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2.1. XSPECIES METHOD FOR COMPARATIVE GENOMIC
The microarray GeneChip technology was used for transcriptomics studies on ginseng and rubber including both different source of bio-material origins and comparing different growth stages through the Xspecies method. Hammond et al. (2005) described the Xspecies method as a useful method to explore genomic DNA hybridization on species that have no GeneChip designed for them, using the Affymetrix ATH1 GeneChip which was initially developed for Arabidopsis thaliana. In other words, the Xspecies method will be implemented in this project as a microarray GeneChip technology for gene expression profiling and comparative genomics in ginseng and rubber.
The earliest Xspecies work was begun using Brassica oleracea where there was no Affymetrix GeneChip intended for this species. The genomic DNA taken from Brassica oleracea was hybridized onto the ATH1-121501 GeneChip array and used as a probe filter. A high number of genes were subsequently identified as important under nutrient stress in comparison to Arabidopsis thaliana as described by Hammond et al., 2005. Following that, several attempts to study the transcriptomics of species that had no specific GeneChip designed for them using this Xspecies approach were successful (Broadley et al. 2008; Davey et al. 2009; Graham et al. 2009).
The Xspecies approach is applied in this experiment as a development of a cross-species tool for assessing the phylogenetic relationship between ginseng and rubber along with other plant species that have been recorded in the Nottingham Arabidopsis Stock Centre, University of Nottingham. Therefore, this method can derive information on great numbers of genes and their functions, which will provide valuable information to increase the commercial crop's quality and yield production.
The ATH1 Affymetrix GeneChip will be used to generate transcriptomics information for heterologous plant species. Genomic DNA (gDNA) hybridized to the chosen Affymetrix ATH1 GeneChip will determine the downstream success of derived transcriptomics results using the Xspecies method.
2.2. THE MECHANISM OF THE XSPECIES METHOD
The Affymetrix ATH1 GeneChip will be applied to investigate transcriptomics data for heterologous species involving ginseng and rubber in this experiment. The first step in Xspecies analysis is to hybridize genomic DNA from the samples to Affymetrix ATH1 GeneChip. Then, the GeneChip will be scanned to produce a .CEL file (Cell Intensity File). The .CEL file will be made through command console using Affymetrix software which stores the result of the strength values on the pixel intensity which is directly derived from the .DAT file (Data file). Therefore, a sole representative intensity value is collected per feature of the image.
To find out which probes are present in the plant species, a .CEL parser script in the PERL programming language will take a .CDF (Chip Definition File) file and convert it into a new Xspecies .CDF file (NASC's International Affymetrix Service. URL: http://affy.arabidopsis.info/xspecies). The .CDF file contains information on each gene arranged on the GeneChip array and details which probes belong to which probe-set and gene.
The PERL script will select the probe pairs with positive hybridisation to the genomic DNA based on the signal intensities generated in genomic DNA-Affymetrix GeneChip hybridizations. The PERL script is a programme in which probe masking is used to remove probe-pairs whose signal intensities is too low from the threshold level set by user. This step would retain the probe-pairs in which the perfect-match probe has a gDNA hybridization intensity greater than the defined threshold (Hammond et al., 2005).
After that, the new Xspecies .CDF file, which has the retained probe-pairs will be used for the rest of the future RNA hybridizations in this experiment for the transcriptomics study. Such that, only probe-pairs originated from each probe-set on the Affymetrix ATH1 GeneChip array that produced good hybridization of genomic DNA will be used in further genetic analysis.
2.3. BACKGROUND AND USES OF GINSENG
Ginseng was first discovered in China thousands of years ago as a food source. The root was also consumed since it was thought to create euphoria, combat fatigue and promote life extension (Wang et al., 2010). Because the plant's root growth resembles a human form, the name "ren shen" was given to it, which means "man" and "herb" in Chinese words. The benefits of ginseng have been recorded throughout Chinese history for medicinal purposes (Wu and Zhong, 1999). Kerns (2009) has mentioned that Korean ginseng is widely distributed in China and Korea regions and generally referred to as Red Panax and White Ginseng in these regions. Meanwhile, American ginseng is native to Eastern North America where it inhabits a range from Southern Ontario to Georgia and stretches as far as Wisconsin.Â Currently, most of the Asian and American ginseng in the market has been cultivated in farms rather than harvested from the wild.
2.3.2. SPECIES INFORMATION
Ginseng such as Korean and American Ginseng belong to the Araliaceae family. The root of ginseng contains components identified as ginsenosides that are widely presumed to be responsible for its medicinal properties (Yap et al., 2008). Ginsenosides are known to be the major active ingredient of Asian ginseng (Korean ginseng) and American ginseng (Ha et al., 2002; Kwok et al., 2010). However, the ginsenocide content of different ginseng species can vary depending on numerous aspects such as the age of the sample, part of the plant, preservation methods, harvesting period and ginsenocide extraction method (Ha et al., 2002; Kennedy and Scholey, 2003).
