Penny M.A. Kianian

Physical mapping resources for large plant genomes: radiation hybrids for wheat D-genome progenitor Aegilops tauschii

BackgroundDevelopment of a high quality reference sequence is a daunting task in crops like wheat with large (~17Gb), highly repetitive (>80%) and polyploid genome. To achieve complete sequence assembly of such genomes, development of a high quality physical map is a necessary first step. However, due to the lack of recombination in certain regions of the chromosomes, genetic mapping, which uses recombination frequency to map marker loci, alone is not sufficient to develop high quality marker scaffolds for a sequence ready physical map. Radiation hybrid (RH) mapping, which uses radiation induced chromosomal breaks, has proven to be a successful approach for developing marker scaffolds for sequence assembly in animal systems. Here, the development and characterization of a RH panel for the mapping of D-genome of wheat progenitor Aegilops tauschii is reported.ResultsRadiation dosages of 350 and 450 Gy were optimized for seed irradiation of a synthetic hexaploid (AABBDD) wheat with the D-genome of Ae. tauschii accession AL8/78. The surviving plants after irradiation were crossed to durum wheat (AABB), to produce pentaploid RH1s (AABBD), which allows the simultaneous mapping of the whole D-genome. A panel of 1,510 RH1 plants was obtained, of which 592 plants were generated from the mature RH1 seeds, and 918 plants were rescued through embryo culture due to poor germination (<3%) of mature RH1 seeds. This panel showed a homogenous marker loss (2.1%) after screening with SSR markers uniformly covering all the D-genome chromosomes. Different marker systems mostly detected different lines with deletions. Using markers covering known distances, the mapping resolution of this RH panel was estimated to be <140kb. Analysis of only 16 RH lines carrying deletions on chromosome 2D resulted in a physical map with cM/cR ratio of 1:5.2 and 15 distinct bins. Additionally, with this small set of lines, almost all the tested ESTs could be mapped. A set of 399 most informative RH lines with an average deletion frequency of ~10% were identified for developing high density marker scaffolds of the D-genome.ConclusionsThe RH panel reported here is the first developed for any wild ancestor of a major cultivated plant species. The results provided insight into various aspects of RH mapping in plants, including the genetically effective cell number for wheat (for the first time) and the potential implementation of this technique in other plant species. This RH panel will be an invaluable resource for mapping gene based markers, developing a complete marker scaffold for the whole genome sequence assembly, fine mapping of markers and functional characterization of genes and gene networks present on the D-genome.

Accelerated evolution of the mitochondrial genome in an alloplasmic line of durum wheat

BackgroundWheat is an excellent plant species for nuclear mitochondrial interaction studies due to availability of large collection of alloplasmic lines. These lines exhibit different vegetative and physiological properties than their parents. To investigate the level of sequence changes introduced into the mitochondrial genome under the alloplasmic condition, three mitochondrial genomes of the Triticum-Aegilops species were sequenced: 1) durum alloplasmic line with the Ae. longissima cytoplasm that carries the T. turgidum nucleus designated as (lo) durum, 2) the cytoplasmic donor line, and 3) the nuclear donor line.ResultsThe mitochondrial genome of the T. turgidum was 451,678 bp in length with high structural and nucleotide identity to the previously characterized T. aestivum genome. The assembled mitochondrial genome of the (lo) durum and the Ae. longissima were 431,959 bp and 399,005 bp in size, respectively. The high sequence coverage for all three genomes allowed analysis of heteroplasmy within each genome. The mitochondrial genome structure in the alloplasmic line was genetically distant from both maternal and paternal genomes. The alloplasmic durum and the Ae. longissima carry the same versions of atp6, nad6, rps19-p, cob and cox2 exon 2 which are different from the T. turgidum parent. Evidence of paternal leakage was also observed by analyzing nad9 and orf359 among all three lines. Nucleotide search identified a number of open reading frames, of which 27 were specific to the (lo) durum line.ConclusionsSeveral heteroplasmic regions were observed within genes and intergenic regions of the mitochondrial genomes of all three lines. The number of rearrangements and nucleotide changes in the mitochondrial genome of the alloplasmic line that have occurred in less than half a century was significant considering the high sequence conservation between the T. turgidum and the T. aestivum that diverged from each other 10,000 years ago. We showed that the changes in genes were not limited to paternal leakage but were sufficiently significant to suggest that other mechanisms, such as recombination and mutation, were responsible. The newly formed ORFs, differences in gene sequences and copy numbers, heteroplasmy, and substoichiometric changes show the potential of the alloplasmic condition to accelerate evolution towards forming new mitochondrial genomes.

