Genetic Assessment of Traits and Genetic Relationshipin Blackgram (Vigna mungo) Revealed by Isoenzymes

Ajay Kumar Singh Æ Avinash Mishra Æ Arvind Shukla

Received: 17 February 2008 / Accepted: 9 May 2009 / Published online: 27 May 2009

� Springer Science+Business Media, LLC 2009

Abstract Sixty blackgram accessions were evaluated and classified into different

clusters to assess genetic diversity and traits using isoenzymes. Trait-specific

expression was assessed, and isoenzyme bands were observed: a peroxidase band

(Rm 0.60) associated with dwarfness and an esterase band (Rm 0.25) with tallness.

Early maturing varieties were characterized by a specific esterase isoenzyme band

of Rm 0.51. All yellow mosaic virus susceptible genotypes had two bands of

esterase isoenzyme, Rm 0.42 and 0.70. Resistant genotypes showed three bands

(0.32, 0.33, and 0.35) of alkaline phosphatase. Peroxidase isoenzyme was helpful to

differentiate green-seeded from black-seeded varieties. Two bands (0.58 and 0.83)

were observed in black-seeded accessions, and two different bands (0.74 and 0.76)

were observed in green-seeded accessions. Clustering of germplasm and assessment

of traits will facilitate the use of germplasm for the improvement of blackgram.

Keywords Isoenzyme � Blackgram � Cluster analysis � Dendrogram �Peroxidase � Esterase � Alkaline Phosphatase

A. K. Singh � A. Mishra � A. Shukla

G. B. Pant University of Agriculture and Technology, Pantnagar 263145, India

Present Address:A. K. Singh

Plant Variety Evaluator, PPV&FR Authority, Department of Agriculture and Cooperation,

Ministry of Agriculture, NAS Complex, New Delhi 110012, India

A. Mishra (&)

Discipline of Marine Biotechnology and Ecology, Central Salt and Marine Chemicals Research

Institute, Council of Scientific and Industrial Research (CSIR), G. B. Marg,

Bhavnagar 364002, Gujarat, India

e-mail: avinash@csmcri.org

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Biochem Genet (2009) 47:471–485

DOI 10.1007/s10528-009-9247-1

Introduction

Blackgram (Vigna mungo), a tropical leguminous plant of the Asiatic Vigna species,

is cultivated for human consumption. Dry mature seeds contain thrice as much

protein as cereals and thus constitute an important source of protein in a vegetarian

diet (Lakhanpaul et al. 2000). Furthermore, blackgram plays a crucial role in

sustaining the productivity of a cropping system by adding atmospheric nitrogen to

the soil. Blackgram is grown in various agroecological conditions and cropping

systems with diverse agricultural practices.

Domesticated in South Asia from V. mungo var. silvestris (Lukoki et al. 1980;

Fuller 2002, 2007), blackgram is widely cultivated on the Indian subcontinent and to

a lesser extent in Australia, Thailand, Asia, and the South Pacific (Poehlman 1991).

In parts of India, a number of traditional landraces are still being cultivated as an

intercrop in rice, sugarcane, cotton, groundnut, and sorghum cultivating season;

these landraces possess unique traits such as disease tolerance, abiotic stress

tolerance, and pest tolerance (Sivaprakash et al. 2004).

In India during 2005–2006, blackgram was cultivated over an estimated area

of 2.9688 Mha, with production estimated at 12.455 metric tonne and an average

yield (productivity) of only 419 kg/ha (Directorate of Pulses Development 2008).

Major constraints on yield are the lack of genetic variability, absence of suitable

idiotypes for different cropping systems, poor harvest index, and susceptibility to

diseases. Research on blackgram lags behind that of cereals and other legumes.

A large number of accessions representing V. mungo coming from diverse

agroclimatic zones are being maintained in the germplasm collection center at

the National Bureau of Plant Genetic Resources, New Delhi, India. Efforts at

genetic improvement of blackgram are still at a low ebb, barring a few efforts to

identify important morphological descriptors and develop advanced breeding

lines for locale-specific cultivars (Gupta et al. 2001). These efforts are impeded

by the lack of distinctive morphological attributes and characterization of diverse

genotypes. Documentation of diversity among the genotypes is of utmost

significance in genetic improvement of blackgram. Therefore, amelioration is

required through the utilization of available genetic diversity. This investigation

was undertaken to analyze as well as to assess genetic diversity among

blackgram genotypes, collected from diversity-rich zones of India, using

isoenzymes.

