Study On Genetic Basis And Molecular Mechanism Of S58-mediated Hybrid Male Sterility Between Asian And African Rice

Hybrids between Asian cultivated rice（Oryza sativa L.）and African cultivated rice（Oryza glaberrima Steud）,which have significantly differentiated genome sequences,show strong heterosis.However,due to hybrid sterility（HS）,the utilization of heterosis in the hybrids is severly limited.Therefore,cloning and study on genetic basis and molecular mechanism for hybrid male sterility（HMS）in rice have important theoretical significance for both basic research and rice breeding practice.In our laboratory,a HMS locus S58 between O.sativa（carrying S58-s locus）and O.glaberrima（carrying S58-g locus）was identified by genetic analysis.The pollen fertility and spike fertility of an O.sativa parent 9522（S58-s S58-s,ss）and a near isogenic line SG42（S58-g S58-g,gg）are full fertility（FF）.While the hybrid F₁（S58-s S58-g,sg）has semi-sterility（SS）in pollen fertility and FF in spike fertility.S58 was finely mapped to a chromosome 7region,corresponding to 22 kb in O.sativa.In this study,to obtain the specific-unkown sequences in mapped region of S58-g,a modified TAIL-PCR method,mhi TAIL-PCR was developed.Subsequently,sequence analysis,candidate gene knockout,functional complementation,gene function and molecular mechanism studies were carried out.The main results of the S58-mediated HMS are as follows:1.Base on sequence analysis,it was found that there was a big difference between O.sativa and O.glaberrima in S58.It is necessary to develop mhi TAIL-PCR for capturing unknown S58-g sequences in mapping region.By using the optimized m LADs primers and introducing the AC0 primer,the efficiency of pre-amplification was great improved.The use of a high-performancy enzyme Phanta DNA polymerase resulted in a substantial increase in the size of target fragments.Then the mhi TAIL-PCR was used to obtain two large insert sequences of approximately 10 kb each in the S58-g allele.2.Sequencing analysis and gene prediction showed that there are 8 ORFs in the mapping area of Asian rice and 14 ORFs in that of African rice.Among them,4 ORFs are Asian rice-specific genes,10 ORFs are African rice-specific genes and 4 ORFs are Asian-African rice common genes.3.RT-PCR analysis showed that in S58 region,7 ORFs（S58G2,S58G3,S58G7,S58C1,S58C2,S58C3 and S58C4）were expressed in anthers.So they were selected as candidate target genes.4.Using CRISPR/Cas9-mediated segment deletion and gene-by-gene knockout,the African rice-specific genes S58G2 and S58G7 were identified as the S58-related genes.5.Knockout of S58G2 or S58G7 in SG42 could only obtain functionally lost heterozygote types（G2’g2 or G7’g7）,and the pollen grains carrying the non-functional g2or g7 were selectively aborted.The g2g2 plants obtained from T₁ generation,showed inhibited growth in vegetative growth stage.q RT-PCR showed that expression levels of some antoimmune-related（AR）genes in g2g2 plants was significantly higher than those in wild type.6.Loss-of-function of S58G2 or S58G7 in F₁（S58-s S58g2 and S58-s S58g7）also leads to growth inhibition,and knockout of S58G2 in F₁eliminated the genetic effect of S58-mediated HMS and cause male compatibility in the hybrids.In addition,S58G2-transgene（S58G2^t）can protect the g2G7-type gamete from abortion in the functionally lost heterozygote line（G2g2）.7.Functional complementation experiments showed that S58G2^t or S58G7^t single-gene transformation in 9522 and F₁ background（G2^t—/S58-s S58-s,G2^t—/S58-s S58-g,G7^t—/S58-s S58-s and G7^t—/S58-s S58-g）did not affect the pollen fertility of the transgenic plants.The S58G2-G7^t double-gene transgenic T₀ plants（G2-G7^t—/S58-s S58-s）in the 9522background could produce the pollen semi-sterility phenotype as F₁,whose pollen carrying transgene survive and pollen not carrying transgene abort.This suggests that S58G2^t or S58G7^t alone did not have the role of sporophytic-killer nor gamete-protector.To kill the male gametes,the combination of S58G2 and S58G7 is needed.8.q RT-PCR analysis indicated that S58G2 and S58G7 had temporal-spatial constitutive expression in SG42 and F₁ plants.This was proved that S58G7 is expressed in both pollen and anther by GUS-transgenic experiment.S58G2 and S58G7 encode an oligomeric peptide respectively,in which S58G2 is localized in nucleus（NLS）and S58G7 is mainly localized in NLS and endoplasmic reticulum（ER）.According to the results of semi-thin section experiments,the abortion of S58-s-pollen in F₁ was determined to be the bicellular pollen stage.9.There is not direct interaction between the S58G2 and S58G7 proteins,but they interacted with S58C4 in yeast two-hybrid（Y2H）assay respectively.Knockout of S58C4could not cause pollen SS in SG42 nor FF in F₁ background.Y2H and pull-down assays confirmed that S58G7 could form homologous oligomers.10.b HLH49 and PDHE1-β,which were screened from Y2H library,could interact with S58G2 and S58G7 respectively.b HLH49,as a transcription factor,is localized in NLS.PDHE1-βis mainly localized in mitochondria.11.Compared with WT,the expression levels of b HLH49 were appreciably decreased in g2g2 and S58-s S58g7 plants,wherase the expression levels of some AR genes were significantly increased.It suggests that expression of b HLH49,which participated as a negative regulator in the pathway related to plant stress response,is affected by S58G2 and S58G7.12.A large number of differentially expressed genes were found in the anther tissues among 9522,SG42 and F₁ by RNA-seq analysis.A considerable part of them are involved in the rice AR process.The expression levels of b HLH49 in SG42 and F₁ are significantly lower than that in 9522.At the same time,the expression levels of AR and reactive oxygen metabolism genes are detected to appreciably increase in the S58-g-containing plants.These results in this study suggest that both S58G2 and S58G7 are important for S58-mediated HMS between O.sativa and O.glaberrima.They participate in the role of sporophytic-killer and gametophytic-protector.The abortion signal,which may be related to the abnormally high expression of AR genes,is possibly produced by S58G2 and S58G7 in the sporophytic tissue of early anther development.In the subsequent pollen developmental process,S58G2 and S58G7 together inhibite the expression of AR genes by interacting with b HLH49,to eliminate the influence of the abortion signal.The S58-s male gametes in F₁background,which lack S58G2 and S8G7,can not eliminate the abortion signal generated in sporophytic tissue,so eventually leading to pollen abortion.Therefore,the special new genes,S58G2 and S58G7,evolved from African cultivated rice both play very important roles in development and reproductive isolation.