Evaluation Of Genetic Diversity In Five Cultivars Of Pigeon Pea Using Cytological Characters And Protein Profiling

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1

Evaluation Of Genetic Diversity In Five Cultivars Of Pigeon Pea Using

Cytological Characters And Protein Profiling

S.A.Shah, G.N.Sheikh, R.Kudesia, M.K, Shrivastava, Z.A.Reshi

Institute of Basic Sciences, Department of Botany, Bundelkhand University, Jhansi (U.P.)-284128, India. Department of Botany, University of Kashmir, 190006

Abstract

Cytological characters and protein profile of 5 cultivars of pigeon pea (AMAR, AZAD, MAL-13, and NDA-1and Pusa-9) of family Fabaceae were investigated through poly acrylamide gel electrophoresis and using cytological characters viz. mitotic index and pollen viability. The present protein profile revealed that experimental accession Amar (A1) is very close to accession Pusa-9(A5) and accessions MAL-13 and NDA-1 are closer at molecular level as compared to other accessions. Similarity index was more i.e., 34.61% in Amar (A1) and Pusa-9 (A5) as compared to other accessions i.e., Amar and NDA-1 where similarity index was only 18.51%. Present investigation revealed that mitotic index was highest in MAL-13 i.e., 18.25% meaning that accession has highest power of division. A dendrogram constructed based on UPGMA clustering method revealed two major clusters, cluster-1 and cluster-11 comprising of two accessions each. The accession Azad (A2) occupies a distinct place as revealed in dendrogram. Jaccards similarity coefficient ranged from 0.23810 - 0.40000.

Keywords: Genetic diversity; mitotic index; pigeon pea; SDS-PAGE; UPGMA.

Introduction.

Pigeon pea is widely grown in semi-arid tropics, particularly in the Indian subcontinent where it accounts for over 70% of the world’s production and coverage (FAO, 2007). The International crop Research Institute for the semi-Arid Tropics (ICRISAT) maintains a large ex-situ collection of over 13,000 acceccions of Cajanus cajan from around 75 countries (http://singer.cgiar.org). It is likely that pigeon pea evolved by interspecific hybridization of Cajanus cajanifolia and Cajanus scarabaeoides (Nadimplli et al., 1992). Pigeonpea (Cajanus cajan L. Millspaugh) is a major grain legume of the tropical and subtropical regions. It is a diploid species (2n = 2x = 22) comprising a genome of 833.1 Mbp arranged into 11 linkage groups (Varshney et al. 2012). India is the centre of origin and largest producer of pigeonpea in the world sharing approximately 70% of the production and covering 74% of the area (Bohra et al. 2012). It plays an important role in food security, balanced diet and subsistence agriculture because of its diverse usages in food, fodder, fuel, soil conservation, integrated farming systems and symbiotic nitrogen fixation (Reddy et al. 2005). Further, pigeon pea offers a rich source of variability in the form of wild relatives, which could be utilized for disease resistance, good agronomic traits, enhancing nutritional quality, identification and diversification of cytoplasmic base of cytoplasmic male sterility (CMS) system etc. Pigeonpea suffers from a number of abiotic (e.g. drought, salinity and water-logging) and biotic (e.g. diseases like Fusarium

wilt, sterility mosaic and pod borer insects) stresses. Among biotic stresses, Fusarium wilt (FW) caused by

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been focused on the use of electrophoretic methods for reliable discrimination and identification of plant varieties (Sammour, 1985; Sammour et al., 1994; Przybylska J, and Zimniak-Przybylska Z. 1995). Electrophoresis adds information to taxonomy and should not be dissociated from morphological, anatomical and cytological observations (Boulter et al., 1966; Ghafoor et al., 2002). Seed protein profiles and molecular markers obtained by electrophoresis have been successfully used to study taxonomical and evolutionary relationships of several crop plants (Gepts and Bliss, 1988; Gepts et al., 1988; Sammour, 1989; Rao et al., 1992Discovery of molecular markers served several functions in pigeonpea including, genetic diversity analysis using restriction fragment length polymorphism (RFLP) (Nadimpalli et al. 1994; Sivaramakrishnan et al. 2002), amplified fragment length polymorphism (AFLP) (Panguluri et al. 2006; Wasike et al. 2005), random amplification of polymorphic DNA (RAPD) (Yadav et al. 2012),

