ROAD MAP

Material and
Introduction Objectives
Methods

Outcomes References
 INTRODUCTION
• DNA barcoding is a standardized approach to identifying
plants and animals by minimal sequences of DNA, called
DNA barcodes.
• Mangoes, often referred to as the "King of Fruits," hold a
position of global importance. Their delectable flavor, rich
nutritional profile, economic significance, and cultural value
make them a cherished fruit worldwide.
• Mangoes belong to genus Mangifera which consists of about
30 species of tropical fruiting trees in the flowering plant
family Anacardiaceae.
• Pakistan exports around 100,000 tons of mangoes yearly,
valuing around $100 million. This number could grow
exponentially.
 • Pharmacological effects • Antispasmodic and
Biological antipyretic activity.
Importance • Anti-oxidant • Immunomodulatory
• Anti-diarrhoeal
of Mango • Anti-diabetic
• Anti-inflammatory
• Anti-viral
• Anti-bacterial and
• Anthelmintic and anti- antifungal activity
allergenic activity
• Anti-microbial
• Anti-parasitic • Hepatoprotective
• Anti-bone resorption • Gastro protective
• Anti-tumer-anti-HIV
Markers used in Plant
DNA barcoding
The chloroplast gene rbcL encodes the large subunit of
ribulose bisphosphate carboxylase.
The role of rbcL gene is widely used in the
reconstruction of phylogenies
RbcL gene is located in the large single copy region of
chloroplast genome
Maturase K (matK) is a plant universal gene
OBJECTIVES

PHYLOGENETIC
MANGIFERA INDICA SPECIES DNA BARCODE GENERATION RELATIONSHIPS AND
DNA ISOLATION AND PCR- FOR SELECTED MANGIFERA MOLECULAR
BASED AMPLIFICATION INDICA SPECIES FOUND IN CHARACTERIZATION OF
USING RBCL AND MATK PAKISTAN. SEVERAL MANGIFERA INDICA
PRIMERS. SPECIES.
Plan of Work

Collection of leaves DNA Extraction PCR

Leaves were collected DNA of all cultivars is Amplification of purified
from Horticulture extracted using CTAB DNA was done by using
department method rbcl and matk primers

Gel electropherosis
Agarose gel Sequencing of Amplified
Mapping
electropherosis was done DNA
to confirm PCR product
Material and Methods

Pestle and CTAB centrifugation

Leaves mortar buffer machine

PCR
Machine AGE Sequencer
DNA
Extraction

Fresh Grinding of
leaves leaves Incubation centrifugation

Washing Overnight
Drying at -20C
 DNA samples from species are loaded into gel in wells from 1 to
14 and ladder of 10kb is loaded on right side. Ladder is loaded
DNA bands confirmation on right side of gel tray at last, while from 1 to 14 are bands of
fourteen mango cultivars. All the bands are shown, 1 represent
of Mango species Sindhri, 2 for White Chaunsa, 3 for Black Chaunsa, 4 for Anwar
Ratol, lane 5 for Anwar Ratol 12, 6 for Summer Bishat, and 7 for
Langra, 8 for Sandhura, 9 for Malda, 10 for Sensation, 11 for
Sunghlafia, 12 for Dosehri, 13 for Fajri, and 14 for Chaunsa
Nawab Puri.
Polymerase Chain
Reaction
• Master Mix
• Forward and Reverse Primers (rbcL and matK)
• Tamplate (DNA samples)
• D3H2O
rbcL Primer
•rbcl-F = 5’ATGTCACCACAAACAGAAAC3’
•rbcl-R = 5’TCGCATGTACCTGCAGTAGC3’

PCR Profile for rbcL

matK Primer
• matK-F=5’CGATCTATTCATTCAATATTTC3’
• matK-R=5’TCTAGCACACGAAAGTCGAAGT 3’

PCR Profile for matK

 Use of rbcL primer based amplified
PCR gel results (750 bp)
Use of matK primer based amplified
PCR gel results (950 bp)
Agarose gel visualization of
rbcL and matK amplicons
• PCR process is carried out in PCR reaction tube
• The PCR reaction contains Master mix, forward primer,
and reverse primer, along with D3 water
• Annealed at 50.5 and 45 °C for amplification
• Confirmation of PCR products through Gel
electrophoresis
• DNA on gel electrophoresis at 60 volt for 80-90
minutes
• All fragments revealed 750bp product on the gel that
show PCR amplification in rbcL gene
• The fragments revealed 950 bp product in 1-14 lanes
that show PCR amplification in matK gene
DNA Purification
Process

• Purification of PCR products by PCR

Clean-Up Mini Kit
• DNA sequencing, Validation, and SNPs
detection
• Phylogenetic tree construction by using
MEGA-X Software
SEQUENCING
Samples are sent for sequencing.
 Mango species identified by BLAST/GenBank
.

