Project description:BACKGROUND:Triticum monococcum L., an A genome diploid einkorn wheat, was the first domesticated crop. As a diploid, it is attractive genetic model for the study of gene structure and function of wheat-specific traits. Diploid wheat is currently not amenable to reverse genetics approaches such as insertion mutagenesis and post-transcriptional gene silencing strategies. However, TILLING offers a powerful functional genetics approach for wheat gene analysis. RESULTS:We developed a TILLING population of 1,532 M2 families using EMS as a mutagen. A total of 67 mutants were obtained for the four genes studied. Waxy gene mutation frequencies are known to be 1/17.6 - 34.4 kb DNA in polyploid wheat TILLING populations. The T. monococcum diploid wheat TILLING population had a mutation frequency of 1/90 kb for the same gene. Lignin biosynthesis pathway genes- COMT1, HCT2, and 4CL1 had mutation frequencies of 1/86 kb, 1/92 kb and 1/100 kb, respectively. The overall mutation frequency of the diploid wheat TILLING population was 1/92 kb. CONCLUSION:The mutation frequency of a diploid wheat TILLING population was found to be higher than that reported for other diploid grasses. The rate, however, is lower than tetraploid and hexaploid wheat TILLING populations because of the higher tolerance of polyploids to mutations. Unlike polyploid wheat, most mutants in diploid wheat have a phenotype amenable to forward and reverse genetic analysis and establish diploid wheat as an attractive model to study gene function in wheat. We estimate that a TILLING population of 5, 520 will be needed to get a non-sense mutation for every wheat gene of interest with 95% probability.
Project description:Mapping and map-based cloning of genes that control agriculturally and economically important traits remain great challenges for plants with complex highly repetitive genomes such as those within the grass tribe, Triticeae. Mapping limitations in the Triticeae are primarily due to low frequencies of polymorphic gene markers and poor genetic recombination in certain genetic regions. Although the abundance of repetitive sequence may pose common problems in genome analysis and sequence assembly of large and complex genomes, they provide repeat junction markers with random and unbiased distribution throughout chromosomes. Hence, development of a high-throughput mapping technology that combine both gene-based and repeat junction-based markers is needed to generate maps that have better coverage of the entire genome.In this study, the available genomics resource of the diploid Aegilop tauschii, the D genome donor of bread wheat, were used to develop genome specific markers that can be applied for mapping in modern hexaploid wheat. A NimbleGen array containing both gene-based and repeat junction probe sequences derived from Ae. tauschii was developed and used to map the Chinese Spring nullisomic-tetrasomic lines and deletion bin lines of the D genome chromosomes. Based on these mapping data, we have now anchored 5,171 repeat junction probes and 10,892 gene probes, corresponding to 5,070 gene markers, to the delineated deletion bins of the D genome. The order of the gene-based markers within the deletion bins of the Chinese Spring can be inferred based on their positions on the Ae. tauschii genetic map. Analysis of the probe sequences against the Chinese Spring chromosome sequence assembly database facilitated mapping of the NimbleGen probes to the sequence contigs and allowed assignment or ordering of these sequence contigs within the deletion bins. The accumulated length of anchored sequence contigs is about 155 Mb, representing?~?3.2 % of the D genome. A specific database was developed to allow user to search or BLAST against the probe sequence information and to directly download PCR primers for mapping specific genetic loci.In bread wheat, aneuploid stocks have been extensively used to assign markers linked with genes/traits to chromosomes, chromosome arms, and their specific bins. Through this study, we added thousands of markers to the existing wheat chromosome bin map, representing a significant step forward in providing a resource to navigate the wheat genome. The database website ( http://probes.pw.usda.gov/ATRJM/ ) provides easy access and efficient utilization of the data. The resources developed herein can aid map-based cloning of traits of interest and the sequencing of the D genome of hexaploid wheat.
Project description:A comprehensive understanding of wheat responses to environmental stress will contribute to the long-term goal of feeding the planet. ALERNATIVE OXIDASE (AOX) genes encode proteins involved in a bypass of the electron transport chain and are also known to be involved in stress tolerance in multiple species. Here, we report the identification and characterization of the AOX gene family in diploid and hexaploid wheat. Four genes each were found in the diploid ancestors Triticum urartu, and Aegilops tauschii, and three in Aegilops speltoides. In hexaploid wheat (Triticum aestivum), 20 genes were identified, some with multiple splice variants, corresponding to a total of 24 proteins for those with observed transcription and translation. These proteins were classified as AOX1a, AOX1c, AOX1e or AOX1d via phylogenetic analysis. Proteins lacking most or all signature AOX motifs were assigned to putative regulatory roles. Analysis of protein-targeting sequences suggests mixed localization to the mitochondria and other organelles. In comparison to the most studied AOX from Trypanosoma brucei, there were amino acid substitutions at critical functional domains indicating possible role divergence in wheat or grasses in general. In hexaploid wheat, AOX genes were expressed at specific developmental stages as well as in response to both biotic and abiotic stresses such as fungal pathogens, heat and drought. These AOX expression patterns suggest a highly regulated and diverse transcription and expression system. The insights gained provide a framework for the continued and expanded study of AOX genes in wheat for stress tolerance through breeding new varieties, as well as resistance to AOX-targeted herbicides, all of which can ultimately be used synergistically to improve crop yield.
