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September 22, 2019  |  

A survey of the complex transcriptome from the highly polyploid sugarcane genome using full-length isoform sequencing and de novo assembly from short read sequencing.

Despite the economic importance of sugarcane in sugar and bioenergy production, there is not yet a reference genome available. Most of the sugarcane transcriptomic studies have been based on Saccharum officinarum gene indices (SoGI), expressed sequence tags (ESTs) and de novo assembled transcript contigs from short-reads; hence knowledge of the sugarcane transcriptome is limited in relation to transcript length and number of transcript isoforms.The sugarcane transcriptome was sequenced using PacBio isoform sequencing (Iso-Seq) of a pooled RNA sample derived from leaf, internode and root tissues, of different developmental stages, from 22 varieties, to explore the potential for capturing full-length transcript isoforms. A total of 107,598 unique transcript isoforms were obtained, representing about 71% of the total number of predicted sugarcane genes. The majority of this dataset (92%) matched the plant protein database, while just over 2% was novel transcripts, and over 2% was putative long non-coding RNAs. About 56% and 23% of total sequences were annotated against the gene ontology and KEGG pathway databases, respectively. Comparison with de novo contigs from Illumina RNA-Sequencing (RNA-Seq) of the internode samples from the same experiment and public databases showed that the Iso-Seq method recovered more full-length transcript isoforms, had a higher N50 and average length of largest 1,000 proteins; whereas a greater representation of the gene content and RNA diversity was captured in RNA-Seq. Only 62% of PacBio transcript isoforms matched 67% of de novo contigs, while the non-matched proportions were attributed to the inclusion of leaf/root tissues and the normalization in PacBio, and the representation of more gene content and RNA classes in the de novo assembly, respectively. About 69% of PacBio transcript isoforms and 41% of de novo contigs aligned with the sorghum genome, indicating the high conservation of orthologs in the genic regions of the two genomes.The transcriptome dataset should contribute to improved sugarcane gene models and sugarcane protein predictions; and will serve as a reference database for analysis of transcript expression in sugarcane.

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September 22, 2019  |  

Ecological genomics of tropical trees: how local population size and allelic diversity of resistance genes relate to immune responses, cosusceptibility to pathogens, and negative density dependence

In tropical forests, rarer species show increased sensitivity to species-specific soil pathogens and more negative effects of conspecific density on seedling survival (NDD). These patterns suggest a connection between ecology and immunity, perhaps because small population size disproportionately reduces genetic diversity of hyperdiverse loci such as immunity genes. In an experiment examining seedling roots from six species in one tropical tree community, we found that smaller populations have reduced amino acid diversity in pathogen resistance (R) genes but not the transcriptome in general. Normalized R gene amino acid diversity varied with local abundance and prior measures of differences in sensitivity to conspecific soil and NDD. After exposure to live soil, species with lower R gene diversity had reduced defence gene induction, more cosusceptibility of maternal cohorts to colonization by potentially pathogenic fungi, reduced root growth arrest (an R gene-mediated response) and their root-associated fungi showed lower induction of self-defence (antioxidants). Local abundance was not related to the ability to induce immune responses when pathogen recognition was bypassed by application of salicylic acid, a phytohormone that activates defence responses downstream of R gene signalling. These initial results support the hypothesis that smaller local tree populations have reduced R gene diversity and recognition-dependent immune responses, along with greater cosusceptibility to species-specific pathogens that may facilitate disease transmission and NDD. Locally rare species may be less able to increase their equilibrium abundance without genetic boosts to defence via immigration of novel R gene alleles from a larger and more diverse regional population.

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September 22, 2019  |  

Transcriptome comparative analysis of salt stress responsiveness in chrysanthemum (Dendranthema grandiflorum) roots by Illumina- and Single-Molecule Real-Time-based RNA sequencing.

