Japanese Society of BreedingActa Medica Okayama1344-76107022020DNA markers based on retrotransposon insertion polymorphisms can detect short DNA fragments for strawberry cultivar identification231240ENChiharuHirataFukuoka Agriculture and Forestry Research Center TakamitsuWakiTochigi Prefectural Agricultural Experiment StationKatsumiShimomuraFukuoka Agriculture and Forestry Research Center TakuyaWadaFukuoka Agriculture and Forestry Research CenterSeiyaTanakaGraduate School of Bioresource and Bioenvironmental Sciences, Kyushu UniversityHidetoshiIkegamiFukuoka Agriculture and Forestry Research CenterYousukeUchimuraFukuoka Agriculture and Forestry Research CenterKeitaHirashimaFukuoka Agriculture and Forestry Research Center YoshikoNakazawaTochigi Prefectural Agricultural Experiment Station KaoriOkadaTochigi Prefectural Agricultural Experiment StationKiyoshiNamaiTochigi Prefectural Agricultural Experiment StationMakotoTaharaGraduate School of Environmental and Life Science, Okayama UniversityYukiMondenGraduate School of Environmental and Life Science, Okayama UniversityIn 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.No potential conflict of interest relevant to this article was reported.Oxford University PressActa Medica Okayama1340-28382652019Development of molecular markers associated with resistance to Meloidogyne incognita by performing quantitative trait locus analysis and genome-wide association study in sweetpotato399409ENRumiSasaiGraduate School of Environmental and Life Science, Okayama UniversityHiroakiTabuchiKyusyu Okinawa Agricultural Research Center, National Agriculture and Food Research OrganizationKentaShirasawaKazusa DNA Research InstituteKazukiKishimotoGraduate School of Environmental and Life Science, Okayama UniversityShuseiSatoGraduate School of Life Science, Tohoku UniversityYoshihiroOkadaKyusyu Okinawa Agricultural Research Center, National Agriculture and Food Research OrganizationAkihideKuramotoGraduate School of Agriculture, Kyoto UniversityAkiraKobayashiKyusyu Okinawa Agricultural Research Center, National Agriculture and Food Research OrganizationSachikoIsobeKazusa DNA Research InstituteMakotoTaharaGraduate School of Environmental and Life Science, Okayama UniversityYukiMondenGraduate School of Environmental and Life Science, Okayama UniversityThe 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.No potential conflict of interest relevant to this article was reported.Oxford University PressActa Medica Okayama1340-28382014Efficient DNA Fingerprinting Based on the Targeted Sequencing of Active Retrotransposon Insertion Sites Using a Bench-Top High-Throughput Sequencing PlatformENYukiMondenAyakaYamamotoAkikoShindoMakotoTaharaIn 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.No potential conflict of interest relevant to this article was reported.NRC Research PressActa Medica Okayama5752014Efficient screening of long terminal repeat retrotransposons that show high insertion polymorphism via high-throughput sequencing of the primer binding site245252ENYukiMondenNobuyukiFujiiKentaroYamaguchiKazuhoIkeoYoshikoNakazawaTakamitsuWakiKeitaHirashimaYosukeUchimuraMakotoTaharaRetrotransposons have been used frequently for the development of molecular markers by using their insertion polymorphisms among cultivars, because multiple copies of these elements are dispersed throughout the genome and inserted copies are inherited genetically. Although a large number of long terminal repeat (LTR) retrotransposon families exist in the higher eukaryotic genomes, the identification of families that show high insertion polymorphism has been challenging. Here, we performed an efficient screening of these retrotransposon families using an Illumina HiSeq2000 sequencing platform with comprehensive LTR library construction based on the primer binding site (PBS), which is located adjacent to the 5′ LTR and has a motif that is universal and conserved among LTR retrotransposon families. The paired-end sequencing library of the fragments containing a large number of LTR sequences and their insertion sites was sequenced for seven strawberry (Fragaria × ananassa Duchesne) cultivars and one diploid wild species (Fragaria vesca L.). Among them, we screened 24 families with a “unique” insertion site that appeared only in one cultivar and not in any others, assuming that this type of insertion should have occurred quite recently. Finally, we confirmed experimentally the selected LTR families showed high insertion polymorphisms among closely related cultivars.No potential conflict of interest relevant to this article was reported.Elsevier B.V.Acta Medica Okayama0168-16561852014A rapid and enhanced DNA detection method for crop cultivar discrimination5762ENYukiMondenKazutoTakasakiSatoshiFutoKousukeNiwaMitsuoKawaseHirotoAkitakeMakotoTaharaIn 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.No potential conflict of interest relevant to this article was reported.岡山大学農学部Acta Medica Okayama2186-77551032014Characterization of a novel retrotransposon TriRe‒1 using nullisomic-tetrasomic lines of hexaploid wheat2130ENYukiMondenTakeruTakaiMakotoTahara 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.No potential conflict of interest relevant to this article was reported.Acta Medica Okayama27212004Isolation of a transcriptionally active element of high copy number retrotransposons in sweetpotato genome116127ENMakotoTaharaTakahiroAokiShinyaSuzukaHirokiYamashitaMasaruTanakaSachiMatsunagaShuheiKokumai<p>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>No potential conflict of interest relevant to this article was reported.岡山大学農学部Acta Medica Okayama0474-02548511996オクラホマ州の硬質冬小麦の育種における遺伝子型と環境の交互作用と遺伝子型の環境反応性の指標化について99108ENMakotoTaharaThe 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.No potential conflict of interest relevant to this article was reported.岡山大学農学部Acta Medica Okayama0474-02548711998Phylogenetic Analyses of Taro (Colocasia esculenta (L.) Schott) and Related Species based on Esterase Isozymes133139ENViet XuanNguyenHiromichiYoshinoMakotoTaharaPhylogenetic 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.No potential conflict of interest relevant to this article was reported.岡山大学農学部Acta Medica Okayama0474-02548912000葉緑体DNAの制限酵素断片長多型(RFLP)を利用したサトイモとその近縁種の類縁関係分析1521ENToshinoriOchiaiMakotoTaharaHiromichiYoshinoPhylogenetic relationshios among 51 accessions of taro (Colocasia esculenta(L.) Schott), C.gigantee Hook, Alocasia macrorrhiza, A.odora, Xanthosoma sagittifolium and Schismatoglottis spp. were investigated using restriction fragment length polymorphisms (RFLPs) of chloroplast DNA. The phylogenetic tree using the Neighbor Joining(NJ) method revealed that Xanthosoma and Schismatoglottis genera were distantly related to Colocasia and Alocasia genera. Among Colocasia and Alocasia accessions, C.esculenta accessions formed a single cluster. However C. gigantea accessions were related DNA among taro accessions were found to be too small to establish significant grouping of the accessions. However, four accessions of taro, which were thought to be inter-generic or inter-specific hybrids, formed an independent cluster. Based on the banding pattern of the RFLP, the plant in genus Colocasia appeared to be the maternal parent of these four accessions.No potential conflict of interest relevant to this article was reported.岡山大学農学部Acta Medica Okayama0474-0254 9612007A Novel Initiation Complex for Reverse Transcription of an Active LTR Retrotransposon in Seeetpotato711ENMakotoTaharaHirokiYamashitaSequence 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).No potential conflict of interest relevant to this article was reported.