biochemical characterization based on sds-page … · 10 br010 dhola chhota gotia (dcg) u.p. land...
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Corresponding author: S. Shukla, National Botanical Research Institute, Rana Pratap Marg,
Lucknow-226001, U.P., India Tel: 91-522-2297936; Fax: 91-522-2205836; e-mail:
UDC 575.630
DOI: 10.2298/GENSR1503029V Original scientific paper
BIOCHEMICAL CHARACTERIZATION BASED ON SDS-PAGE ANALYSIS AND
CORRELATION AMONG TRAITS IN OPIUM POPPY (Papaver somniferum L.)
GERMPLASM
Nidhi VERMA1, *Sudhir SHUKLA
1, Kusum YADAV
2, Brij K. MISHRA
1 and Anu RASTOGI
1
1Genetics and Plant Breeding, National Botanical Research Institute, Lucknow-226001, U.P., India
2Department of Biochemistry, University of Lucknow, Lucknow-226020, U.P., India
Verma N., S. Shukla, K. Yadav, B. K. Mishra
and A. Rastogi (2015):
Biochemical characterization based on SDS-PAGE analysis and correlation among traits
in opium poppy (Papaver somniferum L.) germplasm.- Genetika, Vol 47, No. 3,1029 -
1050.
This research aimed to investigate the genetic diversity based on the pattern of
seed storage protein and to establish correlation between alkaloid and seed protein
content among 124 germplasm lines of opium poppy (Papaver somniferum L.). Twenty
seven polypeptide bands in range of 6 to 205 kDa were recorded. Similarity index was
calculated by using Jaccard’s Similarity index and cluster analysis was performed using
UPGMA model. Polymorphism was observed in three variable regions i.e., high, medium
and low molecular weight, among which bands of 10.4, 20, 22, 24, 30, 32, 33, 49 and 205
kDa’s were common in all the germplasms but other bands showed variation. All the 124
germplasms were broadly grouped into 13 clusters based on average linkage method.
None of the trait showed significant correlation with seed protein content. The differential
protein profile of the germplasms revealed wide variability and diversity among the
studied germplasms which could be further used in hybridization programme to obtain
maximum gain.
Key words: Alkaloids; Correlation; Opium Poppy; Path analysis; Seed storage
protein
.
INTRODUCTION
Opium poppy (Papaver somniferum L.) is a medicinal plant of immense importance and
one of the cheapest sources for raw opium having more than 80 alkaloids of medicinal value,
among which five alkaloids i.e. morphine, codeine, thebaine, narcotine and papaverine are the
1030 GENETIKA, Vol. 47, No.3, 1029-1050, 2015
major ones (FRICK et al., 2005; SHUKLA et al., 2006, 2010). However, raw opium is used as
sedative, antispasmodic, hypnotic and sudorific is also used in diarrhea and dysentery (MISHRA et
al., 2010; CHATERJEE et al., 2010). Apart from being used mainly for pharmaceutical purposes,
poppy seeds are invaluable source of plant based protein supplement for human consumption
(AZCAN et al., 2004; OZCAN and ATALAY, 2006). Poppy seeds contain protein upto 24% and are
used whole or ground as an ingredient in many foods (SINGH et al., 1995, 1998). India has a rich
diversity of opium poppy germplasms (SINGH et al., 1997). The efficient management and
utilization of germplasms requires detailed knowledge of genetic diversity of economic traits for
proper characterization of populations to facilitate designing of efficient breeding strategies to
accomplish specific objectives (SHUKLA et al., 2010). Morphological characterization is the first
step in description and classification of germplasm (BHARGAVA et al., 2007, 2008). Several
attempts have been made to study the diversity in opium poppy based on morphological traits (LAL
et al., 1996; BHANDARI et al., 1997; SAINI and KAICKER, 1987; SINGH et al., 1998, 2003, 2004;
TIWARI et al., 2001). However the efforts to assess the diversity based on chemotypic traits were
also done (SHUKLA et al., 2006, 2010; YADAV et al., 2006). But, it is quite difficult to identify
potentially distinct germplasm lines based only on morphological traits as these are easily
influenced by the environment and thus less reliable. This has led to the development of new stable
parameters such as use of their genetic material (nucleic acids and proteins) as a tool for
varietal/germplasm identification (CRUZ et al., 1994; TAMKOC and ARSLAN, 2010). Many tools are
now available for studying variability and relationships among germplasms/accessions that include
seed protein electrophoresis, isozymes and various types of molecular markers (DOYLE et al.,
1992). Seed proteins are used as biomarkers because they are physiologically stable, easy to
handle and direct gene products where environment has little influence. Seed protein analysis
through sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) is widely used
to resolve inter or intra specific genetic diversity studies (OMONHINMIN and OGUNBODEDE, 2013)
identification and characterization of accessions, and phylogenetic relationship studies of the
accessions (TAMKOC and ARSLAN, 2010). Seed protein variations also provide information on the
relationship among the seeds collected from various geographical regions (SHOTWELL and
LARKINS, 1989; MURPHY et al., 1990; ANWAR et al., 2003; JAVAID et al., 2004). Since so much
efforts have been done to characterize the available germplasms of opium poppy, but till to date
the characterization based on molecular marker is not done. So the present study was undertaken
with the objectives (i) to gather the reliable information of genetic diversity in available
germplasms of opium poppy (ii) to study the association of seed protein with opium and seed yield
and its alkaloids.
MATERIALS AND METHODS
Plant Material
The experimental material for the study comprised of dry seeds of 124 distinct germplasm
lines of opium poppy (Papaver somniferum L.) maintained at Dept. of Genetics and Plant
Breeding, National Botanical Research Institute, Lucknow, India. The description and origin of
each germplasm is presented in Table 1. The germplasm lines were evaluated in randomized block
design with three replications. The rows of each germplasm per replication were grown with row
to row distance 30cm and plant to plant 10cm. Each row was three meter in length.
N. VERMA et al.: CHARACTERIZATION AND CORRELATIONIN OPIUM POPPY 1031
HPLC Analysis
The pooled latex (opium) of four lancing of five plants from each germplasm per
replication was collected. Similarly the seeds of five plants per replication of each germplasm were
collected and weighed. The mean data of of opium yield/plant (mg) and seed yield/plant (g) were
used for statistical analysis. Five major alkaloids viz. morphine, codeine, thebaine, narcotine and
papaverine in opium latex were quantified through HPLC, Waters (Milford, USA) following the
method suggested by KHANNA and SHUKLA (1986).
