Transcriptome analysis of the oriental melon (Cucumis melo L. var. makuwa) during fruit development.

BACKGROUND: The oriental melon (Cucumis melo L. var. makuwa) is one of the most important cultivated cucurbits grown widely in Korea, Japan, and northern China. It is cultivated because its fruit has a sweet aromatic flavor and is rich in soluble sugars, organic acids, minerals, and vitamins. In order to elucidate the genetic and molecular basis of the developmental changes that determine size, color, and sugar contents of the fruit, we performed de novo transcriptome sequencing to analyze the genes expressed during fruit development. RESULTS: We identified a total of 47,666 of representative loci from 100,875 transcripts and functionally annotated 33,963 of the loci based on orthologs in Arabidopsis thaliana. Among those loci, we identified 5,173 differentially expressed genes, which were classified into 14 clusters base on the modulation of their expression patterns. The expression patterns suggested that the differentially expressed genes were related to fruit development and maturation through diverse metabolic pathways. Analyses based on gene set enrichment and the pathways described in the Kyoto Encyclopedia of Genes and Genomes suggested that the expression of genes involved in starch and sucrose metabolism and carotenoid biosynthesis were regulated dynamically during fruit development and subsequent maturation. CONCLUSION: Our results provide the gene expression patterns related to different stages of fruit development and maturation in the oriental melon. The expression patterns give clues about important regulatory mechanisms, especially those involving starch, sugar, and carotenoid biosynthesis, in the development of the oriental melon fruit.

De Novo Transcriptome Analysis of Cucumis melo L. var. makuwa.

Oriental melon (Cucumis melo L. var. makuwa) is one of six subspecies of melon and is cultivated widely in East Asia, including China, Japan, and Korea. Although oriental melon is economically valuable in Asia and is genetically distinct from other subspecies, few reports of genome-scale research on oriental melon have been published. We generated 30.5 and 36.8 Gb of raw RNA sequence data from the female and male flowers, leaves, roots, and fruit of two oriental melon varieties, Korean landrace (KM) and Breeding line of NongWoo Bio Co. (NW), respectively. From the raw reads, 64,998 transcripts from KM and 100,234 transcripts from NW were de novo assembled. The assembled transcripts were used to identify molecular markers (e.g., single-nucleotide polymorphisms and simple sequence repeats), detect tissue-specific expressed genes, and construct a genetic linkage map. In total, 234 single-nucleotide polymorphisms and 25 simple sequence repeats were screened from 7,871 and 8,052 candidates, respectively, between the KM and NW varieties and used for construction of a genetic map with 94 F2 population specimens. The genetic linkage map consisted of 12 linkage groups, and 248 markers were assigned. These transcriptome and molecular marker data provide information useful for molecular breeding of oriental melon and further comparative studies of the Cucurbitaceae family.

Development of SSR markers by next-generation sequencing of Korean landraces of chamoe (Cucumis melo var. makuwa).

The oriental melon (Cucumis melo var. makuwa), called 'chamoe' in Korean, is a popular fruit crop cultivated mainly in Asia and a high-market value crop in Korea. To provide molecular breeding resources for chamoe, we developed and characterized genomic SSR markers from the preliminary Illumina read assemblies of Gotgam chamoe (one of the major landraces; KM) and SW3 (the breeding parent). Mononucleotide motifs were the most abundant type of markers, followed by di-, tri-, tetra-, and pentanucleotide motifs. The most abundant dinucleotide was AT, followed by AG and AC, and AAT was the most abundant trinucleotide motif in both assemblies. Following our SSR-marker development strategy, we designed a total of 370 primer sets. Of these, 236 primer sets were tested, exhibiting 93 % polymorphism between KM and SW3. Those polymorphic SSRs were successfully amplified in the netted and Kirkagac melons, which respectively exhibited 81 and 76 % polymorphism relative to KM, and 32 and 38 % polymorphism relative to SW3. Seven selected SSR markers with a total of 17 alleles (2-3 alleles per locus) were used to distinguish between KM, SW3, and four chamoe cultivars. Our results represent the first attempt to provide genomic resources for Korean landraces for the purposes of chamoe breeding, as well as to discover a set of SSR markers capable of discriminating chamoe varieties from Korea and the rest of Asia, which possess little genetic diversity. This study establishes a highly efficient strategy for developing SSR markers from preliminary Illumina assemblies of AT-rich genomes.

