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Efficient Detection of Novel Nuclear Markers for Brassicaceae by Transcriptome Sequencing.

Stockenhuber R, Zoller S, Shimizu-Inatsugi R, Gugerli F, Shimizu KK, Widmer A, Fischer MC - PLoS ONE (2015)

Bottom Line: Sequence analysis and phylogenetic reconstruction with a subset of these markers on different levels of phylogenetic divergence in the mustard family were compared with previous studies.The results corroborate the usefulness of the newly developed primer pairs, e.g., for phylogenetic analyses or population genetic studies.Thus, our method provides a cost-effective approach for designing nuclear loci across a broad range of taxa and is compatible with current NGS technologies.

View Article: PubMed Central - PubMed

Affiliation: Institute of Evolutionary Biology and Environmental Studies, University of Zurich, Zurich, Switzerland; Institute of Integrative Biology, ETH Zurich, Zurich, Switzerland.

ABSTRACT
The lack of DNA sequence information for most non-model organisms impairs the design of primers that are universally applicable for the study of molecular polymorphisms in nuclear markers. Next-generation sequencing (NGS) techniques nowadays provide a powerful approach to overcome this limitation. We present a flexible and inexpensive method to identify large numbers of nuclear primer pairs that amplify in most Brassicaceae species. We first obtained and mapped NGS transcriptome sequencing reads from two of the distantly related Brassicaceae species, Cardamine hirsuta and Arabis alpina, onto the Arabidopsis thaliana reference genome, and then identified short conserved sequence motifs among the three species bioinformatically. From these, primer pairs to amplify coding regions (nuclear protein coding loci, NPCL) and exon-primed intron-crossing sequences (EPIC) were developed. We identified 2,334 universally applicable primer pairs, targeting 1,164 genes, which provide a large pool of markers as readily usable genomic resource that will help addressing novel questions in the Brassicaceae family. Testing a subset of the newly designed nuclear primer pairs revealed that a great majority yielded a single amplicon in all of the 30 investigated Brassicaceae taxa. Sequence analysis and phylogenetic reconstruction with a subset of these markers on different levels of phylogenetic divergence in the mustard family were compared with previous studies. The results corroborate the usefulness of the newly developed primer pairs, e.g., for phylogenetic analyses or population genetic studies. Thus, our method provides a cost-effective approach for designing nuclear loci across a broad range of taxa and is compatible with current NGS technologies.

No MeSH data available.


Workflow for the identification of conserved nucleotide sequences in multiple Brassicaceae species and subsequent primer design.Blue boxes refer to the three major steps in the workflow, white boxes indicate the general steps taken Explanations on the right provide specific results from this study.
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pone.0128181.g001: Workflow for the identification of conserved nucleotide sequences in multiple Brassicaceae species and subsequent primer design.Blue boxes refer to the three major steps in the workflow, white boxes indicate the general steps taken Explanations on the right provide specific results from this study.

Mentions: To detect conserved sequence regions within the Brassicaceae, we used the available high- quality reference genome of Arabidopsis thaliana and additionally sequenced the transcriptomes of two divergent Brassicaceae species, Cardamine hirsuta and Arabis alpina. Sequencing reads from these two species were then aligned against the A. thaliana reference genome. These three species represent different lineages of the Brassicaceae family and hence should allow to detect shared, conserved regions suitable for designing universally applicable Brassicaceae primers. Cardamine hirsuta as well as A. thaliana are members of lineage I according to Al-Shehbaz [38], and diverged relatively early. Arabis alpina belongs to the Arabideae, the largest tribe of Brassicaceae [38], which is part of the expanded lineage II sensu Franzke et al. [39]. Dating the evolutionary history of Brassicaceae is particularly difficult, mostly due to few fossil records [40] and rapid radiation events [37,39,41]. A recent study [42] dated the split of lineage I and II to 27 million years ago (mya). Divergence time between Cardamine and Arabidopsis has been estimated to be at least 13 mya [35,43]. Therefore, the selection of these three different taxa reflects a divergence of nearly 30 million years and hence they cover a broad evolutionary range across the Brassicaceae, which allowed us to identify conserved regions among these species. The workflow is presented in Fig 1 and explained in detail below.


