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September 22, 2019

Phenotypic diversification by enhanced genome restructuring after induction of multiple DNA double-strand breaks.

DNA double-strand break (DSB)-mediated genome rearrangements are assumed to provide diverse raw genetic materials enabling accelerated adaptive evolution; however, it remains unclear about the consequences of massive simultaneous DSB formation in cells and their resulting phenotypic impact. Here, we establish an artificial genome-restructuring technology by conditionally introducing multiple genomic DSBs in vivo using a temperature-dependent endonuclease TaqI. Application in yeast and Arabidopsis thaliana generates strains with phenotypes, including improved ethanol production from xylose at higher temperature and increased plant biomass, that are stably inherited to offspring after multiple passages. High-throughput genome resequencing revealed that these strains harbor diverse rearrangements, including copy number variations, translocations in retrotransposons, and direct end-joinings at TaqI-cleavage sites. Furthermore, large-scale rearrangements occur frequently in diploid yeasts (28.1%) and tetraploid plants (46.3%), whereas haploid yeasts and diploid plants undergo minimal rearrangement. This genome-restructuring system (TAQing system) will enable rapid genome breeding and aid genome-evolution studies.

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September 22, 2019

Integrated proteomics, genomics, metabolomics approaches reveal oxalic acid as pathogenicity factor in Tilletia indica inciting Karnal bunt disease of wheat.

Tilletia indica incites Karnal bunt (KB) disease in wheat. To date, no KB resistant wheat cultivar could be developed due to non-availability of potential biomarkers related to pathogenicity/virulence for screening of resistant wheat genotypes. The present study was carried out to compare the proteomes of T. indica highly (TiK) and low (TiP) virulent isolates. Twenty one protein spots consistently observed as up-regulated/differential in the TiK proteome were selected for identification by MALDI-TOF/TOF. Identified sequences showed homology with fungal proteins playing essential role in plant infection and pathogen survival, including stress response, adhesion, fungal penetration, invasion, colonization, degradation of host cell wall, signal transduction pathway. These results were integrated with T. indica genome sequence for identification of homologs of candidate pathogenicity/virulence related proteins. Protein identified in TiK isolate as malate dehydrogenase that converts malate to oxaloacetate which is precursor of oxalic acid. Oxalic acid is key pathogenicity factor in phytopathogenic fungi. These results were validated by GC-MS based metabolic profiling of T. indica isolates indicating that oxalic acid was exclusively identified in TiK isolate. Thus, integrated omics approaches leads to identification of pathogenicity/virulence factor(s) that would provide insights into pathogenic mechanisms of fungi and aid in devising effective disease management strategies.

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September 22, 2019

The evolution of genomic and epigenomic features in two Pleurotus fungi.

Pleurotus tuoliensis (Bailinggu, designated Pt) and P. eryngii var. eryngii (Xingbaogu, designated Pe) are highly valued edible mushrooms. We report de novo assemblies of high-quality genomes for both mushrooms based on PacBio RS II sequencing and annotation of all identified genes. A comparative genomics analysis between Pt and Pe with P. ostreatus as an outgroup taxon revealed extensive genomic divergence between the two mushroom genomes primarily due to the rapid gain of taxon-specific genes and disruption of synteny in either taxon. The re-appraised phylogenetic relationship between Pt and Pe at the genome-wide level validates earlier proposals to designate Pt as an independent species. Variation of the identified wood-decay-related gene content can largely explain the variable adaptation and host specificity of the two mushrooms. On the basis of the two assembled genome sequences, methylomes and the regulatory roles of DNA methylation in gene expression were characterized and compared. The genome, methylome and transcriptome data of these two important mushrooms will provide valuable information for advancing our understanding of the evolution of Pleurotus and related genera and for facilitating genome- and epigenome-based strategies for mushroom breeding.

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September 22, 2019

Genomic analyses of unique carbohydrate and phytohormone metabolism in the macroalga Gracilariopsis lemaneiformis (Rhodophyta).

