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SummaryBrassica species exhibit a wide diversity of leaf and floral morphologies.There are many cultivars of heading Chinese cabbage in East Asia, but the genetic mechanism of head formation remains unclear. In this study, we generated an F2 population derived from a cross between two Chinese cabbages of different heading type (i.e., the cylindrical-head type, ‘Chihili 70’, and the round-head type, inbred line ‘Y-54’). The F2 population was grown in 2010 and in 2011 to score morphological traits for head formation [i.e., HT, degree of head top leaf overlap; PW, plant fresh weight (FW); HW, head FW; HW:PW, head FW to plant FW ratio; HH, head height; HD, head diameter; HH:HD, head height to head diameter ratio; and HDS, head density].We constructed linkage maps in both years so that quantitative trait locus (QTL) analyses could be performed. Thirteen and ten QTLs were detected in 2010 and in 2011, respectively. QTL clusters for the different heading traits were found on linkage groups A1, A5, A7, and A8 in ...

Injectable systems can be used in minimally invasive surgical applications. Although chitosan-glycerophosphate hydrogel systems are biodegradable and biocompatible, the long periods of time required for their effective gelation have severely limited their clinical application. The challenges currently facing researchers in this field are therefore focused on shortening the gelation time and biocompatibility of these materials to develop hydrogels suitable for clinical application. Chitosan and γ-glycidoxypropyltrimethoxysilane (GPTMS) hybrids have recently demonstrated good cytocompatibility with respect to human osteoblastic cells (MG63) and human bone marrow cells. Although these precursor sols could form gels under physiological conditions, they required neutralization with a sodium hydroxide solution. In this study, the chitosan-GPTMS hybrid systems were neutralized with glycerophosphate to prepare injectable hydrogels. The results revealed that the gelation time of the hydrogels could be controlled by the amount of GPTMS in the precursor sols. The in vitro cytocompatibility of the hydrogels were evaluated in terms of the proliferation of MG63 cells cultured either directly onto the hydrogels or indirectly onto the cell culture plate under a hydrogel insert. In the former case, the cells showed good attachment and proliferated for up to 7 days. Similar results were observed in the in direct culture. These results suggest that this new chitosan-GPTMS hydrogel could potentially be used as an injectable biomaterial in clinical applications.

Radish, Raphanus sativus L., is a vegetable with high economic importance, especially in Asia. A genetic map with 322 markers and a total length of 673 cM was anchored to chromosomes using a complete set of disomic rapeseed-radish chromosome addition lines. Eighteen SSRs and one CAPS marker, at least two for each linkage group, were assigned to the individual radish chromosomes. Three of the radish chromosomes (C, D and H) were characterized cytogenetically by fluorescence in situ hybridization (FISH). The anchored linkage map can be used for QTL analysis of morphological characters and other traits.

The radish displays great morphological variation but the genetic factors underlying this variability are mostly unknown. To identify quantitative trait loci (QTLs) controlling radish morphological traits, we cultivated 94 F4 and F5 recombinant inbred lines derived from a cross between the rat-tail radish and the Japanese radish cultivar ‘Harufuku’ inbred lines. Eight morphological traits (ovule and seed numbers per silique, plant shape, pubescence and root formation) were measured for investigation. We constructed a map composed of 322 markers with a total length of 673.6 cM. The linkage groups were assigned to the radish chromosomes using disomic rape-radish chromosome-addition lines. On the map, eight and 10 QTLs were identified in 2008 and 2009, respectively. The chromosome-linkage group correspondence, the sequence-specific markers and the QTLs detected here will provide useful information for further genetic studies and for selection during radish breeding programs.

Plant architecture plays an important role in the yield, product quality, and cultivation practices of many crops. Branching pattern is one of the most important components in the plant architecture of melon (Cucumis melo L.). ‘Melon Chukanbohon Nou 4 Go’ (Nou-4) has a short-lateral-branching trait derived from a weedy melon, LB-1. This trait is reported to be controlled by a single recessive or incompletely dominant major gene called short lateral branching (slb). To find molecular markers for marker-assisted selection of this gene, we first constructed a linkage map using 94 F2 plants derived from a cross between Nou-4 and ‘Earl’s Favourite (Harukei-3)’, a cultivar with normal branching. We then conducted quantitative trait locus (QTL) analysis and identified two loci for short lateral branching. A major QTL in linkage group (LG) XI, at which the Nou-4 allele is associated with short lateral branching, explained 50.9 % of the phenotypic variance, with a LOD score of 12.5. We suggest that this QTL corresponds to slb because of the magnitude of its effect. Another minor QTL in LG III, at which the Harukei-3 allele is associated with short lateral branching, explained 9.9 % of the phenotypic variance, with a LOD score of 4.2. Using an independent population, we demonstrated that an SSR marker linked to the QTL in LG XI (slb) could be used to select for short lateral branching. This is the first report of mapping a gene regulating the plant architecture of melon.

Viola grayi, a coastal violet, is found on sandy seashores in limited regions in Japan, but not on rocky sea cliffs or in inland areas. This stemmed violet is classified in the subsection Rostratae. Because of recent human activity on sandy seashores, this species is now recorded as endangered. The present status of this species was surveyed and its genetic diversity was analyzed using 17 newly developed simple sequence repeat markers. The results are compared with those for two commonly found inland violet species in the same subsection, V. grypoceras and V. kusanoana. The coastal violet populations showed markedly lower genetic diversity than those of the common species, V. grypoceras, whereas V. kusanoana showed intermediate genetic diversity. About a half of the total genetic variation of V. grayi is found among populations in analysis of molecular variance. In contrast, only less than 25% of the total variation is found among populations for the two common violets. These three species showed a remarkably high inbreeding coefficient, which indicates high inbreeding under natural mating conditions, although these species have both chasmogamous and cleistogamous flowers. In a population-based dendrogram, V. grayi was clustered in a single and concise group, whereas V. grypoceras and V. kusanoana were found in somewhat discrete positions. In the Bayesian analysis of genetic structure of the coastal violet, delta K was the highest at K = 7, and the seven clusters almost correspond to seven populations studied here. Implications of the conservation of this endangered coastal violet are presented.

