Menu

Auto Tag: Arabidopsis

September 22, 2019

Purge Haplotigs: allelic contig reassignment for third-gen diploid genome assemblies.

Recent developments in third-gen long read sequencing and diploid-aware assemblers have resulted in the rapid release of numerous reference-quality assemblies for diploid genomes. However, assembly of highly heterozygous genomes is still problematic when regional heterogeneity is so high that haplotype homology is not recognised during assembly. This results in regional duplication rather than consolidation into allelic variants and can cause issues with downstream analysis, for example variant discovery, or haplotype reconstruction using the diploid assembly with unpaired allelic contigs.A new pipeline-Purge Haplotigs-was developed specifically for third-gen sequencing-based assemblies to automate the reassignment of allelic contigs, and to assist in the manual curation of genome assemblies. The pipeline uses a draft haplotype-fused assembly or a diploid assembly, read alignments, and repeat annotations to identify allelic variants in the primary assembly. The pipeline was tested on a simulated dataset and on four recent diploid (phased) de novo assemblies from third-generation long-read sequencing, and compared with a similar tool. After processing with Purge Haplotigs, haploid assemblies were less duplicated with minimal impact on genome completeness, and diploid assemblies had more pairings of allelic contigs.Purge Haplotigs improves the haploid and diploid representations of third-gen sequencing based genome assemblies by identifying and reassigning allelic contigs. The implementation is fast and scales well with large genomes, and it is less likely to over-purge repetitive or paralogous elements compared to alignment-only based methods. The software is available at https://bitbucket.org/mroachawri/purge_haplotigs under a permissive MIT licence.

Read more
September 22, 2019

Unexpected patterns of segregation distortion at a selfish supergene in the fire ant Solenopsis invicta.

The Sb supergene in the fire ant Solenopsis invicta determines the form of colony social organization, with colonies whose inhabitants bear the element containing multiple reproductive queens and colonies lacking it containing only a single queen. Several features of this supergene – including suppressed recombination, presence of deleterious mutations, association with a large centromere, and “green-beard” behavior – suggest that it may be a selfish genetic element that engages in transmission ratio distortion (TRD), defined as significant departures in progeny allele frequencies from Mendelian inheritance ratios. We tested this possibility by surveying segregation ratios in embryo progenies of 101 queens of the “polygyne” social form (3512 embryos) using three supergene-linked markers and twelve markers outside the supergene.Significant departures from Mendelian ratios were observed at the supergene loci in 3-5 times more progenies than expected in the absence of TRD and than found, on average, among non-supergene loci. Also, supergene loci displayed the greatest mean deviations from Mendelian ratios among all study loci, although these typically were modest. A surprising feature of the observed inter-progeny variation in TRD was that significant deviations involved not only excesses of supergene alleles but also similarly frequent excesses of the alternate alleles on the homologous chromosome. As expected given the common occurrence of such “drive reversal” in this system, alleles associated with the supergene gain no consistent transmission advantage over their alternate alleles at the population level. Finally, we observed low levels of recombination and incomplete gametic disequilibrium across the supergene, including between adjacent markers within a single inversion.Our data confirm the prediction that the Sb supergene is a selfish genetic element capable of biasing its own transmission during reproduction, yet counterselection for suppressor loci evidently has produced an evolutionary stalemate in TRD between the variant homologous haplotypes on the “social chromosome”. Evidence implicates prezygotic segregation distortion as responsible for the TRD we document, with “true” meiotic drive the most likely mechanism. Low levels of recombination and incomplete gametic disequilibrium across the supergene suggest that selection does not preserve a single uniform supergene haplotype responsible for inducing polygyny.

Read more
September 22, 2019

Out in the cold: Identification of genomic regions associated with cold tolerance in the biocontrol fungus Clonostachys rosea through genome-wide association mapping.

