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This study aimed to identify quantitative trait loci (QTLs) for growth-related traits by constructing a genetic linkage map based on single nucleotide polymorphism (SNP) markers in Japanese quail. A QTL mapping population of 277 F2 birds was obtained from an intercross between a male of a large-sized strain and three females of a normal-sized strain. Body weight (BW) was measured weekly from hatching to 16 weeks of age. Non-linear regression growth models of Weibull, Logistic, Gompertz, Richards, and Brody were analyzed, and growth curve parameters of Richards was selected as the best model to describe the quail growth curve of the F2 birds. Restriction-site associated DNA sequencing developed 125 SNP markers that were informative between their parental strains. The SNP markers were distributed on 16 linkage groups that spanned 795.9 centiMorgan (cM) with an average marker interval of 7.3 cM. QTL analysis of phenotypic traits revealed four main-effect QTLs. Detected QTLs were located on chromosomes 1 and 3 and were associated with BW from 4 to 16 weeks of age and asymptotic weight of Richards model at genome-wide significant at 1% or 5% level. No QTL was detected for BW from 0 to 3 weeks of age. This is the first report identified QTLs for asymptotic weight of the Richards parameter in Japanese quail. These results highlight that the combination of QTL studies and the RAD-seq method will aid future breeding programs identify genes underlying the QTL and the application of marker-assisted selection in the poultry industry, particularly the Japanese quail.

Sorghum [Sorghum bicolor (L.) Moench] grown locally by Japanese farmers is generically termed Takakibi, although its genetic diversity compared with geographically distant varieties or even within Takakibi lines remains unclear. To explore the genomic diversity and genetic traits controlling biomass and other physiological traits in Takakibi, we focused on a landrace, NOG, in this study. Admixture analysis of 460 sorghum accessions revealed that NOG belonged to the subgroup that represented Asian sorghums, and it was only distantly related to American/African accessions including BTx623. In an attempt to dissect major traits related to biomass, we generated a recombinant inbred line (RIL) from a cross between BTx623 and NOG, and we constructed a high-density linkage map based on 3,710 single-nucleotide polymorphisms obtained by restriction-site-associated DNA sequencing of 213 RIL individuals. Consequently, 13 fine quantitative trait loci (QTLs) were detected on chromosomes 2, 3, 6, 7, 8 and 9, which included five QTLs for days to heading, three for plant height (PH) and total shoot fresh weight and two for Brix. Furthermore, we identified two dominant loci for PH as being identical to the previously reported dw1 and dw3. Together, these results corroborate the diversified genome of Japanese Takakibi, while the RIL population and high-density linkage map generated in this study will be useful for dissecting other important traits in sorghum.

White rust caused by Puccinia horiana is one of the most serious diseases of chrysanthemum (Chrysanthemum × morifolium). In this study, we report the DNA markers associated with resistance against P. horiana via a simple approach using the genome of a wild diploid relative, Chrysanthemum seticuspe. First, we identified the important region of the genome in the resistant cultivar “Ariesu” via a genome-wide association study. Simplex single nucleotide polymorphism (SNP) markers mined from ddRAD-Seq were used in a biparental population originating from crosses between resistant “Ariesu” and susceptible “Yellow Queen”. The C. seticuspe genome was used as a reference. For the fine mapping of P. horiana resistance locus 2 (Phr2), a comparative whole genome sequencing study was conducted. Although the genome sequences of chrysanthemum cultivars assembled via the short-read approach were fragmented, reliable genome alignments were reconstructed by mapping onto the chromosome level of the C. seticuspe pseudomolecule. Base variants were then identified by comparing the assembled genome sequences of resistant “Ariesu” and susceptible “Yellow Queen”. Consequently, SNP markers that were closer to Phr2 compared with ddRAD-Seq markers were obtained. These SNP markers co-segregated with resistance in F1 progenies originating from resistant “Ariesu” and showed robust transferability for detecting Phr2-conferring resistance among chrysanthemum genetic resources. The wild C. seticuspe pseudomolecule, a de facto monoploid genome used for ddRAD-Seq analysis and assembled genome sequence comparison, demonstrated this method’s utility as a model for developing DNA markers in hexaploid chrysanthemum cultivars.