Radish (Raphanus sativus L.) belongs to Brassicaceae family and is a close relative of Brassica. This species shows a wide morphological diversity, and is an important vegetable especially in Asia. However, molecular research of radish is behind compared to that of Brassica. For example, reports on SSR (simple sequence repeat) markers are limited. Here, we designed 417 radish SSR markers from SSR-enriched genomic libraries and the cDNA data. Of the 256 SSR markers succeeded in PCR, 130 showed clear polymorphisms between two radish lines; a rat-tail radish and a Japanese cultivar, ‘Harufuku’. As a test case for evaluation of the present SSRs, we conducted two studies. First, we selected 16 SSRs to calculate polymorphism information contents (PICs) using 16 radish cultivars and four other Brassicaceae species. These markers detected 3–15 alleles (average = 9.6). PIC values ranged from 0.54 to 0.92 (average = 0.78). Second, part of the present SSRs were tested for mapping using our previously-examined mapping population. The map spanned 672.7 cM with nine linkage groups (LGs). The 21 radish SSR markers were distributed throughout the LGs. The SSR markers developed here would be informative and useful for genetic analysis in radish and its related species.

The edible berry species Vaccinium sieboldii, Vaccinium ciliatum, and Vaccinium oldhami are deciduous shrubs found growing at low altitudes in Japan. They are classified in the same section. Of these, the former two species are listed as endangered species. The present status of these two species was surveyed. V. sieboldii was found in a very limited range, 65 km wide, in central Japan. Many plants are thought to have been lost because of habitat destruction in the past 50 years. Loss of pine forests from pine-wilt disease is thought to be a major threat to this species. In contrast, V. ciliatum was found over a range of more than 300 km in western Japan. Habitats of both species were heavily fragmented. The genetic diversity and phylogenetic relationships of the three species were studied using nine simple-sequence repeat loci. V. sieboldii and V. ciliatum have a close genetic relationship, although their present growth ranges are widely separated. Despite its limited growth range and the limited number of remaining plants, the genetic diversity of V. sieboldii is similar to that of another endangered species, V. ciliatum, and that of the widely grown species, V. oldhami. Seven populations of the former two species were used to create population-based dendrograms. They were separated into three groups: three populations of V. sieboldii, two mountainous populations of V. ciliatum, and two coastal populations of V. ciliatum. The separation was supported by high bootstrap values. Isolation factors of the two population groups in V. ciliatum are discussed.

The Brassica species displays great variation with regard to morphology. However, the genetic mechanisms that underlie this variability are mostly unknown. We generated 188 F2 plants derived from a cross between a Chinese cabbage and a vegetable turnip, and scored several morphological traits (head formation, leaf lobe, pubescence, and turnip formation) twice (2005 and 2007) in two subsets of 94 individuals. With the exception of pubescence, we did not find any typical discrete segregation of these traits in F2 plants, suggesting that they are controlled by multiple genes. We constructed a linkage map to conduct quantitative trait locus (QTL) analysis, and detected eight and 14 QTLs in 2007 and 2005, respectively, for head formation, leaf lobe, pubescence, turnip size, and turnip weight. The amplified fragment length polymorphism (AFLP) fragments linked to the QTLs were converted into sequence-tagged site (STS) markers. The QTLs detected here and their linkage markers may provide useful information for the selection of traits during the breeding of Chinese cabbage and turnip cultivars.

We crossed a doubled haploid line of the turnip Brassica rapa cv. ‘Iyo-hikabu’, which is pigmented with anthocyanin, with a Chinese cabbage inbred line, ‘Y54’, which lacks anthocyanin pigmentation. The presence and absence of anthocyanin pigmentation was segregated 79: 27 in the F2 population, suggesting monogenic control. A bulked segregant analysis was conducted with random amplified polymorphic DNA (RAPD) markers. A total of 480 primers were used to screen a pair of bulked DNA samples of the F2 plants. We found five RAPD markers closely linked to the pigmentation locus. A partial linkage map around the pigmentation locus was constructed with four RAPD markers, two simple sequence repeat (SSR) markers, and one cleaved amplified polymorphic sequence (CAPS) marker. A 0.84-kb fragment amplified by a random primer, OPU10, was cloned, sequenced, and then converted into a CAPS marker (OPU10C). This CAPS marker was mapped at a distance of 4 cM from the pigmentation locus in the partial linkage map and was also mapped in linkage group R07 of the previously reported reference map of B. rapa. This locus is independent of the previously reported anthocyanin-regulating anl locus mapped in R09. We named the locus Anp. The locus will be useful in plant breeding and genome characterization in B. rapa.

A quantitative trait locus (QTL) analysis of pepper growth traits was performed using a doubled haploid (DH) population derived from a cross between two Capsicum annuum genotypes, a bell-type cultivar ‘California Wonder’ and a Malaysian small-fruited cultivar ‘LS2341’. Simple sequence repeats and amplified fragment length polymorphism markers were used to construct a genetic map for this population. The map spans 1,213 cM, and consists of 15 linkage groups (LGs). The axillary shooting, flowering date, primary axis length, number of leaves on primary axis and mean internode length were evaluated in 94 F1DH families. Twelve QTLs were identified by interval analysis, and each QTL accounts for 14 to 34% of the phenotypic variation. Markers on chromosomes 2, 3, 12, and linkage group 8 (LG8) were associated with QTL for these traits. The present analysis revealed 2 loci for the growth traits in LG 8. One affected mainly axis length and had a minor effect on flowering, while the other had a large effect on flowering date and a smaller effect on axis length.

