Chromosome-scale genome assembly of the transformation-amenable common wheat cultivar ‘Fielder’

We have established a high-quality, chromosome-level genome assembly for the hexaploid common wheat cultivar ‘Fielder’, an American, soft, white, pastry-type wheat released in 1974 and known for its amenability to Agrobacterium tumefaciens-mediated transformation and genome editing. Accurate, long-read sequences were obtained using PacBio circular consensus sequencing with the HiFi approach. Sequence reads from 16 SMRT cells assembled using the hifiasm assembler produced assemblies with N50 greater than 20 Mb. We used the Omni-C chromosome conformation capture technique to order contigs into chromosome-level assemblies, resulting in 21 pseudomolecules with a cumulative size of 14.7 and 0.3 Gb of unanchored contigs. Mapping of published short reads from a transgenic wheat plant with an edited seed-dormancy gene, TaQsd1, identified four positions of transgene insertion into wheat chromosomes. Detection of guide RNA sequences in pseudomolecules provided candidates for off-target mutation induction. These results demonstrate the efficiency of chromosome-scale assembly using PacBio HiFi reads and their application in wheat genome-editing studies.     

Discovery and characterization of two new stem rust resistance genes in <Emphasis Type="Italic">Aegilops sharonensis</Emphasis>

Key message

We identified two novel wheat stem rust resistance genes, Sr-1644-1Sh and Sr-1644-5Sh in Aegilops sharonensis that are effective against widely virulent African races of the wheat stem rust pathogen.

Abstract

Stem rust is one of the most important diseases of wheat in the world. When single stem rust resistance (Sr) genes are deployed in wheat, they are often rapidly overcome by the pathogen. To this end, we initiated a search for novel sources of resistance in diverse wheat relatives and identified the wild goatgrass species Aegilops sharonesis (Sharon goatgrass) as a rich reservoir of resistance to wheat stem rust. The objectives of this study were to discover and map novel Sr genes in Ae. sharonensis and to explore the possibility of identifying new Sr genes by genome-wide association study (GWAS). We developed two biparental populations between resistant and susceptible accessions of Ae. sharonensis and performed QTL and linkage analysis. In an F₆ recombinant inbred line and an F₂ population, two genes were identified that mapped to the short arm of chromosome 1S^sh, designated as Sr-1644-1Sh, and the long arm of chromosome 5S^sh, designated as Sr-1644-5Sh. The gene Sr-1644-1Sh confers a high level of resistance to race TTKSK (a member of the Ug99 race group), while the gene Sr-1644-5Sh conditions strong resistance to TRTTF, another widely virulent race found in Yemen. Additionally, GWAS was conducted on 125 diverse Ae. sharonensis accessions for stem rust resistance. The gene Sr-1644-1Sh was detected by GWAS, while Sr-1644-5Sh was not detected, indicating that the effectiveness of GWAS might be affected by marker density, population structure, low allele frequency and other factors.

     

Whole‐genome profiling and shotgun sequencing delivers an anchored,gene‐decorated,physical map assembly of bread wheat chromosome 6A

Bread wheat (Triticum aestivum L.) is the most important staple food crop for 35% of the world's population. International efforts are underway to facilitate an increase in wheat production, of which the International Wheat Genome Sequencing Consortium (IWGSC) plays an important role. As part of this effort, we have developed a sequence‐based physical map of wheat chromosome 6A using whole‐genome profiling (WGP^?). The bacterial artificial chromosome (BAC) contig assembly tools fingerprinted contig (fpc ) and linear topological contig (ltc ) were used and their contig assemblies were compared. A detailed investigation of the contigs structure revealed that ltc created a highly robust assembly compared with those formed by fpc . The ltc assemblies contained 1217 contigs for the short arm and 1113 contigs for the long arm, with an L₅₀ of 1 Mb. To facilitate in silico anchoring, WGP^? tags underlying BAC contigs were extended by wheat and wheat progenitor genome sequence information. Sequence data were used for in silico anchoring against genetic markers with known sequences, of which almost 79% of the physical map could be anchored. Moreover, the assigned sequence information led to the ‘decoration’ of the respective physical map with 3359 anchored genes. Thus, this robust and genetically anchored physical map will serve as a framework for the sequencing of wheat chromosome 6A, and is of immediate use for map‐based isolation of agronomically important genes/quantitative trait loci located on this chromosome.     

Molecular analysis of the phylogenetic relationships among the diploid <Emphasis Type="Italic">Aegilops</Emphasis> species of the section <Emphasis Type="Italic">Sitopsis</Emphasis>

RAPD analysis was used to study the intraspecific variation and phylogenetic relationships of Sgenome diploid Aegilops species regarded as potential donors of the B genome of cultivated wheat. In total, 21 DNA specimens from six S-genome diploid species were examined. On a dendrogram, Ae. speltoides and Ae. aucheri formed the most isolated cluster. Among the other species, Ae. searsii was the most distant while Ae. longissima and Ae. sharonensis were the closest species. The maximum difference between individual accessions within one species was approximately the same (0.18–0.22) in Ae. bicornis, Ae. longissima, Ae. sharonensis, and Ae. searsii. The difference between the clusters of questionable species Ae. speltoides and Ae. aucheri corresponded to the intraspecific level; the difference between closely related Ae. longissima and Ae. sharonensis corresponded to the interspecific level.     