Table1. Source and origin of 124 germplasm lines of opium poppy (P. somniferum L.)
S. No. Germplasm Collection Origin Source
1 BR001 Ghazipur (GZ) U.P. Land race
2 BR002 Bhakua (BK) U.P. Land race
3 BR003 Kalidandi(KD) U.P. Cultivar
4 BR004 MOP- 47 Raj. Land race
5 BR005 NBRI 2 (NB-2) NBRI Selection
6 BR006 Sugapankhi (SP) U.P. Land race
7 BR007 Kali Dandi Baunia (KDB) U.P. Land race
8 BR009 M -11 U.P. Land race
9 BR008 Telia (TE) U.P. Land race
10 BR010 Dhola Chhota Gotia (DCG) U.P. Land race
11 BR275 NC-57923 NBPGR Selection
12 BR271 NC-57913 NBPGR Selection
13 BR286 NC-57958 NBPGR Selection
14 BR306 MOP-217 M.P.(Mandsaur) Selection
15 BR285 NC-57950 NBPGR Selection
16 BR284 NC-57948 NBPGR Selection
17 BR311 ND -7 NDAUT Selection
18 BR283 NC-57947 NBPGR Selection
19 BR282 NC-57945 NBPGR Selection
20 BR281 NC-57936 NBPGR Selection
21 BR280 NC-57934 NBPGR Selection
22 BR279 NC-57932 NBPGR Selection
23 BR278 NC-57928 NBPGR Selection
24 BR277 NC-57925 NBPGR Selection
25 BR276 NC-57924 NBPGR Selection
26 BR329 Ran Jhatak Raj. Landrace
27 BR027 Te x M-11 U.P. Sel. Intra-specific
28 BR321 Dholia Raj. Landrace
29 BR272 NC-57916 NBPGR Selection
30 BR273 NC-57919 NBPGR Selection
31 BR274 NC-57921 NBPGR Selection
32 BR320 NBPGR-11 NBPGR Selection
33 BR292 UO-1185 Raj.(Udaipur) Selection
1032 GENETIKA, Vol. 47, No.3, 1029-1050, 2015
34 BR294 UO-1385 Raj.(Udaipur) Selection
35 BR293 UO-1285 Raj.(Udaipur) Selection
36 BR295 UO-1485 Raj.(Udaipur) Selection
37 BR296 UO-1585 Raj.(Udaipur) Selection
38 BR297 UO-1785 Raj.(Udaipur) Selection
39 BR298 UO-1885 Raj.(Udaipur) Selection
40 BR299 UO-1985 Raj.(Udaipur) Selection
41 BR304 IC-140 NBPGR Selection
42 BR301 IC-128 NBPGR Selection
43 BR290 UO-590 Raj.(Udaipur) Selection
44 BR289 UO-490 Raj.(Udaipur) Selection
45 BR288 UO-290 Raj.(Udaipur) Selection
46 BR287 UO-190 Raj.(Udaipur) Selection
47 BR303 IC-133 NBPGR Selection
48 BR300 IC-30 NBPGR Selection
49 BR302 IC-131 NBPGR Selection
50 BR331 GP-74 NBRI Sel. Inter-specific
51 BR293 UO-1285 Raj.(Udaipur) Selection
52 BR310 MOP-1072 M.P.(Mandsaur) Selection
53 BR314 ND-1186 NDAUT Selection
54 BR317 NBPGR-3 NBPGR Selection
55 BR305 MOP-4 M.P.(Mandsaur) Selection
56 BR309 MOP-1047 M.P.(Mandsaur) Selection
57 BR315 NDHS-101 NDAUT Selection
58 BR316 NDHS-201 NDAUT Selection
59 BR319 NBPGR-5 NBPGR Selection
60 BR318 NBPGR-4 NBPGR Selection
61 BR322 Shayama CIMAP Selection
62 BR323 Shweta CIMAP Selection
63 BR324 Sanchita CIMAP Selection
64 BR325 Vivek CIMAP Selection
65 BR334 Big Cap CIMAP Selection
66 BR222 Papline NBRI. Selection
67 BR220 NBRI-1 NBRI Sel. Intra-specific
68 BR326 NBRI-5 NBRI Sel. Intra-specific
69 BR327 NBRI-6 NBRI Sel. Intra-specific
70 BR328 BROP-1 NBRI Sel. Intra-specific
71 BR307 MOP-541 M.P.(Mandsaur) Selection
72 BR308 MOP-576 M.P.(Mandsaur) Selection
73 BR312 ND-1001 NDAUT Selection
74 BR313 ND-1002 NDAUT Selection
75 BR291 UO-601 Raj.(Udaipur) Selection
76 BR046 IS-1 NBRI Sel. Inter-specific
77 BR047 IS-2-1 NBRI Sel. Inter-specific
78 BR059 IS-2-12 NBRI Sel. Inter-specific
N. VERMA et al.: CHARACTERIZATION AND CORRELATIONIN OPIUM POPPY 1033
79 BR048 IS-3 NBRI Sel. Inter-specific
80 BR049 IS-4 NBRI Sel. Inter-specific
81 BR050 IS-5 NBRI Sel. Inter-specific
82 BR051 IS-6 NBRI Sel. Inter-specific
83 BR052 IS-7 NBRI Sel. Inter-specific
84 BR053 IS-8 NBRI Sel. Inter-specific
85 BR054 IS-9 NBRI Sel. Inter-specific
86 BR055 IS-10 NBRI Sel. Inter-specific
87 BR056A IS-11A NBRI Sel. Inter-specific
88 BR056B IS-11B NBRI Sel. Inter-specific
89 BR057 IS-12 NBRI Sel. Inter-specific
90 BR058 IS-13 NBRI Sel. Inter-specific
91 BR060 IS-15 NBRI Sel. Inter-specific
92 BR061 IS16 NBRI Sel. Inter-specific
93 BR062 IS-17 NBRI Sel. Inter-specific
94 BR063 IS-18 NBRI Sel. Inter-specific
95 BR064 IS-19 NBRI Sel. Inter-specific
96 BR065 IS-20 NBRI Sel. Inter-specific
97 BR066 IS-21 NBRI Sel. Inter-specific
98 BR067 IS-22 NBRI Sel. Inter-specific
99 BR068 IS-23 NBRI Sel. Inter-specific
100 BR113 Rajasthan Raj. Selection
101 BR330 NBRI-11 NBRI Sel. Intra-specific
102 BR223 Aphuri U.P. Landrace
103 BR247 Jhalawar (Raj.) Raj. Selection
104 BR-248 Jhalawar (Raj.) Raj. Selection
105 BR-249 Jhalawar (Raj.) Raj. Selection
106 BR-250 Jhalawar (Raj.) Raj. Selection
107 BR-251 Jhalawar (Raj.) Raj. Selection
108 BR-253 Garoth (M.P.) M.P. Selection
109 BR-254 Pratapgarh (Raj.) Raj. Selection
110 BR-255 Pratapgarh (Raj.) Raj. Selection