There is an increasing importance for using biocontrol agents in combating plant diseases sustainably and in the long term. As large scale genomic sequencing becomes economically viable, the impact of single nucleotide polymorphisms (SNPs) on biocontrol-associated phenotypes can be easily studied across entire genomes of fungal populations. Here, we improved a previously reported genome assembly of the biocontrol fungus Clonostachys rosea strain IK726 using the PacBio sequencing platform, which resulted in a total genome size of 70.7 Mbp and 21,246 predicted genes. We further performed whole-genome re-sequencing of 52 additional C. rosea strains isolated globally using Illumina sequencing technology, in order to perform genome-wide association studies in conditions relevant for biocontrol activity. One such condition is the ability to grow at lower temperatures commonly encountered in cryic or frigid soils in temperate regions, as these will be prevalent for protecting growing crops in temperate climates. Growth rates at 10°C on potato dextrose agar of the 53 sequenced strains of C. rosea were measured and ranged between 0.066 and 0.413 mm/day. Performing a genome wide association study, a total of 1,478 SNP markers were significantly associated with the trait and located in 227 scaffolds, within or close to (< 1000 bp distance) 265 different genes. The predicted gene products included several chaperone proteins, membrane transporters, lipases, and proteins involved in chitin metabolism with possible roles in cold tolerance. The data reported in this study provides a foundation for future investigations into the genetic basis for cold tolerance in fungi, with important implications for biocontrol.

Read more
September 22, 2019

N6-methyladenine DNA modification in Xanthomonas oryzae pv. oryzicola genome.

DNA N6-methyladenine (6mA) modifications expand the information capacity of DNA and have long been known to exist in bacterial genomes. Xanthomonas oryzae pv. Oryzicola (Xoc) is the causative agent of bacterial leaf streak, an emerging and destructive disease in rice worldwide. However, the genome-wide distribution patterns and potential functions of 6mA in Xoc are largely unknown. In this study, we analyzed the levels and global distribution patterns of 6mA modification in genomic DNA of seven Xoc strains (BLS256, BLS279, CFBP2286, CFBP7331, CFBP7341, L8 and RS105). The 6mA modification was found to be widely distributed across the seven Xoc genomes, accounting for percent of 3.80, 3.10, 3.70, 4.20, 3.40, 2.10, and 3.10 of the total adenines in BLS256, BLS279, CFBP2286, CFBP7331, CFBP7341, L8, and RS105, respectively. Notably, more than 82% of 6mA sites were located within gene bodies in all seven strains. Two specific motifs for 6?mA modification, ARGT and AVCG, were prevalent in all seven strains. Comparison of putative DNA methylation motifs from the seven strains reveals that Xoc have a specific DNA methylation system. Furthermore, the 6?mA modification of rpfC dramatically decreased during Xoc infection indicates the important role for Xoc adaption to environment.

Read more
September 22, 2019

The impact of genome evolution on the allotetraploid Nicotiana rustica – an intriguing story of enhanced alkaloid production.

Nicotiana rustica (Aztec tobacco), like common tobacco (Nicotiana tabacum), is an allotetraploid formed through a recent hybridization event; however, it originated from completely different progenitor species. Here, we report the comparative genome analysis of wild type N. rustica (5 Gb; 2n?=?4x?=?48) with its three putative diploid progenitors (2.3-3 Gb; 2n?=?2x =24), Nicotiana undulata, Nicotiana paniculata and Nicotiana knightiana.In total, 41% of N. rustica genome originated from the paternal donor (N. undulata), while 59% originated from the maternal donor (N. paniculata/N. knightiana). Chloroplast genome and gene analyses indicated that N. knightiana is more closely related to N. rustica than N. paniculata. Gene clustering revealed 14,623 ortholog groups common to other Nicotiana species and 207 unique to N. rustica. Genome sequence analysis indicated that N. knightiana is more closely related to N. rustica than N. paniculata, and that the higher nicotine content of N. rustica leaves is the result of the progenitor genomes combination and of a more active transport of nicotine to the shoot.The availability of four new Nicotiana genome sequences provide insights into how speciation impacts plant metabolism, and in particular alkaloid transport and accumulation, and will contribute to better understanding the evolution of Nicotiana species.

Read more
September 22, 2019

Reconstitution of eukaryotic chromosomes and manipulation of DNA N6-methyladenine alters chromatin and gene expression

