Indian Journal of Biotechnology Vol 4, January 2005, pp. 56-64
Amplified fragment length polymorphism (AFLP) analysis of genetic diversity in Indian mungbean [Vigna radiata (L.) Wilczek] cultivars K V Bhat*, S Lakhanpaul and S Chadha National Research Centre on DNA Fingerprinting, National Bureau of Plant Genetic Resources Pusa Campus, New Delhi 110 012, India Received 30 November 2003; revised 14 January 2004; accepted 28 January 2004 Released cultivars and improved lines of mungbean [Vigna radiata (L.) Wilczek] were subjected to AFLP (amplified fragment length polymorphism) analysis to test its usefulness and also to have an assessment of the genetic diversity and relationships among the cultivars. Relative efficiency of the primers, having three (+3) vs. two (+2) selective nucleotides, was also tested for detecting polymorphism. A total of 731 amplification products were obtained in the 27 cultivars with twelve primer pairs. Higher percent polymorphism was obtained with +3 than with +2 primers, though the number of amplification products was much higher with +2 primers. Consequently, higher average similarity coefficient (0.849) was obtained with +2 primers in comparison to +3 primers (0.751). Overall, a narrow genetic diversity (0.681-0.925) was recorded among the cultivars analysed. Distinct clusters were formed in the dendrogram with some variations in the constituents when data from +3 primers alone was compared with that from +2 primers. Principle coordinates analysis supports the results of UPGMA, as there was general agreement between the clustering patterns in both the analyses. The ‘Eigen’ vectors analysis indicated that the contributions of the first three factors were 12.49, 9.44 and 6.71, respectively. Twenty-seven factors were required to explain the total variation observed. Narrow genetic base observed is likely to be due to the use of limited material in the development of the cultivars analysed. Keywords: AFLP, diversity analysis, green gram, mungbean, Vigna radiata IPC Code: Int. Cl.7 C 12 N 15/10
Introduction The genus Vigna has been of immense importance to Asia as six different pulse-yielding crops belong to this genus. Of them, mungbean [Vigna radiata (L.) Wilczek], also known as green gram, is the most widely spread species. It ranks fourth among pulse crops in India (after chickpea, pigeonpea and black gram) by way of total area (3.08 m ha) under cultivation with a production of 1.31 m tonnes. It is noteworthy that of all the important pulses, such as chickpea, fieldpea, pigeonpea, black gram, lentil etc., productivity of green gram is the lowest at 425 kg per hectare1. During the last six to seven decades, a large number of pulse varieties with desirable characters have been released. However, production of new resistant varieties to biotic and abiotic stresses has been felt to reduce the yield losses in India. Like other pulses, mungbean has also considerable scope for improvement, which is evident by large gap between the actual yield and the potential yield2. The genetic ___________ *Author for correspondence: Tel: 91-11-25789211 ext 247 E-mail:
[email protected]
improvement has a key role to play in addition to improved crop management. In this context, genetic characterization of the improved material available is an essential step. Biochemical and molecular markers provide valuable information, which can be used in a number of ways in any crop improvement programme. Most of these markers, whether hybridization-based (RFLPs and VNTRs) or PCR based (RAPDs and STMS) are easy to adopt in crop improvement programes. Recent studies using RAPD markers reported narrow genetic base of the released Indian mungbean cultivars as only low to moderate polymorphism was observed3. RAPD markers, though easy to employ have often been considered to be inconsistent and lacking in reliability4. Amplified fragment length polymorphism (AFLP)5, a recent technique, offers several advantages over other DNA markers as they combine advantages of PCR based technique in terms of efficiency, high throughput and amenability to automation with the specificity and robustness of RFLP based technique. The technique, being generic in nature, does not require prior sequence information for designing primers or construction of genomic/cDNA libraries
BHAT et al: AFLP MARKER DIVERSITY IN MUNGBEAN CULTIVARS
for developing probes. The feasibility to fine-tune the amplification products obtained and very less quantity of template DNA (100-500 ng) required are some of the additional advantages of the technique. The usefulness of the technique in genetic characterization of the cultivars/germplasm has been demonstrated in a vast array of taxa, viz., rice6, maize7, barley8, soybean9, wild bean10, etc. The present study was undertaken on the improved lines and cultivars of mungbean to test the usefulness of AFLP markers for characterization and assessment of genetic variation, and to determine and compare the genetic relationships among the cultivars using AFLP technique. Materials and Methods Plant Material
Seeds of twenty-seven cultivars of V. radiata (Table 1) released from six different agricultural universities/institutes in India were procured from Indian Agricultural Research Institute (IARI), New
57
Delhi. Plants were raised in pots under controlled conditions in the National Bureau of Plant Genetic Resources, New Delhi. Young and actively growing leaves of 30 day-old-plants were harvested and used for DNA extraction. DNA Extraction
Harvested leaves were immediately stored at −80°C until total genomic DNA was extracted using a modified CTAB DNA extraction protocol11. The DNA concentration was estimated with a DNA fluorometer (Hoeffer Scientific, San Francisco, USA) using Hoechst 33258 as the dye and calf thymus DNA as the standard12. The estimates were confirmed by ethidium bromide staining of the gels after electrophoresis in 0.8% agarose gel at 100 V for 1 hr in TAE buffer (0.04 M Tris-acetate, 0.001 M EDTA, pH 8.0) using known DNA concentration standards (λ DNA, uncut). AFLP Protocol
AFLP analysis was carried out following Zabeau &
Table 1—List of mungbean [V. radiata (L.) Wilczek] cultivars used in the present study along with their places of release S. No.
