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Natural (genetic) parthenocarpy contributes to fruit yield and quality under unfavorable environmental conditions where there are no effective pollinators or fertile pollen grains. Several old melon cultivars and weedy melon in East Asia are known to have strong parthenocarpic ability, but there has been little progress in intentionally introducing this ability into current F1hybrid cultivars. Here, we examined its inheritance and confirmed the selection accuracy of DNA markers linked to it. We conducted QTL analysis using three F2populations derived from crosses between a non-parthenocarpic cultivar and three parthenocarpic accessions, and detected two major QTLs on chromosomes 2 (par2.1) and 3 (par3.1). The parthenocarpic parent allele at both QTLs enhanced parthenocarpic ability. Phenotypic segregation was well explained bypar2.1andpar3.1in two F2populations derived from Japanese weedy melon and an old Korean cultivar and bypar3.1in one from an old Japanese cultivar. This difference suggests that the effects ofpar2.1andpar 3.1depend on genetic background. Both QTL regions contain several phytohormone-related genes, so we randomly selected SNPs in auxin- and ethylene-related genes to confirm the accuracy of selection for parthenocarpic ability. These SNP markers proved sufficient, though not perfect, to select plants with strong parthenocarpic ability. These results provide new insights into the molecular mechanisms of parthenocarpic ability in melon and will contribute to the development of new cultivars with high parthenocarpic ability.Key messageSeveral oriental melons have strong parthenocarpic ability controlled by one or two loci. DNA markers linked to these loci can select individuals with this ability.

As prickles cause labour inefficiency during cultivation and scratches on the skin of fruits during transportation, they are considered undesirable traits of eggplant (Solanum melongena L.). Because the molecular basis of prickle emergence has not been entirely revealed in plants, we mapped an eggplant semi-dominant Prickle (Pl) gene locus, which causes the absence of prickles, on chromosome 6 of a linkage map of the F2 population derived from crossing the no-prickly cultivar ‘Togenashi-senryo-nigo’ and the prickly line LS1934. By performing synteny mapping with tomato, the genomic region corresponding to the eggplant Pl locus was identified. Through bacterial artificial chromosome (BAC) screening, positive BAC clones and the contig sequence that harbour the Pl locus in the prickly eggplant genome were revealed. The BAC contig length was 133 kb, and it contained 16 predicted genes. Among them, a characteristic 0.5-kb insertion/deletion was detected. As the 0.5-kb insertion was commonly identified with the prickly phenotype worldwide, a primer pair that amplifies the insertion/deletion could be used for marker-assisted selection of the no-prickly phenotype. Such findings contribute to map-based-cloning of the Pl gene and the understanding of gene function, ultimately providing new insights into the regulatory molecular mechanisms underlying prickle emergence in plants.

The appearance quality of the eggplant (Solanum melongena L.) fruit is an important trait that influences its commercial value. It is known that quality traits such as anthocyanin composition and fruit surface pattern are categorical and are inherited simply. However, research examples of gene mapping for the composition (anthocyanin accumulation profile) and the surface pattern in eggplant fruit are limited.To map loci for these traits including the accumulation profiles of two anthocyanins, a widely spreading anthocyanin, delphinidin 3-(p-coumaroyl) rutinoside-5-glucoside (nasunin), and the relatively rare delphinidin 3-glucoside (D3G), we used two FOverall, the results of this study suggest that inactivation of key enzymes of anthocyanin metabolism and the gene orthologous to the tomato u gene are potential causes of observed variety in eggplant appearance traits.

Capsanthin, the main carotenoid of red pepper fruits, is beneficial for human health. To breed pepper (Capsicum annuum L.) with high capsanthin content by marker-assisted selection, we constructed a linkage map of doubled-haploid (DH) lines derived from a cross of two pure lines of C. annuum ('S3586' × 'Kyoto-Manganji No. 2'). The map, designated as the SM-DH map, consisted of 15 linkage groups and the total map distance was 1403.8 cM. Mapping of quantitative trait loci (QTLs) for capsanthin content detected one QTL on linkage group (LG) 13 at 90 days after flowering (DAF) and one on LG 15 at 45 DAF; they were designated Cst13.1 and Cst15.1, respectively. Cst13.1 explained 17.0% of phenotypic variance and Cst15.1 explained 16.1%. We grouped DH lines according to the genotypes of markers adjacent to Cst13.1 and Cst15.1 on both sides. The DH lines with the alleles of both QTLs derived from 'S3586' showed higher capsanthin content at 45 and 90 DAF than the other lines. This is the first identification of QTLs for capsanthin content in any plant species. The data obtained here will be useful in marker-assisted selection for pepper breeding for high capsanthin content.

