転移能を有するサツマイモ・レトロトランスポゾン塩基配列から推定される逆転写開始複合体の特徴

Sequence analysis of Rtsp-1, an active LTR retrotransposon in the sweetpotato genome,revealed a possible novel Rtsp-1 RNA/tRNAMet complex for initiation of reverse transcription and the first DNA strand transfer. The Rtsp-1 RNA has a primer binding site (PBS) that is partly complementary to the 3’ end of tRNAMet, and possesses an additional sequence complementary to the 5’ end of tRNAMet downstream of the PBS. These additional base-pairings might stabilize the Rtsp-1 RNA/primer complex. In the free form, the 5’ LTR of Rtsp-1 appears to form a stemloop structure apparently preventing the initiation of reverse transcription. While the stemforming site adjacent to the PBS is complementary to the tRNAMet, the other stem-forming site on the LTR complements a region just upstream of the 3’ LTR. Additionally, another region at the 3’ end of the Rtsp-1 RNA shows sequence complementarity to the tRNAMet. As the 3’ end of Rtsp-1 approaches the tRNAMet bound to the PBS, the stem-forming strands dissociate and basepair with their complementary regions in the tRNAMet and the 3’ end of Rtsp-1, respectively. Consequently, the LTR loop opens, allowing reverse transcription to initiate. After the initial reverse transcription stops at the 5’ end of the Rtsp-1 RNA, the synthesized minus strand DNA needs to be transferred to the 3’ end of the RNA to synthesize internal sequences. The Rtsp-1 RNA/tRNAMet complex may have evolved to facilitate this DNA transfer. Similar RNA/tRNA initiation complexes have been reported from reverse transcription in retroviruses and yeast retrotransposons (Ty1 and Ty3).カルスにおける転移が示されたサツマイモ LTR 型レトロトランスポゾン（Rtsp-1）の塩基配列を調べたところ，逆転写が開始される際，転写された Rtsp-1の RNA と最初の逆転写のプライマーに使われる tRNAMETとの間で，特徴的な逆転写開始複合体を形成し，この複合体が最初の逆転写とその後の過程で必要な逆転写産物（cDNA）の転移などを確実なものとしていることが示唆された．その内容は，１）転写された Rtsp-1の RNA 逆転写開始部位の塩基配列は自身の LTR 配列とステム構造をとること，２）tRNAMETが結合する Rtsp-1の Primer Binding Site 部位には，プライマーの機能を果たす tRNAMETの３'末端の相補配列に加えて，その隣接部位に tRNAMETの５'末端部位と相補的な結合部位が存在するために，tRNAMETの両末端が結合すること，３）Rtsp-1の３'末端側に，tRNAMET及びステム構造に関わる５'LTR の部位との相補配列があり，この３ｾ末端側が転写開始複合体と結合することにより，ステム構造が崩れて逆転写が開始されると推定されること,４）逆転写が開始された後も，tRNAMETの結合によってRtsp-1の５'末端と３'末端側に近接した状態が保たれることである．Rtsp-1の３ｾ末端側の転写開始複合体への結合を転写開始の条件とすることにより，最初に合成される cDNA の３'末端への転移が容易となることなどが示唆された

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

Genotype by Environment Interactions and Linear Regression Analyses in Wheat Grain Yield

Agronom

Genotype by Environment Interactions and Parameters for Genotype Responses to Environments in Wheat Breedig Program of Oklahoma