Materials and Methods

The experimental material comprised 60 germplasm accessions of blackgram

collected from the diversity pockets of Uttar Pradesh and Uttaranchal States of India

(Paroda and Arora 1991); some accessions were acquired from the National Bureau

of Plant Genetic Resources, New Delhi, India (Table 1).

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Table 1 Mean of quantitative and visual characteristics of blackgram genotype accessions, collected

from the diversity-rich zones of India

Accession and source Traitc

Entry

no.aAcc. no. Village/

TownbDistrict/

Research

Institute

Plant

height

(cm)

Days to

maturity

Yellow

mosaic

virus

response

Seed

color

1 ShU-9503 Mishrikh Sitapur 50.2 85 LS G

2 ShU-9505 Sidhauli Sitapur 65.4 92 LS B

3 ShU-9508 Sakaran Sitapur 75.8 93 MS B

4 ShU-9511 Hargaon Sitapur 72.6 88 HR B

5 ShU-9519 Khajuraha Hardoi 50.8 81 (E) HR B

6 ShU-9525 Sandi Hardoi 66.7 89 MR G

7 ShU-9532 Beniganj Hardoi 50.4 88 MS M

8 ShU-9536 Berua Hardoi 54.6 81 (E) MS G

9 ShU-9601 Markara Bareilly 66.4 87 MR G

10 ShU-9603 Jawaharpur Bareilly 58.4 87 HR C

11 ShU-9609 Manpur Bareilly 54.0 89 MS M

12 ShU-9612 Kesarpur Bareilly 64.8 83 MR M

13 ShU-9614 Hazipur Bareilly 53.8 83 (E) MR M

14 ShU-9619 Semikhera Bareilly 78.8 91 HR G

15 ShU-9621 Deorania Bareilly 44.8 81 (E) MR C

16 ShU-9626 Baragaon Bareilly 74.0 84 LS M

17 ShU-9632 Shivpuri Bareilly 116.4 (T) 95 MS Br

18 ShU-9633 Dhaneta Bareilly 68.0 89 HR G

19 ShU-9636 Dhunka Bareilly 62.4 88 LS M

20 ShU-9641 Shiragarh Bareilly 67.8 89 MS C

21 ShU-9642 Manpur Bareilly 48.8 85 LS C

22 ShU-9682 Bhowali Nainital 35.6 (D) 89 MS B

23 ShU-96110 Kalsi Pithoragarh 73.0 90 HS B

24 ShU-9720 Narkota Pauri 41.8 88 HS B

25 ShU-9725 Tigreewala Hardwar 99.4 (T) 96 MR Br

26 ShU-9737 Chandergarh Chamoli 41.8 94 HS B

27 ShU-9797 Narayanpur U.S. Nagar 92.4 (T) 95 MS B

28 ShU-9901 Narayanpur U.S. Nagar 49.8 98 (L) LS G

29 PLU-184 * NBPGR 95.8 (T) 99 (L) HR M

30 PLU-195 * NBPGR 111 96 MR M

31 PLU-199 * NBPGR 78.6 97 LS M

32 PLU-289 * NBPGR 49.4 90 HR Br

33 PLU-305 * NBPGR 57.2 96 MR Br

34 PLU-309 * NBPGR 75.8 97 MR Br

35 PLU-329 * NBPGR 115.3 (T) 98 (L) MR Br

36 PLU-342 * NBPGR 82.8 100 (L) MR B

37 PLU-347 * NBPGR 78.2 99 (L) MR G

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Enzyme Extraction

For each accession, a 1 g sample collected from healthy 8-day-old seedlings was

homogenized in ice-cold 5.0 ml extraction buffer (50 mM Tris–HCl with 1% v/v

b-mercaptoethanol, pH 7.0). The extract/homogenate was centrifuged at 12,000 rpm

for 30 min at 4�C, and the supernatant fluid was used for isoenzyme assay (Li et al.

