Download DNA BARCODING CHILLIES

DNA BARCODING CHILLIES
BIO-NERDS:
Say Wah
Yugraj Singh
Tanja Obradovic
Jenny Pham
Lovita Bharossa
Buai Chuol
Diana Corzo
INTRODUCTION
DNA Barcoding is method that uses a short genetic marker
in an organism's DNA for purposes of fingerprinting to identify
it as belonging to a particular species.
It is important to identify plants in gene banks for study of
crops, ecological and botanical improvement. That is why this
project was focus on Chillies DNA barcoding , which are plants
from the genus Capsicum.
The present project describes different DNA sequences
obtained from chillies samples, which were collected from
house backyards, supermarkets and local vegetable markets in
order to examine, compare and investigate these sequences
and analyse what species they come from.
BACKGROUND
DNA Barcoding first came to the attention of the scientific
community in 2003 when Paul Hebert’s research group at the
University of Guelph published a paper titled "Biological
identifications through DNA barcodes". In it, they proposed a
new system of species identification and discovery using a
short section of DNA from a standardized region of the
genome.
That DNA sequence can be used to identify different species,
in the same way a supermarket scanner uses the familiar
black stripes of the UPC barcode to identify your purchases.
“The two gene regions in the chloroplast, matK and rbcL,
have been approved as the barcode regions for land plants.
BACKGROUND
Chili peppers originated in the Americas. After
the Columbian Exchange, many cultivars of chili
pepper spread across the world, used in both
food and medicine. The five domesticated
species of chili peppers are:
Capsicum
annuum, Capsicum frutescens, Capsicum
chinense,
Capsicum
pubescens
and
Capsicum baccatum.
OBJECTIVES
• To extract DNA from chillies samples
collected from different outlets, markets and
shops around Melbourne, and to compare
different DNA sequences obtained from these
samples.
• To learn and apply the Barcoding protocol
including the DNA Isolation process, PCR,
DNA
sequencing,
following
by
PCR
amplification and DNA sequencing.
OBJECTIVES
• To examine if primers used for plant
extraction work on Chillies sample,
and to analyse if these allow
obtaining good results.
• To investigate how markets are
advertising and
labelling different
varieties of chillies.
METHODOLOGY
DNA isolation
Electrophoresis
Positive Results
(DNA bands
shown)
Negative Results
PCR
Amplification
DNA Sequencing
Blast ( DNA
Subway)
Species
Identification
RESULTS
Genomic DNA of eight chillie samples were
successfully extracted. Also, PCR was done on all
samples using the following primer sequences :
rbcL barcoding (plants)
Forward Primer
5’- TGTAAAACGACGGCCAGTATGTCACCACAAACAGAGACTAAACG- 3’
Reverse Primer
5’- CAGGAAACAGCTATGACGTAAAATCAAGTCCACCRCG- 3’
Cont..
The bands of eight chillie samples
tested were within the expected band
range, around 580BP
Cont..
DNA were cleaned up using QIAquick PCR
Purification kit (Qiagen) and sent to Micromon
Monash Medical Center for sequencing.
Trace Sequence:
GAGTTCCACCTGAAGA BASES
Cont…
By using application sequence viewer
(software), by applied Biosystems, We
trimmed the ends of the sequence
which were having errors with peaks
of different bases overlapping.
As a result we got one continuous
strand of sequence with minimum
error in it.
Cont..
Sequence trace file was trimmed and results were blasted
using DNA Subway. We found that all chillie samples
were members of the CAPSICUM ANNUUM species.
SAMPLE
SEQUENCE
CHILLIE 5
(forward)
TGGAGTTCCACCTGAAGAAGCAGGGGCCGCGGTAGCTGCCGAATCTTCTACTGGTACATGGACAAC
TGTATGGACCGATGGACTTACCAGTCTTGATCGTTACAAAGGGCGATGCTACCGCATCGAGCGTGTT
GTTGGAGAAAAAGATCAATATATTGCTTATGTAGCTTACCCTTTAGACCTTTTTGAAGAAGGTTCCGT
TACCAACATGTTTACTTCCATTGTAGGTAATGTATTTGGGTTCAAAGCCCTGCGCGCTCTACGTCTGG
AAGATCTGCGAGTCCCTACTGCTTATATTAAAACTTTCCAAGGTCCGCCTCATGGGATCCAAGTTGAA
AGAGATAAATTGAACAAGTACGGTCGTCCCCTGTTGGGATGTACTATTAAACCTAAATTGGGGTTAT
CTGCTAAAAACTACGGTAGAGCTGTTTATGAATGTCTTC
CHILLIE 5a
(Reverse)
CGACCGTACTTGTTCAATTTATCTCTTTCAACTTGGATCCCATGAGGCGGACCTTGGAAAGTTTTAAT
ATAAGCAGTAGGGACTCGCAGATCTTCCAAACGTAAAGCGCGCAGGGCTTTGAACCCAAATACATTA
CCTACAATGGAAGTAAACATGTTGGTAACGGAACCTTCTTCAAAAAGGTCTAAAGGGTAAGCTACAT
AAGCAATATATTGATCTTTTTCTCCAACAACACGCTCGATGCGGTAGCATCGCCCTTTGTAACGATCA
AGACTGGTAAGTCCATCGGTCCATACAGTTGTCCATGTACCAGTAAAAGATTCGGCAGCTACCGCGG
CCCCTGCTTCTTCAGGTGGAACTCCAGGTTGAGGAGTTACTCGGAATGCTGCCAATATATCAGTATC
CTTGGTTTGGTACTCAGGAGTATAATAAGTCAATTTGTACTCTTTAACACC
SAMPLE CARACTERISTICS
SCIENTIFIC
NAME
Capsicum
annuum
Capsicum
annuum
DISCUSSION AND
CONCLUSIONS
When performing a Blast search, 8
sequences of 8 different chillie samples
returned matches of 99% (range 9899%) maximum identity, representing
1 species (CAPSICUM ANNUUM)
CONCLUSIONS
Eight out of eight chillie samples come
from
same
species
‘’Capsicum
Annuum’’; however, these are from
different
varieties,
with
different
morphological
characteristics.
Conclusively, it is necessary to have
better and specific primers which can
differentiate between varieties of same
species.
CONCLUSIONS
• For the DNA sequencing , it was
necessary to get a Forward and reverse
sequences in order to fill up the gaps of
the bases errors and to achieve a
maximum identity of samples.
Cont..
As Chillies are consumables as part of human
diet, it is important to identify if shops are
labelling the product correctly, in order to
make sure customers are getting what they
are looking for.
Cont..
Accurate species identification remains a basic
first step in any study of biodiversity,
particularly for global changes and their
consequences.
DNA barcoding has proved to be a powerful
alternative method to traditional morphological
approaches,
allowing
to
complement
identification techniques for living organisms
Cont..
DNA Barcoding is a rapid, cost-effective
and
broadly
applicable
molecular
diagnostic technique, which allows species
identification.
Also, it aid in the planning of regeneration
processes by providing an advance notice
of specialized resources or environmental
conditions that might be required for their
culture.