2. Methods

Materials for biochemical and cytological studies were collected from Indian Institute of Pulse Research (IIPR) Kanpur (U.P.) India

Table-1 The experimental accessions are

S.NO Accession Name Sample Name Area of Collection Date of Collection

1 AZAD A1 IIPR 11-04-2011

2 AMAR A2 IIPR 11-04-2011

3 MAL-13 A3 IIPR 11-04-2011

4 NDA-1 A4 IIPR 11-04-2011

5 PUSA-9 A5 IIPR 11-04-2011

Mitotic index was determined from the root tips of experimental species, root tips were harvested and fixed in 3:1 ratio of alcohol and glacial acetic acid after 6 hours of fixation the root tips were preserved in 70% alcohol.

Formulae used:

Mitotic Index= No. of dividing cells

__________________________x100 Total no. of cells

Total seed proteins were also extracted from 1g of seed flour using 400 μl of extraction buffer that contained 25mM tris HCl pH- 8.3, 10% SDS. 5M urea and 10% mercapto-ethanol. Seed flour was thoroughly mixed with buffer by vortexing. The extracted protein was separated by centrifuging the sample at the rate of 1500 rpm for 10 minutes electrophoresis was carried out in discontinuous SDS-PAGE using 7% acrylamide gel. Electrophoresis was run at 50v. The gels were stained in the staining solution containing 40ml methanol, coomassie blue [1%] 1gm and glacial acetic acid (10 ml) was made up to 100 ml by adding distilled water. Destaining was done in a solution containing 30ml methanol, 6ml glacial acetic acid, 74 ml of distilled water until the background color disappeared, and protein bands were clearly visible.

2.1 Data analysis

The results obtained from cytological characters were analysed. RF value and the molecular weight of each protein band was determined [Table 3].Protein bands were scored depending on their presence [1] or absence [0]. Similarity coefficient was determined and hierarchical clustering was constructed by unweighted pair group method with arithmetic average [UPGMA].

2.3 Mitotic study

The mitotic index in different investigated genera is given in Table 2, determined by the formula as described in Methods.

2.4 Protein profiling

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Table 2: The mitotic index in different investigated genera.

S.NO Accession Name Mitotic Index

1 Amar 7.32%

2 Azad 14.98%

3 MAL-13 18.25%

4 NDA-1 10.79%

5 Pusa-9 15.11%

Table -3

Showing the presence and absence of bands in different accessions of pigeon pen

Band Number

Rf value Mol.Wt A1(Amar) A2(Azad)

A3(MAL-13)

A4(NDA-1)

A5(PUSA-9)

