Genetic Diversity Assessment Across Different ... - Semantic Scholar

Electronic Journal of Plant Breeding, 1(4): 379-383 (July 2010)

Research Article

Genetic Diversity Assessment Across Different Genotypes Of Mungbean And Urdbean Using Molecular Markers Ashwini Narasimhan , B.R.Patil and S. Datta, M. Kaashyap

Abstract Pulses compliment the daily diet of Indians along with cereals. They are rich in proteins with satisfactory proportion of carbohydrates. Mungbean, Vigna radiata and Urd bean, Vigna mungo are the important grain legume crops in agriculture, particularly in India. MYMV (Mungbean Yellow vein Mosaic Virus) is a virus transmitted by whitefly, Bemesia tabaci, the most serious disease of Mungbean and Urdbean. In this study, six each of MYMV resistant and susceptible genotypes in Mungbean and Urbean respectively were selected for the diversity analysis using molecular markers. Twenty four RGA primers from cowpea were used to screen the twenty four genotypes. Dendrogram generated clearly indicated two big clusters at 15% similarity. All mungbean genotypes made one cluster (cluster A) except PS16, which was included in other cluster made by Urdbean genotypes (cluster B). Cluster A contained eleven genotypes while cluster B contained thirteen genotypes. Cluster A and B were further classified into two sub clusters namely A1 and A2, B1 and B2 respectively. A1 consisted of seven genotypes of which five were resistant (PANT MUNG 1, PANT MUNG 5, HUM 12, PUSA 9531, HUM 1) and two were susceptible (TARM 2, KOPERGAON 3), while A2 comprised of remaining four genotypes in which three were susceptible (TAP 7, SML 134 and SML 668), and one (AKM 8803) was resistant. Further, it was found that four mungbean resistant genotypes of A1 namely Pant Mung1, Pant Mung5, HUM 12, and PUSA 9531 made one cluster at 55% similarity. Cluster B, again was subdivided into B1 and B2. B1 consisted a single genotype which was a cross between IPU 99-25* SPS5 while, B2 consisted of the rest of the twelve genotypes. It was interesting to see that two resistant (IPU 02-33 and IPU 6-02) and two susceptible (LBG 20 and T9) genotypes of Urd bean made separate cluster with a similarity of 99 per cent and which indicated that though genotypes are differing at resistant locus, they are highly similar at all other loci. Key words: MYMV, RGA, Bemesia tabaci, diversity analysis, Vigna radiata, Vigna mungo, polymorphism.

Introduction Legumes represent the third largest family of higher plants and comprise more than 650 genera and 18,500 species. Mungbean (Vigna radiata) and Urdbean (Vigna mungo) are two important legumes in Phaseloid clade within Papillionidae and occupies pivotal position in Indian Agriculture. The productivity of pulses is very low as compared to cereals, which have been selected for high grain yield under high input conditions, while the selection pressure in case of pulses have been focused on the adaptation to both biotic and abiotic stresses. Mungbean Yellow Mosaic Virus is a Begamovirus transmitted through white fly, Bemesia tabaci, causing significant yield losses in Vigna species, leading to a yield penalty of cent percent under Department of Genetics and Plant Breeding, College of Agriculture, University of Agricultural Sciences, Dharwad-580 005, Karnataka, India

severe conditions. Despite the efforts, development of sustainable resistance with higher yields has not been successful due to the narrow genetic base of the present cultivars. Genetic variability and divergence present in the materials is an important tool for any breeding programme. The assessment of variation made would provide us a correct picture of the extent of variation, further helping us to improve the genotypes for biotic and abiotic stresses. In the present study an attempt is made to assess the molecular diversity so that a suitable mapping population can be developed to identify and validate markers related to resistant loci. Materials and Methods Six each of MYMV resistant and susceptible varieties in Mungbean and Urd bean were selected for the studies which are represented in Table.1

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DNA was isolated from seeds using cetyl trimethyl ammonium bromide (cTAB) method and checked for its purity by running on 0.8 per cent agarose. Quantification was done by comparing with uncut lambda 500ng/µl (Bangalore Genei). Dilutions were made in order to reduce the concentration to 25ng/µl for fifty reaction mixtures. Twenty four RGA primers of cowpea which were designed using WEBSAT programme by searching in public data bases and downloading in fasta format from ncbi site, were used for the present study. Amplification was carried out in 20µl reaction mixture containing 2 µl (25ng /µl ) of DNA, 2 µl (10X) of Taq buffer, 2 µl (25µM) of dntp’s, 1 µl each of forward & reverse primer(10pmols), 0.3 µl (1 units.) of Taq polymerase ( Bangalore Genei) and 11.8 µl of Double distilled water using a MJ RESEARCH, USA thermo cycler. PCR was processed with the following program: a first cycle of pre denaturation at 94ºC for 3 min, followed by 35 cycles of denaturation for 1 min at 94ºC, 1min at the annealing temperature, followed by 2 min of extension at 72ºC, and a final extension of 7 min at 72ºC. The annealing temperatures for the RGA primers varied between 45 ºC to 50 ºC. The PCR products (20 µl) were subjected to electrophoresis on 2 per cent Agarose gel in 1 X TBE buffer for 3 hours at 50volts. A 100 bp (0.1µg/µl, 50µg) ladder from Fermentas was used as a known standard size marker. The electronic image of ethidium bromide stained gels were captured using Alpha Digi Doc. Of the twenty four RGA primers screened, polymorphism was found in eight primers considered for study. Each amplicon was treated as a separate locus. The fragments (loci) were scored as present (1) and absent (0) in each accession. Dendrogram was prepared using the program NTSYS and the following results were obtained. Results and Discussion Dendrogram (Fig.1) clearly indicated two big clusters at 15 per cent similarity. All mungbean genotypes made one cluster (cluster A) except PS16, which was included in other the cluster B. Cluster A contained eleven genotypes while, cluster B contained thirteen genotypes. The Cluster A and B were further