An NB-LRR gene, TYNBS1, was isolated from Begomovirus-resistance locus Ty-2. Transgenic plant analysis revealed that TYNBS1 is a functional resistance gene. TYNBS1 is considered to be synonymous with Ty-2. Tomato yellow leaf curl disease caused by Tomato yellow leaf curl virus (TYLCV) is a serious threat to tomato (Solanum lycopersicum L.) production worldwide. A Begomovirus resistance gene, Ty-2, was introduced into cultivated tomato from Solanum habrochaites by interspecific crossing. To identify the Ty-2 gene, we performed genetic analysis. Identification of recombinant line 3701 confirmed the occurrence of a chromosome inversion in the Ty-2 region of the resistant haplotype. Genetic analysis revealed that the Ty-2 gene is linked to an introgression encompassing two markers, SL11_25_54277 and repeat A (approximately 200 kb). Genomic sequences of the upper and lower border of the inversion section of susceptible and resistant haplotypes were determined. Two nucleotide-binding domain and leucine-rich repeat-containing (NB-LRR) genes, TYNBS1 and TYNBS2, were identified around the upper and lower ends of the inversion section, respectively. TYNBS1 strictly co-segregated with TYLCV resistance, whereas TYNBS2 did not. Genetic introduction of genomic fragments containing the TYNBS1 gene into susceptible tomato plants conferred TYLCV resistance. These results demonstrate that TYNBS1 is a functional resistance gene for TYLCV, and is synonymous with the Ty-2 gene.

Key messageUsing newly developed euchromatin-derived genomic SSR markers and a flexible Bayesian mapping method, 13 significant agricultural QTLs were identified in a segregating population derived from a four-way cross of tomato.AbstractSo far, many QTL mapping studies in tomato have been performed for progeny obtained from crosses between two genetically distant parents, e.g., domesticated tomatoes and wild relatives. However, QTL information of quantitative traits related to yield (e.g., flower or fruit number, and total or average weight of fruits) in such intercross populations would be of limited use for breeding commercial tomato cultivars because individuals in the populations have specific genetic backgrounds underlying extremely different phenotypes between the parents such as large fruit in domesticated tomatoes and small fruit in wild relatives, which may not be reflective of the genetic variation in tomato breeding populations. In this study, we constructed F2 population derived from a cross between two commercial F1 cultivars in tomato to extract QTL information practical for tomato breeding. This cross corresponded to a four-way cross, because the four parental lines of the two F1 cultivars were considered to be the founders. We developed 2510 new expressed sequence tag (EST)-based (euchromatin-derived) genomic SSR markers and selected 262 markers from these new SSR markers and publicly available SSR markers to construct a linkage map. QTL analysis for ten agricultural traits of tomato was performed based on the phenotypes and marker genotypes of F2 plants using a flexible Bayesian method. As results, 13 QTL regions were detected for six traits by the Bayesian method developed in this study.

To investigate the influence of morphological changes in individual leaves of tomato on light interception and dry matter (DM) production, we altered leaf shape by trimming leaflets of young or mature leaves of the Dutch cultivar Gourmet and the Japanese cultivar Momotaro York. Young leaves 5-cm long were trimmed of their first and second leaflets from the leaf apex. Mature leaves were similarly trimmed at ≥71 days after transplanting (DAT). The individual leaf area (LA) of intact ‘Momotaro York’ leaves was significantly larger than that of ‘Gourmet’. Light–photosynthesis curves of the cultivars were almost identical. Mature-trimmed plants of both cultivars had a smaller individual LA and a smaller leaf area index (LAI), and a greater light-extinction coefficient (LEC). Although there was no significant difference in light-use efficiency (LUE) (i.e., DM production per unit intercepted solar radiation) in ‘Gourmet’ between trimming stages, LUE of ‘Momotaro York’ was decreased significantly by young-leaf trimming. Trimming of young leaves significantly decreased the LEC in ‘Gourmet’ but increased it in ‘Momotaro York’. Although leaf trimming would be impractical for commercial cultivation, these results may provide with a clue for breeding for yield improvement.

Genomic selection (GS), which uses estimated genetic potential based on genome-wide genotype data for a breeding selection, is now widely accepted as an efficient method to improve genetically complex traits. We assessed the potential of GS for increasing soluble solids content and total fruit weight of tomato. A collection of big-fruited F1 varieties was used to construct the GS models, and the progeny from crosses was used to validate the models. The present study includes two experiments: a prediction of a parental combination that generates superior progeny and the prediction of progeny phenotypes. The GS models successfully predicted a better parent even if the phenotypic value did not vary substantially between candidates. The GS models also predicted phenotypes of progeny, although their efficiency varied depending on the parental cross combinations and the selected traits. Although further analyses are required to apply GS in an actual breeding situation, our results indicated that GS is a promising strategy for future tomato breeding design.

Tomato yellow leaf curl virus (TYLCV, genus Begomovirus, family Geminiviridae), poses a serious threat to tomato crops in tropical and subtropical regions. We developed a simple agroinoculation method with an infectious clone of TYLCV. Dipping of excised sections of susceptible tomato shoots in an Agrobacterium suspension successfully introduced the replicating virus with high efficiency. An additional vacuum treatment for 5 min ensured uniform infection without escapes, allowing evaluation of differences in TYLCV resistance among tomato cultivars. The method can be used in laboratory experiments for virological studies and in breeding programs for resistant cultivars.

