Annals of Sri Lanka Department of Agriculture. 2007.9:161-168.
DNA TYPING OF BANANA CULTIVAR ‘AMBON’ (AAA) USING SEQUENCE TAGGED MICROSATELLITE SITES ANALYSIS W.L.G. SAMARASINGHE1, R.S. RANAWEERA2, I.P. WICKRAMASINGHE2, R.A.I.S. ARIYARATNE3, A.L.T. PERERA2, R.G.A.S. RAJAPAKSE4 and D.P. PREMATILAKA4 1 Plant Genetic Resources Center, P.O.Box 59, Peradeniya 2 Faculty of Agriculture, University of Peradeniya, Peradeniya 3 Faculty of Agriculture, University of Ruhuna, Mapalana 4 Horticultural Crops Research and Development Institute, Gannoruwa
ABSTRACT Banana is an important fruit crop in Sri Lanka. The cultivar Ambon is a popular commercial dessert cultivar grown in the wet and intermediate zones of Sri Lanka. So far, no improvement been done with respect to plant morphology, yield and for resistance against pests and diseases. Accurate understanding of the genetic diversity of cultivars is a key step towards the improvement. Molecular techniques such as Sequence Tagged Micro Satellites (STMS) are proven to be ideal in generating genetic diversity information of bananas. In this experiment, 34 samples of ‘Ambon’, representing different bunch characteristics, geographical differences, Panama disease resistance and micro propagated plants were subjected to STMS analysis. PCR products were separated by PAGE and bands were detected by silver staining. Dendrograms derived by cluster analysis of molecular data identified 8 DNA types. Molecular variation was agreeable with some fruit morphology and eco-geographic variations. There were no differences shown between the mother plant and micropropagated plants. The genotypes identified in this work will be useful for evaluation and in variety improvement programs. KEYWORDS: Ambon, Banana, Micro-propagated plants, STMS.
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Banana is cultivated in over 100 countries in the tropical and subtropical regions of the world and is important as a staple food for millions of people and as a cash crop catering for local and international trade. Banana is an most important fruit crop in Sri Lanka. The total production of banana in Sri Lanka is about 468,612t per year in an extent of 51,488ha (Anon., 2005). Morphological characterization of large collections of banana is laborious and time consuming as most of such characteristics are expressed at reproductive stage of the plant. Application of molecular techniques is an useful alternative means of characterization. The application of DNA fingerprinting techniques for Musa germplasm conservation and breeding will allow the identification of duplicates among accessions in the field and in-vitro collection, the monitoring of genetic stability of tissue cultured materials (i.e. Somaclonal variation) and the identification of trait markers for use in breeding programs (Kaemmer et al., 1992). Sequence Tagged Micro-satellite Site (STMS) makers are co-dominant, reliable, universal, and highly polymorphic type of markers, that are applicable for genotyping and genetic mapping of Musa spp. (Kaemmer et al., 1992). In this research, samples representing different agro ecological zones, bunch and other morphological variations, apparently Panama disease tolerant clumps and samples from micro-propagated plants of Musa cultivar Ambon were analyzed using STMS primers such as AGMI and MaSSR. MATERIALS AND METHODS A total of 34 samples, which comprised of morphologically different types (8), collections from different agro ecological regions (16), plants tolerant to Panama disease (5) and tissues culture derived plants (5) together with the mother plant from HORDI were used in the analysis. Passport data were recorded. Cultivars with different vernacular names as well as morphological variants of cultivars were selectively sampled. Random sampling was also done to capture possible ecotypes. DNA extraction was carried out separately from the samples collected from unopened, whitish leaf blades of young leaves, using the modified CTAB method (Samarasinghe et al., 2002) and quantified using a UV spectrophotometer. Twelve MaSSR primers and 4 AGMI primers were tested and 11 were selected for PCR analysis. PCR amplification was done using modified procedure (Kaemmer et al., 1997). Amplification products were separated on 6% and 10% polyacrylamide gels and were stained using modified 0.1% silver nitrate procedure (Samarasinghe et al., 2001). Presence of clear bands representing alleles of each locus was scored as ‘1’ and absence as ‘0’. Empty lanes were scored as ‘.’. The data were statistically analyzed using Nei’s (1978) genetic distance method (=UPGMA) using Pop gene version 1.31 and SPSS software packages and dendrograms were constructed.
