Identification and correction of phase switches with Hi-C data in the Nanopore and HiFi chromosome-scale assemblies of the dikaryotic leaf rust fungus Puccinia triticina

AbstractBackgroundMost animals and plants have more than one set of chromosomes and package these haplotypes into a single nucleus within each cell. In contrast, many fungal species carry multiple haploid nuclei per cell. Rust fungi are such species with two nuclei (karyons) that contain a full set of haploid chromosomes each. The physical separation of haplotypes in dikaryons means that, unlike in diploids, Hi-C chromatin contacts between haplotypes are false positive signals.ResultsWe generate the first chromosome-scale, fully-phased assembly for the dikaryotic leaf rust fungus Puccinia triticina and compare Nanopore MinION and PacBio HiFi sequence-based assemblies. We show that false positive Hi-C contacts between haplotypes are predominantly caused by phase switches rather than by collapsed regions or Hi-C read mis-mappings. We introduce a method for phasing of dikaryotic genomes into the two haplotypes using Hi-C contact graphs, including a phase switch correction step. In the HiFi assembly, relatively few phase switches occur, and these are predominantly located at haplotig boundaries and can be readily corrected. In contrast, phase switches are widespread throughout the Nanopore assembly. We show that haploid genome read coverage of 30-40 times using HiFi sequencing is required for phasing of the leaf rust genome (~0.7% heterozygosity) and that HiFi sequencing resolves genomic regions with low heterozygosity that are otherwise collapsed in the Nanopore assembly.ConclusionsThis first Hi-C based phasing pipeline for dikaryons and comparison of long-read sequencing technologies will inform future genome assembly and haplotype phasing projects in other non-haploid organisms.

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Genetically Divergent Types of the Wheat Leaf Fungus Puccinia triticina in Ethiopia, a Center of Tetraploid Wheat Diversity

Phytopathology ◽

10.1094/phyto-10-15-0247-r ◽

2016 ◽

Vol 106 (4) ◽

pp. 380-385 ◽

Cited By ~ 10

Author(s):

J. A. Kolmer ◽

M. A. Acevedo

Keyword(s):

Leaf Rust ◽

Common Wheat ◽

Hexaploid Wheat ◽

Rust Fungus ◽

Tetraploid Wheat ◽

Low Frequency ◽

Puccinia Triticina ◽

Leaf Rust Resistance ◽

Virulence Phenotype ◽

Wheat Leaf

Collections of Puccinia triticina, the wheat leaf rust fungus, were obtained from tetraploid and hexaploid wheat in the central highlands of Ethiopia, and a smaller number from Kenya, from 2011 to 2013, in order to determine the genetic diversity of this wheat pathogen in a center of host diversity. Single-uredinial isolates were derived and tested for virulence phenotype to 20 lines of Thatcher wheat that differ for single leaf rust resistance genes and for molecular genotypes with 10 simple sequence repeat (SSR) primers. Nine virulence phenotypes were described among the 193 isolates tested for virulence. Phenotype BBBQJ, found only in Ethiopia, was predominantly collected from tetraploid wheat. Phenotype EEEEE, also found only in Ethiopia, was exclusively collected from tetraploid wheat and was avirulent to the susceptible hexaploid wheat ‘Thatcher’. Phenotypes MBDSS and MCDSS, found in both Ethiopia and Kenya, were predominantly collected from common wheat. Phenotypes CCMSS, CCPSS, and CBMSS were found in Ethiopia from common wheat at low frequency. Phenotypes TCBSS and TCBSQ were found on durum wheat and common wheat in Kenya. Four groups of distinct SSR genotypes were described among the 48 isolates genotyped. Isolates with phenotypes BBBQJ and EEEEE were in two distinct SSR groups, and isolates with phenotypes MBDSS and MCDSS were in a third group. Isolates with CCMSS, CCPSS, CBMSS, TCBSS, and TCBSQ phenotypes were in a fourth SSR genotype group. The diverse host environment of Ethiopia has selected and maintained a genetically divergent population of P. triticina.

