Variasi Gen cox1, cox2, dan igs DNA Mitokondria Apis cerana asal Sumatra

Abstract

Distribusi lebah madu Apis cerana sangat luas di Asia dan memiliki adaptasi yang tinggi pada setiap lokasi. Apis cerana di Indonesia dikelompokkan dalam Indo-Malayan yang merupakan morfoklaster VI dengan distribusi di Sumatra, Kalimantan, dan Jawa (Paparan Sunda) hingga ke Timor. Belum ada data dan penelitian molekuler A. cerana asal Sumatra berdasarkan gen cytochrome c oxidase subunits 1 dan 2 (cox1 dan cox2) dan zona intergenic spacer (igs) dari DNA mitokondria (DNAmt). Oleh karena itu, penelitian ini bertujuan menganalisis variasi haplotipe, distribusi, dan stuktur genetik populasi A. cerana asal Sumatra menggunakan tiga marka DNAmt tersebut. Selain itu juga penelitian ini menganalisis kekerabatan A. cerana asal Sumatra dengan A. cerana asal Borneo dan Jawa berdasarkan data DNA di GenBank menggunakan gen cox1, cox2, dan igs dari DNAmt. Koleksi sampel lebah kasta pekerja A. cerana asal Sumatra dilaksanakan oleh tim peneliti dari lima universitas dan BRIN di Sumatra. Koleksi lebah dilakukan di enam provinsi Sumatra di 17 lokasi kecamatan yaitu delapan lokasi lowland (<300 m dpl), tiga lokasi midland (301–1.000 m dpl), dan enam lokasi highland (>1.000 m dpl). Ekstraksi dan amplifikasi DNA A. cerana asal Sumatra dilakukan berturut-turut dengan DNA Mini Kit GenAid dan Polymerase Chains Reaction (PCR). Amplikon DNA A. cerana asal Sumatra dilakukan pengurutan DNA di perusahaan 1st BASE, Selangor, Malaysia. Urutan DNA A. cerana asal Sumatra dilakukan pensejajaran dengan DNA dari GenBank menggunakan BLASTN. Analisis bioinformatika data DNA dilakukan dengan program ClustalX 2.0, MEGAX, DnaSP 5.10, dan Network 10.2.0.0. Analisis multivariat dilakukan menggunakan Canocical Correspondence Analysis (CCA) dengan program Past4.03. Amplikon hasil pengurutan DNA A. cerana asal Sumatra berdasarkan gen cox1 dan cox2 berturut-turut adalah 593 dan 464 pasang basa (pb), sedangkan amplikon igs berkisar antara 84–88 pb. Urutan DNA A. cerana asal Sumatra gen cox1 dan igs-cox2 telah diunggah ke GenBank dengan accession number berturut-turut LC728498–LC728558 dan LC739435–LC739501. Analisis BLASTN menunjukkan bahwa A. cerana asal Sumatra gen cox1 dan igs-cox2 homolog berturut-turut 96,35–99,27% dan 95,45–99,51% dengan A. cerana asal Sabah, Borneo dengan accession number AP018149.1. Penelitian ini menemukan 33 haplotipe A. cerana asal Sumatra berdasarkan gen cox1, cox2, dan igs. Semua haplotipe gen cox1 (12 haplotipe), sembilan dari 12 haplotipe gen cox2, dan delapan dari sembilan haplotipe igs merupakan haplotipe baru dan spesifik pada A. cerana asal Sumatra. Variasi nukleotida ditemukan pada gen cox1, cox2, dan igs A. cerana asal Sumatra berturut-turut sebesar 22/593 pb (3,7%), 28/464 pb (6%), dan 11/88 pb (12,5%). Variasi nukleotida gen cox1 tidak mengubah asam amino putatif. Variasi nukleotida gen cox2 A. cerana asal Sumatra menunjukkan adanya missense mutations pada posisi 289 dan 291. Kedua posisi ini mengubah asam amino Valin menjadi Isoleusin pada haplotipe Sumatra4 cox2 dan posisi 451 mengubah asam amino Isoleusin menjadi Valin pada haplotipe Sumatra9 gen cox2. Perubahan asam amino gen cox2 A. cerana asal Sumatra masih dalam kelompok asam amino nonpolar alifatik. Haplotipe umum (Sumatra1) tertinggi dari A. cerana asal Sumatra adalah berdasarkan igs (66%), cox1 (59%), dan terendah cox2 (37%). Provinsi Sumatra Utara, Lampung, dan Sumatra Selatan menunjukkan variasi haplotipe tertinggi, berdasarkan tiga marka DNAmt. Variasi pada A. cerana asal Sumatra disebabkan oleh ketinggian tempat dan jarak antar apiari yang jauh di seluruh lokasi, serta kemungkinan oleh erupsi super Toba di Sumatra Utara dan dampak antropogenik. Pola yang sama juga ditemukan pada haplotipe spesifik A. cerana asal Sumatra yaitu yang tertinggi igs (27%), cox1 (26%), dan cox2 (22%). Haplotipe spesifik A. cerana asal Sumatra banyak ditemukan di lowland (12 haplotipe), midland (delapan haplotipe), dan highland (tiga haplotipe) berdasarkan tiga marka DNAmt. Haplotipe spesifik tersebut dapat dijadikan marka asal-usul spesies pada lokasi tertentu di Sumatra. Hal yang menarik adalah sebanyak tiga variasi intra-koloni A. cerana asal Sumatra ditemukan di highland, dua variasi di lowland, dan satu variasi di midland. Variasi intra-koloni gen cox1 A. cerana asal Pesisir Selatan, Lampung