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ITS2 Secondary Structure Improves Discrimination between Medicinal "Mu Tong" Species when Using DNA Barcoding.

Zhang W, Yuan Y, Yang S, Huang J, Huang L - PLoS ONE (2015)

Bottom Line: The results showed that the ITS2 structures in our samples had a common four-helix folding type with some shared motifs.As a result, the different species that are often referred to as the herb "Mu tong" were successfully identified using short fragments of 250 bp ITS2 sequences, together with their secondary structure.Thus our analysis strengthens the potential of ITS2 as a promising DNA barcode because it incorporates valuable secondary structure information that will help improve discrimination between species.

View Article: PubMed Central - PubMed

Affiliation: Marine College, Shandong University at Weihai, Weihai, Shandong, China; State Key Laboratory of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China.

ABSTRACT
DNA barcoding is a promising species identification method, but it has proved difficult to find a standardized DNA marker in plant. Although the ITS/ITS2 RNA transcript has been proposed as the core barcode for seed plants, it has been criticized for being too conserved in some species to provide enough information or too variable in some species to align it within the different taxa ranks. We selected 30 individuals, representing 16 species and four families, to explore whether ITS2 can successfully resolve species in terms of secondary structure. Secondary structure was predicted using Mfold software and sequence-structure was aligned by MARNA. RNAstat software transformed the secondary structures into 28 symbol code data for maximum parsimony (MP) analysis. The results showed that the ITS2 structures in our samples had a common four-helix folding type with some shared motifs. This conserved structure facilitated the alignment of ambiguous sequences from divergent families. The structure alignment yielded a MP tree, in which most topological relationships were congruent with the tree constructed using nucleotide sequence data. When the data was combined, we obtained a well-resolved and highly supported phylogeny, in which individuals of a same species were clustered together into a monophyletic group. As a result, the different species that are often referred to as the herb "Mu tong" were successfully identified using short fragments of 250 bp ITS2 sequences, together with their secondary structure. Thus our analysis strengthens the potential of ITS2 as a promising DNA barcode because it incorporates valuable secondary structure information that will help improve discrimination between species.

No MeSH data available.


Related in: MedlinePlus

The predicted ITS2 secondary structure of the “Mu tong” taxa.(a) Consensus secondary structure model of the ITS2 region based on 30 sequences covering four families (Actinidiaceae, Aristolochiaceae, Lardizabalaceae and Ranunculaceae). The four helices, each with a stem–loop, are labeled I–IV. Compatible base pairs are colored and the degree of conservation over the whole alignment is indicated with different degrees of color saturation. (b) One of the example secondary structures of Akebia quinata. (c) The position of the strictly conserved base pair sites (highlight in blue circle) found in the 70% consensus ITS2 secondary structure model.
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pone.0131185.g001: The predicted ITS2 secondary structure of the “Mu tong” taxa.(a) Consensus secondary structure model of the ITS2 region based on 30 sequences covering four families (Actinidiaceae, Aristolochiaceae, Lardizabalaceae and Ranunculaceae). The four helices, each with a stem–loop, are labeled I–IV. Compatible base pairs are colored and the degree of conservation over the whole alignment is indicated with different degrees of color saturation. (b) One of the example secondary structures of Akebia quinata. (c) The position of the strictly conserved base pair sites (highlight in blue circle) found in the 70% consensus ITS2 secondary structure model.

Mentions: ITS2 was aligned into 30 accessions from four families of “Mu tong” herbs. The accessions had a typical four-helix folding consensus structure, among which helix III was the longest, and helix IV was the most variable (Fig 1). Helix II was more stable, and contained a pyrimidine-pyrimidine bulge and a non-canonical U(T)-G base pair. In addition, some conserved motifs were also found in the helices, such as CUCC in helix I, C-G pairs in helix II, and GCGG and CGAUC in helix III (Fig 1a and 1b). The RNAstat analysis showed that the structure sequence ranged from 214 bp to 297 bp in length and the aligned length was 314 bp, among which 15 base pairs were strictly conserved (Fig 1c). The ITS2 nucleotide sequences were aligned, based on these shared structure constraints, and it is interesting that the alignment result was different from the Clustal X alignment, where only the primary homologous sequences were considered. There were differences between the number and position of the indels, and in the whole alignment length (Fig 2).


