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Morphological and Molecular Characterization of Musa Germplasm in Sri Lanka and Selection of Superior Genotypes W.L.G. Samarasinghe1, A.L.T. Perera2, I.P. Wickramasinghe and 2 S. Rajapakse3 1Plant Genetic Resource Center, Department of Agriculture, P.O. Box 59, Peradeniya, Sri Lanka

2Post Graduate Institute of Agriculture, University of Peradeniya, Peradeniya, Sri Lanka 3Department of Biological Sciences, Clemson University, Clemson SC 29634, USA Keywords: banana, genetic characterization, simple sequence repeats Abstract

Genetic variability of Musa spp. in Sri Lanka is rich and needs characterization for its conservation and use. Genetic characterization using simple sequence repeats (SSRs) is a rapid and accurate technique complementary to morphological characterization. In this study, 27 cultivars (M. acuminate (AA genome), M. balbisiana (BB genome), polyploids and interspecific hybrids) were characterized using morphological traits and SSRs. From the SSR analysis, 2-11 alleles were observed, reflecting the genetic diversity among cultivars and hence an important reason for their conservation. Average linkage method (UPGMA) grouping of Musa cultivars showed some similarity to morphological grouping. ‘Angaviaru’ (AAB genome), ‘Seenikesel’ (ABB genome), ‘Kolambaseeni’ (ABB genome) and ‘Embul’ (AAB genome) were very diverse from the other cultivars. Wild ‘Unel’ (AA genome) showed less similarity to Sri Lankan cultivars than the other wild ‘Etikesel’ (BB genome). ‘Ambon’ (AAA genome), which was previously morphologically classified as a ‘Gros Michel’ (AAA genome), was actually closer related to ‘Binkesel’ (AAA genome, ‘Cavendish’ subgroup). Fertile diploids, such as ‘Unel’, ‘Etikesel’ and ‘Navari’ (AA genome), are distinct genotypes useful for genetic mapping and breeding. ‘Muwanethikesel’ (AAB genome), which has no male bud, is a possible mutant of ‘Walsuwandel’ (AAB genome). ‘Alukesel’ (ABB genome) and ‘Etamburu’ (ABB genome), which occasionally produce seeds, are also unique genotypes useful in banana improvement. ‘Puwalu’ (AAB genome), and ‘Seenikesel’ are superior genotypes with respect to bunch, fruit and yield characteristics. INTRODUCTION

Total production of banana and plantains (Musa spp.) in Sri Lanka equals 468,612 t/year, planted on 51,488 ha. Banana and plantains have a high local demand and export potential. In Sri Lanka, a rich genetic variability is found in wild Musa species (M. acuminata (AA genome) and M. balbisiana (BB genome)) and in AA, AAA, AAB and ABB cultivars, some being resistant to diseases such as Fusarium wilt and Mycosphaerella leaf spot (Chandraratne and Nanayakkara, 1951), banana bract mosaic (BBrMV), Banana bunchy top virus (BBTV), Cucumber mosaic virus (CMV) and Banana streak virus (BSV) (Ekanayake et al., 2002). Morphological characterization is laborious and time consuming. Application of molecular markers such as simple sequence repeats (SSR) are useful alternatives for characterization of germplasm and are applicable for Musa genotyping as they produce co-dominant, reliable, universal and highly polymorphic markers (Kaemmer et al., 1997). In this work, AGMI and MaSSR primers (Kaemmer et al., 1997) were used to characterize wild species and cultivars in Sri Lanka, complemented with morphological traits followed by selection of genotypes with superior bunch characteristics. MATERIALS AND METHODS

Twenty-seven cultivars from the collection of the Plant Genetic Resources Center (Peradeniya, Sri Lanka) were characterized at the University of Peradeniya (Peradeniya,

Proc. IC on Banana & Plantain in Africa Eds.: T. Dubois et al. Acta Hort. 879, ISHS 2010

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Sri Lanka) and Clemson University (Clemson, USA) (Table 1). Morphological Characterization

Eighteen morphological characters were recorded and statistically analyzed using SPSS (SPSS, Chicago, USA) (Chandraratne and Nanayakkara, 1951; IPGRI, 1996). A dendrogram was derived from hierarchical cluster analysis. A detailed characterization for 111 descriptors was carried out for the superior ‘Seenikesel’ (ABB genome) and ‘Puwalu’ (AAB genome) using Musa descriptors (IPGRI, 1996). SSR Characterization

