Tropical Agricultural Research & Extension 12(2):2009 INTRODUCTION Banana belongs to the Genus Musa that is classified again into four recognized sections, namely Eumu- sa, Rhodochlamys, Australimusa, and Callimusa. Eumusa includes all species of true bananas (Chandraratna 1951). There is a wide range in culti- vated or true bananas. In Sri Lanka, twenty nine banana cultivars and two wild species have been reported (Chandraratna & Nanayakkara 1951). Five out of twenty nine cultivars are cooking types and the rest are dessert types. Dessert type, „Kolikuttu‟ (Silk, Apple) may be originated in India and has AAB genome. Of the numerous fruit crops grown in Sri Lanka, banana is cultivated in about 48,000 ha contributing 46% of the total fruit pro- duction (AgStat 2004; Rajapakse et al. 2005). „Kolikuttu „is one of the highly prized fruit in Sri Lanka but is highly susceptible to Fusarium wilt disease (Panama wilt) (Rajapakse et al. 2005). Study on the genotypic variation among indi- viduals of a crop is the primary requirement for any crop improvement programme. To perform breed- ing programme it is essential to evaluate genetic variation if it is unknown. Therefore, it is necessary to evaluate genetic diversity of Kolikuttu germplasm in Sri Lanka to exploit genetic variation for desired characters such as Fusarium wilt toler- ance. Simple Sequence Repeat (SSR) markers are routinely used for diversity analysis and molecular breeding in many crops because of their high level of polymorphism, co-dominant nature, efficiency and cost effectiveness. Microsatellite markers have been utilized for numerous applications in Musa (Crouch et al. 1998b). The experiment was carried out with the objectives to estimate the genetic di- *Corresponding author:layanthi.agru2@gmail.com DNA TYPING OF DESSERT BANANA CULTIVAR „KOLIKUTTU‟ („SILK‟) ACCESSIONS BY MICROSATELLITE MARKERS HWL Pushpakumari1* , WLG Samarasinghe2, and SGJN Senanayake1 1Dept of Agricultural Biology, Faculty of Agriculture, University of Ruhuna, Mapalana, Kamburupitiya 2Plant Genetic Resources Centre, Gannoruwa, Peradeniya Accepted: 28th September 2009 ABSTRACT Fourteen Kolikuttu accessions showing morphological variation and Fusarium wilt tolerance were col- lected. Extracted DNA was subjected to PCR using six SSR primers. PCR products were separated by PAGE and bands were visualized, scored and statistically analyzed using POPGENE 1.32 version to obtain the dendrogram by genetic distance matrix. Out of 19 SSR alleles amplified, 12 were polymor- phic resulting in 63.16% loci polymorphism. Based on the dendrogram, three different „Kolikuttu’ ac- cessions known as “Athdath Puwalu”, the accession collected from Telijjawila, and accession collected from Angunakolapelassa were clustered separately. These three accessions could be considered as dif- ferent genotypes. Key words: DNA Typing, Dessert Banana, Cultivar Kolikuttu versity of selected Kolikuttu accessions and to gen- otype selected Kolikuttu accessions. The experiment was carried out at the Plant Ge- netic Resources Centre, Peradeniya, Sri Lanka from March to August 2008. Fourteen „Kolikuttu‟ acces- sions were collected based on morphological varia- tions and Fusarium wilt tolerance from different locations while an „Ambul’ accession was used as the control (Table 1). Immature unopened whitish colour banana cigar leaves were taken for DNA extraction. DNA was extracted according to the CTAB (Centylmethyl Ammonium bromide) procedure (Weising et al. 1995), which was modified for Mu- sa by Samarasinghe et al. (2001). Three grams of each leaf samples were ground in liquid nitrogen until a fine powder was formed. The powder was transferred quickly into 15ml of pre-warmed (600C) 4% CTAB extraction buffer with 0.1% ß–mercaptoethanol. After incubating for 30 min at 600C, 15ml of 24:1 Chloroform: Isoamyl alcohol was added and gently shaken for 20 min. The solution was centrifuged at 5000 rpm (10 min) and supernatant was transferred to another auto- claved “Falcon” centrifuge tube. Then 0.6 volume of ice cold isopropanol was added and mixed gently by inverting. Precipitated DNA was spooled out and placed in an eppendorf tube and washed with washing solution (1M ammonium acetate and 70% ethanol). Mixture was centrifuged at 5000rpm for 10min. The washing solution was drained and DNA pellet was air dried. Pellet was dissolved in 100- 500µl of TE buffer at 40C. Presence of DNA was confirmed on an agarose gel and stained with eth- idium bromide. The extract was further purified from RNA by adding RNAse to 50µg/ml. Paper presented at the 2nd National Symposium, Faculty of Agriculture, University of Ruhuna Quantification of DNA was done using 260nm and 280nm wave lengths in UV spectrophotometer by diluting the stock solution. Finally, a sample of the stock was diluted to 50ng/ µl to be used in PCR. PCR amplification was carried out with six simple sequence repeats (SSR) primers (Table2). Final concentration in the reaction solution was 0.5pM for each forward and reverse primer, 0.2mM for dNTPs each, 2.5mM for MgCl2 and 0.025U/µl of Taq DNA polymerase (Promega Corporation, USA) and 50µg/µl of DNA template in a 15ul reaction volume. PCR was performed using 940C for 2 min for the initial denaturing and then 35cycles of (30 sec 940C denaturing, 30sec at annealing temperature 550C, 30 seconds 720C extension) and a final exten- sion at 720C for 5min for primers MaSSR 18, MaSSR 20 and MaSSR 24. As the annealing tem- peratures of the primers Mb1- 134, Mb1-113 and Mb1-69 ranged from 590C to 620C, two categories of touchdown temperature cycles were used, con- sisting of initial denaturation of 3min at 950C for both profiles, followed by five cycles reduced by 10C per cycle (either 60-550C or 55-450C), culmi- nating with 30 cycles of denaturation for 20s at 940C, annealing for 20s at either 56 or 480C respec- tively, extension for 30s at 720C, and a final exten- sion at 720C for 10 min (Buhariwalla et al., 2005). Eight percent polyacrylamide 40cm gels con- taining 7M urea in 0.5× TBE were prepared. The gel was pre run at (at 50 W) for 45-60 min to pre- warm to 450C. Six micro liters of products were run at 1300V for about one and half hours and at 1500v for about three hours for efficient separation of bands. After electrophoresis, the gels were soaked in 10% acetic acid for 20min, washed 3 times for 3min each in deionized water and stained for 20min in 0.1% AgNO3 containing 0.15% formaldehyde. After a brief rinse (5sec) in de-ionized water signals were developed by soaking the gels in 500ml of 7.5% solution of NaOH and 3.75ml formaldehyde. Development was terminated by soaking gels in HWL Pushpakumari ET AL : DNA TYPING OF “KOLIKKUTTU” BY SSR MARKERS 114 Identity no. Location of sample collected Original collection site (District) Vernacular name Reason for collection 1 HORDI Divulapitiya (Gampaha) Kolikuttu To evaluate for Fusarium wilt disease tolerance 2 HORDI Halpitiya (Kegalle) Kolikuttu do 3 HORDI Sevanagala (Monaragala) Kolikuttu do 4 HORDI Embilipitiya (Ratnapura) Kolikuttu do 5 HORDI Embilipitiya (Ratnapura) Kolikuttu do 6 PGRC Embilipitiya (Ratnapura) Idal Puwalu Had lax bunches 7 PGRC Embilipitiya (Ratnapura) Kutti Puwalu Had compact bunches 8 PGRC Siyambalanduwa (Monaragala) Kotiyagala Type Had compact bunches 9 PGRC Puttlam (Puttlam) Athdath Puwalu Had long fruits 10 PGRC Angunukolapalassa (Hambanthota) Kolikuttu Not affected by