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

Abstract

BackgroundThe 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.ResultsIn 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.ConclusionThis 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.

Physiological characterization, transcriptomic profiling, and microsatellite marker mining of Lycium ruthenicum

“…All trimmed reads were de novo assembled into contigs using the Trinity method with an average contig size exceeding 350 bp in each of the two libraries (Table 2). This can be considered long compared with those reported in certain related studies (Wang et al, 2010;Wei et al, 2014). Among the contigs in each library, about 70% were shorter than 300 bp, about 28% ranged from 300 to 500 bp, and the remaining 2% were longer than 1000 bp (Fig.…”

“…Briefly, (Wang et al, 2010). The sequencing data were generated in Fastq format and deposited in the National Center for Biotechnology Information (NCBI) Sequence Reas Archive (SRA) database (Wheeler et al, 2002).…”

De-novo characterization of the soft-shelled turtle Pelodiscus sinensis transcriptome using Illumina RNA-Seq technology

“…The raw reads were cleaned by removing adaptor sequences, empty reads, and low-quality sequences. The data filtering process was previously described in Wang et al (2010a) and Xie et al (2012). Reads were then assembled using Trinity method (Grabherr et al, 2011).…”

“…Over the past several years, NGS technologies have emerged as powerful tools for high-throughput sequence analysis and dramatically improved the speed and efficiency of gene discovery (Schuster 2008;Metzker, 2010). Moreover, these technologies have shown great potential for expanding sequence databases of not only model species (Hegedűs et al, 2009;Hillier et al, 2009;, but also non-model organisms (Collins et al, 2008;Wang et al, 2010a;Gao et al, 2012). Recently, the development of high-throughput DNA sequencing strategies has provided a new means of both mapping and quantifying transcriptomes, which is the complete set of all transcripts for certain types of cells or tissues in a specific developmental stage or physiological condition.…”

“…The method, termed RNA sequencing (RNA-Seq) is an efficient and powerful method to analyze transcriptome data and has clear advantages over the existing approaches 2010a). A primary platform being used to generate transcriptomic resources is the Illumina short read sequencing platform, followed by de-novo assembly of sequence reads.…”