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April 21, 2020  |  

Benchmarking Transposable Element Annotation Methods for Creation of a Streamlined, Comprehensive Pipeline

Sequencing technology and assembly algorithms have matured to the point that high-quality de novo assembly is possible for large, repetitive genomes. Current assemblies traverse transposable elements (TEs) and allow for annotation of TEs. There are numerous methods for each class of elements with unknown relative performance metrics. We benchmarked existing programs based on a curated library of rice TEs. Using the most robust programs, we created a comprehensive pipeline called Extensive de-novo TE Annotator (EDTA) that produces a condensed TE library for annotations of structurally intact and fragmented elements. EDTA is open-source and freely available: https://github.com/oushujun/EDTA.List of abbreviationsTETransposable ElementsLTRLong Terminal RepeatLINELong Interspersed Nuclear ElementSINEShort Interspersed Nuclear ElementMITEMiniature Inverted Transposable ElementTIRTerminal Inverted RepeatTSDTarget Site DuplicationTPTrue PositivesFPFalse PositivesTNTrue NegativeFNFalse NegativesGRFGeneric Repeat FinderEDTAExtensive de-novo TE Annotator


April 21, 2020  |  

Insect genomes: progress and challenges.

In the wake of constant improvements in sequencing technologies, numerous insect genomes have been sequenced. Currently, 1219 insect genome-sequencing projects have been registered with the National Center for Biotechnology Information, including 401 that have genome assemblies and 155 with an official gene set of annotated protein-coding genes. Comparative genomics analysis showed that the expansion or contraction of gene families was associated with well-studied physiological traits such as immune system, metabolic detoxification, parasitism and polyphagy in insects. Here, we summarize the progress of insect genome sequencing, with an emphasis on how this impacts research on pest control. We begin with a brief introduction to the basic concepts of genome assembly, annotation and metrics for evaluating the quality of draft assemblies. We then provide an overview of genome information for numerous insect species, highlighting examples from prominent model organisms, agricultural pests and disease vectors. We also introduce the major insect genome databases. The increasing availability of insect genomic resources is beneficial for developing alternative pest control methods. However, many opportunities remain for developing data-mining tools that make maximal use of the available insect genome resources. Although rapid progress has been achieved, many challenges remain in the field of insect genomics. © 2019 The Royal Entomological Society.


April 21, 2020  |  

Intercellular communication is required for trap formation in the nematode-trapping fungus Duddingtonia flagrans.

Nematode-trapping fungi (NTF) are a large and diverse group of fungi, which may switch from a saprotrophic to a predatory lifestyle if nematodes are present. Different fungi have developed different trapping devices, ranging from adhesive cells to constricting rings. After trapping, fungal hyphae penetrate the worm, secrete lytic enzymes and form a hyphal network inside the body. We sequenced the genome of Duddingtonia flagrans, a biotechnologically important NTF used to control nematode populations in fields. The 36.64 Mb genome encodes 9,927 putative proteins, among which are more than 638 predicted secreted proteins. Most secreted proteins are lytic enzymes, but more than 200 were classified as small secreted proteins (< 300 amino acids). 117 putative effector proteins were predicted, suggesting interkingdom communication during the colonization. As a first step to analyze the function of such proteins or other phenomena at the molecular level, we developed a transformation system, established the fluorescent proteins GFP and mCherry, adapted an assay to monitor protein secretion, and established gene-deletion protocols using homologous recombination or CRISPR/Cas9. One putative virulence effector protein, PefB, was transcriptionally induced during the interaction. We show that the mature protein is able to be imported into nuclei in Caenorhabditis elegans cells. In addition, we studied trap formation and show that cell-to-cell communication is required for ring closure. The availability of the genome sequence and the establishment of many molecular tools will open new avenues to studying this biotechnologically relevant nematode-trapping fungus.


April 21, 2020  |  

Complete Genome Sequence of Lactococcus lactis subsp. cremoris 3107, Host for the Model Lactococcal P335 Bacteriophage TP901-1.

