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 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 coverage and with faster, cheaper sequencing”sequencing”. Selection of a 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 .”  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 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 thethefield trip, thetheplant with allall ofof itsitsdifferent important parts was collected..Few collected Fewleaves 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 isisattached inin Annexure 11.. Preservation of Plants

The plants collected were preserved byby using thethe following techniques, forfor future examinationexamination::

AA--PressingPressing ofofPlant 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 glue onon thethe sheetsheet..For pasting, thethe plant specimen was inverted and laid onon newspapernewspaper.. The glue was then gently applied onon allall thethe parts ofof thethe plant specimen byby aa small paint brushbrush.. The plant specimen was slowly lifted byby hands oror with thethe help ofofforceps and placed onon thethe herbarium sheet inin desired position .. After pasting thethe plants onon thethe sheets thethe photography ofof allall those plants was accomplished this will bebe helpful later inin providing thethe actual image ofofthat specimen inin online data system ofofBotanic 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) were very fragile, affected by static electricity. To avoid cross- contamination during sampling it was recommend to follow the following techniques. DA Barcoding Procedures: Numerous published methods andandcommercial kits areareavailable forfor tissue lysis andandDNA extractionextraction..Any method that yields quality DNA isis suitable forforPCR andandthus sequencing isis potentially acceptable.. acceptable InIn thetheappendix wewedescribed four methods forforDNA extractionextraction::

1.1. DD Neasy Blood && Tissue Kit Qiagen Catalog NoNo .. 69504 ((5050 )) oror6950669506 ((250250))(U(U..Guelph)Guelph).. 2.2.AutomatedAutomatedDNA extraction ononBeckman/CoulterBeckman/CoulterBiomek FXFX (U(U..Guelph)Guelph).. 3.3.BioSprintBioSprint 9696DNADNA Blood Kit ((QiagenQiagen))(Smithsonian(SmithsonianInst..)).. Inst 4.4.AutoGenPrepAutoGenPrep 965 (Smithsonian Inst..)) Inst 5.5.TheTheabove mentioned methods arearealready being evaluated byby thetheconcerned laboratories andand arearefound acceptable forfor thethe production ofofDNA,DNA, ii..ee..suitable forforPCR andandDNA sequencingsequencing.. Plant Tissue Sample Results and Discussion

One hundred plant species of GCU Botanic Garden were analysed for their DNA Barcoding, using automated DNA Extraction on Beckman/Coulter Biomek FX (U.Guelph). The results are presented in Table 1 and Fig.1.

Table: 1 Phylogenetic analysis of different species with their possible distance to nearest neighbor

Distance Summary ofof 435 Sequences According totorbcla GeneGene:: Table: Comparison on the basis of Base Pair ((bpbp)) distances from class to species level

umber of Taxa Minimum Mean Distance Maximum Distance sequences Comparisons Distance (%) Distance(%) (%) (%) Within 426 92 859 0 0.194 6.077 +0.029 Species

Within Genus 435 75 503 0 2.117 8.059 0.113

Within 435 41 2710 0.181 4.216 15.762 0.063 Family

Within Order 435 23 3791 0.364 6.8 16.312 0.053

Within Class 435 6 50712 0 8.668 23.019 0.009 % Divergance within class

% Divergence within Order % Divergence within Families

% Divergence within Genus % Divergence within Species Sequence Composition onon thetheBasis ofof rbcla Gene Analysis

Composition of Nucleotide Bases at codon position 1, 2 & 3 Nucleotide Bases Minimum Mean Maximum Standard Error

G% 21 22.69 24.82 0.031 C% 18.48 21.1 23.37 0.044 A % 26.2 27.33 29.35 0.029 T% 25.36 28.88 32.07 0.049

GC % All Positions 39.86 43.78 47.64 0.057

GC % Codon Pos 1 53.24 54.89 57.07 0.039

GC % Codon Pos 2 43.48 44.99 47.83 0.036

GC % Codon Pos 3 20.65 31.47 40.76 0.158 GC (%) Composition at Codon position 1

GC (%) Composition at Codon position 2 GC (%) Composition at Codon position 3

GC (%) Composition at All Codon Positions Mean Max Distance Order Family Species earest Species earest eighbor IntraSp IntraSp to

Schefflera Apiales Araliaceae 0 0 Schefflera arboricola GCUBG433-11 1.84 actionophylla Schefflera Apiales Araliaceae 0 0 Schefflera actionophylla GCUBG345-11 1.84 arboricola Dracaena Asparagales Asparagaceae 0 0 Ehretia laevis GCUBG413-11 6.25 marginata

