VI. GENERAL DISCUSSION
Many different DNA markers of various types are available for various purposes. One type of marker that has turned out to be most promising is
microsatellite Roux et al. 2004, Semagn et al. 2006. The present study firstly demonstrated the potential of microsatellite markers for characterizing cultivated
bananas in Indonesia. The result exhibited that microsatellite markers have proved to be an appropriate tool for classifying and estimating genetic diversity of banana
cultivars. Genomic group of each accession could be clearly determined based only on
the primers MaCIR108 and Ma-3-90. Using these primers, banana accessions were divided into 3 species, which are Musa acuminata, M. balbisiana and M. x
paradisiaca . Because cultivated bananas are not a simple and some of them
confirmed to be derived from hybrid between 2 different species, scientific names of M. paradisiaca and M. sapientum could not be applied to banana cultivar
nomenclature. It supported Cheesman 1948 and Simmonds and Shepherd 1955 rejecting the designation of these names for cultivated bananas.
Microsatellites were considered powerful for estimating an intraspecific genetic distance and effective for analyzing polyploidy Bruvo et al. 2004,
especially for allopolyploid. The MaCIR108 produced unique banding pattern for distinguishing M. acuminata A genome from M. balbisiana B genome. Alleles
of the MaCIR108 with the size of more than 270 bp have not been detected in genotypes with only the A genome, however they were always present in
genotypes with the AAB, ABB and BB genomic groups. Allele of the Ma-3-90 with the size of 152 bp was always detected within accessions having two B
genomes BB and ABB, and never detected within accessions having one B genome AAB. Therefore, the genomic groups of banana cultivars especially for
allopolyploidy could be rapidly identified based only on the two primers: MaCIR108 and Ma-3-90.
Conclusion of the present study is 4 alleles of primer MaCIR108 with the size of 295 bp; 289 bp; 287 bp; and 275 bp confirmed as diagnostic characters for
banana containing the B genome, whereas alleles with the size of 270 bp or less were diagnostic characters for banana containing the A genome alone. Those
diagnostic characters for the A and the B genomes were also observed by Creste et al
. 2003 but they had not reported estimation of the range of alleles size. Microsatellite provided precisely identity of each banana cultivar. A cultivar
must be clearly distinct, uniform and stable in its characteristics. Agribusiness commodity particularly in the global market requires information concerning
characteristics of traded cultivars. Based on a specific character or combination of characters, each cultivar can be clearly identified; exhibiting unique differences
and when propagated by suitable methods either reproduced by seeds or cuttings retains its distinguishing characteristics Brickell et al. 2004. Total 267 banana
accessions only 208 genotypes were detected. It indicated that morphological appearances are sometimes difficult to be interpreted Ferreira 2005.
Morphological descriptors are easy to study and relatively low cost to evaluate, but they had some limitations. These characters also have serious drawbacks due
to the time required Cerenak et al. 2004. In the study, several banana accessions with the multiple local names were detected only have a single genotype. The
cases commonly occurred in the centre of banana diversity as reported by Valmayor et al. 2000.
Incorrect morphological identification has been revised by microsatellite markers. All of accessions used in dissertation research 267 accessions collected
from various regions in Indonesia have been effectively classified only based on the primers MaCIR108 and Ma-3-90 into 5 different genomic groups including 3
different species. The number of accessions classified into their genomic groups based on these primers was presented in Table 6.1. The result showed that M
acuminata cultivars AA or AAA; and AAA genomic groups are far exceed than
those the number of M. balbisiana cultivars. Of these, about 50 130 accessions included M. acuminata cultivars. The data support and confirm that Indonesia is
one of main areas of the diversity of M. acuminata Daniells et al. 2001, Pollefeys et al
. 2004. A number of alleles obtained in the study did not exhibit to significantly
increase when a large accession of bananas was examined. It indicated that the number of accesseions used in the study had represented genetic diversity of
cultivated bananas in Indonesia. Commonly in the other cultivated plants that
were supposed to be heterozygous and are clonally propagated, a number of alleles tend to increase when accessions assessed increased Carriero et al. 2002.
