Extrachromosomal circular DNA-mediated spread of herbicide resistance in interspecific hybrids of pigweed.

Extrachromosomal circular DNAs (eccDNAs) are found in many eukaryotic organisms. EccDNA-powered copy number variation plays diverse roles, from oncogenesis in humans to herbicide resistance in crop weeds. Here, we report interspecific eccDNA flow and its dynamic behavior in soma cells of natural populations and F1 hybrids of Amaranthus sp. The glyphosate-resistance (GR) trait is controlled by eccDNA-based amplification harboring the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene (eccDNA replicon), the molecular target of glyphosate. We documented pollen-mediated transfer of eccDNA in experimental hybrids between glyphosate-susceptible Amaranthus tuberculatus and GR Amaranthus palmeri. Experimental hybridization and fluorescence in situ hybridization (FISH) analysis revealed that the eccDNA replicon in Amaranthus spinosus derived from GR A. palmeri by natural hybridization. FISH analysis also revealed random chromosome anchoring and massive eccDNA replicon copy number variation in soma cells of weedy hybrids. The results suggest that eccDNAs are inheritable across compatible species, contributing to genome plasticity and rapid adaptive evolution.

Physical mapping of the wheat genes in low-recombination regions: radiation hybrid mapping of the C-locus.

KEY MESSAGE: This work reports the physical mapping of an important gene affecting spike compactness located in a low-recombination region of hexaploid wheat. This work paves the way for the eventual isolation and characterization of the factor involved but also opens up possibilities to use this approach to precisely map other wheat genes located on proximal parts of wheat chromosomes that show highly reduced recombination. Mapping wheat genes, in the centromeric and pericentromeric regions (~ 2/3rd of a given chromosome), poses a formidable challenge due to highly suppressed recombination. Using an example of compact spike locus (C-locus), this study provides an approach to precisely map wheat genes in the pericentromeric and centromeric regions that house ~ 30% of wheat genes. In club-wheat, spike compactness is controlled by the dominant C-locus, but previous efforts have failed to localize it, on a particular arm of chromosome 2D. We integrated radiation hybrid (RH) and high-resolution genetic mapping to locate C-locus on the short arm of chromosome 2D. Flanking markers of the C-locus span a physical distance of 11.0 Mb (231.0-242 Mb interval) and contain only 11 high-confidence annotated genes. This work demonstrates the value of this integrated strategy in mapping dominant genes in the low-recombination regions of the wheat genome. A comparison of the mapping resolutions of the RH and genetic maps using common anchored markers indicated that the RH map provides ~ 9 times better resolution that the genetic map even with much smaller population size. This study provides a broadly applicable approach to fine map wheat genes in regions of suppressed recombination.

Comprehensive analysis of Q gene near-isogenic lines reveals key molecular pathways for wheat domestication and improvement.

The wheat AP2-like transcription factor gene Q has played a major role in domestication by conferring the free-threshing character and pleiotropically affecting numerous other traits. However, little information is known regarding the molecular mechanisms associated with the regulation of these traits by Q, especially for the structural determination of threshability. Here, transcriptome analysis of immature spike tissues in three lines nearly isogenic for Q revealed over 3000 differentially expressed genes (DEGs) involved in a number of pathways. Using phenotypic, microscopic, transcriptomic, and tissue-specific gene expression analyses, we demonstrated that Q governs threshability through extensive modification of wheat glumes including their structure, cell wall thickness, and chemical composition. Critical DEGs and pathways involved in secondary cell wall synthesis and regulation of the chemical composition of glumes were identified. We also showed that the mutation giving rise to the Q allele synchronized the expression of genes for micro-sporogenesis that affected pollen fertility, and may determine the final grain number for wheat spikes. Transcriptome dissection of genes and genetic pathways regulated by Q should further our understanding of wheat domestication and improvement.

Triple null mutations in starch synthase SSIIa gene homoeologs lead to high amylose and resistant starch in hexaploid wheat.

