DNA markers. Accordingly, this study aimed to establish such a method based on CAPS markers to realize expedient cultivar identification. Thirty-three citrus cultivars and breeding lines were genotyped using nine CAPS markers and the results obtained. The data constitute a source of basic information for the identification of domestic citrus cultivars. Using the genotyping data, we computed the smallest marker set that can distinguish between all 33 cultivars and breeding lines. This showed that all cultivars and breeding lines could be identified using eight CAPS markers. The genotyping data were also used to perform parentage analysis of seven cultivars bred in Japan. This analysis revealed that ‘Shiranui’ was not the male parent of ‘Kanpei’. Further analysis strongly suggested that Ponkan was the male parent of ‘Kanpei’.
Horticultural Research (Japan) 04/2015; 14(2):127-133. DOI:10.2503/hrj.14.127
[Show abstract][Hide abstract] ABSTRACT: Most citrus tristeza virus (CTV) strains infect almost all Citrus species and have caused serious damage on citrus production in many countries. Poncirus trifoliata (trifoliate orange) has a single dominant resistance gene effective against a broad range of CTV strains. P. trifoliata is sexually compatible with Citrus species, and introgression breeding programs have been initiated to introduce the CTV resistance into Citrus. “Kankitsu Chukanbohon Nou 8 Gou” (Nou-8) was developed by crossing Citrus sp. “Kiyomi” with an F1 hybrid derived from Citrus hassaku × P. trifoliata, but it still had undesirable eating traits. In aim to progress marker-assisted introgression breeding for CTV resistance efficiently, we selected 189 DNA markers which could be applied to genotyping for Citrus and P. trifoliata and constructed a genetic linkage map of Nou-8. The map included 189 DNA markers on 10 linkage groups and extended about 722.8 cM in total length. CTV resistance locus was located at 2.4 cM position in linkage group 02_(1), with the same location found when progeny were scored for resistance using an ELISA assay and for DNA marker for CTV resistant (CTV103). Graphical genotypes of Nou-8 and its progeny suggested that (1) 36 % of the chromosome in the BC’2 population were inherited from Nou-8 without any recombination sites, (2) Nou-8 had been already completely substituted by Citrus genotype in two linkage groups, and (3) four of 93 progeny of Nou-8 had recombination sites in the flanking region of the CTV resistance gene locus.
[Show abstract][Hide abstract] ABSTRACT: Three cDNA clones from Satsuma mandarin (Citrus unshiu Marc.) were isolated and expressed in Escherichia coli. CuSTS3-1 and CuSTS3-2 encode linalool synthases and CuSTS4 encodes a nerolidol/linalool synthase. Transcripts of CuSTS3-1, CuSTS3-2 and CuSTS4 were abundant in young fruit at 60 days after flowering (DAF), flowers and leaves, respectively. Treatments with Xanthomonas citri subsp. citri (XCC), the causal agent of citrus canker and Penicillium italicum (PI), the cause of post-harvest fruit decay, and wounding up-regulated CuSTS3-1 in fruit and mainly CuSTS4 in leaves. Linalool, citral, geraniol and citronellol showed strong antibacterial and antifungal activities against XCC and PI in vitro, while most other mono-and sesquiterpenes, including limonene and gamma-terpinene, did not. Linalool, used at levels similar to those present in resistant Ponkan mandarin (Citrus reticulata Blanco) leaves, was able to inhibit growth of XCC in vitro. Compared to other five citrus types, linalool accumulated at extraordinarily high levels in Ponkan mandarin leaves and was released at high amounts from their leaves, while it was hardly detectable in the most susceptible species, indicating that linalool biosynthesis and accumulation might be involved in plant defense against bacterial and fungal pathogens and be associated with field resistance to citrus canker.