Genetic variability and its spatial structure within and among Japanese populations of wild radish Raphanus sativus L. var. hortensis Backer f. raphanistroides Makino were surveyed at regional (

Clubroot disease is a major threat to crops belonging to the Brassicaceae. It is controlled most effectively by the use of resistant cultivars. Plasmodiophora brassicae, the causal agent, shows a wide variation for pathogenicity, which can be displayed by using differential host sets. Except for Brassica juncea and B. carinata, resistant accessions can be found in all major crops. Most resistance sources are race-specific, despite some race-independent resistant accessions which can be found in B. oleracea. European field isolates from P. brassicae display great variation and show a tendency to overcome different resistance sources from either B. rapa or B. oleracea. At present, resistance genes from stubble turnips (B. rapa) are most effective and most widely used in resistance breeding of different Brassica crops. Resistance to P. brassicae from turnips was introduced into Chinese cabbage, oilseed rape, and B. oleracea. Although most turnips carry more than one resistance gene, the resistant cultivars from other crops received primarily a single, dominant resistance gene having a race-specific effect. Populations of P. brassicae that are compatible against most of the used resistance sources have been present in certain European areas for many decades. Such pathogen populations appeared in Japanese Chinese cabbage crops only a few years after the introduction of resistant cultivars. As the spread of virulent P. brassicae pathotypes seems to be slow, resistant cultivars are still a very effective method of control in many cropping areas. Mapping studies have revealed the presence of several clubroot-resistance genes in the Brassica A and C genomes; most of these genes are showing race specificity. Only in B. oleracea was one broad-spectrum locus detected. Two loci from the A genome confer resistance to more than one pathotype, but not to all isolates. Progress made in the determination of resistance loci should be used to formulate and introduce an improved differential set. Future efforts for breeding P. brassicae resistance will focus on durability by broadening the genetic basis of clubroot resistance by using either natural variation or transgenic strategies.

The water lotus, genusNelumbo, contains two species, the sacred (Nelumbo nucifera) and American lotuses (Nelumbo lutea). Hundreds of flowering lotus cultivars are currently known. However, their classification is unclear. For the classification ofNelumbocultivars, in addition to 35 simple sequence repeat (SSR) markers recently developed, we have developed 17 and 16 of newNelumboSSR markers from SSR-enriched genomic libraries and expressed sequence tag (EST) data, respectively. Out of these 68 SSRs, along with SSRs recently published by others, 52 showed clear polymorphisms in 98Nelumbosamples. A total of 300 alleles were observed, ranging from 2 to 11 alleles per locus, with an average of 5.77. Alleles specific for the American lotus-derived cultivars and a cluster of the American lotus-derived cultivars on a neighbour-joining tree confirmed genetic differences betweenN. luteaandN. nucifera. In addition, a possible differentiation between Chinese and Japanese cultivars was also suggested. Parentage analysis using the SSR markers confirmed four known parentages and predicted currently-unknown parentages of six cultivars. The present data have demonstrated that site-specific, co-dominant SSR markers enable more accurate classification, identification and comparison ofNelumbospecies.

Simple sequence repeat (SSR) markers were developed in the water lotus (Nelumbo nucifera Gaertn.) from an SSR-enriched genomic library. Of the SSR markers tested, 11 primer pairs produced clearly distinguishable DNA banding patterns. Forty-three alleles were detected with the 11 markers. The allele number per locus ranged from 2 to 5 with an average of 3.9. Polymorphism values ranged from 0.11 to 0.66 with an average of 0.51. These primers were also applicable to another Nelumbo species, Nelumbo lutea (Willd.) Pers. (American lotus) and hybrids between N. nucifera and N. lutea. These results indicate that the SSR markers developed in this study are informative and will be useful for genetic analysis in Nelumbo species.

A Malaysian pepper accession, ‘LS2341’ (Capsicum annuum L.) is highly resistant to bacterial wilt (BW) caused by Ralstonia solanacearum E. F. Smith (species complex). Quantitative trait locus (QTL) analysis was performed using a double haploid (DH) population derived from a cross between the susceptible cultivar ‘California Wonder’ and ‘LS2341’. A linkage map was constructed using simple sequence repeats (SSRs) and amplified fragment length polymorphism (AFLP). The map spans 974 cM, and consists of 15 linkage groups (LGs). Growth chamber evaluations of BW resistance detected a QTL on LG 11. This LG corresponds to pepper chromosome 1 (P1). The QTL explained 33% of the resistance derived from ‘LS2341’, and was named Bw1. An SSR marker, CAMS451 was mapped in the centre of the QTL. Although BW-resistance is thought to be polygenically controlled, use of this linkage marker may improve the efficiency of breeding BW-resistant cultivars.

l-Galactono-1,4-lactone (GalL) dehydrogenase (GLDH) is an enzyme that catalyzes the last step of l-ascorbate (AsA) biosynthesis in plants. To re-evaluate the importance of the enzyme and the possibility of manipulating the AsA content in plants, a cDNA encoding GLDH from sweet potato was introduced into tobacco plants by Agrobacterium-mediated transformation under the control of a CaMV 35S promoter. Protein blot analysis revealed the elevation of GLDH protein contents in three GLDH-transformed lines. Furthermore, these transgenic lines showed 6- to 10-fold higher GLDH activities in the roots than the non-transformed plants, SR1. Despite the elevated GLDH activity, the AsA content in the leaves did not change in all lines; i.e., the AsA content in GLDH-transformed lines was 3–7 μmol g−1 FW, comparable to that in the non-transformed plants. Incubation of leaf discs in a GalL solution led to a rapid 2- to 3-fold increase in the AsA content in both GLDH-transformed and non-transformed plants in the same manner. These results suggest that the supply of GalL is a crucial factor for determining the AsA pool size and that the upstream genes in the AsA biosynthetic pathway are responsible for enhancing the AsA content in plants.

