Genetic Diversity and The Relationship Between The Indonesian Mangosteen ( Garcinia Mangostana ) and The Related Species Using Isozyme Markers

Indonesia was known to have high diversity of mangosteens (Garcinia mangostana) and the related species. In order to elucidate the genetics variability of the diversity, thirty three accessions were examined by using isozyme analysis. The genetic diversity and relationships among several mangosteens and other Garcinia sp were established by using four isozymes. The level of polymorphism as revealed by isoenzyme was 88%. Although mangosteen is believed to reproduce exclusively through apomixis, our result show that considerable genetic diversity exists within G. mangostana and between other Garcinia species. Based on 27 bands there were 5-42% dissimilarity level among mangosteen accessions, while the other species has 75% dissimilarity. The dendrogram is built based on isozyme marker analysis to separate clusters of mangosteen and other Garcinia sp. The data showed that G. mangostana is a close relative of G. malaccensis, G. porrecta, G. celebica, and G. hombroniana. The concurrence analysis on isozyme analysis result showed the very good fit of Rolf correlation value (0.914). This result indicated that isozymes could group G.mangostana and the related species.


INTRODUCTION
The mangoesteen (Garcinia mangostana L) or known as Queen of Tropical Fruits is one of the best exported commodity from Indonesia.To increase the economic values, the production needs to be increased.This can be reached by the improvement of cultivation techniques and the use of the best seedlings as a result of the correct and strategic breeding.
Genetic improvement of the mangoesteen depends on the genetic diversity sources.Indonesia is centre of diversity of mangoesteen in the South East Asia.
Exploration, identification, and characterization were needed to find out the information about the new genetic diversity resources to improve the genetic characters and production.This study was aimed to trace the presence of variation in Indonesia mangoesteens (Drew, 1997).
Mangoesteen is obligate apomictic that is the seeds were not the product of fertilization and the genetic diversity is narrow, so that it was estimated that there is only one species of mangoesteen on nature.
In facts, there are some variations which may be caused by the environment or genetic factors due to natural mutation following the history thousands of years ago (Ramage et al., 2004), high yields, various color of the seedlings and isozyme band analysis (Supriyanto et al., 1999), and various in morphology (Mansyah et al., 1994).
Based on isozyme glucose phosphateisome-rase (GPI) analysis of West Sumateran mangoesteens with 14 samples examined showed similar band patterns although the phenotypes were variable.On other words, the genetic variability was narrow, but the phenotypes were wide (Mansyah et al., 1999).Genetic analysis with contemporary techniques for wider species could be used to identify the paternal progenitors for breeding with the mangoesteen as the maternal progenitors (Osman & Abdul, 2006).
The mangoesteen has a long life cycle, so that a genetic study is difficult to conduct.Thus estimation of genotype variability by using morphological markers and isozymes was important.Isozyme analyses had been done on plants like Aristolochia manshuriensis (Nakonechnaya et al., 2007), Terminalia paniculata (Thangaraja & Ganesan, 2007).
The use of isozyme has given important contributon for plant breeders to handle the apomixis (Ramage et al., 2004).Variability analysis in the mangoesteen and genotyping the other Garcinia were important to find the best progenitors for breeding.The aims of this research were to find out genetic variability and phylogentics among mangoesteens and related species, as well to find out the progenitors.

MATERIALS AND METHODS
Plant Material.Thirty four samples of mangoesteen were collected from some areas in Indonesia and from Bogor Botanic Garden and Mekarsari Fruit Park (Table 1).
Thirty four samples of fresh leaves were used for isozyme analysis.Before analysis, all leaves were cleaned, dried, and sprayed with alcohol 70%.The rest of alcohol was stored in the freezer (-20 0 C).This analysis was done following Soltis and Soltis (1989).The enzymes analyzed were peroxidase (PER), phosphatase acid (ACP), Malic Dehydrogenase (MDH), and Esterase (EST).The isozyme bands were separated by using electrophoresis with 10% strach gel for 4 hours and 100 volt.
The bands were translated as binary data.These data were used to arrange matrices of genetic diversity based on formula of Nei and Li (1979) with method of UPGMA (Unweighted Pair-Group Method Aritmetic) using NTSYS (Numerical Taxonomy and Multivariate System) version 2.02 (Rolf 1998).The coefficient of Nei dan Li (Nei and Li 1979) calculated genetic similarity (GS) between two samples, i and j, with formula GS(i,j) where Nij is the number of bands presence between i and j, Ni number of bands present in i and absent in j, Nj is the number of bands present in j and absent ini.