Salt response has long been considered a polygenic-controlled character in plants. Under salt stress conditions, plants respond by activating a great amount of proteins and enzymes. To develop a better understanding of the molecular mechanism and screen salt responsive genes in chrysanthemum under salt stress, we performed the RNA sequencing (RNA-seq) on both salt-processed chrysanthemum seedling roots and the control group, and gathered six cDNA databases eventually. Moreover, to overcome the Illumina HiSeq technology’s limitation on sufficient length of reads and improve the quality and accuracy of the result, we combined Illumina HiSeq with single-molecule real-time sequencing (SMRT-seq) to decode the full-length transcripts. As a result, we successfully collected 550,823 unigenes, and from which we selected 48,396 differentially expressed genes (DEGs). Many of these DEGs were associated with the signal transduction, biofilm system, antioxidant system, and osmotic regulation system, such as mitogen-activated protein kinase (MAPK), Acyl-CoA thioesterase (ACOT), superoxide (SOD), catalase (CAT), peroxisomal membrane protein (PMP), and pyrroline-5-carboxylate reductase (P5CR). The quantitative real-time polymerase chain reaction (qRT-PCR) analysis of 15 unigenes was performed to test the data validity. The results were highly consistent with the RNA-seq results. In all, these findings could facilitate further detection of the responsive molecular mechanism under salt stress. They also provided more accurate candidate genes for genetic engineering on salt-tolerant chrysanthemums.

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September 22, 2019  |  

High-quality reference transcript datasets hold the key to transcript-specific RNA-sequencing analysis in plants.

Re-programming of the transcriptome involves both transcription and alternative splicing (AS). Some genes are regulated only at the AS level with no change in expression at the gene level. AS data must be incorporated as an essential aspect of the regulation of gene expression. RNA-sequencing (RNA-seq) can deliver both transcriptional and AS information, but accurate methods to analyse the added complexity in RNA-seq data are needed. The construction of a comprehensive reference transcript dataset (RTD) for a specific plant species, variety or accession, from all available sequence data, will immediately allow more robust analysis of RNA-seq data. RTDs will continually evolve and improve, a process that will be more efficient if resources across a community are shared and pooled.© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

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September 22, 2019  |  

Bacterial diversity and community structure in Chongqing radish paocai brines revealed using PacBio single-molecule real-time sequencing technology.

Traditional Chongqing radish paocai fermented with aged brine is considered to have the most intense flavor and authentic taste. Eight ‘Yanzhi’ (red, RRPB group) and ‘Chunbulao’ (white, WRPB) radish paocai brine samples were collected from Chongqing peasant households, and the diversity and community structures of bacteria present in these brines were determined using PacBio single-molecule real-time sequencing of their full-length 16S rRNA genes.In total, 30 phyla, 218 genera, and 306 species were identified from the RRPB group, with 20 phyla, 261 genera, and 420 species present in the WRPB group. Obvious differences in bacterial profiles between the RRPB and WRPB groups were found, with the bacterial diversity of the WRPB group shown to be greater than that of the RRPB group. This study revealed several characteristics of the bacteria composition, including the predominance of heterofermentative lactic acid bacteria, the species diversity of genus Pseudomonas, and the presence of three opportunistic pathogenic species.This study provides detailed information on the bacterial diversity and community structure of Chongqing radish paocai brine samples, and suggests it may be necessary to analyze paocai brine for potential sources of bacterial contamination and take appropriate measures to exclude any pathogenic species. © 2018 Society of Chemical Industry.© 2018 Society of Chemical Industry.

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September 22, 2019  |  

De novo assembly and characterizing of the culm-derived meta-transcriptome from the polyploid sugarcane genome based on coding transcripts

