Banana is an important fruit crop in the world in terms of production and consumption. Its production in Iran was confined to certain areas along southern regions, especially at Sistan and Baloochestan province. One of the most serious problems limiting the extension of banana plantation in Iran is the salinity of soil and water. Therefore, to extend banana cultivation it is important to improve salinity resistance in this crop (Miri et al., 2003).
The breeding of most banana cultivars is made difficult due to genetic sterility and triploidy, hence, producing hardly any seeds. In addition, it is further complicated by low seed germination of hybrid banana plants (Javed et al., 2004). Induced mutation has been utilized as a tool to generate variation and breeding in a number of vegetatively propagated crops such as banana (Hautea et al., 2004).
Genetic improvement of banana is to some degree limited by poor knowledge of genetic diversity. To make the collection useful for breeders, morphological and molecular characterization of the germplasms is necessary. Differentiation of genomes through morphological characteristics is unreliable. The disadvantages of phenotype-based assays can be overcome by direct identification of genotypes with DNA-based markers (Nsabimana and Staden, 2007). Molecular markers have been employed in Musa genotypes to assess ploidy (Oselebe et al., 2006), phylogenetic relationships (Jain et al, 2007; Nsabimana and Staden, 2007; Uma et al., 2006) and genetic diversity due to somaclonal variation (Lakshmanan et al., 2007; Bairu et al., 2006; Ray et al., 2006) or mutation induction (Hautea et al., 2004; Finalet et al., 2000; Toruan-Mathius and Haris, 1999). Random amplified polymorphic DNA (RAPD) analysis is a polymerase chain reaction (PCR)-based technique, which uses random primers to generate DNA fragments. Polymorphism generated by RAPD analysis has been used for fingerprinting and classification of the Musa genotypes. Linkage of RAPD markers to specific traits such as disease resistance has been possible (Damasco et al., 1996). Because RAPD polymorphisms result from either a nucleotide base change that alters the primers binding site or from an insertion or deletion within the amplified region, polymorphisms usually result in the presence or absence of an amplification product from a single locus. The products of these amplifications can be polymorphic and are useful as genetic markers (Vidal and de Garcia, 2000).
The objective of this study was primarily to assess the level of genetic diversity in mutated plants and to compare RAPDs and morphological dendrogram schemes of the plants. This study also helps identification of RAPD markers that would differentiate salinity resistance from susceptible clones.
MARERIALS AND METHODS
Plant materials: Plant materials (listed in Table 1) were generated in Nuclear Research Center for Agriculture and Medicine, Atomic Energy Organization of Iran (AEOI), Karaj, Iran. This experiment containing 3350 irradiated tissue culture shoot tips of banana (Musa acuminata cv. Dwarf Cavendish (AAA)) irradiated with gamma ray doses of 0, 25, 35, and 45 Gy, were produced after three times of subcultures. Explants were cultured on MS medium (Murashige and Skoog, 1962) containing 2.5 mg/l BAP and NaCl in concentrations of 0, 6, 7, 8, 9 g/l for 60 days. Green buds were then transferred to a similar medium without salt. After 30 days, buds were transferred to a medium similar to the initial stage and incubated for 60 days. Thereafter, all viable buds were rooted and shifted to the pots. Acclimatized plants were irrigated weekly with the solutions containing 0, 6, 7, 8, 9 g/l NaCl for 2 months. Research was conducted in a factorial experiment as randomized complete block design (Data not presented). Finally, 22 greenhouse-grown plants including a non-irradiated clone sensitive to salinity as control (sample No. 1) and twenty one irradiated clones resistance to salinity (No. 2 to 22) were collected for morphological and RAPD analyses.
Morphological characteristics: Morphological characterization of greenhouse-grown plants was carried out using the banana descriptor (IPGRI-INIBAP/CIRAD, 1996). Each clone was characterized using 30 traits taken at the vegetative stage.
