Development of molecular tools for simultaneous analyses of multiple key traits in the cultivated and weedy rice (Oryza sativa L.) / Noraikim Mohd Hanafiah

Noraikim , Mohd Hanafiah (2022) Development of molecular tools for simultaneous analyses of multiple key traits in the cultivated and weedy rice (Oryza sativa L.) / Noraikim Mohd Hanafiah. PhD thesis, Universiti Malaya.

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Abstract

In the twenty-first century, a new paradigm of agriculture that utilises fewer resources than it did during the green revolution is required to feed the constantly expanding population while combating climate change, particularly for global major crops such as rice (Oryza sativa L.), which feeds more than half of the world's population. More improved rice varieties must be developed to meet changing demands, such as fragrant rice with desirable amylose content, as well as to combat the negative effects of global warming, which increases major biotic stressors such as bacterial leaf blight (BLB), sheath blight (SB), and blast. One viable option is to use weedy germplasms that have been reported to contain many valuable genes; however, the challenge is that available literature on the potential use of the genetic resources of these species in the development of climate-resilient varieties is sparse. It is also critical to develop reliable yet affordable methods to assist rice breeding programmes in resource-constrained settings, particularly in developing countries like Malaysia. Although numerous important genes have been reported for major rice diseases and quality, it remains unclear whether these genes work for local cultivated and weedy rice. The aims of this study were to synthesise previous studies on rice biology and genetics, highlighting key research gaps and future directions in rice improvement using weedy rice, and to identify reported genes and molecular markers suitable for simultaneously screening of important biotic stresses and quality traits in rice. This study used the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) protocol to conduct the systematic review, and involved the development of multiplex polymerase chain reaction (PCR)-assays to identify BLB, SB, and blast with fragrance and amylose content in both local cultivated and weedy rice. The amplification products were analysed using high-resolution gel and capillary electrophoresis, and the results were validated using sequencing. A total of 30 primer sets were selected from the literature to be tested, with 12 found to be functional polymorphic via uniplex PCR. Of these, six polymorphic primer sets (pTA248, RM6836, RM8225, RM202, fgr- SNP, and Wx-SSR) were chosen for the development of multiplex assays due to their clear polymorphisms and non-overlapping band sizes. After validation through sequencing, eight multiplex assays that can analyse up to four primer sets were developed in this study. High-resolution gel agarose electrophoresis was found to be less expensive (USD 0.30/sample) and more practical for screening disease resistance and quality genes in rice, particularly in laboratories with limited resources. One of the developed multiplex assays (Multiplex-7: RM8225, RM202, pTA248) was then used to screen 100 weedy rice biotypes, with 12%, 19%, and 89% of the biotypes showing resistance to SB, blast and BLB, respectively. A total of fifteen weedy rice biotypes used in molecular screening were selected for greenhouse phenotyping, and the molecular and phenotyping results were compatible for all three biotic stresses studied. This demonstrates that the multiplex assay developed in this study can be used to effectively screen all the major rice diseases in weedy rice biotypes in a single PCR reaction, which can aid in the exploration of the use of weedy rice. Overall, the findings of this project can assist rice breeding programmes in Malaysia and other rice producing countries.

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