Analisis Keragaman Genetik dan Pengembangan Marka SNAP berbasis Gen Biosintesis Asam Jasmonat pada Phalaenopsis.

Abstract

Phalaenopsis merupakan salah satu genus anggrek yang paling populer di dunia dan memiliki nilai ekonomi tinggi karena keindahan karakter bunganya. Meskipun memiliki permintaan pasar yang tinggi, budidaya anggrek ini masih menghadapi berbagai kendala, terutama penyakit busuk lunak (PBL) yang disebabkan oleh bakteri Dickeya dadantii. Sebagian besar spesies Phalaenopsis diketahui rentan terhadap penyakit tersebut, namun penelitian sebelumnya berhasil mengidentifikasi lima spesies, yaitu P. amboinensis, P. corningiana, P. modesta, P. speciosa, dan P. tetraspis, yang menunjukkan ketahanan terhadap infeksi D. dadantii. Analisis transkriptom P. amboinensis mengungkapkan peningkatan ekspresi gen-gen yang terlibat dalam biosintesis asam jasmonat yang diduga berperan dalam resistensi terhadap serangan D. dadantii. Gen allene oxide synthase 2 (AOS2) dan allene oxide cyclase (AOC) merupakan gen kunci yang mengkode enzim pada tahap awal biosintesis asam jasmonat. Variabilitas single nucleotide polymorphism (SNP) pada gen AOS2 dan AOC di antara spesies Phalaenopsis yang resisten dan rentan terhadap penyakit busuk lunak diduga berkaitan dengan perbedaan respons terhadap infeksi D. dadantii. Variabilitas tersebut berpotensi dimanfaatkan sebagai dasar pengembangan marka single nucleotide amplified polymorphism (SNAP) untuk seleksi ketahanan terhadap penyakit busuk lunak. Penelitian ini bertujuan: 1) menganalisis keragaman genetik dan mengidentifikasi SNP pada gen AOS2 dan AOC sebagai dasar pengembangan marka SNAP; 2) mengevaluasi keefektifan marka SNAP berbasis AOS2 untuk analisis keragaman genetik spesies Phalaenopsis yang resisten atau rentan terhadap penyakit busuk lunak (PBL); 3) menganalisis substitusi residu asam amino pada protein AOS2 serta pengaruhnya terhadap struktur sekunder. Metodologi penelitian diawali dengan isolasi DNA dari sepuluh spesies anggrek Phalaenopsis yang resisten atau rentan terhadap penyakit busuk lunak. Tahap berikutnya mencakup analisis sekuens gen AOS2 dan AOC, desain primer, dan amplifikasi gen, analisis SNP, desain primer SNAP dan amplifikasi PCR untuk uji SNAP pada sepuluh spesies anggrek Phalaenopsis yang resisten atau rentan terhadap penyakit busuk lunak. Hasil amplifikasi SNAP berupa data visual kemudian dikonversi menjadi data alel untuk analisis nilai polymorphic information content (PIC), observed heterozygosity (Ho), expected heterozygosity (He), dan analisis filogenetik. Selain itu, hasil sekuensing akan digunakan untuk analisis filogenetik dan protein. Hasil penelitian ini menunjukkan analisis full length CDS (1530 bp) gen AOS2 berhasil diisolasi dari 10 spesies yang diuji dan ditemukan adanya 150 SNP pada gen tersebut. Sementara pada gen AOC hanya diperoleh fragmen dari gen berukuran 700 bp dan pada fragmen tersebut ditemukan sebanyak 55 SNP. Pada gen AOS2 ditemukan 14 SNP dan pada fragmen gen AOC sebanyak 7 SNP memiliki frekuensi alel tinggi. Sebanyak 6 SNP dari gen AOS2 dan 5 SNP dari gen AOC dipilih sebagai kandidat pengembangan marka SNAP. Validitas marka SNAP AOS2 ditunjukkan oleh nilai PIC, Ho, dan He yang berkisar dari rendah hinggasedang, serta nilai Ho yang lebih rendah daripada He, yang mengindikasikan frekuensi heterozigositas yang rendah. Analisis filogenetik membagi 10 spesies Phalaenopsis menjadi dua klaster utama, tetapi pengelompokan tersebut tidak sesuai dengan tingkat resistensi terhadap D. dadantii. Sementara itu, marka SNAP AOC tidak diuji melalui amplifikasi PCR karena sekuens gen AOC yang tersedia hanya bersifat parsial. Sebanyak 5 SNP non-synonymous dengan frekuensi alel tinggi terdeteksi menyebabkan substitusi residu asam amino, namun substitusi tersebut diprediksi tidak mengubah struktur sekunder protein AOS2 pada spesies Phalaenopsis. Penelitian ini menghasilkan beberapa temuan baru, yaitu karakterisasi gen AOS2 dan AOC pada 10 spesies Phalaenopsis, meliputi identifikasi variasi sekuens, SNP, hubungan filogenetik, serta perubahan residu asam amino yang dikodekan oleh kedua gen tersebut. Berdasarkan SNP yang berhasil diidentifikasi, dikembangkan marka SNAP berbasis gen AOS2 dan AOC yang mampu membedakan variasi genetik antarspesies Phalaenopsis. Marka SNAP yang dihasilkan dapat digunakan untuk mengidentifikasi materi genetik secara cepat dan akurat, mendukung pengelolaan sumber daya genetik, serta membantu pemilihan tetua berdasarkan keragaman genetik dalam perencanaan persilangan yang lebih terarah. Gen AOS2 dan AOC, yang berperan dalam jalur biosintesis asam jasmonat terkait respons pertahanan tanaman, memberikan landasan ilmiah bagi pengembangan hibrida Phalaenopsis yang memiliki ketahanan terhadap penyakit busuk lunak yang disebabkan oleh bakteri D. dadantii.