2007). A sample mix (25 ll), containing isoenzyme extract, glycerol (50%), and

Table 1 continued

Accession and source Traitc

Entry

no.aAcc. no. Village/

TownbDistrict/

Research

Institute

Plant

height

(cm)

Days to

maturity

Yellow

mosaic

virus

response

Seed

color

38 PLU-433 * NBPGR 67.0 91 MR Br

39 PLU-730 * NBPGR 50.6 97 MR Br

40 PLU-820 * NBPGR 56.2 89 MR Br

41 IC-292 * NBPGR 53.2 97 MR M

42 IC-110664 * NBPGR 55.2 89 LS Br

43 IC-201887 * NBPGR 71.8 90 LS Br

44 IC-201886 * NBPGR 44.2 85 MR M

45 IC-201889 * NBPGR 37.6 (D) 88 HR Br

46 IC-201892 * NBPGR 34.6 (D) 89 MR G

47 IC-208468 * NBPGR 56.4 97 MR M

48 IC-37176 * NBPGR 60.8 96 MS Br

49 IC-73264 * NBPGR 105 (T) 98 (L) HR Br

50 NIC-8190 * NBPGR 71.2 98 (L) MR Br

51 NIC-15266 * NBPGR 56.2 88 LS Br

52 NIC-15274 * NBPGR 86.8 91 MR Br

53 NIC-23231 * NBPGR 69.4 97 LS Br

54 NC-73203 * NBPGR 33.6 (D) 88 MR G

55 JBT-9193 * NBPGR 57.4 92 LS B

56 SDI-29 * NBPGR 68.0 90 HR G

57 PUSA-105 * NBPGR 83.0 99 (L) MR Br

58 VL-310 * NBPGR 50.2 86 LS Br

59 NKG-43 * NBPGR 50.2 96 MR Br

60 IC-201893 * NBPGR 72.6 87 LS Br

a Numbers as in Fig. 4b Asterisk (*) indicates acquisition from National Bureau of Plant Genetic Resources, New Delhi, India

(NBPGR)c Highest and lowest values shown in bold. T tall, D dwarf, E early maturing, L late maturing, HR high

resistance, MR medium resistance, HS high susceptibility, MS medium susceptibility, LS low suscepti-

bility, B black, G green, Br brown, C chocolate, M mottled

474 Biochem Genet (2009) 47:471–485

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bromophenol blue (0.05 mg/ml in dH2O) in the ratio of 5:3:2 (Upadhyay et al.

2002) was electrophoresed on 7% native PAGE at 80 V for 4–5 h at 4�C till the

tracking dye migrated 1 cm above the anodal end (Sambrook et al. 1989).

Peroxidase

The gel was incubated in a freshly prepared solution containing 0.1% w/v

tetramethylbenzidine, 50% v/v methanol, and 0.5 M Na acetate (pH 4.7) for about

5 min in the dark. Thereafter, H2O2 (30%) was added dropwise till the bands

became visible. The gel was transferred to a solution of 7% acetic acid for 5 min to

fix the bands (Song et al. 2007; Zhou et al. 2007).

Esterase

After gel electrophoresis, the separating gel was washed twice with 50 mM Tris–

HCl and soaked in 0.02 M sodium phosphate buffer (pH 7.0) with 2% v/v 30 mM

a-naphthyl acetate dissolved in acetone at 30�C for 15 min. Enzyme activity was

developed by adding 0.04% w/v Fast Blue BB salt (4-benzoylamino-2,5-

diethoxybenzenediazonium chloride hemi [zinc chloride] salt) after 30–45 min of

incubation at 37�C (Zhou et al. 2005; Kim et al. 2006). The stained gel was fixed in

methanol, double-distilled water, and acetic acid (5:5:1) for 3 min and washed with

tap water.

Alkaline Phosphatase

The gel was stained in 50 mM Tris–HCl (pH 8.5), 10 mM MgCl2, 10 mM MnCl2,

0.03% v/v Na a-napthyl phosphate, prepared in 50% acetone at 30�C for 1–5 h until

purple or pink bands appeared. The gel was then washed with distilled water and

bands were fixed by soaking in 7% acetic acid for 5 min.

Data Analysis

Sixty genotypes were evaluated in the field. They were sown in six blocks, each

having ten plots. The augmented design was evaluated using the method given by

Feeder (1956, 1961) and elaborated by Feeder and Raghavarao (1975) and Peterson

(1985). Observations were taken on plant and plot basis as per the descriptor’s list

published by IBPGR (1985), which includes both quantitative and visual characters.

An adjusted mean value for characters of 60 accessions was analyzed using the

concept of principal components based on the multivariate technique (Pearson 1901;

Hotelling 1933). For principal component analysis, accessions were characterized

on the basis of specific traits on a single point in a standardized multidimensional

space. The axis of this space was principal components, obtained from the original

data as an orthogonal transformation of the original varieties. In this way, each

principal component becomes a linear combination of varietal scores corresponding

to the original variables. Genetic divergence among genotypes was studied using

Biochem Genet (2009) 47:471–485 475

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nonhierarchical euclidean cluster analysis described by Beale (1969) and elaborated

by Spark (1973).