[Show abstract][Hide abstract] ABSTRACT: 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 com-parison of the genetic map and the Clementine genome sequence. The 708 gene-based markers were used to con-struct a genetic map using the 87 progenies (AG popula-tion) 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×tan-gelo)×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 cen-timorgans (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 tofold_04, Scaffold_08, and Scaffold_05, respectively. LG-08 and LG-09 contained morphological traits controlling em-bryo 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 pro-teins. The 708 gene-based markers and the AGI map are valuable for integrating various citrus genetic maps, align-ment of genomic sequences, chromosome assignment, and understanding the diversity of citrus germplasms.
[Show abstract][Hide abstract] ABSTRACT: The expression profiles of carotenoid metabolism pathway genes are one of the important factors that regulate carotenoid content and composition in the fruit of Citrus cultivars. To identify regulatory factors of gene expressions in the carotenoid pathway, expression quantitative trait loci (eQTL) analysis was applied using a hybrid population. In the analyzed population, significant eQTLs for phytoene synthase (PSY), phytoene desaturase (PDS), zeta-carotene desaturase (ZDS), beta-ring hydroxylase (HYb), and zeaxanthin epoxidase (ZEP) were detected. Among these eQTLs, eQTLs for HYb and ZEP were located on their responsible gene loci, respectively. This result indicates that the expression level of HYb and ZEP was influenced by cis-elements in their up-stream regions. Conversely, eQTLs for PDS and ZDS were located in different loci from those of the responsible genes, indicating that the loci were trans-regulating factors. It also suggested that expression levels of PDS and ZDS were regulated by a common transcription factor because their eQTLs were co-localized. Significant eQTL of lycopene beta-cyclase (LCYb) and 9-cis-epoxycarotenoid dioxygenase (NCED) were not detected in this population. The major factors to control gene expression depend on each carotenoid metabolism gene, and their combination might cause complex carotenoid metabolism and extend the variation of content and composition in citrus varieties. eQTL analysis was demonstrated as a powerful tool to identify cis- or trans-regulation for carotenoid metabolism genes, as well as in generating functionally defined markers for marker-assisted selection to increase the carotenoid content of citrus in breeding programs.
Journal- Japanese Society for Horticultural Science 03/2014; 83(1):32–43. DOI:10.2503/jjshs1.CH-054 · 0.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Fluorescence in situ hybridization (FISH) was used to localize rDNA sites in relation to heterochromatic regions revealed by double chromomycin A3 (CMA) and 4′-6-diamidino-2-phenylindole (DAPI) in somatic chromosomes of Meiwa kumquat (Fortunella crassifolia Swing.). Four sites were observed, all corresponding to CMA positive regions. Two of them were located in the centromeric region of the two chromosomes with three CMA bands (type A chromosome), and the other two, in the long arm's terminal region of the chromosomes with terminal CMA bands on both telomeres (type C). These sites were of different sizes among probable homologous chromosomes, most likely due to a hybrid origin of this accession. The sites located in the type C chromosomes represent a new locus of rRNA genes in these taxa, suggesting that different evolutionary pathways may lead to a similar heterochromatin distribution, and, hence, that chromosomes with the same CMA banding pattern among different species may not always be homoeologous.
[Show abstract][Hide abstract] ABSTRACT: DNA markers are frequently used to analyze crop varieties, with the coded marker data summarized in a computer-generated table. Such summary tables often provide extraneous data about individual crop genotypes, needlessly complicating and prolonging DNA-based differentiation between crop varieties. At present, it is difficult to identify minimal marker sets - the smallest sets that can distinguish between all crop varieties listed in a marker-summary table - due to the absence of algorithms capable of such characterization. Here, we describe the development of just such an algorithm and MinimalMarker, its accompanying Perl-based computer program. MinimalMarker has been validated in variety identification of fruit trees using published datasets and is available for use with both dominant and co-dominant markers, regardless of the number of alleles, including SSR markers with numeric notation. We expect that this program will prove useful not only to genomics researchers but also to government agencies that use DNA markers to support a variety of food-inspection and -labeling regulations.
