Rice Genomics and Genetics 2015, Vol.6, No.1, 1-8
7
the PCR amplification consisted ofa total of 35
cycles of initial denaturation (94°C for 4 min)
followed by denaturation (94°C for 1 min),
annealing (57°C for 2 min), elongation (72°C for 2
min) and final elongation (72°C for 15 min). The 1
X Tris-acetate ethylene diamine tetra acetic acid
(TAE) buffer was used to electrophoreses PCR
amplified products in 2.5% (w/v) agarose gel
(Merck Bioscience, India) along with 3 kb ladder
(Himedia Laboratories Pvt. Ltd., Mumbai). The gel
image was documented using gel documentation
system (UVITECH, Cambridge, UK).
Table 1 Pair wise evolutionary divergence of aromatic rice cultivars
Banikunja Basnasapuri Basna-
parijat
Basumati
dhan
Basaomati
(P)
Chatia-
maki-1
Dhoia-
bankoi
Ganjam
local-2
Gatia
Kalikati
-1
Banikunja
1
Basnasapuni
0.0290
1
Basnaparijat
0.0276
0.0230
1
Basumati dhan
0.0263
0.0250
0.0243 1
Basaomati (P)
0.0263
0.0263
0.0249 0.0216
1
Chatiamaki-1
0.0269
0.0262
0.0256 0.0262
0.0229
1
Dhoiabankoi
0.0243
0.0256
0.0196 0.0243
0.0209
0.0216 1
Ganjam local-2 0.0296
0.0216
0.0236 0.0250
0.0256
0.0283 0.0236 1
Gatia
0.0263
0.0203
0.0223 0.0190
0.0236
0.0276 0.0256 0.0223 1
Kalikati-1
0.0269
0.0229
0.0249 0.0243
0.0216
0.0236 0.0203 0.0263 0.0256 1
Note: Bold font indicates minimum and maximum evolutionary divergence between aromatic rice cultivars.
PCR product purification and sequencing
Large-scale amplification was performed to elute
bright PCR fragment. The single bright amplicon of
1500kb was eluted using Gel Extraction Kit
(Genei
TM
).The PCR amplified DNA fragments were
subjected for two way sequencing in order to get the
full sequence and minimize the error in sequencing
and was sequenced by using both 96 capillary high
throughput sequencer; ABI 3730 XL system to
generate sequences with accurate base calling which
is an extension and refinement of Sanger’s
dideoxy method (Sanger et al., 1977) at SciGenom,
Cochin, Kerala, India.
Sequence Data Analysis
The sequenced nucleotide datasets were manually
edited in BioEdit program (version 5.0.9
.
mbio.ncsu.edu/BioEdit/bioedit.html) and used for
further analysis. Alignment of all ten sequences were
toggled using Multiple Sequence Alignment (MSA)
of muscle programme (www. ebi.ac.uk/Tools/MSA/
muscle/) and alignments were visualized in Jalview.
Meanwhile BlastN program was used for preparation
of dataset based on the alignment results of
nucleotide for all related sequences were
downloaded from the
GeneBank
database and
analyzed in MSA along with ten aromatic rice
cultivars, MEGA (Molecular Evolutionary genetic
analysis) version 6 was used for phylogenetic
analysis and calculations of pair wise distances
] (Tamura et al., 2013).
The phylogenetic tree was generated by bootstrap
test Neighbour joining (NJ) with kimura
two-parameter model (Kimura, 1980). The stability
of internal nodes was assessed by bootstrap
analysis with 1000 replicates.
Acknowledgement
The authors wish to acknowledge to Department of
Biotechnology, Government of India for providing financial
assistance under PG teaching HRD program.
References
Ahuja S. C., Panwar D.V.S., Ahuja U., and Gupta K.R., 1995, Basmati
Rice - The Scented Pearl, CCS Haryana Agricultural University,
Hissar, India, p.63
Ammiraju J., Lu F., and Sanyal A., 2008, Dynamic evolution of
Oryza
genomes is revealed by comparative genomic analysis of a genus-wide
vertical data set. Plant Cell, 20: 3191-3209
Brar D.S., and Khush G.S., 1997, Alien introgression in rice. Plant
Molecular Biology, 35: 35-47
