Cytological Studies And Gene Mapping Of Pollen Sterility In Hybrids Between Ludao And Cultivated Varieties Of Rice (Oryza Sativa L.)

Ludao, a weedy rice strain in lower Yangtze valley, grows naturally in the fields of Lianyungang region (34°33'-34°46'N, 119o13'-119°30'E), east China's Jiangsu province (Jiang et al, 1985). Due to its sepecific geographic distribution and the potential significance on the understanding of the origin and differentiation of Chinese rice cultivars, Ludao has been attached increasing attention since it was discovered in 1960's. However, so far no consensus on its taxonomic status has been reached. Moreover, the ubiquitous reproductive barriers between Ludao and modern cultivar of rice have restrained extremely its application of excellent characters such as high salinity and drought tolerance in rice breeding. In our study, combined with crossing affinity and SSR assay, approach to clarify the relationship between Ludao and modern cultivated varieties was conducted, with emphasis on cytological mechanism and gene mapping of pollen sterility in hybrids between Ludao and a typical japonica variety, Akihikari. The results are as follows:1. The relationship between Ludao and modern cultivated varietiesThe spikelet and pollen fertility of hybrids between Ludao and tested cultivar, indica, japonica as well as wide compatibility varieties showed that crossing affinity of Ludao with japonica varieties was closer compared with indica varieties. SSR assay was used to further investigate the genetic similarity between Ludao and 21 cultivated varieties, and cluster analysis was undertaken based on the genetic distance between them. The results proved that Ludao and japonica varieties were grouped into one type, but the genetic distance of which was greater than that between japonica varieties.2. Cytological mechanism of pollen abortion in the Ludao/Akihikari hybridHybrids pollen of Ludao/Akihikari showed partial pollen sterility, same to the F₁ pollen of reciprocal. During the process of microsporogenesis and uni-nucleate pollen formation, the pollen development of Ludao/Akihikari hybrid is indistinguishable from it's parent, Akihikari. Visible differences of pollens between Ludao/Akihikari hybrid and Akihikari began at the transition from late microspores to mature pollens. Two types of abnormalities were observed in hybrid pollens. In the first type, at the stage of a single nucleus to two nuclei of microspore development, some microspores became abortive as indicated by their irregular shape. In the second type, during the late bicellular pollen stage, a large proportion of pollen generative and vegetative cells began to disaggregate gradually, and shape a bigger chromatin conglomeration across pollen center, eventually leading to abortion of the pollens. No visible abnormality in the development and disintegration of tapetum and other anther walls during microspore formation and development in the Ludao/Akihikari hybrid.3. Construction of molecular linkage map and analysis of distorted segregationA molecular linkage map consisted of 118 SSR markers and 1 EST marker was constructed using 215 individual plants of Akihikari//Ludao/Akihikari, with a total length of 1597.0cM on all 12 chromosomes with an average interval of 13.4cM. Segregation distortions of markers totaled 30 distributed on chromosome 2, 3, 4, 6, 8, 9, 10 and 11, respectively. Among which, the most pronounced one lies in a wide region on chromosome 3.4. Detection and analysis of QTL for hybrid pollen sterilityIn virtue of the constructed molecular linkage map, two putative QTLs controlling hybrid pollen sterility were detected on chromosomes 3 and 11, and designated as qPS-3 and qPS-11 respectively.The former was linked with the EST marker C0729 with LOD score of 52.6 and PVE (phenotypic variance explained) of 57.9% and the later, with LOD score of 32.1 was linked with the SSR marker RM552 accounted for 32.5% of PVE. Both loci are different from those reported previously and then designated as S33(t) and S34(t) respectively. No interaction was detected between the two loci, which affected pollen sterility independently with additive effects, with S33(t) more effect on pollen sterility than S34(t). Interaction between the Ludao and Akihikari alleles at each of the two loci resulted in a reduction in pollen fertility, and wide compatible variety 02428 was considered to possess the neutral allele at loci S33(t) and S34(t) respectively.5. Fine mapping of novel gene S33(t) for hybrid pollen sterilityTo determine the precise map location of S33(t), a major locus for hybrid pollen sterility, individual plants with pollen fertility higher than 80.0% from BC₁F₁ population of Akihikari//Ludao/Akihikari were selected and recombination events surrounding the S33(t) locus were assayed using SSR and InDel markers newly developed. As a result, S33(t) was delimited to an 86 kb region between SSR markers RM15621 and RM15627. Sequence analysis of which revealed that ten ORFs (open reading frame) were included. These results will be valuable for cloning the corresponding gene and for marker-assisted transferring of the related neutral allele in rice breeding programs.