DNA N6-adenine methylation (6mA) has recently been reported in diverse eukaryotes, spanning unicellular organisms to metazoans. Yet the functional significance of 6mA remains elusive due to its low abundance, difficulty of manipulation within native DNA, and lack of understanding of eukaryotic 6mA writers. Here, we report a novel DNA 6mA methyltransferase in ciliates, termed MTA1. The enzyme contains an MT-A70 domain but is phylogenetically distinct from all known RNA and DNA methyltransferases. Disruption of MTA1 in vivo leads to the genome-wide loss of 6mA in asexually growing cells and abolishment of the consensus ApT dimethylated motif. Genes exhibit subtle changes in chromatin organization or RNA expression upon loss of 6mA, depending on their starting methylation level. Mutants fail to complete the sexual cycle, which normally coincides with a peak of MTA1 expression. Thus, MTA1 functions in a developmental stage-specific manner. We determine the impact of 6mA on chromatin organization in vitro by reconstructing complete, full-length ciliate chromosomes harboring 6mA in native or ectopic positions. Using these synthetic chromosomes, we show that 6mA directly disfavors nucleosomes in vitro in a local, quantitative manner, independent of DNA sequence. Furthermore, the chromatin remodeler ACF can overcome this effect. Our study identifies a novel MT-A70 protein necessary for eukaryotic 6mA methylation and defines the impact of 6mA on chromatin organization using epigenetically defined synthetic chromosomes.

Read more
September 22, 2019

Genome sequences of two diploid wild relatives of cultivated sweetpotato reveal targets for genetic improvement

Sweetpotato [Ipomoea batatas (L.) Lam.] is a globally important staple food crop, especially for sub-Saharan Africa. Agronomic improvement of sweetpotato has lagged behind other major food crops due to a lack of genomic and genetic resources and inherent challenges in breeding a heterozygous, clonally propagated polyploid. Here, we report the genome sequences of its two diploid relatives, I. trifida and I. triloba, and show that these high-quality genome assemblies are robust references for hexaploid sweetpotato. Comparative and phylogenetic analyses reveal insights into the ancient whole-genome triplication history of Ipomoea and evolutionary relationships within the Batatas complex. Using resequencing data from 16 genotypes widely used in African breeding programs, genes and alleles associated with carotenoid biosynthesis in storage roots are identified, which may enable efficient breeding of varieties with high provitamin A content. These resources will facilitate genome-enabled breeding in this important food security crop.

Read more
September 22, 2019

Three New Genome Assemblies Support a Rapid Radiation in Musa acuminata (Wild Banana).

Edible bananas result from interspecific hybridization between Musa acuminata and Musa balbisiana, as well as among subspecies in M. acuminata. Four particular M. acuminata subspecies have been proposed as the main contributors of edible bananas, all of which radiated in a short period of time in southeastern Asia. Clarifying the evolution of these lineages at a whole-genome scale is therefore an important step toward understanding the domestication and diversification of this crop. This study reports the de novo genome assembly and gene annotation of a representative genotype from three different subspecies of M. acuminata. These data are combined with the previously published genome of the fourth subspecies to investigate phylogenetic relationships. Analyses of shared and unique gene families reveal that the four subspecies are quite homogenous, with a core genome representing at least 50% of all genes and very few M. acuminata species-specific gene families. Multiple alignments indicate high sequence identity between homologous single copy-genes, supporting the close relationships of these lineages. Interestingly, phylogenomic analyses demonstrate high levels of gene tree discordance, due to both incomplete lineage sorting and introgression. This pattern suggests rapid radiation within Musa acuminata subspecies that occurred after the divergence with M. balbisiana. Introgression between M. a. ssp. malaccensis and M. a. ssp. burmannica was detected across the genome, though multiple approaches to resolve the subspecies tree converged on the same topology. To support evolutionary and functional analyses, we introduce the PanMusa database, which enables researchers to exploration of individual gene families and trees.

Read more
September 22, 2019

Improved nucleic acid extraction protocols for Ganoderma boninense, G. miniatocinctum and G. tornatum.

The first and most crucial step of all molecular techniques is to isolate high quality and intact nucleic acids. However, DNA and RNA isolation from fungal samples are usually difficult due to the cell walls that are relatively unsusceptible to lysis and often resistant to traditional extraction procedures. Although there are many extraction protocols for Ganoderma species, different extraction protocols have been applied to different species to obtain high yields of good quality nucleic acids, especially for genome and transcriptome sequencing. Ganoderma species, mainly G. boninense causes the basal stem rot disease, a devastating disease that plagues the oil palm industry. Here, we describe modified DNA extraction protocols for G. boninense, G. miniatocinctum and G. tornatum, and an RNA extraction protocol for G. boninense. The modified salting out DNA extraction protocol is suitable for G. boninense and G. miniatocinctum while the modified high salt and low pH protocol is suitable for G. tornatum. The modified DNA and RNA extraction protocols were able to produce high quality genomic DNA and total RNA of?~?140 to 160 µg/g and?~?80 µg/g of mycelia respectively, for Single Molecule Real Time (PacBio Sequel® System) and Illumina sequencing. These protocols will benefit those studying the oil palm pathogens at nucleotide level.

