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  |  

Endogenous pararetrovirus sequences are widely present in Citrinae genomes.

Endogenous pararetroviruses (EPRVs) are characterized in several plant genomes and their biological effects have been reported. In this study, hundreds of EPRV segments were identified in six Citrinae genomes. A total of 1034 EPRV segments were identified in the genomes of sweet orange, 2036 in pummelo, 598 in clementine mandarin, 752 in Ichang papeda, 2060 in citron and 245 in atalantia. Genomic analysis indicated that EPRV segments tend to cluster as hot spots in the genomes, particularly on chromosome 2 and 5. Large numbers of simple repeats and transposable elements were identified in the 2-kb flanking regions of the EPRV segments. Comparative genomic analysis and PCR experiments showed that there are highly conserved EPRV segments and species-specific EPRV segments between the Citrinae genomes. Phylogenetic analysis suggested that the integration events of EPRVs could initiate in a common progenitor of Citrinae species and repeatedly occur during the Citrinae divergence.Copyright © 2018 Elsevier B.V. All rights reserved.


April 21, 2020  |  

Full-length transcriptome analysis of Litopenaeus vannamei reveals transcript variants involved in the innate immune system.

To better understand the immune system of shrimp, this study combined PacBio isoform sequencing (Iso-Seq) and Illumina paired-end short reads sequencing methods to discover full-length immune-related molecules of the Pacific white shrimp, Litopenaeus vannamei. A total of 72,648 nonredundant full-length transcripts (unigenes) were generated with an average length of 2545 bp from five main tissues, including the hepatopancreas, cardiac stomach, heart, muscle, and pyloric stomach. These unigenes exhibited a high annotation rate (62,164, 85.57%) when compared against NR, NT, Swiss-Prot, Pfam, GO, KEGG and COG databases. A total of 7544 putative long noncoding RNAs (lncRNAs) were detected and 1164 nonredundant full-length transcripts (449 UniTransModels) participated in the alternative splicing (AS) events. Importantly, a total of 5279 nonredundant full-length unigenes were successfully identified, which were involved in the innate immune system, including 9 immune-related processes, 19 immune-related pathways and 10 other immune-related systems. We also found wide transcript variants, which increased the number and function complexity of immune molecules; for example, toll-like receptors (TLRs) and interferon regulatory factors (IRFs). The 480 differentially expressed genes (DEGs) were significantly higher or tissue-specific expression patterns in the hepatopancreas compared with that in other four tested tissues (FDR <0.05). Furthermore, the expression levels of six selected immune-related DEGs and putative IRFs were validated using real-time PCR technology, substantiating the reliability of the PacBio Iso-seq results. In conclusion, our results provide new genetic resources of long-read full-length transcripts data and information for identifying immune-related genes, which are an invaluable transcriptomic resource as genomic reference, especially for further exploration of the innate immune and defense mechanisms of shrimp. Copyright © 2019 Elsevier Ltd. All rights reserved.


April 21, 2020  |  

Population Genome Sequencing of the Scab Fungal Species Venturia inaequalis, Venturia pirina, Venturia aucupariae and Venturia asperata.

The Venturia genus comprises fungal species that are pathogens on Rosaceae host plants, including V. inaequalis and V. asperata on apple, V. aucupariae on sorbus and V. pirina on pear. Although the genetic structure of V. inaequalis populations has been investigated in detail, genomic features underlying these subdivisions remain poorly understood. Here, we report whole genome sequencing of 87 Venturia strains that represent each species and each population within V. inaequalis We present a PacBio genome assembly for the V. inaequalis EU-B04 reference isolate. The size of selected genomes was determined by flow cytometry, and varied from 45 to 93 Mb. Genome assemblies of V. inaequalis and V. aucupariae contain a high content of transposable elements (TEs), most of which belong to the Gypsy or Copia LTR superfamilies and have been inactivated by Repeat-Induced Point mutations. The reference assembly of V. inaequalis presents a mosaic structure of GC-equilibrated regions that mainly contain predicted genes and AT-rich regions, mainly composed of TEs. Six pairs of strains were identified as clones. Single-Nucleotide Polymorphism (SNP) analysis between these clones revealed a high number of SNPs that are mostly located in AT-rich regions due to misalignments and allowed determining a false discovery rate. The availability of these genome sequences is expected to stimulate genetics and population genomics research of Venturia pathogens. Especially, it will help understanding the evolutionary history of Venturia species that are pathogenic on different hosts, a history that has probably been substantially influenced by TEs.Copyright © 2019 Le Cam et al.


