Kazuhiko Tarora

Garcinia subelliptica Merr. (called “fukugi” in Japanese) is a dioecious tree species, which has traditionally been planted as windbreak forests in the coast of Ryukyu islands. Although effective afforestation and maintenance of G. subelliptica would greatly be assisted by early sex diagnosis and selection at the seedling stage, the genome and the sex-determination system of G. subelliptica are still poorly understood. In this study, we estimated the genome size of G. subelliptica to be approximately ~ 12 Gb for female and male, based on k-mer analysis. Furthermore, we developed a male-specific DNA marker for G. subelliptica, using the restriction site-associated DNA sequencing (RAD-seq) method. The 660 bp marker fragment, named FSDM (“fukugi” sex determination marker), was amplified in all males but not in the female G. subelliptica genome analyzed so far. In addition, the segregation ratio of the marker was 1:1 among the tested population. The DNA sequence of FSDM showed a significant similarity to the antifungal protein ginkbilobin-2 gene. The development of the sex-specific genetic marker of G. subelliptica may allow more effective conservation of traditional landscape and future design of villages around the northern part of the main island of Okinawa, a promising candidate site for World Natural Heritage.

The genetic architecture of quantitative traits is determined by both Mendelian and polygenic factors, yet classic examples of plant domestication focused on selective sweep of newly mutated Mendelian genes. Here we report the chromosome-level genome assembly and the genomic investigation of a nonclassic domestication example, bitter gourd ( Momordica charantia ), an important Asian vegetable and medicinal plant of the family Cucurbitaceae. Population resequencing revealed the divergence between wild and South Asian cultivars about 6,000 y ago, followed by the separation of the Southeast Asian cultivars about 800 y ago, with the latter exhibiting more extreme trait divergence from wild progenitors and stronger signs of selection on fruit traits. Unlike some crops where the largest phenotypic changes and traces of selection happened between wild and cultivar groups, in bitter gourd large differences exist between two regional cultivar groups, likely reflecting the distinct consumer preferences in different countries. Despite breeding efforts toward increasing female flower proportion, a gynoecy locus exhibits complex patterns of balanced polymorphism among haplogroups, with potential signs of selective sweep within haplogroups likely reflecting artificial selection and introgression from cultivars back to wild accessions. Our study highlights the importance to investigate such nonclassic example of domestication showing signs of balancing selection and polygenic trait architecture in addition to classic selective sweep in Mendelian factors.

Simple sequence repeat (SSR) markers provide a reliable tool for the identification of accessions and the construction of genetic linkage maps because of their co-dominant inheritance. In the present study, we developed new SSR markers with next-generation sequencing using the Roche 454 GS FLX+ platform. Five hundred SSR primer sets were tested for the genetic identification of pineapple, including 100 each for the di-, tri-, tetra-, penta-, and hexa-nucleotide motif SSRs. In total, 160 SSR markers successfully amplified fragments and exhibited polymorphism among accessions. The SSR markers revealed the number of alleles per locus (ranging from 2 to 13), the expected heterozygosity (ranging from 0.041 to 0.823), and the observed heterozygosity (ranging from 0 to 0.875). A total of 117 SSR markers with tri- or greater nucleotide motifs were shown to be effective at facilitating accurate genotyping. Using the SSR markers, 25 accessions were distinguished genetically, with the exception of accessions ‘MD-2’ and ‘Yonekura’. The developed SSR markers could facilitate the establishment of efficient and accurate genetic identification systems and the construction of genetic linkage maps in the future.

パパイヤ奇形葉モザイクウイルス（Papaya leaf distortion mosaic virus: PLDMV）抵抗性を持つ属間雑種個体（パパイヤCarica papaya × Vasconcellea cundinamarcensis）にパパイヤを戻し交雑し，胚培養による戻し交雑個体の作出を試みた．戻し交雑後180～210日に供試した5,762の種子から，胚を持つ45の種子が得られ，最終的に32個体の再生植物体が得られた．PCRによって，性染色体型を調査した結果，32個体の内，19個体に戻し交雑親のY染色体の保有が確認できたことから，戻し交雑個体であることが確認できた．全ての戻し交雑個体は，母本として用いた属間雑種の性染色体を持っていたことから，非還元配偶子が形成されている可能性が示唆された．戻し交雑個体のPLDMV抵抗性を評価するため，PLDMVの人工接種を行った結果，66%がPLDMVに対して全く病徴を示さない抵抗性であった．残りの戻し交雑個体は，接種上位葉に壊疽斑点を生じたが，PLDMVの拡大を妨げた．従って，戻し交雑個体は全てPLDMVに対して抵抗性を有していた．これらの戻し交雑個体は，沖縄県で問題となっているPLDMVへのパパイヤ抵抗性品種育成において有望な育種素材と考えられる．

Bitter gourd (Momordica charantia) is an important vegetable and medicinal plant in tropical and subtropical regions globally. In this study, the draft genome sequence of a monoecious bitter gourd inbred line, OHB3-1, was analyzed. Through Illumina sequencing and de novo assembly, scaffolds of 285.5 Mb in length were generated, corresponding to ∼84% of the estimated genome size of bitter gourd (339 Mb). In this draft genome sequence, 45,859 protein-coding gene loci were identified, and transposable elements accounted for 15.3% of the whole genome. According to synteny mapping and phylogenetic analysis of conserved genes, bitter gourd was more related to watermelon (Citrullus lanatus) than to cucumber (Cucumis sativus) or melon (C. melo). Using RAD-seq analysis, 1507 marker loci were genotyped in an F₂ progeny of two bitter gourd lines, resulting in an improved linkage map, comprising 11 linkage groups. By anchoring RAD tag markers, 255 scaffolds were assigned to the linkage map. Comparative analysis of genome sequences and predicted genes determined that putative trypsin-inhibitor and ribosome-inactivating genes were distinctive in the bitter gourd genome. These genes could characterize the bitter gourd as a medicinal plant.

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