CANDIDATE GENE MARKERS BASED AMPLIFICATION PROFILING OF STERILITY MAINTAINERS AND FERTILITY RESTORERS FOR WILD ABORTIVE RICE CYTOPLASM
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Date
2021
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DRPCAU, PUSA
Abstract
Identification of fertility restorers and sterility maintainers among the diverse rice germplasm lines is very important for the development of WA-CMS-based rice hybrids. A study was carried out to discriminate maintainers and restorers among the 24 entries using PPR9-782(M, I) and SF21 candidate genes-based panel of reported primer pairs, designed genic SSR and designed generic primer pairs and to investigate the genetic importance of these candidate genes-based panel of designed generic and microsatellite primers in discrimination of sterility maintainers and fertility restorers for validation of gene specific designed primers. The materials used in this investigation involved a set of 16 known fertility restorers and 8 known sterility maintainers of WA-CMS system. Employing a panel of 9 reported primers, 3 designed genic microsatellite primers and 12 designed candidate gene specific generic primers; amplification of targeted genomic regions was carried out. Statistical parameters utilized during present study included polymorphic per cent, polymorphism information content and similarity coefficient.
Using two candidate genes based nine reported primer pairs, namely, RMS-PPR9-1, RMS-PPR9-2, RMS-PPR9-3, RMS-PPR9-4, RMS-PPR9-5, RMS-SF21-1, RMS-SF21-2, RMS-SF21-3 and RMS-SF21-5, the amplification profiling yielded 56 allelic variants with a total of 45 shared alleles and 11 unique alleles. Polymorphism per cent expressed in terms of the percentage of unique alleles of primers ranged from 0 to 42.9. The PIC value varied from 0.519 to 0.806, with an average of 0.709. The targeted amplification utilizing candidate gene based 3 designed genic SSR primers, namely, RRMS-PPR9-2, RRMS-SF21-1 and RRMS-SF21-2, yielded 16 allelic variants with 13 of them being shared alleles and 3 being unique alleles. Polymorphism per cent varied from 0 to 33.3. The PIC value for these three designed genic SSR primer pairs ranged from 0.660 for to 0.771 with an average of 0.721.
The amplification using candidate gene based 12 designed generic primers, namely, RRCG-PPR9-1, RRCG-PPR9-2, RRCG-PPR9-3, RRCG-PPR9-4, RRCG-PPR9-5, RRCG-PPR9-6, RRCG-SF21-1, RRCG-SF21-2, RRCG-SF21-3, RRCG-SF21-4, RRCG-SF21-5 and RRCG-SF21-6 yielded 76 allelic variants, with 63 of them being shared alleles and 13 being unique alleles. While polymorphism per cent varied from 0 to 40, the PIC value ranged from 0.684 to 0.861, with an average of 0.775.
Hierarchical cluster analysis as well as principal coordinate analysis using candidate gene based 9 reported markers enabled differentiation and classification of 8 sterility maintainers and 16 fertility restorers into two well separated groups with higher level of accuracy. Further, using candidate genes-based panel of three designed SSR primer pairs, reproducible amplification was successfully achieved. These three designed SSR primer pairs clearly differentiated 8 sterility maintainers and 16 fertility restorers into two distinct groups.
Employing the two candidate genes based 12 designed generic primer pairs, the genotypes were differentiated into 3 groups with cluster A having 6 genotypes, cluster B having 8 genotypes and cluster C having 10 genotypes. Fertility restorers were accommodated into two groups, whereas sterility maintainers were conciliated into a separate group. Utilizing the two candidate gene based panel of five selected designed generic primer pairs, namely RRCG-PPR9-1, RRCG-PPR9-2, RRCG-PPR9-3, RRCG-PPR9-4 and RRCG-SF21-2, cluster analysis clearly differentiated the 16 fertility restorers from the 8 sterility maintainers, accommodating them into two well separated clusters.
Analysis of the genetic polymorphism in the fertility restoration related candidate genes specific genomic regions using gene specific reported and designed primers provided the evidence to infer the existence of nucleotide sequence length variation among fertility restorers and sterility maintainers of wild abortive type cytoplasmic male sterility in rice. The differences appeared to be more pronounced between than within each of these two categories of genotypes, which contributed to their differential amplification profiles. Relatively more easily recognizable genetic polymorphism between sterility maintainers and fertility restorers was exhibited by reported primer pairs than designed primer pairs utilized for selective amplification in the present investigation. Experimental results established the genetic importance of fertility restoration related candidate genes-based panel of designed generic and microsatellite primers in discrimination of sterility maintainers and fertility restorers of wild abortive type cytoplasmic male sterility in rice. The usefulness of gene specific panel of three microsatellite primer pairs and gene specific selected panel of five generic primers was validated during the present experimentation. Hence, these markers can be further tested and effectively utilized for discrimination of sterility maintainers and fertility restorers among the rice genotypes.