Genetic purity assessment of hybrid rice using microsatellite markers
Loading...
Date
2015
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Rajendra Agricultural University, Pusa (Samastipur)
Abstract
A study was carried out to evaluate the microsatellite markers based polymorphism for identification of polymorphic and informative markers in order to characterize two WA type CMs lines, four fertility restorer lines, eight experimental hybrids and four genotypic mixtures containing seeds of experimental hybrid and respective fertility restorer male parent and to validate the usefulness of these markers for assessment of genetic purity of hybrid rice genotypes. The experimental hybrids were evaluated on the basis of pollen and spikelet fertility to assess the extent of fertility restoration. Using a set of 18 microsatellite primer pairs, molecular profiling was conducted and amplification pattern based polymorphism was recognized on the basis of presence or absence of bands, besides variation in number and position of bands.
Altogether 73 allelic variants were detected at 29 microsatellite loci with an average of 2.51 alleles per locus. A total of 33 shared and 40 unique allelic variants were generated. The number of unique alleles per primer pairs ranged from one in the cases of RM 114, RM 250, RM 276, RM 280, RM 427, RM 524, RM 538, RM 1108 and RM 5359 to five alleles in the case of RM 17 and RM 201. Remarkably higher polymorphism per cent was exhibited by the primer pairs RM 17, RM 201, RM 558, RM 337, RM 206 and RM 171, since these primer pairs generated considerably greater percentage of unique alleles in descending order of magnitude. Considering the number of alleles generated in conjunction with the level of polymorphism detected, the primer pairs RM 17, RM 171, RM 201 and RM 206 appeared to be highly polymorphic and comparatively more informative for the purpose of molecular characterization of entries under evaluation. These primers generated considerably greater number of allelic and polymorphic variants due to variation in the length of simple sequence repeats.
In general, the microsatellite specific primer pairs flanking di-nucleotide and complex repeat motifs tended to detect lesser number of allelic variants than loci with tri-nucleotide and tetra-nucleotide repeat motifs. Among the microsatellite loci having di-nucleotide repeat motifs, the markers with CT, GA and AT repeat motifs tended to detect greater number of allelic variants than primers targeting microsatellite loci with TG, AG and TC di-nucleotide repeat motifs. Considerably greater ability to differentiate pair-wise combinations of entries was observed in the cases of primer pairs RM 17 followed by RM 152, RM 201, RM 280, RM 171, RM 206, RM 341, RM 276, RM 337, RM 524 and RM 8146 in decreasing order of magnitude. Contrarily, the primer pairs RM 538 RM 558, RM 5359, RM 114, RM 250, RM 427 and RM 1108 exhibited lesser ability to differentiate pair-wise combinations of entries.
Appearance of more than one band in the same entry was noticed revealing the existence of the duplicated region in the genome. The primer pairs RM 17, RM 152, RM 171, RM 201, RM 206, RM 276, RM 280, RM 337, RM 524, RM 558 and RM 8146 generated amplified products due to amplification of only two locus. However, further investigation under more stringent condition is required to confirm it because the intensity of bands was comparatively lower in some of the cases. The total repeat count of microsatellite loci did not appear to be strongly associated with the number of alleles generated by the primers. Results did not reflect that the larger the repeat number involved in the microsatellite locus, larger was the number of identified alleles. Presence of stutter bands mostly for di-nucleotide repeat motifs indicated the presence of minor products amplified that had lower intensity than the main allele and normally lacked or had extra repeat units. Such bands were observed to be present in the case of di-nucleotide repeat sequences detected by primer pairs RM 171, RM 427, RM 524, RM 538, and RM 558.
Analysis of divergence pattern allowed relative assessment of genetic diversity amongst the two WA type CMS lines and four fertility restorer lines. A combination of only five microsatellite primer pairs, namely, RM 17, RM 171, RM 201, RM 206 and RM 558 was equally effective in differentiation of two WA type CMS lines from four fertility restorer lines and genetic purity assessment of their hybrids. Experimental results finally led to validation of five microsatellite primer pairs, which allowed easily recognizable differentiation of WA type CMS lines and fertility restorer lines and provided a basis for genetic fidelity assessment of experimental hybrids. These microsatellite primer pairs, namely, RM 17, RM 171, RM 201, RM 206 and RM 558 may be further validated and utilized for the purpose of genetic purity assessment of three-line hybrid rice. Amongst these five primer pairs, RM 17, RM 171, RM 206 and RM 558 appeared to be more important in differentiating WA type CMS lines from their fertility restorer lines. Results of the present study also revealed that use of RM 17 and RM 171 along with RM 206 or RM 558 was equally effective in differentiation of two WA type CMS lines from four fertility restorer lines and evaluation of genetic purity of their hybrids. Experimental hybrids were not differentiated from the genotypic mixtures containing seeds of experimental hybrid and respective fertility restorer male parent because of the reason that microsatellite primer pairs directed genomic profiling and further analysis was based on bulk seedlings. Therefore, single plant based molecular profiling and analysis appeared to be an essential requirement for ascertaining the genetic purity of three-line hybrid rice.
Description
Keywords
null