Journal of Bioinformatics and Computational Biology 04/2013; 11(2):1250022. DOI:10.1142/S0219720012500229 · 0.78 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Scientia Horticulturae 09/2012; 145:102. DOI:10.1016/j.scienta.2012.08.001 · 1.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Polyembryony, in which multiple somatic nucellar cell-derived embryos develop in addition to the zygotic embryo in a seed, is common in the genus Citrus. Previous genetic studies indicated polyembryony is mainly determined by a single locus, but the underlying molecular mechanism is still unclear. As a step towards identification and characterization of the gene or genes responsible for nucellar embryogenesis in Citrus, haplotype-specific physical maps around the polyembryony locus were constructed. By sequencing three BAC clones aligned on the polyembryony haplotype, a single contiguous draft sequence consisting of 380 kb containing 70 predicted open reading frames (ORFs) was reconstructed. Single nucleotide polymorphism genotypes detected in the sequenced genomic region showed strong association with embryo type in Citrus, indicating a common polyembryony locus is shared among widely diverse Citrus cultivars and species. The arrangement of the predicted ORFs in the characterized genomic region showed high collinearity to the genomic sequence of chromosome 4 of Vitis vinifera and linkage group VI of Populus trichocarpa, suggesting that the syntenic relationship among these species is conserved even though V. vinifera and P. trichocarpa are non-apomictic species. This is the first study to characterize in detail the genomic structure of an apomixis locus determining adventitious embryony.
[Show abstract][Hide abstract] ABSTRACT: Citrus trees alternate between rich and poor crops and are known to be alternate-bearing crops. Alternate bearing results from suppression of flowering due to bearing of fruits. To understand the molecular mechanism how fruit bearing affects flowering, we investigated the relationship between fruits and a flowering-related gene, citrus FLOWERING LOCUS T (CiFT). On trees with different amounts of fruits, the fruit weight/leaf area ratio at harvest was negatively and highly correlated with CiFT expression in the vegetative shoots during fall and winter, which is the period of floral induction. In addition, CiFT expression levels during fall and winter were positively and highly correlated with the flower number the following spring. These results indicate that fruit growth suppresses CiFT expression and decreases the flower number the next spring. In another experiment conducted to determine the effect of the period of fruit bearing on CiFT expression, trees having 3 primary scaffold branches were analyzed. From each branch in 1 tree, all the flowers or fruits were harvested at different times. In November, CiFT was expressed at different levels in each branch, with a tendency to be low in the stem of vegetative shoots from branches that bore fruits for longer periods. This result indicates that a long fruit-bearing period suppresses CiFT expression in vegetative shoots. CiFT expression was detected at much higher levels in fruit-bearing shoots than in vegetative shoots in September. In January, the high levels of CiFT expression in bearing shoots decreased to levels lower than those found in vegetative shoots. Thus, in fruit-bearing shoots, the CiFT expression of an unknown relationship to floral induction is observed during the period before floral induction and the seasonal change of CiFT expression in fruit-bearing shoots is different from that in vegetative shoots.
[Show abstract][Hide abstract] ABSTRACT: In fruit trees and vegetative reproductive plants, viral elimination from mother plants is conducted to produce virus- and viroid-free nursery plants. In citrus, shoot tip culture and semi-micrografting combined with thermotherapy were adopted for viral elimination; however, these methods should be modified for ease or higher efficiency. One solution might be the application of effective antiviral agents. In this study, we tested the efficacy of 4 antiviral agents, ribavirin, foscarnet, zidovudine, and enfuvirtide, against Satsuma dwarf virus (SDV) in Citrus unshiu Marc. 'Ueno-wase' to screen effective agents. The results showed that foscarnet had the highest efficacy against SDV at semi-micrografting combined with thermotherapy, and that the efficacy was significantly different from that of the untreated control. On the other hand, ribavirin, which was thought to be efficient against some citrus viruses, has no significant effect against SDV. There were no harmful effects of foscarnet or ribavirin on re-generation. It was considered that the optimization of treatment method and concentration is required before applying ribavirin as an antiviral agent against SDV. Foscarnet was thought to be highly effective against SDV and was also expected to have high efficacy against other plant viruses. Moreover, our results suggested the possibility that other human antiviral agents that have not been investigated in plants could be effective against plant viruses.