April 21, 2020  |  

Midrib Sucrose Accumulation and Sugar Transporter Gene Expression in YCS-Affected Sugarcane Leaves

Sucrose accumulation and decreased photosynthesis are early symptoms of yellow canopy syndrome (YCS) in sugarcane (Saccharum spp.), and precede the visual yellowing of the leaves. To investigate broad-scale gene expression changes during YCS-onset, transcriptome analyses coupled to metabolome analyses were performed. Across leaf tissues, the greatest number of differentially expressed genes related to the chloroplast, and the metabolic processes relating to nitrogen and carbohydrates. Five genes represented 90% of the TPM (Transcripts Per Million) associated with the downregulation of transcription during YCS-onset, which included PSII D1 (PsbA). This differential expression was consistent with a feedback regulatory effect upon photosynthesis. Broad-scale gene expression analyses did not reveal a cause for leaf sugar accumulation during YCS-onset. Interestingly, the midrib showed the greatest accumulation of sugars, followed by symptomatic lamina. To investigate if phloem loading/reloading may be compromised on a gene expression level – to lead to leaf sucrose accumulation – sucrose transport-related proteins of SWEETs, Sucrose Transporters (SUTs), H+-ATPases and H+-pyrophosphatases (H+-PPases) were characterised from a sugarcane transcriptome and expression analysed. Two clusters of Type I H+-PPases, with one upregulated and the other downregulated, were evident. Although less pronounced, a similar pattern of change was observed for the H+-ATPases. The disaccharide transporting SWEETs were downregulated after visual symptoms were present, and a monosaccharide transporting SWEET upregulated preceding, as well as after, symptom development. SUT gene expression was the least responsive to YCS development. The results are consistent with a reduction of photoassimilate movement through the phloem leading to sucrose build-up in the leaf.


April 21, 2020  |  

The role of genomic structural variation in the genetic improvement of polyploid crops

Many of our major crop species are polyploids, containing more than one genome or set of chromosomes. Polyploid crops present unique challenges, including difficulties in genome assembly, in discriminating between multiple gene and sequence copies, and in genetic mapping, hindering use of genomic data for genetics and breeding. Polyploid genomes may also be more prone to containing structural variation, such as loss of gene copies or sequences (presence–absence variation) and the presence of genes or sequences in multiple copies (copy-number variation). Although the two main types of genomic structural variation commonly identified are presence–absence variation and copy-number variation, we propose that homeologous exchanges constitute a third major form of genomic structural variation in polyploids. Homeologous exchanges involve the replacement of one genomic segment by a similar copy from another genome or ancestrally duplicated region, and are known to be extremely common in polyploids. Detecting all kinds of genomic structural variation is challenging, but recent advances such as optical mapping and long-read sequencing offer potential strategies to help identify structural variants even in complex polyploid genomes. All three major types of genomic structural variation (presence–absence, copy-number, and homeologous exchange) are now known to influence phenotypes in crop plants, with examples of flowering time, frost tolerance, and adaptive and agronomic traits. In this review, we summarize the challenges of genome analysis in polyploid crops, describe the various types of genomic structural variation and the genomics technologies and data that can be used to detect them, and collate information produced to date related to the impact of genomic structural variation on crop phenotypes. We highlight the importance of genomic structural variation for the future genetic improvement of polyploid crops.


April 21, 2020  |  

Metagenomic assembly through the lens of validation: recent advances in assessing and improving the quality of genomes assembled from metagenomes.

