2.1. XSPECIES METHOD FOR COMPARATIVE GENOMIC
The microarray GeneChip engineering was used for transcriptomics surveies on ginseng and gum elastic including both different beginning of bio-material beginnings and comparing different growing phases through the Xspecies method. Hammond et Al. ( 2005 ) described the Xspecies method as a utile method to research genomic DNA hybridisation on species that have no GeneChip designed for them, utilizing the Affymetrix ATH1 GeneChip which was ab initio developed for Arabidopsis thaliana. In other words, the Xspecies method will be implemented in this undertaking as a microarray GeneChip engineering for cistron look profiling and comparative genomics in ginseng and gum elastic.
The earliest Xspecies work was begun utilizing 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 investigation filter. A high figure of cistrons were later identified as of import under alimentary emphasis in comparing to Arabidopsis thaliana as described by Hammond et al. , 2005. Following that, several efforts to analyze the transcriptomics of species that had no specific GeneChip designed for them utilizing this Xspecies approach were successful ( Broadley et al. 2008 ; Davey et al. 2009 ; Graham et Al. 2009 ) .
The Xspecies attack is applied in this experiment as a development of a cross-species tool for measuring the phyletic relationship between ginseng and rubber along with other works species that have been recorded in the Nottingham Arabidopsis Stock Centre, University of Nottingham. Therefore, this method can deduce information on great Numberss of cistrons and their maps, which will supply valuable information to increase the commercial harvest ‘s quality and output production.
The ATH1 Affymetrix GeneChip will be used to bring forth transcriptomics information for heterologic works species. Genomic DNA ( gDNA ) hybridized to the chosen Affymetrix ATH1 GeneChip will find the downstream success of derived transcriptomics consequences utilizing the Xspecies method.
2.2. THE MECHANISM OF THE XSPECIES METHOD
The Affymetrix ATH1 GeneChip will be applied to look into transcriptomics informations for heterologic species affecting ginseng and gum elastic in this experiment. The first measure in Xspecies analysis is to crossbreed genomic Deoxyribonucleic acid from the samples to Affymetrix ATH1 GeneChip. Then, the GeneChip will be scanned to bring forth a.CEL file ( Cell Intensity File ) . The.CEL file will be made through bid console utilizing Affymetrix package which shops the consequence of the strength values on the pel strength which is straight derived from the.DAT file ( Data file ) . Therefore, a exclusive representative strength value is collected per characteristic of the image.
To happen out which investigations are present in the works species, a.CEL parser book in the PERL scheduling linguistic communication will take a.CDF ( Chip Definition File ) file and change over it into a new Xspecies.CDF file ( NASC ‘s International Affymetrix Service. Uniform resource locator: hypertext transfer protocol: //affy.arabidopsis.info/xspecies ) . The.CDF file contains information on each cistron arranged on the GeneChip array and inside informations which probes belong to which probe-set and cistron.
The PERL book will choose the investigation pairs with positive hybridization to the genomic DNA based on the signal strengths generated in genomic DNA-Affymetrix GeneChip hybridisations. The PERL book is a programme in which investigation cover is used to take probe-pairs whose signal strengths is excessively low from the threshold degree set by user. This measure would retain the probe-pairs in which the perfect-match investigation has a gDNA hybridisation strength 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 remainder of the hereafter RNA hybridisations in this experiment for the transcriptomics survey. Such that, merely probe-pairs originated from each probe-set on the Affymetrix ATH1 GeneChip array that produced good hybridisation of genomic DNA will be used in farther familial analysis.
2.3. BACKGROUND AND USES OF GINSENG
Ginseng was foremost discovered in China 1000s of old ages ago as a nutrient beginning. The root was besides consumed since it was thought to make euphory, combat weariness and promote life extension ( Wang et al. , 2010 ) . Because the works ‘s root growing resembles a human signifier, the name “ ren shen ” was given to it, which means “ adult male ” and “ herb ” in Chinese words. The benefits of ginseng have been recorded throughout Chinese history for medicative intents ( Wu and Zhong, 1999 ) . Kerns ( 2009 ) has mentioned that Korean ginseng is widely distributed in China and Korea parts and by and large referred to as Red Panax and White Ginseng in these parts. Meanwhile, American ginseng is native to Eastern North America where it inhabits a scope from Southern Ontario to Georgia and stretches every bit far as Wisconsin.A Currently, most of the Asian and American ginseng in the market has been cultivated in farms instead than harvested from the natural state.