Samples were collected from unopened, whitish young leaf blades. DNA was extracted and purified using the modified CTAB method (Saghai-Maroof et al., 1984; Doyle and Doyle, 1987; Rogers and Bendich, 1988). Five MaSSR primers (MaSSR7, MaSSR18, MaSSR20, MaSSR22 and MaSSR24) and two AGMI primers (AGMI 95 and AGMI 96) (Kaemmer et al., 1997) were selected from 26 primers for analysis. A volume of 2.5 µl of forward primers (10 pmol/µl) were labeled with P33 by adding 1.0 µl of γ33ATP (10 µci/µl), 1.0 µl of 10 x kinase buffer, 0.5 µl of T4 kinase and 5.0 µl of H2O to a final volume of 10.0 µl. The mixture was incubated at 37°C for 1 h and kinase inactivated at 70°C for 10 min. Polymerase chain reaction (PCR) was carried out in 10 µl of reaction volume including 10 x buffer (1.00 µl), 10 mM each of dNTPs (0.20 µl), MgCl2 (0.80µl), Taq DNA polymerase (0.05 u) (Jumpstart, Sigma-Aldrich, St. Louis, USA), labeled primer (F) mixture (0.40 µl), unlabeled reverse primer (0.15 µl), 20 ng/µl of DNA (1.40 ul) and H2O (5.77 µl). PCR was performed at 94°C for 2 min for the initial denaturing followed by nine touchdown cycles (30 s at 94°C denaturing, 30 s at 60–54.5°C annealing and 1 min at 72°C extension). Thereafter, 25 cycles (30 s at 94°C, 30 s at 55°C and 1 min extension) and a final extension (72°C for 10 min) were carried out. Four µl of products were run in 6% 0.2-mm polyacrylamide gels (containing 7 M urea, 0.043% ammonium persulphate, 0.005% TEMED in 1 x TBE) and autoradiographs were developed. Presence of clear bands representing alleles of each loci were identified and scored (1 = present, 0 = absent). The data were analyzed using the average linkage method (UPGMA) with Popgene Version 1.31 (University of Alberta, Edmonton, Canada), based on Nei (1978), and a dendrogram was developed. RESULTS AND DISCUSSION Morphological Characterization of Cultivars

Based on the dendrogram, the 27 cultivars were different from each other (Fig. 1). ‘Muwanethikesel’ (AAB genome) was highly distant from all other cultivars. ‘Unel’ (AA genome) and ‘Navari’ (AA genome) were also distant from the other cultivars. ‘Unel’ possesses distinct morphological characters, which clustered separately. However, the wild ‘Etikesel’ (BB genome) was closely associated to ‘Seenikesel’, ‘Kolambaseeni’ (ABB genome) and ‘Etakesel’ (BB genome). ‘Navari’ was grouped separately from the cultivar group with AAA genomes, indicating morphological dissimilarity from that group. ‘Sapu Anamalu’ (AAA genome), ‘Ambon’ (AAA genome) and ‘Anamalu’ (AAA genome) are morphologically similar cultivars. ‘Williams’ (AAA genome) is a recent introduction, and used in this study for comparison with traditional and other introduced cultivars. ‘Embul’ (AAB genome) and ‘Kolikuttu’ (AAB genome) were grouped together with the latter cultivars with AAA genomes. ‘Walsuwandel’ (AAB genome), ‘Suwandel’ (AAB genome) and ‘Rathkesel’ (AAB genome), which are three indigenous cultivars, were clustered together. ‘Alukesel’ (ABB genome), ‘Etamburu’ (ABB genome) and ‘Mondan’ (ABB genome) grouped separately from other ABB cultivars. The seedy character in ‘Etamburu’ and ‘Alukesel’, which are plantain cooking bananas, are useful in banana breeding (Ekanayake et al., 2002).

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SSR Characterization Six primers amplified a total of 38 alleles in all six polymorphic loci. The number

