Genetic Diversity of Honeybee (Apis cerana F.)

169 Pages·2013·7.46 MB·English

by  Shahnawaz Ahmad Dar

Genetic Diversity of Honeybee (Apis cerana Fabricius)
using Morphological and Molecular Markers

Shahnawaz Ahmad Dar

(2008-211-D)

Division of Entomology
Faculty of Post-graduate Studies
Sher-e-Kashmir University of Agricultural Sciences &
Technology of Kashmir

2012
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Genetic Diversity of Honeybee (Apis cerana Fabricius)
using Morphological and Molecular Markers

Shahnawaz Ahmad Dar

(2008-211-D)

THESIS

Submitted to

The Faculty of Postgraduate Studies
Sher-e-Kashmir University of Agricultural Sciences &
Technology of Kashmir
in partial fulfilment of requirement for the award of the degree of

Doctor of Philosophy in Agriculture
(Entomology)

2012
2
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Sher-e-Kashmir
University of Agricultural Sciences and Technology of Kashmir
Division of Entomology, Shalimar Campus, Srinagar
191121

Certificate – I

This is to certify that the thesis entitled “Genetic Diversity of
Honeybee (Apis cerana F.) using Morphological and Molecular
Markers” submitted in partial fulfillment of the requirements for the
award of the degree of Doctor of Philosophy in Agriculture
(Entomology), to the Faculty of Post-graduate Studies, Sher-e-
Kashmir University of Agricultural Sciences and Technology of
Kashmir, is a record of bonafide research work carried out by Mr.
Shahnawaz Ahmad Dar (Regd. No. 2008-211-D) under my
supervision and guidance. No part of the thesis has been submitted for
any other degree or diploma.
It is further certified that information received during the course
of investigation has duly been acknowledged.

(Dr. Sheikh Bilal Ahmad)
Chairman
Advisory Committee

Endorsed

Professor & Head,
Division of Entomology
SKUAST Kashmir
4
Sher-e-Kashmir
University of Agricultural Sciences and Technology of Kashmir
Division of Entomology, Shalimar Campus Srinagar- 191121

Certificate – II

We, the members of the Advisory committee of Mr. Shahnawaz
Ahmad Dar (Regd. No. 2008-211-D), a candidate for the degree of
Doctor of Philosophy in Agriculture (Entomology), have gone
through the manuscript of the thesis entitled, “Genetic Diversity of
Honeybee (Apis cerana F.) using Morphological and Molecular
Markers” and recommend that it may be submitted by the student in
partial fulfillment of the requirements for award of the degree.
Advisory Committee

Chairman Dr. Sheikh Bilal Ahmad
Professor-cum-Cheif Scientist, Division of
Entomology, SKUAST-Kashmir
Members
Dr. Mushtaq Ahmad
Dean, Faculty of Agriculture, Wadura,
SKUAST-Kashmir

Dr. Nazir Ahmad Wani
Professor-cum-Cheif Scientist, Division of
Entomology, SKUAST-Kashmir

Dr. Showkat Maqbool
Assistant Professor-cum-Jr. Scientist, Division of
Agri. Statistics, SKUAST-Kashmir

Dean PG Nominee Dr. G. N. Malik
Professor & Head, Temperate Sericulture
Research Institute, Mirgund, SKUAST-Kashmir
5
Sher-e-Kashmir
University of Agricultural Sciences & Technology of Kashmir
Division of Entomology, Shalimar Campus Srinagar – 191 121

Certificate – III

This is to certify that the thesis entitled, “Genetic Diversity of
Honeybee (Apis cerana F.) using Morphological and Molecular
Markers” submitted by Shahnawaz Ahmad Dar (Regd. No. 2008-
211-D) to the Faculty of Postgraduate Studies, Sher-e-Kashmir
University of Agricultural Sciences and Technology of Kashmir,
in partial fulfillment of the requirements for the award of the degree of
Doctor of Philosophy in Agriculture (Entomology) was examined
and approved by the Advisory Committee and external examiner on
………………..