Asteranae-lamiids_unplaced Boraginaceae Ehretia laevis 0 0 Dracaena marginata GCUBG330-11 6.25

Chenopodium Caryophyllales Amaranthaceae 0 0 Callicarpa reevesii GCUBG033-11 1.28 album Plumbago Caryophyllales Plumbaginaceae 0 0 Plumbago capensis GCUBG 171 -11 0 auriculata Plumbago Caryophyllales Plumbaginaceae 0 0 Plumbago auriculata GCUBG169-11 0 capensis Maytenus Celastrales Celastraceae 0 0 Lonicera japonica GCUBG220-11 4.49 royleana Cycas Cycadales Cycadaceae 0.2 0.38 Cycas revoluta GCUBG279-11 0 circinalis

Cycadales Cycadaceae Cycas revoluta 0 0 Cycas circinalis GCUBG355-11 0

Lonicera Dipsacales Caprifoliaceae 0 0 Malpighia glabra GCUBG224-11 1.1 japonica Equisetum Equisetales Equisetaceae 0 0 Hypericum oblongifolium GCUBG211-11 18.8 arvense Phylogenetic analysis of different species with theirtheir Possible Distance To Nearest Neighbor

Mean Max Distance Order Family Species earest Species earest eighbor IntraSp IntraSp to

Apiales Araliaceae Schefflera actionophylla 0 0 Scheffleraarboricola GCUBG433-11 1.84

Apiales Araliaceae Scheffleraarboricola 0 0 Scheffleraactionophylla GCUBG345-11 1.84

Asparagales Asparagaceae Dracaena marginata 0 0 Ehretia laevis GCUBG413-11 6.25

Asteranae- Boraginaceae Ehretia laevis 0 0 Dracaena marginata GCUBG330-11 6.25 lamiids_unplaced

Caryophyllales Amaranthaceae Chenopodium album 0 0 Callicarpa reevesii GCUBG033-11 1.28

Caryophyllales Plumbaginaceae Plumbagoauriculata 0 0 Plumbago capensis GCUBG171-11 0

Caryophyllales Plumbaginaceae Plumbago capensis 0 0 Plumbago auriculata GCUBG169-11 0

Celastrales Celastraceae Maytenus royleana 0 0 Lonicera japonica GCUBG220-11 4.49

Cycadales Cycadaceae Cycascircinalis 0.2 0.38 Cycasrevoluta GCUBG279-11 0

Cycadales Cycadaceae Cycas revoluta 0 0 Cycas circinalis GCUBG355-11 0 Mean Max Distance Order Family Species earest Species earest eighbor IntraSp IntraSp to

Dipsacales Caprifoliaceae Lonicera japonica 0 0 Malpighia glabra GCUBG224-11 1.1

Equisetales Equisetaceae Equisetum arvense 0 0 Hypericum oblongifolium GCUBG211-11 18.8

Ericales Primulaceae Jacquinia armillaris 0 0 Jacaranda mimosifolia GCUBG383-11 6.82

Fabales Albizia lebbeck 0 0 Calliandra alba GCUBG164-11 1.28

Fabales Fabaceae Bauhiniavariegata 0 0 Hibiscusschizopetalus GCUBG227-11 1.1

Fabales Fabaceae Calliandra alba 0 0 Albizia lebbeck GCUBG459-11 1.28

Fabales Fabaceae Gleditsia tricanthos 0 0 artemisioides GCUBG349-11 1.84

Fabales Fabaceae Senna artemisioides 0 0 Gleditsia tricanthos GCUBG421-11 1.84

Fabales Fabaceae Sophora griffthii 0 0 Sophora secundiflora GCUBG428-11 3.54

Fabales Fabaceae Sophora secundiflora 0.1 0.19 Senna artemisioides GCUBG349-11 2.41

Gentianales Apocynaceae Acokantheraspectabilis 0 0 Carissa grandiflora GCUBG069-11 1.1 Fig Maximum Intra-Specific Distance (%) to Nearest Neighbor

Fig : Mean Intra-Specific Distance (%) to Nearest Neighbor Fig Individuals per Species with their Maximum Intra-Specific Distances

Fig.: Mean Intra-Specific Divergance (%) Fig .: Percentage Divergence with respect to Nearest Neighbor Thanks

We are immeasurable indebted to Dr. M. Ashfaq (NIBGE) and Dr . Paul Herbert and his associates at University of Guelph, Canada, for their technical assistance in performing our research work.