Ginseng root extract and other cellular forms could be used as antioxidants and delay cell death as reported by Naval et al. (2007). The most widely use ginseng roots in traditional Chinese medicines at present are American ginseng (Panax quinquefolius) and Korean ginseng (Panax ginseng) as reported by Schlag and McIntosh (2006). Sometimes, Korean ginseng is also referred as Asian ginseng in China and many Asian countries (Chen et al., 2010; Li et al., 2010).
2.3.3. USES OF GINSENG
Ginseng root is usually processed into dried form and is used to make tablets, extracts, and teas, as well as for external use. The potential of ginseng to generate positive effects on cognitive function and general well-being have been reported through observations on cardiovascular and haemorrheological (the study of blood flows) effects by Kennedy and Scholey (2003). Meanwhile, Siberian ginseng (Eleutherococcus senticosus) has been promoted as a cheaper alternative yet is claimed to have similar benefits to Asian and American ginseng. However, Siberian ginseng does not contain ginsenosides which is the active ingredients found inÂ Korean and American ginseng. Siberian ginseng is not considered to be one of the true ginseng species by many plant scientists although it comes from the same Araliaceae family as Korean and American ginseng (Kennedy and Scholey, 2003; Kerns, 2009).
Many reports on the uses of ginseng in medicinal purposes in Asian region due to their alleged benefits in general health enhancement, vitality, anti-fatigue, regeneration of the homeostasis system, wound healing etc. have been accredited primarily to ginsenosides. (Schlag and McIntosh, 2006; Naval et al., 2007; Oliver Chen et al., 2009; Wang et al., 2010). Strong evidence of the medicinal effects of ginseng were largely related to its protective properties (ginsenosides) as a counter to free radicals. Recent research suggested that Panax ginseng extracts might shield neuronal cells from oxidative damage and supports neuron survival. It could be concluded that ginseng is capable of combating severe oxidant stress (Naval et al., 2007). Ginseng products that are purchased by consumers might contain little or no detectable ginsenosides if they come from Siberian ginseng, which is not considered as a real ginseng species. Therefore, false marketing information on these products needs to be highlighted since true ginseng only refers to Panax such as Korean and American ginseng (Ha et al. 2002).
Product forensics is a vital point of regulation to ensure the authenticity of food products that are sold at a premium price in the market as described by Spaniolas et al. (2006). In this study, the Xspecies technique is applied to detect product authentication using the Affymetrix Microarray GeneChip, especially on Siberian ginseng and its relationship compared to other ginseng species such as Korean and American ginseng.
2.4. BACKGROUND AND USES OF RUBBER
The first Mokaya culture (Descendant of Maya civilization) in Mexico is believed to have had a rubber ball court established in 1800 B.C. Furthermore, in the sixth century there were Mesoamerican indigenous people in South America that had made a wide range of toys and domestic items from fabric and natural rubber mixtures mostly for ritual sacrifice. Eventually, it is strongly believed that rubber went to Europe by French entrepreneurs during the 17th century even though Columbus brought a rubber ball to Seville, Spain during his explorations in 1523. The first patented process for curing rubber by Thomas Hancock on Goodyear's vulcanized rubber in the 18th century marked the beginning of the booming rubber industry (Loadman, 2005).
Eventually, Sir Henry Wickham from United Kingdom introduced the rubber tree to Eastern regions in 1877. From 2000 seedlings dispatched to Sri Lanka, only twenty two seedlings were further shipped from there to Singapore and Malaya. Since then, the Asian rubber industry expanded from this small amount of seedlings which comprised the whole genetic source in the Asian region (Malaysian Rubber Board, http://lgm.gov.my; Masahuling et al., 2007).
2.4.2. SPECIES INFORMATION
The rubber tree or Hevea brasiliensis belongs to the family Euphorbiaceae and originated in South America primarily in the Amazon rainforest, but it is currently mainly cultivated in South East Asia. Natural rubber is obtained from the latex produced by the Hevea tree in specialized cells called laticifers or latex vessels (Priya et al., 2006). Hevea brasiliensis is the only plant species being cultivated for commercial production of rubber in the world even though there are many closely related wild relatives. In order to achieve ever-increasing rubber demand, it is necessary to identify and select promising characteristics involved in the rubber biosynthesis by many plant breeders in different regions as mentioned by Chotigeat et al. (2009).
2.4.3. USES OF RUBBER
The use of natural rubber is prevalent ranging from domestic to industrial products, covering the production stream as the intermediate material or as final rubber products. Natural rubber has great resilience and tensile strength as well as low heat swelling during manufacturing processes. Recently, protein-carbohydrate interaction and biochemical study in the rubber tree strongly indicate a specific biological characteristic for the latex properties that will affect the quality of end products (van Beilan and Poirier, 2007; Wititsuwannakul et al., 2008)
Nevertheless, natural rubber produces latex that has the excellent capability to stick to itself and to other materials that makes it the best choice for pressure-sensitive adhesives, and has exceptional water resistance in many end products. The future trend in the rubber industry is that natural rubber producing would be concentrated on environmental friendly products like bio-degradable gloves with low protein rubber latex to address the protein allergy issue; carbon sequestration towards reducing global warming; and sustainable yield improvement and agro-ecological plantation management; even exploitation as bio-diesel fuel to replace fossil fuel (Edwin Geo et al., 2008; Malaysian Rubber Board, http://www.lgm.gov.my).
