Identification And Fine Mapping Of Defective Pistil And Stamens 2 In Rice

Rice(Oryza sativa L.)is the model plant of monocotyledonous,one of the crucial crops in China.The formation and development of floral organs are closely related to rice yield.Cloning and functional analysis of organ development genes provide foundation for further study on the molecular mechanism of rice flower development.In this study,a defective pistil and stamens 2(dps2)mutant was identified in the CJ06 and TN1 substitution lines.The main agronomic traits and morphological characteristics of floral organs between wild type CJ06 and dps2 mutant were compared under conventional planting condition in field.The structure of anther was observed by SEM and paraffin section,and the fertility of pollen and embryo sac was observed by staining.Map-based cloning was used for the gene localization.The expression levels of floral development-related genes in wide-type and mutant were analyzed by transcriptome sequencing and RT-PCR.We obtained the following results:Longer heading date,normal palea and lemma flowering and abnormal glume opening were presented in the dps2 mutant.There were flower organs remaining in the spikelet,but no seed was setting on dps2 mutant.Other agronomic traits such as plant height,tiller number,panicle length,primary branch number and secondary branch number were similar.The morphology and pistils of dps2 were shriveled differently,the color was light and pale yellow,the number of stamens and stigmas were increased.Further observation showed that the anthers of dps2 were collapsed and no microspores,the outer epidermal cells were folded and irregularly arranged,the outer surface was smooth and there was no linear material distribution such as wax and keratin.Though some anthers formed chambers and the pollen grains were dry without starch accumulation,a few pollens performed potentially active.In addition,the development of dps2 embryo sac was abnormal,the central anpolar nucleus structure of the embryo sac and the traces of cell degeneration appear.Genetic analysis showed that the mutant phenotype was controlled by a pair of recessive nuclear genes.DPS2 was narrowed to a 91.2 Kb interval in the short arm of chromosome 4,and there were no reported genes related to flower organ development in the region.RNA-Seq analysis indicated that 731 genes were up-regulated and 1679 genes were down-regulated in dps2.The DEGs were involved in biological wetabolism and biological regulation,among which 9 genes were involved in auxin synthesis and signal transduction pathway.RT-PCR indcicated that,the expression of auxin synthesis related gene TDD1 was significantly reduced in dps2,and the auxin response gene OsIAA3,OsARF11,OsARF3,OsARF15 was significantly reduced.The expression levels of OsMADS2、OsMADS4、OsMADS16 associated with class B and OsMADS1、OsMADS6、OsMADS7、OsMADS8 associated with E genes were up-regulated in dps2.The results above indicated that the mutation of DPS2 affected auxin synthesis and metabolism,The abnormal development of stamens and pistils resulted in complete infertility of the dps2 mutant,indicating that DPS2 played an important role in third round of stamen development and the fourth round of pistil development.