BIOSISTEMATIKA PAMELO (Citrus maxima (Burm.) Merr.) DI INDONESIA BERDASARKAN KAJIAN MORFOLOGIS, FITOKIMIA, DAN MOLEKULAR

Dra. Ratna Susandarini, M.Sc., Prof. Sukarti Moeljopawiro, Ph.D.

2014 | Disertasi | S3 Biologi

Pamelo (Citrus maxima (Burm.) Merr.) merupakan tanaman buah asli kawasan tropis Asia Tenggara. Di Indonesia dikenal sejumlah kultivar komersial pamelo yang dibudidayakan secara intensif di daerah sentra perkebunan. Berdasarkan tinjauan literatur diketahui bahwa pamelo memiliki keragaman morfologi yang tinggi khususnya pada organ buah, namun sejauh ini belum ada publikasi yang secara komprehensif mendokumentasikan keragaman tersebut, demikian juga tinjauan mengenai taksonomi di bawah tingkatan spesies. Penelitian sistematika pamelo yang dilaporkan ini menggunakan pendekatan fenetik dan filogenetik untuk mengungkap variasi infraspesifik, hubungan kekerabatan, dan kejelasan identitas taksonomi infraspesifik pamelo berdasarkan kajian morfologi, fitokimia, dan molekular. Penelitian ini bertujuan menghasilkan klasifikasi infraspesifik pamelo, dan memperoleh penanda morfologis, fitokimia, dan molekular untuk dasar klasifikasi infraspesifik. Bahan penelitian berupa organ vegetatif dan reproduktif pamelo, dengan total jumlah sampel 107 individu. Data morfologi daun, bunga, buah, dan biji diperoleh melalui pengamatan dan pengukuran langsung di lapangan dan laboratorium. Analisis hubungan kekerabatan fenetik secara morfologis dilakukan terhadap 60 aksesi berdasarkan 21 karakter. Analisis profil minyak atsiri daun dilakukan terhadap 40 aksesi berdasarkan komposisi dan konsentrasi senyawa hasil kromatografi gas – spektrum massa (GC-MS). Pendekatan fenetik dilakukan menggunakan analisis klaster dan analisis komponen utama. Kajian molekular dilakukan dengan analisis sekuen DNA ITS1 terhadap 70 aksesi dan variasi motif (GA)n pada microsatellite terhadap 80 aksesi. Pendekatan filogenetik berdasarkan sekuen DNA dilakukan menggunakan metode minimum evolution. Hasil analisis klaster terhadap data morfologi menunjukkan ada dua klaster utama, yang merepresentasikan dua grup kultivar. Berdasarkan analisis komponen utama diketahui terdapat enam karakter morfologis dari organ daun dan buah yang berperan besar dalam pengelompokkan aksesi ke dalam dua klaster. Hasil analisis morfologi menjadi dasar penyusunan klasifikasi terbuka pamelo, yang membagi 60 aksesi pamelo menjadi dua grup kultivar, yaitu “Grup Kultivar Gulung” dan “Grup Kultivar Pamelo”. Kombinasi karakter daun dan buah merupakan penanda morfologis untuk membedakan grup kultivar. Hasil analisis terhadap kromatogram GC-MS pada 40 aksesi pamelo menunjukkan variasi infraspesifik minyak atsiri daun pamelo. Variasi kualitatif dan kuantitatif senyawa minyak atsiri tersebut menjadi dasar pengelompokkan aksesi ke dalam tiga kemotipe, yaitu Kemotipe “nerol-loliolide-allo aromadendrene”, Kemotipe “alpha selinene”, dan Kemotipe “alpha pinene-delta carene”. Analisis terhadap sekuen ITS1 yang menunjukkan variabilitas infraspesifik rendah, yaitu 1,83% mendukung peran ITS1 sebagai penanda molekular yang tepat digunakan untuk level interspesies. Analisis sekuen microsatellite yang mengandung ulangan dinukleotida (GA)n mengungkap variasi genotip yang tinggi pada pamelo, yang ditunjukkan dengan terbentuknya 9 clade pada pohon filogeni yang dihasilkan dengan metode minimum evolution.

Pummelo (Citrus maxima (Burm.) Merr.) is native fruit of tropical South East Asia regions. There are a number of commercial pummelo cultivars intensively cultivated in various orchards in Indonesia. Based on the information found in many literature, pummelo is known as having high morphological diversity, especially in fruit characters. Nevertheless, so far there is no comprehensive publication documenting this diversity as well as the taxonomic treatment at infraspecific level. Systematics study reported here used both phenetic and phylogenetic approaches to disclose infraspecific variation, taxonomic affinitity, and clarification of taxonomic identity of pummelo at infraspecific level based on morphological, phytochemistry, and molecular analysis. The objectives of this study were to generate open classification system a infraspecific level for Indonesian pummel, and reveal morphological, phytochemical, and molecular markers as basis for the infraspecific classification. Materials used in this study were vegetative and reproductive organs of 107 individuals. Morphological data from leaves, flowers, fruits and seeds were obtained from observation and measurements either in the field or laboratory. Phenetic affinity based on morphology was conducted on 60 accessions using 21 characters. Meanwhile, phenetic affinity based on leaf essential oils was performed on 40 accessions using compounds’ composition and concentration analyzed using gas chromatography – mass spectrometry. Morphological and essential oil data were subjected to cluster analysis and principal component analysis. The molecular approach was conducted using DNA sequence analysis of ITS1 on 70 accesions and (GA)n repeat motifs of microsatellites on 80 accessions. Phylogenetic relationships was examined using minimum evolution method. Result of cluster analysis on morphological data revealed two main clusters corresponding to the existence of two cultivar groups of pummelo. Six morphological characters from leaves and fruit were identified as the major distinguishing characters for these two clusters based on principal component analysis. Results of morphological analysis as used as a basis for constructing open classification in which 60 accessions of pummelo was divided into two cultivar groups, the “Gulung Cultivar Group” and “Pamelo Cultivar Group”. Combination of leaf and fruit characters was found to be morphological marker for the recognition of cultivar groups. Analysis on the GC-MS chromatograms of 40 pummelo accessions revealed infraspecific variation on leaf essential oil. The qualitative and quantitative infraspecific variation was used to differentiate accessions into three chemotypes. They were recignized as “nerol-loliolide-allo aromadendrene”, “alpha selinene”, and “alpha pinene-delta carene” chemotypes, respectively. Analysis on the ITS1 sequence showed a low infraspecific variation of 1.83%, and thus supported the role of ITS1 as molecular marker for differentiating species. Meanwhile, sequence analysis on microsatellite containing (GA)n dinucleotide repeat discloses high genotypic variation on pummelo. This high genotypic variation was reflected in phylogeny tree constructed by minimum evolution method which showed the existence of 9 clades.

Kata Kunci : Citrus maxima, hubungan kekerabatan, fenetik, filogenetik, Citrus maxima, taxonomic affinity, phenetic, phylogenetic