BACKGROUND: Lack of nutritionally appropriate foods is one of the leading causes of obesity in the US and worldwide. Wheat (Triticum aestivum) provides 20% of the calories consumed daily across the globe. The nutrients in the wheat grain come primarily from the starch composed of amylose and amylopectin. Resistant starch content, which is known to have significant human health benefits, can be increased by modifying starch synthesis pathways. Starch synthase enzyme SSIIa, also known as starch granule protein isoform-1 (SGP-1), is integral to the biosynthesis of the branched and readily digestible glucose polymer amylopectin. The goal of this work was to develop a triple null mutant genotype for SSIIa locus in the elite hard red winter wheat variety 'Jagger' and evaluate the effect of the knock-out mutations on resistant starch content in grains with respect to wild type. RESULTS: Knock-out mutations in SSIIa in the three genomes of wheat variety 'Jagger' were identified using TILLING. Subsequently, these loss-of function mutations on A, B, and D genomes were combined by crossing to generate a triple knockout mutant genotype Jag-ssiia-∆ABD. The Jag-ssiia-∆ABD had an amylose content of 35.70% compared to 31.15% in Jagger, leading to ~ 118% increase in resistant starch in the Jag-ssiia-∆ABD genotype of Jagger wheat. The single individual genome mutations also had various effects on starch composition. CONCLUSIONS: Our full null Jag-ssiia-∆ABD mutant showed a significant increase in RS without the shriveled grain phenotype seen in other ssiia knockouts in elite wheat cultivars. Moreover, this study shows the potential for developing nutritionally improved foods in a non-GM approach. Since all the mutants have been developed in an elite wheat cultivar, their adoption in production and supply will be feasible in future.

Deciphering the Mechanism of Glyphosate Resistance in Amaranthus palmeri by Cytogenomics.

In agriculture, various chemicals are used to control the weeds. Out of which, glyphosate is an important herbicide invariably used in the cultivation of glyphosate-resistant crops to control weeds. Overuse of glyphosate results in the evolution of glyphosate-resistant weeds. Evolution of glyphosate resistance (GR) in Amaranthus palmeri (AP) is a serious concern in the USA. Investigation of the mechanism of GR in AP identified different resistance mechanisms of which 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene amplification is predominant. Molecular analysis of GR AP identified the presence of a 5- to >160-fold increase in copies of the EPSPS gene than in a glyphosate-susceptible (GS) population. This increased copy number of the EPSPS gene increased the genome size ranging from 3.5 to 11.8%, depending on the copy number compared to the genome size of GS AP. FISH analysis using a 399-kb EPSPS cassette derived from bacterial artificial chromosomes (BACs) as probes identified that amplified EPSPS copies in GR AP exist in extrachromosomal circular DNA (eccDNA) in addition to the native copy in the chromosome. The EPSPS gene-containing eccDNA having a size of ∼400 kb is termed EPSPS-eccDNA and showed somatic mosacism in size and copy number. EPSPS-eccDNA has a genetic mechanism to tether randomly to mitotic or meiotic chromosomes during cell division or gamete formation and is inherited to daughter cells or progeny generating copy number variation. These eccDNAs are stable genetic elements that can replicate and exist independently. The genomic characterization of the EPSPS locus, along with the flanking regions, identified the presence of a complex array of repeats and mobile genetic elements. The cytogenomics approach in understanding the biology of EPSPS-eccDNA sheds light on various characteristics of EPSPS-eccDNA that favor GR in AP.

Discovery of a susceptibility factor for Fusarium head blight on chromosome 7A of wheat.

KEY MESSAGE: Discovery and mapping of a susceptibility factor located on the short arm of wheat chromosome 7A whose deletion makes plants resistant to Fusarium head blight. Fusarium head blight (FHB) disease of wheat caused by Fusarium spp. deteriorates both quantity and quality of the crop. Manipulation of susceptibility factors, the plant genes facilitating disease development, offers a novel and alternative strategy for enhancing FHB resistance in plants. In this study, a major effect susceptibility gene for FHB was identified on the short arm of chromosome 7A (7AS). Nullisomic-tetrasomic lines for homoeologous group-7 of wheat revealed dosage effect of the gene, with tetrasomic 7A being more susceptible than control Chinese Spring wheat, qualifying it as a genuine susceptibility factor. Five chromosome 7A inter-varietal substitution lines and a tetraploid Triticum dicoccoides 7A substitution line showed similar susceptibility as that of Chinese Spring, indicating toward the commonality of the susceptibility factor among these diverse genotypes. The susceptibility factor was named as Sf-Fhb-7AS and mapped on chromosome 7AS to a 48.5-50.5 Mb peri-centromeric region between del7AS-3 and del7AS-8. Our results showed that deletion of Sf-Fhb-7AS imparts 50-60% type 2 FHB resistance and its manipulation can be used to enhance resistance against FHB in wheat.