Triploid breeding is an effective way to obtain seedless hybrids. In addition to artificial breeding, some triploid cultivars occur naturally in the genus Citrus. ‘Yellow Bell’, a new lemon cultivar recently patented in Japan was selected from the small seed of an open-pollinated ‘Villafranca’ fruit. Therefore, it has an unknown pollen parent. To characterize the pollen parent of ‘Yellow Bell’, possible parental cultivars were genotyped using simple sequence repeat markers, and then the parental relationships were estimated by establishing the inheritance of tri-allelic marker genotypes. As a result, the pollen-donating parent of ‘Yellow Bell’ was determined to be ‘Summer Fresh’, which was growing 40 m from the maternal tree in the same orchard. The parentage of ‘Tahiti lime’, a lime cultivar having a long cultivation history, was evaluated in a similar manner and it was confirmed that ‘Mexican lime’ and a true lemon cultivar were the parents.

Cultivated chrysanthemum (Chrysanthemum morifolium Ramat.) is one of the most economically important ornamental crops grown worldwide. It has a complex hexaploid genome (2n = 6x = 54) and large genome size. The diploid Chrysanthemum seticuspe is often used as a model of cultivated chrysanthemum, since the two species are closely related. To expand our knowledge of the cultivated chrysanthemum, we here performed de novo whole-genome assembly in C. seticuspe using the Illumina sequencing platform. XMRS10, a C. seticuspe accession developed by five generations of self-crossing from a self-compatible strain, AEV2, was used for genome sequencing. The 2.72 Gb of assembled sequences (CSE_r1.0), consisting of 354,212 scaffolds, covered 89.0% of the 3.06 Gb C. seticuspe genome estimated by k-mer analysis. The N50 length of scaffolds was 44,741 bp. For protein-encoding genes, 71,057 annotated genes were deduced (CSE_r1.1_cds). Next, based on the assembled genome sequences, we performed linkage map construction, gene discovery and comparative analyses for C. seticuspe and cultivated chrysanthemum. The generated C. seticuspe linkage map revealed skewed regions in segregation on the AEV2 genome. In gene discovery analysis, candidate flowering-related genes were newly found in CSE_r1.1_cds. Moreover, single nucleotide polymorphism identification and annotation on the C. × morifolium genome showed that the C. seticuspe genome was applicable to genetic analysis in cultivated chrysanthemums. The genome sequences assembled herein are expected to contribute to future chrysanthemum studies. In addition, our approach demonstrated the usefulness of short-read genome assembly and the importance of choosing an appropriate next genome sequencing technology based on the purpose of the post-genome analysis.

This research was conducted to identify quantitative trait loci (QTL) associated with egg-related traits by constructing a genetic linkage map based on single nucleotide polymorphism (SNP) markers using restriction-site associated DNA sequencing (RAD-seq) in Japanese quail. A total of 138 F2 females were produced by full-sib mating of F1 birds derived from an intercross between a male of the large-sized strain with three females of the normal-sized strain. Eggs were investigated at two different stages: the beginning stage of egg-laying and at 12 weeks of age (second stage). Five eggs were analyzed for egg weight, lengths of the long and short axes, egg shell strength and weight, yolk weight and diameter, albumen weight, egg equator thickness, and yolk color (L*, a*, and b* values) at each stage. Moreover, the age at first egg, the cumulative number of eggs laid, and egg production rate were recorded. RAD-seq developed 118 SNP markers and mapped them to 13 linkage groups using the Map Manager QTX b20 software. Markers were spanned on 776.1 cM with an average spacing of 7.4 cM. Nine QTL were identified on chromosomes 2, 4, 6, 10, 12, and Z using the simple interval mapping method in the R/qtl package. The QTL detected affected 10 egg traits of egg weight, lengths of the long and short axes of egg, egg shell strength, yolk diameter and weight, albumen weight, and egg shell weight at the beginning stage, yellowness of the yolk color at the second stage, and age at first egg. This is the first report to perform a quail QTL analysis of egg-related traits using RAD-seq. These results highlight the effectiveness of RAD-seq associated with targeted QTL and the application of marker-assisted selection in the poultry industry, particularly in the Japanese quail.

