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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.

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 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.

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 (

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.

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.

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.

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.

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.

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.

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 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.

We previously identiffed three random amplified polymorphic DNA (RAPD) markers linked to a major gene conferring clubroot resistance (CR) in Brassica rapa. The markers were cloned and sequenced. A pair of primers were designed for specific amplification of each marker. All three CR markers were specifically amplified as clear single and dominant bands. Identity of the loci of the amplified markers with the original RAPD markers was demonstrated using a segregating F2 population. The marker bands can be amplified by at least two types of PCR machine. Therefore these three markers may be widely used as a reference to CR genes in the genome of B. rapa. Presence of the markers in turnips and Chinese cabbage cultivars was examined. Turnips showed a high degree of DNA polymorphism within cultivars. No relationship between CR and the presence of the marker bands was detected, while Chinese cabbage hybrid cultivars showed a rather low DNA polymorphism. One of the CR markers, RA12-75A was found to be useful for the breeding of CR Chinese cabbage, because no clubroot-susceptible cultivars of Chinese cabbage harbour this marker band. Use of these specifically amplified markers in marker-as-sisted selection (MAS) of Brassica crops is discussed.

In order to transfer the growth habit and the resistance to Pseudomonas solauacearum from S. sanitwongsei to S. melongena, electrofusion was carried out. The fused protoplasts were cultured in KM(8p) medium supplemented with 1, 000ppm wilt-inducing product secreted by a virulent strain of P. solanacearum. The regenerated plants were further screened on the soil contaminated with P. solanacearum. Only one plant survived. Several morphological traits of the surviving plant were intermediate between those of the parents, and the chromosome number was 48. The pollen viability averaged 82.3%, and fruits were set. The hybridity was confirmed using random amplified polymorphic DNA (RAPD) markers. The somatic hybrid exhibited a resistance to P. solanacearum comparable to that of S. sanitwongsei. The juvenile growth of the offsprings (S1 plants) obtained by selfing of the somatic hybrid was superior to that of S. sanitwongsei. The S1 plants were also as resistant to P. solanacearum as S. sanitwongsei. When the S1 plants were used as rootstocks for eggplants, the fruit yields were as high as those of eggplants grafted on S. sanitwongsei. It was considered that the S1 plants could be used as rootstocks for eggplant production.

Mesophyll protoplasts of black mustard and Hakuran were fused by the PEG-high pH ?? igh Ca method. Out of six plants regenerated, one showed hybrid morphology. The hybridity was ascertained by the isozyme pattern of acid phosphatase and chromosome number. This hybrid was estimated to be a trigenomic hexaploid having A. B and C genomes, and showed similar male sterility to Hakuran.

Both biochemical and morphological characters were examined in 85 Japanese cultivars of taro (Colocasia esculenta ) to analyze the phylogenic relationships. Acrylamide gel electrophoresis of the tuber proteins revealed five patterns, designated as E, I, D, T, and A, for the major cultivars. Additional three patterns were found in the minor cultivars. Cluster analysis of taro cultivars based on 40 morphological characters was also performed. The results which were in agreement with those of the above-mentioned biochemical analyses, enabled to identlfy seven groups, i.e. Eguimo, Dodare (Dotare), Hasubaimo, Ishikawa-wase, Kurojiku, Akame, and Tonoimo groups, for the non-fasciculated taro cultivars, while two, Yatsugashira and Shogaimo groups for the fasciculated ones. The Yatsugashira group showed elec-trophoretic patterns common to the non-fasciculated Tonoimo group. Similarly the Shogaimo and Dodare groups exhibited a common electrophoretic pattern. The fasciculated cultivars are, therefore, assumed to be derivecl from the corresponding non-fasciculated cultivars by bud mutation. The Kurojiku group may be at the origin of the Ishikawa-wase group, since these two groups showed a common electrophoretlc pattern. The phylogenic and morphological relationships between the Hasubaimo and Dodare groups were also discussed.

The varietal differences in embryogenic ability in anther culture of Chinese cabbage, Brassica campestris ssp. pekinensis, were investigated. In the first experiment, the anthers of B. campestris were incubated in the modified B5 medium (KELLER and ARMSTRONG 1979) at 30°C for 14 days and then maintained at 25°C in the dark. Only a few embryos were ob-served in a limited number of cultivars in this treatment. In contrast, embryos were formed efficiently by the treatment at 35'C for one day. The embryogenic ability from anthers was remarkably different among the cultivars. Among the cultivars of B. campestris, the anthers of 'ASO Ichigo' belonging to the Kenshin group produced embryos most efficiently. The second experiment was conducted to know the embryogenic ability of cultivars in the Kenshin group. Although some varietal differences in the embryogenic ability were observed, one of the cultivars exhibited the highest culture efficiency (expected yield of embryos from 1, 000 anthers cultured) of over 10, 000. Comparing the data of two experiments, the Kenshin group were thought to have a high embryogenic ability in anther culture of Chinese cabbage.

Isozymes of GOT and MDH in leaves of 105 citrus biotypes were analyzed using acrylamide gel electrophoresis. At least 3 loci were detected on these two enzymes. From the analysis, Natsumikan (Citrus natsudaidai), Hassaku (C. hassaku) and some other citrus cultivars are considered to be hybrids of pummelo (C.grandis) and mandarin as suggested. Moreover, Iyo (C.iyo), which has been assumed to be a hybrid of sweet orange (C.sinensis) and mandarin, was found to have a genetic effect from pummelo. Sour oranges (C.aurantium) had unique allele A at Got-1. In addition to the sour orange, the same allele was found only in lchang lemon (C.wilsoni), Bergamot (C. bergamia) and 'Hirado' pummelo. While, Tachibana (C.tachibana), Shiikuwasha (C. depressa), and Korai-tachibana (C. nippokoreana), which are reported to be native to Japan and its neighboring areas, had an unique allele A at Got-2. This allele was also found in Hyuganatsu (C.tamurana) and some biotypes originated in Japan, but not found in mandarins originated in mainland China, India, Philippines and other areas. While, Yuzu (C.junos) and its relatives had B at Got-2 and B at Mdh-1.

Leaf isozymes of four enzymes in citrus were analyzed using polyacrylamide gel electrophoresis to demonstrate the phylogenic relationship of the cultivars. Two loci for the superoxide dismutase(SOD) (EC1.15.1.1), one locus for the peroxidase (EC 1.11.1.7) and polyphenol oxidase (EC1.10.3.2), respectively were examined. In addition to the two loci of glutamate oxal-acetate transaminase(GOT) (EC2.6.1.1), reported in the former study, a new locus was detected in the present report. These experiments suggest that the mandarin cultivars originated from three genetic sources ; Indian, Chinese, and Japanese ones. Most of the Chinese mandarins including 'Kinokuni' showed identical genotypes, while the 'Ponkan' and 'Dancy tangerine' differed from the Chinese cultivars in their genotypes at the Px locus, and they were assumed to have originated from India as suggested by T. Tanaka. On the other hand, the Japanese mandarins were considered to be genetically affected by the'Chinese mandarins. But some of the Japanese mandarins including 'Yatsushiro' and 'Koji' exhibited genetic effects from Tachibana, the wild type of the mandarin in J.apan. The genotypes of the sweet orange cultivars were homogeneous and similar to those 0L the Chinese mandarins. Sour orange was heterozygous at several loci, and was assumed to be a hybrid between mandarin and pummelo. Among the pummelo cultivars, some heterogeneity was detected at some loci.