Sugarcane biomass has been used for sugar, bioenergy and biomaterial production. The majority of the sugarcane biomass comes from the culm, which makes it important to understand the genetic control of biomass production in this part of the plant. A meta-transcriptome of the culm was obtained in an earlier study by using about one billion paired-end (150 bp) reads of deep RNA sequencing of samples from 20 diverse sugarcane genotypes and combining de novo assemblies from different assemblers and different settings. Although many genes could be recovered, this resulted in a large combined assembly which created the need for clustering to reduce transcript redundancy while maintaining gene content. Here, we present a comprehensive analysis of the effect of different assembly settings and clustering methods on de novo assembly, annotation and transcript profiling focusing especially on the coding transcripts from the highly polyploid sugarcane genome. The new coding sequence-based transcript clustering resulted in a better representation of transcripts compared to the earlier approach, having 121,987 contigs, which included 78,052 main and 43,935 alternative transcripts. About 73%, 67%, 61% and 10% of the transcriptome was annotated against the NCBI NR protein database, GO terms, orthologous groups and KEGG orthologies, respectively. Using this set for a differential gene expression analysis between the young and mature sugarcane culm tissues, a total of 822 transcripts were found to be differentially expressed, including key transcripts involved in sugar/fiber accumulation in sugarcane. In the context of the lack of a whole genome sequence for sugarcane, the availability of a well annotated culm-derived meta-transcriptome through deep sequencing provides useful information on coding genes specific to the sugarcane culm and will certainly contribute to understanding the process of carbon partitioning, and biomass accumulation in the sugarcane culm.

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September 22, 2019  |  

Single-molecule real-time transcript sequencing facilitates common wheat genome annotation and grain transcriptome research.

The large and complex hexaploid genome has greatly hindered genomics studies of common wheat (Triticum aestivum, AABBDD). Here, we investigated transcripts in common wheat developing caryopses using the emerging single-molecule real-time (SMRT) sequencing technology PacBio RSII, and assessed the resultant data for improving common wheat genome annotation and grain transcriptome research.We obtained 197,709 full-length non-chimeric (FLNC) reads, 74.6 % of which were estimated to carry complete open reading frame. A total of 91,881 high-quality FLNC reads were identified and mapped to 16,188 chromosomal loci, corresponding to 13,162 known genes and 3026 new genes not annotated previously. Although some FLNC reads could not be unambiguously mapped to the current draft genome sequence, many of them are likely useful for studying highly similar homoeologous or paralogous loci or for improving chromosomal contig assembly in further research. The 91,881 high-quality FLNC reads represented 22,768 unique transcripts, 9591 of which were newly discovered. We found 180 transcripts each spanning two or three previously annotated adjacent loci, suggesting that they should be merged to form correct gene models. Finally, our data facilitated the identification of 6030 genes differentially regulated during caryopsis development, and full-length transcripts for 72 transcribed gluten gene members that are important for the end-use quality control of common wheat.Our work demonstrated the value of PacBio transcript sequencing for improving common wheat genome annotation through uncovering the loci and full-length transcripts not discovered previously. The resource obtained may aid further structural genomics and grain transcriptome studies of common wheat.

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September 22, 2019  |  

Global analysis of epigenetic regulation of gene expression in response to drought stress in Sorghum.

Abiotic stresses including drought are major limiting factors of crop yields and cause significant crop losses. Acquisition of stress tolerance to abiotic stresses requires coordinated regulation of a multitude of biochemical and physiological changes, and most of these changes depend on alterations in gene expression. The goal of this work is to perform global analysis of differential regulation of gene expression and alternative splicing, and their relationship with chromatin landscape in drought sensitive and tolerant cultivars. our Iso-Seq study revealed transcriptome-wide full-length isoforms at an unprecedented scale with over 11000 novel splice isoforms. Additionally, we uncovered alternative polyadenylation sites of ~11000 expressed genes and many novel genes. Overall, Iso-Seq results greatly enhanced sorghum gene annotations that are not only useful in analyentified differentially expressed genes and splicing events that are correlated with tzing all our RNA-seq, ChIP-seq and ATAC-seq data but also serve as a great resource to the plant biology community. Our studies idhe drought-resistant phenotype. An association between alternative splicing and chromatin accessibility was also revealed. Several computational tools developed here (TAPIS and iDiffIR) have been made freely available to the research community in analyzing alternative splicing and differential alternative splicing.

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September 22, 2019  |  

PacBio for haplotyping in gene families.

