Biology subjects

Shirsekar, G.

Publications and source records attributed to Shirsekar, G..

11 recordsLinked to original sources

bioRxiv2026

Efficient and Robust Genomic DNA Isolation and Next-Generation Sequencing Library Preparation from Recalcitrant Wild Grape Species

This protocol details the extraction of high-molecular-weight genomic DNA from grapevine tissues (wild and cultivated Vitis spp., including pathogen-infected samples) and the subsequent preparation of Illumina(R) whole-genome sequencing libraries using bead-bound Tn5 transposase. It is designed to overcome challenges from polyphenolic compounds and secondary metabolites in wild plants, providing a cost-effective workflow for large-scale population genomics. It includes recipes for buffers, incubation times, critical notes, and troubleshooting tips to maximize yield and library quality. Although designed for the grapevine DNA, this protocol is potentially applicable to other similar wild plant species HighlightsO_LIOptimized CTAB-PTB DNA extraction protocol for field-collected wild plant tissues. C_LIO_LIEffective removal of polyphenols and secondary metabolites associated with DNA using PTB. C_LIO_LICost-effective Illumina DNA Prep library preparation using bead-bound Tn5 transposase (Tagmentation). C_LIO_LIScalable workflow suitable for large-scale population genomics in Vitis species. C_LIO_LIValidated method for high-molecular-weight DNA and high-quality sequencing data. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=195 SRC="FIGDIR/small/713680v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@b637d4org.highwire.dtl.DTLVardef@10b563aorg.highwire.dtl.DTLVardef@14a32caorg.highwire.dtl.DTLVardef@4c9577_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2024

The 1001G+ project: A curated collection of Arabidopsis thaliana long-read genome assemblies to advance plant research

Arabidopsis thaliana was the first plant for which a high-quality genome sequence became available. The publication of the first reference genome sequence almost 25 years ago was already accompanied by genome-wide data on sequence polymorphisms in another accession, or naturally occurring strain. Since then, inventories of genome-wide diversity have been generated at increasingly precise levels. High-density genotype data for A. thaliana, including those from the 1001 Genomes Project, were key to demonstrating the enormous power of GWAS in inbred populations of wild plants, and the comparison of intraspecific polymorphism with interspecific divergence has illuminated many aspects of plant genome evolution. Over the past decade, an increasing number of nearly complete genome sequences have been published for many more accessions. Here, we highlight the diversity of a curated collection of previously published and so far unpublished genome sequences assembled using different types of long reads, including PacBio Continuous Long Reads (CLR), PacBio High Fidelity (HiFi) reads, and Oxford Nanopore Technologies (ONT) reads. This 1001 Genomes Plus (1001G+) resource is being made available at http://1001genomes.org. We invite colleagues with yet unpublished genome assemblies from A. thaliana accessions to contribute to this effort.

genomics↗

bioRxiv2024

Pangenomic context reveals the extent of intraspecific plant NLR evolution

Nucleotide-binding leucine-rich repeat (NLR) proteins are a major component of the plant immune system, which directly or indirectly detect molecular signals of pathogen invasion. Despite their critical role, the processes by which NLR genes diversify remain poorly characterised due to the extraordinary sequence, structural, and regulatory variability of NLRs, even among closely related individuals. To understand the evolution of NLR diversity in Arabidopsis thaliana, we leverage graph-based methods to define pangenomic NLR neighbourhoods in 17 genetically diverse genomes. We integrate full-length transcript and transposable element information to exhaustively annotate all intact and degraded NLRs, enabling exploration of the processes that underpin the birth, death and maintenance of NLR diversity within a species. Our main finding is that many uncorrelated mutational processes create NLR diversity, and that there is no single metric that captures on its own the true extent of NLR structural and sequence variation. This immense diversity in plant immune system diversification allows populations to survive the constant onslaught of pathogens, not unlike vertebrate adaptive immunity, where variation is also generated by a variety of complementary mechanisms, albeit at the level of individuals.

plant biology↗

bioRxiv2023

Atlas of telomeric repeat diversity in Arabidopsis thaliana

Telomeric repeat arrays at the ends of chromosomes are highly dynamic but their repetitive nature and technological limitations have made it difficult to assess the variation in genome diversity surveys. Here we present a comprehensive characterization of the sequence variation immediately adjacent to the canonical telomeric repeat arrays at the very ends of chromosomes in 49 genetically diverse Arabidopsis thaliana accessions. We reveal several types of distinct telomeric repeat units and identify evolutionary processes such as local homogenization and higher-order repeat formation that shape diversity of chromosome ends. The identification of segmental duplications and at least one recombination event suggests a plausible history of telomerase-independent maintenance generation. By comparing largely isogenic samples, we are able to determine variant telomeric repeat number variation at both the germline and somatic levels. Analysis of haplotype structure uncovers chromosome end-specific as well as genetic group-specific patterns in telomeric repeat diversity and provides evidence for linkage disequilibrium between repeat arrays and their adjacent non-coding regions. Together, our findings illustrate the fine-scale telomeric repeat spectrum in A. thaliana, expanding our knowledge of the evolution of chromosome ends.

