In situ and Ex situ Conservation of Commercial Tropical Trees - ITTO

371Materials and MethodsSamplingThis study was conducted in an artificial forest located in the Forest ResearchInstitute Malaysia (FRIM). This 200 ha forest, which is surrounded by primaryand secondary forests, was established in 1927 and it includes D. oblongifoliaand various other dipterocarp and nondipterocarp timber tree species. Thirtynineto 40 open-pollinated seeds were collected from each of eight singleprogeny families using the “shaking-catch” method as described by Lee et al.(2000b).ElectrophoresisEnzyme extraction and starch gel electrophoresis of germinated embryo tissueswas carried out following the procedure described by Lee et al. (2000b). Matingsystem parameters were estimated using four polymorphic allozyme loci (Gpi,Pgm, Me and Ugp) based on their Mendelian segregation patterns. Genotypesof maternal parents were determined directly via electrophoresis of adult innerbark tissue.Genetic AnalysisMating system parameters were estimated by using the MLTR computerprogram of Ritland (1994) for a mixed-mating model. From progeny arraydata and through maximum likelihood procedures, the program simultaneouslyestimated; (1) multilocus population outcrossing rate (t m) by the Newton Raphsonmethod; (2) the average single locus population outcrossing rate (t s): (3) theaverage single locus inbreeding coefficient of maternal parents (F); (4) thepollen and ovule allele frequencies (p and o) by the expectation maximizationmethod; and (5) variances of the above using the bootstrap method where theprogeny array (within families) is the unit of resampling (250 bootstraps wereused). Difference between t mand t s(biparental mating) was also tested usingStudent t-test as t = (D – 0) / S D, where D is the mean difference between t mand t s, and S Das the mean standard deviation. Homogeneity of the pollen poolover all the maternal trees was tested by chi-square distribution statistics as χ 2= NG st(A –1), where N is the total number of pollen gametes, G stis the proportionof among-tree variance in pollen allele frequencies relative to the total variancein pollen allele frequencies, and A is number of alleles at a locus (James et al.