Nobuko Fukino

A consensus linkage map for molecular markers and quantitative trait loci associated with economically important traits in melon (Cucumis melo L.).

BackgroundA number of molecular marker linkage maps have been developed for melon (Cucumis melo L.) over the last two decades. However, these maps were constructed using different marker sets, thus, making comparative analysis among maps difficult. In order to solve this problem, a consensus genetic map in melon was constructed using primarily highly transferable anchor markers that have broad potential use for mapping, synteny, and comparative quantitative trait loci (QTL) analysis, increasing breeding effectiveness and efficiency via marker-assisted selection (MAS).ResultsUnder the framework of the International Cucurbit Genomics Initiative (ICuGI, http://www.icugi.org), an integrated genetic map has been constructed by merging data from eight independent mapping experiments using a genetically diverse array of parental lines. The consensus map spans 1150 cM across the 12 melon linkage groups and is composed of 1592 markers (640 SSRs, 330 SNPs, 252 AFLPs, 239 RFLPs, 89 RAPDs, 15 IMAs, 16 indels and 11 morphological traits) with a mean marker density of 0.72 cM/marker. One hundred and ninety-six of these markers (157 SSRs, 32 SNPs, 6 indels and 1 RAPD) were newly developed, mapped or provided by industry representatives as released markers, including 27 SNPs and 5 indels from genes involved in the organic acid metabolism and transport, and 58 EST-SSRs. Additionally, 85 of 822 SSR markers contributed by Syngenta Seeds were included in the integrated map. In addition, 370 QTL controlling 62 traits from 18 previously reported mapping experiments using genetically diverse parental genotypes were also integrated into the consensus map. Some QTL associated with economically important traits detected in separate studies mapped to similar genomic positions. For example, independently identified QTL controlling fruit shape were mapped on similar genomic positions, suggesting that such QTL are possibly responsible for the phenotypic variability observed for this trait in a broad array of melon germplasm.ConclusionsEven though relatively unsaturated genetic maps in a diverse set of melon market types have been published, the integrated saturated map presented herein should be considered the initial reference map for melon. Most of the mapped markers contained in the reference map are polymorphic in diverse collection of germplasm, and thus are potentially transferrable to a broad array of genetic experimentation (e.g., integration of physical and genetic maps, colinearity analysis, map-based gene cloning, epistasis dissection, and marker-assisted selection).

Glucosinolate metabolism, functionality and breeding for the improvement of Brassicaceae vegetables

Unique secondary metabolites, glucosinolates (S-glucopyranosyl thiohydroximates), are naturally occurring S-linked glucosides found mainly in Brassicaceae plants. They are enzymatically hydrolyzed to produce sulfate ions, D-glucose, and characteristic degradation products such as isothiocyanates. The functions of glucosinolates in the plants remain unclear, but isothiocyanates possessing a pungent or irritating taste and odor might be associated with plant defense from microbes. Isothiocyanates have been studied extensively in experimental in vitro and in vivo carcinogenesis models for their cancer chemopreventive properties. The beneficial isothiocyanates, glucosinolates that are functional for supporting human health, have received attention from many scientists studying plant breeding, plant physiology, plant genetics, and food functionality. This review presents a summary of recent topics related with glucosinolates in the Brassica family, along with a summary of the chemicals, metabolism, and genes of glucosinolates in Brassicaceae. The bioavailabilities of isothiocyanates from certain functional glucosinolates and the importance of breeding will be described with emphasis on glucosinolates.

Fine mapping of the clubroot resistance gene CRb and development of a useful selectable marker in Brassica rapa

In Chinese cabbage (Brassica rapa), the clubroot resistance (CR) gene CRb is effective against Plasmodiophora brassicae isolate No. 14, which is classified as pathotype group 3. Although markers linked to CRb have been reported, an accurate position in the genome and the gene structure are unknown. To determine the genomic location and estimate the structure of CRb, we developed 28 markers (average distance, 20.4 kb) around CRb and constructed a high-density partial map. The precise position of CRb was determined by using a population of 2,032 F2 plants generated by selfing B. rapa ‘CR Shinki.’ We determined that CRb is located in the 140-kb genomic region between markers KB59N07 and B1005 and found candidate resistance genes. Among other CR genes on chromosome R3, a genotype of CRa closest marker clearly matched those of CRb and Crr3 did not confer resistance to isolate No. 14. Based on the genotypes of 11 markers developed near CRb and resistance to isolate No. 14, 82 of 108 cultivars showed a strong correlation between genotypes and phenotypes. The results of this study will be useful for isolating CRb and breeding cultivars with resistance to pathotype group 3 by introducing CRb into susceptible cultivars through marker-assisted selection.