The throughput and read length provided by Pacific Bioscience (PacBio) Single Molecule Real Time (SMRT) sequencing platform makes it feasible to construct contiguous, non-chimeric sequences. This is especially useful for genes with repetitive sequences in their gene bodies in gene families. We illustrate the use of PacBio to sequence and assemble hundreds of transcripts of gluten gene families from different cultivars of wheat using sequence from a single SMRT cell. To this end, we barcoded amplicons from different cultivars, then pooled these into one library for sequencing. Sequencing reads were later separated by the barcodes and further sorted into different gene groups by blast. The reads from each gene are then assembled by SeqmanNGen software. Given the length of 1 kb for each sequence derived from an initial molecule, the phase of the polymorphisms is not lost and can be used to infer also haplotype differences between different cultivars.

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September 22, 2019  |  

Genome-wide analysis of complex wheat gliadins, the dominant carriers of celiac disease epitopes.

Gliadins, specified by six compound chromosomal loci (Gli-A1/B1/D1 and Gli-A2/B2/D2) in hexaploid bread wheat, are the dominant carriers of celiac disease (CD) epitopes. Because of their complexity, genome-wide characterization of gliadins is a strong challenge. Here, we approached this challenge by combining transcriptomic, proteomic and bioinformatic investigations. Through third-generation RNA sequencing, full-length transcripts were identified for 52 gliadin genes in the bread wheat cultivar Xiaoyan 81. Of them, 42 were active and predicted to encode 25 a-, 11 ?-, one d- and five ?-gliadins. Comparative proteomic analysis between Xiaoyan 81 and six newly-developed mutants each lacking one Gli locus indicated the accumulation of 38 gliadins in the mature grains. A novel group of a-gliadins (the CSTT group) was recognized to contain very few or no CD epitopes. The d-gliadins identified here or previously did not carry CD epitopes. Finally, the mutant lacking Gli-D2 showed significant reductions in the most celiac-toxic a-gliadins and derivative CD epitopes. The insights and resources generated here should aid further studies on gliadin functions in CD and the breeding of healthier wheat.

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September 22, 2019  |  

De novo assembly of a Chinese soybean genome.

Soybean was domesticated in China and has become one of the most important oilseed crops. Due to bottlenecks in their introduction and dissemination, soybeans from different geographic areas exhibit extensive genetic diversity. Asia is the largest soybean market; therefore, a high-quality soybean reference genome from this area is critical for soybean research and breeding. Here, we report the de novo assembly and sequence analysis of a Chinese soybean genome for “Zhonghuang 13” by a combination of SMRT, Hi-C and optical mapping data. The assembled genome size is 1.025 Gb with a contig N50 of 3.46 Mb and a scaffold N50 of 51.87 Mb. Comparisons between this genome and the previously reported reference genome (cv. Williams 82) uncovered more than 250,000 structure variations. A total of 52,051 protein coding genes and 36,429 transposable elements were annotated for this genome, and a gene co-expression network including 39,967 genes was also established. This high quality Chinese soybean genome and its sequence analysis will provide valuable information for soybean improvement in the future.

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September 22, 2019  |  

Full-length RNA sequencing reveals unique transcriptome composition in bermudagrass.

Bermudagrass [Cynodon dactylon (L.) Pers.] is an important perennial warm-season turfgrass species with great economic value. However, the reference genome and transcriptome information are still deficient in bermudagrass, which severely impedes functional and molecular breeding studies. In this study, through analyzing a mixture sample of leaves, stolons, shoots, roots and flowers with single-molecule long-read sequencing technology from Pacific Biosciences (PacBio), we reported the first full-length transcriptome dataset of bermudagrass (C. dactylon cultivar Yangjiang) comprising 78,192 unigenes. Among the unigenes, 66,409 were functionally annotated, whereas 27,946 were found to have two or more isoforms. The annotated full-length unigenes provided many new insights into gene sequence characteristics and systematic phylogeny of bermudagrass. By comparison with transcriptome dataset in nine grass species, KEGG pathway analyses further revealed that C4 photosynthesis-related genes, notably the phosphoenolpyruvate carboxylase and pyruvate, phosphate dikinase genes, are specifically enriched in bermudagrass. These results not only explained the possible reason why bermudagrass flourishes in warm areas but also provided a solid basis for future studies in this important turfgrass species. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

