SSR polymorphism among the inbred lines was analyzed using Super Fine Resolution agarose gel electrophoresis, following the procedure suggested by Senior and Heun (1996). Data obtained from 22 polymorphic loci, distributed over all 10 chromosomes, was used for analysis of genetic diversity. Except for chromosome 4, where only one polymorphic SSR marker could be identified in the present study, the rest of the chromosomes were represented by at least 2-3 SSR loci distributed at different map locations. A total of 58 alleles were detected for the 22 polymorphic SSR markers, giving an average of 2.64 alleles per locus. Although a large number of SSR loci used in the present study revealed only 2-3 alleles using the agarose system, a few loci such as bnlg105, bnlg125, bnlg389 and phi116 showed 4-5 alleles per locus in various inbred lines.
Polymorphism Information Content (PIC), a measure of allelic diversity at a locus, was estimated for various SSR loci used during the study. The values ranged from 0.06 (phi042) to 0.72 (bnlg105). The mean PIC value estimated across all SSR loci was 0.43. The PIC values of 10 primers were greater than this mean value. A comparison of the PIC values for SSR loci with different repeats shows that the mean PIC value for 10 SSR loci with di-repeats was 0.47, two loci with tri-repeats was 0.62, five loci with tetra-repeats was 0.32, one locus with penta-repeat was 0.6, two loci with hexa-repeat was 0.32, and one locus with a complex repeat was 0.56. These values do not show any clear association of PIC values with the nature of repeat.
The SSR marker data could facilitate discrimination of various inbred lines on the basis of occurrence of 'rare alleles' (with frequencies less than 0.25) for different SSR loci. The allelic frequencies indicated for various inbreds in Table 1 reveal the occurrence of many alleles with frequencies less than 0.10; this means that these alleles occur in no more than 2 out of the 22 inbreds. Such alleles could be effectively employed as possible diagnostic alleles for discrimination of specific inbred lines either alone or in combination. Rare alleles could not be detected in seven (BIO-1, BIO-4, BIO-5, BIO-6, CM202, CM137 and CM138) of the 22 inbreds analyzed. The present analysis also indicated instances where the SSR profiles for some inbreds showed deviations from the expected patterns. Inbreds are assumed to be highly homozygous, revealing one band per SSR locus. However, double bands could be consistently detected in some of the inbreds for specific SSR loci. An analysis of the frequency of heterozygous SSR loci revealed that eleven inbreds, including CM119, CM123, CM135, CM136, CM137, CM138, CM140, CM111, CM202 and BIO-1 have considerably high frequencies (>10%) of double bands.
The SSR data matrix was utilized to estimate the genetic relationships among the various selected inbreds. The genetic similarity matrix between various inbreds was computed in a pair-wise comparison using Jaccard's coefficient and the resulting similarity matrix was subjected to the UPGMA clustering algorithm; computations were carried out using NTSYS-pc 2.02. The cophenetic correlation coefficient (r) was 0.664, showing a moderate fit of the dendrogram with the similarity matrix generated using SSR data. This indicates the need for further analysis using a greater number of markers to ascertain the genetic relationships among various Indian maize inbreds. The dendrogram obtained in this study (Fig. 1) provides preliminary information about the genetic similarities among the inbreds. On the basis of canonical discriminant analysis, the inbreds could be grouped into five distinct clusters (Fig. 2). It could be observed that CM111 and CM202 (heterotic testers for Indian maize germplasm), CM139 and CM140 (parents of a single-cross hybrid 'Parkash'), BIO-7 and BIO-8 (parental lines of a promising experimental hybrid BH1183) fall into distinct groups. Interestingly, both CM212 and CM141 (parents of single cross hybrid 'Vivek-4') were found to be genetically similar as also LM5 and LM6 (parental lines of the hybrid 'Paras'), for the SSR markers used for analysis. Close genetic relationships could be observed between some of the BIO lines (from Hyderabad) and the LM lines (from Ludhiana). The study also clearly distinguished the NAI lines, NAI123, NAI127 and NAI144, from the other inbreds. This could be because these inbreds were essentially derived from foliar disease resistant maize materials obtained from Thailand and the Philippines (although the exact pedigree is not known), while most other inbreds used in this study were developed either from indigenous maize populations or from materials obtained from the USA or to some extent, CIMMYT, Mexico. Further analysis using a greater number of polymorphic SSR markers with adequate coverage of the entire genome, and methodologies with better resolving power (such as PAGE), will lead to a more comprehensive understanding of the genetic diversity in the Indian maize germplasm.
Table 1. PIC of SSR loci across various
|SSR locus||Bin location||No. of alleles||PIC||SSR locus||Bin location||No. of alleles||PIC|
Figure 1. Dendrogram depicting genetic relationships among the Indian maize germplasm based on SSR analysis.
Group 1 - BIO-1, BIO-8, CM116, CM123, CM115, NAI116, CM111, CM136,
CM138, CM137, CM119, CM139 and CM133
Group 2 - BIO-2, CM202, CM117, CM140, BIO-3, BIO-6, CM135, CM127, BIO-4, BIO-7, LM6, BIO-5 and LM5
Group 3 - CM212, CM141 and CM126
Group 4 - NAI-123 and NAI127
Group 5 - NAI144
2. Canonical discriminant analysis for determining acceptable number
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