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Random Amplified Polymorphic DNA (RAPD) Analysis of Agarwood

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1.0 Introduction

Agarwood is considered as the most expensive wood in the world which can be derived mostly from genera Aquilaria and Gyrinops from the family Thymelaeaceae (Akter, Tanvir, Zulkefeli, & Khan, 2013). According to Subasinghe, Hettiarachchi, and Rathnamalala (2012), some of the species from the Aquilaria and Gyrinops can be found across at least 12 countries including Bangladesh, Bhutan, Cambodia, India, Indonesia, Lao PDR, Malaysia, Myanmar, Philippines, Thailand, Vietnam and Papua New Guinea. Agarwood is the dark resinous heartwood that can be derived from wounded trees of Aquilaria species (Akter et al., 2013). The dark resinous organic compound is actually one type of plant secondary metabolite produced by the heartwood as a plant self-defensive mechanism towards physical or chemical damages to the trees (Blanchette, 2003). In terms of its uses, agarwood can be made into incenses, fragrances, aromatherapy, medicines, and even used in religious ceremonies (Liu et al., 2013).

However, the supply rates of agarwood are only 40% out of the total demands (Gratzfeld & Tan, 2008). Even worst, many naturally grown agarwood have been exploited and traded for own benefits by harvesting the wild agarwood illegally. Agarwood can be produced in natural ways by lightning strike, insects attack, and some other possible wounding that happens in natural ways. Artificial wounds like bark removal, nailing, and many other methods can also induce agarwood formation but each has different rate of success (Pojanagaroon & Kaewrak, 2006). These old inducing methods produce agarwood with low yield after a long period of time and the resin produced often showed inferior quality (Liu et al., 2013). Over time, a more productive way of producing agarwood by which fungi inoculation method is being proposed (Persoon, 2007). Fungi inoculation method enables the production of good quality agarwood and it is one of the best methods to induce resin production (Persoon, 2007).

Prior to fungal induction method, it is important to isolate and study the fungus communities that can cause wounding to the trees before the fungus consortium can be formulated as inoculant media in inducing agarwood formation. Agarwood can be extracted and peeled off the tree from its barks, roots, leaves of the infected tree. The chosen infected part must be dark brown or black color as it possibly indicates the presence of resinous compound. This can be the most challenging part as some of the darken part may only represent the rotten wood.

After isolating all the fungus species, a molecular marker known as random amplified polymorphic DNA (RAPD) can be adopted for genetic diversity studies (Kumar & Gurusubramanian, 2011). RAPD technology is a type of polymerase chain reaction (PCR) because it involves the use of PCR to amplify nanogram amounts of the DNA by using single arbitrary oligonucleotide primer that usually made up of 10 bases long (Hadrys, Balick, & Schierwater, 1992). RAPD technology can be used to reveal the polymorphism of unknown samples because the segments of DNA are amplified at random (Kumar & Gurusubramanian, 2011).

According to Bielikova, Landa, Osborne, and ÄŒurn (2002), the application of RAPD markers can be used to provide characterization of the DNA isolated from the studied sample by creating the fingerprints without going through gene sequencing as well as probe hybridization. In addition to that, the RAPD markers generated can be used for the construction of phylogenetic tree that reveals the relatedness and genetic variability of the studied species. Although some other molecular characterization techniques such as amplified fragment length polymorphism (AFLP) and restriction fragment length polymorphism (RFLP) are more sensitive in revealing the DNA polymorphism but RAPD is certainly an easier tool to be used for large sample handling and its relatively low cost involvement has accounted for its great popularity (Bielikova et al., 2002).

In this study, the 17 isolates of fungus which were previously extracted from the resinous stem agarwood have been revived from the stock culture for RAPD analysis by using two different primers (GTG)5 and OPA-14. The main objectives of this project are:

  1. To select the best primer suitable for RAPD-PCR analysis of fungal DNA that gives high level of polymorphic DNA markers;
  2. To optimize the gel electrophoresis conditions;
  3. To analyze the genomic heterogeneity of fungus by construction of phylogenetic tree.