DNA extraction: Total DNA was isolated by a modified cetyl-trimethyl ammonium bromide (CTAB) extraction technique (IAEA, 2002). Approximately 0.2 g of fresh leaf tissue was ground in liquid nitrogen and added to 700 ml of extraction buffer (2% CTAB, 100 mM Tris-HCl, 20 mM EDTA, 1.4 M NaCl, 0.2% b-mercaptoethanol, pH=8) and incubated at 60ºC for 30 min. The aqueous solution was extracted with 600 ml of chloroform:isoamyl alcohol (24:1) and centrifuged for 20 min at 10000 rpm. The extraction was repeated and 1 ml 96% cold ethanol was added to aqueous phase and left at -20ºC for 20 min to precipitate the nucleic acid. The samples were then centrifuged for 10 min at 10000 rpm. The precipitated nucleic acid was washed with one ml 70% ethanol, left to air dry, rehydrated in 100 ml TE buffer and digested with 1 ml RNase (10 mg/ml, Fermentas, Germany) for 45 min at 37ºC. The quality of genomic DNA was examined by agarose (1%) gel electrophoresis and quantified spectrophotometically at A260 and A280 nm. Each sample was diluted to 50 ng/ml in TE buffer and stored at -20ºC.
DNA amplification: The PCR reaction contained 50 ng of template DNA, 1 U Taq polymerase (Fermentas), 2.5 ml 10X PCR buffer, 0.2 mM dNTP, 4 mM MgCl2 and sterile distilled water to 25 ml. Fourteen arbitrary decamer primers (Metabion, Germany) were used for the PCR. The reactions were incubated in a thermocycler (Biothermal, Germany) programmed for 3 min at 93ºC, followed by 25 cycles of 1 min denaturation at 93ºC, 40s annealing at 36ºC and 1 min extension at 72ºC. After the 25th cycle, the extension was continued for 4 min at 72ºC. The products were held at 4ºC until analyzed. Amplified samples were run on a 1% agarose gel stained with ethidium bromide at 100 V for up to 90 min with 1X TAE as running buffer and gels were visualized under UV light and photographed with Polaroid film. Duplicate reactions were performed to ensure reproducibility.
Data analysis: Amplification product profiles were scored for the presence (1) or absence (0) of bands. Molecular size of the amplified fragments was estimated using 100 bp DNA ladder (Fermentas). RAPD assays generating weak or ambiguous amplification products were repeated to confirm the consistency of these markers. The NTSYS-pc software was used to estimate genetic similarities with the Jaccard’s similarity coefficient. The generated matrix of similarities was analyzed by the unweighted pair-group method with arithmetic average (UPGMA), using the sequential hierarchical agglomerative and nested clustering (SHAN) module. Morphological traits were also analyzed using the same software.
Morphological characterization of the 22 clones using 30 traits was carried out. Of these, 22 distinctive traits were considered for diversity evaluation, which exhibited a total of 69 classifications. Traits of drooping leaf habit and black-purple blotches were only characterized in non-irradiated clone, while the irradiated clones were shown other classifications (Table 2). The dendrogram and clustering pattern are observed in Figure 1. The UPGMA dendrogram based on morphological characterization indicated that all clones were clustered into two major groups: the first group included 13 irradiated clones and the second group contained other irradiated clones plus non-irradiated clone.
Amplification patterns are performed twice in order to evaluate if the RAPD patterns can be reproduced. The results indicate that reproducible RAPD patterns can be obtained under the same amplification conditions for two replicates.
The fourteen primers resulted in 106 scorable bands, ranging from 150-3100 bp in size. Of these, 98 (92.4%) bands were polymorphic (Table 3). The number of bands for each primer varied from 2 (OPH-07 and OPJ-09) to 14 (OPA-04) with an average of 7.5 bands per primer. The average polymorphism exhibited by all 14 primers was 92.1% and the OPAA-14 was the only primer generating an amplification product in less than 70% of the accession tested. A 240 bp band derived from the primer OPA-02 was observed to be present only in the resistant clones (Fig. 2).
To estimate the similarities among clones, the simple matching coefficient provided similarity values ranging from 0.30 to 0.79. According to the UPGMA dendrogram, the twenty-two clones were clustered into two major clusters: the irradiated clones were grouped together and separate from the non-irradiated clone (Fig. 3). Clones No. 20 and 21 at a similarity coefficient of 0.79 were close to each other. Minimum similarity coefficient (0.30) was observed between clones No. 1 and 17.