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September 22, 2019  |  

Assessment of an organ-specific de novo transcriptome of the nematode trap-crop, Solanum sisymbriifolium

Solanum sisymbriifolium, also known as “Litchi Tomato” or “Sticky Nightshade,” is an undomesticated and poorly researched plant related to potato and tomato. Unlike the latter species, S. sisymbriifolium induces eggs of the cyst nematode, Globodera pallida, to hatch and migrate into its roots, but then arrests further nematode maturation. In order to provide researchers with a partial blueprint of its genetic make-up so that the mechanism of this response might be identified, we used single molecule real time (SMRT) sequencing to compile a high quality de novo transcriptome of 41,189 unigenes drawn from individually sequenced bud, root, stem, and leaf RNA populations. Functional annotation and BUSCO analysis showed that this transcriptome was surprisingly complete, even though it represented genes expressed at a single time point. By sequencing the 4 organ libraries separately, we found we could get a reliable snapshot of transcript distributions in each organ. A divergent site analysis of the merged transcriptome indicated that this species might have undergone a recent genome duplication and re-diploidization. Further analysis indicated that the plant then retained a disproportionate number of genes associated with photosynthesis and amino acid metabolism in comparison to genes with characteristics of R-proteins or involved in secondary metabolism. The former processes may have given S. sisymbriifolium a bigger competitive advantage than the latter did. Copyright © 2018 Wixom et al.

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September 22, 2019  |  

Comparative mapping of the ASTRINGENCY locus controlling fruit astringency in hexaploid persimmon (Diospyros kaki Thunb.) with the diploid D. lotus reference genome

Persimmon (Diospyros kaki) is a tree crop species that originated in East Asia, consists mainly of hexaploid individuals (2n = 6x = 90) with some nonaploid individuals. One of the unique characteristics of persimmon is the continuous accumulation of proanthocyanidins (PAs) in its fruit until the middle of fruit development, resulting in a strong astringent taste even at commercial fruit maturity. Among persimmon cultivars, pollination-constant and non-astringent (PCNA) types cease PA accumulation in early fruit development and become non-astringent at commercial maturity. PCNA is an allelic trait to non-PCNA and is controlled by a single locus called the ASTRINGENCY (AST) locus. Previous segregation analyses indicated that the AST locus shows hexasomic inheritance; a recessive allele, ast, at this locus confers PCNA. Here, we report a shuttle mapping approach to delimit the AST locus region in the hexaploid persimmon genome by using D. lotus, a diploid relative of D. kaki, as a reference. A D. lotus F1 population of 333 individuals and 296 D. kaki siblings segregating for the PCNA trait were used to map the AST region using haplotype-specific markers covering the AST region. This indicated that the AST locus is syntenic to an approximately 915-kb region of the D. lotus genome. In this 915-kb region, we found several candidates for AST that were revealed from the fruit transcriptome of a population segregating for the PCNA trait. These results could provide important clues for the isolation of AST in hexaploid persimmon.

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September 22, 2019  |  

An ancient integration in a plant NLR is maintained as a trans-species polymorphism

Plant immune receptors are under constant selective pressure to maintain resistance to plant pathogens. Nucleotide-binding leucine-rich repeat (NLR) proteins are one class of cytoplasmic immune receptors whose genes commonly show signatures of adaptive evolution. While it is known that balancing selection contributes to maintaining high intraspecific allelic diversity, the evolutionary mechanism that influences the transmission of alleles during speciation remains unclear. The barley Mla locus has over 30 described alleles conferring isolate-specific resistance to barley powdery mildew and contains three NLR families (RGH1, RGH2, and RGH3). We discovered (using sequence capture and RNAseq) the presence of a novel integrated Exo70 domain in RGH2 in the Mla3 haplotype. Allelic variation across barley accessions includes presence/absence of the integrated domain in RGH2. Expanding our search to several Poaceae species, we found shared interspecific conservation in the RGH2-Exo70 integration. We hypothesise that balancing selection has maintained allelic variation at Mla as a trans-species polymorphism over 24 My, thus contributing to and preserving interspecific allelic diversity during speciation.

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