RESULTS AND DISCUSSION
Fragment of 34 accessions of mangoesteens and related species with four isozymes results in 28 bands  2).This could be used to determine diversity of the 34 accessions with high polymorphism (85.7%).
The cluster analysis results in a dendrogram which separate the mangoesteen from the other rewlated species with dissimilarity index 43%, except G. malaccensis which grouped with the mangoesteen (Figure 1).
Group  However, the mangoesteen accession still could be mapped separately from the other closely related species except G. malaccensis 1 with 2 (Figure 2).
Two dimension mapping by using component 1 and 3 (Figure 3) tend to be more similar with those produced by the dendrogram.This result showed that the characters playing the most important roles in constructing the dendrigram came from component 1 and 3.While mapping based on component 2 and 3 did (Figure 4) was not similar as those based on morphology.
The analysis results of the principle component on 28 isozyme bands showed that 21 bands had the highest the main component absolute score (Table 4).
Determination of the 21 bands was obtained from the accummulation diversity proportion of each PC i.e. 70% with details contributions as follows: PC1 with diversity proportion 21.81%, PC2 with diversity proportion 15.83%, PC3 13.39%, PC4 9.01%, PC5 8.91% and PC6 9.23%.There were 6 repeated bands within 2 PC which caused total number of bands became 14 not 21.Esterase system enzymes (EST) contributed the majority bands to cluster namely 6 bands, Malate dehydrogenase (MDH) and Peroksidase (PER) with 3 and Acid phosphatase (ACP) with 2 bands.
Mapping with three dimension showed clusters matching those based on dendrogram with a tendency of mangoesteen separated from relatives (Figure 5).
Therefore the use of three components continually parallel with grouping with dendrogram using all characters.
Correlation analysis between bands of each enzyme system can be used to select enzyme system prioritized in isozyme analysis in mangoesteens and allies (  6).Morphological characters such as flower colors, fruit colors, fruit taste, and stigma number (Table 6) showed that mangoesteens was located among twi progenitor characters.Morphologically, the mangoesteen tend to be similar as G. malaccensis rather than G. hombroniana.
Therefore, hypothesis stating that mangoesteens were hybrid of G. malaccensis and G. hombroniana is acceptable.Genetic diversity of mangoesteen and related species based on isozyme (83%) was high for obligate apomixis than Taraxacum (19%) (Ford & Richards, 1985).Variation presence in apomixis accurred more rapidly than mutation (Hughes & Richards, 1985).High polymorphic percentage was incommon in the mangoesteen as an obligate apomixsis.Possibly, this was caused by the fact that G. mangostana was not monohybrid.Repeated hybridization among progenitors had caused the presence of wider genetic variation among the progenitors.
The high genetic variation among mangoesteens was very potential for priducing high quality plants.This can be done by mass selection in some plants to produce new varieties.
Based on isozyme markers, G. celebica and G.
hombroniana which were estimated as the progenitors were located at genetic similarity if 43%, while G.

CONCLUSIONS
Genetic diversity of mangoesteen and closely related species can be traced with four isozymes with 85,7% polymorphism.Genetic variability of mangoesteen and related species was wide (3-86%).
G. mangostana as an obligate apomictic plant have wide genetic variation.They can be grouped into 5 groups, while their closely related species had formed three groups.The hypothesis stating that G. mangostana was the hybrid of G. hombroniana and G. malaccensis based on isozyme markers is acceptable.

Figure 6 .
Figure 6.Proportion of isozyme bands in mangoesteen and G. hombroniana and G. malaccensis

Table 1 .
Accession of mangoesteen and related allies analyzed using isozyme markers

Table 2 .
The number of bands and levels of polymorphism of 4 isoenzymes in 13 accession of mangoesteens and closely related species

Table 5 .
Significant correlation among isozyme bands Figure 5. Analysis of similarity main conponents of mangoesteen and related species mapped in three main axis based on isozyme markers