genomics↗

bioRxiv2023

Transposon-triggered epigenetic chromatin dynamics modulate EFR-related pathogen response

Infectious diseases drive the evolution of wild plants and impact yield in crop plants. Like animals, plants can sense biotic threats via conserved pathogen-associated patterns (PAMPs). Since an overly robust immune response can harm plants, understanding the mechanisms for tuning defense responses to the appropriate level is vital as we endeavor to develop pathogen-resistant crops. In this paper, we studied the Arabidopsis pattern recognition receptor (PRR) EFR, which senses bacterial EF-Tu. An inverted-repeat transposon (Ea-IR) between EFR and the neighboring XI-k locus controls local chromatin organization, promoting the formation of a repressive chromatin loop. Upon pathogen infection, the chromatin landscape around EFR and Xl-k dynamically changes to allow for increased EFR transcription. Chromatin opening facilitates the passage of RNA polymerase II across the neighboring XI-k gene termination site, leading to a longer XI-k transcript that includes Ea-IR sequences. Dicer-like (DCL) enzymes process the longer Xl-k transcript into small RNAs (sRNAs), which reset chromatin topology to a repressive state, attenuating, in turn, the immune response, reminiscent of attenuation of receptor signaling in other systems. From an evolutionary point of view, we found that natural Arabidopsis accessions missing Ea-IR have a constitutive "EFR-open" chromatin configuration that correlates with higher basal EFR levels and higher background resistance to pathogens. Collectively, our study offers evidence for a scenario in which a transposon, chromatin organization, and gene expression interact to fine-tune immune responses, both during the course of infection and in the course of evolution. Similar gene-associated IRs in crops could provide valuable non-coding targets for genome editing or assisted plant breeding programs.

plant biology↗

bioRxiv2022

Drought selection on Arabidopsis populations and their microbiomes

Microbes affect plant health, stress tolerance1 and life history2. In different regions of the globe, plants are colonized by distinct pathogenic and commensal microbiomes, but the factors driving their geographic variation are largely unknown3. We identified and measured the core leaf microbiome of Arabidopsis thaliana in its native range, from almost 300 populations across Europe. Comparing the distribution of the approximately 500 major bacterial phylotypes, we discovered marked, geography-dependent differences in microbiome composition within A. thaliana and between A. thaliana and other Brassicaceae, with two distinct microbiome types segregating along a latitudinal gradient. The differences in microbiome composition mirror the spatial genetics of A. thaliana, with 52-68% of variance in the first two principal coordinates of microbiome type explained by host genotype. Microbiome composition is best predicted by drought-associated metrics that are well known to be a major selective agent on A. thaliana populations. The reproducible and predictable associations between specific microbes and water availability raise the possibility that drought not only directly shapes genetic variation in A. thaliana, but does so also indirectly through its effects on the leaf microbiome.

ecology↗

bioRxiv2021

vcf2gwas - Python API for comprehensive GWAS analysis using GEMMA

We present a new software package vcf2gwas to perform reproducible genome-wide association studies (GWAS). vcf2gwas is a Python API for bcftools, PLINK and GEMMA. Before running the analysis a traditional GWAS workflow requires the user to edit and format the genotype information from commonly used Variant Call Format (VCF) file and phenotype information. Post-processing steps involve summarizing and visualizing the analysis results. This workflow requires a user to utilize the command-line, manual text-editing and knowledge of one or more programming/scripting languages which can be time-consuming especially when analyzing multiple phenotypes. Our package provides a convenient pipeline performing all of these steps, reducing the GWAS workflow to a single command-line input without the need to edit or format the VCF file beforehand or to install any additional software. In addition, features like reducing the dimensionality of the phenotypic space and performing analyses on the reduced dimensions or comparing the significant variants from the results to specific genes/regions of interest are implemented. By integrating different tools to perform GWAS under one workflow, the package ensures reproducible GWAS while reducing the user efforts significantly. Availability and implementationThe source code of vcf2gwas is available under the GNU General Public License. The package can be easily installed using conda. Installation instructions and a manual including tutorials can be accessed on the package website at https://github.com/frankvogt/vcf2gwas

bioinformatics↗

bioRxiv2021

Fine-scale Population Structure of North American Arabidopsis thaliana Reveals Multiple Sources of Introduction from Across Eurasia

