Development of PCR-based Fingerprinting Tool in Banana (Musa sp., AAA) and Conversion of Negative to Positive DNA Marker

Abstract

The banana (Musa sp., AAA) genome is continuously expanding due to the high frequency of somaclonal variation. Because of this increasing diversity, numerical and morphological methods of taxonomic and phylogenetic identification of banana cultivars became laborious, difficult, and often the subject of disagreements. The aim of this study, therefore, is to develop molecular tools for DNA fingerprinting that can discriminate Musa, AAA Cavendish subgroup cultivars. In this paper, we showed that the plastid-subtype identity (PS-ID) sequence of the noncoding region between rpl16 and rpl14 genes of plastid DNA was highly conserved except for single-base substitution and deletion. These differences separated the clones into three groups (G1, G2, and G3) and suggested that clones within groups are closely related maternally. Using arbitrary primer A13, we later identified negative RAPD markers A133.0 and A131.3 specifically for S4 (selection from Giant Cavendish subgroup, AAA) and S11 (`Morado' from `Red' and `Green Red' subgroup, AAA), respectively. Fragments corresponding to the missing bands were sequenced and used as templates to design new primers with overlapping sequences. Two of these primers, Ba3.0A and Ba1.3A, successfully generated positive markers consistently amplified as Ba3.0A0.8 and Ba1.3A0.6 for S4 and S11, respectively. It is proposed that the method just described can be a better alternative over screening more arbitrary primers in generating positive markers in cases when negative ones were already identified. Results of PS-ID subtype analysis likewise suggested potential use in identifying wild maternal progenitor in polyploid bananas.