classified into two sub clusters namely A1 and A2, B1 and B2 respectively. A1 consisted of seven genotypes of which five were resistant (PANT MUNG1, PANT MUNG5, HUM12, PUSA9531, HUM1) and two susceptible (TRAM 2, KOPERGAON3), while A2 consisted of remaining four genotypes in which three were susceptible (TAP7, SML134 and SML 668), and one (AKM 8803) was resistant. Further, it was found that four mungbean resistant genotypes of A1 namely Pant Mung1, Pant Mung5, HUM 12, and PUSA 9531 made one cluster at 55% similarity. Cluster B was subdivided into B1 and B2. The cluster B1 consisted of a single genotype which was a cross between IPU 99-25* SPS5 while the cluster B2 consisted of the rest of the twelve genotypes. It was interesting to see that two resistant (IPU 02-33 and IPU 6-02) and two susceptible (LBG 20 and T9) genotypes of Urd bean made separate cluster with similarity of 99 per cent. The yields of Urdbean and Mungbean have remained low across subtropical and tropical Asia (Lakhanpaul et al. 2004) It is important to estimate genetic diversity in existing cultivars in order to examine if the lack of genetic variability is a constraining factor for genetic improvement. Genetic diversity analysis using RGA primers revealed two big clusters and was subdivided into four sub clusters. A mungbean genotype PS16 was included in the cluster made by Urdbean genotypes. The results indicated that PS 16 was very closely related to Urdbean. As the two resistant (IPU 02-33 and IPU 6-02) and two susceptible (LBG 20 and T9) genotypes of Urd bean made separate cluster with similarity of 99 per it is concluded that though genotypes are differing at resistant locus, they are highly similar at all other loci. The separate cluster made by IPU 99-25* SPS5 suggested that the genotype of this cross is diverse in comparison with the rest of the urdbean genotypes. The phenotypic evaluation of the variety IPU 99-25* SPS5 corroborated with the present findings shall make it suitable in the hybridization programme. The results indicated that there is close genetic similarity between the cultivars of black gram based on the study. This might be due to the high degree of commonness in their pedigrees. The narrow genetic base of the black gram cultivars was revealed in the present study. Analysis emphasizes the need to exploit the large germplasm collections having diverse morphoagronomic traits in cultivar improvement programs as reported by Basak et al . (2005) and Ramasubramanian, (2008).

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The study conducted by earlier workers like Lakhanpaul et al. (2004) , Chattopadhyay et al., (2007) and Souframanien and Gopalakrishna, (2004) analyzed the diversity in Vigna species using RAPD and SSR primers has also shown similar results. Even they have reported the narrow genetic base of Vigna species. While in contrary the diversity obtained in the present study can be used for development of mapping populations. References Basak, J.S. Kundagrami, T. K. Ghose and A. Pal., 2005, Development of Yellow Mosaic Virus (YMV) resistance linked DNA marker in Vigna mungo from populations segregating for YMV-reaction. Molecular Breeding, 14(4): 375-383.

Chattopadhyay,K, S. Bhattacharya, N. Mandal, and H.K. Sarkar., 2007, PCR-based Characterization of Mung Bean (Vigna radiata) Genotypes from Indian Subcontinent at Intra- and Inter-Specific Level. Journal of Plant Biochemistry and Biotechnology, 17(2) Lakhanpaul,S, Sonia Chadha and K.V. Bhat., 2004, Random amplified polymorphic DNA (RAPD) analysis in Indian mung bean (Vigna radiata (L.) Wilczek) cultivars. Genetica, 109(3): 227-234. Ramasubramanian T., 2008, Cloning and characterization of resistance gene analogs from under-exploited plant species. Electronic Journal of Biotechnology, 11(4) Souframanien .J, and T. Gopalakrishna., 2004, A comparative analysis of genetic diversity in black gram genotypes using RAPD and ISSR markers. Theor Appl Genet., 109:1687–1693.

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Figure1: UPGMA based clustering of genotypes using RGH primers

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Table 1 Genotypes varying for MYMV reaction

Mungbean

Mungbean

Resistant (MYMV)

Susceptible MYMV)

1. PANTMUNG 1

1. SML 668

2. PANTMUNG 5

2. TRAM 2

3. PUSA 9531

3. KOPERGAON 03

4. HUM 12

4. TAP 7

5. HUM 1

5. SML 134

6. AKM 8803

6. PS 16

Urdbean

Urdbean

Resistant (MYMV)

Susceptible (MYMV)

1. DPU 88-31

1. AKU 9904

2. UTTARA

2. PDU1

3. IPU 02-43

3. LBG20

4. IPU 99-25* SPS5

4. SHEKHAR 2

5. IPU 02-33

5. T9

6. IPU 6-02

6. BARABANKI LOCAL

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