of alleles observed ranged from 2–11/locus depending on the primers and genotype (Fig. 2). In the dendrogram, SSRs identified 25 different genotypes (Fig. 1). The SSR grouping showed some similarities to but also some differences with morphological grouping. Morphologically different ‘Seenikesel’ and ‘Kolambaseeni’, and morphologically similar ‘Sapu-Anamalu’ and ‘Anamalu’ were identified as duplicate genotypes. Therefore, 26 cultivars including either ‘Sapuanamalu’ or ‘Anamalu’ are recommended for field and in vitro conservation. All dessert cultivars, except ‘Suwandel’, were genetically distinct from plantain cultivars (Fig. 1). The dendrogram shows that the dessert cultivars ‘Kolambaseeni’, ‘Seenikesel’ and ‘Angaviaru’ (AAB genome) (47 units in dendrogram) are followed by ‘Embul’ (32 units) and the wild ‘Unel’ (30 units) were the most distinct genotypes. However, the other wild ‘Etikesel’ grouped within indigenous cultivars, indicating the possibility of contribution of its genome to local cultivars in contrast to ‘Unel’ which had been collected from an isolated forest. Seedy ‘Unel’, ‘Etikesel’ and possibly ‘Navari’ will be useful for gene mapping and banana breeding after evaluation. ‘Navari’ and ‘Anamalu’ showed a close association indicating a common source of origin. ‘Ambon’, a popular commercial cultivar, shows genetic similarity to ‘Binkesel’ (AAA genome, ‘Cavendish’ subgroup). It is doubtful whether ‘Ambon’ can continue to be classified as a ‘Gros Michel’ (Simmonds, 1966). A close association observed between ‘Muwanethikesel’ and ‘Walsuwandel’ suggests the possibility of the former evolving as a mutant from the latter. Selection of Superior Genotypes

Detailed description of cultivars (data not shown) is useful for further evaluation, and for distinctness, uniformity and stability tests (DUST) before release as recommended cultivars by the Department of Agriculture. Morphological and molecular analysis concluded that the selected cultivars are unique genotypes within the germplasm collection. ‘Puwalu’ (syn. ‘C9402-08’) has been selected for superior bunch characters such as large and attractive fruits. This cultivar produced a bunch with 72 fruits in fertile soil indicating its high yield potential. ‘Seenikesel’ (syn. ‘C9403-32’) was also selected as a superior genotype because it produces well-filled, big fruits (100 g/fruit) and attractive bunches (±12 hands with ±188 fruits). ACKNOWLEDGEMENTS

The authors express their sincere thanks to Bioversity International for financial support. for the research component at Clemson University, and to the Asian Development Bank for financial support for the work at the University of Peradeniya.

Literature Cited Chandraratne, M.F. and Nanayakka, K.D.S.S. 1951. Cultivated varieties of banana in

Ceylon. Tropical Agriculturist 107:70–91. International Network for the Improvement of Banana and Plantain. 1997. Musa

Germplasm Information System, Version 1.0: User's Guide. INIBAP, Montpellier. Doyle, J.J. and Doyle, J.L. 1987. A rapid DNA Isolation procedure for small quantities of

leaf tissue. Phytochem. Bull.19:11–15. Ekanayake, E.M.D.S.N., Samarasinghe, W.L.G. and Ariyaratne, I. 2002. Collection,

characterization, evaluation, identification and conservation of Musa genetic resources in Sri Lanka. In: H.P. Singh and N.K. Dadlani (eds.), Global conference on Banana and Plantain. Bangalore, India. 28-31 October, 2002. AIPUB, New Delhi.

International Plant Genetic Resources Institute. 1996. Descriptors for Banana (Musa spp.). IPGRI, Rome.

Kaemmer, D., Fisher, D., Jarret, R.L., Bauvens, F.C.G., Grafin, A., Dambier, D., Noyel, J., Lanaude, L., Kahl, G. and Lagoda, P.J.L. 1997. Molecular breeding in genus Musa: a strong case for SSR marker technology. Euphytica 96:49–62.

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Nei, M. 1978. Estimation of average heterozygosity and genetic distance from a small number of individuals. Genetics 89:583–590.

Rogers, S.O. and Bendich, A.J. 1988. Extraction of DNA from plant tissues. p.1-10. In: S.B. Gelvin and R.A. Schilperoort (eds.), Plant Molecular Biology Manual. Kluwer, Boston.

Saghai-Maroof, M.A., Soliman, K.M., Jorgensen, R.A. and Allard, R.W. 1984. Ribosomal DNA spacer-length polymorphism in barly: Mendalian inheritance, chromosomal location and population dynamics. Proc. Nat. Acad. Sci. 81:8014–8018.