To facilitate marker-assisted breeding and genetic analyses of pepper (Capsicum annuum), we developed non-redundant 2- or 3-base simple sequence repeat (SSR) markers from enriched C. annuum genomic libraries and from C. annuum cDNA sequences in public databases. The SSR-enriched libraries were constructed using combinations of three restriction enzymes (AluI, HaeIII, and RsaI) and two biotinylated oligonucleotides [b(GA)15 and b(CA)15]. Ultimately, we obtained 1,736 genomic SSR markers and 1,344 cDNA-derived SSR markers from 6,528 clones and 13,003 sequences, respectively. We mapped 597 markers, including 265 of the newly developed SSR markers, onto a linkage map by using doubled-haploid (DH) lines derived from an intraspecific cross of two pure lines of C. annuum (K9-11 × MZC-180). The map, designated as the KL-DH map, consisted of 12 linkage groups. The map covered a genetic distance of 2,028 cM, and the average distance between markers was less than 4 cM. The frame structure of the KL-DH map was compared with the published standard conserved ortholog set II (COSII) map, which was derived from an interspecific F2 population (C. frutescens × C. annuum), by using tomato (Solanum lycopersicum) chromosomal sequences to bridge the two maps. The intraspecific KL-DH map constructed in this study and the interspecific COSII map were similar in map length and marker distribution, suggesting that the KL-DH map covers nearly the whole genome of C. annuum.

Introduction: TSU-68 is an oral small-molecule inhibitor that targets vascular endothelial growth factor receptor 2, platelet-derived growth factor receptor β, and fibroblast growth factor receptor 1. An open-label, single-arm, phase I study was performed to evaluate escalating doses of TSU-68 in combination with standard chemotherapy in patients with advanced non-small cell lung cancer. Methods: Eligible patients received TSU-68 at 200 or 400 mg twice daily and continuously in combination with carboplatin (area under the curve, 6 mg · min/mL) plus paclitaxel (200 mg/m 2 ) on day 1 every 21 days. Results: Thirty-seven patients were enrolled at the two dose levels of TSU-68. No dose-limiting toxicities were observed with TSU-68 at the 200 mg twice a day dose level. At 400 mg twice a day, one of six patients experienced a dose-limiting toxicity (anorexia of grade 3) during the first cycle. The 400 mg twice a day dose level was determined to be the recommended dose, and a total of 34 patients were treated at this dose. Overall, adverse events were mild to moderate in severity, with the most frequently observed such events being myelosuppression, neuropathy, and gastrointestinal disorders. No drug-related bleeding was observed. The objective response rate was 39.4% (95% confidence interval, 22.9–57.9%), and median progression-free survival was 5.6 months (95% confidence interval, 3.6–7.2 months). Coadministration of TSU-68, carboplatin, and paclitaxel had no substantial impact on the pharmacokinetics of these drugs. Conclusions: TSU-68 can be safely combined with standard doses of carboplatin-paclitaxel, with the combination manifesting promising antitumor activity.

Parthenocarpy, the ability to set fruits without pollination, is a useful trait for setting fruit under unfavorable conditions. To identify the loci controlling parthenocarpy in eggplant (Solanum melongena L.), we constructed linkage maps by using co-dominant simple sequence repeat and single nucleotide polymorphism markers in F(2) populations derived from intraspecific crosses between two non-parthenocarpic lines (LS1934 and Nakate-Shinkuro) and a parthenocarpic line (AE-P03). Total map distances were 1,414.6 cM (ALF2: LS1934 x AE-P03) and 1,153.8 cM (NAF2: Nakate-Shinkuro x AE-P03), respectively. Quantitative trait locus (QTL) analyses revealed two QTLs on chromosomes 3 and 8, which we denoted as Controlling parthenocarpy3.1 (Cop3.1) and Cop8.1, respectively. The percentage of phenotypic variance explained (PVE) of Cop3.1 was 6.3% in ALF2 (LOD = 4.2) and 10.6% in NAF2 (LOD = 3.0). The PVE of Cop8.1 was 45.7% in ALF2 (LOD = 23.8) and 29.7% in NAF2 (LOD = 7.9). Using a population of backcross inbred lines, we confirmed the effect of Cop8.1, but there was no evidence to support the contribution of Cop3.1. We need to verify the effect of Cop3.1 under various temperature conditions. In addition, we clarified the effectiveness of selective SSR markers, emf21H22 and emh11J10, mapped on each side of Cop8.1 in other F(2) populations derived from various parental combinations. This is the first report concerning QTL analysis of parthenocarpy in eggplant using molecular markers. It will be useful in marker-assisted selection and in revealing the genomic mechanism underlying parthenocarpy in eggplant.