Root hair cells form the primary interface of plants with the soil environment, playing key roles in nutrient uptake and plant defense. In legumes, they are typically the first cells to become infected by nitrogen-fixing soil bacteria during root nodule symbiosis. Here, we report a role for the CELLULOSE SYNTHASE-LIKE D1 (CSLD1) gene in root hair development in the legume species Lotus japonicus. CSLD1 belongs to the cellulose synthase protein family that includes cellulose synthases and cellulose synthase-like proteins, the latter thought to be involved in the biosynthesis of hemicellulose. We describe 11 Ljcsld1 mutant alleles that impose either short (Ljcsld1-1) or variable (Ljcsld1-2 to 11) root hair length phenotypes. Examination of Ljcsld1-1 and one variable-length root hair mutant, Ljcsld1-6, revealed increased root hair cell wall thickness, which in Ljcsld1-1 was significantly more pronounced and also associated with a strong defect in root nodule symbiosis. Lotus japonicus plants heterozygous for Ljcsld1-1 exhibited intermediate root hair lengths, suggesting incomplete dominance. Intragenic complementation was observed between alleles with mutations in different CSLD1 domains, suggesting CSLD1 function is modular and that the protein may operate as a homodimer or multimer during root hair development.

Root hair cells form the primary interface of plants with the soil environment, playing key roles in nutrient uptake and plant defense. In addition, they are typically the first cells infected by nitrogen-fixing soil bacteria during the root nodule symbiosis. Here we report a role for the Cellulose Synthase-Like D1 (CSLD1) gene in root hair development in Lotus japonicus. CSLD1 belongs to the cellulose synthase protein family that includes cellulose synthases, and cellulose synthase-like proteins, the latter thought to be involved in the biosynthesis of hemicellulose. We describe 11 csld1 mutant alleles that have either short (Ljcsld1-1) or variable length roots hairs (Ljcsld1-2 to 11). Examination of Ljcsld1-1 and one variable-length root hair mutant, Ljcsld1-6 showed increased root hair cell wall thickness, which in Ljcsld1-1 was more pronounced, suggesting a possible link with the defect in root nodule symbiosis. In addition, Ljcsld1-1 heterozygotes had intermediate root hair lengths, between those of wild type and the homozygotes. Intragenic complementation was observed between alleles with mutations in the N-terminal domain and other alleles, suggesting modularity of CSLD1 function and that it may operate as a homodimer or multimer.One sentence summaryThis research describes novel gain- and loss-of-function mutations at the Lotus japonicus CELLULOSE SYNTHASE-LIKE D1 locus and analyzes their impact on root hair development.

Impairment of Lhca4, a subunit of LHCI, causes high chlorophyll content and LHCII accumulation in rice, suggesting a novel functional interaction between LHCI and LHCII., Chlorophyll is an essential molecule for acquiring light energy during photosynthesis. Mutations that result in chlorophyll retention during leaf senescence are called ‘stay-green’ mutants. One of the several types of stay-green mutants, Type E, accumulates high levels of chlorophyll in the pre-senescent leaves, resulting in delayed yellowing. We isolated delayed yellowing1-1 (dye1-1), a rice mutant whose yellowing is delayed in the field. dye1-1 accumulated more chlorophyll than the wild-type in the pre-senescent and senescent leaves, but did not retain leaf functionality in the ‘senescent green leaves’, suggesting that dye1-1 is a Type E stay-green mutant. Positional cloning revealed that DYE1 encodes Lhca4, a subunit of the light-harvesting complex I (LHCI). In dye1-1, amino acid substitution occurs at the location of a highly conserved amino acid residue involved in pigment binding; indeed, a severely impaired structure of the PSI-LHCI super-complex in dye1-1 was observed in a blue native PAGE analysis. Nevertheless, the biomass and carbon assimilation rate of dye1-1 were comparable to those in the wild-type. Interestingly, Lhcb1, a trimeric LHCII protein, was highly accumulated in dye1-1, in the chlorophyll–protein complexes. The high accumulation of LHCII in the LHCI mutant dye1 suggests a novel functional interaction between LHCI and LHCII.