1 0.01 99 +

₊

₊

₊

₊

2 0.02 98 -

₊

₊

₊

₊

3 0.03 97 +

₊

₊

₊

₊

4 0.05 95 +

₊

_-

₊

_-

5 0.06 94 +

₊

₊

₊

₊

6 0.07 93 +

_-

_-

_-

_-

7 0.08 92 +

_-

_-

_-

_-

8 0.12 88 _

_-

₊

₊

_-

9 0.15 85 +

₊

_-

_-

_-

10 0.16 84 _

_-

_-

_-

₊

11 0.17 93 _

_-

₊

₊

_-

12 0.18 92 +

_-

_-

_-

_-

13 0.21 79 _

_-

_-

₊

_-

14 0.22 78 _

_-

₊

_-

_-

15 0.23 77 _

₊

_-

_-

_-

16 0.25 75 _

_-

_-

₊

_-

17 0.27 73 +

_-

_-

_-

_-

18 0.3 70 _

₊

₊

₊

_-

19 0.31 69 +

₊

₊

_-

₊

20 0.37 63 +

₊

₊

_-

_-

21 0.4 60 _

_-

_-

_-

₊

22 0.47 53 _

₊

_-

_-

_-

23 0.5 50 +

_-

₊

₊

₊

24 0.53 47 _

₊

_-

_-

_-

25 0.56 44 +

_-

_-

_-

₊

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27 0.6 40 _

_-

₊

_-

_-

28 0.61 39 _

₊

_-

_-

_-

29 0.63 38 _

_-

₊

_-

_-

30 0.67 33 _

_-

₊

_-

_-

31 0.73 27 _

_-

_-

₊

_-

32 0.75 25 _

₊

_-

_-

_-

33 0.78 22 _

_-

₊

_-

_-

Table 4 Showing the Similarity Index of different accessions of Pigeon Pea

Amar Azad Mal-13 NDA-1 Pusa-9

Amar 100%

Azad 25% 100%

MAL-13 20.68% 24.13% 100%

NDA-1 18.51% 22.22% 28.57% 100%

Pusa-9 34.61% 30.76% 25.92% 24% 100%

Where similarity Index = Number of common bands

________________________ X Total no. of bands

Table 5:- Jaccard s similarity coefficient

Amar Azad Mal-13 NDA-1 Pusa-9

Amar

Azad 0.35000

MAL-13 0.27273 0.31818

NDA-1 0.23810 0.28571 0.40000

Pusa-9 0.37500 0.27778 0.3333 0.29412

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Figure 1:- Dendrogram showing the UPGMA showing genetic relationship among five accessions of cajanus cajan based on Jaccards similarity , estimates of protein profiling.

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3. Results and Discussion

Great variations were observed in cytological and biochemical characters among five different experimental accessions of Cajanus cajan. To find out genetic diversity among them cytological and biochemical analysis was done during present investigation. The biochemical analysis through protein profiling helped to find out the correlation between five different accessions of cajanus cajan. Cytological study was done to find out mitotic index. The mitotic index [MI] was highest in case of MAL-13 i.e., 18.25% which revealed that this cultivar has highest power of division. The Pusa-9 i.e., 15.11%, Azad 14.98%, NDA-1 10.79% and lowest mitotic Index in Amar i, e., 7.32%. Earlier, cytological and palynological analysis of three species of genus Solanum at Saudi Arabia was done by Al-Wadi et al (2007). Almost similar were observed by Bhat and Kudesia (2011) in five species of Solanaceae. During protein profiling of total proteins of experimental accessions, maximum 15 protein bands generated in case of NDA-1. The protein band for highest molecular weight i.e., 99 KD generated in case of Amar while that of lowest molecular weight i.e., 22 KD was generated in mal-13.The bands observed in different accessions are fourteen in Amar(A1),fourteen in Azad(A2),13 in Mal-13(A5),fifteen in NDA-1(A4) and twelve in Pusa-9(A2).A total of 33 bands were scored out of them 30 were polymorphic with a total of 90.09% polymorphism and three bands were monomorphic with 9.09% monomorphism.Gafoor and Arshad (2008) got 25 bands and among them 20 were polymorphic with a total of 80% polymorphism. Similarly Kakaei and Kehrizi (2011) got 17 bands. Shrivastava and Gupta (2010) almost got same results. This little change is might be due to the crop change. The similarity index [S.I.] calculated after proteins of five selected experimental accessions of cajanus cajan was maximum i.e., 34.61% between Amar and Pusa-9, and next between Azad and Pusa-9 i.e., 30.76%, the lowest similarity index was found between Amar and NDA-1 i.e., 18.51 %Table[4].

4. Conclusion

The presence of genetic diversity is important for improving any crop species. An understanding of the magnitude and patterns of genetic diversity in crop plants has important implications in breeding programmes and for conservation of genetic resources. A large number of cultivars utilizing limited genetic resources are grown and are being released. Often plant breeders limit their efforts to a narrow range of adapted lines for genetic improvement which were more likely to produce economic gains in the short term but may have enhanced vulnerability to insects pests and other biotic stresses. Therefore, it is concluded that except Amar and Pusa-9, all the experimental accessions show low genetic diversity

Abbreviations

SDS= Sodium dodecyl sulphate

UPGMA= Unweighted pair group method using arithmetic average PAGE = Polyacrylamide gel electrophoresis

RAPD= Random amplified polymorphic DNA

Competing Interests

The authors declare that they have no competing interests.

Authors’ Contributions

The work is original and it has been carried out by SAS for M.Phil. degree under supervision of RK at Bundelkhand University,

Jhansi (U. P.), India, and at Indian Biotechnology Research Institute, Agra (U. P.), India.

Acknowledgement

The authors are thankful to IBRC (Indian Biotechnology Research Institute), Agra, India, for providing lab facilities and the help of Dr. S. Chauhan is greatly acknowledged.

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