MICROSATELLITE MARKERS FOR CLASSIFIYING AND ANALYZING GENETIC RELATIONSHIP OF

IV. MICROSATELLITE MARKERS FOR CLASSIFIYING AND ANALYZING GENETIC RELATIONSHIP OF

BANANA CULTIVARS IN INDONESIA ABSTRACT In addition to be used for identifying banana genomic groups, microsa- tellites were employed to investigate genetic relationships among banana accessions collected from various areas in Indonesia. One hundred and sixteen banana accessions were analyzed using the primers MaCIR108 and Ma-3-90 for identifying genomic groups and additional 6 microsatellite primers for genetic relationship analysis. The results showed that 73 accessions identified as AA or AAA; and AAA genomic groups should be designated under the name M. acuminata , 2 accessions of BB should be under the name M. balbisiana, 21 accessions of AAB and 20 accessions of ABB should be placed under the name M. x paradisiaca . This study also detected 1 allele of the Ma-3-139 with the size of 132 bp which supported the MaCIR108 alleles for separating banana containing the B genome from the A genome alone. Analysis of the 116 accessions using the 8 primers detected only 99 genotypes because 26 accessions clustered within 9 identical genotypes. All banana accessions belonging to BB, AAB, and ABB genomic groups clustered in the first main cluster together with majority of accessions containing the A genome alone. The second main cluster was belonging to 11 accessions consisting of AA or AAA; and AAA genomic groups. The accessions BB, AAB and ABB were clearly distinct from each other. The BB appears closer to the ABB genomic groups. All accessions containing the B genome were clustered according to their genomic groups, except 4 accessions of AAB clustering together with accessions of the A genome alone. These accessions of AA or AAA; and AAA could not be significantly distinguished, although majority of them tend to be clustered according to ploidy level. Key words: genomic group, microsatellite, Musa acuminata, M. balbisiana, M.x paradisiaca INTRODUCTION The application of Musa paradisiaca,- the first scientific name given and published by Linnaeus in his Species Plantarum,- for cultivated bananas is not justifiable. It is because the cultivated bananas consisted of different cultivars that are quite distinct from each other Valmayor et al. 2000. There were three possible morphologically different cultivars, those originating from each of the two of wild species, M. acuminata Colla and M. balbisiana Colla and those which are hybrids Karamura 1998, Valmayor et al. 2000. Simmonds and Sheperd 1955 convincingly argued concerning the origin of cultivated bananas. They refused the cultivated bananas which were designated under the name M. paradisiaca. The bananas, either pure diploid or pure triploid cultivars keep the morphological characteristics of their wild type M. acuminata or M. balbisiana. Therefore, they should be classified in the same species as each of their wild parents Valmayor et al. 2000. The plants brought from the wild into the cultivation are designated under the names that are applied to the same taxa growing in nature Brickell et al. 2004. The hybrid bananas that evolved from the two natural species mostly formed triploid cultivars Heslop-Harison and Schwarzacher 2007. According to International Code of Botanical Nomenclature ICBN Mc Neal et al. 2006 and International Code of Nomenclature of Cultivated Plants ICNCP Brickell et al. 2004 the hybrid bananas should be classified into M. x paradisiaca because they have been proven as the nature hybrids Stover and Shepherd 1987; Jumari 2000. The ICNCP recognized the complex hybrid origins of most crops and classified them in convenience ways to users. Nomenclatural stability is needed for trade Hetterscheid and Brandenburg 1995. In the case, the name M. x paradisiaca should be accepted because this binomial was published before M. sapientum L., and is in fact known as the type species for the hybrid banana Stover and Simmonds 1987. According to Karamura 1998, the name M. x paradisiaca is applicable to all hybrids of banana cultivars notwithstanding their genomic composition. According to ICNCP Brickell et al. 2004, the full name of a cultivar is the accepted botanical name in Latin form of the taxon to which it is assigned, followed by the cultivar epithet. The problems of banana cultivars classification and their nomenclature emerged from description of M. paradisiaca given by Linnaeus. The description could not be applied especially in Southeast Asia Valmayor et al 2000 because many local cultivars having important characteristics were not found in the other regions. There is no publication or consensus that could be used as a reference by the banana