Catálogo de publicaciones - libros

For a purpose of better understanding the genome structure of wheat and accelerating the development of DNA markers for gene isolations and breeding, the Japanese research group, as a member of The International Wheat Genome Sequencing Consortium, is now conducting the physical mapping and genomic sequencing of wheat chromosome 6B of ‘Chinese Spring’ (CS). BAC libraries were constructed respectively using the short and long arm-specific DNAs extracted from the flow-sorted chromosome 6BS and 6BL of double ditelosomic 6B lines of CS. With a sequence-based finger printing method and contig assembly, the BAC physical maps for the 6BS and 6BL have been successfully established. For validation and chromosomal landing of the BAC contigs, we have developed a large number of 6B-specific DNA markers using the public resources including available EST databases, and 6B survey sequence. In parallel, three reference maps, a highresolution radiation hybrid map, a gametocidal system-derived deletion map and a genetic linkage map, are also under construction for anchoring the BAC contigs to their specific genomic regions.

International Wheat Genome Sequencing Consortium (IWGSC) decided to adopt the strategy of chromosome sorting and short read assembly to overcome difficulties of wheat genome sequencing derived from the hexaploid status, the large genome size (about 17 Gb) and high repeat contents (more than 80 %). Our Japanese group was responsible for the sequencing of wheat chromosome 6B. Using DNAs from the flow-sorted chromosome arms, we conducted whole-chromosome shotgun sequencing of chromosome 6B. We sequenced more than 12 million reads obtained from the short and long arms by GS-FLX Titanium, and assembled contigs of 235 Mb for 6BS and 273 Mb for 6BL were generated by GS assembler 2.7 (Roche). These assemblies cover 56.6 % and 54.9 % of estimated sizes of 6BS (415 Mb) and 6BL (498 Mb), respectively. We annotated repetitive regions covering more than 80 % of contigs, 4,798 possible expressed loci, and various kinds of RNA genes using our annotation pipeline. We also found the evolutionary conserved regions among syntenic chromosomes from four grass genomes. For application of the 6B sequences to wheat genomics, various kinds of markers, such as simple sequence repeat (SSR) and insertion site-based polymorphism (ISBP) markers were constructed. Combination of the marker data with the comparative genome analysis will lay a strong foundation of functional genomics of the group-6 chromosomes in wheat.

Wheat is cultivated across more land area than any other grain crops. Wheat cultivars are classified as two general types: winter wheat with variable low temperature requirement for a proper flowering time (vernalization) and spring wheat without the requirement, based on their qualitative vernalization requirement. Winter wheat cultivars are classified as three types, weak winter, semi-winter and strong winter, according to their quantitative vernalization requirement to reach a vernalization saturation point or achieve the maximum vernalization effect. Three vernalization genes, , , and , were cloned using a positional cloning approach and a one-gene model of qualitative variation in vernalization requirement between spring and winter wheat. A major gene for the vernalization requirement duration in winter wheat was mapped using a population of recombinant inbred lines (RILs) that were generated from two winter wheat cultivars, ‘Jagger’ and ‘2174’. Furthermore, the cloning population was developed using a RIL to backcross with 2174, which was segregated in a 3:1 ratio of the early flowered plants and the late flowered plants after the population was vernalized for 3 weeks. The wild type Jagger allele for less vernalization was dominant over the 2174 allele for more vernalization, and the two alleles encoded the vrn-A1 proteins with two point mutations. A third haplotype with one of the point mutations was found in common wheat. Gene markers were developed to direct breeding of semi-winter and strong winter wheat cultivars to adapt to different geographical areas and changing climates.

Recent advances of genomic technologies have opened unprecedented possibilities in building high-quality ultra-dense genetic maps. However, with very large numbers of markers available for a mapping population, most of the markers will remain inseparable by recombination. Real situations are also complicated by genotyping errors, which “diversify” a certain part of the markers that would be identical in error-free situations. The higher the error rate the more difficult is the problem of building a reliable map. In our algorithm, we assume that error-free markers can be selected based on the presence of “twins”. There is also a probability of an opposite effect, when non-identical markers may become “twins” because of genotyping errors. Thus, a certain threshold is introduced for the selection of markers with a sufficient number of twins. The developed algorithm (implemented in MultiPoint software) enables mapping big sets of markers (~10–10). Unlike some other algorithms used in building ultra-dense genetic maps, the proposed “twins” approach does not need any prior information (e.g., anchor markers), and hence can be applied to genetically poorly studied organisms.