The complete genome sequence of Lactococcus lactis subsp. cremoris 3107, a dairy starter strain and a host for the model lactococcal P335 bacteriophage TP901-1, is reported here. The circular chromosome of L. lactis subsp. cremoris 3107 is among the smallest genomes of currently sequenced lactococcal strains. L. lactis subsp. cremoris 3107 harbors a complement of six plasmids, which appears to be a reflection of its adaptation to the nutrient-rich dairy environment.


April 21, 2020  |  

Parallels between natural selection in the cold-adapted crop-wild relative Tripsacum dactyloides and artificial selection in temperate adapted maize.

Artificial selection has produced varieties of domesticated maize that thrive in temperate climates around the world. However, the direct progenitor of maize, teosinte, is indigenous only to a relatively small range of tropical and subtropical latitudes and grows poorly or not at all outside of this region. Tripsacum, a sister genus to maize and teosinte, is naturally endemic to the majority of areas in the western hemisphere where maize is cultivated. A full-length reference transcriptome for Tripsacum dactyloides generated using long-read Iso-Seq data was used to characterize independent adaptation to temperate climates in this clade. Genes related to phospholipid biosynthesis, a critical component of cold acclimation in other cold-adapted plant lineages, were enriched among those genes experiencing more rapid rates of protein sequence evolution in T. dactyloides. In contrast with previous studies of parallel selection, we find that there is a significant overlap between the genes that were targets of artificial selection during the adaptation of maize to temperate climates and those that were targets of natural selection in temperate-adapted T. dactyloides. Genes related to growth, development, response to stimulus, signaling, and organelles were enriched in the set of genes identified as both targets of natural and artificial selection. © 2019 The Authors The Plant Journal © 2019 John Wiley & Sons Ltd.


April 21, 2020  |  

Musa balbisiana genome reveals subgenome evolution and functional divergence.

Banana cultivars (Musa ssp.) are diploid, triploid and tetraploid hybrids derived from Musa acuminata and Musa balbisiana. We presented a high-quality draft genome assembly of M. balbisiana with 430?Mb (87%) assembled into 11?chromosomes. We identified that the recent divergence of M. acuminata (A-genome) and M. balbisiana (B-genome) occurred after lineage-specific whole-genome duplication, and that the B-genome may be more sensitive to the fractionation process compared to the A-genome. Homoeologous exchanges occurred frequently between A- and B-subgenomes in allopolyploids. Genomic variation within progenitors resulted in functional divergence of subgenomes. Global homoeologue expression dominance occurred between subgenomes of the allotriploid. Gene families related to ethylene biosynthesis and starch metabolism exhibited significant expansion at the pathway level and wide homoeologue expression dominance in the B-subgenome of the allotriploid. The independent origin of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) homoeologue gene pairs and tandem duplication-driven expansion of ACO genes in the B-subgenome contributed to rapid and major ethylene production post-harvest in allotriploid banana fruits. The findings of this study provide greater context for understanding fruit biology, and aid the development of tools for breeding optimal banana cultivars.


April 21, 2020  |  

Tools and Strategies for Long-Read Sequencing and De Novo Assembly of Plant Genomes.

The commercial release of third-generation sequencing technologies (TGSTs), giving long and ultra-long sequencing reads, has stimulated the development of new tools for assembling highly contiguous genome sequences with unprecedented accuracy across complex repeat regions. We survey here a wide range of emerging sequencing platforms and analytical tools for de novo assembly, provide background information for each of their steps, and discuss the spectrum of available options. Our decision tree recommends workflows for the generation of a high-quality genome assembly when used in combination with the specific needs and resources of a project.Copyright © 2019 Elsevier Ltd. All rights reserved.


April 21, 2020  |  

Genome sequence of Jatropha curcas L., a non-edible biodiesel plant, provides a resource to improve seed-related traits.