Recombinant inbred lines (RILs) are created by a cross between inbred lines followed by repeated selfing or sib-mating, which include many different types of new inbred lines with many recombination events on the genome. The phenotype of each RIL can be assessed based on multiple individuals within the same line to reduce non-genetic variability. Therefore, RILs are useful tools for QTL mapping allowing effective detection and fine localization of QTL. Usually only two inbred lines are involved to create RILs. In such two-way RILs, however, there are at most two different alleles at QTL and only QTL segregating between two parental lines can be detected. Recently a new crossing design using multiple founders for creating RILs was proposed in Arabidopsis thaliana and mice, where the genome of each RIL is a mosaic of the genomes from multiple parents. Such multi-way RILs are more useful to improve the efficiency in QTL detection than two-way RILs because of the increased chance of QTL segregation among multiple lines, leading to the successful detection of many QTL. In this paper, we propose a Bayesian method for mapping multiple QTL simultaneously in four-way RILs allowing the inference about the equivalence relationship among the four possible alleles descended from the four founder lines as well as the estimation of QTL locations and effects via a MCMC algorithm. Simulation experiments show that our method has the practical ability to detect QTL and to provide the information of equivalence relationship among alleles at detected QTL using four-way RILs.

Anther-specific promoters have been extensively studied in terms of the genetic engineering of male-sterile plants. Here, we fused the 5′ upstream promoter regions of a sucrose synthase gene from tomato (TOMSSF) to the β-glucuronidase (GUS) gene and used the construct to transform Chrysanthemum plants. Histochemical analysis of two transformants showed high GUS activity in ray florets and tubular florets. Analysis of the staining pattern in these florets reveled that staining was stamen-specific. GUS gene expression was highest in the stamen and remained at a steady-state level throughout the experiment. These results suggest that the TOMSSF promoter has high activity in the stamen of tubular florets and would be useful as a high stamen-specific promoter in Chrysanthemum.

Fruit nutritional and flavor components are important targets for breeding new cultivars of tomato (Solanum lycopersicum L.). We developed 108 recombinant inbred lines (the K39 RILs) in the F6 generation from a cross between two phenotypically different breeding lines, K03 and K09. A linkage map was constructed using 172 genome-wide simple sequence repeat markers, 3 single-nucleotide polymorphism markers, and 2 phenotypic markers. The K39 RIL map consists of 12 linkage groups (LGs) and covers a genetic distance of 1089 cM. We measured the fruit soluble solids content (SSC), titratable acidity (TA), glutamic acid content (GLU), and lycopene content (LYC) of each line in four generations (F6, F8, F10, F11), β-carotene content (CAR) in two generations, and pH in one generation. By composite interval mapping that considered yearly variations in components as non-genetic effects, we detected three quantitative trait loci (QTLs) for SSC, four for TA, two for CAR, and one each for GLU, LYC, and pH. Among them, we found two QTLs for TA in LGs 6 and 11, those for GLU and LYC were candidates for novel QTLs. QTLs detected in this study were clustered in five LGs, but we observed no apparent trade-off relationships among the QTLs in each LG. Being derived from an intra-specific cross of tomato breeding materials, these QTLs can be used in practical breeding for improving fruit quality with low risk of linkage drag.

Eggplant (Solanum melongena L.) is a widely grown vegetable crop that belongs to the genus Solanum, which is comprised of more than 1000 species of wide genetic and phenotypic variation. Unlike tomato and potato, Solanum crops that belong to subgenus Potatoe and have been targets for comprehensive genomic studies, eggplant is endemic to the Old World and belongs to a different subgenus, Leptostemonum, and therefore, would be a unique member for comparative molecular biology in Solanum. In this study, more than 60,000 eggplant cDNA clones from various tissues and treatments were sequenced from both the 5'- and 3'-ends, and a unigene set consisting of 16,245 unique sequences was constructed. Functional annotations based on sequence similarity to known plant reference datasets revealed a distribution of functional categories almost similar to that of tomato, while 1316 unigenes were suggested to be eggplant-specific. Sequence-based comparative analysis using putative orthologous gene groups setup by reciprocal sequence comparison among six solanaceous species suggested that eggplant and its wild ally Solanum torvum were clustered separately from subgenus Potatoe species, and then, all Solanum species were clustered separately from the genus Capsicum. Microsatellite motif distribution was different among species and likely to be coincident with the phylogenetic relationships. Furthermore, the eggplant unigene dataset exhibited its utility in transcriptome analysis by the SAGE strategy where a considerable number of short tag sequences of interest were successfully assigned to unigenes and their functional annotations. The eggplant ESTs and 16k unigene set developed in this study would be a useful resource not only for molecular genetics and breeding in eggplant itself, but for expanding the scope of comparative biology in Solanum species.