dan gen cox2 A. cerana asal Sungai Geringging, Sumatra Barat berkontribusi terhadap total haplotipe spesifik A. cerana asal Sumatra. Hasil CCA menunjukkan bahwa haplotipe A. cerana asal Sumatra gen cox1, cox2, dan igs mengelompok berdasarkan ketinggian lowland, midland, dan highland. Faktor ketinggian tempat lebih mempengaruhi distribusi variasi haplotipe A. cerana asal Sumatra untuk tiga DNAmt daripada ragam habitat. Empat haplotipe identik ditemukan di masing-masing lowland-midland-highland dan lowland-highland. Dua haplotipe identik ditemukan di lowland-midland dan tidak terdapat haplotipe identik di midland-highland. Nilai FST A. cerana asal Sumatra gen cox1, cox2, dan igs secara bertahap meningkat seiring dengan jarak lokasi antar populasi dari Aceh ke Lampung, dengan nilai FST moderate hingga great differentiation menunjukkan aliran gen yang rendah. Populasi A. cerana asal Sumatra dari lima provinsi ke Lampung memiliki aliran gen terendah berdasarkan tiga marka DNAmt tersebut. Nilai FST berkorelasi positif dengan jarak geografis antar populasi A. cerana asal Sumatra. Berdasarkan jarak genetik dan pohon filogenetik Maximum likelihood, A. cerana asal Sumatra lebih berkerabat dekat dengan A. cerana asal Borneo daripada A. cerana asal Jawa. Selain itu, haplotipe Sumatra4 gen cox2 A. cerana koloni 2 asal Siantar Sitalasari, Sumatra Utara sama dengan haplotipe Java3 gen cox2 dari A. cerana asal Lebak, Banten, sehingga dikonfirmasi berasal dari Jawa. Median-joining network menunjukkan bahwa A. cerana asal Sumatra Selatan dan Lampung berkerabat dekat, haplotipe Sumatra Utara tersebar hampir di seluruh populasi Sumatra, dan A. cerana koloni 2 asal Siantar Sitalasari memiliki percabangan leluhur yang berbeda dari populasi Sumatra. Penelitian ini telah berhasil menyusun database DNAmt lebah madu A. cerana asal Sumatra berdasarkan gen cox1, cox2, dan igs yang dapat dipakai untuk melacak distribusi lebah madu natif Sumatra. Hasil penelitian ini dapat menjadi bahan kebijakan budidaya dan konservasi A. cerana di Indonesia. Penelitian selanjutnya perlu digabungkan hasil DNA mt ini dengan gen DNA inti dan karakter morfologi untuk mendapatkan hasil yang lebih menyeluruh.

Apis cerana honey bee is widely distributed and highly adapted in Asia. Apis cerana in Indonesia is grouped in Indo-Malayan (morphocluster VI), which is distributed in Sumatra, Kalimantan, and Java (Sundaland) to Timor. There is no data and molecular research of A. cerana from Sumatra based on the cytochrome c oxidase subunits 1 and 2 (cox1 and cox2) genes and intergenic spacer (igs) region from mitochondrial DNA (mtDNA). Therefore, this study aimed to analyze haplotype variations, distribution, and the genetic structure of A. cerana from the Sumatra population using these three mtDNA markers. This study also analyzed the relationship of A. cerana from Sumatra with A. cerana from Borneo and Java from GenBank DNA based on the cox1, cox2 genes, and igs mtDNA. The sample of A. cerana worker bees from Sumatra was collected by a research team that consisted of five universities and the National Research and Innovation Agency in Sumatra. The collection of bees was carried out in six Sumatra provinces at 17 locations. Those were from eight locations in the lowland (<300 m asl), three in the midland (301–1,000 m asl), and six in the highland (>1,000 m asl). DNA extraction and amplification of A. cerana from Sumatra were carried out using DNA Mini Kit GenAid and Polymerase Chains Reaction (PCR), respectively. The amplicons of A. cerana from Sumatra were sequenced in 1st BASE, Selangor, Malaysia. The DNA sequences of A. cerana from Sumatra were aligned with the GenBank secondary data using BLASTN. The nucleotide bioinformatics analysis was done using ClustalX 2.0, MEGAX, DnaSP 5.10, and Network 10.2.0.0 software. The Canonical Correspondence Analysis (CCA) multivariate was done using Past4.03 software. The amplicon sequences of A. cerana from Sumatra cox1 and cox2 genes were 593 and 464 base pairs (bp), respectively, while the igs amplicons ranged from 84–88 bp. The cox1 and igs-cox2 sequences of A. cerana from Sumatra had been submitted to GenBank with accession numbers LC728498–LC728558 and LC739435–LC739501, respectively. BLASTN analysis showed that A. cerana from Sumatra cox1 and igs-cox2 was 96.35–99.27% and 95.45–99.51%, respectively homologous with