Cytological Studies Of A Rice Male-Sterility Mutant And Fine Mapping Of Sterility Gene

Male sterility is a common phenomenon in flowering plant. Rice (Oryza saliva L.) is one of the most important food crop in the world, and a model plant of monocotyledons. It is very important to carry out research in mechanisms of male sterility for agricultural industry and biology developing.Tapetum is the innermost of the four somatic layers of anther wall, and plays a crucial role in the process of pollen development. Tapetum undergoes programmed cell death during late stage of anther development, and it is thought to provide cellular contents supporting pollen development subsequently via degradation. In contrast, abnormal degradation of tapetum cells is a major cause of male sterility. In recent years, through a variety of methods of genetics and molecular biology techniques, people have isolated a series of genes affecting tapetum degradation. These studies will help people more deeply understanding of the degradation mechanisms in tapetum.In this study, we identified a rice male-sterile mutant of indica cultivar IR64 background. The mutant had white anther and failed to produce pollen. After preliminarily studying on its phenotype and cytological, we finished the fine mapping of male sterility gene on the method of map-based cloning, which established foundation for isolating this gene and clarifying the molecular mechanism of pollen development. Following are our results:1. Via the phenotype and cytological observation, we concluded the character of the male-sterility mutant is as follows:Male sterility mutant did not show obvious difference with wild-type in vegetative development, but have no spikelet fertility. The mutant had smaller white anther and failed to produce pollen, while the female gamete was fertile. We observed IR64 wild-type and the male-sterile mutant mature anthers using SEM and found anther epidermal cells in mutant is smaller than wild-type, causing the short size in anther. Carmine acetate dyeing revealed the abortion of pollen happened at the later microspore stage to early bicellular pollen stage. The anther transverse sections demonstrated that tapetum of mutant was abnormally degenerated at the microspore stage, comparing to wild-type, its tapetum became thickened and showed abnormal stained. However, compared with the tapetum of wild type, the observation of TEM indicated that cytoplasm in the mutant tapetum became dispersion seriously. The exine of pollen subsequently showed developmental stagnation in mutant and became abortion without nutrients accumulation. Aniline blue staining exhibited that there were significant difference in pollen germination rate between wild-type and heterozygote in vitro or in vivo.2. We construct two F2 populations from cross between male-sterile mutant and 02428 as well as male-sterile mutant and Nipponbare, selecting 51 recessive extreme individuals, located the sterility gene in the range between the Marker S11-10-4 and N11-36 on the long arm in chromosome 11.Then we picked out 742 extreme individuals and the sterility gene was finally fine mapped to the region between the marker SSR-4 and Osnp2, with a physical distance of 543kb. Further analysis indicate that 143kb fragment deletion detected in this region, containing 15 ORFs. Among them,2 ORFs were determined as the most likely sterile genes. The construction of corresponding vectors and complementation experiments are underway.