Solanum torvum Sw. cv. Torubamubiga (TB) is a low cadmium (Cd)-accumulating plant. To elucidate the molecular mechanisms of the Cd acclimation process in TB roots, transcriptional regulation was analysed in response to mild Cd treatment: 0.1 muM CdCl(2) in hydroponic solution. A unigene set consisting of 6296 unigene sequences was constructed from 18 816 TB cDNAs. The distribution of functional categories was similar to tomato, while 330 unigenes were suggested to be TB specific. For expression profiling, the SuperSAGE method was adapted for use with Illumina sequencing technology. Expression tag libraries were constructed from Cd-treated (for 3 h, 1 d, and 3 d) and untreated roots, and 34 269 species of independent tags were collected. Moreover, 6237 tags were ascribed to the TB or eggplant (aubergine) unigene sequences. Time-course changes were examined, and 2049 up- and 2022 down-regulated tags were identified. Although no tags annotated to metal transporter genes were significantly regulated, a tag annotated to AtFRD3, a xylem-loading citrate transporter, was down-regulated. In addition to induction of heavy metal chaperone proteins, antioxidative and sulphur-assimilating enzymes were induced, confirming that oxidative stress developed even using a mild Cd concentration. Rapid repression of dehydration-related transcription factors and aquaporin isoforms suggests that dehydration stress is a potential constituent of Cd-induced biochemical impediments. These transcriptional changes were also confirmed by real-time reverse transcription-PCR. Further additions of TB unigene sequences and functional analysis of the regulated tags will reveal the molecular basis of the Cd acclimation process, including the low Cd-accumulating characteristics of TB.

Eggplant (Solanum melongena L.), also known as aubergine or brinjal, is an important vegetable in many countries. Few useful molecular markers have been reported for eggplant. We constructed simple sequence repeat (SSR)-enriched genomic libraries in order to develop SSR markers, and sequenced more than 14,000 clones. From these sequences, we designed 2,265 primer pairs to flank SSR motifs. We identified 1,054 SSR markers from amplification of 1,399 randomly selected primer pairs. The markers have an average polymorphic information content of 0.27 among eight lines of S. melongena. Of the 1,054 SSR markers, 214 segregated in an intraspecific mapping population. We constructed cDNA libraries from several eggplant tissues and obtained 6,144 expressed sequence tag (EST) sequences. From these sequences, we designed 209 primer pairs, 7 of which segregated in the mapping population. On the basis of the segregation data, we constructed a linkage map, and mapped the 236 segregating markers to 14 linkage groups. The linkage map spans a total length of 959.1 cM, with an average marker distance of 4.3 cM. The markers should be a useful resource for qualitative and quantitative trait mapping and for marker-assisted selection in eggplant breeding.

We developed nearly 700 non-redundant 2- or 3-base simple sequence repeat (SSR) markers from tomato using sequence data obtained from open genome databases. Among various types of core motifs, AT was most abundant in SSRs derived from cDNAs (~53%) and bacterial artificial chromosome (BAC) ends (~72%). There was a positive correlation between the rate of detection of polymorphic alleles (heterozygosity value; Hv) and the repeat number of the core motif in all markers showing polymorphisms among at least one pair of six cultivars or lines tested (r = 0.566**). The average Hv of BAC-end-derived SSR markers (~0.5) was higher than that of cDNA-derived markers (~0.3). These characteristics of BAC-end-derived SSRs are useful for genetic studies using closely related cultivars and lines. However, BAC-end-derived SSRs tended to cluster in centromeric regions (~80%). A scheme for the construction of a high-density linkage map of tomato is discussed.

This is the first report on genetic mapping of a resistance locus against Fusarium wilt caused by the plant pathogen Fusarium oxysporum f. sp. melongenae in cultivated eggplant. Fusarium wilt, caused by the plant pathogen Fusarium oxysporum f. sp. melongenae, is a major soil-borne disease threatening stable production in eggplant (Solanum melongena). Although three eggplant germplasms, LS1934, LS174, and LS2436, are known to be highly resistant to the pathogen, their resistance loci have not been mapped. In this study, we performed quantitative trait locus analyses in F2:3 populations and detected a resistance locus, FM1, at the end of chromosome 2, with two alleles, Fm1 L and Fm1 E , in the F2 populations LWF2 [LS1934 × WCGR112-8 (susceptible)] and EWF2 [EPL-1 (derived from LS174) × WCGR112-8], respectively. The percentage of phenotypic variance explained by Fm1 L derived from LS1934 was 75.0 % [Logarithm of the odds (LOD) = 29.3], and that explained by Fm1 E derived from EPL-1 was 92.2 % (LOD = 65.8). Using backcrossed inbred lines, we mapped FM1 between two simple sequence repeat markers located ~4.881 cM apart from each other. Comparing the location of the above locus to those of previously reported ones, the resistance locus Rfo-sa1 from an eggplant ally (Solanum aethiopicum gr. Gilo) was mapped very close to FM1, whereas another resistance locus, from LS2436, was mapped to the middle of chromosome 4. This is the first report of mapping of a Fusarium resistance locus in cultivated eggplant. The availability of resistance-linked markers will enable the application of marker-assisted selection to overcome problems posed by self-incompatibility and introduction of negative traits because of linkage drag, and will lead to clear understanding of genetic mechanism of Fusarium resistance.