community. Taxonomy Advisory Group TAG for Musa made an effort to develop nomenclature and glossary of terms which could be accepted by Musa research community so that they can communicate and understand each other INIBAP 2006b. Genomic constitutions play an important role in the banana classification Pillay et al. 2004. Alteration of genomic groups sometimes caused the change of the taxonomic status as exhibited in the first and the second study Chapter II and III. The Genomic groups have usually been determined based on 15 morphological appearances following the scoring system described by Simmonds and Shepherd 1955. Cultivar identification based on these characters is not always correct due to the influences of the environmental factors Vicente et al. 2005. Thus, it is necessary to classify the genomic groups of banana cultivars using molecular approaches. The ability of the molecular techniques to distinguish one cultivar from the others was recognized to be more effective than morphological techniques Ford-Lloyd et al. 1997. Microsatellite is one of the most informative molecular markers to reveal genetic diversity of banana cultivars Kaemmer et al. 1997; Creste et al. 2003, 2004. The markers have been used for genotypic identification of many plant species, such as coconut Perera et al. 2001, rice Chakravarthi and Naravaneni 2006, and Annona senegalensis Kwapata et al. 2007. Microsatellites are DNA regions flanked by 2 conserved sequences and detected in a specific locus Spooner et al. 2004; Semagn et al. 2006. Polymorphisms in the microsatellite regions could be detected using primers designed from these flanking regions Innan et al. 1997. Besides a few microsatellite primers were proven to provide diagnostic characters for banana genomic groups Kaemmer et al. 1997; Creste et al. 2003, 2005, determination key of microsatellite markers have been successfully constructed in the second study Chapter II. The purposes of the study were to classify banana accessions collected from various regions in Indonesia using the microsatellite determination key and to investigate the genetic relationships among these accessions based on microsatelite markers. MATERIAL AND METHODS Plant materials and primers A total of 116 banana accessions collected from various areas in Indonesia were examined Table 4.1. The accessions were classified into their genomic groups using microsatellite determination key. The genetic relationships among banana accessions were investigated based on 8 microsatellite primers Table 4.2. Table 4.1 List of 116 accessions used in the study of cultivated banana classification and genetic relationship analysis Accession Region Sourcecollection Barangan Merah Medan Diperta Barley Cukurgondang Pasuruan Diperta Embuk Putih Giri Banyuwangi Diperta Gintung Tawangsari Sukoharjo Diperta Goplek Unknown Diperta Koja Santen Unknown Diperta Kolis Unknown Diperta Mas Lumut Pakem Sleman Diperta Medan Tegalsari Girimulyo Diperta Morli Unknown Diperta Raja Madu Giri Banyuwangi Diperta Sebrot Cukurgondang Pasuruan Diperta Sembuk Cukurgondang Pasuruan Diperta Siam Paris Unknown Diperta Toklek Sleman Diperta Wangu Cipaku Bogor Diperta Ambon Lumut A Unknown PKBT Ambon Lumut B Unknown PKBT Ampyang Unknown PKBT Angleng Unknown PKBT Anjasmara Yogyakarta PKBT Badak Unknown PKBT Embok Unknown PKBT Ice Yogyakarta PKBT Jepang Unknown PKBT Kapas Tasikmalaya Tasikmalaya PKBT Kepok Amerika Yogyakarta PKBT Kepok Kuning Gowa makasar PKBT Kepok Manggala Unknown PKBT Kutes Wonosobo PKBT Lampung Unknown PKBT Lidi Unknown PKBT Madura Unknown PKBT Mandar Unknown PKBT Mas 40 Hari Yogyakarta PKBT Mas Purbalingga Unknown PKBT Nangka Unknown PKBT Oli Unknown PKBT Table 4.1 List of 116 accessions used in the study of cultivated banana classification and genetic relationship analysis continued Accession Region Sourcecollection Papan Unknown PKBT Potho Bunthek Yogyakarta PKBT Potho Yogya Yogyakarta PKBT Prabumulih Unknown PKBT Raja Bulu Unknown PKBT Raja Sableng Yogyakarta PKBT Raja Sereh Unknown PKBT Rotanhari Yogyakarta PKBT Sabulan Unknown PKBT Segli Unknown PKBT Siam Manggala Unknown PKBT Sigung Unknown PKBT Susu Unknown PKBT Austroli Jasinga Bogor Bogor Badak Jasinga Bogor Bogor Jepang Jasinga Bogor Bogor Kepok Merah Jasinga Bogor Bogor Klutuk Hijau Jasinga Bogor Bogor Pisang Kuning Jasinga Bogor Bogor Raja Bening Jasinga Bogor Bogor Rejang Jasinga Bogor Bogor Udang Jasinga Bogor Bogor Unknown 1 Jasinga Bogor Bogor Unknown 2 Jasinga Bogor Bogor Unknown 3 Jasinga Bogor Bogor Unknown 4 Jasinga Bogor Bogor Unknown 5 Jasinga Bogor Bogor Unknown 6 Jasinga Bogor Bogor Unknown 7 Jasinga Bogor Bogor Unknown 8 Jasinga Bogor Bogor Unknown 9 Jasinga Bogor Bogor Agakher Bokondini Jayawijaya RIF Angleng Unknown RIF Awomen