Global Transcriptome Sequencing Using the Illumina Platform and the Development of EST-SSR Markers in Autotetraploid Alfalfa

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Abstract

Background

Alfalfa is the most widely cultivated forage legume and one of the most economically valuable crops in the world. The large size and complexity of the alfalfa genome has delayed the development of genomic resources for alfalfa research. Second-generation Illumina transcriptome sequencing is an efficient method for generating a global transcriptome sequence dataset for gene discovery and molecular marker development in alfalfa.

Methodology/Principal Findings

More than 28 million sequencing reads (5.64 Gb of clean nucleotides) were generated by Illumina paired-end sequencing from 15 different alfalfa tissue samples. In total, 40,433 unigenes with an average length of 803 bp were obtained by de novo assembly. Based on a sequence similarity search of known proteins, a total of 36,684 (90.73%) unigenes were annotated. In addition, 1,649 potential EST-SSRs were identified as potential molecular markers from unigenes with lengths exceeding 1 kb. A total of 100 pairs of PCR primers were randomly selected to validate the assembly quality and develop EST-SSR markers from genomic DNA. Of these primer pairs, 82 were able to amplify sequences in initial screening tests, and 27 primer pairs successfully amplified DNA fragments and detected significant amounts of polymorphism among 10 alfalfa accessions.

Conclusions/Significance

The present study provided global sequence data for autotetraploid alfalfa and demonstrates the Illumina platform is a fast and effective approach to EST-SSR markers development in alfalfa. The use of these transcriptome datasets will serve as a valuable public information platform to accelerate studies of the alfalfa genome.

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De novo assembly and characterization of root transcriptome using Illumina paired-end sequencing and development of cSSR markers in sweetpotato (Ipomoea batatas)

Zhangying Wang, Boping Fang, Jingyi Chen … (2010)

Background The tuberous root of sweetpotato is an important agricultural and biological organ. There are not sufficient transcriptomic and genomic data in public databases for understanding of the molecular mechanism underlying the tuberous root formation and development. Thus, high throughput transcriptome sequencing is needed to generate enormous transcript sequences from sweetpotato root for gene discovery and molecular marker development. Results In this study, more than 59 million sequencing reads were generated using Illumina paired-end sequencing technology. De novo assembly yielded 56,516 unigenes with an average length of 581 bp. Based on sequence similarity search with known proteins, a total of 35,051 (62.02%) genes were identified. Out of these annotated unigenes, 5,046 and 11,983 unigenes were assigned to gene ontology and clusters of orthologous group, respectively. Searching against the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG) indicated that 17,598 (31.14%) unigenes were mapped to 124 KEGG pathways, and 11,056 were assigned to metabolic pathways, which were well represented by carbohydrate metabolism and biosynthesis of secondary metabolite. In addition, 4,114 cDNA SSRs (cSSRs) were identified as potential molecular markers in our unigenes. One hundred pairs of PCR primers were designed and used for validation of the amplification and assessment of the polymorphism in genomic DNA pools. The result revealed that 92 primer pairs were successfully amplified in initial screening tests. Conclusion This study generated a substantial fraction of sweetpotato transcript sequences, which can be used to discover novel genes associated with tuberous root formation and development and will also make it possible to construct high density microarrays for further characterization of gene expression profiles during these processes. Thousands of cSSR markers identified in the present study can enrich molecular markers and will facilitate marker-assisted selection in sweetpotato breeding. Overall, these sequences and markers will provide valuable resources for the sweetpotato community. Additionally, these results also suggested that transcriptome analysis based on Illumina paired-end sequencing is a powerful tool for gene discovery and molecular marker development for non-model species, especially those with large and complex genome.

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Characterization of the sesame (Sesamum indicum L.) global transcriptome using Illumina paired-end sequencing and development of EST-SSR markers

Wenliang Wei, Xiaoqiong Qi, Linhai Wang … (2011)

Background Sesame is an important oil crop, but limited transcriptomic and genomic data are currently available. This information is essential to clarify the fatty acid and lignan biosynthesis molecular mechanism. In addition, a shortage of sesame molecular markers limits the efficiency and accuracy of genetic breeding. High-throughput transcriptomic sequencing is essential to generate a large transcriptome sequence dataset for gene discovery and molecular marker development. Results Sesame transcriptomes from five tissues were sequenced using Illumina paired-end sequencing technology. The cleaned raw reads were assembled into a total of 86,222 unigenes with an average length of 629 bp. Of the unigenes, 46,584 (54.03%) had significant similarity with proteins in the NCBI nonredundant protein database and Swiss-Prot database (E-value < 10-5). Of these annotated unigenes, 10,805 and 27,588 unigenes were assigned to gene ontology categories and clusters of orthologous groups, respectively. In total, 22,003 (25.52%) unigenes were mapped onto 119 pathways using the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG). Furthermore, 44,750 unigenes showed homology to 15,460 Arabidopsis genes based on BLASTx analysis against The Arabidopsis Information Resource (TAIR, Version 10) and revealed relatively high gene coverage. In total, 7,702 unigenes were converted into SSR markers (EST-SSR). Dinucleotide SSRs were the dominant repeat motif (67.07%, 5,166), followed by trinucleotide (24.89%, 1,917), tetranucleotide (4.31%, 332), hexanucleotide (2.62%, 202), and pentanucleotide (1.10%, 85) SSRs. AG/CT (46.29%) was the dominant repeat motif, followed by AC/GT (16.07%), AT/AT (10.53%), AAG/CTT (6.23%), and AGG/CCT (3.39%). Fifty EST-SSRs were randomly selected to validate amplification and to determine the degree of polymorphism in the genomic DNA pools. Forty primer pairs successfully amplified DNA fragments and detected significant amounts of polymorphism among 24 sesame accessions. Conclusions This study demonstrates that Illumina paired-end sequencing is a fast and cost-effective approach to gene discovery and molecular marker development in non-model organisms. Our results provide a comprehensive sequence resource for sesame research.

