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Tree Genetics and Molecular Breeding, 2013, Vol.3, No.2, 4
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7
throughout the summer.
Planting of grafts in the orchard site and planted in
previously prepared pits of size 50×50×50 cm.
Location of orchards Forest Circles of Kerala With the
onset of monsoon showers the grafts were transferred
to the orchard Three representative site were located
in Southern, Central and Northern was implemented
by Kerala Forest Research Institute and another one
was implemented by Kerala Forest Department at
Kulathupuzha in Southern region. Lay out design of
the grafted ramets were planted in the orchard site in a
randomized polycross design. This is to ensure max-
imum degree of intercrossing among the assembled
plus tree clones and reduce inbreeding between ramets
belonging to the same clone. Spacing in all the orchards
an espacement of 8×8 m (quincuncial) was adopted.
Such wider espacement is meant to ensure open
sun-light condition which is essential for good
flowering and seed production. Isolation of all the four
orchards is isolated by more than 200 meters from the
nearest teak stands.
5 Studies on genetic diversity
Genetic diversity forms the base of biodiversity
hierarchy (Namkoong et al., 1996) and it serves as
building blocks in future selection and breeding (FAO,
1989). In recent years, biochemical and molecular
markers are widely used to study the extent and
pattern of genetic variation in tree species. A few
studies have been conducted to estimate genetic
diversity and outcrossing rates in selected populations
from natural and cultivated range in teak using
isozyme and Random Amplified Polymorphic DNA
(RAPD) markers (Changtragoon and Szmidt, 2000;
Nicodemus et al., 2005; Lowe et al., 2005). However,
genetic diversity information of teak in clonal seed
orchard (CSO) has not been reported.
AFLP marker study on genetic variation in a clonal
seed orchard established in 1985 by Kerala Forest
Department at Kulathupuzha in Kollam district of
Southern Kerala, India consisting of 1 200 trees of 31
clones in the orchard, planted in 8×8 m spacing. These
clones were raised from 31 plus trees selected from
natural teak forests as well as plantations raised in
main teak growing forest divisions of Kerala. Clones
were out planted in clonal seed orchard in randomized
polycross design. Phenological events and seed setting
of each clone were taken. Fruits collected from each
clone were dried and cleaned by removing calyx and
other debris. Fruits were subjected to pre-sowing
treatment of alternate wetting and drying for seven
days. Immediately after pre-sowing treatment, fruits
were sown in germination trays filled with vermiculate.
Each tray contained the fruits from each clone.
Regular watering was provided and daily germination
count was recorded.
DNA was extracted from 0.5 to 1 g of fresh leaf
tissues using the modified CTAB procedure (Doyle
and Doyle, 1990) and AFLP method was carried out
following the standard protocol of (Vos et al., 1995).
The reactions were carried out according to the
manufacturer’s protocol (AFLP
®
Analysis System 1
and Starter Primer Kit; Invitrogen Life Technologies,
Inc.,USA). AFLP
®
DNA ladder of size 30~300 bp
(Invitrogen Life Technologies, Inc., USA) was run on
either side of the denaturing gel as the standard marker.
The ladder was labeled with γ-p32 ATP using T
4
polynucleotide kinase according to the manufacturer’s
protocol. The autoradiogram developed was scanned
using HP Scanjet 3 770 digital flatbed scanner and
transferred each scanned autoradiogram to Kodak
Digital Science 1D Image Analysis Software. These
bands were scored manually as ‘1’ for the presence of
loci and ‘0’ for the absence of loci. Both polymorphic
and monomorphic bands were included in the final
data sets forming a binary matrix.
The total number of DNA bands formed from 31
clones was 653 of which 651 were polymorphic
(99.69%). At the population level, i.e. considering
clones from the same location of origin as separate
group analysis was done using POPGENE 1.32
version software. The polymorphism varied from
71.67 (Arienkavu) to 86.37 percent (Nilambur). Gene
diversity index (H) varied from 0.200 7 (Areinkavu)
to 0.220 8 (Nilambur). The mean total genetic
diversity (HT) was 0.227 4 and the relative magnitude
of genetic differentiation among population (GST)
was 0.078 3 indicating that 7.83 per cent of the total