Red algae are economically valuable for food and in industry. However, their genomic information is limited, and the genomic data of only a few species of red algae have been sequenced and deposited recently. In this study, we annotated a draft genome of the macroalga Gracilariopsis lemaneiformis (Gracilariales, Rhodophyta).The entire 88.98 Mb genome of Gp. lemaneiformis 981 was generated from 13,825 scaffolds (=500 bp) with an N50 length of 30,590 bp, accounting for approximately 91% of this algal genome. A total of 38.73 Mb of scaffold sequences were repetitive, and 9281 protein-coding genes were predicted. A phylogenomic analysis of 20 genomes revealed the relationship among the Chromalveolata, Rhodophyta, Chlorophyta and higher plants. Homology analysis indicated phylogenetic proximity between Gp. lemaneiformis and Chondrus crispus. The number of enzymes related to the metabolism of carbohydrates, including agar, glycoside hydrolases, glycosyltransferases, was abundant. In addition, signaling pathways associated with phytohormones such as auxin, salicylic acid and jasmonates are reported for the first time for this alga.We sequenced and analyzed a draft genome of the red alga Gp. lemaneiformis, and revealed its carbohydrate metabolism and phytohormone signaling characteristics. This work will be helpful in research on the functional and comparative genomics of the order Gracilariales and will enrich the genomic information on marine algae.

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September 22, 2019

Genome-wide analysis of the NAC transcription factor family and their expression during the development and ripening of the Fragaria × ananassa fruits.

NAC proteins are a family of transcription factors which have a variety of important regulatory roles in plants. They present a very well conserved group of NAC subdomains in the N-terminal region and a highly variable domain at the C-terminus. Currently, knowledge concerning NAC family in the strawberry plant remains very limited. In this work, we analyzed the NAC family of Fragaria vesca, and a total of 112 NAC proteins were identified after we curated the annotations from the version 4.0.a1 genome. They were placed into the ligation groups (pseudo-chromosomes) and described its physicochemical and genetic features. A microarray transcriptomic analysis showed six of them expressed during the development and ripening of the Fragaria x ananassa fruit. Their expression patterns were studied in fruit (receptacle and achenes) in different stages of development and in vegetative tissues. Also, the expression level under different hormonal treatments (auxins, ABA) and drought stress was investigated. In addition, they were clustered with other NAC transcription factor with known function related to growth and development, senescence, fruit ripening, stress response, and secondary cell wall and vascular development. Our results indicate that these six strawberry NAC proteins could play different important regulatory roles in the process of development and ripening of the fruit, providing the basis for further functional studies and the selection for NAC candidates suitable for biotechnological applications.

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September 22, 2019

Inpactor, integrated and parallel analyzer and classifier of LTR retrotransposons and its application for pineapple LTR retrotransposons diversity and dynamics.

One particular class of Transposable Elements (TEs), called Long Terminal Repeats (LTRs), retrotransposons, comprises the most abundant mobile elements in plant genomes. Their copy number can vary from several hundreds to up to a few million copies per genome, deeply affecting genome organization and function. The detailed classification of LTR retrotransposons is an essential step to precisely understand their effect at the genome level, but remains challenging in large-sized genomes, requiring the use of optimized bioinformatics tools that can take advantage of supercomputers. Here, we propose a new tool: Inpactor, a parallel and scalable pipeline designed to classify LTR retrotransposons, to identify autonomous and non-autonomous elements, to perform RT-based phylogenetic trees and to analyze their insertion times using High Performance Computing (HPC) techniques. Inpactor was tested on the classification and annotation of LTR retrotransposons in pineapple, a recently-sequenced genome. The pineapple genome assembly comprises 44% of transposable elements, of which 23% were classified as LTR retrotransposons. Exceptionally, 16.4% of the pineapple genome assembly corresponded to only one lineage of the Gypsy superfamily: Del, suggesting that this particular lineage has undergone a significant increase in its copy numbers. As demonstrated for the pineapple genome, Inpactor provides comprehensive data of LTR retrotransposons’ classification and dynamics, allowing a fine understanding of their contribution to genome structure and evolution. Inpactor is available at https://github.com/simonorozcoarias/Inpactor.

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September 22, 2019

A transposable element annotation pipeline and expression analysis reveal potentially active elements in the microalga Tisochrysis lutea.