Variety
1 2 3 4 5 6 7 8 9 10 11 12 13
ML-5 ML-515 ML-688 ML-713 ML-729 SML-100 SML-240 SML-390 GM-86-35 IIPRM-1 PDM-87 K-92-11 K-851
14 15 16 17 18 19 20 21 22 23 24 25
S-8 PUSA BOLD-3 PUSA-118 PUSA-121 PUSA-9072 PUSA-9131 PUSA-9272 PUSA-9631 PUSA-9671 HUM-3 GANGA-1 AKM-9243
26 27
11/395 TAP-7
Pedigree No. 54 × Hy 45 Mutant of ML-267 (ML 1 × No. 987) Pant M-2 × PDM116 20/19 × RS 4 4453-3 × T 44 Pusa 105 × 10-215 M 981 × Pusa 105 ML 9 × MCK 2 Local selection from Varanasi Kopergaon × TARM 2 Amrit × PS 7 Mutant of S 8
Place of release
Punjab Agricultural University (PAU) Ludhiana, India
Indian Institute of Pulses Research (IIPR) Kanpur, India Chandrashekhar Azad University of Agricultural Sciences and Technology (CAUAST), Kanpur, India
Indian Agricultural Research Institute (IARI), New Delhi, India
Banaras Hindu University (BHU) Varanasi, India Punjab Rao Deshmukh Krishi Vishwavidyalaya (PRDKV), Akola, Maharashtra, India Bihar Bhabha Atomic Research Centre (BARC), Mumbai, Maharashtra, India
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Vos13 and Vos et al5 with some minor modifications. 200 ng of total genomic DNA was double digested with two restriction endonucleases, Mse I and Eco RI, one frequent cutter and the other rare cutter in a 25 μl reaction volume containing 5 μl of 5 X reaction buffer (50 mM Tris-HCl, pH 7.5, 50 mM Mg-acetate, 250 mM K-acetate), 2 μl restriction endonucleases mix (1.25 units each in 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 0.1 mM EDTA, 0.1 mg/ml BSA, 50% (v/v) glycerol, 0.1% Triton X-100) and volume made up with sterile distilled water. Digestion mix was incubated at 37°C for 2 hrs, followed by incubation at 70°C for 15 min in order to inactivate the enzymes. 25 μl of ligation mix containing 24 μl of adapter-ligation solution (Eco RI/Mse I adapters, 0.4 mM ATP, 10 mM Tris-HCl, pH 7.5, 10 mM Mg-acetate, 50 mM Kacetate) and 1 μl of T4 DNA ligase (1 unit/μl in 10 mM Tris-HCl, pH 7.5, 1 mM DTT, 50 mM KCl, 50% glycerol v/v) was added to the same solution and ligation was carried out at 20°+2°C for 2 hrs. This was followed by a 1:10 dilution of ligation mixture with TE buffer (10 mM Tris HCl, pH 8.0, 0.1 mM EDTA). Pre-amplification was performed in a Perkin-Elmer Thermal-Cycler (9600 GeneAmp) in 0.2 ml thin walled PCR tubes containing 5 μl of diluted template DNA, 40 μl of pre-amplification primer mix, 5 μl of 10 X PCR buffer containing Mg++, 1 unit of Ampli Taq DNA polymerase in the following PCR conditions: 20 cycles of 94°C for 30 sec, 56°C for 60 sec and 72°C for 60 sec. Eco RI primers were radiolabelled by phosphorylating the 5′ end of the primers with [γ-P32] ATP and T4 kinase. Selective amplification was carried out in a 20 μl reaction volume which contained 5 μl of diluted (1:50) preamplified DNA, 5 μl mix A (5 μl labelled Eco RI primer, 45 μl Mse I primer), 10 μl of mix B (2 μl of 10 X PCR buffer plus Mg), 1 unit of Ampli Taq polymerase and 7.9 μl of water). The PCR conditions were: one cycle at 94°C for 30 sec; 65°C for 30 sec and 72°C for 60 sec, followed by a touchdown