Cucumber (Cucumis sativus L.) is an important vegetable crop grown worldwide, but the number of available and mapped simple-sequence repeat (SSR) markers is insufficient for construction of an SSR-based genetic linkage map. To improve this situation, we developed SSR markers from SSR-enriched genomic libraries for C. sativus. We sequenced 2304 clones, and obtained 101 primer pairs which showed clear amplification and polymorphism among genotypes in either C. sativus or Cucumis melo (melon). In C. sativus, all were amplified and 91 were polymorphic among the three genotypes that we analyzed. In C. melo, 41 were polymorphic among the three genotypes that we analyzed, but 32 were not amplified. In addition to the SSRs developed in this study, we used SSRs and sequence-characterized amplified regions (SCARs) reported previously in cucumber and melon to construct the first SSR-based linkage map of cucumber. The map now comprises 120 SSRs and 6 SCARs. It contains 22 markers from previous cucumber maps and 8 linkage groups, and spans a distance of 625.7 cM. The present map is the most comprehensive one in terms of the number of SSR markers and will therefore be highly useful in future cucumber genetic studies.

Capsicum frutescens is known as a highly pungent chili pepper species, but an accession of C. frutescens showed very low capsaicinoid content. This few-pungent trait is novel and unique to ‘S3212’ in C. frutescens. To investigate the inheritance of the few-pungent trait, segregation of capsaicinoid content in F2 and backcross populations (BC1) crossed with high pungent C. chinense ‘S3010’ were observed. The segregation ratio indicated that few-pungency was controlled by a single recessive gene designated as the cf gene. Isolocus test between cf locus and C locus controlling capsaicinoid production was carried out using cleaved amplified polymorphic sequence (CAPS) marker SCY-800 linked to the C locus. CAPS pattern and few-pungency were not related, so the cf locus is different from the C locus. The single cf gene controlling few-pungency found in this study is expected to be used to develop new cultivars stable in few-pungency.

Membrane microdomains rich in gangliosides are recognized as being critical for proper compartmentalization of insulin signaling. Plasma membrane–associated sialidase, NEU3 , is a key enzyme for ganglioside hydrolysis. We previously reported that mice overexpressing NEU3 mainly in muscles developed severe insulin-resistant diabetes. To examine the possible contributions of NEU3 to in vivo insulin sensitivity and glucose tolerance, NEU3 was expressed by using adenoviral vectors in the livers of C57BL/6 mice on standard and high-fat diets, and insulin-resistant KKAy mice on standard diets. Hepatic NEU3 overexpression paradoxically improved glucose tolerance and insulin sensitivity in the C57BL/6 mice fed standard diets, and glucose tolerance in the C57BL/6 mice fed high-fat diets and in KKAy mice. Hepatic NEU3 overexpression increased hepatic glycogen deposition and triglyceride accumulation, and enhanced the hepatic peroxisome proliferator–activated receptor γ and fetuin expression in the C57BL/6 mice on standard and high-fat diets, and in KKAy mice. Thin-layer chromatographic analysis demonstrated increased levels of GM1 and markedly reduced GM3 in the livers of mice with hepatic NEU3 overexpression ( NEU3 mice). Basal and insulin-stimulated tyrosine phosphorylations of insulin receptor substrate 1 were significantly increased, but tyrosine phosphorylations of the insulin receptor and insulin receptor substrate 2 in the NEU3 liver were unchanged. Insulin-stimulated tyrosine phosphorylations of the insulin receptor were increased in adipose tissues of NEU3 mice. These results suggest that hepatic NEU3 overexpression improves insulin sensitivity and glucose tolerance through modification of ganglioside composition and peroxisome proliferator–activated receptor γ signaling. Our findings also provide further evidence that NEU3 is an important regulator of insulin sensitivity and glucose tolerance.

UV-irradiated cotyledonary protoplasts of Solanum integrifolium and iodoacetamide-treated cotyledonary protoplasts of S. sanitwongsei were electrofused and cultured. Regenerated plants were classified into three groups based on morphology and genomic in situ hybridization data. Morphology of the first group was intermediate between those of parental species. The plants bore fruits with viable seeds and had a chromosome number of 2n� 48, the sum of the parental chromosome numbers, suggesting that they were symmetric fusion hybrids. Morphology of the plants in the second group was more S. integrifolium-like than that of the first group, and had two sets of S. integrifolium chromosomes and one set of S. sanitwongsei chromosomes. In contrast, plants in the third group had one set of S. integrifolium chromosomes and two sets of S. sanitwongsei chromosomes. Plants in the second and third groups were less vigorous than those in the first group, and bore few fruits. Electrophoretic analysis of the isozymes shikimate dehydrogenase, isocitrate dehydrogenase, and phosphoglucomutase, as well as random amplified polymorphic DNA analysis, demonstrated that 23 of regenerated plants from the three groups were somatic hybrids. The plants in the first group grew more vigorously than the parental plants and produced more than 5000 seeds per plant. The fertile somatic hybrids obtained in this study may be suitable candidates for eggplant rootstocks.