A mean of alleles per locus 10.3 was detected in an analysis of the 48 accessions of pure acuminata, 9.4 of the 92 accessions and 11.6 of the 116 accessions
consisting of various genomic groups. The result also explained that genetic diversity of cultivated bananas in Indonesia is determined by acuminata alleles.
Alleles of the A genome contributed by M. acuminata within genetic diversity of cultivated bananas are higher than alleles of the B genome donated by M.
balbisiana . It suggested that actually wild balbisiana did not originate from
Indonesia. Table 6.1 Recapitulation of the accessions number classified into their genomic
groups based on alleles of the MaCIR108 and allele of the Ma-3-90 with the size of 152 bp
Genomic group based on molecular key Genomic group
based on morphology
and unknown Number of
accessions AA or AAA AAA
BB BBB
AAB ABB
AA 25 23
2 AAA 34
25 8
1 BB 8
8 BB or BBB
1 1
AAB 37 4
30 3
ABB 46 5
3 38
Unknown 116 27 46
2 21
20 Total 267
50 80
10 64
63
The competence of molecular techniques in all aspects of plant breeding is essential Ferreirra 2005. They are several advantages of molecular techniques in
breeding and genetic improvement in Musa. The application of molecular techniques may dramatically improve the breeding efficiency in term of both time
and space. Recently, the use of microsatellite markers is favorable and the markers are commonly used as a routine procedure for genetic analysis and
cultivar identification in various plants Mitchell et al. 2001. Because the efficiency of the microsatellite has been determined and proven, reclassification
of doubtful banana accessions based on microsatellites is necessary. Therefore, the use of microsatellite markers could be introduced as a new method in
classification of cultivated bananas in Indonesia. The application of molecular
markers to quantify the genetic diversity among accessions may extensively decrease redundancy of cultivar duplication Ferreira 2005.
The cultivated plants classification based on molecular markers was considered more accurate and stable than morphological characters due to their
ability to detect differences of cultivars Guzow-Krzeminska et al. 2001; Vicente et al
. 2005. Every cultivar could be clearly identified and significantly differentiated from the others Ferreirra 2005; Vicente et al. 2005. The two
hundred and eight genotypes obtained from analysis of 267 accessions based on the 8 primers were detected in the study Figure 6.1.
The genetic similarity coefficients exhibited in the dendrogram ranged from 0.15 to 0.95. Genetic relationship analysis among the 267 banana accessions
showed that all the accessions containing the B genome clustered according to their genomic group, except 15 accessions of AAB genomic groups clustering
together with accessions of the A genome alone. The twelve accessions of AAB grouped in one cluster due to presence of allele d
4
of the MaCIR108 with the size of 275 bp while the remaining 3 accessions of AAB grouped due to the similarity
of allele profiles of all primers minus the Ma-3-90 and Ma-1-132. The AA or AAA accessions could not be clearly distinguished from AAA genomic groups,
although most accessions tend to be clustered according to ploidy levels. Generally, the clustering pattern of the 267 accessions was similar to the
116 accessions in the third study Chapter IV. The general conclusion which could be obtained is BB accessions appear closer to the ABB than AAB
accessions. It supported that the AAB was more divergence from the original ancestors than ABB genomic group Pillay et al. 2004. Banana accessions of AA
or AAA; AAA; and AAB were also more diverse than those BB and ABB genomic groups. It is because the contribution of the A genome is higher than that
of the B genome.
