CONVERSION OF NEGATIVE To POSITIVE DNA MARKERS: DEVELOPMENT OF AUXILIARY TOOL FOR A RAPD-BASED MARKER SYSTEM IN BANANA (MUSA SPP.; AAA)

摘要

The banana (Musa spp.; AAA) genome is continuously expanding due to high frequency of somaclonal variation. Because of this increasing diversity; identification and taxonomic classification of banana cultivars have become difficult and often subject to disagreement. In this paper; we describe the development of an auxiliary protocol in converting negative to positive RAPD markers that can discriminate closely related Musa; AAA cultivars. Using arbitrary primer A13; we identified negative RAPD markersA1330 and A131 :> specifically for S4 (selection from Giant Cavendish subgroup; AAA) and S8 ('Morado' from 'Red'and 'Green Red' subgroup; AAA); respectively. Fragments corresponding to these missing bands were sequenced and used as templates to design new primers targeted around the 3'end of the original primer-annealing site. Two of these primers; BaS.OA and Bal.SA; successfully generated positive markers consistently amplified as BaS.OA08 and Bal.SA06 for S4 and S8; respectively. Since the resultingpositive markers can be sequenced and longer primer pairs can be designed; this marker-like SCAR can become highly reproducible to discriminate S4 and S8 from among genetically related and morphologically indistinguishable Musa; AAA cultivars. The results of the test demonstrate the value of an auxiliary tool that can be used in any RAPD-based marker system using various DNA sources. In addition; this provides an alternative method to screening more arbitrary primers in generating positive markers especially in situations when negative RAPD markers have been already identified.