Project description:The environment can have a decisive influence on the structure of the genome, changing it in a certain direction. Therefore, the genomic distribution of environmentally sensitive transposable elements may vary measurably across a species area. In the present research, we aimed to detect and evaluate the level of LTR retrotransposon intraspecific variability in Aegilops speltoides (2n = 2x = 14), a wild cross-pollinated relative of cultivated wheat. The interretrotransposon amplified polymorphism (IRAP) protocol was applied to detect and evaluate the level of retrotransposon intraspecific variability in Ae. speltoides and closely related species. IRAP analysis revealed significant diversity in TE distribution. Various genotypes from the 13 explored populations significantly differ with respect to the patterns of the four explored LTR retrotransposons (WIS2, Wilma, Daniela, and Fatima). This diversity points to a constant ongoing process of LTR retrotransposon fraction restructuring in populations of Ae. speltoides throughout the species' range and within single populations in time. Maximum changes were recorded in genotypes from small stressed populations. Principal component analysis showed that the dynamics of the Fatima element significantly differ from those of WIS2, Wilma, and Daniela. In terms of relationships between Sitopsis species, IRAP analysis revealed a grouping with Ae. sharonensis and Ae. longissima forming a separate unit, Ae. speltoides appearing as a dispersed group, and Ae. bicornis being in an intermediate position. IRAP display data revealed dynamic changes in LTR retrotransposon fractions in the genome of Ae. speltoides. The process is permanent and population specific, ultimately leading to the separation of small stressed populations from the main group.
Project description:BackgroundHow new forms arise in nature has engaged evolutionary biologists since Darwin's seminal treatise on the origin of species. Transposable elements (TEs) may be among the most important internal sources for intraspecific variability. Thus, we aimed to explore the temporal dynamics of several TEs in individual genotypes from a small, marginal population of Aegilops speltoides. A diploid cross-pollinated grass species, it is a wild relative of the various wheat species known for their large genome sizes contributed by an extraordinary number of TEs, particularly long terminal repeat (LTR) retrotransposons. The population is characterized by high heteromorphy and possesses a wide spectrum of chromosomal abnormalities including supernumerary chromosomes, heterozygosity for translocations, and variability in the chromosomal position or number of 45S and 5S ribosomal DNA (rDNA) sites. We propose that variability on the morphological and chromosomal levels may be linked to variability at the molecular level and particularly in TE proliferation.ResultsSignificant temporal fluctuation in the copy number of TEs was detected when processes that take place in small, marginal populations were simulated. It is known that under critical external conditions, outcrossing plants very often transit to self-pollination. Thus, three morphologically different genotypes with chromosomal aberrations were taken from a wild population of Ae. speltoides, and the dynamics of the TE complex traced through three rounds of selfing. It was discovered that: (i) various families of TEs vary tremendously in copy number between individuals from the same population and the selfed progenies; (ii) the fluctuations in copy number are TE-family specific; (iii) there is a great difference in TE copy number expansion or contraction between gametophytes and sporophytes; and (iv) a small percentage of TEs that increase in copy number can actually insert at novel locations and could serve as a bona fide mutagen.ConclusionsWe hypothesize that TE dynamics could promote or intensify morphological and karyotypical changes, some of which may be potentially important for the process of microevolution, and allow species with plastic genomes to survive as new forms or even species in times of rapid climatic change.
Project description:We determined nucleosome positions throughout the genome in diploid S. cerevisiae undergoing early stages of synchronous meiosis. This study sought to assess if systematic reorganization of nucleosomes occurs during meiotic prophase at or near sites of DNA double strand break formation. Six samples were sequenced, each a separate time point from either of two meiotic time courses. Dataset N1 consists of samples collected at 0, 1, 2, and 3 hr in meiosis; Dataset N2 consists of samples collected at 0 and 4 hr. Time courses, sample preparation, and sequencing were carried out in separate laboratories for the two datasets: Hochwagen lab (Whitehead Institute) for Dataset N1; Keeney lab (Howard Hughes Medical Institute; Memorial Sloan-Kettering Cancer Center) for Dataset N2.
Project description:Determining the genome sequence of an organism is challenging, yet fundamental to understanding its biology. Over the past decade, thousands of human genomes have been sequenced, contributing deeply to biomedical research. In the vast majority of cases, these have been analyzed by aligning sequence reads to a single reference genome, biasing the resulting analyses, and in general, failing to capture sequences novel to a given genome. Some de novo assemblies have been constructed free of reference bias, but nearly all were constructed by merging homologous loci into single "consensus" sequences, generally absent from nature. These assemblies do not correctly represent the diploid biology of an individual. In exactly two cases, true diploid de novo assemblies have been made, at great expense. One was generated using Sanger sequencing, and one using thousands of clone pools. Here, we demonstrate a straightforward and low-cost method for creating true diploid de novo assemblies. We make a single library from ?1 ng of high molecular weight DNA, using the 10x Genomics microfluidic platform to partition the genome. We applied this technique to seven human samples, generating low-cost HiSeq X data, then assembled these using a new "pushbutton" algorithm, Supernova. Each computation took 2 d on a single server. Each yielded contigs longer than 100 kb, phase blocks longer than 2.5 Mb, and scaffolds longer than 15 Mb. Our method provides a scalable capability for determining the actual diploid genome sequence in a sample, opening the door to new approaches in genomic biology and medicine.
Project description:The association of genetic variation with disease and drug response, together with improvements in nucleic acids technologies, has given great optimism for the impact of 'genomic medicine'. However, the formidable size of the diploid human genome has prevented the routine application of sequencing methods to deciphering complete individual human genomes, and has so far limited the realization of the full potential of genomics for science and human health. Working towards the goal of harnessing the power of genomics, we sequenced the diploid genome of a single individual, Dr. James D. Watson, using a massively-parallel method of sequencing in picoliter size reaction vessels. Here we report the results of genotyping the subject's DNA using an Affymetrix 500k GeneChip as well as copy number variations as reported by Agilent 244k comparative genomic hybridization arrays. Keywords: Genotyping, copy number variation (CNV), aCGH