Molecular identification and evolutionary relationships between the subspecies of Musa by DNA barcodes

- Molecular identification and evolutionary relationships between the subspecies of Musa by DNA barcodes.
- Results: Hence, in the present study, we used universal DNA barcode ITS2 region to identify and to find the genetic relationship between the cultivars and varieties of banana.
- Conclusion: Thus, from the results of the present study, it is clear that ITS2 is not only an efficient DNA barcode to identify the banana species but also a potential candidate for enumerating the phylogenetic relationships between the subspecies and cultivars.
- Banana and plantain belong to the family Musaceae and are cultivated throughout tropical and subtropical re- gions of the world [1].
- The edible Musa species and their hybrids and polyploids originated from the two main wild species of banana, viz., Musa acuminata Colla and M.
- There have been extensive discussions related to the identities of the progenitors of domesticated banana.
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- These wild species are extensively distributed in the sub- tropical and tropical regions of Asia.
- balbisiana and these hybrids are found to exhibit sev- eral commonly occurring morphological characters..
- nagensium and was provided with the photograph of the former.
- The occurrence of genetic diversity has been satisfactorily docu- mented in the pool of M.
- The total number of cultivars in bananas and plantains was estimated to be around 300–1000 and their nomenclature and descriptions are found to be highly am- biguous even within a country [13].
- Cultivated bananas are found to differ markedly from their wild relatives due to multiplication through vegetative propagation, exhibiting a high level of morphological diversification [7].
- Accordingly, later formulated classification schemes were found to be unambiguous and coherent and were accepted widely..
- Though the cultivated banana has socio-economic import- ance, genetic studies are found to be limited in this flora due to the occurrence of extensive polyploidy and par- thenocarpy together with complexity associated with sam- ple collection protocols.
- It is realized that correct identification of Musa cultivars is crucial for utilization of this crop species and also important for conservation of the genetic resources.
- DNA finger- printing methodologies are found to be useful in detecting the relationship between parental genotypes with progeny populations [19].
- With the current status of research in this area of foolproof identification of Musa species and cultivars, a simple and accurate method is highly required for determining the genetic variation between the different cultivars of Musa species..
- The internal transcribed spacer 2 (ITS2) is located between the ribosomal 5.8S and 28S, which is ac- tively involved in the regulation of the transcription of ac- tive ribosomal subunits and it is essential for pre-rRNA processing [32].
- Due to this universality, in the current scenario, ITS2 has been considered to be a standard barcode for au- thenticating different medicinal plants [33–36].
- Recently DNA barcoding has been appropriately employed in clear identification of the different varieties of plants, imported teas [37] and small millet land races [38].
- In the present study, DNA barcoding analysis was performed for the ba- nana cultivars and wild Musa accessions using the internal transcribed spacer region ITS2 for a better understanding of the origin and domestication of cultivated banana and to clear the confusions that exist in the nomenclature and varietal synonyms..
- The amplification and sequence success rate of the ITS2 sequences from sampled specimens of Musa sp.
- found to be 100%.
- The lengths of the ITS2 sequences used for the analyses were in the range of 325–375 bp, with an average of 345 bp.
- The inter-specific distance was found to be in the range of equaled 0.002 for only 0.26% and the proportion of inter-specific gen- etic distance <.
- The intra-specific distance ranged from 0.000 to 0.135, and most Musa species with more than two samples in our study had a unique sequence (58.93%) in the ITS2 region.
- ITS2 showed 97.7 and 95.8% identification success rates at the species level for the 321 analysed samples of Musa using BLAST1 and nearest genetic distance, respectively..
- Only two species which were found to fall under the exception category, viz., M.
- ITS2 region showed higher polymorphic sites representing higher genetic diversity in between subspecies and cultivars of Musa.
- Unique haplotypes of Musa species and subspecies were identified by using re- striction enzymes, such as, MseI, PstI and AvaII, respect- ively and are shown in Table 3..
- We found that nucleotide diversity at non- synonymous sites ITS2 region was reduced in the A gen- ome of wild species represented as shown in Table 4.
- However, it was found that the genetic diver- sity of the AAA genome was 4-6 and folds higher than A genome cultivars.
- Thus, the observations of the present study showed no significant departure from the neutral model..
- The molecular classification of Musa species is based on DNA based profiling [39, 40].
- Sequences for the subspecies and hybrids were obtained from the GenBank and the sequence of the species Ensete ventricosum was treated as an outgroup.
- In clade A, cultivar red banana was found to be evolutionarily re- lated to wild species, viz., M.
- Further the cultivar red banana was found to be closer to subspecies M.
- The clade B consisted of 5 cultivars, viz., Pisang lilin, M.
- flava isolate, chemmatti, grandnain and nadan and all those cultivars were found to be closely related to the banana subspecies, viz., M..
- Among the 4 cultivars belonging to clade C, 3 cultivars, viz., Njalipoovan, Matti and Kunnan, were found to be closely related to wild species of banana, viz., M.
- The species M.
- ingens are distinct from the species M.
- maclayi, which exhibited high similarity index and were placed in the clade III..