Fragments of different length (4 kb and 1 kb) of the 5� upstream region of the Wiv-1 (Lin6) gene encoding a wound-inducible cell wall-bound acid invertase in tomato were fused to the beta-glucuronidase (GUS) gene and expressed in tobacco and tomato plants. The GUS activity detected was similar in all transformed lines, suggesting that the 1 kb region contains most of the regulatory motifs responsible for the characteristics of the Wiv-1 promoter. GUS activity was observed in vascular tissues of transgenic tomato and tobacco. In stem sections of transgenic tobaccos, the strongest activity was observed in the internal phloem of the nodes adjacent to the bases of petioles. The GUS activity in leaves of transgenic tobacco was elevated by addition of soluble sugars, especially sucrose. Previous studies suggested that the Wiv-1 enzyme participated in the regulation of carbohydrate contents in tomato leaves (Ohyama and Hirai 1999). The results obtained in the present study suggest that the Wiv-1 enzyme controls the loading of sucrose at the phloem of nodes and Wiv-1 regulation depends on the concentration of sugars in source leaves.

We evaluated the nature of microsatellites in Brassica rapa in order to develop an informative and reliable DNA marker system for Brassica genetic analysis. Microsatellites were isolated by hybridization screening of an unconcentrated small-insert genomic library using tri-and dinucleotide probes. Of 45,000 clones screened, 210 had repeat sequences, in which 228 microsatellites were identified. The most frequent microsatellite motif was (GA)n at a frequency of one every 4.8 × 105 bp, followed by (CAA)n at one every 5.0 × 105 bp. The frequency of the tri- and dinucleotide microsatellites throughout the B. rapa genome was estimated to be one every 120 Kb. The number of repeats and the polymorphism information content of the dinucleotide microsatellites were higher than those of the trinucleotide microsatellites. More than 90 % of the primer pairs successfully amplified the corresponding microsatellite regions in other Brassica species. Furthermore, a considerable portion of them could be used in other Cruciferous species, 78.5 % in Raphanus sativus, 68.6 % in Sinapis alba and 39.8 % in Arabidopsis thaliana. Based on these results, we concluded that B. rapa microsatellites have a high potential for the development of DNA markers that could contribute to the genetic analysis of Brassica and other Cruciferae.

We isolated the CIP353 cDNA, which encodes a novel cold-inducible protein, from cold-stored tubers of potato (Solanum tuberosum L.). The level of CIP353 transcripts began to increase in tubers 2 weeks after storage at 3 degrees C and continued increasing for at least 3 months during storage. This increase was not observed in tubers stored at >or=9 degrees C. The increased level of transcripts in tubers stored at 3 or 6 degrees C decreased when the tubers were shifted to 20 degrees C. These data suggest that CIP353 is a temperature-dependent and slowly responsive cold-inducible gene of potato. The middle of the deduced amino acid sequence of CIP353 cDNA showed high similarity to the AP2/ERF domain, which occurs in some plant regulatory factors. The deduced protein contained a putative basic nuclear-localization signal and potential acidic activation domains. These data suggest that CIP353 protein is a transcription factor of genes expressed in tubers under long-term storage at low temperatures.

A small insert genomic library of eggplant (Solanum melongena L.) was screened with eight trinucleotide probes. Based on the screening of approximately 108,000 plaques, the occurrence of (AAC/TTG) n, (AAG/TTC)n, (ACC/TGG)n, (ACG/TGC)n, (ACT/TGA)n, (AGC/TCG) n, (AGG/TCC)n and (ATC/TAG) n repeats was estimated to be, respectively, 1.2 × 10 3, 1.3 × 102, 4.0 × 102, 7.6 × 10, 1.1 × 102, 5.7 × 10, 1.3 × 102 and 3.8 × 10, i. e. once every 951 kbp, 8.3 Mbp, 2.8 Mbp, 14.5 Mbp, 9.7 Mbp, 19.3 Mbp, 8.3 Mbp and 29 Mbp in the eggplant genome, respectively. Two trinucleotide repeats, (AAC/TTG)n and (ACC/TGG) n, predominated, accounting for 84.5 % of the isolated trinucleotide microsatellites. A total of 83.5 % of the identified trinucleotide repeats contained seven repeat units or less. Inserts of the positive clones were sequenced and 85 PCR primer sequences bordering the microsatellite repeats were designed. In order to assess the polymorphism, 11 S. melongena lines and 11 Solanum relatives were used. The mean number of alleles per polymorphic locus was 2.1 for S. melongena lines and 2.9 for Solanum relatives. Low levels of polymorphism were observed in eggplant, with the average heterozygosity being 0.31 between S. melongena lines and 0.32 between Solanum relatives. Most of the markers that detected polymorphism between S. melongena lines contained eight or more repeat units. The motif most frequently found in Solanum was (AAC/TTG)n in this study. All the primer sets which detected PCR products in S. melongena yielded PCR products in S. incanum, which is in agreement with its very close relationship with S. melongena. Further transferability, however, was rather low, since only half of the primer sets gave products in Solanum relatives except for S. incanum. Analysis of the polymorphic loci in an eggplant F2 population revealed a codominant mendelian segregation.