Asteraceae is the largest family of angiosperms, comprising approximately 24,000 species. Molecular genetic studies of Asteraceae are essential for understanding plant diversity. Chrysanthemum morifolium is the most industrially important ornamental species in Asteraceae. Most cultivars of C. morifolium are autohexaploid and self-incompatible. These properties are major obstacles to the genetic analysis and modern breeding of C. morifolium. Furthermore, high genome heterogeneity complicates molecular biological analyses. In this study, we developed a model strain in the genus Chrysanthemum. C. seticuspe is a diploid species with a similar flowering property and morphology to C. morifolium and can be subjected to Agrobacterium-mediated transformation. We isolated a natural self-compatible mutant of C. seticuspe and established a pure line through repeated selfing and selection. The resultant strain, named Gojo-0, was favorable for genetic analyses, including isolation of natural and induced mutants, and facilitated molecular biological analysis, including whole genome sequencing, owing to the simplicity and homogeneity of its genome. Interspecific hybridization with Chrysanthemum species was possible, enabling molecular genetic analysis of natural interspecific variations. The accumulation of research results and resources using Gojo-0 as a platform is expected to promote molecular genetic studies on the genus Chrysanthemum and the genetic improvement of chrysanthemum cultivars.

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.

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.

Pseudomonas resinovorans strain MO-1, which possesses a high ability to oxidize Mn(II), has been isolated from oligotrophic pond sediment. The draft genome sequence consists of 6,252,942 bp and has a G+C content of 63.4%. Strain MO-1 has 5,694 coding sequences, including 13 putative Mn(II) oxidation genes.

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.

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.

Chlorophyll degradation plays important roles in leaf senescence including regulation of degradation of chlorophyll-binding proteins. Although most genes encoding enzymes of the chlorophyll degradation pathway have been identified, the regulation of their activity has not been fully understood. Green cotyledon mutants in legume are stay-green mutants, in which chlorophyll degradation is impaired during leaf senescence and seed maturation. Among them, the soybean (Glycine max) green cotyledon gene cytG is unique because it is maternally inherited. To isolate cytG, we extensively sequenced the soybean chloroplast genome, and detected a 5-bp insertion causing a frame-shift in psbM, which encodes one of the small subunits of photosystem II. Mutant tobacco plants (Nicotiana tabacum) with a disrupted psbM generated using a chloroplast transformation technique had green senescent leaves, confirming that cytG encodes PsbM. The phenotype of cytG was very similar to that of mutant of chlorophyll b reductase catalyzing the first step of chlorophyll b degradation. In fact, chlorophyll b-degrading activity in dark-grown cytG and psbM-knockout seedlings was significantly lower than that of wild-type plants. Our results suggest that PsbM is a unique protein linking photosynthesis in presenescent leaves with chlorophyll degradation during leaf senescence and seed maturation. Additionally, we discuss the origin of cytG, which may have been selected during domestication of soybean.

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.

Transcription of anthocyanin biosynthetic genes is usually controlled by transcription factors such as basic-helix-loop-helix (bHLH) and R2R3-MYB. To know the regulatory mechanisms of anthocyanin accumulation in Asiatic hybrid lily (Lilium spp.), cDNA clones encoding bHLH transcription factors were isolated from tepals and their expression was examined in this study. This is the first report about cDNAs for bHLH genes among the species in monocots other than Gramineae. Two cDNAs corresponding to LhbHLH1 and LhbHLH2 genes were isolated; the former encoded the protein having the similarity to AmDELILA and PhJAF13 and the latter one did to PhAN1. LhbHLH1 and LhbHLH2 were expressed in tepals, stems and leaves. In filaments and pistils, only LhbHLH2 was transcribed. During tepal development, transcriptions of LhbHLH genes were always detected, and the peak of LhbHLH2 expression preceded the peak of LhDFR expression. When lily buds were exposed to light, anthocyanin accumulation was induced in leaves and peaked at 4 days after light exposure. The transcription of LhbHLH2 peaked at 2 days after light exposure and decreased after that. LhbHLH1 transcription was affected by light to a lesser extent than LhbHLH2 at 2 days. These expressional changes preceded the change of anthocyanin amount. Similarly in tepals, both anthocyanin amount and the transcription of LhDFR and LhbHLH2 decreased in the dark after 6 days, but LhbHLH1 transcription was not affected. These results suggest that LhbHLH1 and LhbHLH2, especially LhbHLH2, are involved in anthocyanin biosynthesis, and that response to light exposure was different between LhbHLH1 and LhbHLH2.

