KARAKTERISASI MOLEKULER KANDIDAT GEN KETAHANAN NBS-LRR DAN PROFIL METABOLIT TERPAUT KETAHANAN MELON (Cucumis melo L.) TERHADAP BEGOMOVIRUS

APRILIA SUFI S, Prof. Dr. Budi Setiadi Daryono, M.Agr.Sc; Dr. Ir. Sedyo Hartono, M.P.; Dr. Tri Rini Nuringtyas, M.Sc.

2019 | Disertasi | DOKTOR BIOLOGI

Begomovirus merupakan salah satu ancaman serius dalam produksi melon. Namun data mengenai jenis Begomovirus yang menginfeksi tanaman melon di Indonesia masih minim dan belum diperbaharui secara periodik. Pengembangan kultivar melon tahan Begomovirus merupakan strategi pengendalian Begomovirus yang dinilai efektif dan ramah lingkungan. Guna menunjang keberhasilan pengembangan kultivar tersebut diperlukan studi tentang ketahanan terhadap Begomovirus secara genomik, transkriptomik, maupun metabolomik. Tujuan penelitian ini adalah untuk mengetahui mekanisme respon ketahanan tanaman melon terhadap Begomovirus pada aras genomik dan metabolomik. Penelitian ini diawali dengan studi keragaman Begomovirus di Yogyakarta dan Jawa Tengah berdasarkan sekuen coat protein. Selanjutnya, dilakukan karakterisasi genom Begomovirus yang akan dijadikan sumber inokulan dengan metode kloning sekuensing. Terdapat 9 kultivar melon yang diinokulasi Begomovirus melalui perantara Bemisia tabaci. Pengamatan dan skoring keparahan infeksi dilakukan 14 hari setelah infeksi. Sampel daun yang dikoleksi digunakan untuk deteksi molekular infeksi Begomovirus, identifikasi kandidat gen resisten NBS-LRR, dan analisis profil metabolit dengan Nuclear Magnetic Resonance. Deteksi infeksi Begomovirus dilakukan dengan PCR menggunakan primer Krusty Homer. Kandidat gen ketahanan NBS-LRR terhadap Begomovirus diidentifikasi dengan 4 kombinasi degenerative primer NBS-LRR. Hasil amplifikasi kemudian disekuensing dan dilakukan analisis amino acid identity, conserved motif, alignment sekuen asam amino, dan filogenetik dengan menggunakan aplikasi bioinformatik. Selanjutnya, dilakukan desain kandidat primer RGA terpaut ketahanan terhadap Begomovirus. Selain itu, dilakukan juga analisis profil metabolit terhadap kultivar paling tahan dan paling rentan berdasarkan hasil uji ketahanan. Sampel daun dikeringkan dengan vakum dan dipreparasi dengan pelarut CD3OD kemudian dianalisis dengan 1H-NMR. Spektra sinyal hasil NMR dianalisis menggunakan Principal Component Analysis (PCA) untuk mengetahui perbedaan respon metabolit antara melon terinfeksi dan tidak terinfeksi serta kultivar tahan dan rentan Begomovirus. Hasil penelitian menunjukkan bahwa infeksi Begomovirus pada tanaman melon di Yogyakarta dan Jawa Tengah disebabkan oleh SLCCV dan ToLCNDV. Hasil karakterisasi genom juga menunjukkan hasil yang serupa bahwa isolat sumber inokulan merupakan ToLCNDV yang menunjukkan similaritas DNA-A sebesar 98,25% dengan ToLCNDV-[IN:JV:Luf;17] dan similaritas DNA-B sebesar 97,7% dengan ToLCNDV [IN:Cu:11]. Setelah melon uji diinokulasi virus diketahui bahwa melon kultivar Tacapa GB dan kultivar Tacapa Silver menunjukkan respon moderate resistant terhadap Begomovirus dengan nilai 10 < VI < 30 % sedangkan kultivar Melona dan kultivar Tacapa Gold menunjukkan ketahanan paling rendah terhadap Begomovirus dengan nilai VI berturut-turut 37,5% dan 47,5%. Melon kultivar Tacapa GB dan kultivar Tacapa Silver digunakan sebagai sampel untuk identifikasi gen ketahanan NBS-LRR. Hasil analisis menunjukkan terdapat 6 motif yang teridentifikasi, yaitu P-Loop, RNBS-A, Kinase-2, Kinase-3a, RNBS-C, dan GLPL. Motif P-Loop dan GLPL merupakan motif yang paling lestari. Gen ketahanan yang teridentifikasi merupakan golongan TIR-NBS-LRR dan menunjukkan hubungan kekerabatan dengan gen N dari tembakau dan L6 dari A.thaliana yang juga merupakan gen TIR-NBS-LRR. Berdasarkan sekuen gen ketahanan tersebut, berhasil didesain sebuah marka RGA terpaut ketahanan terhadap Begomovirus. Disisi lain, secara metabolomik diketahui bahwa terdapat perbedaan antara kultivar terinfeksi dan tidak terinfeksi Begomovirus. Senyawa kolin dan alanin menunjukkan penurunan konsentrasi yang signifikan (P< 0.001) antar kedua perlakuan. Kultivar kontrol tahan dan kultivar tahan menunjukkan perbedaan respon metabolit baik saat terinfeksi maupun tidak terinfeksi Begomovirus, meskipun keduanya menunjukkan fenotip moderate resistant. Kata Kunci : Gen NBS-LRR, Begomovirus, karakterisasi gen, profil metabolit, 1H-NMR

