Improving Adapted Rice (Oryza sativa L.) Varieties for Blast Resistance Using Mutation Breeding Technique

Abstract

Rice blast is a fungal disease caused by Magnaporthe oryzae, a hemibiotrophic ascomycete. It is one of the most damaging and widespread fungal diseases of rice globally causing yield losses of about 30%. In Kenya, most of the varieties cultivated are susceptible to the disease. However, breeding efforts for blast resistance in Kenya have been limited despite the impact of the fungi on rice production in the country. An effective breeding approach is needed to develop cultivars resistant to the rice blast disease, while preserving valuable agronomic traits of elite cultivars. Mutation breeding using gamma irradiation is one of the methods that has been used widely to create new genetic variations. The specific objectives for this study were: to identify effective range of gamma ray doses to be used in a mutation breeding program, to evaluate rice blast resistance in M2 and M3 generations, to evaluate the genetic diversity of selected putative resistant mutants and to determine mutations leading to generation of blast resistance. The effective range of gamma irradiation doses was determined using a radio sensitivity test. This was done by irradiating four rice varieties, namely; Basmati 370, Basmati 217, Komboka, and ITA 310 using eleven doses of gamma rays ranging from 0Gy to 500Gy. The M1 and M0 seedlings were raised for 21 days in soil media contained in seedling trays and data on seedling emergence, survival rate, and seedling height collected. In addition, optimal doses including lethal dose 50 (LD50), growth reduction dose 50 (RD50), and growth reduction dose 30 (RD30) were estimated by regression analysis. Results showed that sensitivity to gamma irradiation differed among varieties, with seedling emergence, survival, and growth reducing with increasing gamma irradiation dose. The LD50 values ranged from 437Gy to 522Gy, RD30 values ranged from 287Gy to 417Gy, while RD50 values ranged from 351Gy to 531Gy. Gamma irradiation doses between RD30 and RD50 were considered effective in creating favorable mutation for use in this study based on specific varieties tested. To evaluate blast disease resistance in M2 and M3 generations, inoculation of the putative mutants with strains of Magnaporthe oryzae in both controlled and field conditions was done. In M2 generation, putative mutants were inoculated with KE0215 isolate of Magnaporthe oryzae at seedling stage in a controlled environment, and disease severity scored at 21 days after inoculation (DAI). It was observed that the un-irradiated parents, Basmati 370, and Basmati 217 were highly susceptible to the isolate, while Komboka and ITA 310 showed moderate resistance. A total of 7,470 putative mutants of the two Basmati varieties were screened for resistance against KE0215. Out of these, 1,566 (~21%) putative mutants showed no blast disease lesions, while 5,904 (~79%) were susceptible to the isolate. Healthy seedlings with no blast lesions were grown to maturity and seed from them harvested to form the M3 generation. In M3 generation, 206 putative mutant families and four checks were grown in blast nursery, and at tillering stage, rice blast infected straws were distributed in the experimental block to act source of fungal inoculum. Putative mutant families were further evaluated for field resistance to the resident strains of Magnaporthe oryzae. Results indicated that there was high variability in response to field leaf blast disease. A total of 2,176 plants exhibited resistance to moderate resistance to field leaf blast disease, while 3,343 showed varied degree of susceptibility to field leaf blast disease. In addition, 2,839 plants showed resistance to moderate resistance to field panicle blast disease, while 2,660 plants had varied degree of susceptibility. Putative mutants of Basmati 217 from gamma irradiation doses of 300 Gy, 350 Gy, and 400 Gy showed significant reduction in field leaf blast disease severity and field panicle blast disease severity compared to the parental genotype. Similarly, putative mutants of Basmati 370 at 300 Gy had significantly reduced leaf blast and panicle blast disease severity scores compared to the parental genotypes. Furthermore, these putative mutant families had improved yield related components compared to the parental genotypes. These results suggest that gamma irradiation doses of 300 Gy, 350 Gy, and 400 Gy were effective in inducing favorable mutations that led to the improved rice blast disease resistance and yield components. Putative mutants in M3 generation were also evaluated for blast disease resistance in controlled environment using KE0215 isolate of Magnaporthe oryzae. About 253 putative mutant families in M3 generation were evaluated at seedling stage for resistance to the isolate. Approximately 75% of the evaluated mutant families exhibited a high degree of resistance to the Magnaporthe oryzae isolate, while 25% remained susceptible. This high frequency of resistant mutants in the M3 generation confirm that the induced resistance to rice blast disease was highly heritable. In M4 generation molecular diversity of selected putative mutants was evaluated using 11,577 high quality DArTseq SNP markers generated from 184 selected genotypes. The genetic diversity indices including polymorphic information content (PIC), expected heterozygosity (He), observed heterozygosity (Ho), and inbreeding coefficient (F) were determined. The results indicated that a moderate level of genetic diversity existed in the population. The population structure and analysis of molecular variance (AMOVA) clustered the population into two genetically differentiated sub-populations, each of which had substantial level of genetic diversity. To identify mutations leading to blast resistance, genome wide association study (GWAS) was done by using M4 generation families. A total of 13,086 SNP markers with known chromosome positions were generated from 184 of the selected genotypes using DArT genotyping-by-sequencing platform. In addition, the genotypes were evaluated for disease resistance in controlled environment by inoculation with KE0215 isolate of Magnaporthe oryzae and in field blast nursery. Putative novel loci associated with the observed rice blast disease resistance in the mutants were identified. Five of the loci were located in chromosome 3, while 4 were located one each in chromosomes 4, 7, 9, and 12. Two transversion mutations, C/G to A/T, and G/C to T/A were identified among the resistant genotypes for two of the loci associated with resistance. Interestingly, in 7 of the loci associated with blast disease resistance, susceptible genotypes lacked the reference SNP variants indicating that there were possible indels induced in the resistant putative mutants. Overall, the resent study resulted in induction of rice blast resistance and selection of elite germplasm that can further be tested across environments for adaptability and potential new varieties.