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Published in Issue No. 138, page 50 to 54 - (15836) characters
Genetic diversity assessment of finger millet, Eleusine coracana (Gaertn), germplasm through RAPD analysisB. Fakrudin R.S. Kulkarni H. E. Shashidhar S. Hittalmani
Finger millet (Eleusine coracana Gaertn.) is an important food crop cultivated widely in India , most parts of central and east Africa and in other Asian countries including Sri Lanka , China and Japan . Finger millet grains are nutrient rich and the straw is a nutritious fodder. As of now, in India , only a very small fraction of the total available collections have been used in the national breeding programmes (Ramakrishna et al. 1996). The project coordination cell on small millets in India has an exhaustive collection of 3940 accessions representing the entire global distribution of finger millet. Other major collections are held by international institutions such as ICRISAT in India (5000 accessions), the National Farming Research Station in Kenya (1500 accessions), the Gene Bank in Kenya (1000 accessions), the Plant Genetic Resource Center in Ethiopia (1000 accessions) and the University of Georgia in USA (1500 accessions). A number of non-governmental organizations such as Green Foundation in India also maintain finger millet collections (Ramakrishna et al. 1996).
Although such large and diverse germplasm collections are available in both public and private organizations no concerted efforts have been made to assess the diversity of this species at the molecular level and integrate these results with morphological characterization in breeding programmes for higher yield and disease resistance. Establishment of core collections based on field evaluation and molecular variation shown by the accessions would obviously be advantageous, but clear and detailed assessment of molecular diversity within finger millet germplasm collections is not available. It is worth emphasizing that the assessment of genetic diversity using DNA markers is one of the key tools of crop improvement and germplasm conservation.
DNA markers have been used to evaluate genetic diversity in different crops species (Cooke 1995). Some of the first DNA markers to be widely used in assessing genetic diversity in plant species were restriction fragment length polymorphisms (RFLPs) (Cox et al. 1986), a highly reproducible assay which, however, detects only a few loci for each experiment, and random amplified polymorphic DNA (RAPD) (Williams et al. 1990; Vierling and Nguyen 1992; Kazan et al. 1993), which detects a large number of markers but is not always as reproducible as RFLP (Cooke 1995). The RAPD system has the advantage of being a fast assay requiring only nanograms of genomic DNA and minimum apparatus. Therefore, it has been used to analyse the genetic relatedness in several crop species (Chalmers et al. 1992; Koller et al. 1993; Zhu et al. 1997). Development of co-dominant markers, such as simple sequence repeats (SSRs), which are not currently available in finger millet will greatly aid in diversity and genome analysis efforts.
The purpose of the present study was to investigate, through the use of RAPD markers, the genetic diversity of selected finger millet germplasm from different origins and pedigree backgrounds.
Materials and methods
Twelve accessions representing different geographical regions and pedigree were selected for the study (Table 1). These accessions were kindly provided by Dr A. Seetharam, Coordinator, Millet scheme, GKVK campus, University of Agricultural Sciences , Bangalore , India . DNA was isolated from a pool of five plants for each accession according to Dellaporta et al. (1983) and its concentration estimated through agarose gel electrophoresis by comparison with a known concentration of l DNA. Thirty-seven random decamer primers with different GC contents were used to survey the genome of the accessions (Table 2). A single primer was used in each PCR reaction. The polymerase chain reaction mixture contained of 50 h g of template DNA, 20 h g of random decamer primer (Operon Technologies, USA), 0.1 mM dNTPs, 0.6U Taq polymerase (Bangalore Genei Pvt., Bangalore, India), 1X PCR buffer (10 mM Tris pH 8.0, 50 mM KCl, 1.8 mM MgCl 2 and 0.01 mg ml –1 gelatin) in a volume of 25 µl. Reaction mixtures were overlaid with one drop of mineral oil (Sigma). Amplifications were performed on an MJ Research PTC 200 thermal cycler. The standardized amplification profile was: initial denaturation temperature 94˚C for 2 min, followed by 45 cycles of denaturation 94˚C for 2 min; primer annealing 36˚C for 1 min; primer extension 72˚C for 2 min; and a final primer extension at 72˚C for 3 min. Electrophoresis of the amplified products was carried out on 1.3% agarose (Sigma) gels and visualized through staining with ethidium bromide. The amplified products were scored as 1 for presence and 0 for absence. Only those gels that showed amplicons upon repetition with each of the primers tested were scored in order to overcome problems of poor repeatability. A control, complete reaction mixture without DNA, was included for every reaction. Measurement of genetic similarity for pairwise accessions was based on Jaccard’s similarity coefficient, which is commonly recommended for dominant data such as the results of RAPD (Sneath and Sokal 1973). A similarity matrix was constructed and subjected to clustering analysis following the UPGMA method using NTSYSpc version 1.8 software (Rohlf 1993).
