Pemanfaatan marka molekuler dalam pemuliaan tanaman untuk mendukung penyediaan kultivar unggul baru jarak pagar (Jatropha curcas L.)

Abstract

The main obstacle in the cultivation and commercialization of physic nut (Jatropha curcas L.) as a biodiesel plant is the unavailability of superior cultivars or hybrids with high yield and oil content. Assessment of molecular genetic diversity is important because it is a pre-requisite for plant breeding programs.This research was conducted to develop and exploit molecular marker in order to support genetic improvement of physic nut. Development of Simple Sequence Repeat (SSR) primer was conducted based on DNA sequences available in the GenBank DNA database and 28 primer pairs were yielded. Twenty four accessions of physic nut from Kebun Induk Jarak Pagar (KIJP) Pakuwon germplasm collection were assessed by SRR marker designed. The results show that there was no variability among accession tested. In addition, Random Amplified Polymorphic DNA (RAPD), Inter Simple Sequence Repeat (ISSR) marker was employed to asses genetic variability. Out of 31 RAPD and ISSR primers evaluated 8 RAPD and 4 ISSR primers produced scorable DNA markers and four primers (UBC 873, OPG 17, OPP 03 and OPQ 11) produced polymorphic bands. Genetic similarity coefficients ranged from the highest of 1.0 (between 3189-2 / PT13-2; MT7-1 / PT15-1; PT3-1, 2555-1 / SP8-1; 2555-1 / PT3-1) to the lowest of 0.6 (between 554-1/HS49-2) with a mean 0.9. SSR primer then tested for cross species amplification ability to the relative species of J. curcas i.e. J. integerrima, J. multifida, J. gossypifolia, J. podagrica) and study the relationship between these species was conducted. Out of 28 primers checked, 11 primers showed cross species amplification in all the species tested. Overall percentage of polymorphism (PP) among all species tested was 95% and the mean genetic similarity (GS) was found to be 0.34. Dendrogram showed that J. integerrima have the closest distance from J. curcas. Interspecific crossing between J. curcas x J. integerrima have been conducted and morphological characters were recorded. Variation between hybrids was not too large and generally intermediate between the parents. Molecular analysis using SSR markers, RAPD and ISSR conducted on 8 hybrids and their parents. Two markers (EU099522 and OPC 10) were polymorphic in both parents and co-inherited to all hybrids. EF612741 and EU099524 were specific to J. integerrima and J. curcas respectively and could be used for identification of hybrids through multiplex PCR. Keywords: primer development, genetic variability, cross specific amplification, With such a low genetic diversity among physic nut accessions, breeding program using the analyzed accessions may not useful. Introduction of new accessions of physic nut and or interspecific crossing with its relative may be necessary to increase genetic diversity and improve genetic gain through breeding program.

Hambatan utama dalam budidaya dan komersialisasi jarak pagar (Jatropha curcas L.) sebagai tanaman penghasil bahan bakar nabati adalah belum tersedianya kultivar unggul yang berdaya hasil dan berkadar minyak tinggi. Modal utama untuk perbaikan genetik adalah tersedianya keragaman genetik dari spesies yang bersangkutan. Informasi keragaman genetik jarak pagar belum memadai karena sebagian besar masih berdasar pengamatan secara morfologis. Penelitian ini dimaksudkan untuk mengembangkan dan memanfaatkan marka molekuler untuk mendukung pemuliaan jarak pagar. Primer SSR telah dikembangkan berdasarkan informasi sekuen DNA yang tersedia pada basis data Genbank. Dua puluh delapan pasang primer spesifik SSR telah berhasil didesain menggunakan aksesi DNA asal jarak pagar yang ada di GenBank DNA database. Ekstraksi DNA pada penelitian ini menggunakan protokol standar CTAB yang dimodifikasi. Separasi dan visualisasi hasil amplifikasi menggunakan marka SSR dilakukan dengan polyacrilamide gel electrophoresis (PAGE) dan pewarnaan perak. Separasi dan visualisasi hasil amlifikasi menggunakan marka RAPD, ISSR dan SCAR dilakukan dengan elektroforesis gel agarosa dan ethidium bromida.