Transposable elements (TEs) are mobile DNA sequences known as drivers of genome evolution. Their impacts have been widely studied in animals, plants and insects, but little is known about them in microalgae. In a previous study, we compared the genetic polymorphisms between strains of the haptophyte microalga Tisochrysis lutea and suggested the involvement of active autonomous TEs in their genome evolution.To identify potentially autonomous TEs, we designed a pipeline named PiRATE (Pipeline to Retrieve and Annotate Transposable Elements, download: https://doi.org/10.17882/51795 ), and conducted an accurate TE annotation on a new genome assembly of T. lutea. PiRATE is composed of detection, classification and annotation steps. Its detection step combines multiple, existing analysis packages representing all major approaches for TE detection and its classification step was optimized for microalgal genomes. The efficiency of the detection and classification steps was evaluated with data on the model species Arabidopsis thaliana. PiRATE detected 81% of the TE families of A. thaliana and correctly classified 75% of them. We applied PiRATE to T. lutea genomic data and established that its genome contains 15.89% Class I and 4.95% Class II TEs. In these, 3.79 and 17.05% correspond to potentially autonomous and non-autonomous TEs, respectively. Annotation data was combined with transcriptomic and proteomic data to identify potentially active autonomous TEs. We identified 17 expressed TE families and, among these, a TIR/Mariner and a TIR/hAT family were able to synthesize their transposase. Both these TE families were among the three highest expressed genes in a previous transcriptomic study and are composed of highly similar copies throughout the genome of T. lutea. This sum of evidence reveals that both these TE families could be capable of transposing or triggering the transposition of potential related MITE elements.This manuscript provides an example of a de novo transposable element annotation of a non-model organism characterized by a fragmented genome assembly and belonging to a poorly studied phylum at genomic level. Integration of multi-omics data enabled the discovery of potential mobile TEs and opens the way for new discoveries on the role of these repeated elements in genomic evolution of microalgae.

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September 22, 2019

Nucleotide-binding resistance gene signatures in sugar beet, insights from a new reference genome.

Nucleotide-binding (NB-ARC), leucine-rich-repeat genes (NLRs) account for 60.8% of resistance (R) genes molecularly characterized from plants. NLRs exist as large gene families prone to tandem duplication and transposition, with high sequence diversity among crops and their wild relatives. This diversity can be a source of new disease resistance, but difficulty in distinguishing specific sequences from homologous gene family members hinders characterization of resistance for improving crop varieties. Current genome sequencing and assembly technologies, especially those using long-read sequencing, are improving resolution of repeat-rich genomic regions and clarifying locations of duplicated genes, such as NLRs. Using the conserved NB-ARC domain as a model, 231 tentative NB-ARC loci were identified in a highly contiguous genome assembly of sugar beet, revealing diverged and truncated NB-ARC signatures as well as full-length sequences. The NB-ARC-associated proteins contained NLR resistance gene domains, including TIR, CC, and LRR, as well as other integrated domains. Phylogenetic relationships of partial and complete domains were determined, and patterns of physical clustering in the genome were evaluated. Comparison of sugar beet NB-ARC domains to validated R genes from monocots and eudicots suggested extensive B. vulgaris-specific subfamily expansions. The NLR landscape in the rhizomania resistance conferring Rz region of Chromosome 3 was characterized, identifying 26 NLR-like sequences spanning 20 MB. This work presents the first detailed view of NLR family composition in a member of the Caryophyllales, builds a foundation for additional disease resistance work in B. vulgaris, and demonstrates an additional nucleic-acid-based method for NLR prediction in non-model plant species. This article is protected by copyright. All rights reserved.This article is protected by copyright. All rights reserved.

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September 22, 2019

Size and content of the sex-determining region of the Y chromosome in dioecious Mercurialis annua, a plant with homomorphic sex chromosomes.

Dioecious plants vary in whether their sex chromosomes are heteromorphic or homomorphic, but even homomorphic sex chromosomes may show divergence between homologues in the non-recombining, sex-determining region (SDR). Very little is known about the SDR of these species, which might represent particularly early stages of sex-chromosome evolution. Here, we assess the size and content of the SDR of the diploid dioecious herb Mercurialis annua, a species with homomorphic sex chromosomes and mild Y-chromosome degeneration. We used RNA sequencing (RNAseq) to identify new Y-linked markers for M. annua. Twelve of 24 transcripts showing male-specific expression in a previous experiment could be amplified by polymerase chain reaction (PCR) only from males, and are thus likely to be Y-linked. Analysis of genome-capture data from multiple populations of M. annua pointed to an additional six male-limited (and thus Y-linked) sequences. We used these markers to identify and sequence 17 sex-linked bacterial artificial chromosomes (BACs), which form 11 groups of non-overlapping sequences, covering a total sequence length of about 1.5 Mb. Content analysis of this region suggests that it is enriched for repeats, has low gene density, and contains few candidate sex-determining genes. The BACs map to a subset of the sex-linked region of the genetic map, which we estimate to be at least 14.5 Mb. This is substantially larger than estimates for other dioecious plants with homomorphic sex chromosomes, both in absolute terms and relative to their genome sizes. Our data provide a rare, high-resolution view of the homomorphic Y chromosome of a dioecious plant.

