Yasunari Ogihara

Applicability of inter-simple sequence repeat polymorphisms in wheat for use as DNA markers in comparison to RFLP and RAPD markers

Abstract Inter-simple sequence repeat polymorphic DNA (ISSR) was evaluated for its applicability as a genetic marker system in wheat. PCR was carried out with primers that annealed to simple sequence repeats. The resultant products were subjected to agarose-gel electrophoresis, and the banding patterns were compared among six wheat accessions containing diploid, tetraploid, and hexaploid members. Out of 100 examined, 33 primers produced distinguishable as well as polymorphic bands in each of the six accessions. Although most of the primers that gave distinct bands (30 primers out of 33) contained dinucleotide repeats, each of the primers with tri-, tetra-, and penta-nucleotide motifs also yielded discrete bands. Primers based on (AC)n repeats gave the most polymorphic bands. In total, 224 polymorphic bands were found in the comparison between Einkorn wheats whereas, on the average, 120 polymorphic bands were detected between common wheats. ISSR primers produced several times more information than RAPD markers. The extent of band polymorphism was similar to that of RFLP markers, and greater than that of RAPDs. The genetic relationships of wheat accessions estimated by the polymorphism of ISSR markers were identical with those inferred by RFLP and RAPD markers, indicating the reliability of ISSR markers for estimation of genotypes. These polymorphic bands are potential candidates as novel markers for use in linkage-map construction in wheat. The characteristic features of ISSR markers, i.e. polymorphism, generation of information and ease of handling, suggest their applicability to the analysis of genotypes as well as to the construction of PCR-based genome maps of wheats.

Development of a chromosomal arm map for wheat based on RFLP markers

SummaryA chromosomal arm map has been developed for common wheat (Triticum aestivum L. em. Thell.) using aneuploid stocks to locate more than 800 restriction fragments corresponding to 210 low-copy DNA clones from barley cDNA, oat cDNA, and wheat genomic libraries. The number of restriction fragments per chromosome arm correlates moderately well with relative DNA content and length of somatic chromosomes. The chromosomal arm locations of loci detected with 6 different clones support an earlier hypothesis for the occurrence of a two-step translocation (4AL to 5AL, 5AL to 7BS, and 7BS to 4AL) in the ancestral wheat genomes. In addition, 1 clone revealed the presence of a 5AL segment translocated to 4AL. Anomalies in aneuploid stocks were also observed and can be explained by intrahomoeologous recombination and polymorphisms among the stocks. We view the development of this chromosomal arm map as a complement to, rather than as a substitute for, a conventional RFLP linkage map in wheat.

Structural dynamics of cereal mitochondrial genomes as revealed by complete nucleotide sequencing of the wheat mitochondrial genome

The application of a new gene-based strategy for sequencing the wheat mitochondrial genome shows its structure to be a 452 528 bp circular molecule, and provides nucleotide-level evidence of intra-molecular recombination. Single, reciprocal and double recombinant products, and the nucleotide sequences of the repeats that mediate their formation have been identified. The genome has 55 genes with exons, including 35 protein-coding, 3 rRNA and 17 tRNA genes. Nucleotide sequences of seven wheat genes have been determined here for the first time. Nine genes have an exon–intron structure. Gene amplification responsible for the production of multicopy mitochondrial genes, in general, is species-specific, suggesting the recent origin of these genes. About 16, 17, 15, 3.0 and 0.2% of wheat mitochondrial DNA (mtDNA) may be of genic (including introns), open reading frame, repetitive sequence, chloroplast and retro-element origin, respectively. The gene order of the wheat mitochondrial gene map shows little synteny to the rice and maize maps, indicative that thorough gene shuffling occurred during speciation. Almost all unique mtDNA sequences of wheat, as compared with rice and maize mtDNAs, are redundant DNA. Features of the gene-based strategy are discussed, and a mechanistic model of mitochondrial gene amplification is proposed.

Discrimination of homoeologous gene expression in hexaploid wheat by SNP analysis of contigs grouped from a large number of expressed sequence tags

Single-nucleotide polymorphisms (SNPs) are useful markers for gene diagnosis and mapping of genes on chromosomes. However, polyploidy, which is characteristic of the evolution of higher plants, complicates the analysis of SNPs in the duplicated genes. We have developed a new method for SNP analysis in hexaploid wheat. First, we classified a large number of expressed sequence tags (ESTs) from wheat in silico. Those grouped into contigs were anticipated to correspond to transcripts from homoeologous loci. We then selected relatively abundant ESTs, and assigned these contigs to each of the homoeologous chromosomes using a nullisomic/tetrasomic series of Chinese Spring wheat strains in combination with pyrosequencing. The ninety genes assigned were almost evenly distributed into seven homologous chromosomes. We then created a virtual display of the relative expression of these genes. Expression patterns of genes from the three genomes in hexaploid wheat were classified into two major groups: (1) genes almost equally expressed from all three genomes; and (2) genes expressed with a significant preference, which changed from tissue to tissue, from certain genomes. In 11 cases, one of the three genes in the allopolyploid was found to be silenced. No preference for gene-silencing in particular genomes or chromosomes was observed, suggesting that gene-silencing occurred after polyploidization, and at the gene level, not at the chromosome or genome level. Thus, the use of this SNP method to distinguish the expression profiles of three homoeologous genes may help to elucidate the molecular basis of heterosis in polyploid plants.