Jatropha curcas (physic nut), a non-edible oilseed crop, represents one of the most promising alternative energy sources due to its high seed oil content, rapid growth and adaptability to various environments. We report ~339 Mbp draft whole genome sequence of J. curcas var. Chai Nat using both the PacBio and Illumina sequencing platforms. We identified and categorized differentially expressed genes related to biosynthesis of lipid and toxic compound among four stages of seed development. Triacylglycerol (TAG), the major component of seed storage oil, is mainly synthesized by phospholipid:diacylglycerol acyltransferase in Jatropha, and continuous high expression of homologs of oleosin over seed development contributes to accumulation of high level of oil in kernels by preventing the breakdown of TAG. A physical cluster of genes for diterpenoid biosynthetic enzymes, including casbene synthases highly responsible for a toxic compound, phorbol ester, in seed cake, was syntenically highly conserved between Jatropha and castor bean. Transcriptomic analysis of female and male flowers revealed the up-regulation of a dozen family of TFs in female flower. Additionally, we constructed a robust species tree enabling estimation of divergence times among nine Jatropha species and five commercial crops in Malpighiales order. Our results will help researchers and breeders increase energy efficiency of this important oil seed crop by improving yield and oil content, and eliminating toxic compound in seed cake for animal feed. © 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.


April 21, 2020  |  

The Genome of Cucurbita argyrosperma (Silver-Seed Gourd) Reveals Faster Rates of Protein-Coding Gene and Long Noncoding RNA Turnover and Neofunctionalization within Cucurbita.

Whole-genome duplications are an important source of evolutionary novelties that change the mode and tempo at which genetic elements evolve within a genome. The Cucurbita genus experienced a whole-genome duplication around 30 million years ago, although the evolutionary dynamics of the coding and noncoding genes in this genus have not yet been scrutinized. Here, we analyzed the genomes of four Cucurbita species, including a newly assembled genome of Cucurbita argyrosperma, and compared the gene contents of these species with those of five other members of the Cucurbitaceae family to assess the evolutionary dynamics of protein-coding and long intergenic noncoding RNA (lincRNA) genes after the genome duplication. We report that Cucurbita genomes have a higher protein-coding gene birth-death rate compared with the genomes of the other members of the Cucurbitaceae family. C. argyrosperma gene families associated with pollination and transmembrane transport had significantly faster evolutionary rates. lincRNA families showed high levels of gene turnover throughout the phylogeny, and 67.7% of the lincRNA families in Cucurbita showed evidence of birth from the neofunctionalization of previously existing protein-coding genes. Collectively, our results suggest that the whole-genome duplication in Cucurbita resulted in faster rates of gene family evolution through the neofunctionalization of duplicated genes. Copyright © 2019 The Author. Published by Elsevier Inc. All rights reserved.


April 21, 2020  |  

Potential of TLR-gene diversity in Czech indigenous cattle for resistance breeding as revealed by hybrid sequencing

A production herd of Czech Simmental cattle (Czech Red Pied, CRP), the conserved subpopulation of this breed, and the ancient local breed Czech Red cattle (CR) were screened for diversity in the antibacterial toll-like receptors (TLRs), which are members of the innate immune system. Polymerase chain reaction (PCR) amplicons of TLR1, TLR2, TLR4, TLR5, and TLR6 from pooled DNA samples were sequenced with PacBio technology, with 3–5×?coverage per gene per animal. To increase the reliability of variant detection, the gDNA pools were sequenced in parallel with the Illumina X-ten platform at low coverage (60× per gene). The diversity in conserved CRP and CR was similar to the diversity in conserved and modern CRP, representing 76.4?% and 70.9?% of its variants, respectively. Sixty-eight (54.4?%) polymorphisms in the five TLR genes were shared by the two breeds, whereas 38 (30.4?%) were specific to the production herd of CRP; 4 (3.2?%) were specific to the broad CRP population; 7 (5.6?%) were present in both conserved populations; 5 (4.0?%) were present solely for the conserved CRP; and 3 (2.4?%) were restricted to CR. Consequently, gene pool erosion related to intensive breeding did not occur in Czech Simmental cattle. Similarly, no considerable consequences were found from known bottlenecks in the history of Czech Red cattle. On the other hand, the distinctness of the conserved populations and their potential for resistance breeding were only moderate. This relationship might be transferable to other non-abundant historical cattle breeds that are conserved as genetic resources. The estimates of polymorphism impact using Variant Effect Predictor and SIFT software tools allowed for the identification of candidate single-nucleotide polymorphisms (SNPs) for association studies related to infection resistance and targeted breeding. Knowledge of TLR-gene diversity present in Czech Simmental populations may aid in the potential transfer of variant characteristics from other breeds.