- lutea were placed in the neighboring section of clade V.
- textilis were found to share same allelic profiles with the wild species M.
- A single cultivar was separated from the wild species M.
- basjoo, which is found to be distinct from the wild species, viz., M.
- Results of the present study showed that the restriction enzymes - MseI and AvaII, provided the best discrimin- atory power to differentiate the haploids of Musa species using ITS2 sequences.
- AvaII showed one, two and three restriction sites in the II group of 3 ge- nomes, respectively..
- A common problem for banana researchers and horti- culturists in Southeast Asia is the presence of numer- ous cultivar names and synonyms in different languages in the region.
- recognition is vital to certify the fruits and plantlets of Musa sp.
- To our knowledge, this is the first report wherein DNA barcoding has been employed in the identification of different species and cultivars of Musa using a large sample size.
- In the present study, ITS2 was found to possess a sufficient variable region between the different species and cultivars for the determination of genetic divergence with high discriminatory ability..
- Morphological characters were found to resolve M..
- PCR-RFLP of the ITS region using RsaI restric- tion endonuclease was used on 68 banana accessions, which showed consistent and distinguishing poly- morphic banding of DNA patterns between the wild species and cultivars of M.
- Based on the results of the present study, we propose that ITS2 can be an ideal DNA barcode candidate for Table 2 Identification efficiency for ITS2 using BLAST1 and DISTANCE methods.
- However, it is pertinent to mention that the phylogeny of the members of the Musaceae remains still controversial.
- To cite a few cases, it is known that the taxonomic position of the species M.
- Hence, the taxonomic assignment of culti- vars of Musa based on the ITS2 and its discriminating power in firmly assigning the identifi- cation and nomenclature of the members of the Musaceae might prove to be conclusive..
- Clade A is found to contain two clusters.
- balbsiana seems to be closer to M..
- Based on BLAST1 and distance-based identi- fication methods the cultivars - red banana and ro- busta of AAA genome was found to be closer to subspecies M.
- malaccensis, on the basis of the above- mentioned two methodologies, respectively..
- In the clade B, the species M.
- banksii x Musa schizocarpa was closely related to the wild species M..
- The left side shows the complete list of Musa species used in this study.
- and distance based identification methods, the A gen- ome of cultivar Pisang lilin was found to show 99 and 98.9% similarity with Musa acuminata var.
- The cultivar chemmatti AA and Grandnain were found to be closely related and identified as Musa campestris and Musa acuminata with a similarity of 97 and 94%, respectively.
- In the clade C, the wild species of banana M.
- Thus, the results and inferences of the present study pinpoint that cultivars used in the present study might have originated from the wild species M.
- In summary, our study demonstrated that ITS2 is an ideal DNA barcode (a) to identify Musa subspecies or cultivars and (b) for the reconstruction of the phylogeny of the genus Musa.
- However, more Musa species need to be included in the future to verify whether these find- ings hold good even if closely related taxa are newly in- cluded.
- Nearly 46 annotated species and subspecies of GenBank sequences were employed in the present study and are shown in supplementary Table 1..
- sequences of Musa species in this study were used as query sequences.
- In the nearest distance method, cor- rect identification means that the hit in our database based on the smallest genetic distance is from the same species as that of the query.
- Ambiguous identification means that sev- eral hits from our database were found to have the same smallest genetic distance to the query sequence.
- AA Wild Species .
- Capital letters following each accession name indicate the previously- recognized genome composition of the cultivar.
- Wild and subspecies of Musa.
- Cultivar species of Musa.
- Table 5 Number of chromosomes and ploidy levels of Musa species /Cultivar names and its GenBank Accessions.
- 3 KY710753 Musa acuminata 22 2n = 2x Wild Species.
- 6 KY710756 Musa acuminata 22 2n = 2x Wild Species.
- 9 KY710759 Musa acuminata 33 3n = 3x Wild Species.
- Numerical taxonomy of the wild bananas (Musa).
- Restriction fragment length polymorphism (RFLP)-based phylogenetic analysis of Musa.
- PCR-RFLP of the ribosomal DNA internal transcribed spacers (ITS) provides markers for the a and B genomes in Musa L.
- Validation of the ITS2 region as a novel DNA barcode for identifying medicinal plant species.
- Application of the ribosomal DNA ITS2 region of Physalis (Solanaceae): DNA barcoding and phylogenetic study.
- The ITS2 of the genus Bulinus: novel secondary structure among freshwater snails and potential new taxonomic markers.
- Identification of medicinal plants in the family Fabaceae using a potential DNA barcode ITS2.
- Application of the ITS2 region for barcoding medicinal plants of Selaginellaceae in Pteridophyta..
- DNA barcode and identification of the varieties and provenances of Taiwan ’ s domestic and imported made teas using ribosomal internal transcribed spacer 2 sequences.
- genetic diversity and population structure of Musa accessions in ex situconservation.
- The ITS1-5.8S-ITS2 sequence region in the Musaceae: structure, diversity and use in molecular phylogeny.
- The evolution of the bananas.
- Evaluation of DNA barcode candidates for the discrimination of the large plant family Apocynaceae

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