cycle profile as follows: 94°C for 30 sec, 65°C (temperature reduced by 0.7°C/cycle) for 30 sec and 72°C for 60 sec, which gives a touchdown phase of 13 cycles. This was followed by 23 cycles at 94°C for 30 sec, 56°C for 30 sec and 72°C for 60 sec. Following selective amplification, the samples were denatured by adding formamide dye (formamide 98%, 10 mM EDTA, bromophenol blue and xylene cyanol) to each sample and heating at 90°C for 5 min and immediately placing them on ice.
The denatured amplification products were separated in 6% denaturing polyacrylamide gel (20:1 acrylamide: bis-acrylamide; 7.5 M urea; 1 X TBE buffer) in BioRad Sequi-Gen GT Sequencing cell. Pre-electrophoresis was carried out at constant 85 watts for 40 min, followed by loading 3 μl of sample and running the gel at 110 watts at 50°C temperature till the tracking dye reached two-thirds the length of gel (approximately 3.30 hrs). The gel was dried under vacuum in a gel dryer and exposed on X-ray film for 1-4 days depending on the intensity of the signal. Primer Survey and AFLP Analysis
Primer survey was carried out using three cultivars namely, ML-5, Pusa-118 and K-851 for selecting useful primer combinations. A total of 42 primer combinations (from GIBCO-BRL, USA) comprising 22 Eco RI primers with three selective nucleotides (hereafter designated as +3 primers) and 20 with two selective nucleotides (hereafter designated as +2 primers) were surveyed. Of those, 12 primers were found suitable (Table 2) and used to analyse all 27 cultivars. Data Analysis
Autoradiograms were scored for the presence (1) and absence (0) of an amplification product across the lanes for each of the primer combinations. The raw data matrix was used to calculate pair-wise Jaccard’s similarity coefficients14 among the varieties. This matrix was subjected to UPGMA to construct a dendrogram. The Jaccard’s similarity coefficients matrix was also subjected to Principal Coordinates (PCO) analysis by extracting the ‘Eigen’ vectors and plotting the three most informative factors as a 3dimensional plot to depict relationships among the varieties. The statistical analyses were conducted using the NTSYS-pc, version 1.70 software package (Exeter Software, New York, USA). Data obtained from +3 and +2 primers were also analysed independently in a similar manner. Results Of 42 primer combinations surveyed, 12 primer combinations were selected for the analyses. The criteria for selecting primer pairs were: a) high-quality of amplification, b) good resolution of the amplification products, and c) polymorphism among the cultivars used for the primer survey. Table 2 gives the list of twelve primer combinations, six each with +3 and +2 selective nucleotides, which were used for the analysis of twenty-seven mungbean cultivars
BHAT et al: AFLP MARKER DIVERSITY IN MUNGBEAN CULTIVARS
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Table 2—List of primer pairs used for analyzing mungbean [V. radiata (L.) Wilczek] cultivars along with salient features of their amplification products S. No.
