Establishing Inter-Simple Sequence Repeats (ISSRs) Protocol for Canarium odontophyllum Miq.

Iris Tia, Mekung (2008) Establishing Inter-Simple Sequence Repeats (ISSRs) Protocol for Canarium odontophyllum Miq. [Final Year Project Report] (Unpublished)

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Abstract

Canarium odontophyllum or locally known as "dabai" is one of the wild fruits that can be found in Sarawak. It is classified under the family of Burseraceae and genus Canarium. Inter-simple sequence repeat (ISSR) is a PCR- based molecular marker which involves amplification of DNA segment present at an amplifiable distance in between two identical microsatellite repeat regions that oriented in opposite direction. It is a quick. simple and highly polymorphic marker that combines most of other markers characteristics. ISSR-PCR technique has been applied to various plans species in studying of genetic diversity, genetic mapping, genomic fingerprinting, and germplasm identification and characterization. Little is known about ISSR markers focused on identification or genotyping of variants in agriculturally important wild fruit plants. The aim of this research is to establish the optimum ISSR protocol for C. odontophyllum. Total genomic DNA was successfully extracted from C. odontophyllum leaves using a modified CTAB extraction protocol. The optimum ISSR-PCR conditions were(i)annealing temperature of 60.4C for (AG)1O and (GTG)6, 57.9C for (AC)10 and 59.6C for (CT)1O; (ii) DNA concentration of 5Ong for (AG)1O and (GTG)6 and 1Ong for (AC)10 and (CT)10; and (iii) MgCl2 concentration of 2.0mM for (AG)10 and (GTG)6, 2.5mM for (AC)10, and 3.0mM for (CT)10. Distinct bands obtained in the ranged 500bp to 1500bp for all of the primers used. The optimum ISSRs protocol for C. odontophyllum established will be applied in deve1oping microsatellite DNA markers specifically for C.Odontophyllum genotyping.

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