Figure 6.1 The 208 genotypes resulted from UPGMA clustering of the 267 banana accessions in Indonesia based on
the 8 microsatellite primers. Coefficient
0.15 0.35
0.55 0.75
0.95
2 S 49
106 163
118 S 86
35 S 54
S 36 76
123 122
14 S 100
113 139
148 133
116 58
S 50 188
189 S 84
16W 36
110 80
S 27 S 35
12 63
13 185
186 187
191 40W
S 53 140
43 S 94
S 46 18
23 29W
33 S 90
16 37
34 156
151 104
S 79 143
54 57
S 44 S 69
S 77 S 78
S 85 4
90 136
17 177
112 126
134 S 108
3 114
127 S 76
20W K 7
68 176
175 75
S 32 S 102
92 20
72 19
164 170
S 93 142
65 89
S 62 152
83 77
107 137
8 119
130 157
11 15
181 S 81
S 75 47
94 S 37
194 128
129 S 29
21 50
S 103 S 67
S 10 S 88
S 41 39
73 39W
95 155
125 S 68
49 59
24W 21W
23W S 105
40 86
S 31 60
S 63 S 96
S 45 S 30
7 S 106
146 24
25 74
S 42 97
78 149
S 33 138
93 30
45 100
1W 7W
51 55
88 38
48 47W
153 64
79 82
84 81
25W 96
6 41
62 131
S 58 161
31W 120
158 S 89
S 60 46
87 5
98 115
S 51 17W
2W 53
32W S 52
K 1 160
178 61
180 26
69 S 34
S 43 S 39
AA or AAA Mas 40 Hari
AA or AAA Raja Muli
AAA Roid AA or AAA
Mulih Hijau AA or AAA
Jepang AA or AAA
Neij Sehi AA or AAA
Barley AAA Tolu
AAA Angleng
AAA Nona AAA Papan
AAA Ice AA or AAA
Rejang AAA Branjut
AAA Lidi AAA Angleng
AAA Angleng AAA Segli
AA or AAA
Sigung AA or AAA
Penjalin AA or AAA
Kole AA or AAA
Unknown 6 Bogor AA or AAA
Unknown 7 Bogor AAA Pogori
AAA Sebrot AA or AAA
Mas Besar AA or AAA
Mas Purbalingga AA or AAA
Lilin AA or AAA
Mas Batangmerah AA or AAA
Ratu AA or AAA
Raja Wligi AA or AAA
Pinang AA or AAA
Cici Kuning AA or AAA
Unknwon 3 Bogor AA or AAA
Unknown 4 Bogor AA or AAA
Unknown 5 Bogor AA or AAA
Unknown 8 Bogor AAA Raja
Madu AA or AAA
Ketan AA or AAA
Oli AAA Ketip
Gunungsari AA or AAA
Lilin AAA Barifta
A AAA Gorohito
AAB Longong
AAB Pulut AAB
Pisang Seribu AA or AAA
Hutan Jambi AA or AAA
Monyet AAA Mas
Lumut AAA Kidang
Ijo AAA Beleum
AAA Udang
AAA Potho Merah
AAA Keikeni AAA Potho
Yogya AAA Gintung
AAA Kolis AAA Aghaker
AAA Ik
Osroc AAA Numbungga
AAA Tegetmolo AA or AAA
Koumus AAA Barangan
AAA Kepok Klutuk
AAA Potho Bunthek
AAA Raja Kriyak
AAA Raja Bening
AAA Sabulan AAA Rotanhari
AAA Kutes
AAA Tanduk Lembu
AAA Ambon Hong
AAA Ambon Lumut B
AAA Ambon Lumut A
AAA Ambonaae AAA Sobo
Madura AAA
Unknown 6 Karimunjawa AAA Cebol
Morosebo AAA Jepang
AAA Unknown 1 Bogor
AAA Bawean AAA Lase
AAA Buai AAA Kepok
Ungu AAA
Angleng Kuning AAA Ambon
Warangan AAA Anjasmara
AAA Ambon AAA Ambon
Lumut AAA Ambon
Putih AAA Anjasmara
AAA Ambon Merah
AAB Ambon Jaran