A Wheat Homolog of MOTHER OF FT AND TFL1 Acts in the Regulation of Germination

Among the environmental signals affecting seed development, temperature is the most influential in the formation of seed dormancy in wheat. In this study, transcriptional profiling of the effects of temperature on seed dormancy formation identified MFT as a candidate gene for seed dormancy regulation. Seed dormancy is an adaptive mechanism and an important agronomic trait. Temperature during seed development strongly affects seed dormancy in wheat (Triticum aestivum) with lower temperatures producing higher levels of seed dormancy. To identify genes important for seed dormancy, we used a wheat microarray to analyze gene expression in embryos from mature seeds grown at lower and higher temperatures. We found that a wheat homolog of MOTHER OF FT AND TFL1 (MFT) was upregulated after physiological maturity in dormant seeds grown at the lower temperature. In situ hybridization analysis indicated that MFT was exclusively expressed in the scutellum and coleorhiza. Mapping analysis showed that MFT on chromosome 3A (MFT-3A) colocalized with the seed dormancy quantitative trait locus (QTL) QPhs.ocs-3A.1. MFT-3A expression levels in a dormant cultivar used for the detection of the QTL were higher after physiological maturity; this increased expression correlated with a single nucleotide polymorphism in the promoter region. In a complementation analysis, high levels of MFT expression were correlated with a low germination index in T1 seeds. Furthermore, precocious germination of isolated immature embryos was suppressed by transient introduction of MFT driven by the maize (Zea mays) ubiquitin promoter. Taken together, these results suggest that MFT plays an important role in the regulation of germination in wheat.

Genetic linkage map of ISSR and RAPD markers in Einkorn wheat in relation to that of RFLP markers

Abstract The potential of PCR-based markers for construction of a genetic linkage map in Einkorn wheat was investigated. From a comparison of polymorphisms between two Einkorn wheats, Triticum monococcum (Mn) and T. boeoticum (Bt), we obtained 49 polymorphic bands produced by 33 primers for inter-simple sequence repeat (ISSR) and 36 polymorphic bands shown by 25 combinations of random amplified polymorphic DNA (RAPD) primers for mapping in 66 individuals in the F2 population. Although 44 ISSR fragments and 29 RAPD fragments statistically showed a 3 : 1 segregation ratio in the F2 population, only 9 markers each of the ISSR and RAPD bands were able to be mapped on the RFLP linkage map of Einkorn wheat. ISSR markers were distributed throughout the chromosomes. The mapped positions of the ISSR markers seemed to be similar to those obtained by the RFLP markers. On the other hand, 4 of the 9 RAPD markers could map the RFLP marker-poor region on the short arm of 3Am, suggesting a potential to map novel regions containing repetitive sequences. Comparisons of the genetic linkage map of Einkorn wheat to the linkage map and cytological map of common wheat revealed that the marker orders between the two maps of Einkorn wheat and common wheat coincided except for 4A, which harbors chromosome rearrangements specific for polyploid wheats, indicating a conservatism between the two genomes. Recombinations in Einkorn wheat chromosomes took place more frequently around the centromere and less at the distal part of chromosomes in comparison to those in common wheat. Nevertheless, recombinations even in Einkorn wheat chromosomes were strongly suppressed around the centromere. In fact, the markers located within 1 cM of the centromere were located almost in the central part of the chromosome arm.

Structural features of a wheat plastome as revealed by complete sequencing of chloroplast DNA

Abstract. Structural features of the wheat plastome were clarified by comparison of the complete sequence of wheat chloroplast DNA with those of rice and maize chloroplast genomes. The wheat plastome consists of a 134,545-bp circular molecule with 20,703-bp inverted repeats and the same gene content as the rice and maize plastomes. However, some structural divergence was found even in the coding regions of genes. These alterations are due to illegitimate recombination between two short direct repeats and/or replication slippage. Overall comparison of chloroplast DNAs among the three cereals indicated the presence of some hot-spot regions for length mutations. Whereas the region with clustered tRNA genes and that downstream of rbcL showed divergence in a species-specific manner, the deletion patterns of ORFs in the inverted-repeat regions and the borders between the inverted repeats and the small single-copy region support the notion that wheat and rice are related more closely to each other than to maize.