111 BR-256 Pratapgarh (Raj.) Raj. Selection
112 BR-257 Pratapgarh (Raj.) Raj. Selection
113 BR-258 Kota (Raj.) Raj. Selection
114 BR-259 Kota (Raj.) Raj. Selection
115 BR-260 Neemuch II (M.P.) M.P. Selection
116 BR-261 Mandsaur III (M.P.) M.P. Selection
117 BR-262 Mandsaur III (M.P.) M.P. Selection
118 BR064 Chittor III (Raj.) Raj. Selection
119 BR-265 Chittor III (Raj.) Raj. Selection
120 BR-266 Jaora I (M.P.) M.P. Selection
121 BR-267 Jaora I (M.P.) M.P. Selection
122 BR-268 Mandsaur I (M.P.) M.P. Selection
1034 GENETIKA, Vol. 47, No.3, 1029-1050, 2015
123 BR-269 Mandsaur I (M.P.) M.P. Selection
124 BR-270 Mandsaur I (M.P.) M.P. Selection
CIMAP Central Institute of Medicinal and Aromatic Plants, Lucknow, U.P.
NBPGR National Bureau of Plant Genetic Resources, New Delhi
NBRI National Botanical Research Institute, Lucknow, U.P.
NDAUT Narendra Deva University of Agriculture and Technology, Faizabad, U.P.
M.P. Madhya Pradesh, Raj. Rajasthan
Protein Extraction
For the extraction of protein, 50mg defatted seeds of each opium poppy germplasm were
crushed and grounded into fine powder in liquid nitrogen using mortar and pestle. Grounded
samples were taken in a 1.5 ml eppendorf and 1ml of ‘Modified Laemmli SDS extraction Buffer’
(LAEMMLI, U.K. 1970) was added in each sample, sonicated for 15 minutes and centrifuged at
15,000 rpm for 10 minutes at 40C temperature. The supernatant was taken and pellet was
discarded. 10µL of Protease Inhibitor (PI) was added in each 500µL of sample supernatant, kept it
for overnight and centrifuged again at 15,000 rpm for 10 minutes at 40C. The final clear
supernatant of treated sample was taken in another eppendorf for further quantification.
Protein Quantification
The concentration of the extracted protein samples were determined by Bradford assay
(BRADFORD, 1976) using different concentrations of the samples against control in microtitre plate
(ELISA plate). Relative concentrations of all the samples were calculated using the following
formula at 595nm.
Protein in Sample (µg/µL) = (O.D. obs – O.D. blank) * Standard Factor
µL of sample taken
Where, Standard factor = 18.3 calculated as per the calculations based on standard.
Protein Profiling
Protein profiling of extracted samples were done through SDS-PAGE using 15%
separating gel and 5% stacking gel. Fixed amount of protein samples were loaded into the stacking
gel wells with prior heating of samples at 90-950C for 15 minutes. Electrophoresis was carried out
at 28mA and 150mV. The gels were stained with 0.25% (w/v) Coomassie brilliant blue R250
solution overnight and destained with methanol and acetic acid for about three hours and were
scanned for binary analysis of bands.
N. VERMA et al.: CHARACTERIZATION AND CORRELATIONIN OPIUM POPPY 1035
Protein Imaging and Data analysis
RF value and molecular weight of each protein band was determined using standard
molecular marker ranged from 3-205 kDa. Electropherogram for each germplasm was scored and
the presence [1] and absence [0] of each band was noted. Presence and absence of bands were
entered in the binary data matrix. Based on the results of electrophoretic band spectra, similarity
index was calculated by using Jaccard’s Similarity index through SPSS software. Further a
dendrogram was constructed using NTSYS software based on the UPGMA model (1973).
I n the present study attempt was made to characterize 124 opium poppy germplasm using SDS-
PAGE protein profiling of the seed storage proteins, simultaneously association of seed protein
content with opium, seed yield and alkaloid content were also established. The results showed that
amount of protein in different germplasm lines ranged from 0.610 to 2.530µg/µl. The highest
protein content was observed in germplasm BR56A followed by BR269, BR270 and BR266
(Table 2). The seed yield and opium yield per plant ranged from 0.55 to 5.13 g/plant and 222.67 to
2573.33 mg/plant respectively. The amount of morphine ranged from 8.82 to 17.59%, codeine
from 0.89 to 4.00%, thebaine from 0.92 to 8.06%, narcotine from 1.24 to 11.40% and papaverine
from 0.00 to 5.16 %.
The seed proteins of all the germplams were separated by SDS-PAGE electrophoresis and
the gel pictures of electrophoresis were used for binary analysis (Figure 1).
Table 2. Total alkaloids, seed and opium yield per plant and seed protein content in different
germplasm lines of opium poppy (P.somniferum L.)