Results and discussion
RAPD markers proved to be very informative and useful in monitoring the genetic diversity present in a sample of 12 selected accessions. The fragment sizes, detected by comparing the amplicons with a DNA Ladder (Genei Pvt., Bangalore, India), ranged from approximately 0.4 to 3.7 kb. An example of a typical RAPD pattern is shown in Figure 1. Analysis of the 12 accessions with 37 primers showing repeatable amplification revealed a total of 254 bands, with an average of 6.86 bands per primer. Overall, finger millet genotypes exhibited a high level of genetic diversity. Out of 254 bands, 218 fragments (85.82%) were polymorphic across the whole set of samples (Table 3). The maximum number of bands generated by a single primer was 13 (OPAT-13), and three different primers, OPA-01, OPA-02 and OPAA-02, generated 12 bands each. The primers OPBH-03 and OPS-02 only amplified two monomorphic bands in each accession; on average 5.89 polymorphic bands per primer were recorded. Some primers such as OPAZ-03, OPBH-16 could detect polymorphism across all 12 accessions. This result shows the ability of RAPD to discriminate among genotypes and suggests their application for cultivar identification.
Variation between finger millet accessions was observed with 35 of the 37 selected primers, which is crucial for valid conclusions owing to problems associated with the reproducibility in RAPD technology. While the pedigree background and the geographical diversity of the 12 finger millet accessions used influence the ease with which variation was revealed in this study, it is clear from our results that variation between more closely related accessions (both by pedigree and geographical origin) is readily identifiable.
One of the immediate uses of this study has been to identify primers which are likely to be efficient in revealing diversity in other accessions of finger millet; for instance out of a set of 40 primers used, only 37 could give consistent repeatability and only 35 could be useful in detecting polymorphism among 12 accessions of finger millet. Short-listing such primers will be useful for further analysis of germplasm (Virk et al. 1995).
It was surprising to note the complete absence of amplicons for some primers in some bred varieties like GPU 28 and Indaf 9. Further, some of the advanced breeding lines, L5 and Sel 14, possessed a reduced number of bands compared with landraces for primers such as OPBA-02 and OPC-02. Reduction in the number of amplified bands in highly bred and cultivated varieties could possibly be due to genetic erosion of DNA sequences during evolution (Zhu et al. 1997) affecting the specific RAPD primer annealing sites. However, the reduced number of bands present in these accessions could also be due to breeding selection, which is known to result in narrowing of the genetic base of the cultivated species (Wang et al. 1998). The use of landraces in breeding programmes could therefore be considered useful in restoring part of the diversity lost. It would be interesting to analyse in more detail the DNA sequences presently missing in higher-yielding cultivated varieties by using co-dominant markers such as microsatellites.
Based on the simple matching coefficient, a genetic similarity matrix was constructed using the RAPD data to assess the genetic relatedness among the 12 accessions. The similarity coefficients ranged from 0.397 to 0.821 for all accessions; the minimum genetic similarity (39.7%) was between an advanced breeding line and a wild accession (S 14 and IE 2912) of African type and the greatest similarity (82.1%) between two African accessions, IE 2881 and IE 2885. Genetic similarity coefficients indicating the extent of relatedness among selected accessions are presented in the Table 4.
The similarity coefficients were used as input data for cluster analysis performed by the NTSYSpc program. The resulting dendrogram is shown in Figure 2. The accessions clustered into two major groups, corresponding largely to African and Indian types at a similarity index corresponding to 0.55, with a intermix of a few accessions. This is in agreement with Ishii’s version (1996) in rice where accessions with geographical proximity clustered together more frequently compared with the ones from different geographic locations; advanced breeding lines derived from different geographic parents (Indica and Japonica types) positioned into both clusters corresponding to Indica and Japonica groups. Two African types, GE 4865 and IE 2912 clustered with the Indian accessions while two Indian types, Indaf 9 and Sel. 14, were grouped along with African types. Within the African-type-dominated cluster (cluster B), the accession IE 1012 was further sub-clustered with Indian accessions as this accession has been used extensively in breeding programmes as a source of neck and leaf blast resistance. The molecular distinction of IE 1012, GE 4865 and IE 2912 corresponds with the significantly different phenotypic distinction of these accessions in terms of plant types and growth habit, as well as their response to biotic stresses, especially neck, finger and leaf blast resistance. Accessions IE 2885 and IE 2881 were found to be the most similar in the selected data set with only 18% dissimilarity between these two accessions. These results are in agreement with the findings of Hilu (1995), who investigated selected accessions of African origin using RAPD, and Hiremath (1997), who revealed the karyotypic discrepancies between Indian and African type of accessions by RAPD and cytogenetic investigations.
Accessions with close geographic relations (IE 2885 and IE 2881, collections from the same provenance in Africa ) or pedigree relations (Indaf 9 and Sel 14 where IE 1012 is the male parent in their pedigree) clustered together. However, several fingerprints were available to distinguish African and Indian groups; several primers produced specific (at least one) fingerprint (amplicon of a specific random primer) to distinguish any two accessions at a time. Accession IE 2881 and IE 2885 had very few primers to produce bands, which could distinguish them. The differences in genetic diversity among all selected accessions provide valuable information for genebank curators in developing core collections.
Based on the accessions selected for use in this study, we have observed fewer RAPD polymorphisms between the Indian accessions compared with the more diverse African accessions. In addition, the highly bred cultivated types were found to be less polymorphic and to contain fewer amplicons, indicating that the selection process of the plant breeding programme could have contributed to the genetic erosion of the cultivated types.
We gratefully acknowledge the Council of Scientific and Industrial Research (CSIR), Government of India for the financial support in the form of JRF and SRF to B. Fakrudin.
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