Begomovirus is one of the serious threats in melon production. However, scientific reports regarding Begomovirus species that infect melons in Indonesia are still limited and have not been updated periodically. The development of Begomovirus-resistant melon cultivars is a Begomovirus control strategy that is considered effective and environmentally friendly. In order to support the success of developing cultivars, a study of resistance to Begomovirus genomic, transcriptomic, and metabolomic level is needed. The purpose of this study was to determine the resistance mechanism of melon to Begomovirus in genomic and metabolomic levels. This study was begun with a study of Begomovirus diversity in Yogyakarta and Central Java based on a protein coat sequence. Furthermore, the characterization of the Begomovirus (as a source of inoculants) genome was carried out by sequencing cloning method. There were 9 melon cultivars inoculated with Begomovirus through the vector-inoculated methods. Observation and scoring of infection severity were carried out 14 days after infection. Then, leaf samples were collected to be used for molecular detection of Begomovirus infections, identification of candidate NBS-LRR-resistant genes, and metabolite profile analysis with Nuclear Magnetic Resonance. Detection of Begomovirus infection was conducted using PCR with a Krusty Homer primer while NBS-LRR resistance gene candidates were identified using 4 combinations of NBS-LRR degenerative primer. The amplification results were then sequenced and analyzed which were included analysis of amino acid identity, conserved motif analysis, alignment of amino acid sequences, and phylogenetic tree construction using bioinformatics applications. Furthermore, the sequence were used as the basis for the construction of RGA markers linked to the resistance of melons to Begomovirus. Finally, the profiling of melon metabolites was carried out by the 1H-NMR method. The samples used were the most resistant cultivars ('Tacapa GB' and 'Tacapa Silver') and the most susceptible ('Tacapa Gold' and 'Melona') (based on the results of the resistance test) and cultivar control 'Gracia'. Leaf samples were vacuum dried and prepared with CD3OD solvents. Signal spectra of NMR results were analyzed using PCA to determine the difference in metabolite response between infected and non-infected melons and between Begomovirus-resistant and susceptible cultivars. The results showed that Begomovirus infection in melon plants in Yogyakarta and Central Java was caused by SLCCV and ToLCNDV. Genomic characterization results also showed similar results that inoculant source isolates were ToLCNDV which showed DNA-A similarity of 98.25% with ToLCNDV- [IN: JV: Luf; 17] and DNA-B similarity of 97.7% with ToLCNDV [ IN: Cu: 11]. After the inoculated test melon, it was found that 'Tacapa GB' and 'Tacapa Silver' melons showed a moderate resistant response to Begomovirus with a value of 15% and 27.5% respectively, while 'Melona' and 'Tacapa Gold' showed the lowest resistance to Begomovirus with VI values 37.5% and 47.5% respectively. Hence, 'Tacapa GB' and 'Tacapa Silver' cultivars were used as samples for identification of NBS-LRR resistance genes. The results showed that there were 6 motif identified, namely P-Loop, RNBS-A, Kinase-2, Kinase-3a, RNBS-C, and GLPL. The P-Loop and GLPL motives are the most conserved motifs among them. The identified resistance genes were the TIR-NBS-LRR group and showed a genetic relationship with the N gene from tobacco and L6 from A.thaliana which is also the TIR-NBS-LRR gene. Based on the sequence of resistance genes, an RGA marker linked to resistance to Begomovirus was constructed successfully. On the metabolomic level, it is known that there was a difference response between infected and not infected with Begomovirus. choline and alanine had been shown a significant decrease in concentration after Begomovirus infection. The metabolite response between control resistant with resistant cultivars also showed a different pattern even though both showed moderate resistant phenotype, either when infected with Begomovirus or not. Keywords: NBS-LRR genes, Begomovirus, gene characterization, metabolite profile, 1H-NMR

Kata Kunci : Gen NBS-LRR, Begomovirus, karakterisasi gen, profil metabolit, 1H-NMR