Genetic and Epigenetic Alteration among Three Homoeologous Genes of a Class E MADS Box Gene in Hexaploid Wheat

Bread wheat (Triticum aestivum) is a hexaploid species with A, B, and D ancestral genomes. Most bread wheat genes are present in the genome as triplicated homoeologous genes (homoeologs) derived from the ancestral species. Here, we report that both genetic and epigenetic alterations have occurred in the homoeologs of a wheat class E MADS box gene. Two class E genes are identified in wheat, wheat SEPALLATA (WSEP) and wheat LEAFY HULL STERILE1 (WLHS1), which are homologs of Os MADS45 and Os MADS1 in rice (Oryza sativa), respectively. The three wheat homoeologs of WSEP showed similar genomic structures and expression profiles. By contrast, the three homoeologs of WLHS1 showed genetic and epigenetic alterations. The A genome WLHS1 homoeolog (WLHS1-A) had a structural alteration that contained a large novel sequence in place of the K domain sequence. A yeast two-hybrid analysis and a transgenic experiment indicated that the WLHS1-A protein had no apparent function. The B and D genome homoeologs, WLHS1-B and WLHS1-D, respectively, had an intact MADS box gene structure, but WLHS1-B was predominantly silenced by cytosine methylation. Consequently, of the three WLHS1 homoeologs, only WLHS1-D functions in hexaploid wheat. This is a situation where three homoeologs are differentially regulated by genetic and epigenetic mechanisms.

Evaluation of allelic diversity at chloroplast microsatellite loci among common wheat and its ancestral species

Abstract Twenty four chloroplast microsatellite loci having more than ten mononucleotide repeats were identified from the entire chloroplast DNA sequence of common wheat, Triticum aestivum cv Chinese Spring. For each microsatellite, a pair of primers were designed to produce specific PCR products in the range of 100– 200 bp. The allelic diversity at the microsatellite loci was evaluated using 43 accessions from 11 Triticum and Aegilops species involved in wheat polyploid evolution. Polymorphic banding patterns were obtained at 21 out of 24 chloroplast microsatellite loci. The three monomorphic microsatellites were found to be located in coding regions. For the polymorphic microsatellites, the number of alleles per microsatellite ranged from 2 to 7 with an average of 4.33, and the diversity values (H) ranged from 0.05 to 0.72 with an average of 0.47. Significant correlations (P<0.01) were observed between the number of repeats and the number of alleles, and between the number of repeats and diversity value, respectively. The genetic diversity explained by chloroplast microsatellites and nuclear RFLP markers were compared using 22 tetraploid accessions. Although the number of alleles for nuclear RFLP markers was found to be higher than that for chloroplast microsatellites, similar diversity values were observed for both types of markers. Among common wheat and its ancestral species, the percentages of common chloroplast microsatellite alleles were calculated to examine their phylogenetic relationships. As a result, Timopheevi wheat species were clearly distinguished from other species, and Emmer and common wheat species were divided into two main groups, each consisting of a series of wild and cultivated species from tetraploid to hexaploid. This indicates that the two types of chloroplast genomes of common wheat might have independently originated from the corresponding types of wild and cultivated Emmer wheat species.

TriFLDB: A Database of Clustered Full-Length Coding Sequences from Triticeae with Applications to Comparative Grass Genomics

The Triticeae Full-Length CDS Database (TriFLDB) contains available information regarding full-length coding sequences (CDSs) of the Triticeae crops wheat (Triticum aestivum) and barley (Hordeum vulgare) and includes functional annotations and comparative genomics features. TriFLDB provides a search interface using keywords for gene function and related Gene Ontology terms and a similarity search for DNA and deduced translated amino acid sequences to access annotations of Triticeae full-length CDS (TriFLCDS) entries. Annotations consist of similarity search results against several sequence databases and domain structure predictions by InterProScan. The deduced amino acid sequences in TriFLDB are grouped with the proteome datasets for Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and sorghum (Sorghum bicolor) by hierarchical clustering in stepwise thresholds of sequence identity, providing hierarchical clustering results based on full-length protein sequences. The database also provides sequence similarity results based on comparative mapping of TriFLCDSs onto the rice and sorghum genome sequences, which together with current annotations can be used to predict gene structures for TriFLCDS entries. To provide the possible genetic locations of full-length CDSs, TriFLCDS entries are also assigned to the genetically mapped cDNA sequences of barley and diploid wheat, which are currently accommodated in the Triticeae Mapped EST Database. These relational data are searchable from the search interfaces of both databases. The current TriFLDB contains 15,871 full-length CDSs from barley and wheat and includes putative full-length cDNAs for barley and wheat, which are publicly accessible. This informative content provides an informatics gateway for Triticeae genomics and grass comparative genomics. TriFLDB is publicly available at http://TriFLDB.psc.riken.jp/.