Unknown RIF Ayam Kb. Pipit Baso, West Sumatera RIF Barifta A Unknown RIF Berlin Unknown RIF Branjut Unknown RIF Buai Unknown RIF Dingin Unknown RIF Hutan Jambi Unknown RIF Ik Osroc Amban Manokwari RIF Jantan Unknown RIF Jari Buaya Unknown RIF Keikeni Amban Manokwari RIF Ketan Unknown RIF Kilita Unknown RIF Klutuk Susu Unknown RIF Kole Unknown RIF Table 4.1 List of 116 accessions used in the study of cultivated banana classification and genetic relationship analysis continued Accession Region Sourcecollection Lase Unknown RIF Lilin Jambi RIF Limpyang Bk Sundi, West Sumatera RIF Longong Unknown RIF Mantra Ho Unknown RIF Mantreken Unknown RIF Mourina Unknown RIF Neij Aubu Ransiki Manokwari RIF Neij Amper Ransiki Manokwari RIF Neij Houbwan Ransiki Manokwari RIF Numbungga Bokondini Jayawijaya RIF Panggang Deli Serdang, North Sumatera RIF Pogori Bokondini Jayawijaya RIF Pup Unknown RIF Ratu Tanah Datar, West Sumatera RIF Raja Muli Unknown RIF Ramehaye Unknown RIF Sramfin Amban Manokwari RIF Tanduk Lembu Haroung Gaol, West Sumatera RIF Udang Unknown RIF Unknown 1 Karimunjawa Central Java Unknown 2 Karimunjawa Central Java Unknown 3 Karimunjawa Central Java Unknown 4 Karimunjawa Central Java Unknown 5 Karimunjawa Central Java Unknown 6 Karimunjawa Central Java Unknown 7 Karimunjawa Central Java Unknown 8 Karimunjawa Central Java Unknown 9 Karimunjawa Central Java 1 Diperta, Dinas Pertanian dan Kehewanan, Yogyakarta 2 RIF, Research Institute of Fruits, Solok West Sumatera 3 PKBT, Pusat kajian Buah Tropika, IPB Molecular key for determination of banana genomic groups 1a. The MaCIR108 alleles with the size of 270 bp or less………………………..2 2a. The number of alleles, one to two…………..AA or AAA genomic group 2b. The number of alleles, three………………………...AAA genomic group 1b. The MaCIR108 alleles with the size of more than 270 bp……………………3 3a. Allele with the size of 295 bp, only…………………...BB genomic group 3b. Alleles combination: 295 bp; 295 bp and 289 bp; 295 bp and 287 bp; and 1 allele of the MaCIR108 with the size of 270 bp or less ……...……………………………………………….ABB genomic group 3c. Alleles combination of 287 bp and alleles of the MaCIR108 with the size of 270 bp or less.……………………………………….…...….………….4 4a. Alleles combination of 287 bp and 2 alleles of 270 bp or less .…………………….…………………………....AAB genomic group 4b. Alleles combination of 287 bp and 1 allele of 270 bp or less…......….5 5a. The Ma-3-90 allele of 152 bp, present .…….ABB genomic group 5b. The Ma-3-90 allele of 152 bp, absent .……..AAB genomic group 3d. Allele combination of 275 bp and alleles of the MaCIR108 with the size of 270 bp or less ............................................................AAB genomic group Table 4.2 Primers used to classify the cultivated bananas and to investigate the genetic relationships among the 116 banana accessions Primers Forward primer sequence Reverse primer sequence, 5’-3’ Optimized annealing temperature ºC MaCIR108 a F: TAAAGGTGGGTTAGCATTAGG R: TTTGATGTCACAATGGTGTTCC 55 Ma-1-132 a F: GGAAAACGCGAATGTGTG R: AGCCATATACCGAGCACTTG 53 MaCIR327b a F: AAGTTAGTCAAGATAGTGGGATTT R: CTTTTGCACCAGTTGTTAGGG 50 MaCIR332a a F: TCCCAACCCCTGCAACCACT R: ATGACCTGTCGAACATCCTTT 53 Ma-1-17 a F: AGGCGGGGAATCGGTAGA R: GGCGGGAGACAGATGGAGT 56 Ma-1-27 a F: TGAATCCCAAGTTTGGTCAAG R: CAAAACACTGTCCCCATCTC 56 Ma-3-90 b F: GCACGAAGAGGCATCAC R: GGCCAAATTTGATGGACT 56 Ma-3-139 b F: ACTGCTGCTCTCCACCTCAAC R: GTCCCCCAAGAACCATATGATT 56 a Primers developed by Kaemmer et al. 1997 b Primers developed by Crouch et al. 1998 DNA extraction and microsatellite region amplification Total DNA was extracted from fresh young leaves using a modified SDS procedure Dixit 1998. The extraction buffer consisted of 100 mM Tris pH 8.0, 50 mM EDTA pH 8.0, 500 mM NaCl, 10 mM beta-mercaptoethanol, and 20 SDS. Precipitation of DNA was carried out without a PEG solution treatment. Quality and concentration of DNA were evaluated by electrophoresis in 0.8 agarose gel stained with ethidium bromide EtBr and by spectrophotometer, respectively. PCR reaction volumes were 10 μl containing 1.5 μl DNA template 15 ng DNA, 1 μl 10 x PCR buffer with 20 mM MgCl 2 , 0.2 μl 10 mM dNTPs, 0.2 μl 10 µM of each primer, and 0.06 μl 5 μl -1 Taq DNA Polymerase Native GenScript Corporation®. The PCR cycling consisted of an in initial denaturation at 95ºC for 4 min, followed by 35 cycles of 30 s at 95ºC for template denaturation, 30 s for primer annealing with the temperature depending on the primer, 30 s at 72ºC for primer extension, ended with an extention of 10 min at 72ºC. PCR reactions were carried out in a Perkin Elmer 2400 thermocycler Applied Biosystems, Foster City, CA, USA®. Electrophoresis and silver staining The PCR products were separated in 6 denaturized polyacrylamide gels 7 M