Primer pair
Total no. of amplification products
1
E-ACG:M-CAC
59
48
81.36
21-39
2
E-AGC:M-CTA
59
45
76.27
24-41
3
E-AGC:M-ACA
70
58
82.85
31-54
4
E-ACA:M-CAC
38
17
44.74
26-35
5
E-ACA:M-CAG
44
21
47.73
30-39
6
E-ACA:M-CTC
56
40
71.43
27-37
7
SE-AA:M-CTA
81
26
32.10
51-68
8
SE-AA:M-CTG
73
34
46.58
50-62
9
SE-AC:M-CAC
68
40
58.88
40-52
10
SE-AG:M-CAA
64
27
42.19
46-55
11
SE-TC:M-CTA
68
44
64.71
43-56
12
SE-AG:M-CAC
51
23
45.10
34-45
along with the characteristics of the amplification products obtained. Fig. 1 is a representative AFLP pattern obtained in the present study. Table 3 is the summary of results obtained. A total of 731 amplification products were obtained which comprised 326 and 405 amplification products with +3 and +2 primer combinations, respectively. Number of polymorphic products obtained was 229 and 194 with +3 and +2 primers, respectively. The average number of products per primer was higher with +2 primer (67.5) than with +3 primer (54.33). However, the average number of polymorphic products was higher in case of +3 primers (38.17) than with +2 primers (32.33). Accordingly, the percent polymorphism was more with +3 primers (67.40%) than with +2 primers (48.26%). Table 2 gives the salient characteristics of the amplification products obtained with each of the 12 primer pairs. The primers E-AGC: M-ACA and SE-TC: M-CTA were the most polymorphic +3 and +2 primers combinations, respectively in terms of percent polymorphism. Jaccard’s similarity coefficient values (SCV) calculated from the data obtained from the selected 12 primer pairs ranged from 0.681 to 0.925 with an average of 0.808. As expected, the SCVs were higher with +2 primers (0.741-0.984, average 0.849) in comparison with +3 primers (0.535-0.897, average 0.751). SCVs were used to construct a dendrogram employing the UPGMA algorithm (NTSYS-pc,
No. of polymorphic products
Percent polymorphism
Bands per primer (range)
Fig. 1—AFLP profiles of 27 mungbean cultivars obtained with the primer pair SE-TC: M-CTA. The sequence of the cultivars in the autoradiogram is as given in Table 1. Lane 28 is the mungbean internal control, PLG 619.
INDIAN J BIOTECHNOL, JANUARY 2005
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Table 3—Summary of the AFLP analyses of mungbean cultivars with six each of the +3 and +2 selective nucleotide primers S. No.
Features
1 2 3 4 5 6 7 8
Primer pairs surveyed Primer pairs analyzed Total no. of amplification products No. of polymorphic products Percent polymorphism Average no. of amplification products/primer Average no. of polymorphic products/primer Primer showing highest polymorphism (percent polymorphism)
9
Primer showing lowest polymorphism (percent polymorphism)
10
Similarity coefficient values Range Average
Version 1.70). Dendrograms are presented using the data from +3 as well as +2 primers (Fig. 2a) and only +3 (Fig. 2b) or +2 primers (Fig. 2c). The +3 dendrogram comprises of mainly two clusters, i.e. I and II. Of 27 cultivars, only two cultivars are grouped in cluster I, viz. ML688 and SML244; whereas, cluster II comprises 23 cultivars that are further grouped in three sub clusters, viz. IIA, IIB and IIC. Cluster IIA comprises three cultivars i.e. IIPRM-1, K92-11 and PDM-87, two of which have been released from IIPR, Kanpur. Out of the nine cultivars included in the cluster IIB, two namely, S8 and 11/395 appear to be the closest amongst all. Six out of these nine cultivars have been released from IARI, New Delhi, one each from PAU, Ludhiana, CAUAST; Kanpur and Bihar. In contrast, cluster IIC predominantly contains cultivars released from PAU, Ludhiana, as five out of the nine cultivars included in the present study are placed here. TAP7 though included in IIC appears to be distinct from all other cultivars in this sub cluster. Dendrogram based on +2 primers (Fig. 2c) also comprises mainly two major clusters. Cluster I contains six cultivars grouped in two sub clusters having three cultivars each. Like +3 dendrogram, most of the cultivars (19) are grouped together in cluster II that further grouped in four sub clusters. Three cultivars namely, GM-86-35, SML100 and Pusa 9272, are included in IIA. Nine cultivars in cluster IIB include the six released from IARI, New Delhi. Cluster IIC and IID comprise three and four cultivars, respectively. Clustering pattern in the composite dendrogram based on all twelve-primer pairs (Fig. 2a) was the sum
Total (+3 and +2 primers)
+3 primers
+2 primers
42 12 731 423 57.87 60.92 35.25 E-AGC: M-ACA (82.85) SE-AA: M-CTA (32.10)
22 6 326 229 70.25 54.33 38.17 E-AGC: M-ACA (82.85) E-ACA: M-CAG (44.74)
20 6 405 194 47.90 67.5 32.33 SE-TC: M-CTA (64.71) SE-AA: M-CTA (32.10)
0.681-0.925 0.808
0.535-0.897 0.751
0.741-0.984 0.849
of the other two independent dendrograms. Therefore, a comparison of only +3 and +2 dendrograms is desirable and logical. Cultivars ML688 and SML 244 have grouped together in both the dendrograms (cluster I). Cultivars IIPRM-1, K-92-11 and PDM87 have also clustered together in both the cases. Similarly, S8, Pusa Bold-3, 11/395, Pusa 118, SML390, Pusa-9631 and K-851 have also grouped together in +3 as well as +2 dendrograms. Three cultivars, namely, ML-5, ML515 and HUM3 have also shown close association in both the dendrograms. Interestingly, AKM 9243 and Pusa 9671 did not group in any of the clusters in +3 dendrogram, but are placed together in the sub cluster IIC in +2 dendrogram (Fig. 2c). Overall, a comparison of three different dendrograms obtained using data from six primers with three selective nucleotides, six primers having two selective nucleotides and all the twelve primer combinations together shows a high degree of similarity. One striking difference in the two dendrograms is the placement of two different cultivars as the outliers, which are Pusa 121 and ML 713 in +3 and +2 dendrograms, respectively. Principal Coordinates Analysis