DNA TYPING OF BANANA CULTIVAR ‘Ambon’ 163
RESULTS AND DISCUSSION Morphological variation of Ambon Variations were observed for appearance of the bunch and fruit characters such as, bunch compactness, brown stripes or pigmentation, fruit shape, fruit size and bunch size. During the 4 exploration trips, 24 samples were collected from 10 districts. Collection sites ranged from 30–1100m above mean sea level. It was observed that Ambon is well adapted to Low Country and Mid Country Wet Zones. Most of the Ambon clumps observed were apparently free from major diseases like Banana Bract Mosaic Virus, Bunchy Top, Cucumber Mosaic Virus and Banana Streak Virus diseases. Fungal diseases similar to Panama disease (Fusarium wilt) and a leaf spotting were observed in some fields. Characterization using many characters at the collection site was ineffective as within Ambon, there was not much morphological variation. STMS analysis of 19 samples Primers MaSSR 12a/b, MaSSR 7a/b, MaSSR 19a/b, (Plate1) MaSSR 8a/b, AGMI 59/60, AGMI 95/96 and AGMI 102/102 showed monomorphic and polymorphic bands between Ambon accessions (Table 1). Primers MaSSR 7a/b and MaSSR 19a/b (Plate 1) exhibited SSR polymorphism. Only 7 clearly visible alleles were scored for statistical analysis. Based on the dendrogram, 5 clusters were identified (Fig. 1). Table 1. PCR amplification data. Primer Product size (bp) MaSSR 7a/b MaSSR 8a/b MaSSR 12a/b MaSSR 18a/b MaSSR 19a/b MaSSR 20a/b AGMI 50/60 AGMI 95/60 AGMI 101/102
210-220 240-250 155-165 160-185 125-150 200-280 309 220 120-140
Total number of alleles 2 3 3 2 3 4 1 1 2
Number of monomorphic alleles 0 3 3 2 0 1 1 1 2
Number of polymorphi c alleles 2 0 0 0 3 3 0 0 0
As STMS loci of individual collections in each cluster were identical, the dendrogram identified accessions within a group as duplicates. In the dendrogram, two major genotypic groups can be recognized.
MaSSR 7 MaSSR 12 MaSSR 19
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Plate 1. Silver stained urea PAGE with PCR products (MaSSR 12. MaSSR 19 and MaSSR 7) of 19 ‘Ambon’ samples. 1-Rambukkana, 2-Rambukkana, 3-Rambukkana, 4-Galagedara, 5-Alawwa, 6Alawwa, 7-Galigamuwa, 8-Kandy, 9-Dehiowita, 10-Dehiowita, 11-Nivitigala, 12Pelmadulla, 13- Imbulpe,14-Bandarawela, 15-Kotmale, 16-Kundasale, 17Kundasale, 18-Wattegama, 19-Ukuwela.
DNA TYPING OF BANANA CULTIVAR â€˜Ambonâ€™ 165 1.Rambukkana 2.Rambukkana
3.Rambukkana 4.Galagedera IV
5.Alawwa 8.Kandy 9.Dehiowita
6.Alawwa 7.Galigamuwa 10.Dehiowita 14.Banadarawela
15.Kotmale 16.Kundasale I
17.Kundasale 18.Wattegama 19.Ukuwela
11.Nivitigala Figure 1. Dendrogram derived from SSR data of 19 Ambon accessions. 12.Pelmadulla
Group I consisting of 3 collections (11, 12 and 13) from 13.Imbulpe Ratnapura district was comparatively distant from the rest of the 16 accessions. Genetic distance of collection no. 17 from collection nos. 11, 12 and 13 was 0.5596. Distinct features of group I were short, stout, curved and dull fruits. No brown stripes were observed in this category, whereas in the other groups (II, III, IV and V) there were brown stripes, long and straight fruits except for collection no.18, which had fruits, curved inside. Group II consisted of 7 samples having identical SSR loci for the primers used in this experiment. These samples were collected from a wide range of agroecological regions. STMS analysis of 21 samples Out of 7 STMS primers used in the analysis, only 4 primers amplified clear bands which can be used to detect the genetic variation in Ambon. Those primers were MaSSR 19a/b, AGMI 101/102, MaSSR 20a/b and MaSSR 18a/b (Table 1). MaSSR 20a/b (Plate 2) and MaSSR 19a/b (Plate 3) amplified polymorphic alleles. Dendrogram (Fig. 2) separated all samples into 7 clusters where samples from Galle and Matara districts were clustered
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(Group I) separately from the rest. According to the dendogram sample collected from Kelanigama, Ginigathhena was also different from other samples, which has an intensive pink pigmentation in the petiole. Samples collected from Alawwa, Pelmadulla and Wattegama clustered as a different group (Group II) in the dendogram had different fruit characters and clustered differently in the former STMS analysis (Fig. 1). Samples collected from Kalutara (Group III), Kelanigama (Group V), Ehaliyagoda (Group VI) and Rambukkana (Group VII) were unique DNA types.
500bp 400bp 300bp
200bp 200 bp Plate 2. Polyacrylamide gel picture of the SSR products of 21 ‘Ambon’ samples amplified by Primer MaSSR 20a/b.
Plate 3. Polyacrylamide gel picture of the SSR products of 21 ‘Ambon’ samples amplified by Primer MaSSR 19a/b. Key: 2-Rambukkana, 5-Alawwa, 8-Kandy, 12-Pelmadulla, 14-Bandarawela, 18Wattegama, 25-Kadugannawa, 26-Ehaliyagoda, 27-Rambukkana, 28-Diwlapitya, 24–Kelanigama, 29-Mother plant of Tissue cultured plants, 30-Tissue cultured (TC), 31-TC, 32-TC, 33-TC, 34-TC, 21-Kalutara, 22-Galle, 20-Colombo, 23-Matata.
