Cytological Studies Of Hybrid Sterility And Fine Mapping Of The Interspecific Crosses Sterility Gene S37 In Rice(Oryza Sativa L.)

Rice(Oryza sativa L.)is one of the major crops in the world and its yield has always been paid much attention.With the increasing of the world population,the improvement of rice yield is the key focus by scientists,especially for rice breeders.In recent years,due to some high yield rice varieties were planted in the large area for the promotion and application,the genetic basis of rice varieties have become more and more narrow.The losing favorable genes resulted in the rice yield stagnant.In order to break this embarrassing situation and the bottleneck effect of rice,combined with the global rice breeding research,the most effective way is through intra-species hybrids or even inter-species hybrids.In fact,Asian cultivated rice(Oryza sativa)and the wild species have abundant genetic variation.Through the hybrid between Asian species and wild species,we would obtain favorable genes from distantly related species,then improved of existing varieties by introduction of beneficial genes of these distantly related species.African cultivated rice(O.glaberrima),another important rice species mainly domesticated and planted in Africa,and it has a lot of good genes which do not exist in Asian cultivated rice,and if those genes can be introduced into Asian cultivated rice,it will have a significant impact on rice breeding.However,in rice improvement process,hybrid sterility and inviability are major obstacles for extensive utilization of closely related species in breeding programs,and impaired the exploitation of the rice genetic diversity and the beneficial effect of the high level of heterosis in these two species.Therefore,it is important to discover sterility locus and overcome reproductive barriers.In this study,we constructed a near-isogenic line(NIL)using the japonica variety Dianjingyou 1(DJY1)as the receptor parent and IRGC102375(O.glaberrima Steud)as the donor parent.The pollens and embryo sacs fertility of DJY1/NIL F1 were semi-sterile,and genetic analysis indicated that it was controlled by S37 gene.We elaborated on the cellular mechanism of hybrid F1 pollen sterility and embryo sac abortion,and fine-mapped of S37 gene,and compared with the genome sequence features near the S37 locus between African cultivated rice and Asian cultivated rice,which laid a solid foundation for further map-based cloning of S37 and eventually elucidating the molecular mechanism of hybrid sterility between rice African cultivated rice and Asian cultivated rice.The main results of this study were as follows:1.When S37 locus was hybrid,the fertility of pollen and spikelet exhibited typical semi-sterility.The type of pollen abortion is stained abortive and happened at the later bicellular pollen stage.The main reason is the abnormal of late secondary mitotic of microspore,leading to abortion of reproductive nuclear.However,the observation of semi-thin sections of anther showed that the development of the F1 anther was normal.The formation and degeneration of tapetum cells in anther inner wall are as normal as DJY1,suggesting that the tapetum has no response to the pollen sterility.The observation of mature embryo sac of parent and F1 by confocal laser scanning showed,the embryo sac of hybrid F1 had varying degree abortion,the main type was no differentiation and empty embryo sac.Continuous sections of development process of F1 hybrid embryo sac by paraffin method showed,the abortion stage of F1 embryo sac is the secondary mitotic of the functional megaspore,which leading the abnormal formation of four-nucleate embryo sac and the further development.Aniline blue staining observation showed that stigma of parents had a lot of attached pollen grains,and pollen tube extended into the style.In addition,the number of pollen attached on the stigma of F1 hybrid was slightly less than parents,and the pollen tube could extend into the style normally.Saturate pollination crosses experiments showed that DJY1 was used as female parent pollinated with NIL-S37/DJY1 pollens,the setting rate of DJY1 was normal.However,when hybrid F1 as female was pollinated with DJY1 pollen,the setting rate of F1 was about 41.6±3.5%.Taken together,these results suggested that the partial abortive embryo sac was the main reason for the hybrid sterility in the F1 hybrids between DJY1 and NIL-S37.2.There is a very severe partial separation phenomenon due to semi-sterility plants in F2 population.Four backcross population are constructed,and combined the molecular data tightly linked with S37 locus and phenotypic data,we found the male and female gametes which carrying the S37j(j:japonica)transfer efficiency was low.3.Linkage analysis found that S37 locus was located at a region of the centromere of chromosome 1,between the molecular marker RM140 and B1-5 of 20.6cM range.A total of 25,600 plants of DJY1/NIL-S37 F2 population were planted for fine-mapping.S37 locus was finally fine-mapped to the region between the InDel marker A16 and the InDel marker S1.The distance between the two markers is 205-kb based on the Nipponbare clones and 108-kb based on the Oryza glaberrima clones respectively.4.DJY1 and Nipponbare are both japonica varieties;the genome sequences of them are similar.In the S37 region,there are 54 ORFs in DJY1 and 29 ORFs in NIL-S37 due to a deletion of 97-kb DNA fragment.Owing to the large difference between DJY1 and Oryza glaberrima,most ORFs of them did not have the same structure and function except for ORF53 and ORF54 in DJY1,corresponding ORF28 and ORF29 in NIL-S37.These two pairs of genes probably were the candidate genes of S37.5.The reduction of recombinant from molecular marker A16 to S4 suggested the difference between Asian cultivated rice and African cultivated rice restrained the normal recombination events.There were significant differences between the 205-kb in DJY1 and 108-kb in the DJY1 and NIL-S37 by sequence comparison,and three large deletion fragments were detected at different position.Primers were designed near the S37 locus based on the Nipponbare to analyse the phylogenetice between 21 African cultivated rice species and 42 Asian cultivated rice species,the result showed that African cultivated rice and Asian cultivated rice were domesticated independently.