Farhad Ghavami

Mixed Model Association Mapping for Fusarium Head Blight Resistance in Tunisian-Derived Durum Wheat Populations

Sources of resistance to Fusarium head blight (FHB) in wheat are mostly restricted to Chinese hexaploid genotypes. The effort to incorporate the resistance from hexaploid wheat or wild relatives to cultivated durum wheat (Triticum turgidum L. var. durum Desf.) have not been successful in providing resistance to the level of the donor parents. In this study, we used 171 BC1F6 and 169 BC1F7 lines derived from crossing of four Tunisian tetraploid sources of resistance (Tun7, Tun18, Tun34, Tun36) with durum cultivars ‘Ben,’ ‘Maier,’ ‘Lebsock,’ and ‘Mountrail’ for association studies. The Tun18 and Tun7 FHB resistances were found to be comparable to the best hexaploid wheat sources. A new significant QTL for FHB resistance was identified on the long arm of chromosome 5B (Qfhs.ndsu-5BL) with both association and classical QTL mapping analysis. Linkage disequilibrium (LD) blocks extending up to 40 cM were evident in these populations. The linear mixed model considering the structure (Q or P) and the kinship matrix (KT) estimated by restricted maximum likelihood (REML) was identified as the best for association studies in a mixture of wheat populations from a breeding program. The results of association mapping analysis also demonstrated a region on the short arm of chromosome 3B as potentially linked to FHB resistance. This region is in proximity of major FHB resistance gene fhb1 reported in hexaploid wheat. A possibility of having susceptibility or suppressor of resistance gene(s) on durum wheat chromosome 2A was further confirmed in this material, explaining the problem in developing resistant genotypes without counter selection against this region.

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.

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.

Fast‐forward genetics by radiation hybrids to saturate the locus regulating nuclear–cytoplasmic compatibility in Triticum

Summary The nuclear‐encoded species cytoplasm specific (scs) genes control nuclear–cytoplasmic compatibility in wheat (genus Triticum). Alloplasmic cells, which have nucleus and cytoplasm derived from different species, produce vigorous and vital organisms only when the correct version of scs is present in their nucleus. In this study, bulks of in vivo radiation hybrids segregating for the scs phenotype have been genotyped by sequencing with over 1.9 million markers. The high marker saturation obtained for a critical region of chromosome 1D allowed identification of 3318 reads that mapped in close proximity of the scs. A novel in silico approach was deployed to extend these short reads to sequences of up to 70 Kb in length and identify candidate open reading frames (ORFs). Markers were developed to anchor the short contigs containing ORFs to a radiation hybrid map of 650 individuals with resolution of 288 Kb. The region containing the scs locus was narrowed to a single Bacterial Artificial Chromosome (BAC) contig of Aegilops tauschii. Its sequencing and assembly by nano‐mapping allowed rapid identification of a rhomboid gene as the only ORF existing within the refined scs locus. Resequencing of this gene from multiple germplasm sources identified a single nucleotide mutation, which gives rise to a functional amino acid change. Gene expression characterization revealed that an active copy of this rhomboid exists on all homoeologous chromosomes of wheat, and depending on the specific cytoplasm each copy is preferentially expressed. Therefore, a new methodology was applied to unique genetic stocks to rapidly identify a strong candidate gene for the control of nuclear–cytoplasmic compatibility in wheat.

Genetic analysis and mapping of seedling resistance to Septoria tritici blotch in ‘Steele-ND’/‘ND 735’ bread wheat population

Septoria tritici blotch (STB) caused by Mycosphaerella graminicola, is one of the most destructive foliar diseases of wheat (Triticum aestivum L.) especially in temperate and humid regions across the world. The susceptibility of recently released varieties, evolution of resistance to fungicides and increasing incidence of STB disease emphasizes the need to understand the genetics of resistance to this disease and to incorporate host resistance into adapted cultivars. This study aimed to decipher the genetics and map the resistance to STB using a recombinant inbred line (RIL) mapping population derived from ‘Steele-ND’ (susceptible parent) and ‘ND 735’ (resistant parent). The RILs were evaluated in three greenhouse experiments, using a North Dakota (ND) isolate of STB pathogen. The mean disease severity of parental genotypes, ‘ND 735’ (11.96%) and ‘Steele-ND’ (66.67%) showed significant differences (p < 0.05). The population segregated for STB and the frequency distribution of RILs indicated quantitative inheritance for resistance. The mean disease severity in RILs ranged from 0 to 71.55% with a mean of 21.98%. The genome map of this population was developed using diversity array technology (DArT) and simple sequence repeat (SSR) markers. The framework linkage map of this population was developed using 469 molecular markers. This map spanned a total distance of 1,789.3 cM and consisted of 17 linkage groups. QTL mapping using phenotypic data and the framework linkage maps detected three QTL through composite interval mapping. One QTL was consistently detected in all experiments on the long arm of chromosome 5B, and explained up to 10.2% phenotypic variation. The other two QTLs, detected in single environments, were mapped to 1D and 7A and explain 13% and 5.5% of the phenotypic variation, respectively. The map position of the consistent QTL on 5BL coincides with the map position of durable resistance gene Stb1 suggesting the importance of this region of ‘ND 735’ as a source of durable STB resistance for the wheat germplasm.

Analysis of ATP6 sequence diversity in the Triticum-Aegilops species group reveals the crucial role of rearrangement in mitochondrial genome evolution.

Mutation and chromosomal rearrangements are the two main forces of increasing genetic diversity for natural selection to act upon, and ultimately drive the evolutionary process. Although genome evolution is a function of both forces, simultaneously, the ratio of each can be varied among different genomes and genomic regions. It is believed that in plant mitochondrial genome, rearrangements play a more important role than point mutations, but relatively few studies have directly addressed this phenomenon. To address this issue, we isolated and sequenced the ATP6-1 and ATP6-2 genes from 46 different euplasmic and alloplasmic wheat lines. Four different ATP6-1 orthologs were detected, two of them reported for the first time. Expression analysis revealed that all four orthologs are transcriptionally active. Results also indicated that both point mutation and genomic rearrangement are involved in the evolution of ATP6. However, rearrangement is the predominant force that triggers drastic variation. Data also indicated that speciation of domesticated wheat cultivars were simultaneous with the duplication of this gene. These results directly support the notion that rearrangement plays a significant role in driving plant mitochondrial genome evolution.

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.