Dataset Information

Comparisons of de novo transcriptome assemblers in diploid and polyploid species using peanut (Arachis spp.) RNA-Seq data.

ABSTRACT: The narrow genetic base and limited genetic information on Arachis species have hindered the process of marker-assisted selection of peanut cultivars. However, recent developments in sequencing technologies have expanded opportunities to exploit genetic resources, and at lower cost. To use the genetic information for Arachis species available at the transcriptome level, it is important to have a good quality reference transcriptome. The available Tifrunner 454 FLEX transcriptome sequences have an assembly with 37,000 contigs and low N50 values of 500-751 bp. Therefore, we generated de novo transcriptome assemblies, with about 38 million reads in the tetraploid cultivar OLin, and 16 million reads in each of the diploids, A. duranensis K38901 and A. ipaënsis KGBSPSc30076 using three different de novo assemblers, Trinity, SOAPdenovo-Trans and TransAByss. All these assemblers can use single kmer analysis, and the latter two also permit multiple kmer analysis. Assemblies generated for all three samples had N50 values ranging from 1278-1641 bp in Arachis hypogaea (AABB), 1401-1492 bp in Arachis duranensis (AA), and 1107-1342 bp in Arachis ipaënsis (BB). Comparison with legume ESTs and protein databases suggests that assemblies generated had more than 40% full length transcripts with good continuity. Also, on mapping the raw reads to each of the assemblies generated, Trinity had a high success rate in assembling sequences compared to both TransAByss and SOAPdenovo-Trans. De novo assembly of OLin had a greater number of contigs (67,098) and longer contig length (N50?= 1,641) compared to the Tifrunner TSA. Despite having shorter read length (2 × 50) than the Tifrunner 454FLEX TSA, de novo assembly of OLin proved superior in comparison. Assemblies generated to represent different genome combinations may serve as a valuable resource for the peanut research community.

SUBMITTER: Chopra R

PROVIDER: S-EPMC4281230 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Comparisons of de novo transcriptome assemblers in diploid and polyploid species using peanut (Arachis spp.) RNA-Seq data.

Chopra Ratan R Burow Gloria G Farmer Andrew A Mudge Joann J Simpson Charles E CE Burow Mark D MD

PloS one 20141231 12

The narrow genetic base and limited genetic information on Arachis species have hindered the process of marker-assisted selection of peanut cultivars. However, recent developments in sequencing technologies have expanded opportunities to exploit genetic resources, and at lower cost. To use the genetic information for Arachis species available at the transcriptome level, it is important to have a good quality reference transcriptome. The available Tifrunner 454 FLEX transcriptome sequences have a ...[more]

PMID: 25551607

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Project description:Roche 454 pyrosequencing has become a method of choice for generating transcriptome data from non-model organisms. Once the tens to hundreds of thousands of short (250-450 base) reads have been produced, it is important to correctly assemble these to estimate the sequence of all the transcripts. Most transcriptome assembly projects use only one program for assembling 454 pyrosequencing reads, but there is no evidence that the programs used to date are optimal. We have carried out a systematic comparison of five assemblers (CAP3, MIRA, Newbler, SeqMan and CLC) to establish best practices for transcriptome assemblies, using a new dataset from the parasitic nematode Litomosoides sigmodontis.Although no single assembler performed best on all our criteria, Newbler 2.5 gave longer contigs, better alignments to some reference sequences, and was fast and easy to use. SeqMan assemblies performed best on the criterion of recapitulating known transcripts, and had more novel sequence than the other assemblers, but generated an excess of small, redundant contigs. The remaining assemblers all performed almost as well, with the exception of Newbler 2.3 (the version currently used by most assembly projects), which generated assemblies that had significantly lower total length. As different assemblers use different underlying algorithms to generate contigs, we also explored merging of assemblies and found that the merged datasets not only aligned better to reference sequences than individual assemblies, but were also more consistent in the number and size of contigs.Transcriptome assemblies are smaller than genome assemblies and thus should be more computationally tractable, but are often harder because individual contigs can have highly variable read coverage. Comparing single assemblers, Newbler 2.5 performed best on our trial data set, but other assemblers were closely comparable. Combining differently optimal assemblies from different programs however gave a more credible final product, and this strategy is recommended.