AAB Raja Sereh
AAB Raja Sereh
AAB Bangkahulu AAB Koja
Susu AAB Ambon
AAB Kepok Amerika
AAB Jambe Saat
AAB Embok AAB Susu
AAB Haseum AAB Triolin
AA or AAA
Cici Gading AA or AAA
Pisang Kuning AA or AAA
Ramehaye AAA Sramfin
AAA Mauli AAA Sri
Nyonya AA or AAA
Jari Buaya AA or AAA
Unknown 9 Bogor AAA Ampyang
AAA Madura
AAA Limpyang AAA Potho
Ijo AAA Koja
Pretel AAA Dingin
AA or AAA Fungkah Mos
AAA Awomen AAA Neij
Houbwan AAA Bole
AAB Ambon
Sepet AAB Agung
Pasuruan AAB Byar
AAB Tanduk Ex Kedondong
AAB Tanduk AAB Mandar
AAB Burlangge
AAB Raja Puser
AAB Koja Santen
AAB Raja Kasman
AAB Austroli AAB Raja
Marto AAB Koumusona
AAB Usuk
AAB Toklek AAB Mantra
Ho AAB Goplek
AAB Neij Amper
AAB Longong AAB Pup
AAB Kilita
AAB Raja Nangka
AAB Nangka
AAB Nangka AAB Raja
Lini AAB Raja
Sabrang AAB Raja
Talun AAB Keja
AAB Raja Bagus
AAB Raja Sereh
AAB Raja Bulu
AAB Raja AAB Raja
Sableng ABB Barangan
Merah ABB Raja
Pendopo ABB Kepok
Asam ABB Kepok
Awu ABB Lempeneng
ABB Kepok
Ladrang ABB Sobo
Kerik ABB Kepok
Gandul ABB Sobo
Londoijo ABB Embuk
Putih ABB Sembuk
ABB Gablok ABB Susu
ABB Kepok Gajih
ABB Sobo Kapuk
ABB Kepok Brot
ABB Sobo Londoputih
ABB Ketan ABB Kepok
Byar ABB Kepok
Kuningan ABB Awak
Rawa ABB Raja
Entog ABB Wangu
ABB Prabumulih ABB
Klutuk Susu ABB Apu
ABB Kaso ABB Siam
Manggala ABB Siem
ABB Selayar ABB Raja
Siem ABB Raja
Bali ABB Comot
ABB Kepok
Kuning ABB Kepok
Lumut ABB Kepok
Manggala
3
ABB Kepok Kuning
ABB Kepok Urang
ABB Raja Wesi
AAB Raja Delima
ABB Raja Kul
ABB Boi ABB
Unknown 1 Karimunjawa ABB Kepok
ABB Kepok Merah
BB Klutuk Warangan
BB Klutuk Hitam
AAB Brentel AAB Kates
AAA Ayam
AAA Neij Aubu
AAA Mourina
I
I I
0.15 0.35 0.55 0.75 0.95 Coefficient
The result also showed that there were several homonymous accessions found within Indonesian banana germplasm Table 6.2. For example, accession
of M. x paradisiaca AAB ’Raja Sereh’ from Diperta, PKBT collection, and Bogor were genetically different. These cultivars most probably possessed high
morphology similarity. There is opportunity for giving a new cultivar name for the cultivars. According to Brickell et al. 2004, a cultivar is not a single plant. A
cultivar is a group of individual plants which collectively is different from any other, which is uniform in its whole appearance and which remain stable in its
attributes. Therefore, a new name of the cultivar could be given if a number of individual plants of its cultivar have been confirmed to be uniform and stable.