Urea Sigma-Aldrich Chemie Germany® at 45-60 W constant power for 2 to 3 hours depending on the size of microsatellites. Alleles of microsatellite were visualized by silver staining according to modified procedure of Creste et al. 2001. All solutions were prepared using distilled water. Data analysis Banana accessions were classified into their genomic groups based on the existence of amplified fragments of the primers MaCIR108 and Ma-3-90. Each variant fragment was assumed as an allele. For the need of genetic relationship analysis using the 8 primers, each allele was scored as present 1 or absent 0. The size of each allele was estimated using a 100-bp DNA ladder Invitrogen®. The microsatelite markers detected 2 alleles in the heterozygous genotype for diploid and triploid or 3 alleles for triploid while in the homozygous genotype the markers detected only 1 allele. Jaccard coefficient using Similarity of Qualitative Data SYMQUAL procedure was used to estimate of similarity between the accessions. The matrix of similarity was then used for clustering analysis to investigate genetic relationships among the accessions. Clustering analysis was performed using Sequential, Agglomerative, Hierarchical and Nested SAHN procedure of Unweighted Pair-grouping Method with Arithmatic Average UPGMA by the Numerical Taxonomy and Multivariate Analysis System NTSYSpc version 2.02 Rohlf 1998. RESULTS AND DISCUSSION Identification of banana genomic groups using the determination keys of microsatellite markers The primers MaCIR108 and Ma-3-90 produced clear alleles and polymorphic banding patterns. Amplification of 2 or 3 alleles was observed in mostly banana accessions. The allele number of more than one within one locus was considered as heterozygous genotypes Cerenak et al. 2004. In the study, the maximum number of alleles from any given accessions was three. It is because tetraploid bananas are rarely found naturally Heslop-Harrison and Schwarzacher 2007. Most cultivated bananas are triploid. The MaCIR108 detected 18 alleles with the size of alleles ranged from 220 to 295 bp, while the Ma-3-90 detected 14 alleles with the size of alleles ranged from 132 to 172 bp. Genomic group identification of the 116 accessions using the primers MaCIR108 and Ma-3-90 determined AA or AAA; AAA; BB; AAB; and ABB genomic groups. Seventy three of the 116 accessions studied only possessed the MaCIR108 alleles with the size of 270 bp or less. In these accessions, allele of the Ma-3-90 with the size of 152 bp was not detected. Therefore, they should be classified into AA or AAA; and AAA genomic groups due to the lacking of diagnostic characters for the B genome. The range of the MaCIR108 alleles in these accessions were approximately from 210 to 268 bp as previously reported in the first study Chapter II. The 46 accessions having 3 alleles of the MaCIR108 were classified into AAA genomic groups whereas the others 27 accessions with one or two alleles were classified into AA or AAA genomic group Table 4.3. Table 4.3 List of 73 accessions classified into AA or AAA; and AAA genomic groups based on the primers MaCIR108 and Ma-3-90 Accession Genomic group Region Source collection Barley AA or AAA Cukurgondang Pasuruan Diperta Morli AA or AAA Unknown Diperta Jepang AA or AAA Unknown PKBT Lampung AA or AAA Unknown PKBT Lidi AA or AAA Unknown PKBT Mas 40 Hari AA or AAA Yogyakarta PKBT Mas Purbalingga AA or AAA Unknown PKBT Oli AA or AAA Unknown PKBT Sigung AA or AAA Unknown PKBT Pisang Kuning AA or AAA Jasinga Bogor Bogor Table 4.3 List of 73 accessions classified into AA or AAA; and AAA genomic groups based on the primers MaCIR108 and Ma-3-90 continued Accession Genomic group Region Source collection Rejang AA or AAA Jasinga Bogor Bogor Unknown 1 AA or AAA Jasinga Bogor Bogor Unknown 3 AA or AAA Jasinga Bogor Bogor Unknown 4 AA or AAA Jasinga Bogor Bogor Unknown 5 AA or AAA Jasinga Bogor Bogor Unknown 6 AA or AAA Jasinga Bogor Bogor Unknown 7 AA or AAA Jasinga Bogor Bogor Unknown 8 AA or AAA Jasinga Bogor Bogor Berlin AA or AAA Unknown RIF Hutan Jambi AA or AAA Unknown RIF Jari Buaya AA or AAA Unknown RIF Ketan AA or AAA Unknown RIF Kole AA or AAA Unknown RIF Lilin AA or AAA Jambi RIF Ratu AA or AAA Tanah Datar, West Sumatera RIF Raja Muli AA or AAA Unknown RIF Ramehaye AA or AAA Unknown RIF Gintung AAA Tawangsari Sukoharjo Diperta Kolis AAA Unknown Diperta Mas Lumut AAA Pakem Sleman Diperta Raja Madu AAA Giri Banyuwangi Diperta Sebrot AAA Cukurgondang Pasuruan Diperta Ambon Lumut A AAA Unknown PKBT Ambon Lumut B AAA Unknown PKBT Ampyang AAA Unknown PKBT Angleng AAA Unknown PKBT Anjasmara AAA Yogyakarta PKBT Badak AAA Unknown PKBT Ice AAA Yogyakarta PKBT Kutes AAA Wonosobo PKBT Madura AAA Unknown PKBT Papan AAA Unknown