Identificaiton of a clubroot resistance locus conferring resistance to a Plasmodiophora brassicae classified into pathotype group 3 in Chinese cabbage (Brassica rapa L.)

In Chinese cabbage (Brassica rapa), the clubroot resistance (CR) genes Crr1 and Crr2 are effective against the mild Plasmodiophora brassicae isolate Ano-01 and the more virulent isolate Wakayama-01, but not against isolate No. 14, classified into pathotype group 3. ‘Akiriso’, a clubroot-resistant F1 cultivar, showed resistance to isolate No. 14. To increase the durability of resistance, we attempted to identify the CR locus in ‘Akiriso’. CR in ‘Akiriso’ segregated as a single dominant gene and was linked to several molecular markers that were also linked to CRb, a CR locus from cultivar ‘CR Shinki’. We developed additional markers around CRb and constructed partial genetic maps of this region in ‘Akiriso’ and ‘CR Shinki’. The positions and order of markers in the genetic maps of the two cultivars were very similar. The segregation ratios for resistance to isolate No. 14 in F2 populations derived from each of the two cultivars were also very similar. These results suggest that the CR locus in ‘Akiriso’ is CRb or a tightly linked locus. The newly developed markers in this study were more closely linked to CRb than previously reported markers and will be useful for marker-assisted selection of CRb in Chinese cabbage breeding.

Identification of quantitative trait loci for downy mildew resistance in cucumber (Cucumis sativus L.)

Downy mildew, caused by Pseudoperonospora cubensis (Berk. & Curt.) Rostov, is one of the most economically important foliar diseases in cucumber (Cucumis sativus L.). Cucumber line CS-PMR1, derived from self-pollination of USDA Plant Introduction 197088, has a high level of resistance to downy mildew and is considered to be promising breeding material. In this study, we performed quantitative trait locus (QTL) analysis for downy mildew resistance using 111 recombinant inbred lines (RILs) derived from a cross between CS-PMR1 and the old Japanese cultivar Santou, which exhibits moderate resistance. The resistance of the RILs and their parents was evaluated by diverse methods using different plant organs (cotyledons, true leaves), stages (seedlings and adult plants), and evaluation criteria (lesion expansion and extent of sporulation). The high resistance of CS-PMR1 was associated with many QTLs with relatively small effects, whereas the moderate resistance of Santou was associated with one major QTL and possibly two others with relatively small effects. In all assays, the major QTL at which the Santou allele was associated with increased resistance had the largest effect. This QTL allele from Santou and several of the most effective QTL alleles identified in CS-PMR1 should be highest priority for selection to efficiently breed new cultivars that carry adequate levels of downy mildew resistance.

Anchoring the consensus ICuGI genetic map to the melon (Cucumis melo L.) genome

Melon (Cucumis melo L.) genetic maps were compiled by the International Cucurbit Genomics Initiative (ICuGI) before the release of the melon genome. However, due to the use of different marker sets, the position of ICuGI markers in the genome remained unknown, complicating the integration of previous genetic mapping studies in the genome. We looked for the genome position of 870 simple sequence repeat and single nucleotide polymorphism (SNP) markers from the ICuGI map, locating 836 of them in the melon pseudochromosomes v3.5.1, and integrating them with previously available SNPs to reach a total of 1850 markers mapped in the genome sequence. The number of markers per scaffold ranged from 1 to 105, with an average of 13, thus improving on the previous studies in melon. Twenty-three of the markers mapped on virtual chromosome “0”, twelve of them being included in the ICuGI map, which could assist in the anchoring of some unanchored contigs and scaffolds. Genetic and physical distance comparison showed a good collinearity between them, confirming the quality of the ICuGI map. A higher recombination rate was also usually found at the ends of the chromosomes, whereas a drastic reduction was observed in the putative pericentromeric regions. Quantitative trait loci (QTL) previously located in the ICuGI map were also anchored in the genome. This work offers the opportunity to supplement the genetic maps available up to now with the genomic resources resulting from the melon genome sequencing to facilitate comparative mapping in melon between past and new studies, and to overcome some of the current limitations in QTL gene identification.

