Assessment of Genetic Diversity in 13 Local Banana (Musa Spp.) Cultivars Using Simple Sequence Repeat (SSR) Markers

ISSN 2349-7823
International Journal of Recent Research in Life Sciences (IJRRLS)
Vol. 2, Issue 1, pp: (65-69), Month: January - March 2015, Available at: www.paperpublications.org
Page | 65
Paper Publications
Assessment of Genetic Diversity in 13 Local
Banana (Musa Spp.) Cultivars Using Simple
Sequence Repeat (SSR) Markers
1
M H Kabir, 2
A N K Mamun, 3
H Fatema, 4
R Amin
1,2
Institute of Food and Radiation Biology, Atomic Energy Research Establishment G. P. O. Box 3787,
Dhaka Bangladesh
3
Department of Biotechnology and Genetic Engineering, Islamic University, Kustia, Bangladesh
4
National Mushroom Development and Extension Centre, Sobhanbag, Savar, Dhaka, Bangladesh
Abstract: A Study was conducted to investigate the genetic variability among 13 local banana cultivars using 3
SSR primers of Mb1-69, Mb1-113 and Mb1-134. All the primer pairs amplified a total of 29 different marker
bands with an average of 9.6 bands per primer. Among the 29 bands only 4 bands were monomorphic and the rest
25 bands were polymorphic. The sizes of the amplified DNA bands in 13 local banana cultivars varied from 200 bp
to 600 bp. The primer Mb1-113 amplified the highest (14) number of DNA bands and the primer Mb1-69
amplified the lowest (7) number of DNA bands whilst primer Mb1-134 amplified 8 DNA bands. The values of pair-
wise genetic distances ranged from 1.00 to 9.00 indicating the presence of wide genetic diversity. The dendogram
constructed based on phylogenetic relationship analysis revealed that the highest genetic diversity (9.00) found
between the cultivars champa and jawayta and also the cultivars champa and jahazy whilst the lowest (1.00)
between the cultivars doubled haploid and kathaly, doubled haploid and sorishafruity, doubled haploid and
amritsagor and doubled haploid and ganasundory. The UPGMA dendogram has segregated the 13 local banana
cultivars into two major clusters. Agnishwar and champa formed in cluster 1 and the rest of the cultivars like sobri
jesore, sobri, anazy, kathaly, jawayta, sorishafruity, amritsagor, jahazy, bangle, ganasundory and doubled haploid
have constituted the cluster 2.
Keywords: Genetic diversity, SSR markers, banana.
1. INTRODUCTION
Banana is one of the most important cash crop in Bangladesh and grown round the year in the country for commercial
purposes and homestead area for local consumption. It stood first position among the fruit producing in the country and
supplies 42% of the total fruit requirement and also financial return is higher compared to other fruits and field crops
(Haque, 1988b). It is a nutritious furit crop in the world and grown in many tropical areas where they are used both as a
staple food and dietary supplements (Assani et al., 2001). There are 32 landraces including dessert and cooking banana
cultivars in the country (Haque, 1988a). The average yield of banana in the country is about 15 t/ha that is far below the
average world yield of 30.63 t/ha in India (FAO, 2006-2007). Edible bananas are mostly sterile polyploids and must be
propagated vegetatively. Genetic variation is the starting point of any breeding program. Therefore, to set up an efficient
strategy for breeding to improve banana cultivars and support the choice of crossing parents, a solid understanding of
genetic diversity of available resources is needed. The crop is mostly damaged by fusarium wilt. It also faces banana
bunchy top virus, banana leaf and fruit beetle, pseudo stem borer and post-harvest diseases such as anthracnose, crown
rot, fruit rot and neck rot (Nik Masdek et al., 1998). Recently, genetic identification has received special attention due to
cultivar protection by the plant breeders and demanding cultivar identity for world trade as well as cultivar registration,
efficient collection and preservation. The enormous increase in the availability of various molecular techniques over the
past decades which has facilitated the classification of new banana cultivars, reassessment of traditional taxonomy,

