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. 2023 Mar 11;14(3):697.
doi: 10.3390/genes14030697.

DNA Barcoding, Phylogenetic Analysis and Secondary Structure Predictions of Nepenthes ampullaria, Nepenthes gracilis and Nepenthes rafflesiana

Nur Azreen Saidon  1 , Alina Wagiran  1 , Abdul Fatah A Samad  1 , Faezah Mohd Salleh  1 , Farhan Mohamed  2 , Jaeyres Jani  3 , Alona C Linatoc  4   5
Affiliations

DNA Barcoding, Phylogenetic Analysis and Secondary Structure Predictions of Nepenthes ampullaria, Nepenthes gracilis and Nepenthes rafflesiana

Nur Azreen Saidon et al. Genes (Basel). .

Abstract

Nepentheceae, the most prominent carnivorous family in the Caryophyllales order, comprises the Nepenthes genus, which has modified leaf trap characteristics. Although most Nepenthes species have unique morphologies, their vegetative stages are identical, making identification based on morphology difficult. DNA barcoding is seen as a potential tool for plant identification, with small DNA segments amplified for species identification. In this study, three barcode loci; ribulose-bisphosphate carboxylase (rbcL), intergenic spacer 1 (ITS1) and intergenic spacer 2 (ITS2) and the usefulness of the ITS1 and ITS2 secondary structure for the molecular identification of Nepenthes species were investigated. An analysis of barcodes was conducted using BLASTn, pairwise genetic distance and diversity, followed by secondary structure prediction. The findings reveal that PCR and sequencing were both 100% successful. The present study showed the successful amplification of all targeted DNA barcodes at different sizes. Among the three barcodes, rbcL was the least efficient as a DNA barcode compared to ITS1 and ITS2. The ITS1 nucleotide analysis revealed that the ITS1 barcode had more variations compared to ITS2. The mean genetic distance (K2P) between them was higher for interspecies compared to intraspecies. The results showed that the DNA barcoding gap existed among Nepenthes species, and differences in the secondary structure distinguish the Nepenthes. The secondary structure generated in this study was found to successfully discriminate between the Nepenthes species, leading to enhanced resolutions.

Keywords: DNA barcode; ITS1; ITS2; Nepenthes; barcoding gap; phylogenetic; rbcL; secondary structure predictions.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The geographical location (a) of the Nepenthes collected from Bukit Janing, Kampung Peta, Endau-Rompin National Park, Johor, Malaysia (GPS location; 2.529908870220549, 103.41185691378324). Nepenthes species collected: (b) Nepenthes ampullaria, (c) Nepenthes gracilis and (d) Nepenthes rafflesiana.
Figure 2
Figure 2
Multiple sequence alignment of rbcL (a), ITS1 (b) and ITS2 (c) barcodes.
Figure 3
Figure 3
Neighbor joining tree of rbcL (a), ITS1 (b) and ITS2 (c) barcode primers depicting the phylogenetic analysis among N. ampullaria, N. gracilis, and N. rafflesiana (indicated in the red boxes).
Figure 3
Figure 3
Neighbor joining tree of rbcL (a), ITS1 (b) and ITS2 (c) barcode primers depicting the phylogenetic analysis among N. ampullaria, N. gracilis, and N. rafflesiana (indicated in the red boxes).
Figure 4
Figure 4
Barcode gap analysis of N. gracilis, N. ampullaria, and N. rafflesiana generated by Automatic Barcode Discovery Gap Discovery (https://bioinfo.mnhn.fr/abi/public/abgd/abgdweb.html, accessed on 23 January 2023) [25] for (a) rbcL, (b) ITS2 and (c) ITS1. Distributions of K2P distances and between each pair of specimens for the histogram of distance.
Figure 5
Figure 5
The variations observed in the predicted minimum free energy (MFE) (a–c), DNA barcodes (d–f), and secondary structures of the ITS1 region for the three Nepenthes species (g–i).
Figure 6
Figure 6
The variations observed in the predicted minimum free energy (MFE) (a–c), DNA barcodes (d–f), and secondary structures of the ITS2 region for the three Nepenthes species (g–i).
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References

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