Read more
September 22, 2019

Whole-genome landscape of Medicago truncatula symbiotic genes.

Advances in deciphering the functional architecture of eukaryotic genomes have been facilitated by recent breakthroughs in sequencing technologies, enabling a more comprehensive representation of genes and repeat elements in genome sequence assemblies, as well as more sensitive and tissue-specific analyses of gene expression. Here we show that PacBio sequencing has led to a substantially improved genome assembly of Medicago truncatula A17, a legume model species notable for endosymbiosis studies1, and has enabled the identification of genome rearrangements between genotypes at a near-base-pair resolution. Annotation of the new M. truncatula genome sequence has allowed for a thorough analysis of transposable elements and their dynamics, as well as the identification of new players involved in symbiotic nodule development, in particular 1,037 upregulated long non-coding RNAs (lncRNAs). We have also discovered that a substantial proportion (~35% and 38%, respectively) of the genes upregulated in nodules or expressed in the nodule differentiation zone colocalize in genomic clusters (270 and 211, respectively), here termed symbiotic islands. These islands contain numerous expressed lncRNA genes and display differentially both DNA methylation and histone marks. Epigenetic regulations and lncRNAs are therefore attractive candidate elements for the orchestration of symbiotic gene expression in the M. truncatula genome.

Read more
September 22, 2019

Desiccation Tolerance Evolved through Gene Duplication and Network Rewiring in Lindernia.

Although several resurrection plant genomes have been sequenced, the lack of suitable dehydration-sensitive outgroups has limited genomic insights into the origin of desiccation tolerance. Here, we utilized a comparative system of closely related desiccation-tolerant (Lindernia brevidens) and -sensitive (Lindernia subracemosa) species to identify gene- and pathway-level changes associated with the evolution of desiccation tolerance. The two high-quality Lindernia genomes we assembled are largely collinear, and over 90% of genes are conserved. L. brevidens and L. subracemosa have evidence of an ancient, shared whole-genome duplication event, and retained genes have neofunctionalized, with desiccation-specific expression in L. brevidens Tandem gene duplicates also are enriched in desiccation-associated functions, including a dramatic expansion of early light-induced proteins from 4 to 26 copies in L. brevidens A comparative differential gene coexpression analysis between L. brevidens and L. subracemosa supports extensive network rewiring across early dehydration, desiccation, and rehydration time courses. Many LATE EMBRYOGENESIS ABUNDANT genes show significantly higher expression in L. brevidens compared with their orthologs in L. subracemosa Coexpression modules uniquely upregulated during desiccation in L. brevidens are enriched with seed-specific and abscisic acid-associated cis-regulatory elements. These modules contain a wide array of seed-associated genes that have no expression in the desiccation-sensitive L. subracemosa Together, these findings suggest that desiccation tolerance evolved through a combination of gene duplications and network-level rewiring of existing seed desiccation pathways.© 2018 American Society of Plant Biologists. All rights reserved.

Read more
September 22, 2019

The genome of the tegu lizard Salvator merianae: combining Illumina, PacBio, and optical mapping data to generate a highly contiguous assembly.

Reptiles are a species-rich group with great phenotypic and life history diversity but are highly underrepresented among the vertebrate species with sequenced genomes.Here, we report a high-quality genome assembly of the tegu lizard, Salvator merianae, the first lacertoid with a sequenced genome. We combined 74X Illumina short-read, 29.8X Pacific Biosciences long-read, and optical mapping data to generate a high-quality assembly with a scaffold N50 value of 55.4 Mb. The contig N50 value of this assembly is 521 Kb, making it the most contiguous reptile assembly so far. We show that the tegu assembly has the highest completeness of coding genes and conserved non-exonic elements (CNEs) compared to other reptiles. Furthermore, the tegu assembly has the highest number of evolutionarily conserved CNE pairs, corroborating a high assembly contiguity in intergenic regions. As in other reptiles, long interspersed nuclear elements comprise the most abundant transposon class. We used transcriptomic data, homology- and de novo gene predictions to annotate 22,413 coding genes, of which 16,995 (76%) likely have human orthologs as inferred by CESAR-derived gene mappings. Finally, we generated a multiple genome alignment comprising 10 squamates and 7 other amniote species and identified conserved regions that are under evolutionary constraint. CNEs cover 38 Mb (1.8%) of the tegu genome, with 3.3 Mb in these elements being squamate specific. In contrast to placental mammal-specific CNEs, very few of these squamate-specific CNEs (<20 Kb) overlap transposons, highlighting a difference in how lineage-specific CNEs originated in these two clades.The tegu lizard genome together with the multiple genome alignment and comprehensive conserved element datasets provide a valuable resource for comparative genomic studies of reptiles and other amniotes.