Genomic features shaping the landscape of meiotic double-strand-break hotspots in maize

Significance Meiotic recombination is a process in plants, animals, and fungi during which chromosomes exchange their parts. It generates new genetic variation in the progeny and is one of the reasons why progeny are both similar to and different from their parents. Recombination is initiated by formation of breaks in chromosomal DNA. We generated a high-resolution map of sites where these breaks are formed in the genome of maize. Surprisingly, we found that DNA breaks are abundant in all genome regions, including sites where recombination was thought to be limited, such as repetitive DNA. The map will allow understanding of how recombination patterns shape the genome and aid development of more efficient breeding methods. Meiotic recombination is the most important source of genetic variation in higher eukaryotes. It is initiated by formation of double-strand breaks (DSBs) in chromosomal DNA in early meiotic prophase. The DSBs are subsequently repaired, resulting in crossovers (COs) and noncrossovers (NCOs). Recombination events are not distributed evenly along chromosomes but cluster at recombination hotspots. How specific sites become hotspots is poorly understood. Studies in yeast and mammals linked initiation of meiotic recombination to active chromatin features present upstream from genes, such as absence of nucleosomes and presence of trimethylation of lysine 4 in histone H3 (H3K4me3). Core recombination components are conserved among eukaryotes, but it is unclear whether this conservation results in universal characteristics of recombination landscapes shared by a wide range of species. To address this question, we mapped meiotic DSBs in maize, a higher eukaryote with a large genome that is rich in repetitive DNA. We found DSBs in maize to be frequent in all chromosome regions, including sites lacking COs, such as centromeres and pericentromeric regions. Furthermore, most DSBs are formed in repetitive DNA, predominantly Gypsy retrotransposons, and only one-quarter of DSB hotspots are near genes. Genic and nongenic hotspots differ in several characteristics, and only genic DSBs contribute to crossover formation. Maize hotspots overlap regions of low nucleosome occupancy but show only limited association with H3K4me3 sites. Overall, maize DSB hotspots exhibit distribution patterns and characteristics not reported previously in other species. Understanding recombination patterns in maize will shed light on mechanisms affecting dynamics of the plant genome.

Radiation Hybrids: A valuable Tool for Genetic, Genomic and Functional Analysis of Plant Genomes

Radiation has been used as a mean to break and transfer fragments of DNA from one plant species to another. Early examples include the experiments by Sears, (Brookhaven Symp Biol 9:1–22, 1956) to transfer rust resistance genes from Aegilops umbellulata to wheat. Radiation found its niche as a mutagen due to advances in nuclear technology and formation of the International Atomic Energy Agency and their sponsorship of developing mutation breeding through “Mutation Enhanced Technologies for Agriculture”. Mutation breeding has resulted in the release of several important cultivars. Although radiation was used in plants for the mutation and introgression of genes from related species (Sears, Brookhaven Symp Biol 9:1–22, 1956; Driscoll and Jensen, Genetics 48:459–468, 1963; Riley and Law, Stadler Genet Symp 16:301–322, 1984; Sears, Crop Sci 33:897–901, 1993), this approach was not used for mapping. This aspect of radiation application was first utilized in animal cell culture lines to generate radiation hybrid (RH) panels. In the beginning these panels were generated for single chromosomes but evolved to the development of whole genome panels. This technology matured in animal systems with the onset of genomics era by its use in the development of high resolution RH-based physical maps for many species before or during the development of complete genome sequence information. The advantages of this system are: (1) radiation-induced breaks are independent of recombination events providing higher and more uniform resolution, (2) radiation dosage could be adjusted to provide varied resolution without greatly affecting the population size and (3) all markers regardless of their polymorphism can be mapped on RH panels. Plant scientists followed these studies by the development of RH panels for individual chromosomes or whole genomes. However, early RH panels in plant systems were of low to medium resolution and of limited use in physical mapping. Recently, RH panels have been produced resulting in map resolutions of 200–400 Kb. These high resolution panels promise the same value as animal systems in helping generate a complete genome sequence with a fraction of the cost of traditional methods. But the use of radiation in plants has matured to go beyond physical mapping by its application to gene cloning and forward/reverse genetic studies. These applications take advantage of plasticity offered by many plant species in tolerating radiation to produce seed and live progeny. This ability allows scientists to phenotype RH lines and to associate the phenotypic data with the genotypic data. The great potential of this system is just being realized.