An approach for high-resolution genetic mapping of distant wild relatives of bread wheat: example of fine mapping of Lr57 and Yr40 genes.

KEY MESSAGE: The article reports a powerful but simple approach for high-resolution mapping and eventual map-based cloning of agronomically important genes from distant relatives of wheat, using the already existing germplasm resources. Wild relatives of wheat are a rich reservoir of genetic diversity for its improvement. The effective utilization of distant wild relatives in isolation of agronomically important genes is hindered by the lack of recombination between the homoeologous chromosomes. In this study, we propose a simple yet powerful approach that can be applied for high-resolution mapping of a targeted gene from wheat's distant gene pool members. A wheat-Aegilops geniculata translocation line TA5602 with a small terminal segment from chromosome 5 Mg of Ae. geniculata translocated to 5D of wheat contains genes Lr57 and Yr40 for leaf rust and stripe rust resistance, respectively. To map these genes, TA5602 was crossed with a susceptible Ae. geniculata 5 Mg addition line. Chromosome pairing between the 5 Mg chromosomes of susceptible and resistant parents resulted in the development of a high-resolution mapping panel for the targeted genes. Next-generation-sequencing data from flow-sorted 5 Mg chromosome of Ae. geniculata allowed us to generate 5 Mg-specific markers. These markers were used to delineate Lr57 and Yr40 genes each to distinct ~ 1.5 Mb physical intervals flanked by gene markers on 5 Mg. The method presented here will allow researchers worldwide to utilize existing germplasm resources in genebanks and seed repositories toward routinely performing map-based cloning of important genes from tertiary gene pools of wheat.

High-resolution mapping of the Mov-1 locus in wheat by combining radiation hybrid (RH) and recombination-based mapping approaches.

KEY MESSAGE: This work reports a quick method that integrates RH mapping and genetic mapping to map the dominant Mov-1 locus to a 1.1-Mb physical interval with a small number of candidate genes. Bread wheat is an important crop for global human population. Identification of genes and alleles controlling agronomic traits is essential toward sustainably increasing crop production. The unique multi-ovary (MOV) trait in wheat holds potential for improving yields and is characterized by the formation of 2-3 grains per spikelet. The genetic basis of the multi-ovary trait is known to be monogenic and dominant in nature. Its precise mapping and functional characterization is critical to utilizing this trait in a feasible manner. Previous mapping efforts of the locus controlling multiple ovary/pistil formation in the hexaploid wheat have failed to produce a consensus for a particular chromosome. We describe a mapping strategy integrating radiation hybrid mapping and high-resolution genetic mapping to locate the chromosomal position of the Mov-1 locus in hexaploid wheat. We used RH mapping approach using a panel of 188 lines to map the Mov-1 locus in the terminal part of long arm of wheat chromosome 2D with a map resolution of 1.67 Mb/cR1500. Then using a genetic population of MOV × Synthetic wheat of F2 lines, we delineated the Mov-1 locus to a 1.1-Mb physical region with a small number of candidate genes. This demonstrates the value of this integrated strategy to mapping dominant genes in wheat.

Extrachromosomal circular DNA-based amplification and transmission of herbicide resistance in crop weed Amaranthus palmeri.

Gene amplification has been observed in many bacteria and eukaryotes as a response to various selective pressures, such as antibiotics, cytotoxic drugs, pesticides, herbicides, and other stressful environmental conditions. An increase in gene copy number is often found as extrachromosomal elements that usually contain autonomously replicating extrachromosomal circular DNA molecules (eccDNAs). Amaranthus palmeri, a crop weed, can develop herbicide resistance to glyphosate [N-(phosphonomethyl) glycine] by amplification of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene, the molecular target of glyphosate. However, biological questions regarding the source of the amplified EPSPS, the nature of the amplified DNA structures, and mechanisms responsible for maintaining this gene amplification in cells and their inheritance remain unknown. Here, we report that amplified EPSPS copies in glyphosate-resistant (GR) A. palmeri are present in the form of eccDNAs with various conformations. The eccDNAs are transmitted during cell division in mitosis and meiosis to the soma and germ cells and the progeny by an as yet unknown mechanism of tethering to mitotic and meiotic chromosomes. We propose that eccDNAs are one of the components of McClintock's postulated innate systems [McClintock B (1978) Stadler Genetics Symposium] that can rapidly produce soma variation, amplify EPSPS genes in the sporophyte that are transmitted to germ cells, and modulate rapid glyphosate resistance through genome plasticity and adaptive evolution.