ITS2 Secondary Structure Improves Discrimination between Medicinal "Mu Tong" Species when Using DNA Barcoding.

Zhang W, Yuan Y, Yang S, Huang J, Huang L - PLoS ONE (2015)

The predicted ITS2 secondary structure of the “Mu tong” taxa.(a) Consensus secondary structure model of the ITS2 region based on 30 sequences covering four families (Actinidiaceae, Aristolochiaceae, Lardizabalaceae and Ranunculaceae). The four helices, each with a stem–loop, are labeled I–IV. Compatible base pairs are colored and the degree of conservation over the whole alignment is indicated with different degrees of color saturation. (b) One of the example secondary structures of Akebia quinata. (c) The position of the strictly conserved base pair sites (highlight in blue circle) found in the 70% consensus ITS2 secondary structure model.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4488503&req=5

pone.0131185.g001: The predicted ITS2 secondary structure of the “Mu tong” taxa.(a) Consensus secondary structure model of the ITS2 region based on 30 sequences covering four families (Actinidiaceae, Aristolochiaceae, Lardizabalaceae and Ranunculaceae). The four helices, each with a stem–loop, are labeled I–IV. Compatible base pairs are colored and the degree of conservation over the whole alignment is indicated with different degrees of color saturation. (b) One of the example secondary structures of Akebia quinata. (c) The position of the strictly conserved base pair sites (highlight in blue circle) found in the 70% consensus ITS2 secondary structure model.
Mentions: ITS2 was aligned into 30 accessions from four families of “Mu tong” herbs. The accessions had a typical four-helix folding consensus structure, among which helix III was the longest, and helix IV was the most variable (Fig 1). Helix II was more stable, and contained a pyrimidine-pyrimidine bulge and a non-canonical U(T)-G base pair. In addition, some conserved motifs were also found in the helices, such as CUCC in helix I, C-G pairs in helix II, and GCGG and CGAUC in helix III (Fig 1a and 1b). The RNAstat analysis showed that the structure sequence ranged from 214 bp to 297 bp in length and the aligned length was 314 bp, among which 15 base pairs were strictly conserved (Fig 1c). The ITS2 nucleotide sequences were aligned, based on these shared structure constraints, and it is interesting that the alignment result was different from the Clustal X alignment, where only the primary homologous sequences were considered. There were differences between the number and position of the indels, and in the whole alignment length (Fig 2).

Bottom Line: The results showed that the ITS2 structures in our samples had a common four-helix folding type with some shared motifs.As a result, the different species that are often referred to as the herb "Mu tong" were successfully identified using short fragments of 250 bp ITS2 sequences, together with their secondary structure.Thus our analysis strengthens the potential of ITS2 as a promising DNA barcode because it incorporates valuable secondary structure information that will help improve discrimination between species.

View Article: PubMed Central - PubMed

Affiliation: Marine College, Shandong University at Weihai, Weihai, Shandong, China; State Key Laboratory of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China.

ABSTRACT
DNA barcoding is a promising species identification method, but it has proved difficult to find a standardized DNA marker in plant. Although the ITS/ITS2 RNA transcript has been proposed as the core barcode for seed plants, it has been criticized for being too conserved in some species to provide enough information or too variable in some species to align it within the different taxa ranks. We selected 30 individuals, representing 16 species and four families, to explore whether ITS2 can successfully resolve species in terms of secondary structure. Secondary structure was predicted using Mfold software and sequence-structure was aligned by MARNA. RNAstat software transformed the secondary structures into 28 symbol code data for maximum parsimony (MP) analysis. The results showed that the ITS2 structures in our samples had a common four-helix folding type with some shared motifs. This conserved structure facilitated the alignment of ambiguous sequences from divergent families. The structure alignment yielded a MP tree, in which most topological relationships were congruent with the tree constructed using nucleotide sequence data. When the data was combined, we obtained a well-resolved and highly supported phylogeny, in which individuals of a same species were clustered together into a monophyletic group. As a result, the different species that are often referred to as the herb "Mu tong" were successfully identified using short fragments of 250 bp ITS2 sequences, together with their secondary structure. Thus our analysis strengthens the potential of ITS2 as a promising DNA barcode because it incorporates valuable secondary structure information that will help improve discrimination between species.

No MeSH data available.


Related in: MedlinePlus