2.3.2. SPECIES INFORMATION
Ginseng such as Korean and American Ginseng belong to the Araliaceae household. The root of ginseng contains constituents identified as ginsenosides that are widely presumed to be responsible for its medicative belongingss ( Yap et al. , 2008 ) . Ginsenosides are known to be the major active ingredient of Asiatic ginseng ( Korean ginseng ) and American ginseng ( Ha et al. , 2002 ; Kwok et al. , 2010 ) . However, the ginsenocide content of different ginseng species can change depending on legion facets such as the age of the sample, portion of the works, saving methods, reaping period and ginsenocide extraction method ( Ha et al. , 2002 ; Kennedy and Scholey, 2003 ) .
Ginseng root infusion and other cellular signifiers could be used as antioxidants and hold cell decease as reported by Naval et Al. ( 2007 ) . The most widely use ginseng roots in traditional Chinese medical specialties at present are American ginseng ( Panax quinquefolius ) and Korean ginseng ( Panax ginseng ) as reported by Schlag and McIntosh ( 2006 ) . Sometimes, Korean ginseng is besides referred as Asiatic ginseng in China and many Asiatic states ( Chen et al. , 2010 ; Li et al. , 2010 ) .
2.3.3. Use OF GINSENG
Ginseng root is normally processed into dried signifier and is used to do tablets, infusions, and teas, every bit good as for external usage. The potency of ginseng to bring forth positive effects on cognitive map and general wellbeing have been reported through observations on cardiovascular and haemorrheological ( the survey of blood flows ) effects by Kennedy and Scholey ( 2003 ) . Meanwhile, Siberian ginseng ( Eleutherococcus senticosus ) has been promoted as a cheaper alternate yet is claimed to hold similar benefits to Asiatic and American ginseng. However, Siberian ginseng does non incorporate ginsenosides which is the active ingredients found inA Korean and American ginseng. Siberian ginseng is non considered to be one of the true ginseng species by many works scientists although it comes from the same Araliaceae household as Korean and American ginseng ( Kennedy and Scholey, 2003 ; Kerns, 2009 ) .
Many studies on the utilizations of ginseng in medicative intents in Asiatic part due to their alleged benefits in general wellness sweetening, verve, anti-fatigue, regeneration of the homeostasis system, wound mending etc. have been accredited chiefly to ginsenosides. ( Schlag and McIntosh, 2006 ; Naval et al. , 2007 ; Oliver Chen et al. , 2009 ; Wang et al. , 2010 ) . Strong grounds of the medicative effects of ginseng were mostly related to its protective belongingss ( ginsenosides ) as a counter to free groups. Recent research suggested that Panax ginseng infusions might screen neural cells from oxidative harm and supports nerve cell endurance. It could be concluded that ginseng is capable of battling terrible oxidant emphasis ( Naval et al. , 2007 ) . Ginseng merchandises that are purchased by consumers might incorporate small or no noticeable ginsenosides if they come from Siberian ginseng, which is non considered as a existent ginseng species. Therefore, false selling information on these merchandises demands to be highlighted since true ginseng merely refers to Panax such as Korean and American ginseng ( Ha et al. 2002 ) .
Merchandise forensics is a critical point of ordinance to guarantee the genuineness of nutrient merchandises that are sold at a premium monetary value in the market as described by Spaniolas et Al. ( 2006 ) . In this survey, the Xspecies technique is applied to observe merchandise hallmark utilizing the Affymetrix Microarray GeneChip, particularly 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 civilization ( Descendant of Maya civilisation ) in Mexico is believed to hold had a rubber ball tribunal established in 1800 B.C. Furthermore, in the 6th century there were Mesoamerican autochthonal people in South America that had made a broad scope of playthings and domestic points from cloth and natural gum elastic mixtures largely for ritual forfeit. Finally, it is strongly believed that gum elastic went to Europe by French enterprisers during the seventeenth century even though Columbus brought a gum elastic ball to Seville, Spain during his geographic expeditions in 1523. The first patented procedure for bring arounding gum elastic by Thomas Hancock on Goodyear ‘s cured gum elastic in the eighteenth century marked the beginning of the flourishing gum elastic industry ( Loadman, 2005 ) .