The phototropic response in Arabidopsis thaliana is initiated by the blue-light photoreceptors, phototropin (phot)1 and phot2. A recent study has shown that one of the phototropic signal transducers, NPH3, is phosphorylated under dark conditions and dephosphorylated by blue-light irradiation. To further understand the function of phosphorylation and dephosphorylation of NPH3 during this phototropic response, we have mapped the phosphorylation sites of NPH3 in our current study. The NPH3 protein has at least three phosphorylation sites at serine residues, Ser212, Ser222, and Ser236, and these sites are dephosphorylated by blue-light irradiation. By immunoblotting analysis, these phosphorylation sites in phot1 mutants are not dephosphorylated following blue-light irradiation at both low and high fluence rates, even though such irradiations induce the phot2-dependent phototropic response in phot1 . These results suggest that the dephosphorylated NPH3 is involved in the phot1-dependent phototropic response and is not essential for the phot2-dependent phototropic response. We generated two types of transgenic nph3 plants expressing a NPH3 S212A/S222A/S232A/S236A protein and a NPH3 Δ212–238 protein in which the phosphorylation region is deleted, and assessed the phototropic phenotype of these. Based upon our present findings, we discuss the role of dephosphorylated and phosphorylated NPH3 in mediating the phototropic response.

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 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 (

Flower pigmentation is one of the most important traits for ornamental plants. To clarify the genetic basis for carotenoid pigmentation in flower tepals of Asiatic hybrid lily ( Lilium sp.), we evaluated the segregation of a tepal-carotenoid content among F 1 plants derived from a cross between ‘Montreux’ (having a small amount of carotenoids) and ‘Connecticut King’ (having a large amount of carotenoids), and mapped genetic loci for the carotenoid pigmentation onto the molecular linkage maps of ‘Montreux’ and ‘Connecticut King’ that we constructed previously. The tepal-carotenoid content among the F 1 plants showed continuous segregation, indicating that several genes are associated with this trait. Quantitative trait loci (QTL) analysis identified one QTL, qCARmon6 , on the sixth linkage group of the ‘Montreux’ map. qCARmon6 explained 58.2% of the total phenotypic variation, that is, this locus had a large effect on the carotenoid accumulation. The result that qCARmon6 was mapped on the linkage group of ‘Montreux’ which has a small amount of carotenoid pigments in tepals indicates that this locus has a dominant negative effect on carotenoid pigmentation.

PCR-based DNA markers in Asiatic hybrid lily were examined. Random primers with various lengths (10-, 12-, 15- and 20-base) were tested in randomly amplified polymorphic DNA (RAPD) reactions with total DNA from two cultivars, ‘Montreux’ and ‘Connecticut King’. Thirty-three out of 61 (54%) 15-base primers and 14 out of 21 (67%) 20-base primers generated polymorphic fragments whereas 17 out of 145 (12%) and 4 out of 24 (17%) of the 10- and 12-base primers, respectively, amplified them. The efficiency of the RAPD reaction increased with increasing primer length. We examined inter-simple sequence repeat (ISSR) reactions using 3′-anchored simple sequence repeat (ASSR) primers, and found 33 out of 63 (52%) primers that amplified polymorphic bands between the two cultivars. We repeated all RAPD and ISSR reactions at least two times and obtained the same DNA fingerprints, indicating high reproducibility obtained with these reactions. All polymorphic fragments were inherited into their F1 hybrids, indicating that these DNA markers are genetically stable.

To understand the genetic background of two floral anthocyanin pigmentation traits, anthocyanin pigmentation in the flower tepals and spot formation, in the Asiatic hybrid lily (2n = 24), segregation of the two traits among 96 F(1) plants derived from a cross between commercial cultivars 'Montreux' and 'Connecticut King' were investigated. 'Montreux' has anthocyanin pigmentation in the tepals with many spots, and 'Connecticut King' has flowers with carotenoid pigmentation without spots. The F(1) plants with or without anthocyanin pigment in the tepals segregated with a 1:1 segregation ratio, indicating that a single gene controls anthocyanin pigmentation in the tepals. The number of spots per square centimeter of all tepals showed continuous distribution in the F(1) plants. To map the loci for the two anthocyanin pigmentation traits, molecular linkage maps in the Asiatic hybrid lily were constructed using a double pseudo-testcross strategy, with the same F(1) plants used for phenotypic evaluation, and 212 PCR-based DNA markers. The trait for anthocyanin pigmentation in tepals was used as a trait marker. The map of 'Montreux' comprised 95 markers in 26 linkage groups, and the map of 'Connecticut King' used 119 markers in 24 linkage groups. The total map lengths were 867.5 and 1,114.8 cM, respectively. The trait locus for anthocyanin pigmentation in the tepals was between markers ASR35-180 and P506-40 in linkage group 1 of the 'Montreux' map with a map distance of 1.2 cM and 2.6 cM, respectively. A single-point analysis of quantitative trait loci (QTLs) for tepal spot number identified two putative QTLs in linkage groups 1 and 19 of the 'Connecticut King' map. One putative QTL in linkage group 19 explained 64% of the total phenotypic variation. Because both putative QTLs were mapped on the linkage map of 'Connecticut King' that has no spots, dominant alleles of them might suppress spot formation.