PKBT Potho Bunthek AAA Yogyakarta PKBT Potho Yogya AAA Yogyakarta PKBT Rotanhari AAA Yogyakarta PKBT Sabulan AAA Unknown PKBT Segli AAA Unknown PKBT Badak AAA Jasinga Bogor Bogor Jepang AAA Jasinga Bogor Bogor Raja Bening AAA Jasinga Bogor Bogor Udang AAA Jasinga Bogor Bogor Unknown 9 AAA Jasinga Bogor Bogor Agakher AAA Bokondini Jayawijaya RIF Angleng AAA Unknown RIF Awomen AAA Unknown RIF Ayam AAA Kb. Pipit Baso, West Sumatera RIF Barifta A AAA Unknown RIF Table 4.3 List of 73 accessions classified into AA or AAA; and AAA genomic groups based on the primers MaCIR108 and Ma-3-90 continued Accession Genomic group Region Source collection Branjut AAA Unknown RIF Buai AAA Unknown RIF Dingin AAA Unknown RIF Ik Osroc AAA Amban Manokwari RIF Keikeni AAA Unknown RIF Lase AAA Unknown RIF Limpyang AAA Bk. Sundi, West Sumatera RIF Mourina AAA Unknown RIF Neij Aubu AAA Ransiki Manokwari RIF Neij Houbwan AAA Ransiki Manokwari RIF Numbungga AAA Bokondini Jayawijaya RIF Pogori AAA Bokondini Jayawijaya RIF Sramfin AAA Amban Manokwari RIF Tanduk Lembu AAA Haroung Gaol, West Sumatera RIF Udang AAA Unknown RIF Unknown 6 AAA Karimunjawa Central Java Two accessions only having allele of the MaCIR108 with the size of 295 bp should be placed into BB genomic group Table 4.4 because they did not have alleles of the MaCIR108 with the size of 270 bp or less. The existence of the BB genomic groups was naturally considered to be higher than BBB Pillay et al. 2004. Twenty accessions possessed allele combination of 295 bp; 295 bp and 289 bp; or 295 bp and 287 bp; and 1 allele of the MaCIR108 with the size of 270 bp or less. These accessions having alleles of diagnostic characters for the A and B genomes should be placed into ABB genomic groups Table 4.4 because the Ma- 3-90 allele of 152 bp was detected. Several of the remaining 21 accessions containing 1 allele of the MaCIR108 of 287 bp and alleles of 270 bp or less should be placed into AAB genomic groups Table 4.4 because they did not have the Ma-3-90 allele of 152 bp. Other AAB possessed 1 allele of the MaCIR108 with the size of 275 bp and alleles of 270 bp or less. This study also detected 1 allele of the Ma-3-139 with the size of 132 bp found in all accessions containing the B genome, and none was observed in all accessions of the A genome alone. This suggested that locus Ma-3-139 also provide a diagnostic character for distinguishing accessions of the B genome from the A genome alone. In the first and the second study, the importance of this allele could not be concluded due to the limitation of wide diversity of the studied materials. According to Cerenak et al. 2004 the number of alleles obtained from an investigation by microsatellites correlated with the number of accessions analyzed. Table 4.4 List of 43 accessions classified into BB, AAB and ABB genomic groups based on the primers MaCIR108 and Ma-3-90 Accession Genomic group Region Source Collection Klutuk Hijau BB Jasinga Bogor Bogor Unknown 2 BB Jasinga Bogor Bogor Goplek AAB Uknown Diperta Koja Santen AAB Uknown Diperta Toklek AAB Sleman Diperta Embok AAB Unknown PKBT Kapas Tasikmalaya AAB Tasikmalaya PKBT Kepok Amerika AAB Yogyakarta PKBT Mandar AAB Unknown PKBT Nangka AAB Unknown PKBT Raja Bulu AAB Unknown PKBT Raja Sableng AAB Yogyakarta PKBT Raja Sereh AAB Unknown PKBT Susu AAB Unknown PKBT Austroli AAB Jasinga Bogor Bogor Jantan AAB Unknown RIF Kilita AAB Unknown RIF Longong AAB Unknown RIF Mantra Ho AAB Unknown RIF Mantreken AAB Unknown RIF Neij Amper AAB Ransiki Manokwari RIF Panggang AAB Deli Serdang, North Sumatera RIF Pup AAB Unknown RIF Barangan Merah ABB Medan Diperta Embuk Putih ABB Giri Banyuwangi Diperta Medan ABB Tegalsari Girimulyo Diperta Sembuk ABB Cukurgondang Pasuruan Diperta Siam Paris ABB Unknown Diperta Wangu ABB Cipaku Bogor Diperta Kepok Kuning ABB Gowa makasar PKBT Kepok Manggala ABB Unknown PKBT Prabumulih ABB Unknown PKBT Siam Manggala ABB Unknown PKBT Kepok Merah ABB Jasinga Bogor Klutuk Susu ABB Unknown RIF Unknown 1 ABB Karimunjawa Central Java Unknown 2 ABB Karimunjawa Central Java Unknown 3 ABB Karimunjawa Central Java Unknown 4 ABB Karimunjawa Central Java Table 4.4 List of 43 accessions classified into BB, AAB and ABB genomic groups based on the primers MaCIR108 and Ma-3-90 continued Accession Genomic group Region Source Collection Unknown 5 ABB Karimunjawa Central Java Unknown 7 ABB Karimunjawa Central Java Unknown 8 ABB Karimunjawa Central Java Unknown 9 ABB Karimunjawa Central Java The seventy three accessions of AA or AAA; and AAA genomic groups should be designated under the name M. acuminata, 2 accessions of BB genomic group under the name M. balbisiana, and 21 accessions of AAB and 20 acessions of ABB genomic groups under the name of nature