Map-Based Cloning And Functional Analysis Of S7 Causing Hybrid Female Sterility In Rice (Oryza Sativa L.)

The Asian cultivated rice consists of two major types or subspecies,indica and japonica,which were also referred to as "Hsien" and "Keng" respectively.Distinct phenotypic variations,from growth and development to environmental adaptations,exist between indica and japonica rice,leading to strong hybrid vigor in F1 progeny.Such heterosis attracted a large amount of research interest,for developing hybrid rice to increase yield potential.However,hybrid sterility frequently occurs in such inter-subspecific crosses and hinders the utilization and exploitation of the heterosis.In this study,we tried to better understand the mechanism of hybrid sterility gene S7 by cytological and genetic analyses.Fine-mapping and interference transgenic demonstrated that a tetratricopeptide repeat?TPR?-containing protein plays significant role in hybrid female sterility causing by S7 locus.These data may have a profound implication in understanding of reproductive isolation and artificial breeding of cultivated rice.Main results are as follows:1.The partial abortive embryo sac was the main reason causing hybrid sterility in Ingra/Cpslo17 F1 and Ingra/IR36 F1.The in vitro germination of pollen grains,the number of pollen grains adhered to stigmas and pollen tube elongation from F1 all showed no distinction between F1 hybrids and parent.However,the spikelet fertility of F1 hybrids was not restored to normal levels but in accordance with open seed-setting rate after hand pollination with pollen from each parent.Further,observation of the embryo sacs from mononuclear stage to mature embryo sac stage showed that abnormal embryo sacs occurred at eight nuclei embryo sac developing stage of which two polar nuclei located in the central cavity with arrangement of horizontal instead of vertical.Analysis of histological sections of mature embryo sacs revealed that the frequency of abnormal embryo sacs in F1 hybrids was significantly higher than in parental controls.There were mainly two different types of abnormalities observed in the mature embryo sacs of the F1 hybrids which include embryo sacs with abnormal position of polar nuclei and small embryo sacs.In the first type,the polar nuclei located either above the egg apparatus with arrangement of vertical instead of horizontal or near the wall of embryo sac.In the second type,the embryo sacs were smaller in which abnormal position of polar nuclei was found sometime.2.Genetic evidences demonstrated that reduced genetic transmission of female gametophytes carrying S7cp and S7i do cause the semi-sterility of Ingra/Cpslol7 F1 and Ingra/IR36 F1,irrelevant to male gametophytic functions.An outline linkage map was constructed previously,which confirmed that the locus found in our study is S7.By genetic analysis of reciprocal crosses conducted between Ingra/Cpslo17 or Ingra/IR36 and Cpslo17 or IR36 separately,we found that the segregation rate was not in accordance with Mendelian segregation theories totally but only when pollinating Cpslo17 and IR36 with their F1 pollen grains respectively.On the contrary,the number of individual with homozygote genotype decreased obviously.The S7cp and S7i allele transmitted efficiently through male gametes?km=0.49 and 0.48 respectively?,nevertheless,blocked significantly while through female gametes?kf=0.97 and 0.94 respectively?.In other words,the S7ai allele exhibited absolutely preferential transmission by promoting the elimination of S7cp and S7i to their progeny only through female gamete.3.S7 was fine mapped in an overlapped 139 kb region which between TI15 and TI53.The S7 locus was located between Y6 and TI53 with 2 recombinant events between Y6 and S7,5 between TI53 and S7 by Ingra/Cpslo17 F2:3.Results from previous work suggested a fragment of overlapped 139 kb might just the targeted S7-containing region which between TI15 and TI53.The recombination rate of the 241 kb interval?TI24 to Y16?and the following 50 kb?Y16 to Y6?decreased suddenly,and the 184 kb interval from Y6 to Y26 had no detectable recombination.Interestingly,the regions of which recombination rate drop sharply was also uncovered a surge of retrotransposons.Taken located in centromere region into consideration,it seems like that less occurrence of recombination events in Ingra/Cpslo17 F2:3 populations were mainly caused by the specific location of S7.4.ORF3,which encoding a tetratricopeptide repeat domain protein was selected as candidate gene.By sequencing and analyze the expression pattern of predicted genes in S7-containing region,ORF3 was highly expressed in mature ovary.On the other hand,parents,especially Ingra had their unique substitutions compared with N22 and Dular which carrying S7n.Moreover,a variant amino acid was detected at amino acid 119 in WCVs?Dular and N22?that has a Met?M?instead of a Leu?L?as in Ingra,Cpslo17 and Ketan Nangka.However,most of the substitutions have their sequence conservation compared with ORF3 homologos in indica,japonica and wild rice.5.ORF3 RNAi could restore spikelet fertility of heterozygote S7ai/S7cp due to functional female reproductive organs.ORF3 RNAi vector was constructed and transferred into parents and Ingra/Cpslo17 heterozygote plants to investigate the function of ORF3.In parents,the reduced expression of ORF3 had no effect on their pollen fertility and spikelet fertility.The pollen grains in the Ingra/Cpslol7 heterozygote transgenic positive plants showed no obvious abortion compared with that in transgenic negative plants,while fertility of embryo sacs and spikelet were significantly restored.RT-PCR of ORF3 RNAi transgenic plants demonstrated that the extent of reduced expression of ORF3 was consistent with the level of spikelet fertility.The frequency of normal embryo sacs in the ORF3 RNAi representative transgenic plants?from three families?increased substantially,which were in accordance with the rate of spikelet fertility.In addition,the offspring of all the RNAi transgenic positive plants exhibited a similar phenotype of restored spikelet fertility from the T₁ to T₂ generation.