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.

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.

We have developed a CiFT co-expression system for the rapid evaluation of transgenic citrus flowers and fruit. In order to co-express a target gene and the CiFT gene, a CiFT co-expression vector was constructed by inserting the fragment containing a chimeric target gene into the binary vector harboring P35S::CiFT. This system was applied to metabolically engineer aroma components in trifoliate orange (Poncirus trifoliata L. Raf). In order to reduce the limonene level, a gene encoding limonene synthase (CitMTSE1) from satsuma mandarin (Citrus unshiu Marc.) was introduced into trifolliate orange in an anti-sense orientation with the CiFT co-expression vector. Transgenic plants flowered extremely early, and began to produce normal fruit within 2 years of Agrobacterium infection. Transcripts of transgenes were accumulated in reproductive tissues of transgenic plants, and endogenous transcripts for limonene synthase were reduced. The ratio of limonene to other monoterpenes was significantly decreased in flowers and fruit of transgenic plants. These results clearly indicated that the CiFT co-expression system is a useful tool for the functional analysis of genes in citrus flowers and fruit. Such analyses are important for several applications, including the metabolic engineering of aroma components in citrus.

The preparation of fluorescence labeled acyl enzymes (Streptomyces griseus trypsin) was successfully carried out using specific trypsin substrates, ‘inverse substrates’. The topographical analysis of the structures of the area around the active site was carried out by measuring the fluorescence spectra of the acyl enzyme preparations and these results were compared with those of bovine trypsin. It was found that the polarity of the active site vicinity of pH 5 was similar to that of bovine trypsin, whereas considerable differences were noticed at lower pH as a result of pH-induced transformation. Conformational changes of the active site induced by the interaction with the specific ligand were analyzed from the fluorescence spectra. In these responses the two enzymes were quite distinguishable. The two enzymes active sites were also different in the energy transfer experiments. The spatial arrangements of the catalytic residues relative to the intrinsic tryptophan residues were suggested to be substantially different for the two enzymes.

Persistent but reversible changes in the phenotype of a tobacco callus are reported in this paper. The callus (S401) was first isolated as a spontaneous variant which differed in pigmentation (dark grayed green) and friability (very friable and wettish) from its parent callus S4 (yellow and firm) during subculture on Murashige and Skoog medium with 2, 4-D. The callus consisted mainly of small embryonic cells and contained a high level of pectin, especially of water soluble forms, as compared with the S4 callus. Almost no lignified vascular elements were observed.2, 4-D removal from the medium was accompanied by an increase in lignin and a decrease in pectin content. Peroxidase activity in the callus was low, but the level increased during subculture on the medium without 2, 4-D. Isozymes of peroxidase found in the callus after 2, 4-D removal were the same as those in the S4 callus. When the tissues of S401 grown on the 2, 4-D free medium were placed back on the medium with 2, 4-D, they recovered its original characters. The regenerated plant originated from the S401 callus produced only the S401 type callus.

The kinetic properties of esters derived from guanidinophenol and aminomethylphenol were investigated with trypsin. These compounds, in which the site-specific groups (positive charge) are of the inverse type compared with normal substrates, were demonstrated to be specific substrates, like amidinophenyl esters. The behavior of these"inverse substrates"with trypsin and pseudotrypsin was also compared. A dramatic decrease in the efficiency of hydrolysis of the"inverse substrates"by pseudotrypsin as compared to that by trypsin was observed, which was comparable in extent to that observed for specific normal-type substrates. All these observations confirm the view that specific interaction between the positive charge at the leaving moiety of"inverse substrates"and the anionic site of the trypsin active center is an essential feature of the catalysis, just as in the case of normal-type substrates.