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September 22, 2019

Signatures of host specialization and a recent transposable element burst in the dynamic one-speed genome of the fungal barley powdery mildew pathogen.

Powdery mildews are biotrophic pathogenic fungi infecting a number of economically important plants. The grass powdery mildew, Blumeria graminis, has become a model organism to study host specialization of obligate biotrophic fungal pathogens. We resolved the large-scale genomic architecture of B. graminis forma specialis hordei (Bgh) to explore the potential influence of its genome organization on the co-evolutionary process with its host plant, barley (Hordeum vulgare).The near-chromosome level assemblies of the Bgh reference isolate DH14 and one of the most diversified isolates, RACE1, enabled a comparative analysis of these haploid genomes, which are highly enriched with transposable elements (TEs). We found largely retained genome synteny and gene repertoires, yet detected copy number variation (CNV) of secretion signal peptide-containing protein-coding genes (SPs) and locally disrupted synteny blocks. Genes coding for sequence-related SPs are often locally clustered, but neither the SPs nor the TEs reside preferentially in genomic regions with unique features. Extended comparative analysis with different host-specific B. graminis formae speciales revealed the existence of a core suite of SPs, but also isolate-specific SP sets as well as congruence of SP CNV and phylogenetic relationship. We further detected evidence for a recent, lineage-specific expansion of TEs in the Bgh genome.The characteristics of the Bgh genome (largely retained synteny, CNV of SP genes, recently proliferated TEs and a lack of significant compartmentalization) are consistent with a “one-speed” genome that differs in its architecture and (co-)evolutionary pattern from the “two-speed” genomes reported for several other filamentous phytopathogens.

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September 22, 2019

Identification of a leucine-rich repeat receptor-like serine/threonine-protein kinase as a candidate gene for Rvi12 (Vb)-based apple scab resistance

Apple scab caused by Venturia inaequalis is the most important fungal disease of apples (Malus × domestica). Currently, the disease is controlled by up to 15 fungicide applications to the crop per year. Resistant apple cultivars will help promote the sustainable control of scab in commercial orchards. The breakdown of the Rvi6 (Vf) major-gene based resistance, the most used resistance gene in apple breeding, prompted the identification and characterization of new scab resistance genes. By using a large segregating population, the Rvi12 scab resistance gene was previously mapped to a genetic location flanked by molecular markers SNP_23.599 and SNP_24.482. Starting from these markers, utilizing chromosome walking of a Hansen’s baccata #2 (HB2) BAC-library; a single BAC clone spanning the Rvi12 interval was identified. Following Pacific Biosciences (PacBio) RS II sequencing and the use of the hierarchical genome assembly process (HGAP) assembly of the BAC clone sequence, the Rvi12 resistance locus was localized to a 62.3-kb genomic region. Gene prediction and in silico characterization identified a single candidate resistance gene. The gene, named here as Rvi12_Cd5, belongs to the LRR receptor-like serine/threonine-protein kinase family. In silico comparison of the resistance allele from HB2 and the susceptible allele from Golden Delicious (GD) identified the presence of an additional intron in the HB2 allele. Conserved domain analysis identified the presence of four additional LRR motifs in the susceptible allele compared to the resistance allele. The constitutive expression of Rvi12_Cd5 in HB2, together with its structural similarity to known resistance genes, makes it the most likely candidate for Rvi12 scab resistance in apple.

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September 22, 2019

The complete chloroplast genome sequence of Actinidia arguta using the PacBio RS II platform.