Fusarium wilt 11 Telijjawila Matara “Agra” Newly released variety and has not affected by Fusarium wilt over ten generations and no hard lumps formation in fruits 12 Telijjawila Matara Kolikuttu Used for consumption 13 Jaffna Jaffna Kolikuttu Used for domestic consumption 14 Angunukolapalassa Hambanthota Kolikuttu Fusarium wilt Susceptible plant 15 HORDI Gannoruwa (Kandy) Ambul Control Table 1. Kolikuttu accessions used in the experiment and their collection sites [HORDI- Horticultural Research and Development Institute, PGRC- Plant Genetic Resources Centre] Table 2. Six SSR primers used in the analysis F- Forward; R- Reverse Source: Samarasinghe et al., 2002 and Buhariwalla et al., 2005 Primer pair Sequence of 5‟ to 3‟primer pairs Annealing Temperature Expected Product size (bp) MaSSR 18a (F) CGTCACAGAAGAAAGCACTTG 550C 200 MaSSR 18b (R) CCTTCTCCATCGTCATCAATC MaSSR 20a (F) GAAATGGAGTTGGAGAAACA 550C 222 MaSSR 20b (R) CACATATCCTTGTCGGAAGT MaSSR 24a (F) GACCCCCTTAAGCTGAACA 550C 172 MaSSR 24b (R) CCGACGGTCAACATACAATACA Mb1-69a (F) CTGCCTCTCCTTCTCCTTGGAA 48 oC 386 Mb1-69b (R) TCGGTGATGGCTCTGACTCA Mb1-113a (F) AGGTGCCACACAGTTCAGACA 56 oC 399 Mb1-113b (R) CAACCCAAACCTGTTCGACCAA Mb1-134a (F) ATGCCCAAGAAGGGAAGGGAA 56 oC 398 Mb1-134b (R) TAATGCCGGAGGATCAGTGTGA Tropical Agricultural Research & Extension 12(2):2009 10% acetic acid for 2-3min. Finally, gels were rinsed with de-ionized water. Each polyarylamide gel was scored visually for each primer and data were analyzed using POP- GENE version 1.32. Presence of a band scored as “1” and absence of a band scored as “0” whereas non amplified bands were considered as missing data and denoted as “.‟ for the software. Three different Kolikuttu genotypes were iden- tified among examined accessions. The accession known as “Athdath Puwalu” collected from Puttlam area (Genetic distances with the accession 11 is 0.1112, with the accession 12 is 0.3054 and with the control is 0.9985) is clustered separately while the accession collected from Telijjawila also is shown as a different genotype (Genetic distances with the accession 13 is 0.1719 and with the control is 0.4595) (Table 3) whereas accessions known as “Kutti Puwalu” , “Idal Puwalu” and “Kotiyagala type” have included in the same group (Fig. 1). Accession collected from Angunakolapelassa which had not affected from Fusarium wilt is unique genotype clustered separately from other Fusarium wilt tolerant accessions. The accession known as “Agra” collected from Telijjawila is a newly released variety [by Dr. (Mrs) Sujatha We- erasinghe of Agriculture Research station, Teli- jjawila, Matara] with neither hard lump formation in fruits nor affected by Fusarium wilt over ten generations. But it has not shown a genetic varia- tion at analyzed SSR loci with reference to either morphological variation or Fusarium wilt tolerance (Fig. 1). Accessions 1 to 5 were collected from Horticultural Research and Development Institute (HORDI) to evaluate Fusarium wilt disease toler- ance. However, they have clustered together with Fusarium wilt susceptible accession (accession No. 14) and therefore, a clear relationship between the- se two types could not obtain and further investiga- tions are needed. Among the analyzed SSR primers, MaSSR 24 a/b was the best primer for genotyping “Kolikuttu” ac- cessions. Despite a considerable morphological variation observed, analyzed accessions showed a lower genetic variation with respect to the analyzed SSR loci. This concludes that the most of the mor- phological variations in „Kolikuttu‟ were due to the environmental effect than the genetic effect. However, three different genotypes were iden- tified among examined Kolikuttu accessions which 115 Plate 1: Polyacrylamide gel (8%) picture of the SSR products of 14 “Kolikuttu” accessions and “Ambul” ac- cession amplified by six primers– [Sample descriptions of lane 1-15 are given in Table 1] 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 L MaSSR 20 a/b MaSSR 18 a/b 100bp 300bp MaSSR 24 a/b Mb1-134 a/b Mb1-113 a/b 400bp Mb1-69 a/b 200bp Accesion 01 Accesion 14 Accesion 09 Accesion 10 Accesion 12 Accesion 15 Accesion 02 Accesion 03 Accesion 04 Accesion 05 Accesion 06 Accesion 07 Accesion 08 Accesion 11 Accesion 13 Figure 1. Hierarchical cluster tree (Dendrogram) in- dicates grouping among accessions [Note: Accessions 1-5 HoRDI collection, 6-Idal puwalu, 7- Kutti puwalu, 8-Kotiyagala type, 9- Athdath puwalu, 10- PGRC[Plant Genetic Resources Centre] (Angunakolapalessa), 11- Agra, 12- Tellijjawila, 13- Jaff- na, 14- Fusarium wilt susceptible, 15- Control(Ambul) HWL Pushpakumari ET AL : DNA TYPING OF “KOLIKKUTTU” BY SSR MARKERS 116 [Nei‟s genetic identity (above diagonal) and genetic distance (below diagonal)] pop ID 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 **** 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 0.8947 0.8947 1.0000 0.8421 1.0000 1.0000 0.4737 2 0.0000 **** 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 0.8947 0.8947 1.0000 0.8421 1.0000 1.0000 0.4737 3 0.0000 0.0000 **** 1.0000 1.0000 1.0000 1.0000 1.0000 0.8947 0.8947 1.0000 0.8421 1.0000 1.0000 0.4737 4 0.0000 0.0000 0.0000 **** 1.0000 1.0000 1.0000 1.0000 0.8947 0.8947 1.0000 0.8421 1.0000 1.0000 0.4737 5 0.0000 0.0000 0.0000 0.0000 **** 1.0000 1.0000 1.0000 0.8947 0.8947 1.0000 0.8421 1.0000 1.0000 0.4737 6 0.0000 0.0000 0.0000 0.0000 0.0000 **** 1.0000 1.0000 0.8947 0.8947 1.0000 0.8421 1.0000 1.0000 0.4737 7 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 **** 1.0000 0.8947 0.8947 1.0000 0.8421 1.0000 1.0000 0.4737 8 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 **** 0.8947 0.8947 1.0000 0.8421 1.0000 1.0000 0.4737 9 0.1112 0.1112 0.1112 0.1112 0.1112 0.1112 0.1112 0.1112 **** 1.0000 0.8947 0.7368 0.8947 0.8947 0.3684 10 0.1112 0.1112 0.1112 0.1112 0.1112 0.1112 0.1112 0.1112 0.0000 **** 0.8947 0.7368 0.8947 0.8947 0.3684 11 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.1112 0.1112 **** 0.8421 1.0000 1.0000 0.4737 12 0.1719 0.1719 0.1719 0.1719 0.1719 0.1719 0.1719 0.1719 0.3054 0.3054 0.1719 **** 0.8421 0.8421 0.6316 13 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.1112 0.1112 0.0000 0.1719 **** 1.0000 0.4737 14 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.1112 0.1112 0.0000 0.1719 0.0000 **** 0.4737 15 0.7472 0.7472 0.7472 0.7472 0.7472 0.7472 0.7472 0.7472 0.9985 0.9985 0.7472 0.4595 0.7472 0.7472 **** Table 3: Nei‟s pairwise Genetic Distances (1972) will be useful in variety improvement. The acces- sion located at Angunakolapallessa can be consid- ered as an important genotype for further evalua- tion for Fusarium wilt tolerance. Analyzing these samples with more primers may help for further genotyping. ACKNOWLEDGEMENT The authors wish to thank Prof. Rohan Rajapakse, Dr (Mrs.) Sudarshanee Geekiyanage, Dr (Mrs.) Disna Rathnasekara, Dr RGAS Rajapakse, Dr (Mrs.) Sujatha Weerasinghe, Dr (Mrs.) Manel Dassanayake, and Mr Mahesh T Gunasena for their valuable suggestions given during this study. Also the assistance given by Miss SLD Jayaweera, Research Assistant is kindly acknowledged. REFERENCES AgStat 2004. Agricultural Statistics , Socio - Eco- nomic and Planning Center. Department of Ag- riculture, Peradeniya Buhariwalla HK, Jarret RL, Jayashree B, Crouch J H and Ortiz R 2005 Isolation and characteriza- tion of microsatellite markers from Musa balbi- siana. Molecular Ecology Notes. 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