Identification of Morphological and Genetic Markers Related to Male Budless Characters on Kepok Banana Mutant.

Abstract

Bananas is an important horticultural commodity in Indonesia. However, the improvement of production capacity of cooking banana is hampered by Blood Disease Bacterium (BDB). Cooking banana such as kepok banana is very important commodity in several areas in eastern part of Indonesia for cash crop as well as for daily consumption. Since late 90s, production of kepok banana from Kalimantan drastically declined as a result of devastating outbreak of BDB. As a result many farmers are losing their main source of income. The disease is quickly spread at about 100 km distance per year as the bacterium is insect transmitted. Disease infection could be reduced by avoiding direct contact between insect vector and male bud by breaking peduncula supporting male bud or by cultivating male budless kepok banana mutant. Recently Centre for Tropical Horticultural Studies (PKHT) at Bogor Agricultural University (IPB) already collected several male budless kepok banana mutants with good eating quality named Unti Sayang (US-1). However, the genetic stability of these mutants is still unknown. Therefore, discovering DNA markers related to male budless character in kepok banana is very important to establish a stable mass propagation system for male budless character on kepok banana, and valuable for science to elucidate male budless mechanism in banana. The objectives of this research were to identify morphological characters and RAPD and ISSR markers and develop Pistillata and Agamous gene based moleculer marker related to male budless characters in kepok banana mutant Unti Sayang (US-1). Identification of morphological characters were carried out based on the International Plant Genetic Research Institute (IPGRI) descriptor for banana. The results showed that there is no morphological variation among wild type, mutants and revertrant other than a male bud character. The revertrant mutants were found among planlets derived from the sixth sub culture and second generation of suckers. Polymerase Chain Reaction (PCR) was used to amplify DNA of 24 plants derived from the first and sixth sub cultures using 20 RAPD and 12 ISSR primer combinations. Those RAPD and ISSR primers produced monomorphic DNA bands with the amount ranged from 2 to 6 and 6 to 8 per plant, respectively. No polymorphism was found between male budless mutants and revertrant mutants. The absence of genetic variation among wild type, mutants and revertrants has been reported in several cases of molecular marker analysis in in vitro cultured banana, although there was a variation in their morphology. Based on the PCR analysis, it was suggested that there was no genetic change between wild type and mutants. However, it is possible that some changes might occurred as point mutation located outside of the priming sites. Male budless characters was suggested to be controlled by Pistillata gene that control petal and stamen formation and Agamous gene for karpel formation. Therefore, the primers based Pistillata and Agamous genes were developed using Musa GeneBank database. PCR analysis using those primer combination have also produced no polymorphic bands among the analyzed banana plants. Carefull nucleotide sequence analysis of both DNA fragments showed three single nucleotide polymorphism between wild type and revertrant mutants at the position of 445, 461 and 507. Unfortunately, the nucleotide variation was located at 3‘UTR (untranslated region), which no any interference into protein sequence expressed by the genes. This result suggested that there might be other genes controlling the male budless variation or possibly an epigenetic variation occurred behind the variation. Further study needs to be conducted to clarify the genetic background underlaying male budless phenomenon occurred in kepok banana mutants.