hybrids M. x paradisiaca. The number of pure acuminata was more dominant than balbisiana accessions and their hybrids. These results also supported the hypothesis that Indonesia is one of main centre of diversity and centre of origin of M. acuminata Daniells et al. 2001 as previously reported in the first study Chapter II. Most accessions were classified into genomic groups in agreement with the common local names. For example, accessions designated under the local name ‘Klutuk Hijau’ were identified as BB genomic group. The accessions using the local names ‘Sobo’, ‘Kepok’, and ‘Siam’ were commonly classified into ABB genomic groups. It confirmed that basically, morphological characters especially in bananas containing the B genomes were sufficiently effective for genomic group identification. However, the use of these characters required a long time waiting for flowers and fruits Stover and Simmonds 1987. Indeed, the morphological characters were sometimes influenced by environmental factors Vicente et al. 2005. Similar to the first study, accessions of M. acuminata only having 1 or 2 alleles were designated as AA or AAA genomic groups. Diploid accessions could not be easily distinguished from triploid accessions because they did not have specific alleles to differentiate AA from AAA genomic groups. Furthermore, the dosage effects of microsatellite allele simplex, duplex, triplex etc. can not be differentiated Zhang et al. 2000 because most cultivated bananas were nature polyploid Stover and Simmonds 1987. The triploid accessions having 2 alleles in a certain locus could not be differentiated from the diploid also having the same alleles. According to Oselebe et al . 2006, the ploidy status of bananas could be identified by the chloroplast number in guard cells. The number of chloroplast increased proportionally with the size of cells in response to increase in ploidy Tenkouano et al. 1998; Oselebe et al. 2006. Dolezel et al. 2004 suggested the use of flow cytometry for determining ploidy level. Two banana cultivars, ‘Kluai Tiparot’ from Thailand and ‘Jambe’ from Indonesia have morphologically been classified into tetraploids. Using the cytometric technique, ploidy level of these cultivars was identified as triploid. It indicated that there was no molecular markers available yet that fulfill all requirements needed by researchers Semagn et al . 2006. The advantage of application of the microsatellite markers is not recognized intermediate characters, thus genotype of each banana cultivar could be precisely identified. The key as well as the scoring system created by Simmonds and Sheperd 1955 and Silayoi and Chamchalow 1987 are only applicable to bananas that contained the A and B genomes. The cultivated bananas consisted of 4 genomes namely A, B, S, and T which were derived from M. acuminata, M. balbisiana, M. schizocarpa and section Callimusa, respectively Pillay et al. 2004. The scoring system based on morphological characters did not consider cultivars with the S and T genomes. Identification of those genomes using microsatellite markers could be performed if the specific primers were available. Analysis of genetic relationships among banana germplasms in Indonesia All microsatellite markers generated clear alleles and polymorphic banding patterns. Total 93 alleles were detected by the 8 markers. The number of alleles per locus varied from 5 to 18, with an average of 11.6 alleles per locus. The size of alleles approximately from 110 to 436 bp with polymorphism degree per primer ranged from 5 to 18 alleles. The highest degree of polymorphism was observed in the MaCIR108 and the lowest was detected in the MaCIR327b Table 4.5. Table 4.5 Alleles size, number of alleles and observed heterozygosity value produced from analysis of the 116 accessions using the 8 microsatellite primers Primer Alleles size range bp Number of alleles Observed heterozygosity MaCIR327b 388-436 5 0.45 Ma-1-132 330-378 9 0.51 MaCIR332a 260-296 11 0.85 MaCIR108 220-295 18 0.84 Ma-3-139 132-177 17 0.83 Ma-3-90 132-172 14 0.86 Ma-1-27 122-142 8 0.57 Ma-1-17 110-154 11 0.91 Total 93 Mean 11.6 0.73 The polymorphism of microsatellite loci detected in this study was consistent with previously studied by Creste et al. 2004, but it was higher than that obtained by Creste et al 2003. Using 9 primers, Creste et al. 2004 observed an average of 12.8 alleles, ranging from 10 to 15 alleles in a group of 58 Musa genotypes, including 49 AA and 9 AAB cultivars. On the other hands, Creste et al 2003 detected an average of 6.1 alleles at each of 11 microsatellite loci studied in a group of 35 genotypes from various genomic composition and ploidy level. One possible reason for this difference is that all accessions used