ISSN 2349-7823
Page | 66
Paper Publications
diversity study and molecular characterization. Simple sequence repeat (SSR) have been successfully applied in the
molecular genotyping of many important crops such as rice (Pessoa-Filho et al., 2007), cereals (Hayden et al., 2007),
grapevine (This et al., 2004), Cacao (Zhang et al., 2006) and banana (Oriero et al., 2006) due to its co-dominant in nature.
Thus, the present study was undertaken to investigate the potentiality of SSR markers for distinguishing individual
accessions to rule out duplications among cultivars and to develop a standardized procedure for Musa genotyping which
could serve as a basis for molecular characterization of newly introduced global Musa gene bank as well as Musa research
and breeding community.
2. MATERIALS AND METHODS
Thirteen local banana cultivars namely sobri jessore (large size), sobri (small size), anazy (cooking, large), kathaly
(cooking, small), jawayta (seeded, large), sorishafruity (seeded, small), amritsagor, jahazy, agnishwar, champa,
ganasundory, bangle and doubled haploid (anther derived plant) were used for molecular characterization through SSR
analysis. The cultivars were collected from the experimental garden of Plant Biotechnology and Genetic Engineering
Division, Atomic Energy Research Establishment, Savar, Dhaka. Young and fresh leaf were used for DNA extraction.
The harvested leaf of 0.25g was cut down into small pieces and isolated DNA using modified CTAB (Abdullah et al.,
2012). The concentration of DNA sample was determined using UV spectrophotometer (PG Instruments Ltd. Korea) at
260/280 nm readings were 1.6 to 1.8. The PCR reaction mixture for 10 μl containing template DNA (50 ng) 1 μl, de-
ionized distilled water 1.8 μl, Taq buffer A 10X (15mM Tris-Hcl with Mgcl2) 1 μl, primer (2 μM) 5 μl, dNTPs (2.5 mM)
1 μl and Taq DNA polymerase (5u/ μl) 0.2 μl. The reaction with each primer was replicated thrice to check the
reproducibility of DNA. DNA amplification was carried out in an oil-free DNA thermal cycler (Finnzymes Instruments,
EU) at the following thermal profile: initial denaturation for 5 minutes at 94ºC followed by denaturation for 60 seconds at
94ºC, annealing at 55ºC for 60 seconds and extension at 72ºC for 120 seconds. A final extension at 72ºC for 7 minutes
was allowed for complete extension of all amplified fragments. Amplified fragments were separated on a 1.4% agarose
gel containing 0.5 μg/ml ethidium bromide in 1X TBE buffer for 1 hour at 100 volts. The gel was visualized by UV-
transilluminator and photographed by gel documentation system (Major Science, USA). The amplified bands were
visually scored as present (1) and absent (0) of bands, size of bands and overall polymorphism of bands. The scores
obtained using all primers in the SSR analysis were then pooled for constructing a single data matrix. This was used to
estimate polymorphic loci, genetic diversity, genetic distance (Nei, 1972) and a UPGMA (Unweighted Pair Group
Method with Arithmatic Means) dendrogram using computer program ‘Statistica’.
3. RESULTS AND DISCUSSION
The three (3) primers generated 29 distinct bands of which 21 were considered as polymorphic. The percentage of
polymorphic loci was 86.20% indicating a higher level of polymorphism. The three primers generated 9.66 score able
bands per primer and 7.0 polymorphic per primer. Oriero et al. (2006) selected 44 SSR primers to assess 40 Musa
accessions and found only 9 primers produced amplified product which is very low number of amplified product
comparing to the present study. Thus, the primers are used in this study is useful for Musa genetic analysis. Creste et al.
(2004) used 33 primers to investigate genetic diversity in Musa spp. and 15 primers gave amplified products. Thus,
selection of primers played an important role in Musa genotyping. A diverse level of polymorphism in different crops has
been reported in tomato (Moonmoon, 2006), eggplants (Biswas et al., 2009) and chili (Para et al., 1998). The values of
pair-wise (Nei’s, 1972) genetic distance ranged from 1.00 to 9.00. The highest genetic distances (9.00) were found
between cultivars champa and jawayta and also champa and jahazy. The lowest (1.00) value were observed between the
cultivars doubled haploid and kathaly, doubled haploid and sorishafruity, Doubled haploid and amrit sagor and doubled
haploid and ganasundory. The difference between the highest and the lowest value of genetic distance revealed that wide
range of variability persisting among the 13 banana cultivars. Moonmoon (2006) reported that assessment of genetic
diversity, molecular markers were superior to morphological, biochemical and other method like heterosis.
Dendrogram based on Nei’s (1972) genetic distance using UPGMA indicated segregation of 13 banana cultivars into two
main clusters. Cultivar agnishwar and champa formed cluster-1 and the remaining 11 cultivars grouped in cluster-2.
Cluster-2 was divided into 4 sub-clusters. Jahazy, jawayta, anazy formed sub-cluster-1, sub-cluster-2 and sub-cluster-3
respectively. Sub-cluster-4 includes sobri jesore, kathaly, sorishafruity, amrit sagor, ganasundory, doubled haploid, bangla
and sobri. Sub-cluster-4 was again divided into more sub-clusters forming sub-sub-clusters. From this investigation, it