S.S. No. Germplasm M% C% T% N% P%
SY
(g/plt)
OY
(mg/plt)
SP (µg/µl)
1 BR001 12.46 4.23 2.00 11.36 0.00 2.61 187.00 1.590
2 BR002 15.96 3.72 1.50 10.54 0.00 3.64 216.00 1.790
3 BR003 17.33 3.47 1.75 10.31 0.60 3.45 169.33 1.530
4 BR004 15.08 2.26 1.92 10.96 0.00 3.42 192.67 1.590
5 BR005 20.79 1.82 1.52 9.76 0.00 3.16 173.53 1.730
6 BR006 15.33 2.95 1.93 9.59 0.00 2.78 162.00 1.720
7 BR007 14.35 2.73 2.06 8.38 0.00 2.54 243.33 1.360
8 BR009 15.96 3.19 2.12 10.56 0.00 3.10 226.40 1.709
9 BR008 12.37 2.80 1.61 9.79 0.00 3.70 156.40 1.550
10 BR010 12.72 3.87 1.93 8.93 0.00 5.13 171.33 1.750
11 BR275 11.68 4.51 2.32 10.34 0.00 4.29 187.23 1.570
12 BR271 13.06 3.37 2.47 9.99 0.00 2.86 218.33 1.820
13 BR286 15.68 3.43 1.58 7.67 0.00 3.73 180.00 1.360
14 BR306 14.26 2.81 1.54 9.94 0.63 1.20 82.00 1.400
15 BR285 14.87 3.48 1.38 10.80 0.80 2.80 150.13 0.732
16 BR284 18.45 3.04 5.30 17.92 4.08 3.77 197.50 1.270
1036 GENETIKA, Vol. 47, No.3, 1029-1050, 2015
17 BR311 14.49 4.44 1.69 13.12 1.67 2.50 162.90 1.610
18 BR283 13.99 4.69 2.90 15.34 5.27 2.97 171.20 1.430
19 BR282 11.36 6.76 4.85 15.09 5.78 2.99 197.40 1.990
20 BR281 14.48 4.38 7.03 12.70 6.04 2.25 224.00 2.450
21 BR280 16.79 5.19 2.13 13.40 2.30 1.66 189.40 2.210
22 BR279 18.65 3.23 3.97 10.10 3.49 2.83 137.30 2.404
23 BR278 16.62 2.59 3.30 3.41 0.00 3.88 242.60 2.050
24 BR277 13.51 4.27 2.17 6.92 0.14 2.69 243.80 1.940
25 BR276 14.35 4.89 1.57 8.11 2.60 2.69 215.50 2.220
26 BR329 18.31 3.62 1.69 8.28 0.33 2.73 393.20 1.680
27 BR027 14.62 5.04 2.91 9.23 0.00 3.30 259.50 1.850
28 BR321 14.33 5.84 2.46 8.95 0.00 2.76 235.70 1.850
29 BR272 20.40 2.56 1.86 9.69 0.30 2.70 213.50 1.840
30 BR273 17.60 3.03 2.05 8.68 0.00 2.13 162.80 1.830
31 BR274 18.96 4.20 1.95 10.67 0.00 2.21 160.80 2.203
32 BR320 17.83 2.94 1.83 10.08 0.00 2.60 119.50 2.020
33 BR292 18.51 3.20 1.44 11.37 0.00 1.89 112.40 2.002
34 BR294 15.86 2.37 2.44 8.37 2.79 2.53 157.40 2.310
35 BR293 12.62 4.96 3.12 8.21 0.36 3.07 200.40 2.130
36 BR295 18.76 3.48 3.66 6.11 0.00 3.07 229.00 2.130
37 BR296 16.87 3.28 2.03 11.04 3.89 3.62 175.80 1.440
38 BR297 17.73 3.10 1.31 9.66 1.39 3.65 136.50 1.570
39 BR298 19.25 2.89 1.52 8.98 0.23 2.89 150.70 1.550
40 BR299 16.33 3.16 2.50 9.58 0.26 3.65 227.33 1.760
41 BR304 16.45 4.68 4.52 5.90 0.59 3.05 126.33 1.250
42 BR301 12.98 3.48 3.52 9.16 0.59 2.51 248.00 2.220
43 BR290 14.70 3.43 3.18 7.97 2.22 3.16 211.33 1.650
44 BR289 18.70 2.48 2.66 10.30 3.97 2.94 205.67 1.380
45 BR288 18.77 1.85 1.34 7.99 1.56 2.69 184.50 1.630
46 BR287 14.27 2.01 2.57 7.89 2.18 4.07 154.80 1.870
47 BR303 13.39 2.10 1.84 8.40 1.86 2.17 164.40 1.620
48 BR300 18.43 2.75 1.31 13.94 0.50 3.20 195.50 1.840
49 BR302 15.24 2.97 2.52 13.88 0.25 2.49 266.00 1.360
50 BR331 15.41 3.48 3.16 11.50 0.57 3.33 191.30 1.770
51 BR293 14.82 3.68 3.01 6.67 1.19 2.11 247.10 1.810
52 BR310 12.79 2.28 2.28 5.51 5.06 2.58 186.00 1.660
N. VERMA et al.: CHARACTERIZATION AND CORRELATIONIN OPIUM POPPY 1037
53 BR314 14.42 3.21 3.16 8.21 4.06 2.16 194.50 1.600
54 BR317 15.00 2.77 2.23 7.89 0.59 4.34 193.40 2.000
55 BR305 12.12 3.27 2.33 10.31 3.19 2.75 270.37 2.220
56 BR309 15.18 3.47 2.45 6.06 0.27 3.25 213.07 2.260
57 BR315 13.69 3.50 8.37 8.08 0.00 2.39 215.37 1.290
58 BR316 14.10 3.89 3.49 9.26 0.00 3.31 230.50 1.800
59 BR319 20.86 3.66 1.80 8.21 0.00 2.48 257.40 1.860
60 BR318 17.15 3.44 2.67 8.95 0.00 2.29 159.80 1.710
61 BR322 17.66 3.10 1.60 8.22 0.00 2.12 232.00 1.750
62 BR323 20.69 3.24 2.03 7.93 0.00 2.39 157.70 1.790
63 BR324 17.35 3.39 2.37 10.81 0.00 3.02 163.50 1.920
64 BR325 17.28 3.42 1.16 8.00 0.28 2.75 179.10 1.480
65 BR334 11.66 3.04 1.94 8.32 5.98 4.01 128.70 0.890
