IDENTIFIKASI GEN PENANDA MOLEKULER KADAR ISOFLAVON KEDELAI HITAM ADAPTIF PERUBAHAN IKLIM IDENTIFICATION OF MOLECULAR MARKER GENES FOR ISOFLAVONE CONTENT ON BLACK SOYBEAN ADAPTIVE TO CLIMATE CHANGE Tati Suryati Syamsudin Subahar1), Adi Pancoro2), Agung Karuniawan3), Joko Prasetiyono4), Dadang Sumardi2) 1)
Institut Teknologi Bandung Sekolah Ilmu dan Teknologi Hayati 3) Universitas Padjajaran Badan Penelitian dan Pengembangan Pertanian 2)
4)
ABSTRAK Kedelai hitam mengandung metabolit sekunder, di antaranya isoflavon yang bermanfaat untuk mengatasi penyakit kanker, kardiovaskuler, osteoporosis, dan efek menopause. Kandungan isoflavon pada tanaman juga berperan penting untuk pertahanan terhadap cekaman biotik dan abiotik dan sebagai chemoatractant bakteri Rhizobium. Kadar isoflavon mudah berubah karena pengaruh lingkungan, terutama perubahan suhu. Kondisi seperti ini menyulitkan proses pemuliaan secara konvensional sehingga diperlukan pemuliaan berbantuan marka. Identifikasi gen pengatur kadar isoflavon kedelai pada kondisi suhu lingkungan yang berbeda diperlukan sebagai tahap awal proses pemuliaan. Penelitian ini merupakan tahap pertama dari tiga tahap penelitian yang
bertujuan untuk (1) memperoleh informasi mengenai gen pengendali kadar isoflavon yang berekspresi stabil pada kondisi suhu yang berbeda, dan (2) memperoleh genotipe kedelai yang menunjukkan kadar isoflavon stabil pada kondisi suhu yang berbeda. Penelitian dilaksanakan di Kebun Percobaan Fakultas Pertanian Unpad, lahan petani, Laboratorium Genetika ITB, Laboratorium Biologi Molekuler BB Biogen, dan Laboratorium Farmasi Unpad. Penentuan genotipe terpilih dilakukan melalui evaluasi karakter hasil, analisis kekerabatan dalam populasi koleksi kedelai hitam lokal, dan analisis kadar isoflavon 39 genotipe kedelai hitam. Tiga puluh sembilan genotipe kedelai hitam ditanam di kebun percobaan Fakultas Pertanian Unpad dengan menggunakan rancangan acak kelompok, dua ulangan. Evaluasi karakter hasil dilakukan dengan analisis varian dan uji beda Least Significant Increase. Analisis kekerabatan dilakukan dengan analisis fragmen terhadap hasil PCR dan analisis kadar isoflavon dengan HPLC. Analisis ekspresi gen dilaksanakan melalui penanaman 35 genotipe kedelai hitam pada lingkungan dataran menengah dan dataran rendah. RNA untuk analisis ekspresi gen diisolasi dari sampel daun dan sampel biji dan ekspresi gen dianalisis menggunakan PCR kuantitatif. Hasil penelitian menunjukkan gen CHS7, CHS8, IFS1, dan IFS2 merupakan gen pengendali utama kadar isoflavon biji yang ekspresinya dipengaruhi oleh kondisi lingkungan dan genotipe. Genotipe UP128 (KBI 4), UP117(KA-2) dan UP130 (KH-3) memiliki kadar isoflavon tinggi, sedang dan rendah pada lokasi dataran menengah, sedangkan genotipe UP114, UP134, UP135, dan Malika menunjukkan hasil yang stabil. Genotipe-genotipe tersebut dapat
digunakan sebagai penelitian tahap II.
calon
tetua
persilangan
pada
Kata kunci: Kedelai hitam, penanda molekuler, gen, isoflavon, hasil.
ABSTRACT Black soybean contains secondary metabolites including isoflavones that have benefits to cope with some types of cancer, cardiovascular disease, osteoporosis and menopause syndrome. Isoflavones are also beneficial for soybean plant as chemical defense against biotic and abiotic stress and as chemoatractant for Rhizobium. Isoflavone content prone to changes due to environmental influences, especially temperature. Hightemperature stress can affect soybean isoflavone content. Plant breeding using conventional methods to improve the isoflavones character will take a long time. Breeding using molecular markers can help to solve these problems. Identification of genes controlling the levels of isoflavones at the different temperature conditions, is still at the initial stage of the breeding process. At the first of the three-year study aimed to (1) obtain the information about the genes that showed stable expression at different temperature conditions, and (2) obtain soybean genotypes showed stable levels of isoflavones in different temperature conditions. The study was conducted at the Research Station of Faculty of Agriculture of Padjadjaran
University, farmer’s land, Genetics Laboratory of ITB, Molecular Biology Laboratory of ICABIOGRAD, and Pharmacy Laboratory of Padjadjaran University during February-November 2013. The genotypes were selected based on yield character, genetic diversity analysis within black soybean collection, and isoflavone content of 39 genotypes. The genotypes were planted at the experimental garden using a randomized block design with two replications. Evaluation of yield character used analysis of variance and least difference test. Genetic diversity was analyzed using fragment analysis and isoflavone content was analyzed by HPLC. Gene expression was analyzed using quantitative PCR. The results showed that genes CHS7, CHS8, IFS1, and IFS2 were the primary genes controlling isoflavone content, and their expreessions were influenced by environmental conditions and soybean genotypes. Genotypes UP128 (KBI 4), UP117 (KA-2) and UP130 (KH-3) respectively had high, medium and low isoflavone contents in medium elevation areas, while genotypes UP114, UP134, UP135 and Malika had stable yield. These genotypes could be used as parents of crossing in the second year study.
Keywords: Black soybean, molecular marker, genes, isoflavones, yields.
Gambar 2. Genotipe UP128 (KBI-4)