The present study had two aims. The first was to evaluate the reliability and the validity of the Japanese version of the World Health Organization (WHO)-Five Well-Being Index (WHO-5-J) as a brief well-being scale. The second was to examine the discriminatory validity of this test as a screening tool for current depressive episodes in diabetic patients. A sample of 129 diabetic patients completed the WHO-5-J. Of these, 65 were also interviewed by psychiatrists to assess whether they had any current depressive episodes according to DSM-IV. The internal consistency was evaluated using Cronbach's alpha, the Loevinger coefficient of homogeneity, and factor analysis. The external concurrent validity was evaluated by correlations with the external scales potentially related to subjective well-being. Discriminatory validity was evaluated using receiver operating characteristic (ROC) analysis. Cronbach's alpha and the Loevinger coefficient were estimated to be 0.89 and 0.65, respectively. A factor analysis identified only one factor. The WHO-5-J was significantly correlated with a number of major diabetic complications, depression, anxiety, and subjective quality of life. ROC analysis showed that the WHO-5-J can be used to detect a current depressive episode (area under curve: 0.92; 95% confidence interval: 0.85-0.98). A cut-off of

A segregating doubled haploid (DH) population (n = 96) was developed by anther culture of an F1 plant crossed between susceptible (‘Manganji’) and resistant (‘Criollo de Morelos 334’) lines of pepper (Capsicum annuum L.) to conduct a genetic analysis of resistance to Phytophthora rot caused by Phytophthora capsici. In order to perform a quantitative trait locus (QTL) analysis, we constructed a high density simple sequence repeat (SSR)-based map with a total length of 878 cM. Sixteen linkage groups (LGs) and 118 SSR markers were located using the 626 SSR markers that we previously developed. Resistance was evaluated in two root inoculation tests. Interval mapping for the resistance to P. capsici detected a common major QTL in the duplicate tests and a minor QTL specific to the first test. The major QTL was located on LG15 and flanked with an SSR marker, CAMS420. In addition, seven SSR markers were located within 21 cM intervals from the peak of this QTL. In contrast, the QTL on LG3 was detected with small effects in the first test, the nearest marker was a dominant amplified fragment length polymorphism (AFLP) marker, and the QTL was surrounded by eight SSR markers within a distance of 10 cM. Since some of the linkage markers for agriculturally valuable traits cannot detect polymorphism within breeding populations in C. annuum, the present linkage markers may widen the choice in marker-assisted selection in breeding programs for Phytophthora rot resistant pepper cultivars.

The FLC gene product is an inhibitor of flowering in Arabidopsis. FLC homologs in Brassica species are thought to control vernalization. We cloned four FLC homologs (BoFLCs) from Brassica oleracea. Three of these, BoFLC1, BoFLC3 and BoFLC5, have been previously characterized. The fourth novel sequence displayed 98% sequence homology to the previously identified gene BoFLC4, but also showed 91% homology to BrFLC2 from Brassica rapa. Phylogenetic analysis showed that this clone belongs to the FLC2 clade. Therefore, we designated this gene BoFLC2. Based on the segregation of RFLP, SRAP, CAPS, SSR and AFLP loci, a detailed linkage map of B. oleracea was constructed in the F(2) progeny obtained from a cross of B. oleracea cv. Green Comet (broccoli; non-vernalization type) and B. oleracea cv. Reiho (cabbage; vernalization type), which covered 540 cM, 9 major linkage groups. Six quantitative trait loci (QTL) controlling flowering time were detected. BoFLC1, BoFLC3 and BoFLC5 were not linked to the QTLs controlling flowering time. However, the largest QTL effect was located in the region where BoFLC2 was mapped. Genotyping of F(2 )plants at the BoFLC2 locus showed that most of the early flowering plants were homozygotes of BoFLC-GC, whereas most of the late- and non-flowering plants were homozygotes of BoFLC-Rei. The BoFLC2 homologs present in plants of the non-vernalization type were non-functional, due to a frameshift in exon 4. Moreover, duplications and deletions of BoFLC2 were detected in broccoli and a rapid cycling line, respectively. These results suggest that BoFLC2 contributes to the control of flowering time in B. oleracea.

A linkage map of Chinese cabbage (Brassica rapa) was constructed to localize the clubroot resistance (CR) gene, Crr3. Quantitative trait loci analysis using an F(3) population revealed a sharp peak in the logarithm of odds score around the sequence-tagged site (STS) marker, OPC11-2S. Therefore, this region contained Crr3. Nucleotide sequences of OPC11-2S and its proximal markers showed homology to sequences in the top arm of Arabidopsis chromosome 3, suggesting a synteny between the two species. For fine mapping of Crr3, a number of STS markers were developed based on genomic information from Arabidopsis. We obtained polymorphisms in 23 Arabidopsis-derived STS markers, 11 of which were closely linked to Crr3. The precise position of Crr3 was determined using a population of 888 F(2) plants. Eighty plants showing recombination around Crr3 locus were selected and used for the mapping. A fine map of 4.74 cM was obtained, in which two markers (BrSTS-41 and BrSTS-44) and three markers (OPC11-2S, BrSTS-54 and BrSTS-61) were cosegregated. Marker genotypes of the 21 selected F(2) families and CR tests of their progenies strongly suggested that the Crr3 gene is located in a 0.35 cM segment between the two markers, BrSTS-33 and BrSTS-78.

There are five cultivated species of pepper, of which Capsicum annuum is the most widely cultivated as a vegetable or spice and the main experimental material of most pepper breeding programs. However, the number of simple sequence-repeat (SSR) markers known for C. annuum is limited. To develop SSR markers for Capsicum species, we constructed four SSR-enriched libraries from the genomic DNA of C.␣annuum, sequenced 1873 clones, and isolated 626 unique SSR clones. A higher percentage of these SSR markers were taken from dinucleotide motif libraries than from trinucleotide motif libraries. Primer pairs for the 626 SSR clones were synthesized and tested for polymorphisms; 594 amplified products were detected with the expected size. However, only 153 products were polymorphic between the parents of our mapping population. Using 106 highly reproducible pairs from the primer pairs, we constructed a linkage map of C. annuum in an intraspecific doubled haploid population (n=117) that contains nine previously reported SSRs as well as AFLP, CAPS, and RAPD markers and the trait of fruit pungency. The map contains 374 markers, including 106 new SSR markers distributed across all 13 linkage groups, and covers 1042 cM. The polymorphism information content (PIC) of these new SSR markers was calculated using 14 lines of Capsicum species. The average number of alleles per locus was 2.9 and the average PIC value was 0.46, even within C. annuum. The SSR markers developed in this study will be useful for mapping and marker-assisted selection in pepper breeding, and the linkage map provides a reference genetic map for Capsicum species.