66 BR222 14.25 3.14 1.22 6.50 0.00 3.92 260.50 1.520
67 BR220 17.73 3.40 1.71 9.11 1.42 2.66 288.40 0.810
68 BR326 14.03 3.00 1.12 5.06 1.53 3.67 303.00 1.660
69 BR327 12.52 4.06 1.12 3.74 0.00 3.27 203.13 0.620
70 BR328 15.39 3.48 2.04 4.88 3.72 2.53 207.50 0.680
71 BR307 10.78 2.99 1.00 4.68 0.00 2.14 245.50 1.620
72 BR308 9.20 3.72 0.70 5.31 0.00 2.49 257.33 0.610
73 BR312 13.78 3.11 1.27 6.28 0.00 3.26 211.50 1.470
74 BR313 15.93 3.03 1.76 4.33 3.86 3.02 162.10 1.990
75 BR291 14.82 3.07 1.47 8.39 2.03 2.36 218.50 1.540
76 BR046 14.52 4.57 2.37 7.82 0.14 0.63 223.97 1.480
77 BR047 10.49 2.63 4.03 3.29 2.30 0.58 185.00 1.520
78 BR059 11.47 3.64 2.80 8.11 0.00 2.18 125.00 1.520
79 BR048 11.98 3.71 1.55 5.54 2.68 3.22 231.00 2.060
80 BR049 12.92 4.92 2.19 7.84 0.61 3.52 208.00 2.360
81 BR050 11.02 3.39 2.67 8.75 4.20 3.42 215.70 1.570
82 BR051 9.35 4.79 2.22 7.61 0.95 4.91 183.33 1.700
83 BR052 15.91 2.40 2.44 6.57 0.00 4.64 178.60 1.890
84 BR053 16.28 2.37 1.62 9.47 0.00 1.92 216.00 1.420
85 BR054 13.00 2.73 2.67 7.19 4.77 2.64 264.30 1.800
86 BR055 14.90 2.61 1.37 9.95 0.00 2.66 149.50 1.780
87 BR056A 17.16 2.76 1.08 8.04 0.00 2.25 130.33 2.520
88 BR056B 16.03 2.71 2.83 8.43 3.10 2.31 192.33 2.370
1038 GENETIKA, Vol. 47, No.3, 1029-1050, 2015
89 BR057 14.27 2.29 0.77 11.36 2.61 2.64 141.30 1.640
90 BR058 11.99 3.98 3.05 12.99 4.07 2.79 171.60 1.080
91 BR060 15.13 3.40 0.72 10.70 0.32 3.79 153.50 1.590
92 BR061 16.14 2.88 1.96 7.84 0.67 3.23 237.00 1.402
93 BR062 11.47 1.95 0.81 7.34 0.66 2.77 263.60 2.020
94 BR063 12.64 1.74 0.61 6.31 0.00 3.21 218.60 1.705
95 BR064 11.03 2.16 1.74 5.72 0.00 3.03 173.30 1.910
96 BR065 11.64 1.68 2.10 8.75 0.00 4.14 120.00 2.030
97 BR066 10.50 2.45 2.79 6.34 0.00 2.87 126.20 1.970
98 BR067 16.19 1.96 1.55 3.78 0.00 2.28 146.00 2.180
99 BR068 16.64 3.54 1.64 5.84 1.29 2.42 159.67 2.170
100 BR113 18.48 3.03 1.68 5.66 1.35 1.86 137.60 1.600
101 BR330 18.68 2.09 1.08 5.41 0.25 3.54 134.33 1.500
102 BR223 20.26 1.68 0.82 5.40 0.51 4.71 112.53 2.200
103 BR247 16.11 2.00 0.78 6.82 3.30 3.12 158.67 1.900
104 BR-248 15.62 3.69 1.26 5.31 1.53 3.79 134.73 1.900
105 BR-249 16.01 3.44 1.32 4.55 2.21 3.05 142.67 1.800
106 BR-250 16.44 2.07 1.23 6.51 1.17 4.00 131.33 1.300
107 BR-251 17.30 2.21 0.85 4.76 2.81 3.78 138.13 1.800
108 BR-253 16.35 2.65 1.16 5.45 2.37 1.74 152.13 1.600
109 BR-254 15.09 3.17 1.07 5.44 2.58 2.80 159.87 1.400
110 BR-255 17.80 3.02 0.91 7.18 0.09 3.12 164.80 1.800
111 BR-256 19.38 1.57 1.04 6.37 0.06 2.33 192.73 1.400
112 BR-257 18.49 3.16 1.11 6.48 1.50 3.38 151.53 2.000
113 BR-258 15.09 3.26 1.35 5.53 1.40 1.82 152.47 1.600
114 BR-259 17.90 3.58 1.75 4.42 2.43 2.74 200.83 1.400
115 BR-260 16.82 3.60 1.55 5.80 0.88 3.13 151.33 1.300
116 BR-261 18.21 3.61 1.57 5.58 1.47 2.74 143.60 1.600
117 BR-262 17.72 2.95 0.89 5.17 0.00 2.72 151.17 1.600
118 BR064 14.55 2.49 0.61 6.11 2.19 4.96 191.90 1.400
119 BR-265 15.87 2.52 0.80 5.78 0.36 3.59 149.67 2.000
120 BR-266 17.93 2.74 1.12 6.35 0.94 3.11 125.33 2.500
121 BR-267 18.13 3.01 1.26 3.70 1.50 2.93 220.00 1.900
122 BR-268 15.15 1.66 0.86 7.04 2.27 3.04 200.00 1.800
123 BR-269 17.63 3.06 1.09 3.47 1.37 2.92 392.67 2.500
124 BR-270 15.08 1.62 0.90 7.14 2.31 2.95 396.20 2.500
N. VERMA et al.: CHARACTERIZATION AND CORRELATIONIN OPIUM POPPY 1039
Mean+
SE
15.43+
0.23
3.20+
0.08
2.03+
0.10
8.13+
0.24
1.26+
0.14
2.95+
0.07
191.66+
4.80
1.73+
0.03
M, morphine; C, codeine; T, thebaine; N, narcotine; P, papaverine; SY, seed yield (g) per plant; OY, opium yield (mg)
per plant; SP, seed protein (µg) per µl
a) (b) (c)
(d) (e) (f)
(g) (h) (i)
Contd.