[Show abstract][Hide abstract] ABSTRACT: 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 ofCTV 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 ofthe linkage groups so that these markers could be used to distinguishP. trifoliata genome regions remaining in the hybrid progenies. The set ofPCR primers developed in this study will be useful for marker assisted backcrossing to introduce the P. trifoliata CTV resistance gene into Citrus.
Journal- Japanese Society for Horticultural Science 01/2011; 80(3):295-307. DOI:10.2503/jjshs1.80.295 · 0.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To obtain genetic information on carotenoid content in Citrus fruit for a breeding program, we investigated quantitative trait loci (QTL) in a mapping population derived from a cross between ‘Okitsu-46’ and ‘Nou-5’. Among individual progeny, total and each carotenoid contents were segregated in a transgressive manner. QTLs for total and each carotenoid contents were detected by Kruskal-Wallis analysis (P < 0.01) and interval mapping (LOD (logarithm (base 10) of odds) > 2.0) from 51 individuals. Most of the QTLs for each carotenoid component were mapped to different locations on the linkage map. In β-cryptoxanthin, the strongest QTL (LOD 3.4) was detected on linkage group 6 of ‘Nou-5’, and accounted for 26.9% of explainable variance. LOD scores of QTLs for total carotenoid content were lower than those for each carotenoid content. There were one QTL for total carotenoid content at the criteria of LOD > 2.0, and seven weaker QTLs, which overlapped with QTLs for each carotenoid component. The effects of QTLs detected for each carotenoid content worked cumulatively to increase total carotenoid content. This study provides preliminary data because we obtained data in only one season and from a limited number of individuals (n = 51). However, the results suggested that QTL information could be used to generate DNA markers to select progeny with higher carotenoid content.
Journal- Japanese Society for Horticultural Science 01/2011; 80(2):136-144. DOI:10.2503/jjshs1.80.136 · 0.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Tree Physiology 03/2010; 30(3):431-9. DOI:10.1093/treephys/tpp122 · 3.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We transformed the European pear (Pyrus communis L.) 'La France' and 'Ballade' with the Citrus FLOWERING LOCUS T (CiFT) gene, which induces early flowering. Subsequent DNA blot analysis of the transformed and wild-type plants indicated that the transformed plants carried 1-4 copies of CiFT. Of the 8 transformed 'La France' and 7 transgenic 'Ballade' plant lines obtained, 7 and 5 lines respectively, flowered early when cultivated in vitro in a micropropagation medium. No correlation was found between the CiFT copy number and early flowering in vitro, however, the results of RNA gel blot analysis indicated a strong correlation between the expression of the CiFT gene and floral bud differentiation in vitro. Flowers that developed in vitro differed from wild-type flowers in terms of phenotypic traits, such as the number of pistils, petals, sepals, and stamens. To investigate the inheritance pattern of the CiFT gene, we obtained 15 seeds from the fruits of P. communis L. 'Bartlett' plants that were cross-pollinated with transgenic line No. 6, which had the second highest expression of the transgene. Of 7 seedlings that expressed the CiFT gene, 5 flowered within 10 months after they were transferred to a greenhouse. This result indicated that the CiFT gene induced early flowering in the transformed pear plants and that their progeny inherited the early flowering phenotype. JSHS
[Show abstract][Hide abstract] ABSTRACT: 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.
Tree Physiology 05/2009; 29(7):921-6. DOI:10.1093/treephys/tpp021 · 3.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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. JSHS
Journal- Japanese Society for Horticultural Science 01/2009; 78(1):84-89. DOI:10.2503/jjshs1.78.84 · 0.74 Impact Factor