April 21, 2020  |  

Genetic map-guided genome assembly reveals a virulence-governing minichromosome in the lentil anthracnose pathogen Colletotrichum lentis.

Colletotrichum lentis causes anthracnose, which is a serious disease on lentil and can account for up to 70% crop loss. Two pathogenic races, 0 and 1, have been described in the C. lentis population from lentil. To unravel the genetic control of virulence, an isolate of the virulent race 0 was sequenced at 1481-fold genomic coverage. The 56.10-Mb genome assembly consists of 50 scaffolds with N50 scaffold length of 4.89 Mb. A total of 11 436 protein-coding gene models was predicted in the genome with 237 coding candidate effectors, 43 secondary metabolite biosynthetic enzymes and 229 carbohydrate-active enzymes (CAZymes), suggesting a contraction of the virulence gene repertoire in C. lentis. Scaffolds were assigned to 10 core and two minichromosomes using a population (race 0 × race 1, n = 94 progeny isolates) sequencing-based, high-density (14 312 single nucleotide polymorphisms) genetic map. Composite interval mapping revealed a single quantitative trait locus (QTL), qClVIR-11, located on minichromosome 11, explaining 85% of the variability in virulence of the C. lentis population. The QTL covers a physical distance of 0.84 Mb with 98 genes, including seven candidate effector and two secondary metabolite genes. Taken together, the study provides genetic and physical evidence for the existence of a minichromosome controlling the C. lentis virulence on lentil. © 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.


April 21, 2020  |  

Investigating the bacterial microbiota of traditional fermented dairy products using propidium monoazide with single-molecule real-time sequencing.

Traditional fermented dairy foods have been the major components of the Mongolian diet for millennia. In this study, we used propidium monoazide (PMA; binds to DNA of nonviable cells so that only viable cells are enumerated) and single-molecule real-time sequencing (SMRT) technology to investigate the total and viable bacterial compositions of 19 traditional fermented dairy foods, including koumiss from Inner Mongolia (KIM), koumiss from Mongolia (KM), and fermented cow milk from Mongolia (CM); sample groups treated with PMA were designated PKIM, PKM, and PCM. Full-length 16S rRNA sequencing identified 195 bacterial species in 121 genera and 13 phyla in PMA-treated and untreated samples. The PMA-treated and untreated samples differed significantly in their bacterial community composition and a-diversity values. The predominant species in KM, KIM, and CM were Lactobacillus helveticus, Streptococcus parauberis, and Lactobacillus delbrueckii, whereas the predominant species in PKM, PKIM, and PCM were Enterobacter xiangfangensis, Lactobacillus helveticus, and E. xiangfangensis, respectively. Weighted and unweighted principal coordinate analyses showed a clear clustering pattern with good separation and only minor overlapping. In addition, a pure culture method was performed to obtain lactic acid bacteria resources in dairy samples according to the results of SMRT sequencing. A total of 102 LAB strains were identified and Lb. helveticus (68.63%) was the most abundant, in agreement with SMRT sequencing results. Our results revealed that the bacterial communities of traditional dairy foods are complex and vary by type of fermented dairy product. The PMA treatment induced significant changes in bacterial community structure.Copyright © 2019 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.


April 21, 2020  |  

Two large reciprocal translocations characterized in the disease resistance-rich burmannica genetic group of Musa acuminata.