2.0 Literature Review

2.1 Agarwood Production

According to Akter and Neelim (2008), agarwood could be induced by three possibilities, which were through pathological, wounding, or non-pathological processes. However, the exact evidence of the responsibility of each method was not well understood. According to Ng et al. (1997), fungus infection was also responsible for agarwood formation which triggered plant self-defensive mechanism to produce the resinous product. However, fungus infection is not the sole reason for agarwood formation because other environmental factors such as the ecological interactions need to be taken into consideration (Akter & Neelim, 2008). In the study conducted by Akter and Neelim (2008), the age of the tree and genetic variation of Aquilaria could also affect different agarwood production rates. In naturally grown trees, the infected Aquilaria could produce agarwood slowly from the age of 20 years and above. But, the Tropical Rainforest Project (TRP) in Vietnam showed that the agarwood could be formed from a cultivated Aquilaria tree at age of three years (Akter & Neelim, 2008).

2.2 Endophytic Fungus Communities found in Agarwood

Tian et al. (2012) stated that agarwood grown in natural environment was often associated with the exposure to damage or attack from pathogens or insects. The infected Aquilaria host tree would then produce uneven black and fragrant resin to retard the fungi growth. The resinous compound would deposit in the trucks or main branches of the trees (Tian et al., 2012). In order to induce agarwood from the Aquilaria, wild agarwood have been extracted to determine the microorganisms that are responsible to trigger the host trees to produce resin.

According to Bhattacharyya (1952), the fungus isolated from infected Aquilaria agallocha was Epicoccum granulatum and the South China Botanical Garden found that Menanotus flavoliven was responsible to induce agarwood in an infected Aquilaria sinensis. In a study reported in year 2000, two species of fungi namely Fusarium oxysporum and Chaetum globosum had successfully induced agarwood (Tamuli et al., 2000). Later in 2005, Fusarium sp. was again found to be one of the inducer of agarwood formation (Subehan et al., 2005). These fungi were endophytes that lived within plant tissues and their roles in inducing agarwood were differentiated by each of their inoculating effects (Tabata et al., 2003). The presence of endophytes could offer noteworthy benefits to the host plants, as in the case of agarwood formation. These endophytes had successfully triggered the formation of pathological products in diseased trees.

2.3 Principles of the RAPD Technique

According to Kumar and Gurusubramanian (2011), the fundamental working principle of the RAPD-PCR technique depends on the use a single, short, and arbitrary oligonucleotide that will bind to many loci of the chromosome and amplify the segments of DNA randomly from the DNA template. The amplified fragments of DNA will be generated based on the length and size of primer and the target genome. The oligonucleotide primer used is usually 10 bases long and prior knowledge of the DNA sequence of the studied species is not required. Under suitable annealing temperate, the primer will bind to several priming sites on the complementary DNA template to produce the amplified fragments of DNA (Kumar and Gurusubramanian, 2011). The successful amplification of DNA depends on whether these priming sites are within an amplifiable distance to each other and the primers must anneal in the orientation of pointing towards each other (Kumar and Gurusubramanian, 2011). After PCR, the amplified products are separated on the agarose gel for visualization of the RAPD markers and continuous analysis thereafter.

2.4 RAPD Analysis for Identification and Authentication of Genetic Origin

According to Congiu, Chicca, Cella, Rossi, and Bernacchia (2000), random amplified polymorphic DNA (RAPD) technique could be used in identification of different varieties of plants. In the study, RAPD analysis was applied to settle a lawsuit regarding unauthorized commercialization of a patented strawberries variety “Marmolada”. The “Marmolada” strawberries were registered in 1984 by the Consorzio Italiano Vivaisti (CIV) in Italy. Soon after that, farmers that farmed strawberries had been suspected of reproducing the relevant species of “Marmolada” without any permission from the cultivar. The RAPD identification was performed as added information to traditional morphological examination. For crops reproduced by micropropagation, all the individuals came from the same family should have identical genome information. The RAPD was directly applied in strawberries by estimating the genetic distance among the varieties of strawberries obtained from the suspected farm with the “Marmolada” strawberries. At the end of the testing, 13 plants out of 31 total plants were detected similar to the “Marmolada” strawberries by using two different primer sets (Congiu et al., 2000).

