コムギ染色体欠損系統を用いた新規活性型レトロトランスポゾン TriRe-1 の分子遺伝学的解析

　Retrotransposons constitute the large fraction (～80%) of the wheat genome where numerous and diverse retrotransposon families exist, where especially the long terminal repeat (LTR) retrotransposon family is known to be predominant. Thus, they have been considered to contribute to the genome expansion, sequence diversification and the genome structure alternation in the wheat genome. In addition, the insertion polymorphism of the LTR retrotransposon family among the cultivars has been known to be quite useful for the genetic analysis such as the linkage mapping and the phylogenetic studies. Here, we report the characteristics of a novel active LTR retrotransposon family TriRe‒1, which belongs to the Ty1‒copia group in the hexaploid wheat (Triticum aestivum L.) genome. This retroelement appears to encode all proteins required for the transposition and showed high insertion polymorphism among the hexaploid wheat cultivars, suggesting its potential of transpositional activity with at least recent transposition during wheat evolution. We studied the chromosomal localization of the TriRe‒1 insertion site based on the genome-wide comparative analysis using the nullisomic-tetrasomic lines of the cultivar Chinese Spring. The results showed that although the majority of the TriRe‒1 insertion sites exist across the homoeologous chromosomes of A, B or D genomes, a higher number of insertions in the B genome was detected compared to A or D genome, suggesting a specific amplification in the history of B genome progenitors. In conclusion, a novel LTR retrotransposon TriRe‒1 should be valuable for the development of molecular markers based on insertion polymorphism among the cultivars, and also the genome-specific TriRe‒1 insertion site can be utilized to study evolutional history of wheat genomes.　レトロトランスポゾンは植物ゲノムの主要な構成要素であり，コムギゲノムにおいてはその80ｵを占める．特に LTR 型レトロトランスポゾンの割合が高く，ゲノムの拡大，配列の多様性およびゲノム構造変異等に大きく寄与し てきたと考えられている．これら配列は自身のコピー配列を複製し増幅するため，ゲノム中には数百，数千に及ぶコ ピー配列をもつ．また，ゲノム進化の過程において多数のファミリーを形成してきた．これら多数のファミリーのう ち，現在でも転移活性を示す活性型ファミリーは，品種間において高い挿入多型を示すことが知られている．このよ うな挿入多型は，連鎖解析および系統解析等各種遺伝解析に利用可能である. 本研究では，コムギにおける新規活性 型レトロトランスポゾンファミリー TriRe-1 の特徴を詳細に解析した．TriRe-1 は転移に必要なタンパク質をコー ドする内部配列をもち，また日本で育成されたコムギ近縁品種間においても高い挿入多型を示したため，現在でも転 移活性を有している，もしくはごく最近まで転移していた可能性が高いと考えられた．一方で，コムギ染色体欠損系 統（ナリソミックテトラソミック系統）を用い，TriRe-1 の挿入箇所を比較解析した．その結果，大部分の挿入箇所 は複数の同祖染色体に存在すると考えられたが，Ｂゲノムにおいて最も多くの特異的な挿入箇所が同定された．よっ て，Ｂゲノム祖先種において活発に増幅してきた可能性が示唆された．今回の結果により，新規活性型レトロトラン スポゾン TriRe-1 の品種間挿入多型を利用した DNA マーカー，また，各ゲノム（Ａ，Ｂ，Ｄゲノム）特異的な挿 入箇所を利用したゲノム識別性に優れた DNA マーカーの開発の可能性が期待される

Efficient DNA Fingerprinting Based on the Targeted Sequencing of Active Retrotransposon Insertion Sites Using a Bench-Top High-Throughput Sequencing Platform

In many crop species, DNA fingerprinting is required for the precise identification of cultivars to protect the rights of breeders. Many families of retrotransposons have multiple copies throughout the eukaryotic genome and their integrated copies are inherited genetically. Thus, their insertion polymorphisms among cultivars are useful for DNA fingerprinting. In this study, we conducted a DNA fingerprinting based on the insertion polymorphisms of active retrotransposon families (Rtsp-1 and LIb) in sweet potato. Using 38 cultivars, we identified 2024 insertion sites in the two families with an Illumina MiSeq sequencing platform. Of these insertion sites, 91.4% appeared to be polymorphic among the cultivars and 376 cultivar-specific insertion sites were identified, which were converted directly into cultivar-specific sequence-characterized amplified region (SCAR) markers. A phylogenetic tree was constructed using these insertion sites, which corresponded well with known pedigree information, thereby indicating their suitability for genetic diversity studies. Thus, the genome-wide comparative analysis of active retrotransposon insertion sites using the bench-top MiSeq sequencing platform is highly effective for DNA fingerprinting without any requirement for whole genome sequence information. This approach may facilitate the development of practical polymerase chain reaction-based cultivar diagnostic system and could also be applied to the determination of genetic relationships