A. cerana from Sabah, Borneo, with accession number AP018149.1. This research found a total of 33 haplotypes of A. cerana from Sumatra based on the cox1, cox2 genes, and igs. All haplotypes of the cox1 genes (12 haplotypes), nine out of 12 and eight out of nine haplotypes of the cox2 gene and igs, respectively, were new and specifically found in A. cerana from Sumatra. The nucleotide variations in the cox1, cox2, and igs genes of A. cerana from Sumatra were 22/593 bp (3.7%), 28/464 bp (6%), and 11/88 bp (12.5%), respectively. The cox1 gene nucleotide variations did not change putative amino acids. Nucleotide variations in the cox2 gene A. cerana from Sumatra showed missense mutations at positions 289 and 291. The mutations changed the amino acid Valine to Isoleucine in the Sumatra4 cox2 haplotype, and position 451 changed the amino acid Isoleucine to Valine in the Sumatra9 cox2 haplotype. Changes in the amino acid of the cox2 gene A. cerana from Sumatra were still in the same aliphatic nonpolar amino acid group. The highest common haplotypes (Sumatra1) of A. cerana from Sumatra were on igs (66%), cox1 (59%), and the lowest was cox2 (37%). The provinces of North Sumatra, Lampung, and South Sumatra showed the highest haplotype variation based on these three mtDNA markers. The variations in A. cerana from Sumatra were caused by altitudes and the long distance of apiaries in all locations, then presumably by the super-eruption of Toba and anthropogenic impact. The same pattern was also found in the specific haplotypes of A. cerana from Sumatra, i.e., the highest was igs (27%), cox1 (26%), and the lowest was cox2 (22%). Specific haplotypes of A. cerana from Sumatra based on cox1, cox2 genes, and igs were found in the lowland (12 haplotypes), midland (eight haplotypes), and highland (three haplotypes), respectively. Specific haplotypes can be used as the origin marker of A. cerana in distinguished locations of Sumatra. Three intra-colony variations of A. cerana from Sumatra were found in the highland, two variations in the lowland, and one colony in the midland. Intra-colony variations of A. cerana from Pesisir Selatan, Lampung based on the cox1 gene and A. cerana from Sungai Geringging, West Sumatra, based on the cox2 gene, contributed to the specific haplotype total number of A. cerana from Sumatra. The CCA result showed that the haplotypes of A. cerana from Sumatra based on the cox1, cox2 genes, and igs were grouped based on the lowland, midland, and highland altitudes. The altitudes contribute more than habitat variety to the haplotype diversity distribution of A. cerana from Sumatra based on the cox1, cox2 genes, and igs. Four identical haplotypes were found in the lowland-midland-highland and the lowland-highland. Two identical haplotypes were found in the lowland-midland, and no identical haplotype in the midland-highland. The FST values of A. cerana from Sumatra of the cox1, cox2 genes, and igs gradually increased along the distance of the population from Aceh to Lampung, with moderate to great FST values indicating the low gene flow. Apis cerana from the Sumatra population from five provinces to Lampung had the lowest gene flow based on these three mtDNA markers. The FST value positively correlates with the geographic distance among populations of A. cerana from Sumatra. Based on the genetic distance and the Maximum likelihood phylogenetic tree, A. cerana from Sumatra was more closely related to A. cerana from Borneo than A. cerana from Java. Moreover, the haplotype Sumatra4 cox2 gene of A. cerana colony 2 from Siantar Silatasari, the same as the haplotype Java3 cox2 gene of A. cerana from Lebak, Banten, thus was confirmed from Java. The median-joining network showed that A. cerana from South Sumatra and Lampung were closely related, haplotypes of North Sumatra were spread almost in all populations, and A. cerana colony 2 from Siantar Sitalasari is in different ancestral branches. This research successfully provides the mtDNA database of A. cerana honey bee from Sumatra based on the cox1, cox2, and igs, which can be used to trace the distribution of Sumatran native honey bees. The results can support A. cerana beekeeping and conservation. Further, an analysis combining these mtDNA results with nuclear genes and morphological characters is needed to have more comprehensive data on A. cerana in Sumatra.