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Virulence and Molecular Polymorphism in International Collections of the Wheat Leaf Rust Fungus Puccinia triticina

Phytopathology ◽

10.1094/phyto.2000.90.4.427 ◽

2000 ◽

Vol 90 (4) ◽

pp. 427-436 ◽

Cited By ~ 43

Author(s):

J. A. Kolmer ◽

J. Q. Liu

Keyword(s):

New Zealand ◽

Leaf Rust ◽

Resistance Genes ◽

Rust Fungus ◽

Puccinia Triticina ◽

Western Canada ◽

Wheat Leaf Rust ◽

Wheat Leaf ◽

Rapd Variation ◽

Wheat Lines

Collections of Puccinia triticina, the wheat leaf rust fungus, were obtained from Great Britain, Slovakia, Israel, Germany, Australia, Italy, Spain, Hungary, South Africa, Uruguay, New Zealand, Brazil, Pakistan, Nepal, and eastern and western Canada. All single-uredinial isolates derived from the collections were tested for virulence polymorphism on 22 Thatcher wheat lines that are near-isogenic for leaf rust resistance genes. Based on virulence phenotype, selected isolates were also tested for randomly amplified polymorphic DNA (RAPD) using 11 primers. The national collections were placed into 11 groups based on previously established epidemiological zones. Among the 131 single-uredinial isolates, 105 virulence phenotypes and 82 RAPD phenotypes were described. In a modified analysis of variance, 26% of the virulence variation was due to differences in isolates between groups, with the remainder attributable to differences within groups. Of the RAPD variation, 36% was due to differences in isolates between groups. Clustering based on the average virulence distance (simple distance coefficient) within and between groups resulted in eight groups that differed significantly. Collections from Australia-New Zealand, Spain, Italy, and Britain did not differ significantly for virulence. Clustering of RAPD marker differences (1 - Dice coefficient) distinguished nine groups that differed significantly. Collections from Spain and Italy did not differ significantly for RAPD variation, neither did collections from western Canada and South America. Groups of isolates distinguished by avirulent/virulent infection types to wheat lines with resistance genes Lr1, Lr2a, Lr2c, and Lr3 also differed significantly for RAPD distance, showing a general relationship between virulence and RAPD phenotype. The results indicated that on a worldwide level collections of P. triticina differ for virulence and molecular backgrounds.

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Fusion Body Formation, Germ Tube Anastomosis, and Nuclear Migration During the Germination of Urediniospores of the Wheat Leaf Rust Fungus, Puccinia triticina

Phytopathology ◽

10.1094/phyto-99-12-1355 ◽

2009 ◽

Vol 99 (12) ◽

pp. 1355-1364 ◽

Cited By ~ 24

Author(s):

Xiben Wang ◽

Brent McCallum

Keyword(s):

Leaf Rust ◽

Germ Tube ◽

Rust Fungus ◽

Puccinia Triticina ◽

Rust Fungi ◽

Wheat Leaf Rust ◽

Virulence Phenotype ◽

Close Proximity ◽

Wheat Leaf ◽

Germ Tubes

Vegetative or parasexual recombination is thought to be a key mechanism for the genetic diversity of cereal rust fungi. The process of germ tube fusion leading to hyphal anastomosis and nuclear recombination was analyzed in wheat leaf rust fungus, Puccinia triticina. Germ tube anastomosis was observed in 27 P. triticina isolates, each representing a different virulence phenotype. Germ tube fusion bodies (GFBs), which appeared as viscid globules formed at tips of germ tubes, were essential for germ tube anastomosis. The formation of GFBs was affected by the urediniospore density and the length of illumination during germination. GFBs were formed at the highest frequency when urediniospores were spread to a concentration of 1 × 106 urediniospores/ml and incubated in dark for 12 to 24 h during germination. GFB attached to either the side of another germ tube (“tip to side”) or to another GFB formed at the tip of a second germ tube (“tip to tip”). In “tip to side” anastomosis, two nuclei in the germ tube bearing the GFB migrated into the second germ tube through the GFB which resulted in four nuclei within this germ tube. In “tip to tip” anastomosis, nuclei in both germ tubes migrated into the fused GFB and all four nuclei came into close proximity. Urediniospores of isolates MBDS-3-115 and TBBJ-5-11 were stained with DAPI (4′,6′diamine-2-phenylindole) and Nuclear Yellow (Hoechst S769121), respectively, and then mixed and germinated on water agar. Some fused GFBs contained nuclei stained with DAPI and nuclei stained with Nuclear Yellow in close proximity, demonstrating the fusion between genetically different P. triticina isolates. In some fused GFBs, “bridge-like” structures connecting different nuclei were observed.