Comparative analysis of chromosome 2A molecular organization in diploid and hexaploid wheat.

Diploid A genome wheat species harbor immense genetic variability which has been targeted and proven useful in wheat improvement. Development and deployment of sequence-based markers has opened avenues for comparative analysis, gene transfer and marker assisted selection (MAS) using high throughput cost effective genotyping techniques. Chromosome 2A of wheat is known to harbor several economically important genes. The present study aimed at identification of genic sequences corresponding to full length cDNAs and mining of SSRs and ISBPs from 2A draft sequence assembly of hexaploid wheat cv. Chinese Spring for marker development. In total, 1029 primer pairs including 478 gene derived, 501 SSRs and 50 ISBPs were amplified in diploid A genome species Triticum monococcum and T. boeoticum identifying 221 polymorphic loci. Out of these, 119 markers were mapped onto a pre-existing chromosome 2A genetic map consisting of 42 mapped markers. The enriched genetic map constituted 161 mapped markers with final map length of 549.6 cM. Further, 2A genetic map of T. monococcum was anchored to the physical map of 2A of cv. Chinese Spring which revealed several rearrangements between the two species. The present study generated a highly saturated genetic map of 2A and physical anchoring of genetically mapped markers revealed a complex genetic architecture of chromosome 2A that needs to be investigated further.

Structural variation and rates of genome evolution in the grass family seen through comparison of sequences of genomes greatly differing in size.

Homology was searched with genes annotated in the Aegilops tauschii pseudomolecules against genes annotated in the pseudomolecules of tetraploid wild emmer wheat, Brachypodium distachyon, sorghum and rice. Similar searches were performed with genes annotated in the rice pseudomolecules. Matrices of collinear genes and rearrangements in their order were constructed. Optical BioNano genome maps were constructed and used to validate rearrangements unique to the wild emmer and Ae. tauschii genomes. Most common rearrangements were short paracentric inversions and short intrachromosomal translocations. Intrachromosomal translocations outnumbered segmental intrachromosomal duplications. The densities of paracentric inversion lengths were approximated by exponential distributions in all six genomes. Densities of collinear genes along the Ae. tauschii chromosomes were highly correlated with meiotic recombination rates but those of rearrangements were not, suggesting different causes of the erosion of gene collinearity and evolution of major chromosome rearrangements. Frequent rearrangements sharing breakpoints suggested that chromosomes have been rearranged recurrently at some sites. The distal 4 Mb of the short arms of rice chromosomes Os11 and Os12 and corresponding regions in the sorghum, B. distachyon and Triticeae genomes contain clusters of interstitial translocations including from 1 to 7 collinear genes. The rates of acquisition of major rearrangements were greater in the large wild emmer wheat and Ae. tauschii genomes than in the lineage preceding their divergence or in the B. distachyon, rice and sorghum lineages. It is suggested that synergy between large quantities of dynamic transposable elements and annual growth habit have been the primary causes of the fast evolution of the Triticeae genomes.

Gene Duplication and Aneuploidy Trigger Rapid Evolution of Herbicide Resistance in Common Waterhemp.

An increase in gene copy number is often associated with changes in the number and structure of chromosomes, as has been widely observed in yeast and eukaryotic tumors, yet little is known about stress-induced chromosomal changes in plants. Previously, we reported that the EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) gene, the molecular target of glyphosate, was amplified at the native locus and on an extra chromosome in glyphosate-resistant Amaranthus tuberculatus Here, we report that the extra chromosome is a ring chromosome termed extra circular chromosome carrying amplified EPSPS (ECCAE). The ECCAE is heterochromatic, harbors four major EPSPS amplified foci, and is sexually transmitted to 35% of the progeny. Two highly glyphosate resistant (HGR) A. tuberculatus plants with a chromosome constitution of 2n = 32+1 ECCAE displayed soma cell heterogeneity. Some cells had secondary ECCAEs, which displayed size polymorphisms and produced novel chromosomal variants with multiple gene amplification foci. We hypothesize that the ECCAE in the soma cells of HGR A. tuberculatus plants underwent breakage-fusion-bridge cycles to generate the observed soma cell heterogeneity, including de novo EPSPS gene integration into chromosomes. Resistant soma cells with stable EPSPS amplification events as de novo insertions into chromosomes may survive glyphosate selection pressure during the sporophytic phase and are plausibly transmitted to germ cells leading to durable glyphosate resistance in A. tuberculatus This is the first report of early events in aneuploidy-triggered de novo chromosome integration by an as yet unknown mechanism, which may drive rapid adaptive evolution of herbicide resistance in common waterhemp.