Finally, Sir Henry Wickham from United Kingdom introduced the gum elastic tree to Eastern parts in 1877. From 2000 seedlings dispatched to Sri Lanka, merely twenty two seedlings were farther shipped from at that place to Singapore and Malaya. Since so, the Asiatic gum elastic industry expanded from this little sum of seedlings which comprised the whole familial beginning in the Asiatic part ( Malayan Rubber Board, hypertext transfer protocol: //lgm.gov.my ; Masahuling et al. , 2007 ) .
2.4.2. SPECIES INFORMATION
The gum elastic tree or Hevea brasiliensis belongs to the household Euphorbiaceae and originated in South America chiefly in the Amazon rain forest, but it is presently chiefly cultivated in South East Asia. Natural gum elastic is obtained from the latex produced by the Hevea tree in specialised cells called laticifers or latex vass ( Priya et al. , 2006 ) . Hevea brasiliensis is the lone works species being cultivated for commercial production of gum elastic in the universe even though there are many closely related wild relations. In order to accomplish ever-increasing gum elastic demand, it is necessary to place and choose promising features involved in the gum elastic biogenesis by many works breeders in different parts as mentioned by Chotigeat et Al. ( 2009 ) .
2.4.3. Use OF RUBBER
The usage of natural gum elastic is prevailing runing from domestic to industrial merchandises, covering the production watercourse as the intermediate stuff or as concluding gum elastic merchandises. Natural gum elastic has great resiliency and tensile strength every bit good as low heat swelling during fabrication procedures. Recently, protein-carbohydrate interaction and biochemical survey in the gum elastic tree strongly indicate a specific biological feature for the latex belongingss that will impact the quality of terminal merchandises ( new wave Beilan and Poirier, 2007 ; Wititsuwannakul et al. , 2008 )
However, natural gum elastic produces latex that has the first-class capableness to lodge to itself and to other stuffs that makes it the best pick for pressure-sensitive adhesives, and has exceeding H2O opposition in many terminal merchandises. The future tendency in the gum elastic industry is that natural gum elastic bring forthing would be concentrated on environmental friendly merchandises like bio-degradable baseball mitts with low protein rubber latex to turn to the protein allergy issue ; carbon segregation towards cut downing planetary heating ; and sustainable output betterment and agro-ecological plantation direction ; even development as bio-diesel fuel to replace fossil fuel ( Edwin Geo et al. , 2008 ; Malayan Rubber Board, hypertext transfer protocol: //www.lgm.gov.my ) .
The gum elastic tree produces its latex by a specialised secretory system in latex vass distributed throughout the bast and harvested through minor cuts or tapping of its bark beds ( Yoonram et al. , 2008 ) . The most of import characteristic in the gum elastic tree is that the latex is a renewable resource that fit absolutely into the intent of sustainable agribusiness. Rubber is extremely H2O resistant, electrically non-conductive and lasting in most of its terminal merchandises due to the alone molecular construction of gum elastic ( Wititsuwannakul et al. , 2008 ) .
Therefore, the look of cistrons and transcriptomics in the gum elastic tree are of involvement to works breeders who would wish to progress the output of the gum elastic tree. In this experiment, the findings concentrate on phyletic relationships between gum elastic trees as a commercial works and other high commercial value works species like ginseng utilizing Affymetrix GeneChip arrays. This survey will supply an early cogent evidence of construct of transcriptomics in gum elastic that will research its cistrons and most of the molecular maps involved in foliages peculiarly during different turning phases.