ISSN 2349-7823
Page | 67
Paper Publications
was found that the highest genetic identity (9.00) remains between the cultivar champa and jawayta and champa and
jahazy. On the other hand, the lowest (1.00) genetic identity was observed between the cultivar doubled haploid and
kathaly, doubled haploid and sorishafruity, doubled haploid and amrit sagor, doubled haploid and ganasundory indicating
these cultivars might be beneficial for banana improvement program. In this study, it is also revealed that each of the 13
banana cultivars possessed specific marker which could be used for their authentic identification. From the table, it was
observed that most of the cultivars is linked with each other as they have equal genetic distance. Some of the cultivars also
maintained close relationship with each other due to nearest genetic distance among the cultivars. Similar observation was
reported by Alam et al. (2012) in tomato. Many authers namely Uma et al., 2006, Jain et al., 2007, Noyer et al., 2005,
Hautea et al., 2004, Pillay et al., 2001 and 2000, Crouch et al., 2000, Newbury et al., 2000, Sunchez et al., 2000, Howell
et al., 1994 studied relationships among banana populations.
The banding patterns of different banana cultivars using 3 primers are shown in Figs. 1 to 3. Band size ranging from 200
to 600 bp in PCR amplification products scored for primers. Strong and weak bands were produced in the PCR reactions.
Weak bands produced from low homology between the primer and the pairing site on the DNA strand (Thormann et al.,
1994). This study shows the genetic diversity within Musa germplasm collection in Atomic Energy Research
Establishment. There are several limitation in breeding and genetic improvement in Musa genotypes. Therefore, selection
the most suitable parents that would result in higher diversity is crucial for significant genetic progress.
Linkage Distance
champa
agnishwar
jahazy
jawayta
anazy
sobri
bangla
doubledhaploid
ganasundory
amritsagor
sorishafruity
kathaly
sobrijesore
0 1 2 3 4 5 6
Figure1: UPGMA dendogram based on Nei’s (1972) genetic distance, summarizing data on differentiation in 13 local banana
cultivars according to SSR analysis.

ISSN 2349-7823
Page | 68
Paper Publications
A B
C
Figure2: SSR profiles of 13 banana cultivars DNA with different primers (a)Mb1-69, (b)Mb1-113 & (c)Mb1-134. LaneM-1.0 kb
DNA ladder, Lane1-Sobri jessore (large size), Lane2-Sobri (small size), Lane3-Anazykola, Lane4-Kathaly, Lane5-Jawayta
(large size seeded), Lane6-Sorishafruity (small size seeded), Lane7-Amrit sagor, Lane8-Jahazykola, Lane9-Agnishwar, Lane10-
Champa, Lane11-Ganasundory, Lane12-Banglakola, Lane13-Doubled haploid.
REFERENCES
[1] Alam, S.K.S., E. Ishrat, M.Y. Zaman and M.A. Habib. 2012. Comparative karyotype and RAPD analysis for
characterizing three varieties of Lycopersicon esculentum Mill. Bangladesh J. Bot. 41(2): 149-154.
[2] Assani, A.R., G. Haicour, F. Wenzel, F. Cote, F. Bakry, B. Foroughiwehr, G. Ducreux, M.E. Aguillar and A. Grapin.
2001. Plant regeneration from protoplasts of dessert banana cv. Grande Naine (Musa spp., Cavendish sub-group
AAA) via somatic embryogenesis. Plant Cell Rep. 20: 482-488.
[3] Biswas, T.G., A.A.Y. Akhond, M. Alamin, M. Khatun and M.R. Kabir. 2009. Genetic relationship among ten
promising eggplant varieties using RAPD markers. Plant Tissue Cult.& Boitech. 19(2): 119-126.
[4] Crouch, H.K., J.H. Crouch, S. Madsen, D.R. Vuylsteke and R. Ortiz. 2000. Comparative analysis of phenotypic and
genotypic diversity among plantain landraces (Musa spp. AAB group). Theo Appl Gen. 101: 1056-1065.
[5] FAO. 2007. Production Yearbook 2006, Rome.
[6] Hautea, D.M., G.C. Molina, C.H. Balatero, N.B. Coronado, E.B. Perez, M.T.H. Alvarez, A.Q. Canama, R.H. Akuba,
R.B. Quilloy, R.B. Frankie and C.S. Caspillo. 2004. Analysis of induced mutants of Philippine with molecular
markers. In: Banana improvement: Cellular, Molecular Biology and Induced Mutations. Jain, S.M. and R. Swennen
(eds.) www.scipub.net.