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De novo sequencing and analysis of the American ginseng root transcriptome using a GS FLX Titanium platform to discover putative genes involved in ginsenoside biosynthesis

Chao Sun, Ying Li, Qiong Wu … (2010)

Background American ginseng (Panax quinquefolius L.) is one of the most widely used herbal remedies in the world. Its major bioactive constituents are the triterpene saponins known as ginsenosides. However, little is known about ginsenoside biosynthesis in American ginseng, especially the late steps of the pathway. Results In this study, a one-quarter 454 sequencing run produced 209,747 high-quality reads with an average sequence length of 427 bases. De novo assembly generated 31,088 unique sequences containing 16,592 contigs and 14,496 singletons. About 93.1% of the high-quality reads were assembled into contigs with an average 8-fold coverage. A total of 21,684 (69.8%) unique sequences were annotated by a BLAST similarity search against four public sequence databases, and 4,097 of the unique sequences were assigned to specific metabolic pathways by the Kyoto Encyclopedia of Genes and Genomes. Based on the bioinformatic analysis described above, we found all of the known enzymes involved in ginsenoside backbone synthesis, starting from acetyl-CoA via the isoprenoid pathway. Additionally, a total of 150 cytochrome P450 (CYP450) and 235 glycosyltransferase unique sequences were found in the 454 cDNA library, some of which encode enzymes responsible for the conversion of the ginsenoside backbone into the various ginsenosides. Finally, one CYP450 and four UDP-glycosyltransferases were selected as the candidates most likely to be involved in ginsenoside biosynthesis through a methyl jasmonate (MeJA) inducibility experiment and tissue-specific expression pattern analysis based on a real-time PCR assay. Conclusions We demonstrated, with the assistance of the MeJA inducibility experiment and tissue-specific expression pattern analysis, that transcriptome analysis based on 454 pyrosequencing is a powerful tool for determining the genes encoding enzymes responsible for the biosynthesis of secondary metabolites in non-model plants. Additionally, the expressed sequence tags (ESTs) and unique sequences from this study provide an important resource for the scientific community that is interested in the molecular genetics and functional genomics of American ginseng.

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Author and article information

Contributors

Shu-Biao Wu: Role: Editor

Journal

Journal ID (nlm-ta): PLoS One

Journal ID (iso-abbrev): PLoS ONE

Journal ID (publisher-id): plos

Journal ID (pmc): plosone

Title: PLoS ONE

Publisher: Public Library of Science (San Francisco, USA )

ISSN (Electronic): 1932-6203

Publication date Collection: 2013

Publication date (Electronic): 12 December 2013

Volume: 8

Issue: 12

Electronic Location Identifier: e83549

Affiliations

[1 ]State Key Laboratory of Grassland Agro-ecosystems, School of Pastoral Agricultural Science and Technology, Lanzhou University, Lanzhou, China

[2 ]Key Laboratory of Cell Activities and Stress Adaptations, Ministry of Education, School of Life Sciences, Lanzhou University, Lanzhou, China

[3 ]Plant Biology Division, The Samuel Roberts Noble Foundation, Ardmore, Oklahoma, United States of America

University of New England, Australia

Author notes

* E-mail: lzp@ 123456lzu.edu.cn (ZPL); yrwang@ 123456lzu.edu.cn (YRW)

Competing Interests: The authors have declared that no competing interests exist.

Conceived and designed the experiments: YRW ZPL. Performed the experiments: ZPL TLC LCM. Analyzed the data: ZPL ZGZ PXZ. Contributed reagents/materials/analysis tools: ZPL YRW. Wrote the manuscript: ZPL YRW ZBN.

Article

Publisher ID: PONE-D-13-11992

DOI: 10.1371/journal.pone.0083549

PMC ID: 3861513

PubMed ID: 24349529

SO-VID: bd9897e0-c153-434c-9fd6-9efb9362a307

License:

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

History

Date received : 20 March 2013

Date accepted : 5 November 2013

Funding

This research was supported by the National Basic Research Program of China (2014CB138704), the National Natural Science Foundation of China (31072072 and 31272492), and the Fundamental Research Funds for the Central Universities (lzujbky-2012-94). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Global Transcriptome Sequencing Using the Illumina Platform and the Development of EST-SSR Markers in Autotetraploid Alfalfa

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Abstract

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Most cited references 23

De novo assembly and characterization of root transcriptome using Illumina paired-end sequencing and development of cSSR markers in sweetpotato (Ipomoea batatas)

Characterization of the sesame (Sesamum indicum L.) global transcriptome using Illumina paired-end sequencing and development of EST-SSR markers

De novo sequencing and analysis of the American ginseng root transcriptome using a GS FLX Titanium platform to discover putative genes involved in ginsenoside biosynthesis

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