Chairman External Examiner
Advisory Committee

Professor & Head
Division of Entomology

Director Resident Instruction-cum-Dean
Postgraduate Studies, SKUAST Kashmir

6
Sher-e-Kashmir
University of Agricultural Sciences & Technology of Kashmir
Division of Entomology, Shalimar Campus Srinagar – 191 121

Name of the student : Shahnawaz Ahmad Dar
Registration No. : 2008- 211-D
Major subject : Entomology
Minor subject : Plant Pathology
Major advisor : Dr. Sheikh Bilal Ahmad
Professor-cum-Cheif Scientist
Division of Entomology
Title of the Thesis : Genetic Diversity of Honeybee (Apis
cerana F.) using Morphological and
Molecular Markers
ABSTRACT
The investigations entitled “Genetic diversity of honey bee, Apis cerana F.
using morphological and molecular markers” were undertaken to estimate the
extent of genetic relationship by morphological and molecular characterization
during 2010-2011. 100 genotypes of A. cerana F. from feral/wild colonies were
collected from 5 locations viz, Pahalgam, Tral, Srinagar, Sonamarg and Bandipora
for studying both morphometric and genetic variations in the population.
Morphometric studies revealed that 22 morphological characters showed
significant inter-locality variations. However, other morphological characters did
not show any significant differences among the bee samples collected from these
locations. On the other hand, the comparative morphometric study of A. cerana
F. collected from areas of mountainous zone (Pahalgam and Sonamarg) showed
significant higher value of several morphological characters as compared to those
found in sub mountainous zone (Srinagar and Bandipora). A. cerana F. collected
from mountainous zone of Kashmir region were darker in colour as compared to
those found in sub-mountainous zone, which indicated that the colour pattern of
A. cerana F. probably varies with the altitude. A significant positive correlation
between altitude and several morphological characters was observed indicating
that morphometry of A. cerana F. changes with altitude. Most of the
morphological characters were significantly correlated among themselves either
positively or negatively. Thus change in the size of one morphological part may
affect the size of other parts. Multivariate analysis which includes ‘Cluster
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Analysis’ was employed to examine the extent of phenotypic variation in A.
cerana F. based on the morphological data. The dendrogram generated by
UPGMA for morphological data of different genotypes collected from different
locations were grouped into six clusters. The cluster II consisted of highest
number of genotypes (32) followed by cluster IV (22) and cluster III (16). The
clusters I, V, and VI contained the lowest number genotypes (10 each). While
dendrogram generated for different locations of Kashmir region revealed that the
five locations are grouped into two clusters. The cluster I comprised of locations,
Pahalgam and Sonamarg and the cluster II comprised of locations, Tral, Srinagar
and Bandipora.
Genetic diversity of honey bee, A. cerana F. in different locations of
Kashmir region was examined by PCR- RFLP analysis. Three primers and five
restriction enzymes were initially used to identify the genetic diversity of A.
cerana F. All the enzymes showed restriction digestion with appropriate primers
except Alu1 which does not show digestion with any of the primers used. The
molecular data was used to calculate percentage similarity matrix of bees using
Jaccards coefficient which ranged from 10.0% between (Ac-55 and Ac-99) to
93.3 % between (Ac-5 and Ac-10) with an average percentage of 46.5%.
Dendrogram generated by UPGMA of molecular data revealed that all the
genotypes were grouped into seven clusters. Maximum number of genotypes was
observed in cluster III (24) and minimum number in cluster VI and VII (2 each).
DNA fragments generated from the amplified PCR products digested with
different enzymes showed polymorphism percentage ranging from 77.77% to
100%. Analysis of molecular variance (AMOVA) also illustrated significant
genetic differentiation among populations (55.87%) and within populations
(44.13%). The Mantel matrix correspondence test was used to compare the
morphological data with molecular data on the basis of cluster analyses. The
molecular data did not show any correlation with morphological data (r = 0.005, P
= 0.44). Present study revealed that more number of RFLP markers must be used
to depict the genetic relationships among A. cerana F. genotypes before reaching
to a comprehensive conclusion. The morphometric variations found in A. cerana
F. viz-a-viz altitudes could have a significant impact in future bee breeding
programmes with respect to A. cerana F. in Kashmir.
Key words: Honeybees, Apis cerana F., PCR-RFLP, Morphological diversity,
Molecular variability, Correlation, Mantel test