Metagenomic samples are snapshots of complex ecosystems at work. They comprise hundreds of known and unknown species, contain multiple strain variants and vary greatly within and across environments. Many microbes found in microbial communities are not easily grown in culture making their DNA sequence our only clue into their evolutionary history and biological function. Metagenomic assembly is a computational process aimed at reconstructing genes and genomes from metagenomic mixtures. Current methods have made significant strides in reconstructing DNA segments comprising operons, tandem gene arrays and syntenic blocks. Shorter, higher-throughput sequencing technologies have become the de facto standard in the field. Sequencers are now able to generate billions of short reads in only a few days. Multiple metagenomic assembly strategies, pipelines and assemblers have appeared in recent years. Owing to the inherent complexity of metagenome assembly, regardless of the assembly algorithm and sequencing method, metagenome assemblies contain errors. Recent developments in assembly validation tools have played a pivotal role in improving metagenomics assemblers. Here, we survey recent progress in the field of metagenomic assembly, provide an overview of key approaches for genomic and metagenomic assembly validation and demonstrate the insights that can be derived from assemblies through the use of assembly validation strategies. We also discuss the potential for impact of long-read technologies in metagenomics. We conclude with a discussion of future challenges and opportunities in the field of metagenomic assembly and validation. © The Author 2017. Published by Oxford University Press.


April 21, 2020  |  

Hybrid sequencing reveals insight into heat sensing and signaling of bread wheat.

Wheat (Triticum aestivum L.), a globally important crop, is challenged by increasing temperatures (heat stress, HS). However its polyploid nature, the incompleteness of its genome sequences and annotation, the lack of comprehensive HS-responsive transcriptomes and the unexplored heat sensing and signaling of wheat hinder our full understanding of its adaptations to HS. The recently released genome sequences of wheat, as well as emerging single-molecular sequencing technologies, provide an opportunity to thoroughly investigate the molecular mechanisms of the wheat response to HS. We generated a high-resolution spatio-temporal transcriptome map of wheat flag leaves and filling grain under HS at 0 min, 5 min, 10 min, 30 min, 1 h and 4 h by combining full-length single-molecular sequencing and Illumina short reads sequencing. This hybrid sequencing newly discovered 4947 loci and 70 285 transcripts, generating the comprehensive and dynamic list of HS-responsive full-length transcripts and complementing the recently released wheat reference genome. Large-scale analysis revealed a global landscape of heat adaptations, uncovering unexpected rapid heat sensing and signaling, significant changes of more than half of HS-responsive genes within 30 min, heat shock factor-dependent and -independent heat signaling, and metabolic alterations in early HS-responses. Integrated analysis also demonstrated the differential responses and partitioned functions between organs and subgenomes, and suggested a differential pattern of transcriptional and alternative splicing regulation in the HS response. This study provided comprehensive data for dissecting molecular mechanisms of early HS responses in wheat and highlighted the genomic plasticity and evolutionary divergence of polyploidy wheat. © 2019 The Authors. The Plant Journal published by John Wiley & Sons Ltd and Society for Experimental Biology.


April 21, 2020  |  

Development of CRISPR-Cas systems for genome editing and beyond

The development of clustered regularly interspaced short-palindromic repeat (CRISPR)-Cas systems for genome editing has transformed the way life science research is conducted and holds enormous potential for the treatment of disease as well as for many aspects of biotech- nology. Here, I provide a personal perspective on the development of CRISPR-Cas9 for genome editing within the broader context of the field and discuss our work to discover novel Cas effectors and develop them into additional molecular tools. The initial demonstra- tion of Cas9-mediated genome editing launched the development of many other technologies, enabled new lines of biological inquiry, and motivated a deeper examination of natural CRISPR-Cas systems, including the discovery of new types of CRISPR-Cas systems. These new discoveries in turn spurred further technological developments. I review these exciting discoveries and technologies as well as provide an overview of the broad array of applications of these technologies in basic research and in the improvement of human health. It is clear that we are only just beginning to unravel the potential within microbial diversity, and it is quite likely that we will continue to discover other exciting phenomena, some of which it may be possible to repurpose as molecular technologies. The transformation of mysterious natural phenomena to powerful tools, however, takes a collective effort to discover, characterize, and engineer them, and it has been a privilege to join the numerous researchers who have contributed to this transformation of CRISPR-Cas systems.