1040 GENETIKA, Vol. 47, No.3, 1029-1050, 2015
(j) (k) (l)
(m) (n) (o)
Figure 1. Seed protein pattern in 124 germplasm lines of opium poppy (P. somniferum L.) (a-o)
Genetic Diversity Evaluation
The protein profiling of 124 germplasms gave a total of 27 bands. Polymorphism was
observed in three variable regions i.e., high, medium and low molecular weight, among which
bands of 10.4, 20, 22, 24, 30, 32, 33, 49 and 205 kDa’s were common in all the germplasms while
other bands showed variation. The protein profile of the germplasm also showed that seeds of most
of the germplasm lines contained majorly low molecular weight proteins (<75kDa). Some protein
bands showed high expressions and their expression pattern also varied in different germplasm
lines. It was noticed that germplasm lines BR-321, BR-320, BR-287, BR-302, BR-331, BR-247,
BR-249, BR-268 and BR-269 do not have higher molecular weight proteins. The germplasms
namely BR-001, BR-002, BR-006, BR-007, BR-009, BR-008, BR-276, BR-029, BR-027, BR-273,
BR-320, BR-292, BR-293 and BR-049 possessed majorly expressing seed protein bands of 49kDa,
43 kDa, 33 kDa, 32 kDa and 30 kDa. The protein bands of size 26 kDa, 25kDa and 27kDa were
unique bands which were present in few germplasm lines viz., BR-283, BR-282, BR-272, BR-273,
BR-320, BR-298, BR-299, BR-304, BR-301, BR-290, BR-289, BR-309, BR-315, BR-308, BR-
249, BR-250, BR-251, BR-253, BR-254, BR-256, BR-257, BR-258, BR-259, BR-260, BR-261,
N. VERMA et al.: CHARACTERIZATION AND CORRELATIONIN OPIUM POPPY 1041
BR-262, BR-064, BR-265, BR-266, BR-267, BR-268 and BR-270. The 14.3kDa band was a
unique band with low molecular weight present in germplasms BR-319, BR-318, BR-322, BR-
323, BR-324, BR-325, BR-312, BR-313, BR-291, BR-046, BR-047, BR-059, BR-048, BR-049,
BR-050, BR-051, BR-056B, BR-057, BR-068 and BR-248 only.
Cluster Analysis
All the 124 germplasms were broadly grouped into 13 clusters based on average linkage
method (Figure 2). The mean values of germplasms falling in each cluster are presented in Table
2. Cluster I had 17 different germplasm lines and Cluster II had largest number of germplasms
(44). Cluster III, IV and V comprised of 32, 5 and 2 germplasm lines respectively. Cluster VI had
six germplasms and cluster VII had two germplasms while cluster VIII and IX had one germplasm
in each. Cluster X had five germplasms and Clusters XI, XII and XIII had three germplasms in
each cluster.
Figure 2. Dendrogram showing 13 major and various other sub clusters in opium poppy (P. somniferum L.).
1042 GENETIKA, Vol. 47, No.3, 1029-1050, 2015
Similarity Index
The similarity index between germplasm ranged from 0-1%. Based on the similarity
index, the similarities between germplasms is classified as exact similar (1.00), very similar (0.9
and 0.8), moderate similar (0.7 and 0.6) and less similar (<0.3).
Correlation Studies
Table 3. Genotypic and phenotypic (in parenthesis) correlation among various traits in opium poppy (P.
somniferum L.) Characters Codeine Thebaine Narcotine Papaverine Seed Yield
(mg)
Opium Yield
(mg)
Seed Protein (µg/µl)
Morphine -0.3422**
(-0.2679)
0.0183
(0.0529)
0.1396
(0.1852)
-0.3673**
(-0.3150)
0.0908
(0.0770)
-0.4022**
(-0.3677)
-0.1196
(-0.1097)
Codeine 0.0716
(0.0823)
-0.0509
(-0.0207)
-0.1761
(-0.1557)
-0.1830
(-0.1517)
0.0763
(0.0695)
-0.1177
(-0.1078)
Thebaine 0.1526
(0.2099)
0.1001
(0.1099)
-0.2004
(-0.1651)
0.0127
(0.0112)
-0.0224
(-0.0205)
Narcotine 0.1608
(0.1681)
-0.0222
(0.0016)
0.1284
(0.1154)
0.0049
(0.0053)
Papaverine -0.0002
(0.0086)
0.2131*
(0.2055)
0.1214
(0.1188)
Seed Yield (mg)
0.1381 (0.1191)
0.0224 (0.0202)
Opium
Yield (mg)
0.1849
(0.1840)
* & ** Significance at 5% and 1% respectively
The genotypic correlation coefficient between different pair was similar in sign and
nature to the corresponding phenotypic correlation coefficient (Table 3). However, genotypic
correlation coefficient was generally higher in magnitude for all the traits than the corresponding
phenotypic correlation coefficient. In the present study, none of the trait showed significant
correlation with seed protein content. Seed protein was negatively correlated with morphine (-
0.1196), codeine (-0.1177) and thebaine (-0.0224) while positively correlated with narcotine
(0.0049), papaverine (0.1214), seed yield (0.0224) and opium yield (0.1849). Seed yield was
positively correlated with morphine (0.0908), opium yield (0.1381) and seed protein (0.0224)
while negatively correlated with codeine (-0.1830), thebaine (-0.2004), narcotine (-0.0222) and
papaverine (-0.0002). Opium yield had positive significant correlation with papaverine (0.2131)
and negative significant correlation with morphine (-0.4022) while positive correlation with all the
traits. Morphine had negative significant correlation with codeine (-0.3422), papavarine (-0.3673)
and opium yield (-0.4022) while had positive but non-significant correlation with thebaine
(0.0183), narcotine (0.1396) and seed yield (0.0908). Codeine had positive correlation with
thebaine (0.0716) and opium yield (0.0763). Thebaine had positive correlation with narcotine
N. VERMA et al.: CHARACTERIZATION AND CORRELATIONIN OPIUM POPPY 1043
(0.1526), papaverine (0.1001) and opium yield (0.0127) while narcotine had positive correlation
with papaverine (0.1608), opium yield (0.1284) and seed protein (0.0049). Papaverine had
significant negative correlation with morphine (-0.3673) but positive correlation with opium yield
(0.2131).
Path coefficient analysis
Figure 3. Path diagrams showing path coefficients and correlation of Seed protein, Seed yield and Opium
yield with different traits in Opium Poppy (P.somniferum L.)