Similarity Query Accession Similarity Query Accession

Common Name Common Name
Index Cover Number Index Cover Number

Sindhri 99.77% 100% BAO71756.1

Sensation 99.87% 100% AAX57170.1
White Chaunsa 89% 94% BAO71900.1
Sunglafia 88.8% 94% AFI14901.1
Black Chaunsa 100% 100% AIK19512.1

Anwar Ratol 99.77% 100% QBA96915.1 Dosehri 100% 100% AFI14902.1

Anwar Ratol 12 99.40% 99% QBA96919.1 Fajri 99.78% 100% AYN75109.1

Summer Bishat 99.32% 99% AKP98892.1
Chaunsa Nawab puri 99.45% 99% QDE14070.1
Langra 98.86% 99% AKP98893.1

Sandhura 100% 100% QBC73629.1 Malda 99.37% 99% ALI16494.1

 Nucleotide Discrimination

Sr. No. Species Name A T G C Total GC% AT%

1 Sindhri 166 186 160 137 649 45.76 54.23

2 White Chaunsa 280 312 151 162 905 34.58 65.41

3 Black Chaunsa 341 362 327 251 1281 45.12 54.87

4 Anwar Ratol 111 118 101 108 438 47.71 52.28

5 Anwar Ratol 12 338 355 254 327 1274 45.60 54.39

6 Summer Bishat 337 351 329 251 1268 45.74 54.25

7 Lanagra 338 356 328 257 1279 45.73 54.26

8 Sandhura 342 333 326 253 1282 44.12 55.67

Sr. No. Species Name A T G C Total GC% AT%

9 Malda 166 186 160 137 689 45.56 54.23

10 Sensation 280 312 151 162 985 34.50 65.41

11 Sunghlafia 341 362 327 251 1281 45.18 54.87

12 Dosehri 131 122 161 198 488 47.91 52.38

13 Fajri 336 352 256 337 1284 45.80 54.49

14 Chaunsa Nawab puri 335 371 330 261 1269 45.73 52.85
Phylogenetic
analysis

Phylogenetic tree of Mango species for matK

marker.
References

• Antonius-Klemola, K. 1999. Molecular markers in Rubus (Rosaceae)

research and breeding. J. Hortic. Sci. Biotechnol. 74:149–160.
• Azeem, F., A. Bilal, M.A. Rana, A.A. Muhammad, N. Habibullah, H. Sabir,
R. Sumaira, M. Hamid, A. Usama and A. Muhammad. 2019. Drought affects
aquaporins gene expression in important pulse legume chickpea (Cicer
arietinum L.). Pakistan J. Bot. 51:81–88.
• Babaei, A., S.R. Tabaei-Aghdaei, M. Khosh-Khui, R. Omidbaigi, M.R.
Naghavi, G.D. Esselink and M.J.M. Smulders. 2007. Microsatellite analysis
of Damask rose (Rosa damascena Mill.) accessions from various regions in
Iran reveals multiple genotypes. BMC Plant Biol. 7:1–6.
References

• Centifolia, R., P. Review, J. Jitendra, T. Vineeta, K. Ashok, K. Brijesh and P.

Singh. 2014. ROSA CENTIFOLIA : PLANT REVIEW.
• Cui, W.-H., X.-Y. Du, M.-C. Zhong, W. Fang, Z.-Q. Suo, D. Wang, X. Dong,
X.-D. Jiang and J.-Y. Hu. 2022. Complex and reticulate origin of edible roses
( Rosa , Rosaceae) in China . Hortic. Res. 9.
• DeSalle, R. and P. Goldstein. 2019. Review and Interpretation of Trends in
DNA Barcoding. Front. Ecol. Evol. 7:1–11.
• Fiser Pecnikar, Z. and E. V. Buzan. 2014. 20 years since the introduction of
DNA barcoding: From theory to application. J. Appl. Genet. 55:43-52.