Project description:BackgroundAquaporins (AQPs) facilitate transport of water and small solutes across cell membranes and play an important role in different physiological processes in plants. Despite their importance, limited data is available about AQP distribution and function in the economically important oilseed crop peanut, Arachis hypogea (AABB). The present study reports the identification and structural and expression analysis of the AQPs found in the diploid progenitor genomes of A. hypogea i.e. Arachis duranensis (AA) and Arachis ipaensis (BB).ResultsGenome-wide analysis revealed the presence of 32 and 36 AQPs in A. duranensis and A. ipaensis, respectively. Phylogenetic analysis showed similar numbers of AQPs clustered in five distinct subfamilies including the plasma membrane intrinsic proteins (PIPs), the tonoplast intrinsic proteins (TIPs), the nodulin 26-like intrinsic proteins (NIPs), the small basic intrinsic proteins (SIPs), and the uncharacterized intrinsic proteins (XIPs). A notable exception was the XIP subfamily where XIP1 group was observed only in A. ipaensis genome. Protein structure evaluation showed a hydrophilic aromatic/arginine (ar/R) selectivity filter (SF) in PIPs whereas other subfamilies mostly contained a hydrophobic ar/R SF. Both genomes contained one NIP2 with a GSGR SF indicating a conserved ability within the genus to uptake silicon. Analysis of RNA-seq data from A. hypogea revealed a similar expression pattern for the different AQP paralogs of AA and BB genomes. The TIP3s showed seed-specific expression while the NIP1s' expression was confined to roots and root nodules.ConclusionsThe identification and the phylogenetic analysis of AQPs in both Arachis species revealed the presence of all five sub-families of AQPs. Within the NIP subfamily, the presence of a NIP2 in both genomes supports a conserved ability to absorb Si within plants of the genus. The global expression profile of AQPs in A. hypogea revealed a similar pattern of AQP expression regardless of the subfamilies or the genomes. The tissue-specific expression of AQPs suggests an important role in the development and function of the respective organs. The AQPs identified in the present study will serve as a resource for further characterization and possible exploitation of AQPs to understand their physiological role in A. hypogea.

Project description:BackgroundCultivated peanut (Arachis hypogaea) is an allotetraploid species whose ancestral genomes are most likely derived from the A-genome species, A. duranensis, and the B-genome species, A. ipaensis. The very recent (several millennia) evolutionary origin of A. hypogaea has imposed a bottleneck for allelic and phenotypic diversity within the cultigen. However, wild diploid relatives are a rich source of alleles that could be used for crop improvement and their simpler genomes can be more easily analyzed while providing insight into the structure of the allotetraploid peanut genome. The objective of this research was to establish a high-density genetic map of the diploid species A. duranensis based on de novo generated EST databases. Arachis duranensis was chosen for mapping because it is the A-genome progenitor of cultivated peanut and also in order to circumvent the confounding effects of gene duplication associated with allopolyploidy in A. hypogaea.ResultsMore than one million expressed sequence tag (EST) sequences generated from normalized cDNA libraries of A. duranensis were assembled into 81,116 unique transcripts. Mining this dataset, 1236 EST-SNP markers were developed between two A. duranensis accessions, PI 475887 and Grif 15036. An additional 300 SNP markers also were developed from genomic sequences representing conserved legume orthologs. Of the 1536 SNP markers, 1054 were placed on a genetic map. In addition, 598 EST-SSR markers identified in A. hypogaea assemblies were included in the map along with 37 disease resistance gene candidate (RGC) and 35 other previously published markers. In total, 1724 markers spanning 1081.3 cM over 10 linkage groups were mapped. Gene sequences that provided mapped markers were annotated using similarity searches in three different databases, and gene ontology descriptions were determined using the Medicago Gene Atlas and TAIR databases. Synteny analysis between A. duranensis, Medicago and Glycine revealed significant stretches of conserved gene clusters spread across the peanut genome. A higher level of colinearity was detected between A. duranensis and Glycine than with Medicago.ConclusionsThe first high-density, gene-based linkage map for A. duranensis was generated that can serve as a reference map for both wild and cultivated Arachis species. The markers developed here are valuable resources for the peanut, and more broadly, to the legume research community. The A-genome map will have utility for fine mapping in other peanut species and has already had application for mapping a nematode resistance gene that was introgressed into A. hypogaea from A. cardenasii.

Dataset Information

Comparisons of de novo transcriptome assemblers in diploid and polyploid species using peanut (Arachis spp.) RNA-Seq data.

Publications

Comparisons of de novo transcriptome assemblers in diploid and polyploid species using peanut (Arachis spp.) RNA-Seq data.

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