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Altered cell and RNA isoform diversity in aging Down syndrome brains

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2114326118 ◽

2021 ◽

Vol 118 (47) ◽

pp. e2114326118

Author(s):

Carter R. Palmer ◽

Christine S. Liu ◽

William J. Romanow ◽

Ming-Hsiang Lee ◽

Jerold Chun

Keyword(s):

Down Syndrome ◽

Large Scale ◽

Cell Types ◽

Chromosome 21 ◽

Specific Cell ◽

Sequencing Technologies ◽

Isoform Diversity ◽

Long Read ◽

Single Nucleus ◽

Altered Cell

Down syndrome (DS), trisomy of human chromosome 21 (HSA21), is characterized by lifelong cognitive impairments and the development of the neuropathological hallmarks of Alzheimer’s disease (AD). The cellular and molecular modifications responsible for these effects are not understood. Here we performed single-nucleus RNA sequencing (snRNA-seq) employing both short- (Illumina) and long-read (Pacific Biosciences) sequencing technologies on a total of 29 DS and non-DS control prefrontal cortex samples. In DS, the ratio of inhibitory-to-excitatory neurons was significantly increased, which was not observed in previous reports examining sporadic AD. DS microglial transcriptomes displayed AD-related aging and activation signatures in advance of AD neuropathology, with increased microglial expression of C1q complement genes (associated with dendritic pruning) and the HSA21 transcription factor gene RUNX1. Long-read sequencing detected vast RNA isoform diversity within and among specific cell types, including numerous sequences that differed between DS and control brains. Notably, over 8,000 genes produced RNAs containing intra-exonic junctions, including amyloid precursor protein (APP) that had previously been associated with somatic gene recombination. These and related results illuminate large-scale cellular and transcriptomic alterations as features of the aging DS brain.

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‘Sneezing’ plants: pathogen transport via jumping-droplet condensation

Journal of The Royal Society Interface ◽

10.1098/rsif.2019.0243 ◽

2019 ◽

Vol 16 (155) ◽

pp. 20190243 ◽

Cited By ~ 4

Author(s):

Saurabh Nath ◽

S. Farzad Ahmadi ◽

Hope A. Gruszewski ◽

Stuti Budhiraja ◽

Caitlin E. Bisbano ◽

...

Keyword(s):

Boundary Layer ◽

Surface Energy ◽

Leaf Rust ◽

Laminar Boundary Layer ◽

Rust Fungus ◽

Puccinia Triticina ◽

The Self ◽

Pathogen Transport ◽

Wheat Leaves ◽

Droplet Condensation

We show that condensation growing on wheat leaves infected with the leaf rust fungus, Puccinia triticina , is capable of spontaneously launching urediniospores off the plant. This surprising liberation mechanism is enabled by the superhydrophobicity of wheat leaves, which promotes a jumping-droplet mode of condensation powered by the surface energy released from coalescence events. We found that urediniospores often adhere to the self-propelled condensate, resulting in liberation rates of approximately 10 cm −2 h −1 for leaves infected with rust. Urediniospores were catapulted up to 5 mm from the leaf’s surface, a distance sufficient to clear the laminar boundary layer for subsequent dispersal even in gentle winds.

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Rust Control in Glyphosate Tolerant Wheat Following Application of the Herbicide Glyphosate

Plant Disease ◽

10.1094/pd-89-1136 ◽

2005 ◽

Vol 89 (11) ◽

pp. 1136-1142 ◽

Cited By ~ 51

Author(s):

J. A. Anderson ◽

J. A. Kolmer

Keyword(s):

Leaf Rust ◽

Rust Fungus ◽

Puccinia Triticina ◽

Surfactant Solution ◽

Field Trials ◽

Systemic Effect ◽

Soybean Rust ◽

Application Rates ◽

Infection Types ◽

Herbicide Glyphosate

In greenhouse and field trials, transgenically modified wheat (Triticum aestivum) genotypes with tolerance to glyphosate had extremely low infection types to leaf rust caused by Puccinia triticina when treated with a labeled rate of the herbicide glyphosate prior to inoculation with leaf rust. A surfactant solution and a nonglyphosate herbicide had no effect on leaf rust development on the glyphosate tolerant wheat. Glyphosate had a systemic effect in reducing leaf rust development. The leaf rust control by glyphosate decreased with reduced application rates and longer periods of time between glyphosate application and leaf rust infections. The field and greenhouse tests indicated that control of leaf rust in wheat conditioned by glyphosate is transitory and is effective for at least 21, but not more than 35, days after application. Application of glyphosate also reduced infection types on wheat caused by the stem rust fungus, Puccinia graminis f. sp. tritici. Given these results and evidence from the literature that glyphosate can have adverse effects on other pathogens, including other rust fungi, additional investigation of the fungicidal properties of glyphosate are warranted, with particular attention to the timing of glyphosate application relative to fungal infection. The effects of glyphosate on the soybean rust fungus, Phakopsora pachyrhizi, an emerging pathogen in North America, merit immediate investigation.