Clubroot disease is caused by an obligate parasite, Plasmodiophora brassicae, and is one of the most serious diseases of Brassica crops worldwide. The pathogen is a eukaryote, and is a member of Plasmodiophorales. Recent phylogenic studies did not reveal its close relationship to fungi, but suggest a classification in Protozoa or Protoctista. The infection is thought to occur through two phases. The pathogen induces massive clubs on root, and prevents host growth. A number of clubroot resistance (CR) Chinese cabbage (Brassica rapa) cultivars have been bred using European turnips as sources of CR genes. Field populations of the pathogen have a wide variation of virulence, which causes the infection of CR Chinese cabbage cultivars. The CR gene of B. rapa is extensively studied with the aid of molecular markers. At least four independent loci are identified and mapped. They are all derived from European turnips. Crr1, and Crr2 are derived from Siloga and mapped in linkage groups R8 and R6, respectively. Crr3 from Milan White, and CRb from Gelria R are both mapped in R3. The molecular markers linked to Crr1, Crr2 and CRb showed homology to the central part of the long arm of chromosome 4 of Arabidopsis thaliana. These findings suggest that these CR genes are derived from the same region of the ancestral genome. In contrast, Crr3 is thought to have different origin, since its linkage markers showed homology to chromosome 3 of A. thaliana. Although another CR gene CRa is reported, the relationship between above mentioned four CR loci and CRa is not yet clarified. Genetic studies on CR genes in B. oleracea revealed a polygenic nature of the trait in this species and also suggest the presence of one CR gene having large effect. However, correspondence among the CR genes reported in these studies is not clarified because of the lack of information on base sequence of the linkage markers in this species. Strategies for breeding of more resistant Chinese cabbage cultivars to overcome the variation on virulence of field populations of the pathogen are presented.

Defective function of antigen-presenting cells has been postulated to be one of the non-HLA-linked susceptibility factors for type 1 diabetes mellitus, though the underlying genetic factors remain unclear. SLC11A1 (formerly NRAMP1), a divalent cation transporter, plays a crucial role in macrophage activation. We performed a case-control study in 224 healthy and 95 type 1 diabetic Japanese subjects, examining the length polymorphisms in the promoter region (−377 to −222) of SLC11A1, which may influence transcriptional activity. Alleles designated 2, 3, and 7 have been identified in Japanese subjects. The frequency of allele 7 was significantly higher in subjects with type 1 diabetes (9.47%) than in the healthy controls (4.46%). The difference is more marked in the subpopulation of Japanese subjects with type 1 diabetes; diabetic subjects with at least one protective HLA class II allele and those without any susceptibility HLA class II haplotypes, DR4-DQ4 or DR9-DQ9, had a much higher allele 7 frequency than controls. These findings suggest that the novel promoter polymorphism of SLC11A1 influences the susceptibility to type 1 diabetes in Japanese subjects.

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.

An inbred turnip ( Brassica rapa syn. campestris) line, N-WMR-3, which carries the trait of clubroot resistance (CR) from a European turnip, Milan White, was crossed with a clubroot-susceptible doubled haploid line, A9709. A segregating F(3) population was obtained by single-seed descent of F(2) plants and used for a genetic analysis. Segregation of CR in the F(3) population suggested that CR is controlled by a major gene. Two RAPD markers, OPC11-1 and OPC11-2, were obtained as candidates of linkage markers by bulked segregant analysis. These were converted to sequence-tagged site markers, by cloning and sequencing of the polymorphic bands, and named OPC11-1S and OPC11-2S, respectively. The specific primer pairs for OPC11-1S amplified a clear dominant band, while the primer pairs for OPC11-2S resulted in co-dominant bands. Frequency distributions and statistical analyses indicate the presence of a major dominant CR gene linked to these two markers. The present marker for CR was independent of the previously found CR loci, Crr1 and Crr2. Genotypic distribution and statistical analyses did not show any evidence of CR alleles on Crr1 and Crr2 loci in N-WMR-3. The present study clearly demonstrates that B. rapa has at least three CR loci. Therefore, the new CR locus was named Crr3. The present locus may be useful in breeding CR Chinese cabbage cultivars to overcome the decay of present CR cultivars.

In an analysis of 114 F(2) individuals from a cross between clubroot-resistant and susceptible lines of Brassica rapa L., 'G004' and 'Hakusai Chukanbohon Nou 7' (A9709), respectively, we identified two loci, Crr1 and Crr2, for clubroot (caused by Plasmodiophora brassicae Woronin) resistance. Each locus segregated independently among the F(2) population, indicating that the loci reside on a different region of chromosomes or on different chromosomes. Genetic analysis showed that each locus had little effect on clubroot resistance by itself, indicating that these two loci are complementary for clubroot resistance. The resistance to clubroot was much stronger when both loci were homozygous for resistant alleles than when they were heterozygous. These results indicate that clubroot resistance in B. rapa is under oligogenic control and at least two loci are necessary for resistance.

