I. GENERAL INTRODUCTION
Backgrounds
Indonesia is one of the main areas of banana diversity. Besides 9 subspecies of wild Musa acuminata recorded by Daniells et al. 2001, there were more than
325 banana cultivars, 14 cultivars of them have been commercially cultivated Setyadjit et al. 2003. Pollefeys et al. 2004 recorded 15 subspecies but some of
them were not entered in Musa Germplasm Information System MGIS due to the lacking of geographical data. The cultivars are not simple but rather complex
hybrids and have been further modified by various mutations Pillay et al. 2003. The specific ancestor of these cultivars was difficult to be elucidated. Therefore,
the taxonomic status and nomenclature of banana cultivars have long been a complicated issue Valmayor et al. 2000.
M. paradisiaca L. and M. sapientum L., the original scientific names
applied to cultivated edible bananas were refused either by Cheesman 1948 or by Simmonds and Shepherd 1955 because the cultivars are quite distinct from
each other. M. paradisiaca and M. sapientum which were known as plantain and dessert bananas were described using very limited specimens Valmayor et al.
2002. The descriptions which were very simple could not be used to deal with the high variability of cultivated bananas in the centre of diversity. Therefore, the
adoption of Linnaeus classification in Southeast Asia often led to misunderstanding.
Edible bananas were believed arise from wild and seedy M. acuminata Colla 2n, AA genome and M. balbisiana Colla 2n, BB genome Simmonds
and Shepherd 1955. The balbisiana species is also widely distributed in Indonesia, although some authors believed that it did not originate from this
region. Sterility and parthenocarpy occurring in both species yielded the diploid primitive clones. The clones retained the unique morphological characteristics of
their wild ancestors due to the seedless of the clones. Therefore they should be classified under the same species as their ancestors Valmayor et al. 2002.
Seedless triploid cultivars developed through chromosome restitution. These cultivars should also be designated under the same species because
autopolyploidy did not change the genetic constitution of the clones Valmayor et
al . 2000. The cultivars which have intermediate characteristics between the two
species should be named under the hybrid species M. x paradisiaca L. This name is an agreement with the priority of usage principles as Linnaeus first applied this
term to a hybrid clone in 1753 ahead of his description of M. sapientum in 1759. The prefix x is inserted for indicating their hybrid nature as instructed by the
International Code of Nomenclature for Cultivated Plants ICNCP. Two natural species and a hybrids complex make up the edible bananas in
existence Stover and Simmonds 1987; Espino et al. 1997. M. acuminata and M. balbisiana
are recognized in their wild and cultivated forms. A number of AA diploid cultivars was derived from intraspecific hybridization between various
subspecies of M. acuminata Simmonds 1962, while AAA triploids have arisen either from the AA diploid cultivars or subspecies of AA wild by meiotic
chromosome restitution. A similar development also occurred to lead appearance of diploid and triploid balbisiana cultivars. On the other hands, the hybrids of M.
x paradisiaca emerged in different genomic combination and ploidy levels. The
AAB and ABB cultivars were originated from interspecific hybridization between M. acuminata
AA and or maybe AAA; and M. balbisiana BB Stover and Simmonds 1987; Pillay et al. 2004. Hybridization between heterogenomic
parents also produced a range of genotypes. Crossing between the AAB and a wild diploid parent of AA or BB could produce progeny within various genomic
groups Karamura 1998. Banana taxonomic status and nomenclature could be determined by their
genomic groups Pilley et al. 2004. After determining genomic groups, the species can be easily identified or vice versa. The cultivars then are described
following the Descriptors for Banana Musa spp published by IPGRI- INIBAPCIRAD 1996. Finally, the banana cultivars are classified following the
three tier system consisting of species, genomic group and cultivar Valmayor et al
. 2002. The banana cultivars were morphologically classified into genomic groups
based on scoring system of 15 diagnostic characters. Using these characters, wild M. acuminata
could be clearly distinguished from wild M. balbisiana. Simmonds and Sheperd 1955; Silayoi and Chamchalow 1987; Stover and Simmonds 1987.
Each character similar to acuminata was scored 1 and this with extreme balbisiana
appearance was scored 5. Intermediate expressions were scored 2, 3 and 4 depending on intensity of each character. The scorecard provides a range
from 15 to 75. The AA and AAA genomic groups of pure acuminata cultivars should have scores from 16 to 25 whereas the score of pure balbisiana BB and
BBB cultivars ranged from 70 to 74. The hybrids between M. acuminata and M. balbisiana
should have scores from 26 to 69. Based on morphological characters, several banana accessions in the study
of Jumari 2000 have been classified into different genomic groups from those reported by Pudjoarinto et al. 1994 and Hadisunarso et al. 1995.
Morphological characters have high heritability and easily observed with naked eyes Ferreira 2005, but they are sometimes hardly interpreted because they are
mostly qualitative and do not have distinct boundaries. Morphological characters are also affected by environmental factors, so that the phenotypic identification is
frequently incorrect. It is clear that morphological characters alone could not be employed to precisely determine banana genomic groups.
Characterization and evaluation of cultivars based on molecular markers were more accurate than morphological characters Guzow-Krzeminska et al.
