Genetic Diversity Studies in Ragi (Eleusine coracana (L.) Gaertn.) With SSR and ISSR Mrkers

Investigation was carried out on a collection of forty ragi genotypes to study the genetic diversity using ten SSR and 30 ISSR markers. There were successful amplification SSR and ISSR markers in ragi. Of the ten SSR primers six showed polymorphism, the number of alleles produced by SSR primers ranged from two to five with an average of 2.3 allele per primer with total of 23 alleles. The percentage of amplification ranged from 58 (RM 570) to 92 (RM 3351). Of the thirty ISSR primers used for molecular analysis, twenty primers showed polymorphism among forty ragi genotypes. The twenty polymorphic primers generated a total of 101 alleles. The number of alleles produced by different primers ranged between three and ten with an average of 5.05 alleles per primer. The percentage of amplification ranged from 33 (ISSR 20) to 85 (ISSR 29). Forty genotypes grouped into thirteen clusters (SSR) and five clusters (ISSR), respectively in UPGMA analysis.


Background
Finger millet or Ragi (Eleusine coracana (L.) Gaertn.) 2n=4x=36, is a poor man's crop, originated in Ethiopia (Vavilov, 1951). It belongs to the tribe Chloridae of the family Poaceae. Finger millet is the primary food for millions in dry lands of east and central Africa and southern India. Finger millet is third in importance among millets in the country in area and production after sorghum and pearl millet. Genetic diversity is normally assessed by common morphological traits. However, such traits are affected by effects of environment, development stage of the plant and the type of plant material and also it require several replications to establish the genotypic contributions. Hence, there is a need to go in for a highly reliable and precise method for assessment of genetic variability with no environmental effects. Assessment of genetic diversity with molecular markers overcomes this problem. The use of molecular markers allows the direct assessment of genotypic variation at the DNA level. Marker analysis helps to understand the genetic makeup of the accessions and also make it possible to analyze the global organization of genetic diversity within a species. Several statistical techniques are available for the analysis of genetic diversity using DNA fingerprinting data. The present stud, a set of ten SSR and 30 ISSR markers were used asses the genetic diversity in ragi.

Result and Discussion
Ten SSR and thirteen ISSR primers were tested for their ability to amplify in the forty ragi genotypes. Of the ten SSR primers used, six were found to be polymorphic. The ten primer pairs generated a total of 23 alleles. SSR Marker profile of forty ragi genotypes generated by RM 2 (Figure 1). The number of alleles produced by different primers ranged between two and five with an average of 2.3 alleles per primer. The allele number per SSR locus in this study, was lower than the earlier studies finger millet (6.42 markers per locus, Dida et al., 2008 andBharathi, 2011), sorghum (7.3 markers per locus, Assar et al., 2005) and pearl millet (5 markers per locus, Budak et al., 2004). The percentage of amplification ranged from 58 (RM 570) to 92 (RM 3351). Of the thirty ISSR primers used for molecular analysis, twenty primers showed polymorphism. ISSR Marker profile of forty ragi genotypes generated by ISSR 30 given in Figure 2. The twenty polymorphic primers generated a total of 101 alleles. This is quite higher than the other report in finger millet (Gupta et al., 2010). The number of alleles produced by different primers ranged between three and ten with an average of 5.05 markers per primer. The percentage of amplification ranged from 33 (ISSR 20) to 85 (ISSR 29).

Polymorphic Information Content (PIC)
Polymorphism Information Content (PIC) value was calculated for ten SSR primers and given in the (Table  1). PIC value was highest for the primer RM 440 (0.729) followed by RM 492 (0.726) while, the lowest PIC value recorded by the primer RM 244 (0.32). The mean PIC value for ten SSR primers was 0.323 which was quite lower than the other reports (0.196 (UGEP107) to 0.834 (UGEP12) with an average of 0.530 in finger millet by Bharathi, 2011;0.064 (RM 274) to 0.72 (RM 580) with an average of 0.46 in rice by Seetharam et al., 2008). The higher PIC value indicated the informativeness of the primer pairs. Hence, the primer pairs RM 440, RM 492 and RM 2 are considered to be worth in future studies in the field of taxonomical and genetic resource management. Polymorphism Information Content (PIC) was calculated for twenty ISSR primers (Table 2). PIC value was highest for the primer ISSR 44 (0.761) followed by primer ISSR 99 (0.756) while, the lowest PIC value was recorded by the primer ISSR 58 (0.345). The mean PIC value for ten ISSR primers was 0.588. Of all the primers, the primer ISSR 45 generated a total of ten alleles which was recorded to be the highest, which was quite lower than the results

Cluster analysis
In the present study, 40 genotypes were subjected to cluster analysis for assessing the molecular diversity based on UPGMA analysis. Using SSR data the 40 genotypes were grouped into thirteen clusters (Table 3) at genetic similarity of 72%, which is quite higher than the earlier reports. Babu et al (2007)  The genotypes AF 459, AF 269, TNAU 1008 and GS 159, which showed a higher level of salinity tolerance at both 3000 and 6000 ppm for one or several seedling  characters came under the cluster II formed by the study using 30 ISSR markers. However, the above genotypes were not present in the one cluster formed by the study using 10 SSR markers.

Material and Methods
The material for this study included 40 different genotypes of ragi (Table 5). The materials were obtained from department of millet, Centre for Plant Breeding and Genetics, TNAU, Coimbatore. The

ISSR amplification
ISSR amplification reaction were carried out in a volume of 15 μL containing 50 ng of genomic DNA, 0.5 units of Taq DNA polymerase, 0.1 m mol/L each dNTP, 3 mmol/L Primer, 1.5 μL Taq buffer and amplification was performed in PTC thermal (AB PCR). Amplification conditions were, initial denatureation at 94℃ for 5 minutes followed by 35 cycles of denaturation at 94℃ for 30 seconds, annealing at 45℃~60℃ for 1 seconds and extension 72℃ for 2 minute and a final extension at 72℃ for 10 minutes.
PCR amplified products were subjected to gel electrophoresis in a 3% agarose gel in 1XTBE at 90 V for 3 hours using gel electrophoresis unit. The ethidium bromide stained gels were visualized under UV and documented using Alpha Imager 1200.

Statistical analysis for construction of dendrogram using molecular data
The SSR and ISSR gels were scored and represented by their allele sizes as allelic data. Using the DARwin -5.0 software package (Perrier and Jacquemoud-Collet, 2005), a simple matching dissimilarity index was calculated from the allele-size data set with 100 bootstraps, and this matrix was then subjected to UPGMA analysis.

Dissimilarity between units I and j, d ij = 1-
Where, L = number of loci; π = ploidy; m l = number of matching alleles for locus l.
Cluster analysis was performed using UPGMA strategy to obtain a dendrogram.

Polymorphic Information Content (PIC) value
Polymorphic Information Content (PIC) values were calculated for SSR markers in order to characterize the capacity of each primer to reveal or detect polymorphic loci among the genotypes. It is the sum total of polymorphism information content values of all the markers produced by a particular primer. PIC value was calculated using the formula PIC=1-∑pi 2 , where, pi is the frequency of the 'i'th allele (Smith et al., 1997).