International Journal of Horticulture, 2015, Vol.5, No.21, 1-45
16
1.2.7 PCR Conditions
Twenty arbitrary decamer primers (Bangalore Genei, India) were used for polymerase chain reaction (PCR). PCR
reaction was performed in 20 mL reaction mixture containing 5ng template DNA, 1 unit of Taq DNA polymerase,
100 μM dNTPs, 1.0 μM primer, 2.5 mM MgCl
2,
10 mM Tris-HCl (pH-9.0), 50 mM KCl, 0.01% gelatin. PCR
amplification was performed using a PTC-100 Peltier Thermal Cycler (MJ Research, USA) using the following
conditions: preheating of 4 min at 94
℃
; 45 cycles of 15 sec at 94
℃
, 45 sec at 36
℃
and 1.5 min at 72
℃
and
elongation was completed by a final extension of 4 min at 72
℃
. The final reaction mixture was cooled down to
4
℃
. After amplification, the PCR product was resolved by electrophoresis in 1% agarose gel with 1X TAE buffer.
Bands were visualized by staining with ethidium bromide (0.5
g/mL) under UV light and photographed. Only
distinct bands were counted for data analysis, and faint bands were not considered. The size of the amplification
products was estimated from a 100 bp DNA ladder (Sigma). All the reactions were repeated at least twice and only
those bands reproducible on all runs were considered for analysis.
1.2.8 DATAAnalysis
DNA fragment profiles were scored in binary fashion with ‘0’ indicating absence and ‘1’ indicating presence of
band. Genetic distance was calculated by Jaccard’s coefficient (Jaccard, 1908) which is as follows:S
ij
=N
ij
/(N
ii
+N
ij
+N
jj
)
Where S
ij
is the similarity index between the i
th
and j
th
genotype, N
ij
is the number of bands present in both
genotype, N
ii
is the number of bands present in the i
th
genotype but absent in the j
th
genotype, and N
jj
is the
number of bands absent in the i
th
genotype and present in the j
th
genotype. The similarity matrix was converted to
dissimilarity matrix (1- S
ij
), and a dendrogram was constructed using the Neighbor Joining Tree method using
RAPDistance Package version 2.0(Armstrong et al., 1998).
2 Results
Cytological investigations with special reference to the role of alteration of chromosome morphology in evolution
are well known for a long time. The literature available on these aspects is voluminous. To mention a few,
comparative study of karyotypes in many plants like
Ornithogalum
(Cleland, 1950),
Crepis
(Babcock, 1947),
Lilium
(Stewart, 1948) etc. have revealed interrelationship between species, varieties and even strains. These
aspects were reviewed by Sharma and Sharma (1959). Evidences were presented to show that in all possibilities,
structural changes of chromosomes play a distinct role in the evolution of agricultural strains of crop plants
(Hagberg and Tjio, 1950; Tijio and Hagberg, 1951; Wellhausen, 1994). Cytological investigations were mainly
concentrated for understanding the genetic-evolutionary race history, inter-relationship and breeding of different
species and cultivars of
Alstromeria
(Tsuchiya et al., 1987),
Anthurium
(Marutani et al., 1988; 1993; Sheffer and
Croat, 1983),
Bougainvillea
(Sharma and Bhattacharya, 1960; Ohri and Khoshoo, 1982; Ohri and Zadoo, 1979;
1986; Zadoo et al., 1975a, 1975b, 1975c, 1975d, 1975e, 1975f)
Chrysanthemum
(Tanaka, 1960; Datta and Banerji,
1995; Rana, 1964; Heywood and Humphries, 1977; Nazeer, 1983; Nazeer and Khoshoo, 1982, 1983),
Dianthus
(Brooks, 1960), Marigold (Banerji, 1994; Jalil and Pal, 1980; Jalil and Khoshoo, 1974) and
Polyanthes tuberose
(Ayangar, 1963; Datta and Banerji, 1995; Laxmi et al., 1984; Sato, 1938; Schira and Lanteri, 1986; Sharma and
Ghosh, 1956; Sharma and Bhattacharya, 1956; Sharma and Bal, 1956). No chromosomal changes could be
detected in new ornamental varieties of chrysanthemum developed through induced mutagenesis (Datta and
Banerji, 1995; Datta, 1997).
Heslot (1968) studied the pigments of induced mutants and that of original cultivars of rose and found that usually
the nature of pigments did not alter but the mutants showed either an increase or decrease of one or several of the
pigments found in the control. The anthocyanin pigments in mutant and non-mutant Coleus plants have been
studied by Love and Malone (1967) and they have reported that colour differences between mutant and
non-mutant plants are due to a variation in amount of one anthocyanin pigment rather than change in structure of
the pigment molecule itself. Extensive spectrophotometric studies on pigment analysis in original and gamma ray
induced flower colour mutants of chrysanthemum and rose clearly indicated that somatic flower colour mutations
are due to qualitative and/or quantitative changes in the pigment/s as a result of mutation during pigment
