MOLECULAR DIVERSITY OF Spodoptera spp.

IN
COTTON BASED ON MITOCHONDRIAL CYTOCROME C
OXIDASE I ( COI ) GENE SEQUENCES
Presented by:
CONTENTS
 INTRODUCTION
 INSECT SAMPLING
 DNA ISOLATION
 PCR AMPLICATION
 DNA SEQUENCING
 INSILICO WORK -
PHYLOGENETICS
INTRODUCTION
(1) COTTON Why is cotton so important?
•Mainly grown for textile, food and
Scientific clasification
Kingdom : Plantae cosmetics.
Order : Malvales •Its fibre are strong and comfortable.
Family : Malvacea •Bandages are usually cotton.
Genus : Gossypium •Used to produce cottonseed oil.

•King of fibres
•Major cash crop India’s cotton production report during last
•Backbone of Indian rural economy two years
especially in dry land areas. •India’s cotton production in 2021-22 drop by
Figure 1 10.51% to 315.43 lakh bales , compare to
Characteristics of cotton plant: 352.48 lakh bales. in 2020- 21 season.
•Annual shrub crop •Due to unseasonal rains with the threat of
•Height is about 1.2 m Spodoptera, production of the fibre in Gujarat
•Sowing : June – July may be around 10-15% lower than the estimate.
•Harvesting : September - December
INTRODUCTION
(2) Spodoptera litura
 Taxonomy category [ Scientific Nomenclature ]
Kingdom: Animalia
Phylum: Arthropoda
Class: Insecta
Order: Lepidoptera
Superfamily: Noctuoidea
Family: Noctuidae
Genus: Spodoptera
Species: S. litura

Figure 2
o The Spodoptera litura is a serious polyphagous agricultural pest of numerous crops . It
damages cotton, maize, soybeans, and ornamental plants.

o Generally, S.litura larvae feed on host back side of leaves and the instar begins to feed a
large number of leaves, petals, and pods.
 ADULT
(MOTH) EGGS
Forewings are gray to dark- Eggs are spherical and slightly
brown with a complex pattern flattened.
of lines. The eggs are covered by
Hindwings are grayish-white. brown hairs.

Life cycle
+ Morphological
identification

PUPA
LARVA
Its colour is red-brown.
The larva has bright yellow
Presence of two small spines lines on the back and side of
at the tip of the abdomen is the the body.
characteristic feature.

Figure 3
 Which factors cause resistance to insecticide ?

Repeated use
of the same
class of
insecticide

Development of
Overuse of artificial RESISTANC
pesticide selection of E
insecticide

Frequent
application of
insecticide

 Choice of molecular marker

A variety of molecular markers have been used

To determine the species genetic variation and phylogenetics

Cytochrome C oxidase subunit I ( COI )gene is preferred as a molecular

marker
MATERIALS AND
1)METHOD
Insect Sampling / Collection of Sample
o The sample larvae were collected from the infested leaves
of cotton growing at cotton farm.

o The location was designated as Main Cotton research

Centre (21°10'19.8"N 72°48'14.6"E ).

o Larvae were collected throughout the flowering season

during October – November , 2022.

o The collected larvae were stored in 70% ethanol.

Figure 4
Identification of Sample Two methods
1. Morphological Identification Of Larva
2. Molecular Identification based on COI gene

Morphological Identification of Larva

The larva has bright yellow lines on the back and side
of the body
Figure 6.Damaged fibre

Skin is smooth and free of hairs

with numerous round dark spots
Head is dark brown to black and
scattered over tops and sides
has inverted Y-shaped suture
between the eyes

Figure 5

Maximum larva length is one inch

 Molecular Identification
Steps of Molecular Identification :

 DNA Isolation
 Quantitative and qualitative analysis
 PCR amplification
 DNA sequencing

DNA extraction protocol

1. Collection of required materials
Mortar and pestle
Micropipette ,Eppendorf tube
CTAB buffer
Petri plate Figure 7
2. Formulation of solutions
CTAB buffer ( 2% ) - 100ml

• CTAB - 2g
• NaCl - 1.4M Adjusted all to pH 8.0 and make up to 100 ml
• 1M Tris-HCl - 10.0 ml with deionized water
• EDTA - 4.0 ml

Procedure of DNA extraction

1.Grinding of sample + 200 ul CTAB = CTAB / insect extract mixture
2 .Incubation at 3 .Inversion for 10 – 4 .Centrifugation at 13000 5 .Addition of Phenol : chloroform :
60˚c for 35 min 15 min rpm ,4˚ c for 20 min IsoAmyl Alcohol (25:24:1) +
Inversion ( 10 min)

8 .Amount of 6 .Incubation at
supernatant = ice-cold room temperature +
7 .Collection of
isopropanol + Inversion Centrifugation
supernatant

9 .Incubation at -20 ˚c 11 .Collection of pellet

10 .Centrifugation Air-dried + Dissolved in
overnight for DNA
precipitation 100_150 of TE buffer

Figure 8
Quantitative analysis
No. of sample DNA conc.at
absorbance ratio
A260/A280
1.Loading of blank 2.Loading of DNA
Sample 1 1.568
sample sample
Sample 1 1.795

Sample 1 1.682

Average ratio = 1.675

Display showing the conc. of Display showing the conc. of
blank sample DNA sample

Figure 9
Qualitative analysis

Loading of DNA sample into Running of DNA

the well of Agarose gel DNA bands under UV
Transilluminator
Figure 10
 We are still
PCR amplification working on these
remaining steps to
DNA sequencing get the desired
Insilico work – Phylogenetic Analysis result

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