Out of 21 samples, 12 were grouped to form group IV in the dendogram (Fig. 2), reflecting their close genetic relationship with respect to the analyzed STMS loci. This group consisted of tissue cultured plants, some
DNA TYPING OF BANANA CULTIVAR â€˜Ambonâ€™ 167
samples collected based on panama disease resistance and some collected to represent different regions of the country. STMS variation in micro-propagated plants Tissue cultured plants were used in this experiment to check their genetic uniformity, as there is tendency of changing genetic stability due to somaclonal variation. Therefore tissue culture derived plants were compared with their mother plant. Four primers Ma SSR 19a/b, AGMI 101/102, MaSSR 20a/b and MaSSR 18a/b amplified monomorphic loci in these samples. Therefore, with respect to the analyzed SSR loci, somaclonal variation was not detected. This supports the fact that the micro propagated Ambon plants grown at HORDI are morphologically uniform. It reflects that the current protocol adapted is good enough for mass propagation using tissue culture techniques. STMS variation in panama disease tolerant Ambon plants Three samples from Panama disease tolerant clumps were identical and one collected from Ehalyagoda showed STMS variation for the tested loci. These Ambon clumps were tested at HORDI by growing them in sick plots. It was found that the causal organism Fusarium oxiporirum is detected in the plants even though they did not show symptoms. For identification of genotypes among Panama disease tolerant plants further investigations are proposed using the genotypes identified in this STMS analysis. CONCLUSIONS MaSSR 12a/b, MaSSR 18a/b, MaSSR 19a/b and MaSSR 20a/b primers were useful to detect the genetic variation of Ambon. Ecogeographic variation of Ambon is low but samples from Galle and Matara districts are possible eco-geographic types. Based on these results, three distinct genotypes can be identified: I. Group with typically long (>20cm), straight and shiny fruits with brown stripes on mature fruits, II. Group with typically short (<20cm), curved fruits, peel surface is dull and with no brown stripes or pigmentation and III. Type having severe red pigmentation on the petiole margins. Tissue cultured Ambon plants developed at HORDI did not show any variation in contrast to the mother plant and it reflects the accuracy of the protocol.
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Collections made as panama tolerant showed two genotypes and the genotypes identified by this STMS analysis can be used for disease evaluation. Genotypes identified in this analysis can be used for further evaluation and genetic improvement of Ambon. 0
Sample num Galle Matara Pelmadulla Wattegama Alawwa Kalutara
22 23 12 18 5 21
Tissue cultured Colombo Kandy Tissue cultured Tissue cultured Tissue cultured Tissue cultured Diwlapitiya Mother plant Kadugannawa Rambukkana Bandarawela
34 20 8 32 33 30 31 28 29 25 27 14
Kelanigama Ehaliyagoda Rambukkana
24 26 2
Figure 2. Dendogram based on hierarchical cluster analysis (average linkage between groups) of STMS data of 21 Ambon samples.
ACKNOWLEDGEMENTS Authors acknowledge Asian Development Bank for the financial support and Dr. P.K. Samarajeewa (Head/Biotechnology and Dr. D.H. Muthukudaarachchi (DD/PGRC) for the support given for carrying out the research work at Plant Genetic Resources Center, Gannoruwa. REFERENCES Anon., 2005. AgStat. Pocket book of Agricultural statistics, Socio Economics and Planning Center, Department of Agriculture, Peradeniya, Vol. II: 8p.
DNA TYPING OF BANANA CULTIVAR â€˜Ambonâ€™ 169 Kaemmer, D., R. Afza, K. Weising, G. Khal and F.J. Norvak. 1992. Oligonucleotide and amplification finger printing of wild species and cultivars of banana (Musa spp.) Biotechnology 10: 1030-1035. Kaemmer, D., D. Fischer, D. Jarret, F.C. Baurens, A. Grapin, D. Dambier, J.L. Noyer, C. Lanaud, G. Khal and P.J.L. Lagoda. 1997. Molecular breeding in the genus Musa: A strong case for STMS marker technology.96: 49-63. Samarasinghe, W.L.G., A.L.T. Perera, I.P. Wickramasinghe and A.M. Nahfees. 2002. Molecular Characterization of Musa spp. By Simple Sequence Repeats (SSR). Tropical Agricultural Research Vol. 14:1-10. Samarasinghe, W.L.G., J.P.D. Ruckshanthi., A.M. Nahfees, R. Muhunthan and A.L.T. Perera. 2001. A laboratory Manual on DNA typing using RAPD and SSR techniques with silver stained PAGE. Board of study in Agric. Biology, PGIA, University of Peradeniya. Pp.5-14.