RESULT AND DISCUSSION 1 Genetic diversity

Bogor, 21-22 October 2015 481 Three SSR primers developed for Shorea curtisii Ujino et al., 1998 were selected and used to analyze genetic diversity of three Shorea species producing tengkawang based on the level of heretozygosity. Those primers were Shc-02, Shc-07, and Shc-09 Table 2. Nurtjahjaningsih et al. 2012 reported the amplification of the three SSR primers for the three Shorea species. Tabel 2: Three 3 Shorea curtisiimicrosatellite primers, its motifs and primer sequences used in this study Primer Repeat motif Primer sequence 5′→3′ Shc-02 CT 2 CACT 5 F: CACGC TTTCC CAATC TG R: TCAAGA GCAGA ATCCA G Shc-07 CT 8 CACT 5 CACCCCTCA 3 CTCA 10 F: ATGTC CATGT TTGAG TG R: CATGG ACATA AGTGG AG Shc-09 CT 12 F: TTTCT GTATC CGTGT GTTG R: GCGATT AAGCG GACCT CAG Analysis of molecular variance AMOVA was used to partition the total genetic variance into components due to differences between regions, populations, and individuals. That was calculated using the software POPGENE 1.32 Yeh et al., 2000 and GenAlEx 6.5 Peakall Smouse, 2012. 3. RESULT AND DISCUSSION 3.1 Genetic diversity Number of alleles of each population was varied between 16 to 23 for the three loci. Shorea stenoptera from Ketapang has the higest number of alleles 23, and the lowest was belong to S. macrophylla from Haurbentes 16. However, S. macrophylla from Haurbentes has the highest number of alleles for Shc-02 primer. Number of haplotypes of each population also revealed the similar condition with the number of alleles. Shorea stenoptera from Ketapang has the higest number of haplotypes 96, however, the lowest number of haplotypes was belong to S. stenoptera from Sintang 59 Table 1. The three SSR primer produced clear bands in electrophoresis using agarose gel. Numbers of alleles of the 3 primers varied between 10 and 15. Total of 36 alleles were obtained from the four SSR primers, and used to analyze genetic diversity of the six populations. Based on the 36 alleles, expected heterozygosity of the six populations was between 0.5838 and 0.8246. The highest genetic diversity was S. stenoptera from Ketapang 0.8246 followed by S. pinanga from Sanggau 0.7752 Table 3. The smallest genetic diversity was S. macrophylla from Haurbentes 0.5838. Genetic diversity of 5 populations in West Kalimantan were higher than the samples of each species those reported by Nurtjahjaningsih et al. 2012. Based on the analyzed data, mean expected heterozygosity of the 6 population He was 0.7266, varied between 0.5838 and 0.8246. The value of this He was quite high. The result showed that genetic diversity of Shorea producing tengkawang in the natural forest especially the 6 populations was still high. The mean genetic diversity of the three Shorea species was higher than S. laevis from Kalimantan 0.4443; Mulyadiana et al., 2010 and similar with natural population of S. leprosula 0.733; Sulistyawati et al., 2014. The collected samples from each population also represented the genetic diversity of the original population. The wildlings collected from each population is was predicted from random mating among mother trees in the each population. Shorea macrophylla from Haurbentes, West Java, was a plantation. According to the lowest genetic diversity compared with the other 5 population from West Bogor, 21-22 October 2015 482 kalimantan, it is most likely that the genetic materials planted in Haurbentes was collected from one population with limited number of mother trees. Table 3: Genetic diversity parameters of the six populations Population N A ne H O H E S. stenoptera Ketapang 20 27 5.09 0.6316 0.8246 S. pinanga Sanggau 20 26 4.24 0.8167 0.7752 S. stenoptera Sanggau 20 35 3.44 0.7833 0.7269 S. macrophylla Sanggau 21 30 3.38 0.7778 0.7089 S. stenoptera Sintang 19 25 3.60 0.8070 0.7402 S. macrophylla Haurbentes 20 23 2.65 0.6500 0.5838 N: sample size, A: number of detected alleles, ne = Mean effective number of alleles [Kimura Crow 1964], H O : observed heterozygosity, H E : expected heterozygosity 3.2 Genetic relationship between populations In order to clarify the genetic relationship among population, AMOVA and a UPGMA dendrogram were constructed from the genetic distances data. Genetic distances among regions and among populations were 0 and 9, respectively Table 4; Fig. 1. The high variation was found within individu 91. No genetic distance was found among regions. In this study, the 6 populations were divided into 4 regions, namely Ketapang, Sanggau, Sintang and Haurbentes. However, genetic distance between populations was 9. This result describe higher effect of species rater than location. For example, Sanggau as one region was consisted of 3 different species. High variation within individual trees revealed that each species in their population has different genetic structure, and the individual trees were produced by random mating. Table 4: AMOVA of the six Shorea populations Source df SS MS Among Regions 3 17.025 5.675 Among Populations 2 11.117 5.558 9 Among Individu 114 121.725 1.068 Within Individu 120 134.000 1.117 91 Total 239 283.867 100 Figure 1: Percentages of Molecular Variance Among Regions Among Pops 9 Among Indiv Within Indiv 91 Percentages of Molecular Variance Bogor, 21-22 October 2015 483 According to the dendrogram, two distinct clusters were identified. The first cluster comprised S. stenoptera Ketapang and S. pinanga Sanggau, and the second cluster comprised the remaining 4 populations [S. stenoptera Sanggau, S. macrophylla Sanggau, S. stenoptera Sintang, S. macrophylla Haurbentes] Figure 2. Grouping of the 6 populations did not reveal relation with geographic distance and species. Geographically closed population and the same species did not cluster in the same grup. Higher variability of microsatellite marker can affect the clustering of populations based on geographic distance. Microsatellite marker is not a gene, thus the mutation is faster than gene markers. Figure 2: Dendrogram of genetic relationship between six populations of three Shorea species based on UPGMA Nei, 1978 3.3 Recommendation for ex-situ conservation plot Information of genetic diversity and distribution, also genetic relationship between populations is important to develop strategy of conservation and breeding program, especially for establishing ex-situ conservation plot of Shorea producing tengkawang. From six populations used in this study, diversity of three Shorea species producing tengkawangis still high; meaning that the species can be conserved genetically and it is possible to improve them to obtain better individual trees. Analyzing of genetic diversity of each species and population thathave been planted in ex-situ conservation plot was done in order to collect information and evaluate the genetic diversity of the plot. This information was important to evaluate the materials collected and planted in the ex-situ conservation plot to determine the adequacy of genetic diversity, or whether it representsthe genetic diversity of its original population. Based on this study, the information of genetic diversity of the plot are as follows: 1. Genetic diversity of the six population was relatively high 2. SSR alleles of the three primers in the six populations are evently distributed and no specific allel was found in all populations. S. stenoptera Ketapang S. pinanga Sanggau S. stenoptera Sanggau S. macrophylla Sanggau S. stenoptera Sintang S. macrophylla Haurbentes Bogor, 21-22 October 2015 484 According to the genetic diversity information obtained from this study, some recommendation can be proposed for ex-situ conservation plot of Shorea species producing tengkawang in KRUS as the following: 1. Increase number of population, especially for S. pinanga. 2. Collect more number of genetic materials from other populations. If possible, the materials are collected when high flowering season of each species. The materials are collected for minimal of 20 mother trees, and each mother tree is represented by 10-20 wildlings or seeds. 3. Maintain the remaining individual tree that is still survived as maximum as possible.

4. CONCLUSION