April 21, 2020  |  

Wild relatives of maize

Crop domestication changed the course of human evolution, and domestication of maize (Zea mays L. subspecies mays), today the world’s most important crop, enabled civilizations to flourish and has played a major role in shaping the world we know today. Archaeological and ethnobotanical research help us understand the development of the cultures and the movements of the peoples who carried maize to new areas where it continued to adapt. Ancient remains of maize cobs and kernels have been found in the place of domestication, the Balsas River Valley (~9,000 years before present era), and the cultivation center, the Tehuacan Valley (~5,000 years before present era), and have been used to study the process of domestication. Paleogenomic data showed that some of the genes controlling the stem and inflorescence architecture were comparable to modern maize, while other genes controlling ear shattering and starch biosynthesis retain high levels of variability, similar to those found in the wild relative teosinte. These results indicate that the domestication process was both gradual and complex, where different genetic loci were selected at different points in time, and that the transformation of teosinte to maize was completed in the last 5,000 years. Mesoamerican native cultures domesticated teosinte and developed maize from a 6 cm long, popping-kernel ear to what we now recognize as modern maize with its wide variety in ear size, kernel texture, color, size, and adequacy for diverse uses and also invented nixtamalization, a process key to maximizing its nutrition. Used directly for human and animal consumption, processed food products, bioenergy, and many cultural applications, it is now grown on six of the world’s seven continents. The study of its evolution and domestication from the wild grass teosinte helps us understand the nature of genetic diversity of maize and its wild relatives and gene expression. Genetic barriers to direct use of teosinte or Tripsacum in maize breeding have challenged our ability to identify valuable genes and traits, let alone incorporate them into elite, modern varieties. Genomic information and newer genetic technologies will facilitate the use of wild relatives in crop improvement; hence it is more important than ever to ensure their conservation and availability, fundamental to future food security. In situ conservation efforts dedicated to preserving remnant populations of wild relatives in Mexico are key to safeguarding the genetic diversity of maize and its genepool, as well as enabling these species to continue to adapt to dynamic climate and environmental changes. Genebank ex situ efforts are crucial to securely maintain collected wild relative resources and to provide them for gene discovery and other research efforts.


April 21, 2020  |  

Meiotic sex in Chagas disease parasite Trypanosoma cruzi.

Genetic exchange enables parasites to rapidly transform disease phenotypes and exploit new host populations. Trypanosoma cruzi, the parasitic agent of Chagas disease and a public health concern throughout Latin America, has for decades been presumed to exchange genetic material rarely and without classic meiotic sex. We present compelling evidence from 45 genomes sequenced from southern Ecuador that T. cruzi in fact maintains truly sexual, panmictic groups that can occur alongside others that remain highly clonal after past hybridization events. These groups with divergent reproductive strategies appear genetically isolated despite possible co-occurrence in vectors and hosts. We propose biological explanations for the fine-scale disconnectivity we observe and discuss the epidemiological consequences of flexible reproductive modes. Our study reinvigorates the hunt for the site of genetic exchange in the T. cruzi life cycle, provides tools to define the genetic determinants of parasite virulence, and reforms longstanding theory on clonality in trypanosomatid parasites.


April 21, 2020  |  

Extensive intraspecific gene order and gene structural variations in upland cotton cultivars.

Multiple cotton genomes (diploid and tetraploid) have been assembled. However, genomic variations between cultivars of allotetraploid upland cotton (Gossypium hirsutum L.), the most widely planted cotton species in the world, remain unexplored. Here, we use single-molecule long read and Hi-C sequencing technologies to assemble genomes of the two upland cotton cultivars TM-1 and zhongmiansuo24 (ZM24). Comparisons among TM-1 and ZM24 assemblies and the genomes of the diploid ancestors reveal a large amount of genetic variations. Among them, the top three longest structural variations are located on chromosome A08 of the tetraploid upland cotton, which account for ~30% total length of this chromosome. Haplotype analyses of the mapping population derived from these two cultivars and the germplasm panel show suppressed recombination rates in this region. This study provides additional genomic resources for the community, and the identified genetic variations, especially the reduced meiotic recombination on chromosome A08, will help future breeding.