Transposable element junctions in marker development and genomic characterization of barley

Barley is a model plant in genomic studies of Triticeae species. However, barleys large genome size and high repetitive sequence content complicate the whole‐genome sequencing. The majority of the barley genome is composed of transposable elements (TEs). In this study, TE repeat junctions (RJs) were used to develop a large‐scale molecular marker platform, as a prerequisite to genome assembly. A total of 10.22 Gb of barley nonassembled 454 sequencing data were screened with RJPrimers pipeline. In total, 9,881,561 TE junctions were identified. From detected RJs, 400,538 polymerase chain reaction (PCR)‐based RJ markers (RJMs) were designed across the genome, with an average of 39 markers/Mb. The utility of designed markers was tested using a random subset of RJMs. Over 94% of the markers successfully amplified amplicons, among which ∼90% were genome specific. In addition to marker design, identified RJs were utilized to detect 1190,885 TEs across the genome. In gene‐poor regions of the genome Gypsy elements comprised the majority of TEs (∼65%), while in gene‐rich regions Gypsy, Copia, and Mariner were the main transposons, each representing an average ∼23% of total TEs. The numerous RJ primer pairs developed in this study will be a valuable resource for barley genomic studies including genomic selection, fine mapping, and genome assembly. In addition, the results of this study show that characterizing RJs provides insight into TE composition of species without a sequenced genome but for which short‐read sequence data is available.

Radiation hybrid map of barley chromosome 3H

Assembly of the barley (Hordeum vulgare L.) genome is complicated by its large size (5.1 Gb) and proportion of repetitive elements (84%). This process is facilitated by high resolution maps for aligning bacterial artificial chromosome (BAC) contigs along chromosomes. Available genetic maps, however, do not provide accurate information on the physical position of a large portion of the genome located in recombination‐poor regions. Radiation hybrid (RH) mapping is an alternative approach, which is based on radiation‐induced deletions along the length of chromosomes. In this study, the first RH map for barley chromosome 3H was developed. In total, 373 in vivo RH lines were generated by irradiating wheat (Triticum aestivum L.)–barley chromosome 3H addition lines and crossing them to a normal wheat cultivar. Each RH informative line (containing deletions) had, on average, three deletions. The induced deletion size varied from 36.58 Kb to 576.00 Mb, with an average length of 52.42 Mb. This initial chromosome 3H radiation hybrid (3H‐RH) map had a 9.53× higher resolution than an analogous genetic map, reaching a maximum of >262.40× resolution in regions around the centromere. The final RH map was 3066.1 cR in length, with a 0.76 Mb resolution. It was estimated that the map resolution can be improved to an average of 30.34 Kb by saturating the 3H‐RH map with molecular markers. The generated RH panel enabled alignment of BAC and sequenced contigs as small as 1.50 Kb in size. The high resolution and the coverage of poor‐recombination regions make RH maps an ideal resource for barley genome assembly, as well as other genetic studies.

Mitochondrial dynamics and the cell cycle

Nuclear-mitochondrial (NM) communication impacts many aspects of plant development including vigor, sterility, and viability. Dynamic changes in mitochondrial number, shape, size, and cellular location takes place during the cell cycle possibly impacting the process itself and leading to distribution of this organelle into daughter cells. The genes that underlie these changes are beginning to be identified in model plants such as Arabidopsis. In animals disruption of the drp1 gene, a homolog to the plant drp3A and drp3B, delays mitochondrial division. This mutation results in increased aneuploidy due to chromosome mis-segregation. It remains to be discovered if a similar outcome is observed in plants. Alloplasmic lines provide an opportunity to understand the communication between the cytoplasmic organelles and the nucleus. Examples of studies in these lines, especially from the extensive collection in wheat, point to the role of mitochondria in chromosome movement, pollen fertility and other aspects of development.

High-resolution crossover mapping reveals similarities and differences of male and female recombination in maize

Meiotic crossovers (COs) are not uniformly distributed across the genome. Factors affecting this phenomenon are not well understood. Although many species exhibit large differences in CO numbers between sexes, sex-specific aspects of CO landscape are particularly poorly elucidated. Here, we conduct high-resolution CO mapping in maize. Our results show that CO numbers as well as their overall distribution are similar in male and female meioses. There are, nevertheless, dissimilarities at local scale. Male and female COs differ in their locations relative to transcription start sites in gene promoters and chromatin marks, including nucleosome occupancy and tri-methylation of lysine 4 of histone H3 (H3K4me3). Our data suggest that sex-specific factors not only affect male–female CO number disparities but also cause fine differences in CO positions. Differences between male and female CO landscapes indicate that recombination has distinct implications for population structure and gene evolution in male and in female meioses.Sex-specific meiotic crossover (CO) landscapes have been identified in multiple species. Here, the authors show that male and female meioses in maize have similar CO landscapes, and differences between COs in the two sexes only exists in their location relative to transcription start sites and some chromatin marks.

Dissecting Plant Chromosomes by the Use of Ionizing Radiation.

Radiation treatment of genomes is used to generate chromosome breaks for numerous applications. This protocol describes the preparation of seeds and the determination of the optimal level of irradiation dosage for the creation of a radiation hybrid (RH) population. These RH lines can be used to generate high-resolution physical maps for the assembly of sequenced genomes as well as the fine mapping of genes. This procedure can also be used for mutation breeding and forward/reverse genetics.