54 J. Hortl. Sci. Vol. 12(1) : 54-58, 2017 Genetic diversity studies among AAB group Indian banana cultivars using ISSR markers K.V. Ravishankar1, A. Rekha*2 Rema Menon3, C.K. Anoopa1, K. Sudeepa1 and R. Poornima1 1Division of Bio-Technology, ICAR-IIHR, Hessaraghatta, Bengaluru - 560 089 2Division of Fruit Crops, ICAR-IIHR, Hessaraghatta, Bengaluru, - 560 089 3 Banana Research Station, Kerala Agricultural University, Kannara, Marakkal Thrissur, Kerala - 680 652 * E-mail: arekha@iihr.res.in ABSTRACT Banana and plantains are generally classified based on morphological characteristics namely AA, AAA, AB, AAB and ABB. Further, there are four sub-groups in AAB genomic group. Presently we analyzed diversity using ISSR markers in 18 cultivars of AAB genomic group along with two each of AA types and BB wild accessions for comparison. The results have shown that AAB cultivars form a separate group. Dendrogram analysis showed that the subgroups ‘Plantain’ ‘Silk’ and ‘Mysore’ were placed in between AA and BB type. Whereas ten cultivars of ‘Pome’ sub group of the cultivars were unique and was placed in a separate cluster. In this study using ISSR markers, we are able to identify the subgroups clearly and their genetic relationships within the AAB group. The cultivars Rasthali and Nendran were clearly separated. The Pome sub-group cultivars found to be in a group which may be based on their geographical origin. Key words: Banana and Plantain, Diversity, ISSR markers, AAB group. INTRODUCTION Banana cultivars were classified based on a taxonomical scoring method developed by Simmonds and Shepherd (1955). There are about 30 Musa species grouped into four sections. South East Asia could be a primary center of diversity of the AAB group of bananas with distinct varieties De Langhe (2000). It appears that a first set of edible diploids and polyploids were identified through selection and domestication, which gradually lost the ability of seed set and were clonally propagated. A second range of edible bananas would have evolved through hybridization between closely related species M. acuminata (AA genomic group) and M. balbisiana (BB genomic group), early to evolution of AB; AAB &ABB type cultivars later lot of mutations might have accumulated in these types. Among AAB triploids, there are 11 subgroups as reported by Uma et.al, (2005). A total of 66% diversity was identified in India for AAB groups, which is represented by seven subgroups based on stable mutants present. Generally, this AAB group has been divided into four sub-groups based on morphological traits, namely ‘Plantains’ group with ‘Nendran’; cultivar ‘Rasthali’ a highly susceptible genotype to Fusarium wilt disease in ‘Silk’ group; ‘Pome’ group which comprises of ‘Virupakshi’ or Hill banana cultivars and ‘Mysore’group with cultivar ‘Palayan Kodan’ having high yield & tolerance to Fusarium wilt disease. Reports on diversity analysis of banana accessions involving different genomic groups using different markers like RFLP, RAPD, AFLP were reported by several authors Kaemmer et.al, (1997); Jarret et.al, (1993); Howell et.al, (1994); Bhat & Jarret (1995) and Bhat et.al, (2004). Visser (1996) was able to distinguish AAB and ABB group genotypes using random primers. Creste (2003) and group used microsatellite markers to differentiate the banana genotypes. However, there are no studies exclusively focused on diversity analysis of AAB group cultivars and analysis of the genetic basis among these sub- groups using ISSR markers. MATERIAL AND METHODS Eighteen AAB cultivars were collected from Banana Resea rch Sta tion (Ker ala Agr icultura l University), Kannara, Kerala; two each of AA (Anai Original Research Paper 55 J. Hortl. Sci. Vol. 12(1) : 54-58, 2017 ISSR markers among AAB cultivars of India Komba n & M. acuminata ssp burmannicoides- Calcutta -4) a nd BB (M. balbisiana & Wild-3) genotypes were included for comparison (Table 1) which were maintained in the field germplasm block of ICAR-Indian Institute of Horticultural Research, Bangalore. Total genomic DNA from all 22 cultivars was isolated using the modified CTAB method, Ravishankar et.al, (2000). A few SSR’s were screened and selected 16 ISSR primers, which amplified multiple r epr oducible ba nds (Ta ble-2), wer e used for amplification of DNA. The 25µl PCR reaction consisted of 2.5 µl of reaction buffer, 3.0 µM concentration (2.5 µl of primer), dNTPs 1mM (2.5 µl) Taq polymerase 3 µ/µl (0.5 µl), 20ng/ µl DNA template (2.5 µl) and volume was made up with sterile water. PCR was carried out using a Master Cycler Gradient (Eppendorf AG, Ha mbur g, Ger ma ny) ther ma l cycler with the temperature profile of; initial denaturation at 94°C for 1 min, then 35 cycles of 30 s at 94°C for denaturation, 45s at an annealing temperature of 52°C, and elongation at 720C for 1 min; with a final extension at 72°C for 5 min. The amplified PCR products were separated on 1.5 percent agarose gel along with 1kb DNA ladder. The gel was visualized and documented using UV pro gel documentation system. For ISSR markers, the presence of a band in each position was recorded, the presence as ‘1’ and the absence as ‘0’. The bands for each primers were scored separately. Using this data, squared Euclidian distances were calculated to estimate all pair wise differences in the markers for all cultivars Sneath and Sokal (1973). Based on distance matrix, Cluster analysis was done using a minimum variance algorithm (Ward, 1963). Table 1: List of cultivars AAB genomic groups Table 2: ISSR primers used RESULTS AND DISCUSSION Sixteen ISSR primers amplified 96 markers for 18 AAB cultivars, out of which 71 were polymorphic bands (Table 3). Based on cluster analysis, the genotypes used here were found to be divided in two ma jor gr oups. First subgr oup consisted of AA (M. acuminata) type along with two subgroups ‘Mysore’, ‘Silk’ and BB (Musa balbisiana) types. The second major cluster consisted of cultivars from the subgroup ‘Pome’. The first cluster was further divided into four sub-clusters where, ‘Anai komban’ a cultivated AA type and M. acuminata ssp. burmaniccoides- Calcutta-4, wild AA type, formed one group. The cultivars ‘Mysore Ethan’ and ‘Karim Kadali’ showed a close relation to AA types. ‘Thiruvananthapuram’, and ‘Pisang seribu’, was placed close to ‘Palayan kodan’ cultivar, belonging to ‘Mysore’ sub group, the 56 J. Hortl. Sci. Vol. 12(1) : 54-58, 2017 cultivars ‘Thiruvanathapuram’ and ‘Pisang seribu’ are grouped as unique cultivars based on morphological parameters. ‘Thiruvananthapuram’ has small bunch with 7-8 hands, fruits are long with 12-13cm in length and pulp like that of Pome group cultivars resembling ‘Pisang kelat’. ‘Pisang seribu’ has a very long bunch of 2M long with numerous small fingers (nearly 1000 fruits). The fruits and pulp resemble fruits of ‘Mysore’ subgroup; it is said to be a mutant of Musa chiliocarpa (Stover & Simmonds, 1987). The ‘silk’ subgroup cultivars ‘Poovan’, ‘Martman’ and ‘Madhranga’ were found to be closely related. This group has characteristic yellowish green psuedostem and pink pigmentation all along the petiole margin, the fruit pulp is starchy and very sweet. The second major cluster consisted of ‘Pome’ subgroup cultiva rs na mely ‘Siruma lai’, ‘Kali’, ‘Kaliagali’, ‘Padathi’, ‘Virupakshi’, ‘Krishnavazha’ are grouped together. The cultivars ‘Mannan’ and ‘Kullan’ resembled each other and were placed together. Among the pome group cultivars, ‘Krishnavazha’ is morphologically distinct with dark blackish pigmentation on the psedostem. The fruits of all the cultivars are invariability angular with a distinct apex. The peel is bright yellow when ripe, easily peels off, pulp has a sweet aroma. This subgroup was placed close to BB Musa balbisiana wild types. AAB accessions could have arisen as a result of either AB x AA or AA x AB cross (De Langhe et.al,2010) as contribution of A or B genome in this group does not agree with simple allopolyploid genome formulae of Simmonds and Shepherd. Deviations were observed in the required total score of 35-37 based on morphological characterization, in ‘Pome’ and ‘Silk’ group cultivars. This was further strengthened by study of Careel where inheritance of organelle (Chloroplast and Mitochondria) DNA was used Carreel et.al, (2002). They also described the probable evolution of interspecific hybrids through (BB x AA) x AA cross or (AA x BB) x AA cross combinations to get BAA or AAB type cultivars where the cultivars have ‘B’ type or ‘A’ type cytoplasm and hence dosage of A or B genome varies. Ravishankar et al Table 3: Markers generated by the ISSR primers 57 J. Hortl. Sci. Vol. 12(1) : 54-58, 2017 ISSR markers among AAB cultivars of India These studies have helped in categorizing the AAB cultivars based on their genetic relationship with AA and BB genotypes (Fig.1). However, the Pome types are totally distinct from other three subgroups, these are comparatively more robust, bunch orientation is either horizontal or at an angle. Male phase is short with whip like rachis ending with a ‘top’ shaped male bud. Anthers abort and turn black; fruits are angular with thick peel (Singh et.al, 2001). Further, molecular studies are required to study the evolution of AAB types and its progenitors. Fig 1: Dendro gram of 18 AAB cultivars along with AA and BB types. Us e of IS SR ma r ker s gener a lly gives higher polymorphism than RAPD (Ning et.al, 2007) or RFLP. There are reports on diversity analysis by Ning and co-workers using 216 banana accessions using microsatellite (SSR) markers; the accessions could be separated based on the genomic groups. However, it could not separate the somatic mutants. The ISSR method is widely used in ascertaining the genetic fidelity among the in vitro raised plants (Rout et.al, 2009). In our work diversity analysis was studied involving AAB genomic group using ISSR markers, the results revealed variability within cultivars of this group. Earlier, diversity in this group was reported based on RAPD studies by Menon, 2004 and team. Clustering of AAB group cultivars was based on their sub gr oup cla ssification. The cultivar Rasthali of sub-group ‘Silk’, which is distinct, and Nendran of sub-group ‘Plantain’ a unique dual- purpose cultivar were separated from others. The cultivars of two sub-groups ‘Mysore’ and ‘Pome’ were found to be separated based on geographical locations. Pome types were distinctly separated from other three subgroups. It appears that these subgroups have a distinct genetic base; therefore they form a separate group in cluster analysis. RAPD analysis within 23 ‘Pome’ group cultivars ha s r evea led 3 ma jor gr oups a mong differ ent accessions and did not give a clear difference based on morphologica l traits (Uma et. al, 2005). In gener a l I S S R ma r ker s wer e a b l e t o gr ou p morphologically similar types in a cluster more efficiently than RAPD markers. 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