Large-scale movement of organisms across their habitable range, or migration, is an important evolutionary process that can contribute to observed patterns of genetic diversity and our understanding of the adaptive spread of alleles. While human migrations have been studied in great detail with modern and ancient genomes, recent anthropogenic influence on reducing the biogeographical constraints on the migration of non-native species has presented opportunities in several study systems to ask the questions about how repeated introductions shape genetic diversity in the introduced range. We present here the most comprehensive view of population structure of North American Arabidopsis thaliana by studying a set of 500 (whole-genome sequenced) and over 2800 (RAD-seq genotyped) individuals in the context of global diversity represented by Afro-Eurasian genomes. We use haplotype-sharing, phylogenetic modeling and rare-allele sharing based methods to identify putative sources of introductions of extant N. American A. thaliana from the native range of Afro-Eurasia. We find evidence of admixture among the introduced lineages that has resulted in the increased haplotype diversity and reduced mutational load. Further, we also present signals of selection in the immune-system related genes that impart qualitative disease resistance to pathogens of bacterial and oomycete origins. Thus, multiple introductions to a non-native range can quickly increase adaptive potential of a colonizing species by increasing haplotypic diversity through admixture. The results presented here lay the foundation for further investigations into the functional significance of admixture.

evolutionary biology↗

bioRxiv2020

Measuring both microbial load and diversity with a single amplicon sequencing library

The ratio of microbial population size relative to the amount of host tissue, or "microbial load", is a fundamental metric of colonization and infection, but it cannot be directly deduced from microbial amplicon data such as 16S rRNA gene counts. Because conventional methods to determine load, such as serial dilution plating or quantitative PCR, add substantial experimental burden, they are only rarely paired with amplicon sequencing. Alternatively, whole metagenome sequencing of DNA contributed by host and microbes both reveals microbial community composition and enables determination of microbial load, but host DNA typically greatly outweighs microbial DNA, severely limiting the cost-effectiveness and scalability of this approach. We introduce host-associated microbe PCR (hamPCR), a robust amplicon sequencing strategy to quantify microbial load and describe interkingdom microbial community composition in a single, cost-effective library. We demonstrate its accuracy and flexibility across multiple host and microbe systems, including nematodes and major crops. We further present a technique that can be used, prior to sequencing, to optimize the host representation in a batch of libraries without loss of information. Because of its simplicity, and the fact that it provides an experimental solution to the well-known statistical challenges provided by compositional data, hamPCR will become a transformative approach throughout culture-independent microbiology.

bioengineering↗

bioRxiv2019

The relationship between microbial biomass and disease in the Arabidopsis thaliana phyllosphere

A central goal in microbiome research is to learn what distinguishes a healthy from a dysbiotic microbial community. Shifts in diversity and taxonomic composition are important indicators of dysbiosis, but a full understanding also requires knowledge of absolute microbial population sizes. In addition to the number of microbial cells, information on taxonomic composition can provide important insight into microbiome function and disease state. Here we use shotgun metagenomics to simultaneously assess microbiome composition and microbial load in the phyllosphere of wild populations of the plant Arabidopsis thaliana. We find that wild plants vary substantially in the load of colonizing microbes, and that high loads are typically associated with the proliferation of single taxa, with only a few putatively pathogenic taxa achieving high abundances in the field. Our results suggest (i) that the inside of a plant leaf is on average sparsely colonized with an estimated two bacterial genomes per plant genome and an order of magnitude fewer eukaryotic microbial genomes, and (ii) that higher levels of microbial cells often indicate successful colonization by pathogens. Lastly, our results show that load is a significant explanatory variable for loss of estimated Shannon diversity in phyllosphere microbiomes, implying that reduced diversity may be a significant predictor of microbial dysbiosis in a plant leaf.

plant biology↗

bioRxiv2019

Combining whole genome shotgun sequencing and rDNA amplicon analyses to improve detection of microbe-microbe interaction networks in plant leaves

Microorganisms from all domains of life establish associations with plants. Although some harm the plant, others antagonize pathogens or prime the plant immune system, acquire nutrients, tune plant hormone levels, or perform additional services. Most culture-independent plant microbiome research has focused on amplicon sequencing of 16S rDNA and/or the internal transcribed spacer (ITS) of rDNA loci, but the decreasing cost of high-throughput sequencing has made shotgun metagenome sequencing increasingly accessible. Here, we describe shotgun sequencing of 275 wild Arabidopsis thaliana leaf microbiomes from southwest Germany, with additional bacterial 16S rDNA and eukaryotic ITS1 amplicon data from 176 of these samples. The shotgun data were dominated by bacterial sequences, with eukaryotes contributing only a minority of reads. For shotgun and amplicon data, microbial membership showed weak associations with both site of origin and plant genotype, both of which were highly confounded in this dataset. There was large variation among microbiomes, with one extreme comprising samples of low complexity and a high load of microorganisms typical of infected plants, and the other extreme being samples of high complexity and a low microbial load. We use the metagenome data, which captures the ratio of bacterial to plant DNA in leaves of wild plants, to scale the 16S rDNA amplicon data such that they reflect absolute bacterial abundance. We show that this cost-effective hybrid strategy overcomes compositionality problems in amplicon data and leads to fundamentally different conclusions about microbiome community assembly.

genomics↗