in the study were originated from natural cultivars, thus they have a relatively wide genetic base. The high genetic diversity and genetic relationships of banana accessions in Indonesia were exhibited by dendrogram in Figure 4.1. The dendrogram generated by UPGMA from similarity data based on Jaccard coefficient separate the 116 banana accessions into 2 main clusters at coefficient of 0.13. All accessions belonging to BB, AAB, and ABB genomic groups clustered in the first main cluster I together with mostly accessions of the A genome alone. The second main cluster II was belonging to 11 accessions consisting of acuminata cultivars, namely ‘Raja Madu’, ‘Ketan’, ’Oli’, ‘Lilin’, ‘Barifta’ A, ‘Ratu’ ‘Hutan Jambi’ and 4 unknown cultivars from Bogor. The two of them, ‘Raja Madu’and ‘Barifta’ A were identified as AAA triploids due to the presence of 3 alleles of the MaCIR108 with the size of 270 bp or less. Coefficient 0.13 0.35 0.57 0.78 1.00 37 15 S9 S7 14 54 57 17 12 11 13 S1 13 S4 S6 S7 11 12 K7 13 17 17 17 S3 S1 14 13 14 11 13 18 S8 S7 12 12 S2 S1 S8 S8 16 19 S3 S5 S1 S3 S4 S3 11 11 70 S1 10 S4 11 35 S3 12 12 16 S1 11 13 11 13 18 18 93 10 K2 K4 K9 38 48 11 12 K1 K3 K6 K8 K1 17 14 59 18 60 12 S3 S3 S8 S9 86 S6 S4 S3 12 13 13 S9 16 18 S5 10 12 62 13 40 S5 14 S9 S4 S3 S9 18 18 18 19 AAA Mas Lumut AAA Udang AAA Udang AAA Keikeni AAA Potho Yogya AAA Gintung AAA Kolis AAA Raja Bening AAA Rotanhari AAA Sabulan AAA Kutes AAA Tanduk Lembu AAA Potho Bunthek AAA Aghaker AAA Ik Osroc AAA Numbungga AAA Ambon Lumut A AAA Ambon Lumut B AAA Unknown 6 Karimunjawa AAA Badak AAA Badak AAA Jepang AAA Unknown 9 Bogor AAA Lase AAA Buai AAA Anjasmara AAB Kepok Amerika AAB Raja Sereh AAB Embok AAB Susu AA or AAA Pisang Kuning AA or AAA Ramehaye AAA Sramfin AAA Ampyang AAA Madura AAA Lympyang AAA Awomen AAA Neij Houbwan AAA Pogori AAA Sebrot AA or AAA Unknown 8 Bogor AA or AAA Jari Buaya AA or AAA Kole AAA Dingin AAA Ayam AAA Neij Aubu AAA Mourina AA or AAA Mas Purbalingga AA or AAA Mas 40 hari AA or AAA Morli AA or AAA Berlin AA or AAA Lampung AA or AAA Raja Muli AA or AAA Jepang AA or AAA Barley AAA Angleng AAA Ice AAA Papan AA or AAA Rejang AAA Branjut AA or AAA Lidi AAA Angleng AA or AAA Sigung AAA Segli AA or AAA Unknown 5 Bogor AA or AAA Unknown 6 Bogor ABB Barangan Merah ABB Medan ABB Unknown 2 Karimunjawa ABB Unknown 4 Karimunjawa ABB Unknown 8 Karimunjawa ABB Embuk Putih ABB Sembuk ABB Kepok Manggala ABB Kepok Kuning ABB Unknown 9 Karimunjawa ABB Unknown 3 Karimunjawa ABB Unknown 5 Karimunjawa ABB Unknown 7 Karimunjawa ABB Unknown 1 Karimunjawa ABB Kepok Merah AAB Nangka AAB Koja Santen AAB Austroli AAB Goplek AAB Kapas AAB Mantra Ho AAB Jantan AAB Mantreken AAB Panggang AAB Toklek AAB Neij Amper AAB Pup AAB Kilita AAB Mandar AAB Raja Sableng AAB Raja Bulu AAB Longong BB Klutuk Hijau BB Unknown 1 Bogor ABB Klutuk Susu ABB Siam Paris ABB Siam Manggala ABB Wangu ABB Prabumulih AAA Raja Madu AA or AAA Ketan AA or AAA Oli AA or AAA Lilin AAA Barifta A AA or AAA Ratu AA or AAA Hutan Jambi AA or AAA Unknown 2 Bogor AA or AAA Unknown 3 Bogor AA or AAA Unknown 4 Bogor AA or AAA Unknown 7 Bogor Ia Ib II I Figure 4.1 UPGMA clustering of the 116 banana accessions in Indonesia based on the 8 microsatellite primers 0.13 0.35 0.57 0.78 1.00 Coefficient The other 9 accessions except ‘Ketan’, ‘Ratu’, ‘Oli’ and ‘Lilin’ were most probably wild diploid bananas because they had a small fruit with full seeds and almost without pulp Stover and Simmonds 1987. Genetic relationship analysis among banana accessions of various genomic groups, especially in the first main cluster I demonstrated that the accessions containing the B genome namely BB, AAB and ABB were clearly distinct from each other. The result was similar to the study of Pillay et al. 2006. The BB genomic group appears closer to the ABB genomic group. All banana accessions containing the B genome were clustered according to their genomic groups, except 4 accessions of AAB, ‘Kepok Amerika’, ‘Raja Sereh’, ‘Embok’ and ‘Susu’ clustering together with accessions of the A genome alone. Similarity coefficient at 0.18, dendrogram showed that the main cluster I was divided into 2 subclusters Ia and Ib. The subcluster Ia was belong to AA or AAA; and AAA accessions and the 4 accessions of AAB. Clustering analysis based on microsatellite alleles could not significantly distinguished AA or AAA from AAA genomic group. However, majority of these accessions tend to be clustered according to ploidy levels. Similar results were reported by Creste et al. 2004 and previous study in the first study Chapter II investigating genetic relationships among M. acuminata cultivars. The variations of microsatellites within accessions of the A genome alone was higher than those