Actinidia arguta is the most basal species in a phylogenetically and economically important genus in the family Actinidiaceae. To better understand the molecular basis of the Actinidia arguta chloroplast (cp), we sequenced the complete cp genome from A. arguta using Illumina and PacBio RS II sequencing technologies. The cp genome from A. arguta was 157,611 bp in length and composed of a pair of 24,232 bp inverted repeats (IRs) separated by a 20,463 bp small single copy region (SSC) and an 88,684 bp large single copy region (LSC). Overall, the cp genome contained 113 unique genes. The cp genomes from A. arguta and three other Actinidia species from GenBank were subjected to a comparative analysis. Indel mutation events and high frequencies of base substitution were identified, and the accD and ycf2 genes showed a high degree of variation within Actinidia. Forty-seven simple sequence repeats (SSRs) and 155 repetitive structures were identified, further demonstrating the rapid evolution in Actinidia. The cp genome analysis and the identification of variable loci provide vital information for understanding the evolution and function of the chloroplast and for characterizing Actinidia population genetics.

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September 22, 2019

Amplification and adaptation of centromeric repeats in polyploid switchgrass species.

Centromeres in most higher eukaryotes are composed of long arrays of satellite repeats from a single satellite repeat family. Why centromeres are dominated by a single satellite repeat and how the satellite repeats originate and evolve are among the most intriguing and long-standing questions in centromere biology. We identified eight satellite repeats in the centromeres of tetraploid switchgrass (Panicum virgatum). Seven repeats showed characteristics associated with classical centromeric repeats with monomeric lengths ranging from 166 to 187 bp. Interestingly, these repeats share an 80-bp DNA motif. We demonstrate that this 80-bp motif may dictate translational and rotational phasing of the centromeric repeats with the cenH3 nucleosomes. The sequence of the last centromeric repeat, Pv156, is identical to the 5S ribosomal RNA genes. We demonstrate that a 5S ribosomal RNA gene array was recruited to be the functional centromere for one of the switchgrass chromosomes. Our findings reveal that certain types of satellite repeats, which are associated with unique sequence features and are composed of monomers in mono-nucleosomal length, are favorable for centromeres. Centromeric repeats may undergo dynamic amplification and adaptation before the centromeres in the same species become dominated by the best adapted satellite repeat.© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.

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September 22, 2019

The genome of Artemisia annua provides insight into the evolution of Asteraceae family and artemisinin biosynthesis.

Artemisia annua, commonly known as sweet wormwood or Qinghao, is a shrub native to China and has long been used for medicinal purposes. A. annua is now cultivated globally as the only natural source of a potent anti-malarial compound, artemisinin. Here, we report a high-quality draft assembly of the 1.74-gigabase genome of A. annua, which is highly heterozygous, rich in repetitive sequences, and contains 63 226 protein-coding genes, one of the largest numbers among the sequenced plant species. We found that, as one of a few sequenced genomes in the Asteraceae, the A. annua genome contains a large number of genes specific to this large angiosperm clade. Notably, the expansion and functional diversification of genes encoding enzymes involved in terpene biosynthesis are consistent with the evolution of the artemisinin biosynthetic pathway. We further revealed by transcriptome profiling that A. annua has evolved the sophisticated transcriptional regulatory networks underlying artemisinin biosynthesis. Based on comprehensive genomic and transcriptomic analyses we generated transgenic A. annua lines producing high levels of artemisinin, which are now ready for large-scale production and thereby will help meet the challenge of increasing global demand of artemisinin. Copyright © 2018 The Author. Published by Elsevier Inc. All rights reserved.

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September 22, 2019

Diversity and evolution of the emerging Pandoraviridae family.

With DNA genomes reaching 2.5?Mb packed in particles of bacterium-like shape and dimension, the first two Acanthamoeba-infecting pandoraviruses remained up to now the most complex viruses since their discovery in 2013. Our isolation of three new strains from distant locations and environments is now used to perform the first comparative genomics analysis of the emerging worldwide-distributed Pandoraviridae family. Thorough annotation of the genomes combining transcriptomic, proteomic, and bioinformatic analyses reveals many non-coding transcripts and significantly reduces the former set of predicted protein-coding genes. Here we show that the pandoraviruses exhibit an open pan-genome, the enormous size of which is not adequately explained by gene duplications or horizontal transfers. As most of the strain-specific genes have no extant homolog and exhibit statistical features comparable to intergenic regions, we suggest that de novo gene creation could contribute to the evolution of the giant pandoravirus genomes.

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