A sweet pea (Lathyrus odoratus L.) cultivar, ‘Grace’, which has tendrils, was crossed with ‘Snoopea purple’ lacking tendrils. The resultant F2 population was used to identify RAPD (random amplified polymorphic DNA) markers linked to a tendril gene. The presence of tendrils was found to segregate in a dominant fashion. A total of 302 random primers were used to screen a pair of bulked DNA samples of the F2 plants. Only two primers, WB32 and WB67, showed polymorphism between the bulked samples. The former generated a DNA fragment specific to the bulked sample with tendrils, while the latter amplified a fragment in the bulked sample without tendrils. Segregation of these RAPD markers was examined in the F2 population. One of them, WB32a was found to be linked to the tendril gene. The marker was then cloned and sequenced. A pair of primers was designed for specific amplification of this marker. The primer pairs amplified a clear and dominant band, SWB32a, and the band was specific to individuals with tendrils. The linkage between the marker, SWB32a and the gene for tendrils was demonstrated in the F2 population in a distance of 7.7 cM. Use of this genetic marker in the breeding of sweet pea cultivars without tendrils was discussed.

The increased oxidative stress in diabetes is known to contribute to the progression of diabetes and its complications. We have reported a significant relation between the content of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a product of oxidative DNA damage in urine or leukocytes and the severity of diabetic nephropathy and retinopathy [1]. We investigated whether 8-oxodG in urine or leukocytes is associated with the progression of diabetic nephropathy.We measured urinary 8-oxodG contents at entry and carried out a prospective longitudinal study to assess the progression of nephropathy over 5 years.There was a significant progression of diabetic nephropathy in the patients with higher excretion of 8-oxodG in urine compared with the patients with moderate or lower excretion of 8-oxodG. There was no significant association between the leukocyte 8-oxodG contents and the development of nephropathy. The multivariate logistic regression analysis suggests that the urinary 8-oxodG was the strongest predictor of nephropathy among several known risk factors.This study provides evidence that increased oxidative stress has a primary role in the pathogenesis of diabetic nephropathy. A local enhancement of oxidative stress in diabetic kidney might explain the possible linkage between the increased urinary excretion of 8-oxodG and the development of nephropathy. 8-oxodG in urine is a useful clinical marker to predict the development of diabetic nephropathy in diabetic patients.

Tomato plants were transformed with a truncated replicase gene encoded by RNA 2 of cucumber mosaic virus (CMV) strain GT, subgroup II. The truncated replicase gene does not retain a C-terminal region of the gene that contains the GDD amino acid motif and the NTP binding motif. These motifs are considered to correspond to active and/or recognition domains of RNA-dependent RNA polymerase. Upon transformation via Agrobacterium tumefaciens, 137 individual transgenic lines were obtained. Each transgenic line was evaluated for resistance to CMV. About 10 % of the transgenic lines were highly resistant and the remaining 90 % showed a moderate resistance or were susceptible. The 15 lines were selected as resistant lines. Chenopodium amaranticolor was used to analyze the multiplication of CMV in the symptom-less plants. Among the selected lines, three lines did not appear to show any multiplication of CMV in both inoculated and non-inoculated leaves. Based on transgene amplification by PCR and the kanamycin resistance assay, the T1 progeny of the selected lines harbored the transgene. Several resistant lines of the T1 generation were resistant to viral inoculation. These resistant lines could become suitable breeding materials for resistance to CMV.

DNA markers linked to a locus controlling an extreme late bolting trait, which was originally found in a local cultivar of a non-heading leafy vegetable,‘Osaka Shirona Bansei’ (Brassica rapa L. ssp. pekinensis syn. campestris L.) were identified using bulked segregant analysis. A doubled haploid (DH) line, DH27, which is a progeny of ‘Osaka Shirona Bansei’, shows extreme late bolting, and bolts without vernalization. DH27 was crossed with a normal bolting DH line, G309. The plantlets of the parents, F1 and F2, were vernalized and then grown in a greenhouse. The bolting time of F2 plants showed a continuous distribution from 19 to 231 days after vernalization (DAV), suggesting the effects of a few major genes and polygenes. Possible linkage markers for this trait were screened by modified bulked segregant analysis (BSA). The BSA using four bulks suggested that a 530-bp RAPD band RA1255C was linked to a locus controlling the bolting trait. The RAPD band was cloned and used as a probe to detect RFLP. The fragment detected a single locus, BN007-1,the segregation of which in the F2 population matched that of RA1255C. Three other RAPDs were found to be linked to BN007-1. A quantitative trait locus(QTL) affecting the bolting time was detected around BN007-1 using MAPMAKER/QTL. Since the difference between bolting times of both the parental genotypes in the F2 was 138 days, these markers may be useful for a marker-assisted selection (MAS) in the breeding program for late bolting or bolting-resistant cultivars in B. rapa crops.

We have constructed a linkage map of eggplant (Solanum melongena L.) using an F2 population derived from a cross between a breeding line, EPL-1 and an introduced line, WCGR112-8, from India. The two parental lines showed contrasting responses to several pathogens and differences in several morphological traits. Parental lines were screened with 1, 232 random primers for RAPD and 64 primer combinations for AFLP. The link-age map consisted of 181 loci, comprising 88 RAPD and 93 AFLP markers. These markers identified 21 linkage groups spanning 779.2cM with an average distance of 4.9cM. The linkage groups ranged from 1.9 to 95.6cM in length and included 2 to 32 markers, respectively. The fruit shape and color development trait were scored and the linkage to the markers was investigated. The fruit shape showed a significant association with markers on linkage group 2. Color development in fruit, stem and calyx showed a significant association with markers on linkage group 7. These markers may provide valuable information for eggplant breeding.

