Small variation of glucosinolate composition in Japanese cultivars of radish (Raphanus sativus L.) requires simple quantitative analysis for breeding of glucosinolate component

To reveal varietally differing glucosinolate (GSL) contents in radish (Raphanus sativus L.) cultivated in Japan, the total and individual GSLs of 28 cultivars were analyzed using high-performance liquid chromatography. In these cultivars, GSL types including three aliphatic GSLs (glucoraphenin, glucoerucin, and 4-methylthio-3-butenyl GSL (4MTB-GSL)) and three indolyl GSLs (4-hydroxyglucobrassicin, glucobrassicin, and 4-methoxy-glucobrassicin) were detected. No cultivar-specific type of GSL was identified. The dominant GSL was 4MTB-GSL, but its contents differed remarkably: 8.6 μmol/g in ‘Koushin’ to 135.7 μmol/g in ‘Karami 199’. Over about 90% of all GSLs in Japanese radish type are 4MTB-GSL, a higher percentage than in Chinese or European garden radish cultivars. A simple, rapid method for estimating total GSL contents in crude extracts was established because of the small variation of glucosinolate composition in Japanese cultivars. The total GSL content can be estimated using an equation for prediction with absorbance at 425 nm in a mixture of GSL crude extract and palladium (II) chloride solution: Total GSL (μmol/g) = 305.47 × A₄₂₅ − 29.66. Its coefficient of determination (R²) and standard error of prediction (SEP) are 0.968 and 8.052. This method enables total GSL content estimation from more than 200 samples per person per day.     

Diallel analysis of floral morphology in radish (Raphanus sativus L.)

Patterns of photoperiodic-response curve (PRC) of heading time in rice were examined using the five near-isogenic lines (NILs) of Taichung 65, in which insensitive alleles was replaced by photosensitive alleles through backcrossings. In the first experiment, the NILs showed the diversified patterns of PRC, depending on genotype, under the controlled conditions (9-, 10-, 12- and 14-h daylengths). In addition, the two NILs with different Se1 alleles showed the different patterns although they showed similar heading times under natural daylength (NDL) conditions. The cryptic difference gives some clues as to the relationship between the patterns and heading-time behaviors under NDL conditions. In the second experiment, behaviors of vegetative and reproductive developments under 20-h daylength condition with high- and low-nutrient conditions were examined to know whether or not they behave as expected from their PRC obtained in the first experiment. Under the extremely long-day condition, the photosensitive alleles brought about long-growth durations where the internal changes for senescence might drastically occur with increases of total number of phytomers (TNP) and decrease potentials for reproductive developments. The nutrient levels might not affect the pattern of internal changes but might affect TNP, depending on genotype, and as a result, the nutrient levels might bring about different developmental fates in the same genotype under the long-day condition. The present results suggest the importance of genotype-by-environment interaction in relation to the cryptic variation of PRC in rice.     

Hybridization of radish (Raphanus sativus L.) and oilseed rape (Brassica napus L.) through a flower-culture method

Summary Hybridization between radish and oilseed rape has been cumbersome, requiring elaborate embryo rescue techniques. With a modified flower culture method, we have achieved successful hybridization between radish and (transgenic) oilseed rape without the laborious and technically demandingin vitro ovule or embryo rescue techniques.The hybrid nature of the intergeneric hybrids was demonstrated using morphological traits, and DNA analyses. The described method will facilitate the generation ofRaphanobrassica hybrids useful for biosafety studies of the potential for transgenes to spread in weedyCruciferae as well as for breeding programs aimed at introducing useful radish genes, e.g. nematode resistance genes, into oilseed rape.     

Development of PCR-based markers linked to a restorer gene for cytoplasmic male sterility in radish (Raphanus sativus L.)

A random amplified polymorphic DNA (RAPD) marker named OPC06-1900 was previously found linked to a fertility restorer gene (Rfw) for cytoplasmic male sterility (CMS) in radish (Raphanus sativus L.). The RAPD marker was converted to a dominant sequence characterized amplified region (SCAR) marker SCC06-1894 by molecular cloning and nucleotide sequencing. A BLAST search revealed that the SCAR marker SCC06-1894 showed significant homology to the corresponding regions of Arabidopsis and Brassica sulfate transporter genes. The presence of the intron and exon of the DNA fragment SCC06-1894 was demonstrated by comparing RT-PCR and PCR products. Thus, allele-specific oligonucleotide primers were designed to amplify the SCAR marker SCC06-415. PCR test with F₂ plants and sequence analysis showed that SCC06-1894 and SCC06-415 were allelic, linked to Rfw/rfw gene at 8.0 cM. Nine oligonucleotide primers were designed based on a single radish nuclear restorer gene mRNA. A survey of these primer combinations by bulked segregant analysis (BSA) identified three polymorphisms. The three PCR-based markers were co-segregant in the coupling phase and distant from the Rfw gene by 1.4 cM. These specific markers distributed on both sides of the Rfw gene and will be helpful for breeding new rapseed (Brassica napus L.) restorer lines.     