Unlike other important Solanaceae crops such as tomato, potato, chili pepper, and tobacco, all of which originated in South America and are cultivated worldwide, eggplant (Solanum melongena L.) is indigenous to the Old World and in this respect it is phylogenetically unique. To broaden our knowledge of the genomic nature of solanaceous plants further, we dissected the eggplant genome and built a draft genome dataset with 33,873 scaffolds termed SME_r2.5.1 that covers 833.1 Mb, ca. 74% of the eggplant genome. Approximately 90% of the gene space was estimated to be covered by SME_r2.5.1 and 85,446 genes were predicted in the genome. Clustering analysis of the predicted genes of eggplant along with the genes of three other solanaceous plants as well as Arabidopsis thaliana revealed that, of the 35,000 clusters generated, 4,018 were exclusively composed of eggplant genes that would perhaps confer eggplant-specific traits. Between eggplant and tomato, 16,573 pairs of genes were deduced to be orthologous, and 9,489 eggplant scaffolds could be mapped onto the tomato genome. Furthermore, 56 conserved synteny blocks were identified between the two species. The detailed comparative analysis of the eggplant and tomato genomes will facilitate our understanding of the genomic architecture of solanaceous plants, which will contribute to cultivation and further utilization of these crops.

Partial Wiv-1 cDNA for a cell-wall-bound acid invertase isolated from wounded leaves of tomato (Lycopersicon esculentum) was introduced into tomato plants in an antisense orientation. The enzyme activity was markedly decreased in wounded leaves of 3 of 9 transformants. The soluble sugars and starch contents in the source leaves of these 3 transformants were reduced compared with control plants. These results suggest that the cell-wall-bound acid invertase encoded by Wiv-1 regulates the carbohydrate content in source leaves of tomato. Most of the transformants showed low fertility. It is possible that the enzyme encoded by Wiv-1 participates also in sink metabolism in tomato flowers.

International audience; The genotype data of 7054 single nucleotide polymorphism (SNP) loci in 40 tomato lines, including inbred lines, F1 hybrids, and wild relatives, were collected using Illumina's Infinium and GoldenGate assay platforms, the latter of which was utilized in our previous study. The dendrogram based on the genotype data corresponded well to the breeding types of tomato and wild relatives. The SNPs were classified into six categories according to their positions in the genes predicted on the tomato genome sequence. The genes with SNPs were annotated by homology searches against the nucleotide and protein databases, as well as by domain searches, and they were classified into the functional categories defined by the NCBI's eukaryotic orthologous groups (KOG). To infer the SNPs' effects on the gene functions, the three-dimensional structures of the 843 proteins that were encoded by the genes with SNPs causing missense mutations were constructed by homology modelling, and 200 of these proteins were considered to carry non-synonymous amino acid substitutions in the predicted functional sites. The SNP information obtained in this study is available at the Kazusa Tomato Genomics Database (http://plant1.kazusa.or.jp/tomato/).

Expression of structural proteins of human immunodeficiency virus type 1 requires the direct interaction of the viral Rev trans-activator with its cis-acting RNA target sequence, the Rev response element (RRE). The originally defined 234-residue RRE, however, failed to show the full HIV-1 regulator of virion expression (Rev) response when inserted into a test plasmid that lacked the flanking env sequences. Here, we demonstrate that an alternative 351-residue complete RRE, which carries an extended Stem I structure, is required for the full Rev-responsiveness in vivo. Mutagenesis studies revealed that the association of Rev with RRE was initiated by the recognition of a functional bubble structure in stem-loop II, followed by interaction with the neighbouring duplex RNA, including the elongated Stem I structure. We propose that, as has been previously shown in vitro, the Rev-RRE interaction in vivo is an ordered assembly process and is based on the association of Rev molecules with the double-stranded region of RRE.

