Map-based Cloning And Functional Analysis Of The Interspecific Hybrid Male Sterility Locus QHMS1 In Rice(Oryza Sativa L.)

Rice was the most important food crops and feed about a quarter of the population in the world.In the mid-20th century,Chinese scientists have developed the three-lines hybrid rice technology,which makes good use of the heterosis between rice subspecies and increase the rice yield,meeting people’s demand for food.Along with the accelerated urbanization process in our country,the arable land area has decreased.Increasing the rice yield again to meet current food security would be the most important thing for scientists.Oryza meridionalis（Mer）is a kind of annual wild rice coming from Australia,which has a wide genetic diversity and many excellent genes（like some insect resistance,disease resistance and lodging resistance genes）.Due to its distant relationship with Oryza sativa,Mer is a very important rice germplasm gene bank for improving O.sativa varieties.However,the interspecific hybrid sterility（HS）obstructs the gene flow between O.meridionalis and O.sativa.Therefore,overcoming interspecific HS in rice is a necessary condition for improving rice varieties by using elite alleles in wild rice.In previous studies,four major male hybrid sterility loci were identified between Oryza meridionalis（Mer）and Oryza sativa japonica variety DJY1（Dianjingyou1）.In this study,we focused on qHMS1 on chromosome 1,and the results were showed as follows:1.The BC₅F₁ backcross line was constructed by crossing F₁ with recurrent parent DJY1 for five generations.The near isogenic lines NIL-qHMS1 which had homozygois Mer fragment insertion at the chromosome1 was got from the BC₅F₁ inbred offspring by molecular maker-assistion selection.NIL-qHMS1 was consistent with the recurrent parent DJY1 in the plant height,the spikelet length,the heading date and other agronomic traits.2.Phenotypic identification revealed that pollen fertility of hybrids F₁（DJY1×NIL-qHMS1）showed a semi-sterile phenotype,while pollen fertility of parents DJY1 and NIL-qHMS1 were fully fertile.A series of cytological experiment showed that the abnormal microspores firstly appeared at the mononuclear pollen stage and eventually had no nucleus and starch grains filling.Genetic analysis showed that pollen with DJY1 genotype（D）were selectively aborted while the seed setting rate and female gametes developed normally in hybrid F₁.3.11,800 plants of the BC₅F₃ populations were used to narrow this region at 43-kb,containing five predictive genes.The gene editing technology was used to knock out all the candidate genes and found that the pollen fertility of F₁plants was changed from semi-sterility to fully fertile after ORF3^M were knockouted,and the genotype separation ratio of its self-bred progenies returned to nomal,suggesting that ORF3 could induce pollen abortion at hybrid sterility sites;When knock-out ORF5^M,the pollen fertility of F₁plants was completely aborted,in addition,the pollen fertility of F₁ plants（T₀）inserted by single exogenous ORF5^M fragment restore to 75%.Meanwhile,F₁ plants with homozygous exogenous fragment insertion and the normal pollen fertility were isolated from the T₁generation and the separation ratio of its self-bred progenies also completely returned,suggesting that ORF5^M could rescue pollen in qHMS1.4.qRT-PCR experiment was performed to analyze the expression of ORF3 and ORF5,and the results showed that these two gene were all expressed in different tissues and at different stages of anther development.RNA-Seqresults showed that the c DNA of ORF5was different in the two parents because of the transposon insertion,which was all different with the NIP-predicted.The promoter activity was detected and found that the promoter activity of ORF5^D was lost.GUS staining and RNA in situ hybridization found that ORF5^Mwas expressed in pollen.ORF3 is predicted as helicase with RNA-recognition motif.Compared to ORF3-M,ORF3-D lacks the HELICc domain and CC domain.ORF5 is predicted as a F-box protein and interacted with SKP-6 which was a comporlant of SCF complex.ORF3-D and ORF3-M were localized in cytoplasm and nucleus,while ORF5-M was only localized in nucleus.5.To analyze the evolutionary track of this locus,ORF3 and ORF5 from the different sources was sequenced and found that both ORF3 and ORF5 were functional in most of wild and cultivated rice varieties,which indicated that the ancestral species carried（ORF3+ORF5+）genotype.Then,in some wild rice varieties that differentiated earlier,ORF3 firstly lost its function by single-base variation（ORF3-ORF5+）.At the period of Oryza rufipogon,ORF5 lost its function due to transposon insertion in the case of ORF3mutation（ORF3-ORF5-）,and then this genotype fixed in some O.sativa temperate japonica rice varieties.Other two genotypes were also preserved in O.sativa.This study preliminarily analyzed the genetic and molecular mechanism of hybrid sterility locus qHMS1,and found the method to overcome the hybrid sterility and the wide compatibility varieties of this locus,which was helpful for improving existed rice varieties and breeding new super rice varieties by utilizing the interspecific heterosis and wild rice germplasm resources.