Table 6.2 Homonymous cultivars observed in the 267 Indonesian banana accessions based on the 8 microsatellite primers
Homonymous accession group
Number of accession
Accession Genomic group
Originsource 1 2
Ambon AAA Diperta
Ambon AAA
Bogor 2 3
Ambon Lumut
A AAA
PKBT Ambon
Lumut B
AAA PKBT
Ambon Lumut
AAA Bogor
3 3 Angleng
AAA RIF
Angleng AAA
PKBT Angleng
AAA Bogor
4 2 Anjasmara
AAA Diperta
Anjasmara AAA
PKBT 5 2
Jepang AA or
AAA PKBT
Jepang AAA Bogor
6 2 Kepok
Kuning ABB
Diperta Kepok
Kuning ABB
RIF 7 2
Ketan ABB Diperta
Ketan AA or
AAA RIF
8 2 Nangka AAB
RIF Nangka
AAB PKBT
9 3 Raja
Sereh AAB
PKBT Raja
Sereh AAB
Diperta Raja
Sereh AAB
Bogor 10 2
Susu AAB
PKBT Susu ABB
Bogor
Reclassification of ambiguous cultivated bananas using molecular markers urgently needs due to the high genetic diversity of bananas in Indonesia Daniells
et al . 2001. Genotype of each cultivar can be accurately determined, so the
duplication of accessions can be avoided. The knowledge of the genetic resources
to be limited due to the existence of local names, synonymous, homonymous, and somatics mutants in banana cultivars Creste et al. 2003. The present study
exhibited that the microsatellites to be suitable for classification of Musa into genomic group and to identify synonymous and homonymous accessions as
reported by Creste et al. 2003. Information of synonymous is important for promote understanding and communication as well as banana trade and commerce
Valmayor et al. 2000. On the other hands, the homonymous showed that the accessions of multiple genotypes have a single name. Moreover, the use of
microsatellite markers for identifying cultivars even can be conducted long before the crops were commercially cultivated in order to keep away from wrong
identification Mitchell et al. 2001; Priolli et al. 2002; Novelli et al. 2006. One of disadvantage of microsatellite is not able to significantly
differentiate between different ploidy of pure acuminata. It is due to the polyploidy nature of bananas and the dosage effects of microsatellite alleles.
However in the present study, microsatellite markers were new evidences which could complete the morphological classification. Therefore, the markers should
not replace the morphological characters Guzow-Krzeminska et al. 2001. The attempts to create database of banana germplasm in Indonesia using
morphological characters should be carried out as Cruz et al. 2007 collecting morphological data to obtain comprehensive catalog for introduced and local
cultivars in the Philippines. Plastid genome is important for investigating of cultivated banana parental,
especially for hybrid bananas. The maternal transmission of cpDNA provides an excellent opportunity for studying the maternal of clones Carreel et al. 2002.
Therefore, the noncoding regions from cpDNA, has been widely used to understand plant phylogenies at different taxonomic levels Zhang et al. 2003;
Shaw et al. 2005. Interspesific hybridization events have been reported in the cultivated bananas Stover and Simmonds 1987. In order to understand
phylogenetic relationships and genetic divergence among these hybridizing species, molecular phylogenetic analysis was needed Baumel et al. 2002. The
main result showed that the sequences of the trnL-F intergenic spacer could clearly distinguish accessions having the A-type chloroplast from those accessions
having the B-type chloroplast. It means that there are 2 groups of banana cultivars which are distinguished by their female parents. Firstly, the group having the A-
type chloroplast is derived from female M. acuminata or female gamet containing the A genome. Secondly, the group having the B-type chloroplast is derived from
female M. balbisiana. In the study, the specific acuminata ancestor of each cultivar remains unidentified because wild subspecies studied did not specifically
grouped into clade with certain banana accession. The information concerning of female parent is very important especially for breeders and management of
banana germplasm Sergio and Gianni 2005. The knowledge of the origin of chloroplast type of each cultivar is needed for elucidating evolution of cultivated
bananas Swangpol et al. 2007. In order to obtain precisely parent of cultivated bananas could be conducted through analysis of sequences of chloroplast DNA
which is high variation. Naturally, the tetraploid cultivars with genome constitutions of AAAA; AAAB; AABB; and ABBB are rare Pillay et al. 2004.
On the other hands, many of the domesticated bananas have proven to be triploid AAA; AAB; or ABB which may be derived from different female parents. These
cultivated bananas have been collected from numerous independent sources in the wild. Where fertile plants live together, hybridizations continue to produce new
diversity Pollefeys et al. 2004 from several parental combinations.
VII. CONCLUSION
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