The results of UPGMA and PCO analyses (Fig. 3) were comparable. The pattern of clustering of the varieties was similar with both the analyses. The ‘Eigen’ vectors analysis indicated that the contributions of the first three factors were 12.49, 9.44 and 6.71, respectively (explaining a total of only 28.64% of total variability). In order to explain 100% of the variations observed, 27 factors were required
BHAT et al: AFLP MARKER DIVERSITY IN MUNGBEAN CULTIVARS
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Fig. 3—Three-dimensional plot of the first three factors from the principal coordinates analysis of the combined AFLP data of 27 mungbean cultivars obtained with 12 primer pairs. These three factors explain 12.49, 9.44 and 6.71 percent of the total variations, respectively.
genotypes has assumed greater importance due to reaffirmation of sovereign rights of the countries on their genetic resources15. Analysis of the cultivars and improved lines using efficient and robust DNA markers such as AFLP is valuable in this regard. Diversity Among Cultivars
Fig. 2. UPGMA dendrograms of 27 mungbean cultivars constructed from the analysis of AFLP data obtained from (a) 12 AFLP primers, (six ‘+3’ and six ‘+2’ primers), (b) only (+3) primers and (c) only (+2) primers.
thereby indicating the smaller contributions of each of the variables towards total variability. This was also indicative of the lower genetic diversity among the varieties with respect to the AFLP loci studied. Discussion An assessment of genetic diversity is important for the breeders in order to select the diverse types in crossing programmes and also for the gene bank managers to devise strategies for their conservation. Recently, the identification and characterization of
The study revealed rather low genetic diversity in the improved lines/cultivars. In general, the improved lines/ cultivars released from the same Institute showed greater homologies indicating their closeness. For example, the cultivars from IARI, New Delhi clustered together in the dendrogram indicating closer genetic similarity. Likewise, varieties released from PAU, Ludhiana also show a higher degree of similarity in comparison to other cultivars. These results are in agreement with our earlier findings based on RAPD markers (3). A survey of the pedigrees and source materials of a large number of cultivars clearly shows that repeated use of the same material, having few desirable characters, has taken place in most of the improvement programs3,16. For example, Pusa 9671 and K851 have been developed from ‘Type 1’, a selection from local collections from Bihar state in
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eastern part of India. Similarly, S-8, which is also a source of TAP-7, has been developed from another local collection of Bihar. Pedigree of another cultivar, viz. 11/395, could also be traced to a local germplasm of Bihar, designated as BR-2. Interestingly, Bihar in addition to being the place where mungbean improvement was initiated in India is also located in the Indo-Gangetic plains, which is recognized as the secondary centre of diversity for mungbean. Historically, the organized attempts to identify improved strains of mungbean useful for different regions were made nearly six to seven decades ago in the Botany Section of Imperial Agricultural Research Institute, Pusa, Bihar, (Presently, Indian Agricultural Research Institute located at New Delhi) from the original mixed samples collected from different parts of the country17. According to Sharma16, the first record of a released mungbean variety was ‘Type 1’ in 1948, which was a selection from a local collection of Bihar. Thereafter, a series of varieties were commercialized but all of them were selections of well-adapted local materials though few exotic germplasm has also been included. After early 1960’s, many hybrid derivatives were developed and released as commercial varieties as a result of the All India Coordinated Pulse Improvement Project. However, there has been repeated use of same cultivars and lines having wider acceptance or with one or more economically important traits16. Consequently, the extent of genetic variation in the improved lines and released cultivars is likely to be low which is revealed in the present analysis. Reports on the analysis of genetic diversity present in the vast mungbean germplasm collections18,19 using molecular markers are lacking. However, significant diversity has been reported on the basis of morphoagronomic traits18-20. Therefore, a large extent of variability present in the germplasm seems to have been excluded from the improved material. However, it is important to pointout that some of the diversity reported in the above studies was accounted by accessions sampled from exotic material and from southern zone of India. By contrast, the material analysed in the present study did not have any representation from South India and other countries. Analysis of the diverse germplasm available using molecular markers is highly desirable in order to select the diverse types for breeding programmes. Though, pedigrees of all the cultivars could not be made available, it appears that limited source material has been used for the improvement of the mungbean