Simmonds, N.W. 1966. Bananas. Second Edition. Longman, London. Tables Table 1. Twenty-seven Musa cultivars used for characterization. Musa spp.1 International

classification2 Genomic group3 Unel (W) M. acuminate AA Navari (D) Not identified AA Bin kesel (D) Cavendish AAA Sapu Anamalu (D) Cavendish AAA Anamalu (D) Gros Michel AAA Ambon (D) Pisang Ambon AAA Williams (D) Cavendish AAA Ratambala (D) Red AAA Galanamalu (D) Green AAA Ambul (D) Mysore AAB Puwalu (D) Pome AAB Watupalu (D) Not identified AAB Muwanethikesel (D) Not identified AAB Walsuwandel (D) Not identified AAB Rathkesel (D) Not identified AAB Kolikuttu (D) Silk AAB Angaviaru (P) French plantain AAB Bahu (P) Not identified ABB Suwandel (D) Pisang Kelet AAB Mondan (P) Bluggoe ABB Seenikesel (D) Pisang awak ABB Kolambaseeni (D) Pisang awak ABB Alukesel (P) Not identified ABB Etamburu (P) Not identified ABB Etakesel (Semi-W) Not identified BB? Etikesel (W) M. Balbisiana BB Unlabeled introduction from Australia Not recorded ABBB? 1W = wild, D = dessert, P = plantain. 2Source: INIBAP (1997). 3Souce: Simmonds (1966).

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Figures

Fig. 1. A. Dendrogram of 27 banana (Musa spp.) cultivars based on average linkage

method (UPGMA) with Popgene Version 1.31 (University of Alberta, Edmonton, Canada), using simple sequence repeats (SSR) data (Nei, 1978). B. Dendrogram using hierarchical cluster analysis of morphological data. 1 = ‘Unel’ (AA genome), 2 = ‘Navari’ (AA genome), 3 = ‘Binkesel’ (AAA genome), 4 = ‘Sapu anamalu’ (AAA genome), 5 = ‘Anamalu’ (AAA genome), 6 = ‘Ambon’ (AAA genome), 7 = ‘Williams’ (AAA genome), 8 = ‘Ratambala’ (AAA genome), 9 = ‘Galanamalu’ (AAA genome), 10 = ‘Ambul’ (AAB genome), 11 = ‘Puwalu’ (AAB genome), 12 = ‘Watupalu’ (AAB genome), 13 = ‘Muwanethikesel’ (AAB genome), 14 = ‘Walsuwandel’ (AAB genome), 15 = ‘Rathkesel’ (AAB genome), 16 = ‘Kolikuttu’ (AAB genome), 17 = ‘Angaviaru’ (AAB genome), 18 = ‘Bahu’ (ABB genome), 19 = ‘Suwandel’ (AAB genome), 20 = ‘Mondan’ (ABB genome), 21 = ‘Seenikesel’ (ABB genome), 22 = ‘Kolambaseeni’ (ABB genome), 23 = ‘Alukesel’ (ABB genome), 24 = ‘Etamburu’ (ABB genome), 25 = ‘Etakesel’ (BB genome), 26 = ‘Etikesel’(BB genome), 27 = Introduction from Australia.

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Fig. 2. Autoradiograph of polymerase chain reaction (PCR) products using primer

MaSSR 20a/b of 27 banana (Musa sp.) cultivars. Lanes: 1 = ‘Unel’ (AA genome), 2 = ‘Navari’ (AA genome), 3 = ‘Binkesel’ (AAA genome), 4 = ‘Sapu anamalu’ (AAA genome), 5 = ‘Anamalu’ (AAA genome), 6 = ‘Ambon’ (AAA genome), 7 = ‘Williams’ (AAA genome), 8 = ‘Ratambala’ (AAA genome), 9 = ‘Galanamalu’ (AAA genome), 10 = ‘Ambul’ (AAB genome), 11 = ‘Puwalu’ (AAB genome), 12 = ‘Watupalu’ (AAB genome), 13 = ‘Muwanethikesel’ (AAB genome), 14 = ‘Walsuwandel’ (AAB genome), 15 = ‘Rathkesel’ (AAB genome), 16 = ‘Kolikuttu’ (AAB genome), 17 = ‘Angaviaru’ (AAB genome), 18 = ‘Bahu’ (ABB genome), 19 = ‘Suwandel’ (AAB genome), 20 = ‘Mondan’ (ABB genome), 21 = ‘Seenikesel’ (ABB genome), 22 = ‘Kolambaseeni’ (ABB genome), 23 = ‘Alukesel’ (ABB genome), 24 = ‘Etamburu’ (ABB genome), 25 = ‘Etakesel’ (BB genome), 26 = ‘Etikesel’(BB genome), 27 = Introduction from Australia.

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