Fine Mapping of the Wheat Leaf Rust Resistance Gene Lr42.

Leaf rust caused by Puccinia triticina Eriks is one of the most problematic diseases of wheat throughout the world. The gene Lr42 confers effective resistance against leaf rust at both seedling and adult plant stages. Previous studies had reported Lr42 to be both recessive and dominant in hexaploid wheat; however, in diploid Aegilops tauschii (TA2450), we found Lr42 to be dominant by studying segregation in two independent F2 and their F2:3 populations. We further fine-mapped Lr42 in hexaploid wheat using a KS93U50/Morocco F5 recombinant inbred line (RIL) population to a 3.7 cM genetic interval flanked by markers TC387992 and WMC432. The 3.7 cM Lr42 region physically corresponds to a 3.16 Mb genomic region on chromosome 1DS based on the Chinese Spring reference genome (RefSeq v.1.1) and a 3.5 Mb genomic interval on chromosome 1 in the Ae. tauschii reference genome. This region includes nine nucleotide-binding domain leucine-rich repeat (NLR) genes in wheat and seven in Ae. tauschii, respectively, and these are the likely candidates for Lr42. Furthermore, we developed two kompetitive allele-specific polymorphism (KASP) markers (SNP113325 and TC387992) flanking Lr42 to facilitate marker-assisted selection for rust resistance in wheat breeding programs.

Major structural genomic alterations can be associated with hybrid speciation in Aegilops markgrafii (Triticeae).

During evolutionary history many grasses from the tribe Triticeae have undergone interspecific hybridization, resulting in allopolyploidy; whereas homoploid hybrid speciation was found only in rye. Homoeologous chromosomes within the Triticeae preserved cross-species macrocolinearity, except for a few species with rearranged genomes. Aegilops markgrafii, a diploid wild relative of wheat (2n = 2x = 14), has a highly asymmetrical karyotype that is indicative of chromosome rearrangements. Molecular cytogenetics and next-generation sequencing were used to explore the genome organization. Fluorescence in situ hybridization with a set of wheat cDNAs allowed the macrostructure and cross-genome homoeology of the Ae. markgrafii chromosomes to be established. Two chromosomes maintained colinearity, whereas the remaining were highly rearranged as a result of inversions and inter- and intrachromosomal translocations. We used sets of barley and wheat orthologous gene sequences to compare discrete parts of the Ae. markgrafii genome involved in the rearrangements. Analysis of sequence identity profiles and phylogenic relationships grouped chromosome blocks into two distinct clusters. Chromosome painting revealed the distribution of transposable elements and differentiated chromosome blocks into two groups consistent with the sequence analyses. These data suggest that introgressive hybridization accompanied by gross chromosome rearrangements might have had an impact on karyotype evolution and homoploid speciation in Ae. markgrafii.

Homoeologous recombination in the presence of Ph1 gene in wheat.

A crossover (CO) and its cytological signature, the chiasma, are major features of eukaryotic meiosis. The formation of at least one CO/chiasma between homologous chromosome pairs is essential for accurate chromosome segregation at the first meiotic division and genetic recombination. Polyploid organisms with multiple sets of homoeologous chromosomes have evolved additional mechanisms for the regulation of CO/chiasma. In hexaploid wheat (2n = 6× = 42), this is accomplished by pairing homoeologous (Ph) genes, with Ph1 having the strongest effect on suppressing homoeologous recombination and homoeologous COs. In this study, we observed homoeologous COs between chromosome 5Mg of Aegilops geniculata and 5D of wheat in plants where Ph1 was fully active, indicating that chromosome 5Mg harbors a homoeologous recombination promoter factor(s). Further cytogenetic analysis, with different 5Mg/5D recombinants, showed that the homoeologous recombination promoting factor(s) may be located in proximal regions of 5Mg. In addition, we observed a higher frequency of homoeologous COs in the pericentromeric region between chromosome combination of rec5Mg#2S·5Mg#2L and 5D compared to 5Mg#1/5D, which may be caused by a small terminal region of 5DL homology present in chromosome rec5Mg#2. The genetic stocks reported here will be useful for analyzing the mechanism of Ph1 action and the nature of homoeologous COs.