Efficient Detection of Novel Nuclear Markers for Brassicaceae by Transcriptome Sequencing.

Stockenhuber R, Zoller S, Shimizu-Inatsugi R, Gugerli F, Shimizu KK, Widmer A, Fischer MC - PLoS ONE (2015)

Workflow for the identification of conserved nucleotide sequences in multiple Brassicaceae species and subsequent primer design.Blue boxes refer to the three major steps in the workflow, white boxes indicate the general steps taken Explanations on the right provide specific results from this study.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4465667&req=5

pone.0128181.g001: Workflow for the identification of conserved nucleotide sequences in multiple Brassicaceae species and subsequent primer design.Blue boxes refer to the three major steps in the workflow, white boxes indicate the general steps taken Explanations on the right provide specific results from this study.
Mentions: To detect conserved sequence regions within the Brassicaceae, we used the available high- quality reference genome of Arabidopsis thaliana and additionally sequenced the transcriptomes of two divergent Brassicaceae species, Cardamine hirsuta and Arabis alpina. Sequencing reads from these two species were then aligned against the A. thaliana reference genome. These three species represent different lineages of the Brassicaceae family and hence should allow to detect shared, conserved regions suitable for designing universally applicable Brassicaceae primers. Cardamine hirsuta as well as A. thaliana are members of lineage I according to Al-Shehbaz [38], and diverged relatively early. Arabis alpina belongs to the Arabideae, the largest tribe of Brassicaceae [38], which is part of the expanded lineage II sensu Franzke et al. [39]. Dating the evolutionary history of Brassicaceae is particularly difficult, mostly due to few fossil records [40] and rapid radiation events [37,39,41]. A recent study [42] dated the split of lineage I and II to 27 million years ago (mya). Divergence time between Cardamine and Arabidopsis has been estimated to be at least 13 mya [35,43]. Therefore, the selection of these three different taxa reflects a divergence of nearly 30 million years and hence they cover a broad evolutionary range across the Brassicaceae, which allowed us to identify conserved regions among these species. The workflow is presented in Fig 1 and explained in detail below.

Bottom Line: Sequence analysis and phylogenetic reconstruction with a subset of these markers on different levels of phylogenetic divergence in the mustard family were compared with previous studies.The results corroborate the usefulness of the newly developed primer pairs, e.g., for phylogenetic analyses or population genetic studies.Thus, our method provides a cost-effective approach for designing nuclear loci across a broad range of taxa and is compatible with current NGS technologies.

View Article: PubMed Central - PubMed

Affiliation: Institute of Evolutionary Biology and Environmental Studies, University of Zurich, Zurich, Switzerland; Institute of Integrative Biology, ETH Zurich, Zurich, Switzerland.

ABSTRACT
The lack of DNA sequence information for most non-model organisms impairs the design of primers that are universally applicable for the study of molecular polymorphisms in nuclear markers. Next-generation sequencing (NGS) techniques nowadays provide a powerful approach to overcome this limitation. We present a flexible and inexpensive method to identify large numbers of nuclear primer pairs that amplify in most Brassicaceae species. We first obtained and mapped NGS transcriptome sequencing reads from two of the distantly related Brassicaceae species, Cardamine hirsuta and Arabis alpina, onto the Arabidopsis thaliana reference genome, and then identified short conserved sequence motifs among the three species bioinformatically. From these, primer pairs to amplify coding regions (nuclear protein coding loci, NPCL) and exon-primed intron-crossing sequences (EPIC) were developed. We identified 2,334 universally applicable primer pairs, targeting 1,164 genes, which provide a large pool of markers as readily usable genomic resource that will help addressing novel questions in the Brassicaceae family. Testing a subset of the newly designed nuclear primer pairs revealed that a great majority yielded a single amplicon in all of the 30 investigated Brassicaceae taxa. Sequence analysis and phylogenetic reconstruction with a subset of these markers on different levels of phylogenetic divergence in the mustard family were compared with previous studies. The results corroborate the usefulness of the newly developed primer pairs, e.g., for phylogenetic analyses or population genetic studies. Thus, our method provides a cost-effective approach for designing nuclear loci across a broad range of taxa and is compatible with current NGS technologies.

No MeSH data available.