Quantitative evaluation of the three-dimensional fruit shape and size of Citrus species based on spherical harmonic descriptors

A measurement system, which consisted of a CCD laser displacement sensor and two pulse stages, was constructed to obtain three-dimensional (3-D) surface data of Citrus fruit. Fruits of 58 species of three specimens each were measured. By expanding the data measured in a series of spherical harmonic functions, a set of expansion coefficients to characterize the object shape was obtained. The data was normalized to make the coefficients be independent of the x- and y-coordinates of the object. To summarize the information about shape contained in the62 coefficients, a principal component analysis of the standardized spherical harmonic coefficients of each fruit was made. The contribution of each of the first seven principal components was found to exceed that of a single variable. The first three principal components had a cumulative contribution of 0.823.Analysis of variance showed that the variance due to between-species as tested against the variance due to within-species was significant for the 1^st,2^nd, 3^rd, 4^th, 7^thcomponent score and the volume. A scatter diagram of the 1^st and 2^nd principal components failed to group the species studied. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification of a sequence-tagged site (STS) marker linked to a restorer gene for Ogura cytoplasmic male sterility in radish (Raphanus sativus L.) by non-radioactive AFLP analysis

To identify DNA markers linked to a fertility restorer (Rf) genefor Ogura cytoplasmic male sterility in radish (Raphanus sativus L.),a non-radioactive, amplified fragment length polymorphism (AFLP) analysiswas performed on bulked DNA samples from male-sterile and male-fertileradishes. Ten male-fertile and 10 male-sterile plants selected arbitrarilyfrom an F₂ population made by selfing of F₁ plant from a crossbetween a male-sterile (`MS-Gensuke') plant and a restorer (`Comet') plantwere used as material. Using 32 AFLP primer pairs, one AFLP fragment(AFLP190) which is specific to the bulked DNA samples from male-fertileF₂ plants was identified. AFLP190 was characterized by molecularcloning and nucleotide sequencing, and was converted to a sequence-taggedsite (STS) marker, STS190. A linkage analysis performed in 126individuals of two independent F₂ populations showed tight linkageof STS190 to the Rf gene. The rate of recombination between themarker and Rf was estimated to be less than 1%, making STS1901.2 cM from the gene.     

Evaluation of fineness of wrinkles on husks of malting barley (Hordeum vulgare L.) by texture analysis of digital image data

Summary It has been suggested that the selection based on the wrinkles on the husks of malting barley is useful especially during the early generation in breeding program, because the wrinkles can be an indicator of malting quality of grains. However, it can be done only visually and requires much labor. In this study, the evaluation of the wrinkles by texture analysis of digital image data, was examined for the quantitative evaluation of the fineness of the wrinkles and its automatic selection. The texture analysis based on co-occurrence matrices was applied and it was revealed that finely wrinkled grains can be clearly discriminated by discriminant analysis on several parameters estimated from the co-occurrence matrices.     

Identification of quantitative trait loci for powdery mildew resistance in highly resistant cucumber (Cucumis sativus L.) using ddRAD-seq analysis

Powdery mildew, caused by Podosphaera xanthii (syn. Sphaerotheca fuliginea ex Fr. Poll.), is one of the most economically important foliar diseases in cucumber (Cucumis sativus L.). Cucumber parental line ‘Kyuri Chukanbohon Nou 5 Go’, developed from weedy cucumber line CS-PMR1, is highly resistant to powdery mildew and is promising breeding material. We performed quantitative trait locus (QTL) analysis using double-digest restriction-site-associated DNA sequencing (ddRAD-Seq) in a population from a cross between ‘Kyuri Chukanbohon Nou 5 Go’ and the Japanese native cultivar ‘Kaga-aonaga-fushinari’, which is susceptible to powdery mildew. The resistance of the population and its parents was evaluated using leaf disc assays and image analysis. We detected one major QTL on Chr. 5 that was effective at both 20°C and 25°C and one minor QTL on Chr. 1 effective at 20°C. We detected two additional QTLs in subpopulation: one on Chr. 3 effective at 20°C and one on Chr. 5 effective at both 20°C and 25°C in a position different from the major QTL. The resistance alleles at all four QTLs were contributed by ‘Kyuri Chukanbohon Nou 5 Go’. The results of this study can be used to develop practical DNA markers tightly linked to genes for powdery mildew resistance.     

Variation among maize (Zea mays L.) accessions of Bendel State,Nigeria. Multivariate analysis of agronomic data

Summary Two multivariate techniques were used to characterize 30 maize accessions collected from three ecological zones of Bendel State, Nigeria. Differentiation of the 30 accessions into five distinct groups was achieved with the unweighted variable group method of the average linkage cluster analysis of 34 agronomic characters. Four of the taxonomic groups contained at least three accessions each, while a fifth group contained only one. The single accession contained in the fifth group was characterised by very early maturity, deeply pigmented leaves and ear husks and short statured plants. Clustering of the accessions into different phenetic groups followed substantially along geographical and traditionally trading routes. A few cases of overlapping of accessions from different geographical locations were obtained.Principal component analysis revealed that days to 50% tasseling and silking, number of nodes/plant, ear length, ear weight, leaf width, and kernel colour were the principal discriminatory characters that differentiated the accessions. Sixty-four percent of the total variation among the 34 characters were accounted for by the first five principal components while the first and second components accounted for 26 and 14 respectively.     

Molecular and biological studies on male sterile cytoplasm in Cruciferae. II. The origin of Ogura male sterile cytoplasm inferred from the segregation pattern of male sterility in the F1 progeny of wild and cultivated radishes (Raphanus sativus L.)

Summary To determine the origin of Ogura male sterile cytoplasm in radish (Raphanus sativus L.), wild and cultivated radishes were crossed. Three types of progeny resulted from the F₁ hybrids between the wild radish from Kushikino with Ogura-type mtDNA and the cultivars (Uchiki-Gensuke or Comet). The segregation patterns of the male sterility were compared with those of Ogura cytoplasm. The male sterility induced in the F₁ hybrid was maintained by crossing with Uchiki-Gensuke, that maintains Ogura male sterility. In the two types of progeny, in which Comet (a restorer of Ogura cytoplasm) was used as one of the parents, both fertile and sterile plants segregated at the predicted ratio on the assumption that a single dominant fertility restoring gene exists in the restorer. From these results, we concluded that the Ogura cytoplasm is identical to that of the wild radish, and the former originated in a population of Japanese wild radish.