Signature of Student Signature of Major Advisor
Dated: ___________ Dated: _______________
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Acknowledgement

In the name of “ALLAH” Most gracious, Most merciful
R
esearch is an evolving concept. Any endeavor, in this regard is
challenging as well as exhilarating. It implies the testing of our nerves.
It brings to light our patience, vigour and dedication. Every result
arrived at is a modest beginning for a higher goal and no work can be termed as
a one-man show. It needs the close cooperation and guidance of experts in the
field to achieve something worthwhile and substantial.
It is indeed a great honour and pleasure for me to express my deep sense
of gratitude and indebtness to my major Advisor, Dr. Sheikh Bilal Ahmad,
Professor-cum-Chief Scientist, Division of Entomology. His soft and sober
attitude, inspiring guidance, whole hearted encouragement, facile suggestions
and constructive criticism with a calm exposure, led this uphill task to its
successful completion and shall remain a lifelong gifted memory for me.
It is my privilege to place on record my profound gratitude and thanks to
the members of my Advisory committee viz., Dr. N. A. Wani, Associate
Professor, Division of Entomology, Dr. Mushtaq Ahmad, Dean, Faculty of
Agriculture, Wadura, Dr. G. N. Malik, Professor & Head, Temperate
Sericulture Research Institute, Mirgund (Dean P.G. Nominee), Dr. Showkat
Maqbool, Assistant Professor, Division of Agri. Statistics for their valuable and
learned suggestions, keen interest, exorbitant help during the entire course of
study and review of this manuscript.
It is a genuine opportunity to offer my sincere thanks to Dr. M. I. S.
Waliullah, Professor and Head, Division of Entomology, whose unceasing
whole hearted support and time to time technically valuable suggestions could
enable me to take this research to its logical conclusion.
My special thanks from core of my heart to Dr. Z. H. Khan, Associate
Professor, Division of Entomology, Dr. B.A.Padder, Assistant Professor,
Division of Plant Pathology and Dr. M.D. Shah, Assistant Professor, Division
of Plant Pathology for their learned advice, valuable guidance and help during
the course of investigation.
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Sincere thanks are due to Dr. G. M. Mir, Professor, Division of
Entomology, Dr. S. S. Pathiania, Assistant Professor, Entomology, Dr. Barkat
Hussain, Assistant Professor, Division of Entomology, Dr. Malik Mukhtahar,
Assistant Professor, Division of Entomology, for their timely help and constant
encouragement.
I express my deep regards and heartiest thanks to my dear friends and
colleagues: Mr. Shabir, Mr. Zaffar, Dr. Aflaq, Mr. Showkat, Dr. Muzafar, Dr.
Sheeraz, Dr. Tariq, Dr. Farooq, Mr. Talat, Mr. Samoon, Dr. Malik Ahsaf, Dr.
Zia ul haq, Mr. Imtiyaz, Mr. Asif, Mr. Arsad Abass, Mr. G. I. Hassan, and
other elder and young brothers: Muzafar, Javaid for their all possible help,
enthusiastic and inspiring guidance, strong moral support, valuable suggestions,
intellectual inputs, inspiring ideas of higher studies , constructive criticism,
association rendered by them and careful supervision during my entire post-
graduation programme and whose memories will surely remain as an esteemable
wealth in future life.
My heartfelt devotion is due for my loving Parents, Abdul Gani and
Fatima and other family members, Abida, Shafiqa and Afroza for their
affectionate help and blessings. All those who care for me may not have got a
mention, but none shall ever be forgotten.
The learning scenario for pursuit of academic health provided by Central
Library and ARIS Cell, SKUAST-K is sincerely acknowledged.

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Place: Shalimar, Srinagar
Dated:

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