Phalaenopsis is one of the world’s most popular orchid genera and has high economic value due to the beauty of its flowers. Despite high market demand, the cultivation of this orchid still faces various challenges, particularly soft rot disease (SRD) caused by the bacterium Dickeya dadantii. Most Phalaenopsis species are known to be susceptible to this disease; however, previous research has successfully identified five species, namely P. amboinensis, P. corningiana, P. modesta, P. speciosa and P. tetraspis, which exhibit resistance to infection by D. dadantii. Transcriptome analysis of P. amboinensis revealed increased expression of genes involved in jasmonic acid biosynthesis, which are thought to play a role in resistance to D. dadantii infection. The allene oxide synthase 2 (AOS2) and allene oxide cyclase (AOC) genes are key genes encoding enzymes in the early stages of jasmonic acid biosynthesis. Single nucleotide polymorphism (SNP) variability in the AOS2 and AOC genes amongst Phalaenopsis species that are resistant and susceptible to soft rot disease is thought to be associated with differences in response to D. dadantii infection. This variability has the potential to be utilised as a basis for the development of single nucleotide amplified polymorphism (SNAP) markers for the selection of resistance to soft rot disease. The objectives of this study are: 1) to analyse genetic diversity and identify SNPs in the AOS2 and AOC genes as a basis for the development of SNAP markers; 2) to evaluate the effectiveness of AOS2-based SNAP markers for analysing the genetic diversity of Phalaenopsis species that are resistant or susceptible to soft rot disease (SRD); 3) to analyse amino acid residue substitutions in the AOS2 protein and their effect on secondary structure. The research methodology began with the isolation of DNA from ten species of Phalaenopsis orchids that are either resistant or susceptible to soft rot disease. The next stage involved sequence analysis of the AOS2 and AOC genes, primer design and gene amplification, SNP analysis, SNAP primer design and PCR amplification for SNAP testing on ten Phalaenopsis orchid species that are resistant or susceptible to soft rot disease. The SNAP amplification results, in the form of visual data, were then converted into allele data for the analysis of polymorphic information content (PIC), observed heterozygosity (Ho), expected heterozygosity (He), and phylogenetic analysis. In addition, the sequencing results will be used for phylogenetic and protein analysis. The results of this study show that the full-length CDS (1530 bp) of the AOS2 gene was successfully isolated from the 10 species tested, and 150 SNPs were identified in this gene. Meanwhile, for the AOC gene, only a 700 bp fragment was obtained, in which 55 SNPs were identified. Of these, 14 SNPs in the AOS2 gene and 7 SNPs in the AOC gene fragment exhibited high allele frequencies. A total of 6 SNPs from the AOS2 gene and 5 SNPs from the AOC gene were selected as candidates for the development of SNAP markers. The validity of the AOS2 SNAP marker was demonstrated by PIC, Ho and He values ranging from low to moderate, as well as an Ho value lower than He, indicating a low heterozygosity frequency.Phylogenetic analysis divided the 10 Phalaenopsis species into two main clusters; however, this grouping did not correlate with the level of resistance to D. dadantii. Meanwhile, the AOC SNAP marker was not tested via PCR amplification as the available AOC gene sequence was only partial. A total of 5 non-synonymous SNPs with high allele frequencies were detected, causing amino acid residue substitutions; however, these substitutions are predicted not to alter the secondary structure of the AOS2 protein in Phalaenopsis species. This study yielded several new findings, namely the characterisation of the AOS2 and AOC genes in 10 Phalaenopsis species, including the identification of sequence variations, SNPs, phylogenetic relationships, and changes in the amino acid residues encoded by these two genes. Based on the SNPs successfully identified, SNAP markers based on the AOS2 and AOC genes were developed, capable of distinguishing genetic variation amongst Phalaenopsis species. The resulting SNAP markers can be used to identify genetic material rapidly and accurately, supporting the management of genetic resources, and assisting in the selection of parents based on genetic diversity for more targeted cross-breeding programmes. The AOS2 and AOC genes, which play a role in the jasmonic acid biosynthesis pathway associated with plant defence responses, provide a scientific basis for the development of Phalaenopsis hybrids that are resistant to soft rot disease caused by the bacterium D. dadantii.