Development of an Efficient Preparation and Verifying System for Rice Gametophytic Male Sterility using RNAi on Candidate Gene and OsMYB86R as a Reporter

Abstract

Background: Gametophytic male sterility plays an important role in the study of pollen development mechanism and the breeding of three-generation hybrid rice. Because of the inherent phenotypic and genetic characteristics of gametophytic male sterility, it is very hard to find and identify gametophytic male sterility, which makes the related research lay behind the sporophytic male sterility. On the other hand, with the abundance of gene transcription data, a large number of pollen-specific genes have been found, and most of them are possibly associated with gametophytic male sterility. In order to promote the research of these genes in pollen development and heterosis utilization, it is necessary to construct an efficient, economical and accurate method to test these genes and identify if they are gametophytic male sterility related genes.Results: In this study, at first, the OsC1/OsMYB86 gene involved in anthocyanin synthesis in rice was modified and identified, and the function of revised OsMYB86R gene was created as the same as OsMYB86, and OsMYB86R is more convenient to be used as a reporter gene because the restriction site is removed. Then, an ascorbic acid oxidase gene OsPTD1 was downregulated using RNAi driven by its own promoter, which resulted in abnormal pollen tube growth; Lastly, the RNAi elements were linked with OsMYB86R and together-transferred into an osmyb86 mutant, and the purple color segregation of T1 and F1 derived from positive plants were distorted, which showed that the OsPTD1 related gametogenic male sterility was successfully prepared and determined.Conclusion: In this study, an easy and efficient method to prepare and identify gametophyte male sterility was established by using the strategy of RNAi and OsMYB86R as reporter. Comparing with the current methods, the advantages of this method are: (1) in material preparation, save time, a generation less comparing with conventional method, also a lot of mutation screening maybe avoided; (2) in identification, less cost, no PCR, electrophoresis, and other processes needed, and no expensive chemicals and instruments required; (3) in the results, more accurate, much less background affection, no damage on the plant. As a result, it is an easy, efficient, low-cost, accurate method to prepare and identify gametophytic male sterility genes. It is very important in elucidating the mechanism of pollen development and promoting the utilization of heterosis.