Read more
September 22, 2019

The genomic landscape of molecular responses to natural drought stress in Panicum hallii

Environmental stress is a major driver of ecological community dynamics and agricultural productivity. This is especially true for soil water availability, because drought is the greatest abiotic inhibitor of worldwide crop yields. Here, we test the genetic basis of drought responses in the genetic model for C4perennial grasses, Panicum hallii, through population genomics, field-scale gene-expression (eQTL) analysis, and comparison of two complete genomes. While gene expression networks are dominated by local cis-regulatory elements, we observe three genomic hotspots of unlinked trans-regulatory loci. These regulatory hubs are four times more drought responsive than the genome-wide average. Additionally, cis- and trans-regulatory networks are more likely to have opposing effects than expected under neutral evolution, supporting a strong influence of compensatory evolution and stabilizing selection. These results implicate trans-regulatory evolution as a driver of drought responses and demonstrate the potential for crop improvement in drought-prone regions through modification of gene regulatory networks.

Read more
September 22, 2019

Evolution of host support for two ancient bacterial symbionts with differentially degraded genomes in a leafhopper host.

Plant sap-feeding insects (Hemiptera) rely on bacterial symbionts for nutrition absent in their diets. These bacteria experience extreme genome reduction and require genetic resources from their hosts, particularly for basic cellular processes other than nutrition synthesis. The host-derived mechanisms that complete these processes have remained poorly understood. It is also unclear how hosts meet the distinct needs of multiple bacterial partners with differentially degraded genomes. To address these questions, we investigated the cell-specific gene-expression patterns in the symbiotic organs of the aster leafhopper (ALF), Macrosteles quadrilineatus (Cicadellidae). ALF harbors two intracellular symbionts that have two of the smallest known bacterial genomes: Nasuia (112 kb) and Sulcia (190 kb). Symbionts are segregated into distinct host cell types (bacteriocytes) and vary widely in their basic cellular capabilities. ALF differentially expresses thousands of genes between the bacteriocyte types to meet the functional needs of each symbiont, including the provisioning of metabolites and support of cellular processes. For example, the host highly expresses genes in the bacteriocytes that likely complement gene losses in nucleic acid synthesis, DNA repair mechanisms, transcription, and translation. Such genes are required to function in the bacterial cytosol. Many host genes comprising these support mechanisms are derived from the evolution of novel functional traits via horizontally transferred genes, reassigned mitochondrial support genes, and gene duplications with bacteriocyte-specific expression. Comparison across other hemipteran lineages reveals that hosts generally support the incomplete symbiont cellular processes, but the origins of these support mechanisms are generally specific to the host-symbiont system.Copyright © 2018 the Author(s). Published by PNAS.

Read more
September 22, 2019

N6-methyladenine DNA methylation in Japonica and Indica rice genomes and its association with gene expression, plant development, and stress responses.

N6-Methyladenine (6mA) DNA methylation has recently been implicated as a potential new epigenetic marker in eukaryotes, including the dicot model Arabidopsis thaliana. However, the conservation and divergence of 6mA distribution patterns and functions in plants remain elusive. Here we report high-quality 6mA methylomes at single-nucleotide resolution in rice based on substantially improved genome sequences of two rice cultivars, Nipponbare (Nip; Japonica) and 93-11 (Indica). Analysis of 6mA genomic distribution and its association with transcription suggest that 6mA distribution and function is rather conserved between rice and Arabidopsis. We found that 6mA levels are positively correlated with the expression of key stress-related genes, which may be responsible for the difference in stress tolerance between Nip and 93-11. Moreover, we showed that mutations in DDM1 cause defects in plant growth and decreased 6mA level. Our results reveal that 6mA is a conserved DNA modification that is positively associated with gene expression and contributes to key agronomic traits in plants. Copyright © 2018 The Author. Published by Elsevier Inc. All rights reserved.

Read more

Subscribe for blog updates:

Talk with an expert

If you have a question, need to check the status of an order, or are interested in purchasing an instrument, we're here to help.