Intl. J. of Mol. Evol. and Biodivers. 2015, Vol. 5, No. 1, 1-5
2
observations in the parental palm pool of the Sri
Lanka Yellow Dwarf coconut variety indicate
morphological differences within the variety. In
addition to the palms with typical dwarf features
certain palms are observed to have taller stature, larger
crowns and well-formed root boles which are specific
features of the tall coconuts while the nuts in all of
them are yellow coloured.
In addition to morphological differences, the palms in
this population show a varying degree of
tolerance/susceptibility to the coconut mite
Aceria
guerreronis
(Keifer).
Aceria
mite is a pest that
inhabits underneath the perianth of the fruit. It causes
economic losses to the coconut by reducing the nut
size due to its feeding on the soft tissues of the
immature nut (Fernando et al., 2000).
The current study was conducted with the objectives
of discriminating the different morphological groups
within the Sri Lanka Yellow Dwarf coconut
population by molecular means at microsatellite
marker loci and to identify associations, if there are
any between the same marker loci and the status of
tolerance or susceptibility to
Aceria
mite infestation.
1 Methods and Materials
1.1 Sampling of palms
Sample palms were selected from a pool of Sri Lanka
Yellow Dwarf parental palm population. They were
categorised into four groups, and random sampling
was performed within each group to select a total of
25 experimental palms (Table 1). In addition 01 Sri
Lanka Tall (SLT) palm and 01 Sri Lanka Green Dwarf
(SLGD) palm were included in the study for
comparison purposes.
Table 1 Sampling of palms for molecular evaluation
Group
Number of palms and the
code
Tall-like
Aceria
mite tolerant
Tall-like
Aceria
mite susceptible
Dwarf- like
Aceria
mite tolerant
Dwarf-like
Aceria
mite susceptible
07 (TLMT1 to TLMT7)
05 (TLMS1 to TLMS5)
06 (DLMT1 to DLMT6)
07 (DLMS1 to DLMS7)
Tall stature, larger crown with non-drooping frond tips
and the presence of a root bole were the specific
features used in identifying the tall-like category.
Dwarf stature, smaller crown with drooping frond tips
and the absence of a root bole were the criteria used
for identifying the dwarf-like category.
Palms having more than two damaged nuts in the
crown were classified as
Aceria
mite susceptible,
while the palms with two or less damaged nuts in all
bunches were classified as
Aceria
mite tolerant.
1.2 DNA extraction and PCR amplification
Genomic DNA of the 25 sample palms were extracted
from the spear leaf tissues using a modified C-TAB
DNA extraction method. PCR amplification was
performed with 07 coconut specific microsatellite
markers (Perera et al., 1999; Rivera et al., 1999)
(Table 2). Amplified PCR products were visualized by
Silver staining upon band separation by
poly-acrylamide gel electrophoresis and the genotypes
were scored.
Table 2 Forward (F) and Reverse (R) SSR primer sequences and their annealing temperatures
Primer
Sequence
Annealing Temperature (
o
C)
CAC 8
F 5’- ATC ACC CCAATA CAA GGA CA-3’
57
R 5’- AAT TCT ATG GTC CAC CCA CA-3’
CAC 20
F 5’- CTC ATG AAC CAAACG TTA GA-3’
54
R 5’- CAT CAT ATA CAT ACA TCG AAC A-3’
CAC 23
F 5’- TGAAAA CAAAAG ATA GAT GTC AG-3’
56
R 5’- GAA GAT GCT TTG ATA TGG ACC-3’
CAC 65
F 5’- GAAAAG GAT GTAATAAGC TGG-3’
56
R 5’- TTT GTC CCC AAA TAT AGG TAG-3’
CNZ 12
F 5’- TAG CTT CCT GAG ATAAGA TGC-3’
54
R 5’- GAT CAT GGAACG AAAACA TTA-3’
CNZ 40
F 5’- CTT GAT TGC TAT CTC AAA TGC-3’
53
R 5’- CTG AGA CCAAAT ACC ATG TGT-3’
CNZ 44
F 5’- CAT CAG TTC CAC TCT CAT TTC-3’
53
R 5’- CAA CAAAAG ACA TAG GTG GTC-3’