This study was conducted to determine the extent of genetic diversity among irradiated (resistance to salinity) and non-irradiated (sensitive to salinity) ‘dwarf Cavendish’ banana based on the morphological and RAPD systems.
In plant breeding, genetic variation is essential for the creation of plants with superior traits. Conventional banana breeding is complicated by low seed fertility, triploidy, slow and vegetative propagation, time (2 years from seed to seed) and space (6 m2/plant) requirements. Consequently, alternative approaches such as mutation have also been considered (Okole et al., 2000; Vuylsteke, 2000). The mutation is a powerful technique, which may well produce desired variation in well established clones (Hautea et al., 2004). The frequency of polymorphism obtained by RAPD technique was 98 markers/14 selected primers. This is much higher than the ratio obtained in banana by Javed et al. (2004) of 84/15 and Muhammad and Othman (2005) of 44/15. This high polymorphism indicates that there is considerable variation at the DNA level within radiation-induced mutants. Furthermore, the data obtained from both morphological and molecular analyses indicate related to genome alterations in these accessions. Our study supports the earlier reports that mutation causes genetic variation in banana (Chun Hai et al., 2000; Toruan-Mathius and Haris, 1999; Domingues et al., 1994).
All irradiated tolerant clones were clustered separately from non-irradiated sensitive clone based on RAPD analyses. This result is in agreement with Gomes et al. (2005) who reported that the varieties of higher tolerance to saline stress were genetically distant compared to most of the salt sensitive ones.
Morphology markers have been used extremely to determine the relationships among banana populations (Uma et al., 2006; Noyer et al., 2005; Hautea et al., 2004; Pillay et al., 2001, 2000; Crouch et al., 2000, Newbury et al., 2000; Sanchez et al., 2000; Howell et al., 1994). Based on the results of UPGMA cluster analysis, non-irradiated and irradiated clones share a large number of morphological characterizations and cannot be distinguished on the basis of morphology (Fig. 1). Leaf habit and blotches color were the only traits that could be used to differentiate between mutants and wild type (Table 2). Nevertheless, tracking the mutants even using these two traits was near to impossible. Although induced mutants are usually differentiated from the original plant by phenotypic analysis, but it can be severely limited by the large size of mutagenized populations, particularly in the case of banana, and developmental and environmental effects. The application of molecular markers may overcome this limitation (Hautea et al., 2004). DNA amplification of polymorphic markers (presence and absence of bands) could be the result of base deletion or insertions at priming sites (Muhammad and Othman, 2005). In this study, RAPDs were chosen because it amplify different region of the genome allowing better analysis of genetic stability/variation of clones, as well as their simplicity and cost-effectiveness (Ray et al., 2006). Similar to earlier studies that were implemented RAPD in analyzing irradiated banana genotypes (Hautea et al, 2004; Finalet et al, 2000; Toruan-Mathius and Haris, 1999), we were able to efficiently examine and differentiate genotypic changes in gamma irradiated banana clones.
Primer OPA-02240 was only observed to be present in the resistant clones and it can be used as a salinity resistance marker. Damasco et al. (1996) and Bairu et al. (2006) successfully demonstrated the use of RAPD markers and detected a marker linked with dwarfness in bananas. Vidal and de Garcia (2000) and Javed et al. (2004) have also detected similar results with a resistant marker to yellow Sigatoka and Fusarium wilt diseases in bananas, respectively.
Our results suggest that RAPD could be considered as an alternative molecular marker tool for rapid and inexpensive evaluation of genetic variability in the bananas obtained by mutation. Sequencing of the marker band present in tolerant clones determines its base composition and it can be used to study the molecular basis and mechanisms that might be related to the resistance to salinity, which may be beneficial to the crop improvement.
The authors are thankful to M. Rahimi and C. Vedadi for providing plant materials. This research has been supported by Agricultural, Medical and Industrial Research School, Atomic Energy Organization of Iran (AEOI).