cultivars in India. Such narrow genetic base of the improved cultivars has also been reported earlier21-23. In fact, Steiner et al22 reported that all North American crimson clover cultivars could be traced back to only two germplasm lines. Small Genome Primers (+2) vs. Large Genome Primers (+3)
For small genome size (2C DNA content, 1.67 pg) reported in mungbean24, +2 primers were expected to be more useful as has been suggested by Vos et al5. It is interesting to note that though the number of amplification products formed are more with the +2 primers, +3 primers reveal higher percentage polymorphism (Tables 2 & 3). Therefore, +3 primers may be considered more useful for detecting polymorphism, as only the proportion of monomorphic amplification products were higher in +2 primers. Further, lower SCVs with +3 primers in comparison to +2 primers, in terms of range as well as average, show the higher discrimination efficiency of the +3 primers. Nevertheless, the dendrogram constructed independently with +3 and +2 primers and with the combined data are highly comparable (Fig. 2). Only minor variations in the three dendrograms indicate robustness of the AFLP technique. The variations encountered are expected due to sampling of different genomic regions with different primer combinations depending upon their selective nucleotide sequences. It is important to point out that SE-AC: M-CAC is the only +2 primer pair which is “nested” or “contained” for +3 primer pairs E-ACG: M-CAC and E-ACA: M–CAC as the latter two differ only in the third selective nucleotide for one primer (E) from the former. Thus, the genome fractions analysed by the different primer pairs are not likely to be the same but randomly distributed. Congruence in the patterns, obtained from different and independent sets of primers, may further be considered to substantiate the results obtained25. Comparison with RAPD Analysis
The results obtained in the present study show a high overall genetic similarity (0.68-0.92) between the cultivars analysed and therefore agree with RAPD analysis done earlier3 on the same cultivars, where genetic similarity ranged between 0.65-0.92. It is important to point out that the percent polymorphism obtained in the present study was less in comparison to that with RAPD markers. Russell et al26 have also reported higher percent polymorphism with RAPDs (66.3%) than with AFLPs (46.8%) but still reported
BHAT et al: AFLP MARKER DIVERSITY IN MUNGBEAN CULTIVARS
AFLPs to be more useful than RAPDs in characterizing barley accessions. In fact, they have reported that a comparison of RFLP, AFLP, SSR and RAPD revealed highest diversity index with AFLPs in spite of their lowest level of polymorphism in the barley germplasms analysed. This has been ascribed to a much higher number of bands obtained per lane in case of AFLP, which results in a significant increase in the value of marker index though a large fraction of the bands show monomorphism. A comparison of the clustering pattern obtained from RAPD and AFLP analyses also shows conspicuous similarities. For example, cultivars ML 688, IIPRM-1 and SML-244 are found to be quite distinct from all other cultivars as they form a wellseparated cluster in both the studies. Further, eight of the nine cultivars, namely S8, Pusa Bold-3, 11/395, Pusa 118, Pusa 9072, SML-390, Pusa 9631 and K851, that were grouped together in one cluster in AFLP study were in a single cluster even with the RAPD-based analysis. Similarly, cultivars ML-5, ML-515, Hum-3, Ganga-1, SML-100 and Pusa-9272 that have clustered together in the present study were placed in the same cluster RAPD study. Interestingly, cultivars Pusa 9131 and AKM 9243, which have not been grouped in any cluster and thus are outliers in AFLP study, were placed similarly in the earlier RAPD study3. An exception was the grouping of TAP-7 and Pusa 9631 in a loose cluster in AFLP analysis, while these were placed in two different clusters in the RAPD studies. In conclusion, the present study reveals the narrow genetic base of the improved cultivars and lines of mungbean. However, reports pertaining to the presence of highly diverse germplasms18,19 and India being proposed centre of diversity27-29 show that mungbean does not lack variability in the germplasm material. Thus, a very close similarity among the cultivars analysed may be ascribed to limited use of material in the development of these improved types. Therefore, the need for strengthening germplasm enhancement cannot be overemphasized in this crop. The present data also supports our earlier study where the same cultivars were analyzed using RAPD markers. A significant agreement is found in the two analyses regarding the extent of genetic diversity revealed as well as the clustering pattern of the cultivars. Nevertheless, distinct advantages offered by AFLPs over RAPD and other DNA based techniques make them desirable markers of choice.
