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 isolationElectrophoresis DNA Sequencing PCR Amplification Positive Results (DNA bands shown) Negative Results 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.. SAMPLESEQUENCESAMPLE CARACTERISTICS SCIENTIFIC NAME CHILLIE 5 (forward) TGGAGTTCCACCTGAAGAAGCAGGGGCCGCGGTAGCTGCCGAATCTTCTACTGGTACATGGACAAC TGTATGGACCGATGGACTTACCAGTCTTGATCGTTACAAAGGGCGATGCTACCGCATCGAGCGTGTT GTTGGAGAAAAAGATCAATATATTGCTTATGTAGCTTACCCTTTAGACCTTTTTGAAGAAGGTTCCGT TACCAACATGTTTACTTCCATTGTAGGTAATGTATTTGGGTTCAAAGCCCTGCGCGCTCTACGTCTGG AAGATCTGCGAGTCCCTACTGCTTATATTAAAACTTTCCAAGGTCCGCCTCATGGGATCCAAGTTGAA AGAGATAAATTGAACAAGTACGGTCGTCCCCTGTTGGGATGTACTATTAAACCTAAATTGGGGTTAT CTGCTAAAAACTACGGTAGAGCTGTTTATGAATGTCTTC Capsicum annuum CHILLIE 5a (Reverse) CGACCGTACTTGTTCAATTTATCTCTTTCAACTTGGATCCCATGAGGCGGACCTTGGAAAGTTTTAAT ATAAGCAGTAGGGACTCGCAGATCTTCCAAACGTAAAGCGCGCAGGGCTTTGAACCCAAATACATTA CCTACAATGGAAGTAAACATGTTGGTAACGGAACCTTCTTCAAAAAGGTCTAAAGGGTAAGCTACAT AAGCAATATATTGATCTTTTTCTCCAACAACACGCTCGATGCGGTAGCATCGCCCTTTGTAACGATCA AGACTGGTAAGTCCATCGGTCCATACAGTTGTCCATGTACCAGTAAAAGATTCGGCAGCTACCGCGG CCCCTGCTTCTTCAGGTGGAACTCCAGGTTGAGGAGTTACTCGGAATGCTGCCAATATATCAGTATC CTTGGTTTGGTACTCAGGAGTATAATAAGTCAATTTGTACTCTTTAACACC Capsicum annuum 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.

DISCUSSION AND CONCLUSIONS  When performing a Blast search, 8 sequences of 8 different chillie samples returned matches of 99% (range %) 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.