2.5. XSPECIES ANALYSIS AND PHYLOGENETIC RELATIONSHIP
Affymetrix GeneChips allow a really big figure of cistrons and their merchandises e.g. messenger RNA to be assessed together as a whole. It is a really utile tool to look into the genome in a systematic manner to study DNA and RNA differentiations. GeneChips by Affymetrix engineering are a signifier of DNA microarray. These GeneChips comprise legion 25 base brace cistron fragments ( investigations ) per cistron attached in an array signifier ( Hammond et al. , 2005 )
By utilizing a vitreous silica wafer, oligonucleotide arrays are printed through photolithography and combinative chemical science. The wafer is washed with a barricading composite that is removed by exposure to light beams in a progressive form. The design measure is to utilize a ‘mask ‘ with Windowss that allows visible radiation to go through through where a peculiar base is identified. Then, the wafer is splashed with a fluid of the targeted bases, which causes the base to attach to the widening investigations. Finally, the array is re-blocked and exposed to a fresh ‘mask ‘ and followed by a imperfect luging up of new bases. This process is perennial until investigations are completed ( Schena, 2003 ; DKFZ. German Cancer Research Centre. Uniform resource locator: hypertext transfer protocol: //www.dkfz.de/gpcf/24.html )
At this minute, there are no GeneChip arrays available for analysing genomics and transcriptomics for ginseng or gum elastic. Harmonizing to Broadley et Al. ( 2008 ) , the ATH1 GeneChip is effectual in cross-taxa transcriptomics analysis through a selective focal point on genomic DNA-based cover and standardization to extinguish the effects of DNA polymorphisms. Arabidopsis thaliana as popular theoretical account works has been to the full sequenced ( hypertext transfer protocol: //www.affymetrix.com ) and established for the fiction of usage microarrays such as the ATH1 GeneChip manufactured by Affymetrix, USA. Therefore, the Affymetrix ATH1 GeneChip designed for Arabidopsis thaliana will be used to look into both genomics and transcriptomics in ginseng and gum elastic.
GeneChip microarray analysis varies from other conventional transcript analysis methods in several ways, one of which is the volume of clip required and the measure of informations attained in the experiment ( Schena, 2003 ) . Designation of oligonucleotides on a microarray derived from Arabidopsis thaliana can be applied to analyse and place a corresponding nucleotide sequence in ginseng and rubber despite being a different species to Arabidopsis. The Affymetrix ATH1 GeneChip provides a platform to research cistron and transcriptomics maps that will hold important effects on other workss through homologous cistron survey ( Boyle et al. , 2001 ; Bevan et al. , 2001 ) .
It is by and large accepted that the fluctuation that occurs in the great measure of transcripts among persons within a population will be the consequence of divergency chiefly through phenotypic choice. However, stochastic procedures that result in a impersonal genome development for most of the transcript copiousness differences among persons are selectively impersonal harmonizing to Broadley et Al. ( 2008 ) . It is further reported that fluctuation in the look of a transcript among persons as a effect of impetus will maneuver look divergency among populations and taxa in a additive manner. Therefore, similarities and differences at the cistron and transcript degree among the ginseng species and gum elastic could be used to garner phyletic relationships every bit good as for species authentication probe in ginseng.
A phyletic tree is a straightforward manner to exemplify an evolutionary relationship where species are represented by nodes and lines of descent are represented by links. Normally these are demonstrated utilizing cladograms in which no ancestral nodes have more than two subdivisions ( Chen et al. , 2009 ) . Genes that encode courier RNA ( messenger RNA ) could be assayed to sort an being ‘s systematic group, estimation linked groups and assessment rates of species divergency. This affair can be achieved by utilizing Affymetrix ATH1 GeneChips via the Xspecies method.
This experiment will research the evolutionary relationships between ginseng species every bit good as the hallmark of ginseng through nutrient forensics. It will besides pull on and lend to the current literature with respect to old phyletic surveies on Arabidopsis, Brassicaceae, Musa spp, Aloe spp, Oil Palm and others, utilizing Affymetrix GeneChips, where no specific GeneChips were designed for them. The probes on both genomic and transcriptomic divergency of ginseng will be auxiliary to an bing phylogenetics undertaking of comparings between legion works species as mentioned earlier.