Gels were analyzed and zymograms were prepared using relative mobility of

each band visualized on the gel. A similarity coefficient was obtained by presence

and/or absence of each isoenzyme. The similarity between accessions was

calculated using Jaccard’s coefficient (Sokal and Sneath 1963), and UPGMA

cluster analysis was performed using NTsys-PC software (Rohlf 1992).

Results

Peroxidase Activity

The banding patterns obtained through zymograms reveal that the number of

peroxidase bands in different accessions ranges from three to nine, and genotypes

can be classified into six groups (Fig. 1). The number of peroxidase bands, in

groups, and the intensity of bands, in zones A, B, and C, show that accessions vary

sufficiently with regard to the relative number, mobility, and intensity of bands.

About two-thirds of genotypes were included in the group of six and seven bands.

Zone A, irrespective of the group, was found to have two bands in all genotypes.

Zones B and C consist of bands that differ in number, position, and intensity among

the genotypes. Dark intensity bands were absent in most of the genotypes, but if

present were only one or two in number. Bands showing variation were mostly

medium to light intensity. A wild accession was placed in a separate group with

three bands. Two genotypes, ShU 9505 and ShU 9633, had a faint band with Rm

value 0.35, which did not occur in any other genotype, making it unique. PLU 289

was unique in that it had six bands in zone B but no bands in zone C.

Esterase Activity

Characterization and identification of 60 blackgram genotypes by zymograms of the

esterase banding pattern show three to nine bands, categorized into four zones

(A–D) and six groups. Groups were characterized by the number of bands and their

relative mobility and intensity (Fig. 2). An eight-band group was the largest, with 23

genotypes. Like peroxidase, the wild accession IC 201893 was placed separately in

a three-band group, with no bands in zones B and C. Zone A was the least variant,

and zones B and C together composed the rest of the genotypes; however, bands

were absent in zone D. Bands with dark and medium intensity were very few in

number, and variation was observed in light and faint bands. Genotypes possessing

a higher number of bands also had light to faint bands. IC 201887 was the only

genotype in this enzyme group that had no band in zone A, but with a maximum of

four bands in zone D.

Fig. 1 Electrophoretic banding patterns for peroxidase isoenzyme in 60 blackgram genotypes. Bandintensity: Solid black bar, dark; striped bar, medium dark; gray bar, light; open bar, faint

c

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Biochem Genet (2009) 47:471–485 477

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Alkaline Phosphatase Activity

The banding pattern of alkaline phosphatase isoenzyme consists of three to seven

bands, distributed in three zones (A–C) and five groups. Each group was

characterized by total number, relative position, and intensity of bands (Fig. 3). A

variable number of bands was also included in zones that differ in intensity. In the

three-band group, genotype ShU 9797 possesses no bands in zone C, but all other

genotypes had one to a few bands in zone C. The five-band group was the largest,

including half of the evaluated genotypes (30). Zone A was common to most of the

genotypes, and variation in relative position and intensity was observed in zones B

and C. Dark and medium bands were absent in most of the accessions. The

accessions were differentiated by light and faint bands. In this particular isoenzyme,

the wild accession IC 201893 was not placed separately, but was included in the

four-band group.

Genetic Relationship Based on Isoenzyme Markers

Data scored from 60 blackgram genotypes using the three isoenzymes were used to

generate euclidean distance matrix dissimilarity coefficients, ranging from 2.45 to

4.55 (data not shown). On the basis of this matrix, a dendrogram was drawn, which

classified genotypes into 14 clusters (Fig. 4). Clusters 6, 8, 13, and 14 contain only

one genotype; clusters 4, 11, and 12 have two genotypes each. Cluster 1 contains

three genotypes, cluster 5 has eight, and clusters 2 and 9 both contain nine

genotypes. Clusters 3 and 7 are the largest, with ten genotypes each.

Clusters 1 and 2 interrelate to each other with a coefficient of 4.07. Together they

are correlated to cluster 3 by a 4.10 coefficient. Clusters 4 and 5, 7 and 8, 9 and 10,

and 11 and 12 coincide with each other at a coefficient of 3.90. A group of clusters,

1, 2, and 3, interlink with another group, clusters 4 and 5, by a 4.20 coefficient.

