3/8/11

1

Cotton whitefly in Pakistan; a single species or species complex?

Dr. Shahid Mansoor, SI

Agricultural Biotechnology Division,

National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad,

Pakistan

Cotton leaf curl disease; the three players

What are important issues related to whitefly Important pest and vector for begomoviruses Issues 1. Why we have lass disease in Sindh? Whitefly biotypes or environmental conditions 2. Role of endosymbionts in virus-vector relationships 3. Prospects of genetically-engineered resistance

DNA based methods can help to answer these questions

Bemisia tabaci Faimly Aleyrodidae Order Hemiptera Suborder Homoptera or

Sterorrhyncha v 1200 Species and 126 Genera v Bemisia tabaci first described as pest of tobacco in Greece v About 100 years ago v Grave impact on tropical and subtropical agriculture system by causing decreased production in food and fibre crops.

Biotyping

Samples from Punjab and Sind were Collected. In colleberation with Jose Cenis Biotyping was performed on the basis of Cytochrome Oxidase I gene amplification and Sequencing.

Clades/biotypes of Bemisia tabaci in Pakistán

Biotype B Clade/biotype Indian Clade South East Asia (biotype K)

Clades/Biotypes of B. tabaci samples collected in Pakistan on cotton and vegetables and analised by sequencing of mtCOI gene.

Code Origin Clade/Biotipe 3 Ahmad Pur K 4 Mirpur Mathelo B 5 Ubaro K 7 Tando Muhammad Khan K 8 Multan indian 9 Multan indian 10 Multan indian 11 Sanghar K 12 Larkana B 13 Multan K 20 Kroore indian 21 Fateh Pur indian 22 Chak 99 indian 23 Khairpur B 24 Kot Digi B 25 Moro B 26 Sumar Chanar B

3/8/11

2

Origin

Code

Individuals analyzed

Biotype Rahim Yar Khan

RYK

2

Indian Kot Addu

KA

2

Indian Chichawatni

CW

2

Indian Khenewal

KW

4

Indian Sumundri

SI

4

Indian Bahawalpur

BP

2

Indian Rajanpur

RP

2

Indian Bahawalnagar

BN

4

Indian Layyah

LH

1

Indian Faisalabad

FD

4

Indian Khanpur

KP

1

Indian Ahmedpur Sharkia

APS

3

Indian Burewala

BW

1

Indian Nurpur

NP

4

Indian Chandri Goth

CG

5

Indian Liaqatpur

LP

1

Indian Sukrand

SK

1

Unknown Tandojam TJ

3

Biotype B Total

46

Analysis of the samples of B. tabaci from Pakistan by mt COI gene

Bemisia tabaci; A single species or species complex?

B. tabaci is a complex of 11 well-defined high-level groups containing at least 24 morphologically indistinguishable species Paul J. De Barro, Shu-Sheng Liu, Laura M. Boykin and Adam B. Dinsdale Annual Review of Entomology, 2011

What are important consequences? Most begomoviruses are transmitted by most biotypes Confusion over the whitefly species that transmit begomoviruses

How to resolve the issue?

A DNA barcode is a short gene sequence

taken from standardized portions

of the genome, used to identify species

The Mitochondrial genome

850 bp

COI Gene

658 bp

Barcode regions

3/8/11

3

Wider Impacts of Barcoding

•  Catalyzing interoperability of databases –  Barcode data standards link sequences,

specimens, species names and publications •  Improving the information

infrastructure –  Digital library initiative in taxonomy

•  Renewing the mission of museums –  DNA recovery from formalin-fixed

specimens –  Promoting the growth of DNA banks

•  Expanding analytical toolbox for taxonomy

Characteristics of Barcode Regions

•  Flanked by conserved regions •  Easy to amplify •  Low intraspecies variability •  Discontinuous variation between species •  Long enough to work in all groups •  Short enough for single reads

Why mtDNA?

ü Ease of isolation ü High copy number ü Lack of recombination ü Conservation of sequence and structure

across metazoa ü Range of mutational rates in different

regions of the molecule

Using DNA Barcodes •  Establish reference library of barcodes

from identified voucher specimens •  If necessary, revise species limits •  Then:

–  Identify unknowns by searching against reference sequences

–  Look for matches (mismatches) against ‘library on a chip’

–  Before long: Analyze relative abundance in multi-species samples

Size: 16324 bp Size: 16568 bp

Why COI? ü Relatively well studied at the

biochemical level ü Size and structure conserved across all

aerobic organisms ü Mix of variable and conserved regions ü Largest of the three CO subunits ü Broad spectrum of substitutional rates

3/8/11

4

Methods

Taxon ID Tree

Sanghar   ***   99.1   85.9   85.9   100   99.4   85.8   Sanghar      

Matyari   0.9   ***   85.6   85.5   99.1   99   85.9   Matyari      

T.Allahyar   15.9   16.1   ***   99.5   85.9   85.4   99.3   T.Allahyar  

T.Jam   15.8   16.3    0.5        ***   85.9   85.5   99.4   T.Jam      

Hyderabad   0   0.9   15.9   15.8                ***   99.4   85.8   Hyderabad  

Ghotki   0.6   1.1   16.3   16.3   0.6            ***   85.4   Ghotki      

Mirpur  Khas   16   15.8   0.7   0.6   16   16.4   ***   Mirpur  Khas  

Sanghar   Matyari   T.Allahyar   T.Jam   Hyderabad   Ghotki  Mirpur  Khas  

   

Sequence identity based on biotype sequence

BADIN   B.NAGAR   B.NAGAR   CHISHTIAN   MULTAN   MULTAN   HYDERABAD   FAISALABAD   KHANEWAL  

***        100          98.9            100            100   100   85.7   100   100                        BADIN  

     ***          98.9            100   100   100   86   100   100   BAHAWALNAGAR  

       ***            98.9   98.9   98.9   85   98.9   98.9   BAHAWALNAGAR  

         ***   100   100   85.7   100   100   CHISHTIAN  

         ***   100   85.8   100   100   MULTAN  

         ***   85.7   100   100   MULTAN  

               ***   85.7   85.7   HYDERABAD  

               ***   100   FAISLABAD  

   ***   KHANEWAL  

DNA sequence identity based on barcode region

Conclusions –  COI can be reliably used for species identifications

–  DNA barcoding data support a single species status for Bemisia tabaci, the insect vector of begomovirus from Pakistan

–  Data on diversity of endosymbionts and GroEL is being generated