In the other study conducted by Matsubara, Shindo, Watanabe, and Ikegami (2013), RAPD analysis had been applied in authentication of a medicinal Angelica acutiloba. Some but not all the species of Angelica are used in medicine, particularly the roots of Angelica acutiloba var. acutiloba known as “Toki” and A. acutiloba var. sugiyamae known as “Hokkai Toki” (Matsubara et al., 2013). The purity of traditional medicine made from roots of angelica had become a question since many of these varieties have outcrossed with each other. The use of RAPD method with decamer primer OPD-15 had been successfully authenticate the “Toki” and “Hokkai Toki” from other Angelica varieties via the analysis of RAPD markers.

2.5 RAPD Analysis for Species Relatedness and Heterogeneity

RAPD analysis was also used to evaluate the genetic variability among the Ponkan mandarin as stated in the study conducted by Coletta Filho, Machado, Targon, and Pompeu (2000). The analysis was conducted based on 19 Ponkan mandarin accessions by using 25 different primers. There were 112 amplified products of which only 32 were polymorphic across the five accessions. In other words, there was no genetic variability found in the other 14 accessions that might result from either clonal propagation of the Ponkan mandarin or under-detected genetic variation.

RAPD analysis was also used to reveal species relatedness and extend of diversity within each type of betel vine cultivars. According to Ranade, Verma, Gupta, and Kumar (2002), two different types of betel vines (Piper betle L.) namely Kapoori and Bangla were used as the test samples. The Kapoori and Bangla betal vines each had been analyze by the RAPD analysis and the results showed that Kapoori cultivars were more heterogenous as compared to Bangla cultivars that were very similar to each other (Ranade et al., 2002). The RAPD profile analysis would tell the relatedness of these two different cultivars whereby the two different cultivars were clearly separated with only six common bands out of a total of 60 bands. The bands produced from the RAPD analysis was also being constructed into a phenogram as a summary of the comparting the species relatedness between the two cultivars.

According to Doherty, Zweifel, Elde, McKone, and Zweifel (2003), RAPD markers were useful to determine the degree of genetic variation within the symbiotic fungus Leucoagaricus gongylophorus associated with the lead-cutting ants Atta cephalotes. The fungi were isolated from ant nests in two different geographical areas, of which two isolates are from Panama and the five isolates from Trinidad. Ten decamer primers were used to amplify the fungus DNA of the seven isolates, and their banding patterns were compared. Pairwise comparisons of the DNA bands on agarose gel had been studied which resulted in 36 % of the bands were shared among the isolates while an average of 72 % was for within sites (Doherty et al., 2003).

Genetic variability among Coleus sp. was also analyzed by RAPD banding pattern analysis as reported in the study conducted by Govarthanan, Guruchandar, Arunapriya, Selvankumar, and Selvam (2011). The RAPD markers were used to identify the variability and find out the best quality of Coleus sp. for human consumption. Coleus sp. is a kind of medicinal plant that plays a role in protecting and preserving human health. In order for its survival in long terms and conservation management, it is important to access the genetic profile. According to Govarthanan et al. (2011), several types of primers from OPW and OPU had been used to determine the molecular variation and similarities among the three species of Coleus amboinicus, Coleus aromaticus and Coleus forskohlii. The variability was then used for a selection program and reliable information can also be deduced from the pattern of the bands.

In a study conducted by Nkongolo (1999), RAPD analysis was used to determine the genetic relationship between the black, red, and white spruce namely Picea mariana, Picea rubens and Picea glauca and to determine the degree of polymorphism within the population when compared to the hybrid spruce. Eleven arbitrary primers were used to amplify the genomic content of spruce extracted from the embryonic cultures and seedlings. From the RAPD profile, Picea mariana and Picea rubens showed high similarity with one another suggesting their close genetic relationship. Besides that, RAPD profile could also be used in displaying the level of polymorphism between the pure and hybrid spruce. According to Nkonglo (1999), the level of polymorphism is greater in the hybrid population when compared to the pure hybrid.


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