Transcriptome Analysis Reveals Key Genes Involved in Weevil Resistance in the Hexaploid Sweetpotato

Because weevils are the most damaging pests of sweetpotato, the development of cultivars resistant to weevil species is considered the most important aspect in sweetpotato breeding. However, the genes and the underlying molecular mechanisms related to weevil resistance are yet to be elucidated. In this study, we performed an RNA sequencing-based transcriptome analysis using the resistant Kyushu No. 166 (K166) and susceptible Tamayutaka cultivars. The weevil resistance test showed a significant difference between the two cultivars at 30 days after the inoculation, specifically in the weevil growth stage and the suppressed weevil pupation that was only observed in K166. Differential expression and gene ontology analyses revealed that the genes upregulated after inoculation in K166 were related to phosphorylation, metabolic, and cellular processes. Because the weevil resistance was considered to be related to the suppression of larval pupation, we investigated the juvenile hormone (JH)-related genes involved in the inhibition of insect metamorphosis. We found that the expression of some terpenoid-related genes, which are classified as plant-derived JHs, was significantly increased in K166. This is the first study involving a comprehensive gene expression analysis that provides new insights about the genes and mechanisms associated with weevil resistance in sweetpotato

Mapping of Nematode Resistance in Hexaploid Sweetpotato Using an Next-Generation Sequencing-Based Association Study

The southern root-knot nematode (SRKN; Meloidogyne incognita) is a typical parasitic nematode that affects sweetpotato [Ipomoea batatas (L.) Lam.], causing a significant decrease in crop yield and commercial value. In Japan, the SRKN is classified into 10 races: SP1-SP5, SP6-1, SP6-2, and SP7-SP9, with the dominant race differing according to the cultivation area. Soil insecticides have previously been used to reduce the soil density of SRKNs; however, this practice is both costly and labor intensive. Therefore, the development of SRKN-resistant sweetpotato lines and cultivars is necessary. However, due to the complexity of polyploid inheritance and the highly heterogeneous genomic composition of sweetpotato, genetic information and research for this species are significantly lacking compared to those for other major diploid crop species. In this study, we utilized the recently developed genome-wide association approach, which uses multiple-dose markers to assess autopolyploid species. We performed an association analysis to investigate resistance toward SRKN-SP2, which is the major race in areas with high sweetpotato production in Japan. The segregation ratio of resistant and susceptible lines in the F-1 mapping population derived from the resistant "J-Red" and susceptible "Choshu" cultivars was fitted to 1: 3, suggesting that resistance to SP2 may be regulated by two loci present in the simplex. By aligning the double digest restriction-site associated DNA sequencing reads to the published Ipomoea trifida reference sequence, 46,982 single nucleotide polymorphisms (SNPs) were identified (sequencing depth > 200). The association study yielded its highest peak on chromosome 7 (Chr07) and second highest peak on chromosome 3 (Chr03), presenting as a single-dose in both loci. Selective DNA markers were developed to screen for resistant plants using the SNPs identified on Chr03 and Chr07. Our results showed that SRKN-SP2-resistant plants were selected with a probability of approximately 70% when combining the two selective DNA markers. This study serves as a model for the identification of genomic regions that control agricultural traits and the elucidation of their effects, and is expected to greatly advance marker-assisted breeding and association studies in polyploid crop species

Leukemia autopsies in Japan

For the purpose to know whether the annual increase of leukemia incidence in Japan is due to some leukemogenic factors or due to the increased detection rate, the authors made some statistical survey of autopsy cases in which the diagnosis is reliable and not any type of leukemias escape the detection. The results showed that acute leukemias, which are found mostly in younger age, is actually increasing. In addition, it has been deduced that among the suspected factors the increase in ionizing radiation will be one of the most probable factors for the increase in leukemia incidence</p

Development of molecular markers associated with resistance to Meloidogyne incognita by performing quantitative trait locus analysis and genome-wide association study in sweetpotato

The southern root-knot nematode, Meloidogyne incognita, is a pest that decreases yield and the quality of sweetpotato [Ipomoea batatas (L.) Lam.]. There is a demand to produce resistant cultivars and develop DNA markers to select this trait. However, sweetpotato is hexaploid, highly heterozygous, and has an enormous genome (similar to 3 Gb), which makes genetic linkage analysis difficult. In this study, a high-density linkage map was constructed based on retrotransposon insertion polymorphism, simple sequence repeat, and single nucleotide polymorphism markers. The markers were developed using F-1 progeny between J-Red, which exhibits resistance to multiple races of M. incognita, and Choshu, which is susceptible to multiple races of such pest. Quantitative trait locus (QTL) analysis and a genome-wide association study detected highly effective QTLs for resistance against three races, namely, SP1, SP4, and SP6-1, in the Ib01-6 J-Red linkage group. A polymerase chain reaction marker that can identify genotypes based on single nucleotide polymorphisms located in this QTL region can discriminate resistance from susceptibility in the F-1 progeny at a rate of 70%. Thus, this marker could be helpful in selecting sweetpotato cultivars that are resistant to multiple races of M. incognita

