Map-based Cloning And Functional Analysis Of OsRAD51D.1A Regulating Rice Male Reproductive Development

Male reproductive development is a complex and highly ordered phenomenon which demands comprehensive understandings of underlying molecular mechanism to expand its scope for crop improvement.The study of male sterility is an excellent thoroughfare to critically understand the regulatory mechanisms essential for the complicated male reproductive developmental process.The unique trait of male sterility also provides valuable resources and convenience for the genetic improvement of rice hybrids.Therefore deeper and broader understandings about the genetic causes of male sterility are necessary for rice breeding.In this study,we identified a complete male sterile mutant osrad51d from a mutant library developed by the treatment of Zhonghui8015(ZH8015)with EMS.The mutant was further subjected to phenotypic,cytological and histological analysis to identify the underlying cause of male sterile phenotype.The major out-coms of the study are summarized as below;1.By comparing the phenotype with wild-type,osrad51d mutant had normal vegetative growth but the anthers were shorter,weaker,light yellow to whitish and produced complete sterile pollen while the carpel was fertile.Cytological observations through semi-thin sections revealed no obvious differences during early anther developmental stages because anther underwent normal developmental process to produce MMCs and on subsequent meiosis stage displayed normal dyads and tetrads formation.However after meiosis,the microspores were collapsed and were observed to be abnormal throughout the rest of the stages.Other than pollen development,tapetum also showed delayed degradation.Further SEM and TEM analysis identified completely collapsed pollen with abnormally developed pollen wall characterized by unusual thickness of exine and endexine.Analyzing the meiocytes with callose staining identified the imprecise callose deposition around microsporocytes during the meiosis stage which was probably the leading cause of pollen collapse after the microspores were released from tetrads.These data suggest the indispensible role of OsRAD51D for microspore development.2.An F₂ mapping population developed by crossing osrad51d mutant with 02428(japonica)was used for genetic analysis and to fine map the OsRAD51D locus.Genetic analysis confirmed that a recessive gene was responsible for the phenotype.By using F₂ segregating population,the candidate locus was restricted to a region of 123.32 kb between the markers AB-1 and AB-27.Sequencing analysis identified an intronic point mutation(G-to-A)in the 3?end of second intron of OsRAD51D and its candidacy was confirmed by functional complementation analysis.How an intronic mutation caused phenotype changes,was confirmed by the cloning and sequencing the mutant c DNA.The sequencing results identified that point mutation in splice acceptor site(AG)at 3?end of intron caused aberrant pre-m RNA splicing and produced multiple variants to encode for truncated proteins and altered the open reading frame which led to the phenotype change in osrad51d mutant.3.OsRAD51D is member of Recombinase A protein family which is involved in homologous recombination for double strands break repair process.DAPI analysis identified that the chromatin underwent condensation but failed to observe synapsis,homologous chromosomes pairing and Synaptonemal complex formation during early meiosis.On the subsequent stages of meiosis?and?,the meiocytes produced increased number of univalent,displayed failure of arrangement at equatorial plate,produced lagging chromosomes,abnormal segregation and ultimately resulted in the production of microspores with unequal chromosome number in the daughter cells.Moreover,the abnormally segregated chromosomes produced micronuclei.These data suggest that loss of OsRAD51D.1A caused chromosomal aberration and eventually caused male sterile phenotype.4.Expression analysis by q RT-PCR depicted that OsRAD51D.1A expresses in all the tissues including leaf,root,leaf sheath,stem,and pistil but shows predominant expression in anthers from early meiosis to late meiosis stages while gradually decrease as the anther developmental stages proceed.GUS analysis identified the GUS signals in all the tested parts including leaf,stem,leaf sheath,node and pistil.But consistent with q PCR analysis,strong GUS signal were observed in anthers at meiosis stages.Sub-cellular location analysis found that OsRAD51D localizes in both the cytoplasm and nucleus.5.Y2H analysis detected that OsRAD51D.1A interacts with OsRAD51B which was further verified through Bi FC analysis.OsRAD51B is involved in somatic and meiotic recombination for double strand break repair process.These results provide an indication of involvement of OsRAD51D in homologous recombination pathways.Collectively,our results clearly indicate that OsRAD51D.1A is essential for MMCs meiosis and microspore development in rice.The mutation in the gene triggered abnormal pre-m RNA splicing which ultimately led to chromosomal aberrations and impaired callose biosynthesis pathway resulting in male sterility.