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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: 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.

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.


Phylogenetic inference at the family level.Best Maximum Likelihood phylogram of concatenated gene sequences are shown. Bootstrap support values and posterior probabilities are given above or below the corresponding branches, respectively. Values below 50/0.5 are omitted. "Lineage"-brackets refer to lineages sensu Al-Shehbaz (2012). Percentage amplification success per species is given in brackets next to each species name.
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pone.0128181.g002: Phylogenetic inference at the family level.Best Maximum Likelihood phylogram of concatenated gene sequences are shown. Bootstrap support values and posterior probabilities are given above or below the corresponding branches, respectively. Values below 50/0.5 are omitted. "Lineage"-brackets refer to lineages sensu Al-Shehbaz (2012). Percentage amplification success per species is given in brackets next to each species name.

Mentions: The phylogenetic tree at the family level based on eight concatenated nuclear markers (3,154 bp in total) showed good support for the three major Brassicaceae lineages (Fig 2). "Lineages I", "II" and "III" were strongly supported (BS ≥ 81; PP ≥ 0.98), but basal nodes of "lineage II" were not well resolved with either maximum likelihood or posterior probability inferences. The obtained phylogeny was compared with Couvreur et al. [35] and with the BrassiBase phylogeny [59]. Overall, we obtained good support for all branches identified in these studies. Incongruences were found in the placement of Cochlearia, as well as the non-basal position of Biscutella in our results, whereas traditionally the corresponding tribe (Biscutelleae) was placed as a sister to all major lineages.


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)

Phylogenetic inference at the family level.Best Maximum Likelihood phylogram of concatenated gene sequences are shown. Bootstrap support values and posterior probabilities are given above or below the corresponding branches, respectively. Values below 50/0.5 are omitted. "Lineage"-brackets refer to lineages sensu Al-Shehbaz (2012). Percentage amplification success per species is given in brackets next to each species name.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0128181.g002: Phylogenetic inference at the family level.Best Maximum Likelihood phylogram of concatenated gene sequences are shown. Bootstrap support values and posterior probabilities are given above or below the corresponding branches, respectively. Values below 50/0.5 are omitted. "Lineage"-brackets refer to lineages sensu Al-Shehbaz (2012). Percentage amplification success per species is given in brackets next to each species name.
Mentions: The phylogenetic tree at the family level based on eight concatenated nuclear markers (3,154 bp in total) showed good support for the three major Brassicaceae lineages (Fig 2). "Lineages I", "II" and "III" were strongly supported (BS ≥ 81; PP ≥ 0.98), but basal nodes of "lineage II" were not well resolved with either maximum likelihood or posterior probability inferences. The obtained phylogeny was compared with Couvreur et al. [35] and with the BrassiBase phylogeny [59]. Overall, we obtained good support for all branches identified in these studies. Incongruences were found in the placement of Cochlearia, as well as the non-basal position of Biscutella in our results, whereas traditionally the corresponding tribe (Biscutelleae) was placed as a sister to all major lineages.

Bottom Line: 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.

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.