Mapping of a gene that confers short lateral branching (slb) in melon (Cucumis melo L.)

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.

Image-based phenotyping: use of colour signature in evaluation of melon fruit colour

Fruit colour, both external and internal, is important because it relates directly to the commercial value of the product. In breeding and in pre- and postharvest studies of fruit colour, an effective method for evaluating colour is needed to replace subjective evaluations by eye. We used a series of data processing and statistical analyses used in content-based image retrieval to evaluate melon flesh colour, and assessed the efficacy of this approach. This method relies on summarizing colour information from images into colour signatures, calculating the earth mover’s distance (EMD) between colour signatures, and multi-dimensional scaling (MDS) based on an EMD matrix. Performing MDS on a set of fruit flesh images revealed important colour features, such as the yellowish-green strength in green-fleshed melons and the relative size of the green and red parts in red-fleshed melons, without the need for an explicit definition of these features. The proportion of variance due to differences among cultivars was higher by MDS than by traditional evaluation, indicating that this new method performed best at detecting colour differences among cultivars. The method provides effective, objective indicators of fruit colour, and shows considerable promise for use in research and breeding programs.

A 2-oxoglutarate-dependent dioxygenase mediates the biosynthesis of glucoraphasatin in radish

Biosynthesis of glucoraphasatin, a major glucosinolate in radish, is mediated by 2-oxoglutarate-dependent dioxygenase. Glucosinolates (GSLs) are secondary metabolites whose degradation products confer intrinsic flavors and aromas to Brassicaceae vegetables. Several structures of GSLs are known in the Brassicaceae, and the biosynthetic pathway and regulatory networks have been elucidated in Arabidopsis (Arabidopsis thaliana). GSLs are precursors of chemical defense substances against herbivorous pests. Specific GSLs can act as feeding blockers or stimulants, depending on the pest species. Natural selection has led to diversity in the GSL composition even within individual species. However, in radish (Raphanus sativus), glucoraphasatin (4-methylthio-3-butenyl glucosinolate) accounts for more than 90% of the total GSLs, and little compositional variation is observed. Because glucoraphasatin is not contained in other members of the Brassicaceae, like Arabidopsis and cabbage (Brassica oleracea), the biosynthetic pathways for glucoraphasatin remain unclear. In this report, we identified and characterized a gene encoding GLUCORAPHASATIN SYNTHASE 1 (GRS1) by genetic mapping using a mutant that genetically lacks glucoraphasatin. Transgenic Arabidopsis, which overexpressed GRS1 cDNA, accumulated glucoraphasatin in the leaves. GRS1 encodes a 2-oxoglutarate-dependent dioxygenase, and it is abundantly expressed in the leaf. To further investigate the biosynthesis and transportation of GSLs in radish, we grafted a grs1 plant onto a wild-type plant. The grafting experiment revealed a leaf-to-root long-distance glucoraphasatin transport system in radish and showed that the composition of GSLs differed among the organs. Based on these observations, we propose a characteristic biosynthesis pathway for glucoraphasatin in radish. Our results should be useful in metabolite engineering for breeding of high-value vegetables.

Quantitative trait locus analysis of cucumber fruit morphological traits based on image analysis

Fruit morphology is one of the most important traits in cucumber because it directly affects its commercial value. The cucumber line CS-PMR1 is a promising breeding material for improving disease resistance, but its fruit morphology is not ideal for the Japanese market, and this hampers breeding. We used recombinant inbred lines derived from a cross between CS-PMR1 and the Japanese cultivar ‘Santou’ for genetic analysis of fruit morphological traits. Fruit shape variation was evaluated in detail by means of image analysis, and quantitative trait locus (QTL) analyses were performed by composite interval mapping or interval mapping with a nonparametric model. We detected 41 QTLs: 5 for length, 6 for diameter, 5 for the ratio of length to diameter, 4 for the ratio of placenta diameter to fruit diameter, 16 for principal component scores of elliptic Fourier descriptors related to fruit shape, 2 for wart size and 3 for wart density. Some QTLs were detected in the same chromosomal regions at different fruit stages and for different morphological traits. In particular, several QTLs with large effects on fruit size were located in nearly the same regions as genes for resistance to powdery mildew and downy mildew, indicating that the linkage between fruit shape and the resistance genes is likely to limit breeding efficiency. The results of this study will contribute to the development of informative DNA markers tightly linked to genes for requisite fruit morphological traits, which are urgently required for efficient breeding of new cultivars.