1044 GENETIKA, Vol. 47, No.3, 1029-1050, 2015
Correlation coefficient measure the mutual association between two variables but doesn’t
permit the cause and effect relationship of traits contributing directly or indirectly towards the
economic yield, whereas path coefficient is partially standardized regression coefficient and as
such measure the direct influence of one variable upon another and specifies the causes and
measure their relative importance (SINGH et al., 2004). Path coefficient analysis was carried out as
direct and indirect effect using genotypic correlation coefficient among various characters to
estimate the direct and indirect effect of seven characters of manifestation on seed protein content,
seed yield and opium yield separately (Figure 3). Path analysis based on seed protein content
exhibited that morphine, codeine, thebaine, narcotine and seed yield had negative direct effect
towards seed protein while morphine and seed yield indirectly affected via codeine but codeine,
thebaine and narcotine indirectly affected via seed yield, however seed protein exhibited negative
genotypic correlation. Papaverine and opium yield had positive direct effect towards seed protein
while indirectly affected via narcotine, however seed protein exhibited positive genotypic
correlation. The path analysis based on genotypic correlation of seed yield showed morphine,
narcotine and opium yield had positive direct effect towards seed yield while morphine and opium
yield indirectly affected via narcotine, however seed yield exhibited positive genotypic correlation.
Codeine, thebaine, papaverine and seed protein had negative direct effect towards seed yield while
codeine and thebaine indirectly affected via seed protein, however seed yield exhibited negative
genotypic correlation except for seed protein. Path analysis of opium yield revealed that morphine
and codeine had negative direct effect towards opium yield while morphine indirectly affected via
codeine and papaverine, however opium yield exhibited negative genotypic correlation with
morphine and positive with codeine. Thebaine, narcotine, papaverine, seed yield and seed protein
had positive direct effect towards opium yield while narcotine and seed yield indirectly affected
via morphine, however opium yield exhibited positive genotypic correlation.
DISSCUSSION
The frequent occurrences of insufficient discrimination of germplasms by grow out test which
consequently results inability to confirm distinctness. This fact encouraged us to investigate
complementary method of characterizing germplasms based on the comparison of genetic diversity
following conventional method. Seed protein electrophoresis was performed by total soluble seed
proteins separated by SDS-PAGE electrophoresis. The knowledge of genetic diversity is a useful
tool in gene-bank management and breeding experiments like tagging of germplasm, identification
and/or elimination of duplicates in the gene stock, establishment of core collections and sorting of
populations for genome mapping experiments (KAGA et al., 1996). Since many cultivars are
closely and genetically related, but it is difficult to distinguish the germplasms on the basis of
morphological characters alone due to interaction of environment (TAMKOC and ARSLAN, 2010).
So the hereditary materials are used as genetic markers for the studies on genetic diversity. The
hereditary information (DNA) is expressed in distinguishable traits as RNA and proteins
(STOYANOVA et al., 2011). Proteins are generally polymorphic and heritable and are direct
products of active genes due to which they are the source of good genetic markers and thus
polymorphism in protein profiles reflects the changes in the active part of the genome (GARIMA et
al., 2013). These are independent of environment and display variations through polymorphisms.
Hence, seed protein electrophoresis are being used successfully as a biochemical marker in the
assessment of diversity and confirming cultivar identity (STOYANOVA et al., 2011). The use of
seed storage proteins as biochemical markers to screen the germplasms is cost, time and labour
N. VERMA et al.: CHARACTERIZATION AND CORRELATIONIN OPIUM POPPY 1045
effective in assessing the genetic variations of wild species germplasms and even though have
been used as genetic markers to study the genetic diversity, identifying variations among species,
screening of cultivars and establishing genome relationships (RADWAN et al., 2013). Various
techniques are available to analyze the protein polymorphism but polyacrylamide gel
electrophoresis (PAGE) is preferred due to rapid analysis and simplicity for unfolding genetic
variations among different plant species (CHITTORA and PUROHIT, 2012). In the present study seed
protein electrophoresis was performed by total soluble seed proteins separated by SDS-PAGE. The
differential protein profile pattern for most of the germplasms revealed diversity among the studied
germplasms. Similar findings were also observed by SINGH et al. (2004) and YADAV et al. (2007a,
b) based on their studies on morphological traits.
Polymorphism was observed in three variable regions i.e. high, medium and low
molecular weight which suggested that the origin of these germplasms is from the same genetic
background. Earlier, similar findings based on chemotypic traits were also observed in the same
set of germplasm lines (SHUKLA et al., 2010). The similarity in seed storage protein banding
pattern for some germplasm lines confirmed that the investigated germplasms were collected in
narrow restricted area which is also reflected by significant and higher genotypic correlation than
phenotypic correlation (SINGH et al., 2003, 2004; SHUKLA et al., 2010). Major expressing protein
bands of different molecular weights found in most of the germplasm also had higher amount of
protein content which could be attributed to higher metabolic activity in the germplasm lines. Only
four protein bands of molecular weights 14.3, 25, 26 and 27 from total protein bands were
uniquely present in ample number of germplasm which could ease in identification of these
distinct germplasm.
The clustering grouped all the 124 germplasms into 13 distinct clusters depending upon
the similarity of seed protein profile. The diversity analysis based on morphological similarity also
clustered the same germplasm lines into 13 clusters (SINGH et al., 2004). In the present study,
cluster size varied from 1 to 50 germplasm lines. All clusters contained accessions of different
origin except cluster VIII and IX having single germplasm each. The germplasms involved in
clustering are group of Indian landraces, improved varieties and selections obtained through
different breeding programmes and maintained at National Botanical Research Institute, Lucknow
for the last four decades (SINGH et al., 2004). The germplasm lines of common geographical origin
were distributed over several clusters which indicated partial role of geographical distribution in
the genetic diversity of opium poppy germplasms of Indian origin (SINGH, 1991; SINGH et al.,2003,
2004). Thus, it could be assumed that geographical diversity though important but may not be a
crucial factor in determining genetic divergence.