A rapid and enhanced DNA detection method for crop cultivar discrimination

In many crops species, the development of a rapid and precise cultivar discrimination system has been required for plant breeding and patent protection of plant cultivars and agricultural products. Here, we successfully evaluated strawberry cultivars via a novel method, namely, the single tag hybridization (STH) chromatographic printed array strip (PAS) using the PCR products of eight genomic regions. In a previous study, we showed that genotyping of eight genomic regions derived from FaRE1 retrotransposon insertion site enabled to discriminate 32 strawberry cultivars precisely, however, this method required agarose/acrylamide gel electrophoresis, thus has the difficulty for practical application. In contrast, novel DNA detection method in this study has some great advantages over standard DNA detection methods, including agarose/acrylamide gel electrophoresis, because it produces signals for DNA detection with dramatically higher sensitivity in a shorter time without any preparation or staining of a gel. Moreover, this method enables the visualization of multiplex signals simultaneously in a single reaction using several independent amplification products. We expect that this novel method will become a rapid and convenient cultivar screening assay for practical purposes, and will be widely applied to various situations, including laboratory research, and on-site inspection of plant cultivars and agricultural products

DNA markers based on retrotransposon insertion polymorphisms can detect short DNA fragments for strawberry cultivar identification

In this study, DNA markers were developed for discrimination of strawberry (Fragaria × ananassa L.) cultivars based on retrotransposon insertion polymorphisms. We performed a comprehensive genomic search to identify retrotransposon insertion sites and subsequently selected one retrotransposon family, designated CL3, which provided reliable discrimination among strawberry cultivars. Through analyses of 75 strawberry cultivars, we developed eight cultivar-specific markers based on CL3 retrotransposon insertion sites. Used in combination with 10 additional polymorphic markers, we differentiated 35 strawberry cultivars commonly cultivated in Japan. In addition, we demonstrated that the retrotransposon-based markers were effective for PCR detection of DNA extracted from processed food materials, whereas a SSR marker was ineffective. These results indicated that the retrotransposon-based markers are useful for cultivar discrimination for processed food products, such as jams, in which DNA may be fragmented or degraded

Comparative Gene Analysis Focused on Silica Cell Wall Formation: Identification of Diatom-Specific SET Domain Protein Methyltransferases

Silica cell walls of diatoms have attracted attention as a source of nanostructured functional materials and have immense potential for a variety of applications. Previous studies of silica cell wall formation have identified numerous involved proteins, but most of these proteins are species-specific and are not conserved among diatoms. However, because the basic process of diatom cell wall formation is common to all diatom species, ubiquitous proteins and molecules will reveal the mechanisms of cell wall formation. In this study, we assembled de novo transcriptomes of three diatom species, Nitzschia palea, Achnanthes kuwaitensis, and Pseudoleyanella lunata, and compared protein-coding genes of five genome-sequenced diatom species. These analyses revealed a number of diatom-specific genes that encode putative endoplasmic reticulum-targeting proteins. Significant numbers of these proteins showed homology to silicanin-1, which is a conserved diatom protein that reportedly contributes to cell wall formation. These proteins also included a previously unrecognized SET domain protein methyltransferase family that may regulate functions of cell wall formation-related proteins and long-chain polyamines. Proteomic analysis of cell wall-associated proteins in N. palea identified a protein that is also encoded by one of the diatom-specific genes. Expression analysis showed that candidate genes were upregulated in response to silicon, suggesting that these genes play roles in silica cell wall formation. These candidate genes can facilitate further investigations of silica cell wall formation in diatoms

Elucidation of genetic variation and population structure of melon genetic resources in the NARO Genebank, and construction of the World Melon Core Collection

Numerous genetic resources of major crops have been introduced from around the world and deposited in Japanese National Agriculture and Food Research Organization (NARO) Genebank. Understanding their genetic variation and selecting a representative subset (“core collection”) are essential for optimal management and efficient use of genetic resources. In this study, we conducted genotyping-by-sequencing (GBS) to characterize the genetic relationships and population structure in 755 accessions of melon genetic resources. The GBS identified 39,324 single-nucleotide polymorphisms (SNPs) that are distributed throughout the melon genome with high density (one SNP/10.6 kb). The phylogenetic relationships and population structure inferred using this SNP dataset are highly associated with the cytoplasm type and geographical origin. Our results strongly support the recent hypothesis that cultivated melon was established in Africa and India through multiple independent domestication events. Finally, we constructed a World Melon Core Collection that covers at least 82% of the genetic diversity and has a wide range of geographical origins and fruit morphology. The genome-wide SNP dataset, phylogenetic relationships, population structure, and the core collection provided in this study should largely contribute to genetic research, breeding, and genetic resource preservation in melon