Genome differentiation in Aegilops. 4. Evolution of the U-genome cluster

Phylogenetic relationships of polyploid Aegilops species sharing the U-genome were investigated by analyzing heterochromatin banding patterns of their somatic metaphase chromosomes as revealed by C-banding and fluorescence in situ hybridization (FISH) with the heterochromatin-limited repetitive DNA probes pSc119, pAs1, as well as the distribution of NOR and 5S DNA loci revealed by pTa71 (18S-26S rDNA), and pTa794 (5S rDNA) probes. Seven tetraploid (Ae. triuncialis, Ae. peregrina, Ae. kotschyi, Ae. geniculata, Ae. biuncialis, Ae. columnaris, and 4x Ae. neglecta) and one hexaploid (6x Ae. neglecta) Aegilops species of the U-genome cluster were studied. The U^t and C^t chromosomes of 4x Ae. triuncialis (U^tC^t) were similar to the diploid donors Ae. umbellulata (U) and Ae. caudata (C). However, the size of the NOR locus on chromosome 5U^t was reduced. Karyotypic analyses confirmed that 4x Ae. peregrina (S^pU^p) was derived from a hybridization of the diploid species Ae. umbellulata with Ae. longissima, whereas Ae. umbellulata and Ae. sharonensis (or an immediate precursor) were the diploid progenitor species of Ae. kotschyi (S^kU^k). In both 4x species, the NORs on S-genome chromosomes were inactivated and were accompanied with a decrease or loss of rDNA sequences. Karyotypes of the tetraploid species, Ae. geniculata (U^gM^g) and Ae. biuncialis (U^bM^b) differed from each other and from the putative diploid progenitors Ae. umbellulata and Ae. comosa indicating that various types of chromosomal alterations occurred during speciation. Inactivation of major NORs on the M-genome chromosomes, redistribution of 5S rDNA sites, and loss of some minor 18S-26S rDNA loci were observed in Ae. geniculata and Ae. biuncialis. Significant differences in the total amount and distribution of heterochromatin, the number and location of 5S and 18S-26S rDNA loci observed between Ae. columnaris (U^cX^c)/4x Ae. neglecta (UⁿXⁿ) and Ae. geniculata/Ae. biuncialis indicate that these species have different origins. Similarities in C-banding and FISH patterns of most Ae. columnaris and 4x Ae. neglecta chromosomes suggest that they were probably derived from a common ancestor, whereas distinct differences of three chromosome pairs may indicate that the divergence of these species was probably associated with chromosomal rearrangements and/or introgressive hybridization. Ae. umbellulata contributed the U genome, however, the source of their second genomes remains unknown. The formation of 6x Ae. neglecta (UⁿXⁿNⁿ) was not associated with large modifications of the parental genomes.     

Syntenic Relationships between the U and M Genomes of Aegilops,Wheat and the Model Species Brachypodium and Rice as Revealed by COS Markers

Diploid Aegilops umbellulata and Ae. comosa and their natural allotetraploid hybrids Ae. biuncialis and Ae. geniculata are important wild gene sources for wheat. With the aim of assisting in alien gene transfer, this study provides gene-based conserved orthologous set (COS) markers for the U and M genome chromosomes. Out of the 140 markers tested on a series of wheat-Aegilops chromosome introgression lines and flow-sorted subgenomic chromosome fractions, 100 were assigned to Aegilops chromosomes and six and seven duplications were identified in the U and M genomes, respectively. The marker-specific EST sequences were BLAST-ed to Brachypodium and rice genomic sequences to investigate macrosyntenic relationships between the U and M genomes of Aegilops, wheat and the model species. Five syntenic regions of Brachypodium identified genome rearrangements differentiating the U genome from the M genome and from the D genome of wheat. All of them seem to have evolved at the diploid level and to have been modified differentially in the polyploid species Ae. biuncialis and Ae. geniculata. A certain level of wheat–Aegilops homology was detected for group 1, 2, 3 and 5 chromosomes, while a clearly rearranged structure was showed for the group 4, 6 and 7 Aegilops chromosomes relative to wheat. The conserved orthologous set markers assigned to Aegilops chromosomes promise to accelerate gene introgression by facilitating the identification of alien chromatin. The syntenic relationships between the Aegilops species, wheat and model species will facilitate the targeted development of new markers specific for U and M genomic regions and will contribute to the understanding of molecular processes related to allopolyploidization.