For hexaploid wheat to be highly fertile, only true homologues must pair at meiosis, rather than the highly related chromosomes present. The mechanism, which restricts this pairing, must have arisen rapidly on wheat’s polyploidisation, to ensure stability and fertility. From the analysis of , which is the major locus restricts this pairing, tweaking Cdk-type phosphorylation levels is one way to provide such a control.

Anthocyanins are flavonoid pigments important for plant adaptation under biotic and abiotic stress conditions. In bread wheat ( L.), purple pigmentation caused by anthocyanins can be present on leaves, culm, auricles, glumes, grains, coleoptile, and anthers. Since the first mentions on expression of purple color traits in wheat, the studies into inheritance of these characters have made big steps toward revealing molecular-genetic mechanisms of anthocyanin pigment biosynthesis and its regulation in wheat. Most of the structural genes, encoding enzymes of the biosynthesis, have been cloned and localized in wheat genome. The genetic mapping data suggest that different pigmentation patterns in wheat are determined by genetic loci, distinct from the enzyme encoding loci. The data on functional role of the genes underpinning phenotypic variation together with results of inter-genera comparative mapping suggest these genes to encode transcriptional activators of the anthocyanin biosynthesis structural genes. Here, a brief review is provided of recent findings in the genetic regulation of anthocyanin biosynthesis in wheat.

For successful speciation through allopolyploidization, normal growth and fertility of interspecific hybrids are essential. However, hybrid plants frequently fail to produce a next generation due to lethality and sterility. Such hybrid incompatabilities are considered a postzygotic reproductive barrier, and play important roles in differentiation and establishment of new genealogical lineages in plants. The Dobzhansky-Müller (DM) model simply explains the process for generating genetic incompatibility in hybrids between two diverging lineages (Bomblies and Weigel 2007). This model proposes that reduction of fitness in hybrids generally occurs due to interaction between at least two epistatic loci derived from divergent parents.

Wheat is one of the most important cereals for humans but quite recalcitrant in transformation. We have thoroughly examined every aspect of the wheat transformation protocols mediated by and we were able to identify and optimize the key factors. Immature embryos isolated from healthy plants grown in a greenhouse were pre-treated with centrifuging and co-cultivated with . The frequency of transformation (independent transgenics/explant) was between 50 % and 60 % were routinely observed and higher than 90 % were recorded in the best cases. Not surprisingly, the key factors did not differ much from those in other cereal plants such as rice and maize. Both bar and hpt genes were good as selection markers. Fielder, a spring wheat cultivar, constantly showed high efficiency of transformation by our protocol. We have been able to obtain transgenic plants from the embryos harvested from the greenhouses throughout the year. Most of the transformed plants were normal in morphology and fully fertile. More than 40 % of the transformants had a single copy of the transgenes, which were inherited in a Mendelian fashion in most of the lines analyzed. Transgenic wheat has been generated at high frequency by several research groups by our protocol by now. Therefore, wheat has finally joined the list of cereals that can be efficiently transformed.

Four extra early-heading mutants, named (), , , and , were identified in diploid einkorn wheat ( L.) following heavy-ion beam mutagenesis. Based on their phenotypes in the field, the four mutants were classified into two groups: Type I (moderately extra early-heading type; and ) and Type II (extremely extra early-heading type; and ). Analysis of (), a flowering promoter gene, showed that it was more highly expressed at earlier stages of vegetative growth in Type II mutants than in Type I mutants. Our analyses indicate that the difference in earliness between Type I and Type II mutants is associated with differences in the expression level of .

Stem rust, caused by f.sp. (), is a destructive disease of wheat that has historically caused significant yield losses in much of the global wheat production area. Over the past 50 years, stem rust has been effectively controlled by deploying cultivars carrying stem rust resistance () genes. With the emergence of new races, namely Ug99 and its variants, there has been a reinvestment in stem rust research. This includes discovery, characterization, genetic mapping, and cloning of genes. Here we investigated two such examples of genetic characterization and mapping of stem rust resistance. In the first example, a region on chromosome 6DS harbouring resistance to Ug99 was examined in several populations and from several sources. In the second example, a less typical genetic model of resistance was studied in which seedling resistance was activated by an independent locus exhibiting an apparent “nonsuppressing” effect. The knowledge gained by these and other lines of research will contribute to the goal of durable resistance to stem rust.