ISSN 2349-7823
Page | 69
Paper Publications
[7] Hayden, M.J., H. Kuchel and K.J., Chalmers. 2004. Sequence tagged microsatellites for the xgwm 533 locus provide
new diagnostic markers to select for the presence of stem rust registance gene Sr2 in bread wheat (Triticum aestivum
L.) Theoritical and applied genetics. 109: 1641-1647.
[8] Haque, M.A. 1988a. Studies of the irrigation requirement of banana during dry months. Bangladesh J. Agril.,
13(1):59-60.
[9] Haque, M.A. 1988b. ‘Kolar Bagan’ (3rd
edn.). Banana Research Project, Bangladesh Agricultural University,
Mymensingh. P. 24.
[10] Howell E.C., H.J. Newbury, R.L. Swennen, L.A. Withers and B.V. Ford-liod. 1994. The use of RAPD for
identifying and classifying Musa germplasm. Genome. 37: 328-332.
[11] Jain, P.K., M.L. Saini, H. Pathak and V.K. Gupta. 2007. Analysis of genetic variation in different banana (Musa
spesies) variety using random amplified polymorphic DNAs (RAPDs). Afr J Biotech. 6: 1987-1989.
[12] Marco, P.F.P., A.N.A. Antonio, E.F. Marcio, B. Andre, H.N.R. Paulo. 2007. A set of multiplex panels of
microsatellite markers for rapid molecular characterization of rice accessions. BMC Plant Biology. 7(23): 1-10.
[13] Moonmoon, S. 2006. Random amplified polymorphic DNA markers (RAPD) for genetic variation study among
tomato varieties. M.S. Thesis. Dept. of Biotechnology. Bangladesh Agricultural University, Mymensingh. pp. 34-51.
[14] Nei, M. 1972. Genetic distance between populations. American Nat. 106: 283-292.
[15] Newbury, H.J., E.C. Howell, H.J. crouch and B.V. Ford-lioyd. 2000. Natural and culture-induced genetic variation
in plantains (Musa spp. AAB group). Aus J Bot. 48: 493-500.
[16] Nik Masdek, N.H., Y. Doon, A.K. Sidam, M.S. Osman and S. Muid. 1998. Disease and insect management in
malaysia. Procedings of the first national banana seminar at awana genting golf and country resort. ed., Zakaria
wahab. Pp. 87-94.
[17] Noyer, J.L., S. Causse , K. Tomekpe, A. Bouet and F.C. Baurens. 2005. A new image of plantain diversity assessed
by SSR, AFLP and MSAP markers. Genetica. 124: 61-69.
[18] Oriero, C.E., O.A. Odunola, Y. Lokkoy and I. Ingelbrecht. 2006. Analysis of B-genome derived simple sequence
repeat (SSR) markers in Musa spp. African journal of biotechnology. 5(2): 126-128.
[19] Paran, I., E. Aftergoot and C. Shifriss. 1998. Variation in Capsicum annuum revealed by RAPD and AFLP markers.
Euphytica. 99(3): 167-173.
[20] Pillay, M., D.C. Nwakanma and A. Tenkouano. 2000. Identification of RAPD markers linked to A and B genome
sequences in Musa L. Genome 43: 763-767.
[21] Pillay, M., E. Ogundiwin, D.C. Nawakanma, G. Ude and A. Tenkouano. 2001. Analysis of genetic diversity and
relationships in East African banana germplasm. Theo Appl Gen. 102: 965-970.
[22] Sanchez, I., D. Gaviria, G. Gallego, D. Fajardo, J.A. Valencia, M. Lobo, J. Thome and W. Roca. 2000. Molecular
characterization for the management of the Colombian collection of Musaceae. Proc. Int Soc Trop Hort. 42: 252-
259.
[23] This, P., A. Jung, P. Boccacci, J. Borrego, R. Botta, L. Costantinl, M. Crespan, G.S. Dangl, C. Eisenheld, F.M.
Ferreira, S. Grand, J. Ibanez, T. Lacombe, V. Laucou, R. Magalhaes, C.P. Milani, N. Peterlunger, E. Regner, F.L.
Zulini, E. Maul. 2004. Development of standard set of microsatellite reference alleles for identification of grape
cultivars. Theor. Appl. Genet. 109: 1448-1458.
[24] Uma, S., S.A. Siva, M.S. Saraswathi, M. Manickavasagam, P. Durai, R. Selvarajan and S. Sathiamoorthy. 2006.
Variation and intraspecific relationships in India wild Musa balbisiana (BB) population as evidenced by random
amplified polymorphic DNA. Genet Resour Crop Evol. 53: 349-355.
[25] Zhang, D., S. Mischke, R. Goenaga, A.A. Hemeida, A.J. Saunders. 2006. Accuracy and reliability of high-
throughput microsatellite genotyping for cacao clone identification. 46(5): 2084-2092.

Assessment of Genetic Diversity in 13 Local Banana (Musa Spp.) Cultivars Using Simple Sequence Repeat (SSR) Markers

More Related Content

What's hot (20)

Viewers also liked (11)

Similar to Assessment of Genetic Diversity in 13 Local Banana (Musa Spp.) Cultivars Using Simple Sequence Repeat (SSR) Markers (20)

Recently uploaded (20)

Assessment of Genetic Diversity in 13 Local Banana (Musa Spp.) Cultivars Using Simple Sequence Repeat (SSR) Markers