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Virulence and molecular polymorphisms of the wheat leaf rust fungus Puccinia triticina in Canada from 1997 to 2007

Botany ◽

10.1139/b10-034 ◽

2010 ◽

Vol 88 (6) ◽

pp. 575-589 ◽

Cited By ~ 16

Author(s):

Xiben Wang ◽

Guus Bakkeren ◽

Brent McCallum

Keyword(s):

Leaf Rust ◽

Expressed Sequence Tag ◽

Rust Fungus ◽

Puccinia Triticina ◽

Leaf Rust Resistance ◽

Significant Shift ◽

Wheat Leaf Rust ◽

Rust Disease ◽

The Pacific ◽

Wheat Leaf

Populations of Puccinia triticina , one of the casual agents of wheat leaf rust disease, in the pacific (British Columbia and Alberta), prairie (Manitoba and Saskatchewan), and eastern regions (Quebec and Ontario) of Canada from 1997 to 2007 were analyzed for virulence and genetic diversity by revealing expressed sequence tag derived simple sequence repeat (EST-SSR) polymorphisms. Since 1997, a significant shift in the virulence of P. triticina occurred across Canada. The diversity of P. triticina virulence phenotypes in Manitoba and Saskatchewan, as measured by Shannon and Simpson indexes, decreased due to the directional selection toward predominant virulence phenotypes, whereas it remained relatively constant in Quebec and Ontario. The clustering of P. triticina virulence phenotypes from 1997 to 2007 was similar to that found in previous years, and was correlated with virulence to leaf rust resistance genes Lr2a, Lr2c, and Lr17a. Distinct EST-SSR profiles were found in different groups of P. triticina virulence phenotypes based on virulence to Lr2a, Lr2c, and Lr17a. In addition, the population of P. triticina in Manitoba and Saskatchewan was different from that in Quebec and Ontario from 1997 to 2007, based on both virulence characteristics and EST-SSR genotypes.

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Virulence phenotypes of the wheat leaf rust pathogen, Puccinia triticina, in the United States 2018-2020

Plant Disease ◽

10.1094/pdis-10-21-2321-re ◽

2022 ◽

Author(s):

James Kolmer ◽

Oluseyi Fajolu

Keyword(s):

United States ◽

Winter Wheat ◽

Leaf Rust ◽

Great Plains ◽

Rust Fungus ◽

Puccinia Triticina ◽

The United States ◽

Northern Great Plains ◽

Ohio Valley ◽

Red Winter Wheat

Collections of wheat leaves infected with the leaf rust fungus, Puccinia triticina, were obtained from the southeastern states, the Ohio Valley, the Great Plains, and Washington in 2018, 2019 and 2020 to determine the prevalent virulence phenotypes in the wheat growing regions of the United States. In the hard red winter wheat region of the southern and mid Great Plains, MNPSD, and MPPSD were the two most common phenotypes in 2018 and 2019. In 2020 BBBQD with high virulence to durum wheat was the most common phenotype in the southern Great Plains. In the hard red spring wheat region of the northern Great Plains, MNPSD, MPPSD, MBDSD, and TBBGS were the predominant phenotypes. In the soft red winter wheat region of the southeastern states and Ohio Valley region, MBTNB, MCTNB, and MNPSD were the three most common phenotypes. Collections in Washington had phenotypes LBDSG, LCDSG, LCDJG, and MBDSB that were not found in any other region. Isolates with virulence to Lr11 were most frequent in the southeastern states, and Ohio Valley regions. The frequency of isolates with virulence to Lr39 was highest in the Great Plains region and frequency of isolates with virulence to Lr21 was highest in the northern Great Plains region. Selection of virulence phenotypes by leaf rust resistance genes in the different market classes of wheat, combined with the effects of clonal reproduction, overwintering in southern regions, and low migration between the Great Plains region and eastern wheat producing regions, has maintained the different P. triticina populations in the United States.

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