by: Prof. Dr. Zaeer--udud--dinZaeer din Khan && Ms. Zaib un Nisa What is DNA Barcoding?Barcoding ? Large Scale High Throughput Standardized Approach toto identifying species using aashortshort fragment ofof their DNA Introduction The use ofof nucleotide sequence variations toto investigate evolutionary relationships isis not aa new conceptconcept.. Carl Woese used sequence differences inin ribosomal RNA ((rRNArRNA)) toto discover archaeaarchaea,, which inin turn ledled toto thethe redrawing ofof thethe evolutionary tree, and molecular markers (e(e ..gg..,, allozymes ,, rDNA ,, and mtDNAvage )) have been successfully used inin molecular systematics forfor decadesdecades.. DNA barcoding provides aastandardisedstandardised method forfor this process viavia thethe use ofof aashortshort DNA sequence from aa particular region ofof thethe genome toto provide aa'barcode''barcode' forfor identifying speciesspecies.. In 2003, Paul D.N. Hebert from the University of Guelph, Ontario, Canada, proposed the compilation of a public library of DNA barcodes that would be linked to named specimens. This library would “provide a new master key for identifying species, one whose power will rise with increased taxon coverage and with faster, cheaper sequencing”sequencing”. Selection of a Plant Barcode From 77plastidplastid loci, 33werewere shortshort--listedlisted rbcL easy toto use, but modest discriminatory power matK higher discrimination and coding (close toto COCO11),), lower universality trnH --psbA good universality, higher discrimination, but length variable and frequent termination ofof sequencing reads byby SSRs Selecting aabarcodebarcode from these loci was aacloseclose call, and there isis nono perfect solutionsolution.. Majority recommendation ofof aa corecore--barcodebarcode ofof two coding genesgenes::rbcLrbcL ++matKmatK Conti…… “The Executive Committee therefore concludes that only rbcL and matK are approved and required barcode regions for land plants.” However, the Executive Committee accepted the review panel’s recommendation to reassess the situation in 18 months. The current inability of the proposed plant barcode to resolve more than 70 % of species indicates that improvement in the approach is needed, along with more rbcL and matK data. A reassessment in 18 months would evaluate progress being made on matK primers and sequences assembly techniques for non-coding regions such as trnH-psbA” Objectives The present study will lead to : the compilation of molecular data on hundred plants of GCU Botanic Garden that will facilitate as an additional way in the rapid and accurate identification of the plant species. The registration of the gene pool of pakistani plants into the World’s gene bank. Provide a data that may help the research workers in the identification of different plant life stages, e.g. seeds and seedlings, fragments of plant material for forensic investigation, verification of herbal medicines/ food stuffs, bio-security and trade in controlled species, inventory and ecological surveys, etc.. Above mentioned objectives have been devised by doing extensive literature survey viz., Conti…… Ragupathy et al. (2009) carried out a research to “bring together traditional aboriginal knowledge (TK) and scientific knowledge (SK) to explore the relationship between scientific and aboriginal systems of botanical classification and the corresponding valorization(s) of biological diversity in the Western Ghats of southern India. They worked with two aboriginal cultures namely ‘Irulas’ and ‘Malasars’ of the Nilgiri Biosphere Reserve with an objective of evaluating the ability of different knowledge systems (SK and TK) to distinguish grass species belonging to the genus Tripogon, and assess the ability of DNA barcoding to discriminate a new cryptic species ‘Tripogon cope’ as deciphered by the hill tribes. They discovered that the aboriginal informants identified a common ethnotaxa ‘Sunai pul’, which is a cryptic species of grass not recognized by the SK classification. • Wood and Nakazato (2009) examined patterns of morphological and genetic differentiation and cross ability in the Giliopsis group of Ipomopsis (Polemoniaceae). Analysis of phenotypic variation established that the three species two perennials, I. guttata and I. tenuifolia, and one annual, I. effusa are distinct for floral characters and this differentiation is maintained in genealogical relationships with AFLPs . It means that plant species, as defined by morphological characters are often not genomically cohesive. Kress et al . (2009) applied barcode of three-locus DNA to different 296 woody plant trees’ shrubs and palm plants in Barro Colorado Island (BCI), Panama within the 50-hacters Forest Dynamics plot. More than 98% correct identifications