April 21, 2020  |  

Comprehensive identification of the full-length transcripts and alternative splicing related to the secondary metabolism pathways in the tea plant (Camellia sinensis).

Flavonoids, theanine and caffeine are the main secondary metabolites of the tea plant (Camellia sinensis), which account for the tea’s unique flavor quality and health benefits. The biosynthesis pathways of these metabolites have been extensively studied at the transcriptional level, but the regulatory mechanisms are still unclear. In this study, to explore the transcriptome diversity and complexity of tea plant, PacBio Iso-Seq and RNA-seq analysis were combined to obtain full-length transcripts and to profile the changes in gene expression during the leaf development. A total of 1,388,066 reads of insert (ROI) were generated with an average length of 1,762?bp, and more than 54% (755,716) of the ROIs were full-length non-chimeric (FLNC) reads. The Benchmarking Universal Single-Copy Orthologue (BUSCO) completeness was 92.7%. A total of 93,883 non-redundant transcripts were obtained, and 87,395 (93.1%) were new alternatively spliced isoforms. Meanwhile, 7,650 differential expression transcripts (DETs) were identified. A total of 28,980 alternative splicing (AS) events were predicted, including 1,297 differential AS (DAS) events. The transcript isoforms of the key genes involved in the flavonoid, theanine and caffeine biosynthesis pathways were characterized. Additionally, 5,777 fusion transcripts and 9,052 long non-coding RNAs (lncRNAs) were also predicted. Our results revealed that AS potentially plays a crucial role in the regulation of the secondary metabolism of the tea plant. These findings enhanced our understanding of the complexity of the secondary metabolic regulation of tea plants and provided a basis for the subsequent exploration of the regulatory mechanisms of flavonoid, theanine and caffeine biosynthesis in tea plants.


April 21, 2020  |  

Multi-platform discovery of haplotype-resolved structural variation in human genomes.

The incomplete identification of structural variants (SVs) from whole-genome sequencing data limits studies of human genetic diversity and disease association. Here, we apply a suite of long-read, short-read, strand-specific sequencing technologies, optical mapping, and variant discovery algorithms to comprehensively analyze three trios to define the full spectrum of human genetic variation in a haplotype-resolved manner. We identify 818,054 indel variants (<50?bp) and 27,622 SVs (=50?bp) per genome. We also discover 156 inversions per genome and 58 of the inversions intersect with the critical regions of recurrent microdeletion and microduplication syndromes. Taken together, our SV callsets represent a three to sevenfold increase in SV detection compared to most standard high-throughput sequencing studies, including those from the 1000 Genomes Project. The methods and the dataset presented serve as a gold standard for the scientific community allowing us to make recommendations for maximizing structural variation sensitivity for future genome sequencing studies.


April 21, 2020  |  

Platanus-allee is a de novo haplotype assembler enabling a comprehensive access to divergent heterozygous regions.

The ultimate goal for diploid genome determination is to completely decode homologous chromosomes independently, and several phasing programs from consensus sequences have been developed. These methods work well for lowly heterozygous genomes, but the manifold species have high heterozygosity. Additionally, there are highly divergent regions (HDRs), where the haplotype sequences differ considerably. Because HDRs are likely to direct various interesting biological phenomena, many genomic analysis targets fall within these regions. However, they cannot be accessed by existing phasing methods, and we have to adopt costly traditional methods. Here, we develop a de novo haplotype assembler, Platanus-allee ( http://platanus.bio.titech.ac.jp/platanus2 ), which initially constructs each haplotype sequence and then untangles the assembly graphs utilizing sequence links and synteny information. A comprehensive benchmark analysis reveals that Platanus-allee exhibits high recall and precision, particularly for HDRs. Using this approach, previously unknown HDRs are detected in the human genome, which may uncover novel aspects of genome variability.


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