The Wheat breeding program at Oklahoma State University(OSU) is introduced with reference to genotype by environment interactions and linear regression analyses. Oklahoma is the second largest producer of hard red winter wheat in the US. The breeding porgram is conducted by the wheat breeding personnel of the Agronomy Department in collaboration with plant pathologists, entomologists and biochemists of OSU and wheat geneticists of the US Department of Agriculture. The main-stream breeding procedures are F2 or F3 progeny methods, which are modifications of pedigree and bulk breeding methods. The procedures for source population development,selection practice and field trials are discussed. The major objective of the projict is to develop wheat varieties with supperior yield and yield stability. Drought stress is a serious constraint to wheat crop and frequently causes substantial yield reduction in Okrahoma. Other major obstacles to wheat production are disease and insect damage which include leaf rust, mosaic diseses, septoria and green bug. Research and breeding activities to overcome thesse obstacles are briefly reviewed.Genotype by environment interactions are commonly found and cause serious problems in identifying superior genotype over a wide range of environments in the wheat breeding program. Linear regression analyses and other yield stability parameters are proposed to characterize genotype responses to varying environments. The grain yield data from cultivar trials during 1971-1982 were analyzed by an analysis of variance method and linear regression method. The analysis of varince indicated substantial genotype by environment interactions. The linear regression analyses could adequately explain much of the interaction and provided parameters to compare yield responses of genotypes over environments. Other stability parameters were also estimated and their relationships were discussed. The linear regression analyses revealed that selection toward higher average yield over environments favored genotypes adapted for high yeilding environments.著者は、1981・1982年と1985～1988年の二度にわたり、米国オクラホマ州立大学の大学院に留学し、硬質冬小麦（Hard Red Winter Wheat）の育種プロジェクトの下、修士（Master of Science）と博士号（Ph.D.in Crop Science）を取得した。本稿では、同州の硬質冬小麦育種プロジェクトについて触れるとともに、量的形質の選抜に際して最大の課題の一つである遺伝子型と環境の交互作用と、その交互作用をそれぞれの遺伝子型の環境との反応性によって説明しようとする方法（直線回帰分析）を用いて解析した例を紹介する

Utilization of Physiological Traits as Selection Criteria for Drought Resistance of Winter Wheat

Crop Scienc

エステラーゼ・アイソザイムによるサトイモの系統分類

Phylogenetic relationships among the 84 accessions of taro (Colocasia esculenta (L) Schott), C gigantea Hook Alocasia macrorrhiza, A odora, Xanthosoma sagittifolium (L.) Schott and X. violaceum Schott were investigated using isozyme polymorphism of esterase. The phylogenetic tree estimated by the UPGMA analyses revealed that taro accessions formed a single cluster and C. gigantea was more closely related to Alocasia species than to taro. Taro accessions from Yunnan tended to share band patterns with those from various areas, which indicates that the Yunnan area might have been important for taro evolution.東アジアを中心に採取したサトイモ(Colocasia esculenta (L) Schott)とその近緑野生種C.gigantea Hook, Alocasia macrorrhiza, A. odora, Xanthosoma sagittifolium (L.) Schott and X. violaceum Schottの84系統について、エステラーゼのアイソザイムの多型を基に類縁関係の推定を行った。UPGMA法によって系統樹を求めたところ、サトイモ69系統は一つの独立したクラスターを形成した。また、ハスイモ(C. gigantea Hook)は、サトイモよりもAlocasia属の種とより近縁であることを示唆する結果を得た。サトイモについて、東アジアの各系統のアイソザイムのバンドパターンを比較したところ、中国雲南省で収集したものは、東アジア各地のサトイモと共通のパターンを示すものが多かったことから、この地域がサトイモ進化に重要な役割を果たしていることが推察された

Isolation of a transcriptionally active element of high copy number retrotransposons in sweetpotato genome

Many plant retrotransposons have been characterized, but only three families (Tnt1, Tto1 and Tos17) have been demonstrated to be transpositionally competent. We followed a novel approach that enabled us to identify an active element of the Ty1-copia retrotransposon family with estimated 400 copies in the sweetpotato genome. DNA sequences of Ty1 -copia reverse transcriptase (RTase) from the sweetpotato genome were analyzed, and a group of retrotransposon copies probably formed by recent transposition events was further analyzed. 3’RACE on callus cDNA amplified transcripts containing long terminal repeats (LTR) of this group. The sequence -specific amplification polymorphism (S-SAP) patterns of the LTR sequence in the genomic DNA were compared between a normal plant and callus lines derived from it. A callus -specific S-SAP product was found into which the retrotransposon detected by the 3’RACE had been transposed apparently during cell culture. We conclude that our approach provides an effective way to identify active elements of retrotransposons with high copy numbers.</p

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