International Journal of Horticulture, 2016, Vol.6, No.2, 1-10
2
VTH711/4, Cross G - RP-2 x Kalyanpur Bold Nut, Cross H - M-44/3 x Kalyanpur Bold Nut, Cross I - Vittol- 44/3
x VTH 711/4 and Cross J- BPP-30/1 x Kalyanpur Bold Nut) along with their parents following Rout et al.
(2002).
Each of the above 20 cashew hybrids was designated in terms of alphabetical letters followed by numerical
numbers to refer cross combination and hybrid clone number. The plant materials were homogenized in liquid
Nitrogen and extracted with extraction buffer (1 M Boric acid pH 8.0, 2 mM EDTA, 1.4 M NaCl, 1.5%
Cetyltrimethyl ammonium bromide (CTAB), 0.2% β-mercaptoethanol) at 60
o
C for one hour with occasional
shaking and an equivalent volume of phenol-chloroform-isoamyl alcohol (25:24:1) mixture was added and
centrifuged at 8,000 rpm for 15 min. at 4
o
C. The supernatant was added with equal amount of ice cold absolute
ethanol and kept for overnight to precipitate DNA. The intact genomic DNA was hooked out and washed with
70% ethanol and finally re-dissolved in TE buffer (10 mM Tris-HCl, pH-8.0 and 1mM EDTA). The DNA was
purified by DNase free RNase-A (GeNei, Bangalore, India) @ 20 µg per ml of DNA extract to remove
contaminating RNAs. Finally the quality of DNA was checked using the ratio of absorbance at 260nm and 280nm
and also rechecked by running each sample in 0.8% agarose gel. The DNA was quantified through UV-VIS
Nanodrop-2000 spectrophotometer (Thermo Electron Scientific Instruments LLC, USA) at 260 nm and diluted to
a working concentration of 10 ng/µL for PCR analysis.
Genomic DNA sample of each genotype was individually primed and amplified using 25 random decamer RAPD
primers (Chromos Biotech. Pvt. Ltd., Bangalore, India). The amplification was performed in a reaction volume of
25 µL containing 1X reaction buffer (10 mM Tris HCl, pH 9.0, 1.5 mM MgCl
2
, 50 mM KCl, 0.01% gelatin), 2.5
mM each of dNTPs, 10 ng of single random primer, 20 ng of genomic DNA and 1 unit of Taq polymerase (Genei,
Bangalore). DNA amplification was performed in the Gene-Pro Thermocycler (Bioer Tech. Co., Ltd, Japan),
programmed for 4 min at 94
o
C, 40 cycles of 1 min at 94
o
C, 1 min at 37
o
C and 2 min at 72
o
C and final extension
for 10 min at 72
o
C followed by storing at 4
o
C till loading to the agarose gel. The amplified products were loaded
in 1.6% agarose gel containing 1 µg/ml ethidium bromide and electrophoresed in a constant voltage at 60V. The
amplifications were checked for their reproducibility.
The gels were documented by gel doc system (Fire Reader-Uvtec, Cambridge, UK) for scoring the bands. The
amplification products were checked for their reproducibility using each primer at least twice. Reproducible bands
at about 0.5mm apart or more were considered for scoring. The presence and absence of bands were scored as 1
and 0 respectively across all the genotypes. The size of amplicons was determined by comparing with the lambda
DNA ladder (500bp) with known size (bp) fragments. Polymorphism information content (PIC) and resolving
power (Rp) of a primer were estimated as PIC=∑(1 –pi
2
)/n and Rp=Σ Ιb, where pi is the proportion of genotypes
showing i
th
band amplified by the primer, Ιb (band informativeness) =1–[2×(0.5–pi)] and n = total no. of bands
produced by the primer (Prevost and Wilkinson, 1999).
The binary data matrix of RAPD score was analysed using the multivariate analysis program NTSYS-PC
2.1(2000-01) to estimate Jaccard’s similarity coefficient (Jaccard, 1908) values. The dendrogram was constructed
using Unweighted Paired Group Method with Arithmetic averages (UPGMA) (Sneath and Sokal, 1973)
employing Sequential Agglomerative Hierarchic and Non-overlapping clustering (SAHN).
Results and Discussion
Cashew nut is amenable for molecular analysis owing to its small genome size (1.11pg/2C) (Aliyu, 2012). DNA
profiling using RAPD markers have been standardized and employed successfully by different workers (Samal,
2002; Croxford et al.
,
2005 and Thimmappaiah et al., 2009) to analyze samples of
Anacardium
species. The
success in generating polymorphic loci depends on proper choice of primers for DNA amplification and extent of
genetic variation among the test genotypes. Dhanaraj et al. (2002) used only five primers for DNA amplification
of 90 cashew nut accessions, while only 2-3 primers and even a single primer were sufficient to distinguish
between cultivars of broccoli (Hu and Quaros, 1991) and jack fruit (Gopalsamy et al., 2012) . However, it is
in
vogue
to use more number of primers to differentiate closely related cultivars in cashew nut. In the present
investigation, initially twenty five random primers were examined out of which four RAPD primers gave smeared