BAC-derived markers converted from RFLP linked to Phytophthora capsici resistance in pepper (Capsicum annuum L.).

Phytophthora capsici Leonian, an oomycete pathogen, is a serious problem in pepper worldwide. Its resistance in pepper is controlled by quantitative trait loci (QTL). To detect QTL associated with P. capsici resistance, a molecular linkage map was constructed using 100 F(2) individuals from a cross between Capsicum annuum 'CM334' and C. annuum 'Chilsungcho'. This linkage map consisted of 202 restriction fragment length polymorphisms (RFLPs), 6 WRKYs and 1 simple sequence repeat (SSR) covering 1482.3 cM, with an average interval marker distance of 7.09 cM. QTL mapping of Phytophthora root rot and damping-off resistance was performed in F(2:3) originated from a cross between resistant Mexican landrace C. annuum 'CM334' and susceptible Korean landrace C. annuum 'Chilsungcho' using composite interval mapping (CIM) analysis. Four QTL explained 66.3% of the total phenotypic variations for root rot resistance and three 44.9% for damping-off resistance. Of these QTL loci, two were located close to RFLP markers CDI25 on chromosome 5 (P5) and CT211A on P9. A bacterial artificial chromosome (BAC) library from C. annuum 'CM334' was screened with these two RFLP probes to obtain sequence information around the RFLP marker loci for development of PCR-based markers. CDI25 and CT211 probes identified seven and eight BAC clones, respectively. Nine positive BAC clones containing probe regions were sequenced and used for cytogenetic analysis. One single-nucleotide amplified polymorphism (SNAP) for the CDI25 locus, and two SSRs and cleaved amplified polymorphic sequence (CAPS) for CT211 were developed using sequences of the positive BAC clones. These markers will be valuable for rapid selection of genotypes and map-based cloning for resistance genes against P. capsici.

Development of a molecular marker specific to a novel CMS line in radish (Raphanus sativus L.).

In this study, we have investigated the cytoplasmic male sterility (CMS) of a novel male sterile radish line, designated NWB CMS. The NWB CMS was crossed with 16 fertile breeding lines, and all the progenies were completely male sterile. The degree of male sterility exhibited by NWB CMS is more than Ogura CMS from the Cruciferae family. The NWB CMS was found to induce 100% male sterility when crossed with all the tested breeding lines, whereas the Ogura CMS did not induce male sterility with any of the breeding lines. PCR analysis revealed that the molecular factor that influenced Ogura CMS, the orf138 gene, was absent in the NWB CMS line, and that the orf138 gene was not also expressed in this CMS line. In order to identify the cytoplasmic factors that confer male sterility in the NWB CMS line, we carried out RFLP analyses with 32 mitochondrial genes, all of which were used as probes. Fourteen genes exhibited polymorphisms between the NWB CMS line and other radish cultivars. Based on these RFLP data, intergenic primers were developed in order to amplify the intergenic regions between the polymorphic genes. Among these, a primer pair at the 3' region of the atp6 gene (5'-cgcttggactatgctatgtatga-3') and the 5' region of the nad3 gene (5'-tcatagagaaatccaatcgtcaa-3') produced a 2 kbp DNA fragment as a result of PCR. This DNA fragment was found to be specific to NWB CMS and was not present in other CMS types. It appears that this fragment could be used as a DNA marker to select NWB CMS line in a radish-breeding program.