The rubber tree produces its latex by a specialized secretory system in latex vessels distributed throughout the phloem and harvested through minor cuts or tapping of its bark layers (Yoonram et al., 2008). The most important feature in the rubber tree is that the latex is a renewable resource that fit perfectly into the purpose of sustainable agriculture. Rubber is highly water resistant, electrically non-conductive and durable in most of its end products due to the unique molecular structure of rubber (Wititsuwannakul et al., 2008).
Therefore, the expression of genes and transcriptomics in the rubber tree are of interest to plant breeders who would like to advance the yield of the rubber tree. In this experiment, the findings concentrate on phylogenetic relationships between rubber trees as a commercial plant and other high commercial value plant species like ginseng using Affymetrix GeneChip arrays. This study will provide an early proof of concept of transcriptomics in rubber that will explore its genes and most of the molecular functions involved in leaves particularly during different growing stages.
2.5. XSPECIES ANALYSIS AND PHYLOGENETIC RELATIONSHIP
Affymetrix GeneChips allow a very large number of genes and their products e.g. mRNA to be assessed together as a whole. It is a very useful tool to investigate the genome in a systematic way to survey DNA and RNA distinctions. GeneChips by Affymetrix technology are a form of DNA microarray. These GeneChips comprise numerous 25 base pair gene fragments (probes) per gene attached in an array form (Hammond et al., 2005)
By using a quartz wafer, oligonucleotide arrays are printed through photolithography and combinatorial chemistry. The wafer is washed with a blocking complex that is removed by exposure to light beams in a progressive pattern. The design step is to use a 'mask' with windows that allows light to pass through where a particular nucleotide is identified. Then, the wafer is splashed with a fluid of the targeted nucleotides, which causes the nucleotide to attach to the extending probes. Finally, the array is re-blocked and exposed to a fresh 'mask' and followed by a progressive toting up of new nucleotides. This procedure is recurrent until probes are completed (Schena, 2003; DKFZ. German Cancer Research Centre. URL: http://www.dkfz.de/gpcf/24.html)
At this moment, there are no GeneChip arrays available for analyzing genomics and transcriptomics for ginseng or rubber. According to Broadley et al. (2008), the ATH1 GeneChip is effective in cross-taxa transcriptomics analysis through a selective focus on genomic DNA-based masking and normalization to eliminate the effects of DNA polymorphisms. Arabidopsis thaliana as popular model plant has been fully sequenced (http://www.affymetrix.com) and established for the fabrication of custom microarrays such as the ATH1 GeneChip manufactured by Affymetrix, USA. Therefore, the Affymetrix ATH1 GeneChip designed for Arabidopsis thaliana will be used to investigate both genomics and transcriptomics in ginseng and rubber.
GeneChip microarray analysis varies from other conventional transcript analysis methods in several ways, one of which is the volume of time required and the quantity of data attained in the experiment (Schena, 2003). Identification of oligonucleotides on a microarray derived from Arabidopsis thaliana can be applied to analyze and identify a corresponding nucleotide sequence in ginseng and rubber despite being a different species to Arabidopsis. The Affymetrix ATH1 GeneChip provides a platform to explore gene and transcriptomics functions that will have crucial effects on other plants through homologous gene study (Boyle et al., 2001; Bevan et al., 2001).
It is generally accepted that the variation that occurs in the great quantity of transcripts among individuals within a population will be the result of divergence mainly through phenotypic selection. However, stochastic processes that result in a neutral genome evolution for most of the transcript abundance differences among individuals are selectively neutral according to Broadley et al. (2008). It is further reported that variation in the expression of a transcript among individuals as a consequence of drift will steer expression divergence among populations and taxa in a linear fashion. Therefore, similarities and differences at the gene and transcript level among the ginseng species and rubber could be used to gather phylogenetic relationships as well as for species authentication investigation in ginseng.
A phylogenetic tree is a straightforward way to illustrate an evolutionary relationship where species are represented by nodes and lines of descent are represented by links. Commonly these are demonstrated using cladograms in which no ancestral nodes have more than two branches (Chen et al., 2009). Genes that encode messenger RNA (mRNA) could be assayed to classify an organism's taxonomic group, estimate linked groups and assessment rates of species divergence. This matter can be achieved by using Affymetrix ATH1 GeneChips via the Xspecies method.
This experiment will explore the evolutionary relationships between ginseng species as well as the authentication of ginseng through food forensics. It will also draw on and contribute to the current literature with regard to previous phylogenetic studies on Arabidopsis, Brassicaceae, Musa spp, Aloe spp, Oil Palm and others, using Affymetrix GeneChips, where no specific GeneChips were designed for them. The investigations on both genomic and transcriptomic divergence of ginseng will be supplementary to an existing phylogenetics project of comparisons between numerous plant species as mentioned earlier.