DNA BARCODING

Presented To: Dr. Nadeem Abass

Presented By : Shakeela Mahwish Rana

13061714-015

INTRODUCTION

DNA barcoding was first proposed by Paul Herbert in 2003.

Basic PrincipleDna Barcoding is based on premise that a short standardized sequence can distinguish individuals of a specie because genetic variation between specie exceeds that within specie.

Utility Of Dna Barcoding For Rapid And Accurate Assessment Of Bat Diversity In

Malaysia In The Absence Of Formally Described Species

( Wilson et al., 2014)

ABSTRACT

• Bats are important species for biodiversity research.

• Scientist investigated undescribed bat taxa in Peninsular Malaysia and Pasir Raja

• Barcode library provides a mean of recognizing and recording these taxa across biodiversity inventories.

ABSTRACT

Tissues were sampled from wing membrane of bats trapped at Pasir Raja and Peninsular

Malaysia

Dna was extracted

COI barcode region amplified

Sequenced

ABSTRACT

9 Species were identified, based on analysis of DNA Barcodes. This study confirms the high diversity of bats within Peninsular Malaysia :

9 Species in 13 Samples

INTRODUCTION

• Bats ( Order Chiroptera) play a crucial role in nature conservation.

• Action to protect bat species and specifically their habitat , confers protection to broad range of taxa that are endemic to natural area under threat.

INTRODUCTION

• The incorporation of DNA Barcoding into bat surveys has suggested the frequent occurrence of cryptic taxa.

• They are so-called “ dark ˋ̏ taxa.So in this research paper writer discuss that How “darkˋ̏ taxa are encountered in Peninsular Malaysia? How barcode library provides a mean of recognizing these taxa?

MATERIALS AND METHODS

Bats were trapped at Pasir Raja, and Peninsular Malaysia.

Tissues were sampled from wing membrane into 99% ethanol.

Dna extracted from 13 specimens using Nucleospin Kit

COI mtDNA was PCR amplified

Sampling and extraction using Nucleospin kit

Fig : Nucleospin kit

COI (648 bp)

MATERIAL AND METHODS

The primer pair C_ VFILF and C_ VILR are used.

PCR product were sent for sequencing at a commercially available service (MYTACG-Kuala Lumpur, Malaysia) using, M13R Tail primer.

Resulting DNA barcodes were uploaded to the BOLD project

A neighbor-joining tree of K2P distances was plotted in MEGA 5.

VR1d

VF1d

RESULTS

DNA barcodes were successfully amplified and sequenced from 13 specimens. The samples contained 9 species. Of these species, 4 species (44%) were dark taxa within the genus Hipposideros.

Table 1. Name, similarity and GenBank accession number of the closest matching barcodes to our thirteen specimens

Field ID Name of the closest match

Similarity with closest Match %

Gene Bank accession No. of closest match

b2 Phoniscus atrox 99.82 HM541211

b3 Hipposideros cervinus 100 HM540358

b4 Rhinolophus lepidus 98.55 HM541573

b9 Rhinolophus affinis 100 HM541414

b14 Rhinolophus lepidus 99.81 HM541573

b21 Hipposideros cervinus 100 HM540358

b30 Hipposideros bicolor 31 99.64 HM540344

b56 Phoniscus atrox 99.82 HM541211

b60 Hipposideros cf. bicolor 99.82 HM540379

b67 Rhinolophus affinis 98.71 HM541414

b69 Hipposideros cf. larvatus 100 HM240403

b74 Murina aenea 99.64 HM540928

b91 Hipposideros cf. larvatus 100 HM540403

B3 and B21 Hipposideros cervinus (100%)

B69 and B91 Hipposideros cf. larvatus ( 100%)

B9 and B 67 Rhinolophus affinis (100% and 98.71% respectively)

Similarity With Closest Match (%)

b4 and b14 Rhinolophus lepidus (98.55 and 99.81%)

B2 and B56 Phoniscus atrox (99.82%)

B60 Hipposideros cf. bicolor (99.82%)

b74 Murina aenea (99.64%)

b30 Hipposideros bicolor31 (99.64%)

Figure 1. Neighbor-joining tree of K2P distances for all the publicly available Hipposideros DNA barcodes from BOLD. Some clades have been compressed to triangles due to the large number of individuals.

Neighbor-

Joining Tree

DISCUSSION

Definitive identification require analysis of internal morphology (skull, dentation) and comparison with reference material.Field identifications remain questionable unless voucher specimens are retained which is impossible due to government controls and ethical concerns. An alternative is the bat detector- a device that detect the presence of bats and attempts specie determination based on echolocation ultrasound signals.

DISCUSSION…

In our study, Dark Taxa are frequently encountered during routine biodiversity inventory of bats in Peninsular Malaysia.

Our records of Hipposideros cf. larvatus-CMF04, H.bicolor 142, H.bicolor 131 and H. cervinus CMF02 would not have been possible without the integration of DNA barcoding into our inventory.

DISCUSSION…

Due to rapid technological advances, a DNA barcode can be obtained very easily and inexpensively for less than US$10 per specimen in Malaysia, making the method easily accessible to biodiversity researchers.