of the B genome. The result is supported by the existence of 9 subspecies of M. acuminata in Indonesia Daniells et al. 2001 which were clearly dominant in number over those in other regions of Southeast Asia Pollefeys et al. 2004. The fourth banana accessions of AAB within subcluster Ia were separated from the subcluster Ib consisting of all accessions containing the B genome. It is because the 4 accessions of AAB had a specific allele of the MaCIR108 with the size of 275 bp. This allele was not detected in the other genomic groups. These AAB accessions were closely related to accessions of the A genome alone. Seventeen accessions of AAB had allele of the MaCIR108 with the size of 287 bp which were also observed in several accessions of ABB. Thus, these AAB accessions were closely related to ABB. Clustering analysis showed that microsatellite markers could discriminate each accession from others, except accessions which were known to be similar. Only ninety nine genotypes resulted from analysis of the 116 accessions using the 8 primers because 26 accessions grouped within 9 identical genotypes Table 4.6. Some of them were detected under the same local name and some the other were detected to be synonymous. In the study, there was also detected homonymous because multiple genotypes with a single name were found, such as ‘Ambon Lumut’ A and ‘Ambon Lumut’ B from PKBT collection, ‘Jepang’ from Bogor and ‘Jepang’ from PKBT collection, ‘Angleng’ from RIF and ‘Angleng’ from PKBT collection. Table 4.6 Nine identical genotypes observed in the 116 banana accessions based on the 8 microsatellite primers Identical genotypes Number of accessions Accession Group genome based on microsatellites Source 1 2 Udang AAA Bogor Udang AAA RIF 2 2 Badak AAA PKBT Badak AAA Bogor 3 4 Mas 40 Hari AA or AAA PKBT Morli AA or AAA Diperta Berlin AA or AAA RIF Lampung AA or AAA PKBT 4 4 Medan ABB Diperta Unknown 2 ABB Karimunjawa Unknown 4 ABB Karimunjawa Unknown 8 ABB Karimunjawa 5 3 Kepok Manggala ABB PKBT Kepok Kuning ABB PKBT Unknwon 9 ABB Karimunjawa 6 3 Unknown 3 ABB Karimunjawa Unknown 5 ABB Karimunjawa Unknown 7 ABB Karimunjawa 7 4 Kapas AAB PKBT Mantra Ho AAB RIF Jantan AAB RIF Mantreken AAB RIF 8 2 Klutuk Hijau BB Bogor Unknown 2 BB Bogor 9 2 Siam Paris ABB PKBT Siam Manggala ABB PKBT CONCLUSION One hundred and sixteen banana accessions were firstly identified using the primers MaCIR108 and Ma-3-90. These accessions were effectively classified into their genomic groups, which are 27 accessions of AA or AAA; 46 accessions of AAA; 2 accessions of BB, 21 accessions of AAB, and 20 accessions of ABB. The accessions of AA or AAA; and AAA should be placed into M. acuminata due to the presence of only the MaCIR108 alleles with the size of 270 bp or less. The accessions of BB should be placed into M. balbisiana as they only have one allele of the MaCIR108 with the size of 295 bp without allele of 270 bp or less. The accessions of AAB and ABB should be classified into M. x paradisiaca due to the presence of the MaCIR108 alleles with the size of more than 270 bp and 270 bp or less as diagnostic characters for the B and the A genomes. The Ma-3-90 allele was employed to distinguish AAB from ABB genomic groups. One allele of the Ma-3- 139 with the size of 132 bp was also confirmed as diagnostic character for the B genome which can separate banana accessions containing the B genome from accessions with the A genome alone. Ploidy level of several accessions of the A genome alone could not be definitely determined due to the dosage effect of microsatellite and the absence of a specific allele for each ploidy level. Analysis of the 116 accessions based on the 8 primers detected only 99 genotypes because 26 accessions grouped within 9 identical genotypes. The dendrogram generated by UPGMA split the 116 accessions into 2 main clusters at coefficient of 0.13. All accessions belonging to BB, AAB, and ABB clustered in the first main cluster together with most accessions of the A genome alone. The second main cluster was belonging to 11 accessions consisting of AA or AAA; and AAA. The first main cluster demonstrated that the accessions containing the B genome: BB, AAB and ABB were clearly distinct from each other. The BB appears closer to the ABB than AAB genomic groups and distantly separated from AA or AAA; and AAA. The dendrogram of genetic relationships showed that the banana accessions clustered according to their genomic groups especially for accessions containing the B genomes, while the accessions with the A genomes alone could not be differentiated significantly, although majority of them tend to be clustered according to ploidy level.

V. PHYLOGENETIC RELATIONSHIPS OF INDONESIAN BANANA CULTIVARS INFFERED FROM