A strong correlation and higher heritability for economically-important characters are
highly desirable in breeding and selection programme. Correlation measures the mutual
association between two variables, which aids in determining the most effective procedures for
selection of desirable trait (UDENSI and IKPEME, 2012). The magnitude of genotypic and
phenotypic correlation coefficient observed similar trends for most of the characters and also
showed higher genotypic correlation coefficient than phenotypic correlation which could be
explained by low environmental effect on investigated agronomic traits (FALCONER, 1989; MEHTA
et al., 2006; AKRAM et al., 2008; YADAV et al., 2006). The higher values of genotypic correlation
coefficient might also be due the fact that the germplasm lines are superior but their expressions
are lessened under the influence of environment (MEHTA et al., 2006).
1046 GENETIKA, Vol. 47, No.3, 1029-1050, 2015
The present study also explored the probable reasons of increase or decrease of different
alkaloids based on their biosynthetic pathways. The morphine content in general was the major
constituent followed by narcotine, codeine, thebaine and papaverine (Table 2). The seed protein
content was positively correlated with seed and opium yield, while opium yield had positive
correlation with codeine, thebaine, narcotine and papaverine which is due to the fact that the
increase in quantitative characters can elevate the quality traits as also reported by CALIMAN et al.
(2008). However, negative correlation between yield and quality trait was also prominent as in
case of opium and morphine content which has been reported earlier also (SHUKLA et al., 2003;
YADAV et al., 2004; BHANDARI et al., 1997). The significant and negative correlation of morphine
with codeine has occurred in the present study was also reported in our earlier studies (YADAV et
al., 2006). Contrary to our findings, BAJPAI et al., (2000) reported positive correlation between
morphine and codeine. Morphine is synthesized from thebaine via codeine (PSENAK, 1998). A
valid reason for the negative association among these alkaloids has been reported earlier (YADAV
et al., 2006). However, codeine was positively and significantly correlated with thebaine and
narcotine which might be due to the fact that the content of reticuline, the key branch point
intermediate of opium alkaloid synthesis was significantly correlated to that of codeinone, but had
negative correlation with codeine content as also reported by PRAJAPATI et al. (2002). This
suggests that codeinone reductase, the key enzyme which converts codeinone to codeine was
probably rate limiting and controls the accumulation of morphine in germplasm lines. The increase
in codeine and thebaine content in some of the identified germplasm lines may be due to less
enzymatic activity involved at a particular conversion step causing partial blocking of morphinane
pathway (Thebaine Codeine Morphine, SHUKLA et al., 2006).
The study also confirmed the findings of PRAJAPATI et al. (2002) that the total alkaloids
remain low in the germplasm lines deficient in both papaverine and narcotine as narcotine is one
of the major constituent of opium poppy (HOSZTFI, 1998). Since in mature seeds, type and amount
of proteins are more constant than other plant tissues therefore, the SDS-PAGE pattern of seed
storage proteins of opium poppy showed polymorphism on the basis of difference in protein
intensity among germplasm lines. The enhancement in seed protein content can be achieved
through selection of high seed and opium yielding plant types rich in morphine and thebaine from
the germplasm lines as there exist positive genotypic correlation and direct effect of these traits on
seed protein content.
CONCLUSIONS
Based on the present study desirable germplasm lines for seed storage protein have been
isolated which can be used in future hybridization programmes aimed to develop varieties rich in
seed storage proteins for commercial cultivation in various opium growing locations of the states
of India. Beside this, it was also concluded that the electrophoresis (SDS-PAGE) of seed storage
proteins can be economically used to assess genetic variation and relation in germplasm and also
to differentiate mutants from their parent genotypes. It was viewed that the seed storage protein
profiles could be a useful marker in germplasm identification.
Contribution of each Author: Nidhi Verma, Anu Rastogi conducted the experimentation, Sudhir
Shukla provided the idea and checking of the manuscript, Brij Kishore Mishra and Nidhi Verma
wrote the manuscript and Kusum Yadav provided specific comments, intellectual inputs on the
manuscript and checking of the manuscript.
N. VERMA et al.: CHARACTERIZATION AND CORRELATIONIN OPIUM POPPY 1047
ACKNOWLEDGEMENTS
The authors are thankful to the Director for encouragement and facilities provided during the
investigation. Nidhi Verma, Brij Kishore Mishra and Anu Rastogi thank the Council of Scientific
and Industrial Research, New Delhi for providing Research Fellowships. Financial assistance
provided from Narcotics Dept., Ministry of Finance, Govt. of India is duly acknowledged.
Received February02nd, 2015
Accepted October 20th, 2015
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1050 GENETIKA, Vol. 47, No.3, 1029-1050, 2015
BIOHEMIJSKA KARAKTERIZACIJA BAZIRANA NA SDS–PAGE ANALIZI
KORELACIJE IZMEĐU OSOBINA GERMPLAZME MAKA
(Papaver somniferum L.)
Nidhi VERMA1, *Sudhir SHUKLA
1, Kusum YADAV
2, Brij K MISHRA
1 i Anu RASTOGI
1
1 Genetika i oplemenjivanje biljaka, nacionalni botanički istraživački Instiut, Lucknow-226001,
U.P., India 2 Odelenje za biohemiju, Univerzitet Lucknow, Lucknow-226020, U.P., India
Izvod
Cilj ovih istraživanja je spiivanje genetičkog diverziteta među 124 linije germplazme, zasnovanog
na elektroforegramu rezervnihb proteina zrna i uspostavljanje korelacije između sadržaja
alkaloida i rezervnih roteina zrna maka (Papaver somniferum L.). Dobijeno je 27 polipeptidnih
traka molekulske težine od 6 do 205 kD. Indeks sličnosti je izračunat korišćenjem Jaccard’s
indeksa sličnosti a analiza klastera preko UPGMA modela. Utvrđen je polimorfizam u tri različita
regiona; visoke, srednje i male molekulske težine čije trake težine 10.4, 20, 22, 24, 30, 32, 33, 49 i
205 kDa’s su bile zajedničke kod svih germplazmi ali su ostale trake pokazale varijabilnost. Sve su
široko grupisane u 13 klastera zasnovanih na meetodi prosečne ukopčanosti. Ni jedna od osobina
nije pkazala značajne korelacije sa sadržajem proteina.Razlčiti profili proteina germplazme
potvrđuju široku varijabilnost i diverzitet između ispitivanih germplazmi, što može dalje da se
koristi u programima hibridizacije da bi se ostvarila maksimalna dobit.
. Primljeno 02.II 2015.
Odobreno 20. X. 2015.