A high association between type 2 diabetes mellitus and depressive illness has been reported. Insulin resistance during depressive illness might contribute to the linkage between depression and type 2 diabetes. To determine whether the genetic polymorphisms of the tyrosine hydroxylase ([TH] HUMTH01) and insulin (INS-VNTR) genes contribute to insulin resistance in depressive illness, we analyzed the association between the polymorphisms and insulin resistance in 41 Japanese patients with depressive disorder, 204 normal control subjects, 161 cohort subjects with normal glucose tolerance (NGT) and without depressive symptomatology, and 59 NGT subjects with depressive symptomatology. The depressive patients had a significantly lower insulin sensitivity index (SI) than the control subjects (P = .016). Depressive NGT subjects had a significantly higher homeostasis model assessment (HOMA) insulin resistance index [HOMA(R)] than the nondepressive NGT subjects (P < .0001). The depressive patients and NGT subjects had more HUMTH01 allele 7 (TH7) than the controls and nondepressive NGT subjects. SI was significantly lower in patients with the TH7/7 homozygote versus patients with the other genotypes and the controls. TH7 was associated with higher HOMA(R) as compared with the other alleles in the NGT subjects. Insulin resistance was associated with depressive disorders. The HUMTH01 and INS-VNTR were associated with insulin resistance and depressive symptoms.

Fifteen cultivars of sweet pea (Larthyrus odoratus L.) and one of everlasting pea (Lathyrus latifolium L.) were analysed using 12-mer Random Amplified Polymorphic DNA markers (RAPD). UPGMA clustering analysis indicated that individuals of Lathyrus odoratus L. from various collections belonged to the same cluster at 0.925 level of similarity coefficient. Lathyrus latifolium L. is supposedly an ancestor of Lathyrus odoratus but the similarity coefficient between the two is 0.352, indicating a very distant relationship. These results indicate that the sweet pea cultivars are closely related with a few polymorphic DNA patterns. However, to create new cultivars of sweet pea by crossing, one should use linkage markers for heritable characters.

Bacterial wilt caused by Ralstonia solanacearum is one of the most serious diseases in pepper (Capsicum annuum) crops in warm-temperate, subtropical, and tropical areas, including Japan. Resistant lines are a prerequisite for breeding resistant cultivars but are not well studied. Eight pepper accessions previously described as resistant to the pathogen were selected and inoculated with a highly virulent strain, KP9547. Among them, Malaysian accession LS2341 exhibited the highest resistance. Accession LS2341 was then challenged with 14 virulent strains collected from various areas in Japan. The strains cover biovars 2T, 3, and 4 and include isolates from the host plants of tomato, eggplant, and pepper. Results indicated that accession LS2341 had the highest level of resistance to all the strains of R. solanacearum examined. Therefore, accession LS2341 was confirmed as an appropriate source for the breeding of resistant cultivars of pepper in Japan and is a candidate for a potential source of resistance in other areas.

Increased oxidative stress induced by hyperglycemia may contribute to the pathogenesis of diabetic complications. Oxidative stress is known to increase the conversion of deoxyguanosine (dG) to 8-hydroxydeoxyguanosine (8-OHdG) in DNA, which is linked to increased mitochondrial DNA (mtDNA) deletions. We investigated mtDNA deletions and 8-OHdG in the muscle DNA of non-insulin-dependent diabetes mellitus (NIDDM) patients. mtDNA deletion of 4977 bp (delta mtDNA4977) and the content of 8-OHdG in the muscle DNA of the NIDDM patients were much higher than those of the control subjects. There was a significant correlation between delta mtDNA4977 and the 8-OHdG content (P < 0.0001). Both delta mtDNA4977 and the 8-OHdG content were also correlated with the duration of diabetes. Delta mtDNA4977 and the 8-OHdG content in muscle DNA increased in proportion to the severity of diabetic nephropathy and retinopathy. This is the first report that an increase in delta mtDNA4977 and 8-OHdG is proportional to the severity of diabetic complications. Oxidative mtDNA damage is speculated to contribute to the pathogenesis of diabetic complications though a defect in mitochondrial oxidative phosphorylation or other mechanisms. 8-OHdG and delta mtDNA4977 are useful markers to evaluate oxidative mtDNA damage in the diabetic patients.

A Iinkage map of Brassica rapa was constructed based on random amplified polymorphic DNAs (RAPDs), isozymes and restriction fragment length polymorphism (RFLP), using an F2 population derived from the crossing of a Chinese cabbage line, ‘Homei 09' and a club-root-resistant turnip line, ‘Siloga S2'. The map covered 851 cM, about half of the genome, and consisted of 16 linkage groups. ‘Homei 09' is highly responsive to microspore culture unlike ‘Siloga S2'. To identify the loci affecting embryogenesis in microspore culture, frequencies of RAPD in a microspore-derived population obtained from an F1 of the same parentage were scored. Several parts of the linkage map showed a conspicuous distortion toward ‘Homei 09' alleles. The microspores of another F2 population from the same parentage were cultured. Embryogenic ability and RAPD genotype of the each F2 plant were scored. Embryogenic ability of the plants for two marker types (presence and absence) was determined at each RAPD Iocus. In some parts of the linkage nrap, the plants carrying ‘Homei 09' alleles showed a higher ability of embryogenesis than those of ‘Siloga S2' homozygotes. Comparison of the distortion data with those in the embryogenesis of the F2 suggested that at least one part of the linkage map affected the embryogenic process in microspore culture. Genes in these areas of ‘Homei 09' may play a key role in triggering the embryogenesis or in embryo development at the early stage of microspore culture.

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.

This work was undertaken to investigate the relationship between water status and sucrose metabolism of suspension culture of carrot (Daucus carota L.) cells. Embryogenic callus was induced on carrot hypocotyl in vitro. The cell suspension culture was then produced from this callus.In suspension culture, water potential and pH of the culture solution were decreased immediately after culture initiation to -0.62MPa and 4.5 respectively, but gradually increased from 2 days after culture initiation. Sucrose in suspension culture solution was found to be immediately hydrolyzed into glucose and fructose after the initiation of culture and disappeared within 2 days from culture initiation. The disappearance of sucrose from the culture solution within 2 days of culture initiation corresponds with increases in the activity of cell-wall-bound acid invertase. These results indicate that the decline of water potential of the culture solution at the early stages of carrot suspension culture was associated with sucrose metabolism as a result of an increase in acid invertase activity.