ASSESSMENT OF MOLECULAR DIVERSITY IN RICE GERM PLASM IN NUCLEAR, MITOCHONDRIAL AND CHLOROPLAST GENOMES

Abstract

Rice has twenty three species but out of these only two species Oryza sativa L and Oryza glaberrima are being cultivated around the world. Asian cultivated rice (O. sativa L.) grasps an exclusive place among domesticated crop species. It is the first fully sequenced crop genome and a critical staple food. Rice along with its wild relatives is being grown on diverse series of agroecological environments and climates. Pakistan is famous for special Basmati rice varieties mainly grown in the Punjab province. Along with Basmati rice, Indica varieties are also grown throughout Pakistan and being exported to different countries. The comparison of genetic diversity of rice varieties grown in Pakistan along with other countries has been given little attention. Information and genetic characterization of rice accessions would be useful in determining present trends in rice breeding and in assessing alternative for improving cultivars. In the present study microsatellite and SNP markers were used to characterize rice accessions from different countries along with Pakistan to provide information about their genetic diversity, which may help for maintenance or expansion of diversity in future breeding programs and to investigate rice adulteration for export purposes. The first study was carried out to evaluate the genetic diversity within a diverse collection of rice (O. sativa L.) accessions and to determine differences in the patterns of diversity within the aromatic and non-aromatic rice varieties. Forty rice accessions were evaluated by using 24 microsatellite markers distributed over the whole rice genome. A total of 66 alleles were detected at 24 SSR loci and the number of alleles per marker ranged from 2 to 4, with an average of 2.75. Polymorphism Information Content (PIC) value ranged from 0.0476 (RM315) to 0.5993 (RM252), with an average of 0.3785 per marker. The average genetic diversity over all SSR loci for the 40 genotypes was 0.4477, ranging from 0.0488 to 0.6638. Major allele frequency ranged from 0.4250 (RM252) to 0.9750 (RM315), with an average of 0.6472. The dendrogram based on the cluster analysis by microsatellite polymorphism grouped 40 rice cultivars into three groups, effectively differentiating Basmati cultivars from non-basmati cultivars. xiii Understanding genetic diversity and population structure in rice populations are of great importance and a prerequisite for genetic characterization and crop improvement. Ninety five accessions from sixteen countries were characterized by one hundred and fifty nine SSR markers. SSR makers were well distributed on the nuclear, mitochondrial and chloroplast genome. The overall results revealed that Indian rice accessions were more divergent than International Rice Research Institute (IRRI) Philippines and Pakistan. Cluster-analysis based on genetic differences coefficients clearly separated all the genotypes into three major groups but was unable to separate the accessions based on their geographical area. The population structure analyses showed that most accessions exhibit some degree of admixture, with many individuals within a population sharing the same introgressed segment due to artificial selection. The model-based structure analysis revealed the presence of three subpopulations. This was basically consistent with clustering based on genetic distance. SSRs proved to be an efficient tool in assessing the genetic diversity of rice genotypes. The same ninety-five accessions of rice were also analyzed in order to profile SSR variation in mitochondrial and chloroplast genomes as well, by using 62 pairs of SSR primers. Out of 42 microsatellite loci for mitochondria only 12 SSR markers showed polymorphism whereas in case of chloroplast 6 out of 20 were found polymorphic. The average gene diversity for both mitochondrial and chloroplast was 0.32 oscillating between 0.041 to 0.620. The PIC value ranged from 0.040 to 0.543 with an average of 0.282. While allelic richness ranged from 2-4 alleles with an average of 2.779 alleles. Mononucleotide repeats stood first (50% polymorphic) for detecting polymorphism for organelle genome followed by tri- (25%), tetra- (14.29%) and dinucleotide (12.5%). Cluster and population analysis revealed two groups of accessions but principal coordinate analysis disagreed with this grouping pattern. The overall results revealed the monophyletic origin of rice. Single nucleotide polymorphism is the base pair change most frequently present in the eukaryotes and are responsible for major diversity. Multiplex single nucleotide polymorphism has been acknowledged as having great potential to find out genetic xiv diversity and structure of diverse rice accessions. One hundred and five accessions of rice were analyzed by RiceOPA2.1 from diverse environment. Twenty accessions representing all five groups of rice were used as reference. Overall call percentage was 97.7%. A total of 746 alleles were detected in this study. The lowest polymorphism information content (PIC) was 0.0706 for SNP id3002191 as compared to the highest PIC value 0.5222 for SNP ud4000438 with an average of 0.3353. The average genic diversity (H) over all SNPs loci for the 105 genotypes was 0.4196, ranging from 0.0733 to 0.5919. Neighbor Joining and Unweighted Pair Group Method with Arithmetic mean cluster analysis clearly separated all the genotypes in two major clusters. Structure analysis based on admixture model and principal coordinate analysis produced the same results. The results revealed that the Indian accessions are more diverse followed by IRRI and Pakistan. This comprehensive and comparative study based on microsatellites and SNPs provides a clear image of genetic diversity of Pakistani rice accessions in comparison with those of other countries accessions. A narrow genetic base has been reported for rice cultivars in Pakistan as compared to that in other countries. So there is a strong need to introduce diverse material in breeding program keeping in view preference of the farmers’ community. The overall results derived from analyses of genetic diversity could be used for designing effective breeding programs aimed at broadening the genetic bases of commercially grown varieties particularly in Pakistan. These results could also be useful for monitoring purity, genotype identification and for plant variety protection.