were observed. The barcode data obtained by this study was enough to establish a phylogenetic relationship amoung the plant taxa present in the plat. These results illustrated that the phylogenies based on the DNA Barcodes sequences would enhance research on the interface between community ecology and evolution Methodology The technique, generally used by many plant scientists in the field of molecular taxonomy as an identification tool was employed in the present study for the DNA Barcoding of the plants collected from GCU Botanic Garden, Lahore. The plan of work used in the present study was as follows: Field Trips ofof GCU Botanic Garden, Lahore Collection ofof Plants Preservation ofof Plants Voucher Specimens Plant Tissue Sampling DNA Barcoding technique Field Trips of GCU Botanic Garden Lahore For the collection of the plants, botanic garden trips were arranged to the Botanic Garden GCU Lahore. As planning a field trip for the study and collection of flora of Botanic Garden GCU Lahore the following items were found necessary for plant collection . •Notebook for recording the Data ( date of collection, habitat, GPS, elevation etc.) in the field. •Air tight plastic bags along with silica gel made excellent containers for fresh specimen, i.e. leaves for DNA extraction and keeping plants fresh for a long time when tightly closed. •A sharp cutter to cut the required plant sample from the source plant in the field. Collection of Plants During thethe field trip, thetheplant with allall ofof itsitsdifferent important parts was collected..Few collected Few leaves ofofthat plant sample were preserved inin thethe airair tight plastic bags along with small amount ofof silica gelgel .. Each specimen was numbered asas itit was collected and thetheGPS reading and other notes were entered inin thethenotebook.. notebook The listlist ofofcollected plants with their proper binomials, author citation, family name and voucher numbers isis attached inin Annexure 11.. Preservation of Plants The plants collected were preserved byby using thethe following techniques, forfor future examinationexamination:: AA--PressingPressing ofof Plant Specimens Plants initially collected from thethe field were placed inin thethe polythene bags oror metallic vasculum and then transferred toto aastandardstandard plant press prior toto dryingdrying.. Each specimen was carefully displayed onon thethe paper sheet (newspaper oror blotting paper) exposing it’sit’s allall thethe parts after avoiding folding oror hiding ofof partsparts.. These paper sheets with plant specimens were placed one over another and then tightly sandwitched inin aa plant presspress.. BB--DryingDrying ofofPlant Specimens For drying the press containing the specimens was placed in the sun. The press was opened after 24 hours. The specimens were rearranged by changing/replacing newspaper or blotting papers and tightly sandwitching again, until the complete dryness of specimens. CC--MountingMountingand Photography ofofPlant Specimens After drying the plant specimens were mounted on herbarium sheets of standard size, i.e. 11.5 wide X 16.5 long, along with a documentation label glued to the lower right corner of the sheet. The purpose of mounting was to neatly and uniformly spread and fix a plant specimen on the sheet so that it’s all parts were easily exposed and thus accessible for the morphological studies. The plant specimen was pasted with standard white tehglue teesh no tehsheet.. lgeu no Forteh pasting, altpn ,gstnaipplant teh specimen was inverted and no laid swenprepanewspaper.. adli no The glue was then gentlyteh applied ratps all noteh ppaleidalleht paltn no fo ratpsplant eht fo ybspecimen cespmneia a ybmlalssmall paint brushbrush.. The plant specimen was slowlyyb lifted sdnahhands lfitde yb ororteh with lehp tihwhelp teh ofof forceps andteh placed rraebhmui no tehcalpedherbarium no sheet inin desired position .. After teh pasting nigstap ltpnsateh noltpnsa teh teseshteh tehno tesesh teh photography ofof allall those plants was accomplished thiseb will efhpullhelpful iwll eb later teh inin providing lctaau drvipoignactual teh image ofof that specimen inin online data system ofof Botanic GardenGarden.. Voucher Numbers The properly pressed and mounted plant specimens on the herbarium sheets were deposited in Dr. Sultan Ahmad Herbarium, GC University, Lahore as voucher specimen after alloting the voucher numbers Plant Tissue Sampling The Canadian Centre of DNA Barcoding provided the boxes for the dry plant tissues to be used directly for the semi-automated DNA extraction (Ivanova et al., 2007). They were arranged in strips of 8 tubes with strip caps, plate format friendly. Prior to sampling the tubes were labelled by marker. The dry plant tissue (freshly dried in silica-gel)