Morphological and Molecular Assessment of Durian Germplasm in Malaysia
Santoso, Panca Jarot (2004) Morphological and Molecular Assessment of Durian Germplasm in Malaysia. Masters thesis, Universiti Putra Malaysia.
Genetic resource management is essential for the continuity of improvement and production of durian. It involves maintaining associated data, and characterising and assaying highly heritable morphological and molecular traits for taxonomic study, genetic improvement, quality assurance and management purposes. The objective of this study was to determine the genetic variations of durian germplasm in Malaysia, based on morphological traits and PCR-RFLP on chloroplast DNA and ribosomal DNA regions. For study of genetic variation based on morphological traits, a total of 65 durian accessions, consisting of 60 cultivated clones and five wild species were compared. A total of 12 distinct clusters were identified separating the accessions. Low to moderately high genetic variations were found among the cultivated clones, while moderately high to high genetic variations were found between accessions of the cultivated clones and the wild species, and also among the wild species. Some cultivated clones were found to have revealed high level of similarities, and were placed separately within six different clusters. Three available protocols, those by Gawel and Jarret (1991), Husain (1994) and Mathius and Hutabarat (1997), were used to extract genomic DNA from the leaves of the durian accessions. Mathius and Hutabarat’s (1997) protocol was found able to produce the highest quantity of genomic DNA, however, with low quality. Prolongation of incubation period from 30 min to 75 min, addition of TE buffer following cool isopropanol precipitation and CIA (24:1) re-extraction, were found effective to improve Mathius and Hutabarat’s (1997) protocol to produce high quality genomic DNA from the durian leaves. Six pairs of primers i.e. psbC, rpoB, rbcL, A, N and IGS were used to amplify six specific regions: psbC, rpoB, rbcL, and intergenic spacers of ndhC-trnV, atpB-rbcL and IGS-rDNA, respectively. Two primers, rbcL and N, were found able to consistently amplify the two specific regions, IGS inconsistently amplified the specific and non-specific regions, A inconsistently amplified non-specific region, while rpoB and psbC were found unable to amplify any region at all. Six restriction enzymes, Eco RI, Bam HI, Bsu RI, Hind III, Pst I and Taq I, were used to digest the PCR products. Four enzymes, Bsu RI, Hind III, Pst I and Taq I, were found able to digest the PCR products into smaller fragments, while two enzymes, Eco RI and Bam HI, were unable to do so. Generally, enzymes having four-base recognition sites were found able to digest broader range of PCR products than those having six-base recognition sites. PCR-RFLP on the ndhC-trnV and rbcL regions, using eight restriction enzymes, was conducted to study genetic variation among 11 accessions belonging to 10 species in the genus Durio. The polymorphism produced from ndhC-trnV region showed higher variations than those from rbcL region. Based on the results of PCR-RFLP on ndhC-trnV region, the accessions were grouped into five distinct clusters, while based on results of PCR-RFLP on the rbcL region, they were grouped into only three distinct clusters. PCR-RFLP on the IGS-rDNA and ndhC-trnV regions in durian DNA from 71 cultivated clones were found to have produced only monomorphic bands, indicating that, no alteration occurred on the pattern of sequences in both regions. Thus, neither genetic distance matrix nor dendrogram tree could be constructed.
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