VARIABILITAS GENETIK DAN HUBUNGAN KEKERABATAN TEMU GLENYEH (Curcuma soloensis Val.) DI PULAU JAWA BERDASARKAN KARAKTER MORFOLOGIS, SITOLOGIS, DAN MOLEKULER

MUHAMAD JALIL, Prof. Dr. Purnomo, M.S.; Prof. Dr. Budi Setiadi Daryono, M.Agr.Sc.; Dr. Ir. Aziz Purwantoro, M.Sc.

2021 | Disertasi | DOKTOR BIOLOGI

Temu Glenyeh (Curcuma soloensis Val.) merupakan herba tahunan dari keluarga Zingiberaceae yang berpotensi sebagai tanaman obat, Jamu, dan tanaman ornamental. Rimpang mengandung senyawa kimia penting untuk kesehatan seperti kurkuminoid dan seskuiterpen ar-tumeron. Tanaman ini hidup liar di daerah Pegunungan Seribu, yakni Pegunungan Jawa bagian selatan. Karakter morfologi Temu Glenyeh sulit dibedakan dengan anggota marga Curcuma L. yang lain. Di samping itu, informasi mengenai keragaman dan hubungan kekerabatan Temu Glenyeh di Jawa masih sangat terbatas. Tujuan penelitian ini adalah menemukan variasi morfologis, sitologis, dan genetik pada Temu Glenyeh di Pulau Jawa. Pengambilan sampel penelitian dilakukan di 13 Kabupaten/Kota di Pulau Jawa. Data morfologis dilakukan melalui pengamatan di lapangan. Identifikasi karakter morfologis, pembuatan deskripsi, dan kemiripan fenetik dilakukan berdasarkan 45 karakter. Data sitologis berdasarkan pengamatan ujung akar dengan metode squash yang dimodifikasi. Data molekuler ISSR menggunakan 10 primer, sedangkan molekuler ITS-rDNA menggunakan ITS-1 sebagai primer forward dan ITS-4 sebagai primer reverse. Karakter morfologis dianalisis dengan metode analisis klaster dan Principal Component Analysis (PCA). Data sitologis dianalisis dengan metode deskriptif. Data ISSR dianalisis dengan metode numerik yang diperoleh berupa data biner. Hasil sekuen nukleotida daerah ITS diverifikasi dengan program Basic Local Alignment Search Tool (BLAST). Perunutan sekuen DNA dengan Clustal Omega W. Penyusunan pohon filogenetik dilakukan dengan menggunakan metode Neighbour Joining. Uji statistik kehandalan menggunakan metode bootstrap 1000 replikasi. Model subtitusi nukleotida yang digunakan adalah Kimura-2 Parameter. Hasil penelitian menunjukkan terdapat variasi morfologis C. soloensis di Jawa, terutama pada karakter habitus, bentuk rimpang, warna daging rimpang, dan bentuk daun. Dendrogram membentuk klaster A (C. xanthorrizha dan klaster B (C. soloensis dan C. longa) dengan indeks similaritas 62-96%. Hasil identifikasi menunjukkan bahwa C. soloensis memiliki formula kariotipe yaitu 2n=2x=14=12m+2sm. Bentuk kromosom C. soloensis terdiri atas metasentrik dan submetasentrik. Ukuran kromosom pada C. soloensis adalah 045-1,37 mikrometer. Analisis karakter molekuler dengan analisis ISSR, menunjukkan keragaman yang tinggi dengan koefisien similaritas 57-97%. Dendrogram menunjukkan terdapat dua grup besar yaitu grup A (C. xanthorrizha) dan B (C. soloensis dan C. longa) dengan koefisien 0,566. Variasi genetik terdapat pada panjang sekuens dan perbedaan nukleotida pada daerah ITS r-DNA. Analisis ITS-rDNA, terbagi menjadi dua cabang utama yaitu cabang A (C. soloensis, C. longa, dan C. xanthorrizha) dan cabang B (kelompok outgroup) dengan jarak genetik 1,122 -1,708 (tinggi). Analisis karakter morfologi, sitologis, dan molekuler ISSR pada C. soloensis menunjukkan adanya kongruensi yang jelas, namun pengelompokan secara molekuler ITS-rDNA tidak kongruen dengan pola pengelompokan secara morfologis yang disebabkan karena aksesi CS-02 bergabung dengan aksesi C. longa, sedangkan CS-06 bergabung dengan C. xanthorrizha dalam filogram.

Temu Glenyeh (Curcuma soloensis Val.) is an annual herb belonging to the Zingiberaceae family and considered as a medicinal plant, herbal medicine, and ornamental plant. Rhizome contains important chemical compounds for health such as curcuminoids and sesquiterpenes ar-tumeron. This plant lives wild in the Seribu Mountains, southern Java. The morphological character of Temu Glenyeh is overlapped so that it is very difficult to identify and distinguish from other members of the genus Curcuma L. In addition, information on the diversity and relationship of Temu Glenyeh in Java is still very limited. The purpose of this study was to find morphological, cytological, and genetic variations in Temu Glenyeh on Java Island. The research sample was taken in 13 districts/cities on the island of Java. Morphological data were carried out through field observations. Identification of morphological characters, descriptions and phenetic resemblance was carried out based on 45 characters. Cytological data were conducted based on root tip observations with the modified squash method. The ISSR analysis was conducted using 10 primers, while the ITS-rDNA analysis was used ITS-1 as the forward primer and ITS-4 as the reversed primer. Morphological characters were analyzed using the cluster analysis method and Principal Component Analysis (PCA). Cytological data were analyzed using descriptive methods. ISSR data were analyzed using numerical methods obtained in the form of binary data. The similarity of ITS regional nucleotide sequences was performed using the Basic Local Alignment Search Tool (BLAST) program and tracing using Clustal Omega W. The phylogenetic tree was performed using the Neighbor-Joining method. The reliability statistical test was using the bootstrap 1000 replication method. The nucleotide substitution model was using Kimura-2 Parameters. The results showed that there were morphological variations of C. soloensis in Java, especially in the habitus character, rhizome shape, rhizome flesh color, and leaf shape. Dendrogram formed cluster A (C. xanthorrizhaC. xanthorrizha) and cluster B (C. soloensis and C. longa) with a similarity index of 62-96%. C. soloensis has a karyotype formula 2n=2x=14=12m+2sm. The chromosome of C. soloensis only consisted of two shape, including metacentric and submetacentric. Chromosome size in C. soloensis is 0,45-1,37 mikrometer. Analysis of molecular characters with ISSR analysis showed high diversity with a similarity coefficient of 57-97%. The dendrogram showed that there are 2 large groups, namely, grup A (C. xanthorrizha) and B (C. soloensis and C. longa) with a coefficient of 0,566. Genetic variations were found in the length of the sequence and nucleotide divergences in the ITS rDNA region. Two main branches were formed on the phylogenetic tree of ITS-rDNA, namely branch A (C. soloensis, C. longa, and C. xanthorrizha) and branch B (outgroup) with a genetic distance of 1,122 -1,708 (high). Analysis of the morphological, cytological, and molecular characteristics of ISSR in C. soloensis showed a clear congruence, but the molecular clustering of ITS-rDNA was not congruent with the morphological clustering pattern caused by CS-02 accession joining C. longa accession, while CS-06 joins C. xanthorrizha in the phylogram.

Kata Kunci : Variabilitas genetik, Hubungan kekerabatan, Curcuma soloensis, morfologis, sitologis, molekular