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Acknowledgement Authors are grateful to the Indian Council of Agricultural Research, Government of India, for the financial support to The National Research Centre on DNA Fingerprinting. Dr J L Tickoo and Dr Naresh Chandra, Pulses Division, Indian Agricultural Research Institute, New Delhi, are gratefully acknowledged for provided seeds. Dr P L Gautam, Director, National Bureau of Plant Genetic Resources and Dr J L Karihaloo, Project Director, NRC on DNA Fingerprinting, are acknowledged for providing the facilities and for their encouragement in undertaking this work. References 1
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Asthana A N & Chaturvedi S K, Pulses—A little impetus needed, in The Hindu—Survey of Indian agriculture 1999, edited by N Ravi (National Press, Chennai, India) 1999, 6166. Subbarao G V, Johansen C, Slinkard A E, Rao N, Saxena N P et al, Strategies for improving drought resistance in grain legumes, Crit Rev Plant Sci, 14 (1995) 469-523. Lakhanpaul S, Chadha S & Bhat K V, Random amplified polymorphic DNA (RAPD) analysis in Indian mungbean [Vigna radiata (L.) Wilczek] cultivars, Genetica, 109 (2000) 227-234. Karp A, Seberg O & Buiatti M, Molecular techniques in the assessment of botanical diversity, Ann Bot, 78 (1996) 143149. Vos P, Hogers R, Bleeker M, Reijans M, Theo van dee L et al, AFLP: A new technique for DNA fingerprinting, Nucleic Acids Res, 23 (1995) 4407-4414. Subudhi P K, Nandi S, Casal C, Virmani S S & Huang N, Classification of rice germplasm III. High resolution fingerprinting of cytoplasmic male sterile (CMS) lines with AFLP, Theor Appl Genet, 96 (1998) 941-949. Ajmon-Marsan P, Castglioni P, Fusari F, Kuiper M & Motto M, Genetic diversity and its relationship to hybrid performance in maize as revealed by RFLP and AFLP markers, Theor Appl Genet, 96 (1998) 219-227. Ellis R P, McNicol J W, Baird E, Booth A, Lawrence P et al, The use of AFLPs to examine genetic relatedness in barley, Mol Breed, 3 (1997) 359-369. Maughan P J, Saghai-Maroof M A, Buss G R & Huestis G M, Amplified fragment length polymorphism in soybean: Species diversity, inheritance and near isogenic line analysis, Theor Appl Genet, 93 (1996) 393-401. Tohme J, Gonzales D O, Beebe S & Duque O, AFLP analysis of gene pools of a wild bean core collection, Crop Sci, 36 (1996) 1375-1384. Saghai-Maroof M A, Soliman K M, Jorgenson R A & Allard R W, Ribosomal spacer-length polymorphisms in barley: Mendelian inheritance, chromosomal location and population dynamics, Proc Natl Acad Sci (USA), 81 (1984) 8014-8018. Brunk C F, Jones K C & James T W, Assay for nanogram quantities of DNA in cellular homogenates, Anal Biochem, 92 (1979) 497-500.
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