Banana cultivars are derived from hybridizations involving Musa acuminata subspecies. The latter diverged following geographical isolation in distinct South-east Asian continental regions and islands. Observation of chromosome pairing irregularities in meiosis of hybrids between these subspecies suggested the presence of large chromosomal structural variations. The aim of this study was to characterize such rearrangements.Marker (single nucleotide polymorphism) segregation in a self-progeny of the ‘Calcutta 4’ accession and mate-pair sequencing were used to search for chromosomal rearrangements in comparison with the M. acuminata ssp. malaccensis genome reference sequence. Signature segment junctions of the revealed chromosome structures were identified and searched in whole-genome sequencing data from 123 wild and cultivated Musa accessions.Two large reciprocal translocations were characterized in the seedy banana M. acuminata ssp. burmannicoides ‘Calcutta 4’ accession. One consisted of an exchange of a 240 kb distal region of chromosome 2 with a 7.2 Mb distal region of chromosome 8. The other involved an exchange of a 20.8 Mb distal region of chromosome 1 with a 11.6 Mb distal region of chromosome 9. Both translocations were found only in wild accessions belonging to the burmannicoides/burmannica/siamea subspecies. Only two of the 87 cultivars analysed displayed the 2/8 translocation, while none displayed the 1/9 translocation.Two large reciprocal translocations were identified that probably originated in the burmannica genetic group. Accurate characterization of these translocations should enhance the use of this disease resistance-rich burmannica group in breeding programmes. © The Author(s) 2019. Published by Oxford University Press on behalf of the Annals of Botany Company.


April 21, 2020  |  

PacBio full-length cDNA sequencing integrated with RNA-seq reads drastically improves the discovery of splicing transcripts in rice.

In eukaryotes, alternative splicing (AS) greatly expands the diversity of transcripts. However, it is challenging to accurately determine full-length splicing isoforms. Recently, more studies have taken advantage of Pacific Bioscience (PacBio) long-read sequencing to identify full-length transcripts. Nevertheless, the high error rate of PacBio reads seriously offsets the advantages of long reads, especially for accurately identifying splicing junctions. To best capitalize on the features of long reads, we used Illumina RNA-seq reads to improve PacBio circular consensus sequence (CCS) quality and to validate splicing patterns in the rice transcriptome. We evaluated the impact of CCS accuracy on the number and the validation rate of splicing isoforms, and integrated a comprehensive pipeline of splicing transcripts analysis by Iso-Seq and RNA-seq (STAIR) to identify the full-length multi-exon isoforms in rice seedling transcriptome (Oryza sativa L. ssp. japonica). STAIR discovered 11 733 full-length multi-exon isoforms, 6599 more than the SMRT Portal RS_IsoSeq pipeline did. Of these splicing isoforms identified, 4453 (37.9%) were missed in assembled transcripts from RNA-seq reads, and 5204 (44.4%), including 268 multi-exon long non-coding RNAs (lncRNAs), were not reported in the MSU_osa1r7 annotation. Some randomly selected unreported splicing junctions were verified by polymerase chain reaction (PCR) amplification. In addition, we investigated alternative polyadenylation (APA) events in transcripts and identified 829 major polyadenylation [poly(A)] site clusters (PACs). The analysis of splicing isoforms and APA events will facilitate the annotation of the rice genome and studies on the expression and polyadenylation of AS genes in different developmental stages or growth conditions of rice. © 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.


April 21, 2020  |  

High Quality Draft Genome of Arogyapacha (Trichopus zeylanicus), an Important Medicinal Plant Endemic to Western Ghats of India.

Arogyapacha, the local name of Trichopus zeylanicus, is a rare, indigenous medicinal plant of India. This plant is famous for its traditional use as an instant energy stimulant. So far, no genomic resource is available for this important plant and hence its metabolic pathways are poorly understood. Here, we report on a high-quality draft assembly of approximately 713.4 Mb genome of T. zeylanicus, first draft genome from the genus Trichopus The assembly was generated in a hybrid approach using Illumina short-reads and Pacbio longer-reads. The total assembly comprised of 22601 scaffolds with an N50 value of 433.3 Kb. We predicted 34452 protein coding genes in T. zeylanicus genome and found that a significant portion of these predicted genes were associated with various secondary metabolite biosynthetic pathways. Comparative genome analysis revealed extensive gene collinearity between T. zeylanicus and its closely related plant species. The present genome and annotation data provide an essential resource to speed-up the research on secondary metabolism, breeding and molecular evolution of T. zeylanicus. Copyright © 2019 Chellappan et al.


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