Dataset Information

Single-molecule real-time sequencing identifies massive full-length cDNAs and alternative-splicing events that facilitate comparative and functional genomics study in the hexaploid crop sweet potato.

ABSTRACT: Background:Sweet potato (Ipomoea batatas (L.) Lam.) is one of the most important crops in many developing countries and provides a candidate source of bioenergy. However, neither a complete reference genome nor large-scale full-length cDNA sequences for this outcrossing hexaploid crop are available, which in turn impedes progress in research studies in I. batatas functional genomics and molecular breeding. Methods:In this study, we sequenced full-length transcriptomes in I. batatas and its diploid ancestor I. trifida by single-molecule real-time sequencing and Illumina second-generation sequencing technologies. With the generated datasets, we conducted comprehensive intraspecific and interspecific sequence analyses and experimental characterization. Results:A total of 53,861/51,184 high-quality long-read transcripts were obtained, which covered about 10,439/10,452 loci in the I. batatas/I. trifida genome. These datasets enabled us to predict open reading frames successfully in 96.83%/96.82% of transcripts and identify 34,963/33,637 full-length cDNA sequences, 1,401/1,457 transcription factors, 25,315/27,090 simple sequence repeats, 1,656/1,389 long non-coding RNAs, and 5,251/8,901 alternative splicing events. Approximately, 32.34%/38.54% of transcripts and 46.22%/51.18% multi-exon transcripts underwent alternative splicing in I. batatas/I. trifida. Moreover, we validated one alternative splicing event in each of 10 genes and identified tuberous-root-specific expressed isoforms from a starch-branching enzyme, an alpha-glucan phosphorylase, a neutral invertase, and several ABC transporters. Overall, the collection and analysis of large-scale long-read transcripts generated in this study will serve as a valuable resource for the I. batatas research community, which may accelerate the progress in its structural, functional, and comparative genomics studies.

SUBMITTER: Ding N

PROVIDER: S-EPMC6859871 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Single-molecule real-time sequencing identifies massive full-length cDNAs and alternative-splicing events that facilitate comparative and functional genomics study in the hexaploid crop sweet potato.

Ding Na N Cui Huihui H Miao Ying Y Tang Jun J Cao Qinghe Q Luo Yonghai Y

PeerJ 20191115

<h4>Background</h4>Sweet potato (<i>Ipomoea batatas</i> (L.) Lam.) is one of the most important crops in many developing countries and provides a candidate source of bioenergy. However, neither a complete reference genome nor large-scale full-length cDNA sequences for this outcrossing hexaploid crop are available, which in turn impedes progress in research studies in <i>I. batatas</i> functional genomics and molecular breeding.<h4>Methods</h4>In this study, we sequenced full-length transcriptome ...[more]

PMID: 31741783

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Project description:Purple-fleshed sweet potato (PFSP) is an important food crop, as it is a rich source of nutrients and anthocyanin pigments. Drought has become a major threat to sustainable sweetpotato production, resulting in huge yield losses. Therefore, the present study was conducted to identify drought stress-responsive genes using next-generation (NGS) and third-generation sequencing (TGS) techniques. Five cDNA libraries were constructed from seedling leaf segments treated with a 30% solution of polyethylene glycol (PEG-6000) for 0, 1, 6, 12, and 48 h for second-generation sequencing. Leaf samples taken from upper third of sweet potato seedlings after 1, 6, 12, and 48 h of drought stress were used for the construction of cDNA libraries for third-generation sequencing; however, leaf samples from untreated plants were collected as controls. A total of 184,259,679 clean reads were obtained using second and third-generation sequencing and then assembled into 17,508 unigenes with an average length of 1,783 base pairs. Out of 17,508 unigenes, 642 (3.6%) unigenes failed to hit any homologs in any databases, which might be considered novel genes. A total of 2, 920, 1578, and 2,418 up-regulated unigenes and 3,834, 2,131, and 3,337 down-regulated unigenes from 1 h, 6 h, 12 h, and 48 h library were identified, respectively in drought stress versus control. In addition, after 6, 12, and 48 h of drought stress, 540 up-regulated unigenes, 486 down-regulated unigenes and 414 significantly differentially expressed unigenes were detected. It was found that several gene families including Basic Helix-loop-helix (bHLH), basic leucine zipper (bZIP), Cystein2/Histidine2 (C2H2), C3H, Ethylene-responsive transcription factor (ERF), Homo domain-leucine zipper (HD-ZIP), MYB, NAC (NAM, ATAF1/2, and CUC2), Thiol specific antioxidant and WRKY showed responses to drought stress. In total, 17,472 simple sequence repeats and 510,617 single nucleotide polymorphisms were identified based on transcriptome sequencing of the PFSP. About 96.55% of the obtained sequences are not available online in sweet potato genomics resources. Therefore, it will enrich annotated sweet potato gene sequences and enhance understanding of the mechanisms of drought tolerance through genetic manipulation. Moreover, it represents a sequence resource for genetic and genomic studies of sweet potato.

Project description:BackgroundCrop plants such as rice, maize and sorghum play economically-important roles as main sources of food, fuel, and animal feed. However, current genome annotations of crop plants still suffer false-positive predictions; a more comprehensive registry of alternative splicing (AS) events is also in demand. Comparative genomics of crop plants is largely unexplored.ResultsWe performed a large-scale comparative analysis (ExonFinder) of the expressed sequence tag (EST) library from nine grass plants against three crop genomes (rice, maize, and sorghum) and identified 2,879 previously-unannotated exons (i.e., novel exons) in the three crops. We validated 81% of the tested exons by RT-PCR-sequencing, supporting the effectiveness of our in silico strategy. Evolutionary analysis reveals that the novel exons, comparing with their flanking annotated ones, are generally under weaker selection pressure at the protein level, but under stronger pressure at the RNA level, suggesting that most of the novel exons also represent novel alternatively spliced variants (ASVs). However, we also observed the consistency of evolutionary rates between certain novel exons and their flanking exons, which provided further evidence of their co-occurrence in the transcripts, suggesting that previously-annotated isoforms might be subject to erroneous predictions. Our validation showed that 54% of the tested genes expressed the newly-identified isoforms that contained the novel exons, rather than the previously-annotated isoforms that excluded them. The consistent results were steadily observed across cultivated (Oryza sativa and O. glaberrima) and wild (O. rufipogon and O. nivara) rice species, asserting the necessity of our curation of the crop genome annotations. Our comparative analyses also inferred the common ancestral transcriptome of grass plants and gain- and loss-of-ASV events.ConclusionsWe have reannotated the rice, maize, and sorghum genomes, and showed that evolutionary rates might serve as an indicator for determining whether the identified exons were alternatively spliced. This study not only presents an effective in silico strategy for the improvement of plant annotations, but also provides further insights into the role of AS events in the evolution and domestication of crop plants. ExonFinder and the novel exons/ASVs identified are publicly accessible at http://exonfinder.sourceforge.net/ .

Dataset Information

Single-molecule real-time sequencing identifies massive full-length cDNAs and alternative-splicing events that facilitate comparative and functional genomics study in the hexaploid crop sweet potato.

Publications

Single-molecule real-time sequencing identifies massive full-length cDNAs and alternative-splicing events that facilitate comparative and functional genomics study in the hexaploid crop sweet potato.

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