The membrane traffic of human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins has been investigated in COS-1 cells transiently expressing the HIV-1 env, vpu, and rev genes. Analysis of oligosaccharide processing revealed that the majority of gp160 remained fully endo-H sensitive throughout a 21-h chase period, and hence cleavage of gp160 to gp120-gp41 took place prior to the creation of hybrid and complex oligosaccharides on gp120. Immunofluorescence microscopy demonstrated that in the absence of CD4 both gp160 and Vpu are targeted to the Golgi apparatus, that can be stained with wheat germ agglutinin or antibodies to the human KDEL receptor. In contrast, gp160 complexed with CD4 was retained in the ER and thus failed to reach the cis-Golgi compartment. Although gp160-bound CD4 has its own half life of 4 h 35 min in the endoplasmic reticulum (ER), co-expression of Vpu accelerated the turnover of CD4 by 5.5-fold and thereby enabled gp160 to be translocated out of the ER to the cis-Golgi compartment. We concluded that Vpu prevents the formation of stable CD4-gp160 complexes in the ER and thus indirectly allows gp160 to accumulate in the Golgi apparatus, where it is selectively retained to produce gp120-gp41.

We constructed an integrated DNA marker linkage map of eggplant (Solanum melongena L.) using DNA marker segregation data sets obtained from two independent intraspecific F(2) populations. The linkage map consisted of 12 linkage groups and encompassed 1,285.5 cM in total. We mapped 952 DNA markers, including 313 genomic SSR markers developed by random sequencing of simple sequence repeat (SSR)-enriched genomic libraries, and 623 single-nucleotide polymorphisms (SNP) and insertion/deletion polymorphisms (InDels) found in eggplant-expressed sequence tags (ESTs) and related genomic sequences [introns and untranslated regions (UTRs)]. Because of their co-dominant inheritance and their highly polymorphic and multi-allelic nature, the SSR markers may be more versatile than the SNP and InDel markers for map-based genetic analysis of any traits of interest using segregating populations derived from any intraspecific crosses of practical breeding materials. However, we found that the distribution of microsatellites in the genome was biased to some extent, and therefore a considerable part of the eggplant genome was first detected when gene-derived SNP and InDel markers were mapped. Of the 623 SNP and InDel markers mapped onto the eggplant integrated map, 469 were derived from eggplant unigenes contained within Solanum orthologous (SOL) gene sets (i.e., sets of orthologous unigenes from eggplant, tomato, and potato). Out of the 469 markers, 326 could also be mapped onto the tomato map. These common markers will be informative landmarks for the transfer of tomato's more saturated genomic information to eggplant and will also provide comparative information on the genome organization of the two solanaceous species. The data are available from the DNA marker database of vegetables, VegMarks (http://vegmarks.nivot.affrc.go.jp).

Anthocyanins were detected in extracts from the peels of 123 accessions of eggplant (Solanum melongena) and its related species. Their anthocyanin profiles were classified into four types, including known Japanese eggplant type (type 1) and non-Japanese eggplant type (type 2). Although most of the eggplant accessions had one of the two known profiles, one accession had a novel profile (type 3). Two accessions of related species showed another novel profile (type 4). The major anthocyanins were identified as delphinidin 3-(p-coumaroylrutinoside)-5-glucoside (nasunin) (type 1), delphinidin 3-rutinoside (type 2), delphinidin 3-glucoside (type 3), and petunidin 3-(p-coumaroylrutinoside)-5-glucoside (petunidin 3RGc5G) (type 4). Delphinidin 3-caffeoylrutinoside-5-glucoside (delphinidin 3RGcaf5G) was isolated from the hybrid (F1) plants of a type 1 cultivar and a type 3 germplasm. Among the five purified anthocyanins, delphinidin 3RGcaf5G showed the highest radical-scavenging activities toward both 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and linoleic acid radical, followed in order by nasunin and petunidin 3RGc5G.

A cDNA, Wiv-1, for an isozyme of acid invertase (EC 3.2.1.26) was cloned from wounded leaves of tomato (Lycopersicon esculentum). The encoded protein had a basic isoelectric point and strong similarity to the amino acid sequences of plant cell wall-bound invertases. The conserved sequence WECPD that is found in all plant cell wall-bound invertases was also found in the deduced protein. These results suggested that Wiv-1 encoded a cell wall-bound acid invertase of tomato. Wounding increased the levels of mRNAs for soluble and cell wall-bound invertases and the activities of these invertases in leaves of L. esculentum and of a related species, L. peruvianum. The induction of Aiv-1 mRNA for the soluble enzyme in wounded leaves was not very strong, while that of Wiv-1 mRNA for the wall-bound enzyme was prominent. The level of Aiv-1 mRNA reached a maximum 48 h after wounding while that of Wiv-1 mRNA continued to rise for up to 96 h. These findings suggested that the genes for the two isozymes responded independently to wounding. The levels in various organs of Aiv-1 and Wiv-1 mRNAs were higher in L. esculentum than in L. peruvianum. Possible roles of cell wall-bound acid invertase in wound response and in developing plant are discussed.

The sequence of a novel cDNA clone, Aiv-1, for tomato acid invertase was similar to that of TIV1 (Klann et al., 1992) for the enzyme except for a unique intron-like insertion. It is considered that Aiv-1 is derived from either an alternatively spliced mRNA for an isozyme or a pre-mRNA of TIV1.