Chromosome Rearrangements Caused by Double Monosomy in Wheat-Barley Group-7 Substitution Lines.

Interspecific or introgressive hybridization is one of the driving forces in plant speciation, producing allopolyploids or diploids with rearranged genomes. The process of karyotype reshaping following homoploid interspecific hybridization has not been studied experimentally. Interspecific hybridization is widely used in plant breeding to increase genetic diversity and introgress new traits. Numerous introgression stocks were developed for hexaploid wheat Triticum aestivum L. (2n = 6x = 42, genome AABBDD). Double monosomic lines, containing one alien chromosome from the tertiary gene pool of wheat and one homoeologous wheat chromosome, represent a simplified model for studying chromosome rearrangements caused by interspecific hybridization. The pairing of a chromosome from the tertiary gene pool with a wheat homoeologue is restricted by the activity of the wheat Ph1 gene, thus, rearrangements caused by chromosome breakage followed by the fusion of the broken arms can be expected. We analyzed chromosome aberrations in 4 sets of lines that originated from double monosomics of barley (Hordeum vulgare L.) chromosome 7H and wheat group-7 chromosomes with dicentric or ring chromosomes. The dynamics of wheat-barley dicentric chromosomes during plant development was followed and an increased diversity of rearrangements was observed. Besides the targeted group-7 chromosomes, other wheat chromosomes were involved in rearrangements, as chromosomes broken in the centromeric region fused with other broken chromosomes. In some cells, multi-centric chromosomes were observed. The structure and dosage of the introgressed barley chromatin was changed. The transmission of the rearrangements to the progenies was analyzed. The observed aberrations emphasize the importance of cytogenetic screening in gene introgression projects.

Physical Mapping of Amplified Copies of the 5-Enolpyruvylshikimate-3-Phosphate Synthase Gene in Glyphosate-Resistant Amaranthus tuberculatus.

Recent and rapid evolution of resistance to glyphosate, the most widely used herbicides, in several weed species, including common waterhemp (Amaranthus tuberculatus), poses a serious threat to sustained crop production. We report that glyphosate resistance in A tuberculatus was due to amplification of the 5-enolpyruvylshikimate-3-P synthase (EPSPS) gene, which encodes the molecular target of glyphosate. There was a positive correlation between EPSPS gene copies and its transcript expression. We analyzed the distribution of EPSPS copies in the genome of A tuberculatus using fluorescence in situ hybridization on mitotic metaphase chromosomes and interphase nuclei. Fluorescence in situ hybridization analysis mapped the EPSPS gene to pericentromeric regions of two homologous chromosomes in glyphosate sensitive A tuberculatus In glyphosate-resistant plants, a cluster of EPSPS genes on the pericentromeric region on one pair of homologous chromosomes was detected. Intriguingly, two highly glyphosate-resistant plants harbored an additional chromosome with several EPSPS copies besides the native chromosome pair with EPSPS copies. These results suggest that the initial event of EPSPS gene duplication may have occurred because of unequal recombination mediated by repetitive DNA. Subsequently, gene amplification may have resulted via several other mechanisms, such as chromosomal rearrangements, deletion/insertion, transposon-mediated dispersion, or possibly by interspecific hybridization. This report illustrates the physical mapping of amplified EPSPS copies in A tuberculatus.

Development of a complete set of wheat-barley group-7 Robertsonian translocation chromosomes conferring an increased content of ß-glucan.

KEY MESSAGE: A complete set of six compensating Robertsonian translocation chromosomes involving barley chromosome 7H and three chromosomes of hexaploid wheat was produced. Grain ß-glucan content increased in lines containing 7HL. Many valuable genes for agronomic performance, disease resistance and increased yield have been transferred from relative species to wheat (Triticum aestivum L.) through whole-arm Robertsonian translocations (RobT). Although of a great value, the sets of available translocations from barley (Hordeum vulgare L.) are limited. Here, we present the production of a complete set of six compensating RobT chromosomes involving barley chromosome 7H and three group-7 chromosomes of wheat. The barley group-7 long-arm RobTs had a higher grain ß-glucan content compared to the wheat control. The ß-glucan levels varied depending on the temperature and were higher under hot conditions. Implicated in this increase, the barley cellulose synthase-like F6 gene (CslF6) responsible for ß-glucan synthesis was physically mapped near the centromere in the long arm of barley chromosome 7H. Likewise, wheat CslF6 homoeologs were mapped near the centromere in the long arms of all group-7 wheat chromosomes. With the set of novel wheat-barley translocations, we demonstrate a valuable increase of ß-glucan, along with a resource of genetic stocks that are likely to carry many other important genes from barley into wheat.