本報はゴボウに見出される1種のclosterovirus,ゴボウ黄化ウイルス(burdock yellows virus, BdYV,以前‘Bd-F’と仮称していたもの)の性状の記載ならびにBdYVを含む4種のclosterovirusの血清反応試験について述べたものである。 BdYVはゴボウでほとんど無病徴か,下葉の葉縁部にわずかに黄化病徴をあらわすほか,エゾギク,サンシキカミツレおよびNicotiana clevelandiiに全身感染した。BdYVはゴボウヒゲナガアブラムシ(Dactynotus gobonis)で半永続的に伝搬され, N. clevelandiiだけに汁液接種で移すことができた。粗汁液中での不活化温度は45～50C (10分),保存限度は1～2日(20C)であった。N. clevelandii病葉から得たBdYV純化標品のUV吸収曲線は245nmに極小, 260nmに極大値があり, A260/280=1,73であった。ウイルス粒子は屈曲した長いひも状で1700～1750×12nm,構造らせんのピッチは約3.6nmであった。感染植物超薄切片でウイルス粒子は節部細胞に局在してみられ,しばしば束状の大きな粒子集塊として認められた。また,これらの細胞内には特徴的な小胞構造体がみられ,篩部細胞のえ死も認められた。 BdYV,カーネーションえそ斑(CNFV), カンキツトリステザ(CTV)およびコムギ黄葉(WYLV)の4種のclosterovirusにつきSDS-寒天二重拡散法を行ったところ,それぞれ同種のウイルス-抗血清の組合せで血清反応があらわれたが,寒天中のSDS濃度はBdYVで0.5%, 他の3種のウイルスでは0.1%の場合にもっとも明瞭であった。ミクロプレシピチンテストにより4種closterovirus相互の血清関係を調べたところ, CNFVとWLYVとの間に血清学的類縁のあることが確かめられた。

Trypsin-specific substrate analogs, “inverse substrates,” carrying the fluorescent dimethylaminonaphthalene group were synthesized. Preparation of acyl trypsins in which the fluorescent group is attached to the catalytic residue through a spacer group of various chain lengths was successfully carried out by use of these inverse substrates. The topographical structure of the trypsin active site vicinity was estimated on the basis of the fluorescence spectra of these acyl trypsins.

An "inverse substrate" for trypsin, p-amidinophenyl acetylglycinate, has been synthesized and applied to mechanistic studies of tryptic hydrolysis. The compound reacts to form the intermediate acetylglycyl trypsin in a very specific manner, analogous to the reactions of p-amidinophenyl alkanoates with trypsin to give alkanoyl trypsins (Tanizawa, K., Kasaba, Y., & Kanaoka, Y. (1977) J. Am. Chem Soc. 99, 4485). The effects of alkylammonium and alkylguanidinium ions on tryptic hydrolysis of this "inverse substrate" have been investigated, and all alkylamines and alkylguanidines tested behaved as activators. n-Propylamine, n-propylguanidine, and n-butylguanidine, which have been reported to inhibit tryptic hydrolysis of ethyl or p-nitrophenyl acetylglycinate, were activators with p-amidinophenyl acetylglycinate. The activating properties of these amines and guanidines were demonstrated for the first time by making use of the specific characteristics of this inverse substrate. Tryptic mechanisms proposed in the literature are discussed in the light of these observations.

A rapid and sensitive method for determination of kanamycin (KM) and fradiomycin (FM) in the urine and bile of cattle by high performance liquid chromatogrphy (HPLC) was developed.A urine or bile sample was diluted with 0.1M phosphate buffer (pH 8.0), and a portion of the diluted sample was adsorbed on an SP-Sephadex C-25 mini-column (SO3H form) and eluted with 0.07% sodium hydroxide. KM and FM in the eluate were derivatized with 2, 4-dinitrofluorobenzene and determined by HPLC using a TSK80TM-ODS column with 60% acetonitrile containing 0.1% acetic acid as the mobile phase (UV monitoring at 365nm).The recoveries of KM and FM added to the urine and bile of cattle at the level of 25μg/ml were 97.3 and 90.8 (KM), and 92.2 and 83.3% (FM), respectively. The limit of determination was 0.3μg/ml for KM and FM.By this method, KM and FM in biological samples can be determined simultaneously.