Cluster 13 concurs with 11 and 12 at a 4.25 coefficient. Clusters 7 and 8 coincide

with clusters 9 and 10 at a 4.10 coefficient; together they are allied to cluster 4 with

a 4.20 coefficient. A major cluster, including clusters 1–5, coincides with another

major cluster, containing clusters 6–10, at a coefficient of 4.25; cumulatively, they

concur with the group of clusters 11–13 at a coefficient of 4.30. Cluster 14, being

the farthest one, coincides with the rest of the clusters at 4.40.

Isoenzymic Characterization of Specific Traits

Apart from the assessment of genetic variability through isoenzyme banding

patterns, a few traits were also studied among the genotypes, including plant height,

days to maturity, response to yellow mosaic virus, and seed-coat color; they were

characterized by an agromorphological mean value for quantitative and visual

characteristics (Table 1). Each trait was divided into two categories, with genotypes

Fig. 2 Electrophoretic banding patterns for esterase isoenzyme in 60 blackgram genotypes. Bandintensity: Solid black bar, dark; striped bar, medium dark; gray bar, light; open bar, faint

c

478 Biochem Genet (2009) 47:471–485

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Biochem Genet (2009) 47:471–485 479

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having maximum mean values grouped in one category and those with minimum

mean values grouped in another.

The two categories for plant height were tall and dwarf. Tall genotypes were ShU

9632, ShU 9725, ShU 9797, PLU 184, PLU 195, PLU 329, and IC 73264. Dwarf

genotypes were ShU 9682, IC 201889, IC 201892, and NC 73203. With regard to

the peroxidase isoenzyme, bands with higher Rm values (0.84, 0.88, and 0.90) were

obtained in taller accessions; however, a band with Rm value 0.60 was observed in

all dwarf accessions but was absent in tall genotypes. In contrast, the esterase

isoenzyme bands with high Rm values (0.68, 0.70, and 0.72) were present in dwarf

genotypes but absent in tall genotypes, except for ShU 9632. One band with Rm

value 0.25 was common to all tall genotypes but absent in dwarf accessions. Four to

six bands of alkaline phosphatase isoenzyme were observed in dwarf genotypes and

three to six bands in tall genotypes. No specific band was found in dwarf genotypes;

however, in tall genotypes a band with Rm value 0.55 was observed.

The two categories for days to maturity were early and late maturing. The early

maturing genotypes were ShU 9519, ShU 9536, ShU 9614, and ShU 9621; the late

maturing group included eight genotypes, ShU 9901, PLU 184, PLU 329, PLU 342,

PLU 347, IC 73264, NIC 8190, and Pusa 105. The range of bands of peroxidase

isoenzyme was 6–7 in the early group and 6–9 in the late group. Two bands with

Rm value 0.26 and 0.55 were common in all early genotypes. Bands with higher Rm

values (0.68–0.75) were found in the late genotypes. For esterase, a band with Rm

value 0.33 was common to all genotypes of both groups, and a band with Rm value

0.51 was common to early genotypes but absent in late types. In the late types,

bands with low Rm values (0.02, 0.05, and 0.08) were frequent, but they were

absent in early types. A band of alkaline phosphatase with Rm value 0.48 was

present in all early genotypes but absent in most of the late genotypes. In the late

types, bands with low Rm values (0.01, 0.04, 0.08, 0.10, and 0.16) were present.

One band with Rm value 0.50 was present in six out of eight late genotypes but

completely absent in early types.

The two groups characterized in response to yellow mosaic virus were tolerant

and susceptible. The tolerant group included genotypes ShU 9511, ShU 9519, ShU

9603, ShU 9619, ShU 9633, PLU 184, PLU 289, IC 73264, IC 201889, and SDI 29.

The susceptible group included only three genotypes, ShU 96110, ShU 9720, and

ShU 9737. The number of peroxidase isoenzyme bands observed in tolerant

genotypes (6–8) was large compared with the susceptible types (5–6). For the other

isoenzymes, the tolerant genotypes had five to nine bands (esterase) and four to fiver

bands (alkaline phosphatase); the susceptible genotypes had six esterase bands and

four to five alkaline phosphatase bands. Esterase isoenzyme bands with Rm values

0.42 and 0.70 were observed in all susceptible genotypes. In contrast, three bands of

alkaline phosphatase isoenzyme with Rm values 0.32, 0.33, and 0.35 were present in

the tolerant group only.