2001; Vicente et al. 2005. The occurrence of intra and interspecific hybridization, clonal propagation, and polyploidization sometimes exhibited different variation
patterns which are difficult to be recognized Spooner et al. 2003. Therefore, cultivar identification to obtain characteristics such as differences, uniformity, and
stability Rifai 2006 need conducting through molecular approaches Vicente et al
. 2005. Stability and predictability of cultivars are important for agronomist and horticulturist. Based on a specific character or combination of characters, each
cultivar should be clearly identified and exhibit unique differences. When propagated by suitable methods either reproduced by seeds or cuttings, the plant
retains its distinguishing characteristics Brickell et al. 2004. The cultivar characteristics were recognized through a classification system
providing information of natural variation of the cultivar or culton units and information of its phylogenetic relationships Brandenberg 1986. The
classification of cultivated plants in Indonesia so far is still neglected Rifai 2006.
Currently, cultivars were only identified with scientific names of the species. Several important crops have been developed up to the cultivar level, but the
nomenclature given is usually not following the international codes. Since many cultivated plants in Indonesia provide a large contribution to the national
economy, Rifai 2006 proposed the priority of the cultivated plant classification system should be given to agricultural commodities which can be mass produced
and prospective to be exported. Bananas are a strategic commodity to the export market Areas et al. 2003
which is potential to increase income and foreign exchange Deptan 2005. Besides it grows easily, banana is also cheap sources of energy during the hunger
gap between crop harvests. Therefore it contributes significantly to food and income security of people Lustig 2004; UNCST 2007. The banana produces
fruit in any season with a relatively short period of a life cycle, can be propagated using in vitro technique, and cultivated in a mass Deptan 2005. Therefore, the
studies on classification of cultivated bananas in Indonesia become urgent today. The molecular markers provide an objective way for genomic classification
in Musa Pillay et al. 2006. Microsatellites or simple sequence repeats SSRs are DNA sequences with repeat lengths of a few base pairs Innan et al. 1997. This
marker analysis is one of the most useful methods to expose diversity of banana cultivars Kaemmer et al. 1997; Crouch et al. 1998. They were abundant and
widely dispersed throughout the whole Musaceae genomics Lagoda et al. 1997 and they have been used for genotypic identification of many plant species Perera
et al . 2001; Chakravarthi and Naravaneni 2006. Microsatellites are flanked by
two conserved sequences and detected in a specific locus Spooner et al. 2004; Semagn et al. 2006. Their polymorphisms could easy to be detected using
primers designed from these flanking regions Innan et al. 1997. In previous study of banana, a few microsatellite primers were proven to provide diagnostic
characters for genomic groups Kaemmer et al. 1997; Creste et al. 2003, 2005. Banana cultivars could be incorrectly identified may be due to the plasticity of
morphological characters. Therefore, a classification of cultivated bananas using microsatellite markers is needed.
The banana production around the world trends is triploid banana cultivars Sharrock and Frison 1999. These cultivars are currently threatened by several
pests and diseases, so the new resistant cultivars are urgently needed. There is a continuing effort to improve the new resistant cultivars by introducing useful
genes from their wild diploid parent, hence the phylogenetic relationships between the wild diploid progenitors and the cultivars need to be elucidated
Carreel et al. 2002. The use of molecular genetic analysis may significantly improve breeding efficiency Tenkouano et al. 1999. According to Carreel et al.
2002 chloroplast DNA cpDNA which is sitoplasmic DNA was considered an essential tool for phylogeny analysis and may suggest a powerful tool to conform
hybrid origins of banana cultivars. The transmission monoparental of cpDNA provide an exceptional opportunity for studying maternal lineages. In banana,
cpDNA heredity could be used for distinguishing maternal from paternal lineages. According to Gielly and Taberlet 1996, the intergenic spacer of the trnL-F is
noncoding regions in cpDNA which was suitable for phylogenetic studies due to its small size. The size only ranged from 120 to 350 bp in flowering plant. This
region is also uniparentally inherited, not recombining and structurally relatively stable Barcaccia et al.2007. As compared to coding regions in cpDNA, the trnL-
F intergenic spacer may develop at rates three times faster than that the coding
regions. Therefore, this region may give reasonable resolution within Musa, although it was lower than that of a nuclear based phylogeny Gielly et al. 1996.
Objectives
The main objective of this study is to obtain a classification which is stable and accurate and a phylogenetic inference of cultivated bananas in Indonesia
based on molecular approaches. The banana accessions previously classified based on morphological characters and accessions that have not been classified
were characterized and identified, verified, classified, and analyzed their genetic relationships using molecular characters. The phylogenetic inference of cultivated
bananas was elucidated using the noncoding of cpDNA. To achieve the goals of the study, the research was performed following
four steps. In this work, the classification of cultivated bananas using
morphological characters was tested. In the first study, the microsatellite primers included primer which provided diagnostic characters for the B genome
Kaemmers et al. 1997 was employed for analyzing M. acuminata cultivars containing the A genome alone. The accessions were correctly classified into AA
or AAA genomic groups when the allele indicating the B genome were absent in the acuminata accessions. In the second study, the primers were employed to
confirm banana accessions containing the B genome and to create a determination key, and then in the third study, the microsatellite determination key was used to
classify banana accessions which were previously unidentified yet. Finally, in the fourth study, several accessions representing various genomic groups from the
first, the second, and the third study were used for phylogenetic analyzing of cultivated bananas which are elucidated using the trnL-F intergenic spacer of
cpDNA.
II. CHARACTERIZATION AND EVALUATION OF Musa acuminata CULTIVARS IN INDONESIA BASED ON
Kategori