The Agropyron cristatum karyotype, chromosome structure and cross-genome homoeology as revealed by fluorescence in situ hybridization with tandem repeats and wheat single-gene probes.

KEY MESSAGE: Fluorescence in situ hybridization with probes for 45 cDNAs and five tandem repeats revealed homoeologous relationships of Agropyron cristatum with wheat. The results will contribute to alien gene introgression in wheat improvement. Crested wheatgrass (Agropyron cristatum L. Gaertn.) is a wild relative of wheat and a promising source of novel genes for wheat improvement. To date, identification of A. cristatum chromosomes has not been possible, and its molecular karyotype has not been available. Furthermore, homoeologous relationship between the genomes of A. cristatum and wheat has not been determined. To develop chromosome-specific landmarks, A. cristatum genomic DNA was sequenced, and new tandem repeats were discovered. Their distribution on mitotic chromosomes was studied by fluorescence in situ hybridization (FISH), which revealed specific patterns for five repeats in addition to 5S and 45S ribosomal DNA and rye subtelomeric repeats pSc119.2 and pSc200. FISH with one tandem repeat together with 45S rDNA enabled identification of all A. cristatum chromosomes. To analyze the structure and cross-species homoeology of A. cristatum chromosomes with wheat, probes for 45 mapped wheat cDNAs covering all seven chromosome groups were localized by FISH. Thirty-four cDNAs hybridized to homoeologous chromosomes of A. cristatum, nine hybridized to homoeologous and non-homoeologous chromosomes, and two hybridized to unique positions on non-homoeologous chromosomes. FISH using single-gene probes revealed that the wheat-A. cristatum collinearity was distorted, and important structural rearrangements were observed for chromosomes 2P, 4P, 5P, 6P and 7P. Chromosomal inversions were found for pericentric region of 4P and whole chromosome arm 6PL. Furthermore, reciprocal translocations between 2PS and 4PL were detected. These results provide new insights into the genome evolution within Triticeae and will facilitate the use of crested wheatgrass in alien gene introgression into wheat.

Reassessment of the evolution of wheat chromosomes 4A, 5A, and 7B.

KEY MESSAGE: Comparison of genome sequences of wild emmer wheat and Aegilops tauschii suggests a novel scenario of the evolution of rearranged wheat chromosomes 4A, 5A, and 7B. Past research suggested that wheat chromosome 4A was subjected to a reciprocal translocation T(4AL;5AL)1 that occurred in the diploid progenitor of the wheat A subgenome and to three major rearrangements that occurred in polyploid wheat: pericentric inversion Inv(4AS;4AL)1, paracentric inversion Inv(4AL;4AL)1, and reciprocal translocation T(4AL;7BS)1. Gene collinearity along the pseudomolecules of tetraploid wild emmer wheat (Triticum turgidum ssp. dicoccoides, subgenomes AABB) and diploid Aegilops tauschii (genomes DD) was employed to confirm these rearrangements and to analyze the breakpoints. The exchange of distal regions of chromosome arms 4AS and 4AL due to pericentric inversion Inv(4AS;4AL)1 was detected, and breakpoints were validated with an optical Bionano genome map. Both breakpoints contained satellite DNA. The breakpoints of reciprocal translocation T(4AL;7BS)1 were also found. However, the breakpoints that generated paracentric inversion Inv(4AL;4AL)1 appeared to be collocated with the 4AL breakpoints that had produced Inv(4AS;4AL)1 and T(4AL;7BS)1. Inv(4AS;4AL)1, Inv(4AL;4AL)1, and T(4AL;7BS)1 either originated sequentially, and Inv(4AL;4AL)1 was produced by recurrent chromosome breaks at the same breakpoints that generated Inv(4AS;4AL)1 and T(4AL;7BS)1, or Inv(4AS;4AL)1, Inv(4AL;4AL)1, and T(4AL;7BS)1 originated simultaneously. We prefer the latter hypothesis since it makes fewer assumptions about the sequence of events that produced these chromosome rearrangements.