Two important seed-coat colors were studied for their isoenzymic characteriza-

tion. The group of green-seeded genotypes included ShU 9503, ShU 9525, ShU

Fig. 3 Electrophoretic banding patterns for alkaline phosphatase isoenzyme in 60 blackgram genotypes.Band intensity: Solid black bar, dark; striped bar, medium dark; gray bar, light; open bar, faint

c

480 Biochem Genet (2009) 47:471–485

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Biochem Genet (2009) 47:471–485 481

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9536, ShU 9601, Shu 9619, ShU 9633, ShU 9901, PLU 347, IC 201892, NC 73203,

and SDI 29. The black-seeded group had 11 genotypes: ShU 9505, ShU 9508, ShU

9511, ShU 9519, ShU 9682, ShU 96110, ShU 9720, ShU 9737, ShU 9797, PLU 342,

and JBT 9193. Two bands (Rm 0.58 and 0.83) were associated with black-seeded

accessions and two (Rm 0.74 and 0.76) with green-seeded accessions; they were

Fig. 4 Dendrogram for isoenzymes (peroxidase, esterase, and alkaline phosphatase) of blackgramaccessions constructed using UPGMA-based Jaccard’s similarity coefficient. Genotypes as numbered inTable 1

482 Biochem Genet (2009) 47:471–485

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absent in one group but present in the other, but no particular banding pattern was

observed for esterase and alkaline phosphatase isoenzymes.

Discussion

Characterization of diversity among genotypes is of immense importance to crop

improvement programs. It involves descriptions of variation in morphological traits,

particularly agromorphological characteristics of direct interest to users. The genetic

information provided by morphological traits has certain limits. These limitations

can be reduced by biochemical markers such as isoenzymes. Identification of crop

cultivars through biochemical markers has been used to measure the genetic

diversity and genetic relationships among individuals and populations. It has been

widely used to establish phylogenetic relationships among taxa, genetic markers for

hybrid confirmation, and for convenient screening of seedlings with target

characters (Bleeker and Hurka 2001; Upadhyay et al. 2002; Fromm and Hattemer

2003; Stecconi et al. 2004; Yu et al. 2005; Vasylenko et al. 2006). Isoenzymic

variations have also been extensively used as genetic markers for evolutionary

studies (Gomez Ros et al. 2007), characterization of species and cultivars

(Nakonechnaia et al. 2007; Curtu et al. 2007), and genetic studies in plants (Yu

et al. 2005; Roy et al. 2006; Grundmann et al. 2007).

Mittal et al. (1993) studied eight Vigna radiata cultivars for enzyme activities

and concluded that there was a positive correlation of acid phosphatase with 100

seed weight but a negative correlation with number of pods per plant and seed yield.

Seven isoenzymes, extracted from seed, seedling, stem, root, or leaf tissues of six

closely related lines of black bean, were compared for polymorphism (Driedger

et al. 1994), and variation was observed for acid phosphatase, peroxidase, and

esterase, with some lines possessing unique banding patterns. In this study, bands

were identified for trait-specific expression using different isoenzymes that can

discriminate among plant accessions. A peroxidase isoenzyme band with Rm value

0.60 was associated with dwarfness; an esterase isoenzyme band with Rm value

0.25 was associated with tallness. Early maturing varieties showed a specific

esterase isoenzyme band with Rm value 0.51. All susceptible genotypes had two

bands of esterase isoenzyme, with Rm values 0.42 and 0.70. In resistant genotypes,

however, three bands of alkaline phosphatase were observed, with Rm values 0.32,

0.33, and 0.35. Peroxidase isoenzyme distinguished green-seeded from black-

seeded varieties. Two bands (Rm 0.58 and 0.83) were associated with black-seeded

accessions, and two bands with different Rm values (0.74 and 0.76) were observed

for green-seeded accessions. From this study it may be concluded that the

isoenzyme banding patterns associated with traits can be explored extensively for

breeding programs.

In this study, data obtained from 60 blackgram genotypes using three isoenzymes

were used to generate a euclidean distance matrix, and a dendrogram was drawn,

classifying genotypes into 14 clusters. The genotypes included in the clusters were

quite similar to each other. This reflects that biochemical analysis is more authentic

for clustering patterns, as two accessions look alike in field performance but differ

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genetically. These differences can be easily explored through biochemical and

molecular methods. Since these characters are environmentally and developmen-

tally more stable, they therefore can facilitate the establishment of genetic

relationships quickly and efficiently.

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