Genetic Analysis And Fine Mapping Of Long Sterile Lemma Lsl59 And Panicle Twisted Pt63 In Rice

The normal development of panicle is closely related to grain yield.Therefore,the discovery of genes controlling the development of panicle traits plays an important role in analyzing the genetic network of multiple genes regulating panicle traits.In this study,two mutants,lsl59（long sterile lemma 59）and pt63（panicle twisted 63）mutagenized by EMS from an indica maintainer backbone parent Yixiang1B（YXB）,were used as materials.Two mutants were genetic stable after several generations selfing.The mutants was hybridized with japonica rice variety 02428 and YXB to establish the F2 genetic isolation population,and then combined with SSR molecular marker and Mutmap technology to carry out genetic analysis and gene localization for the traits of mutants.Ⅰ.The specific research results of lsl59 are as follows:1.Through the observation of agronomic characters,it was found that the plant height,1000-grain weight and seed setting rate of the mutant decreased to some extent,while the sterile lemma was significantly elongated.2.Electron microscope scanning showed that,unlike the wild type,the long sterile lemma of lsl59 was not smooth,but had an orderly arrangement of protruding structure,which was similar to the surface of the normal palea and lemma,indicating that the long sterile lemma might have the similar characteristics of the palea and lemma.The qRT-PCR analysis of hull,palea and lemma marker genes showed that in lsl59,the expression of hull marker genes increased,palea marker genes were almost not expressed,and lemma marker genes were also increased,which further proved that long sterile lemma might have the characteristics of lemma.3.Scanning electron microscope was used to observe the development of sterile lemma at booting stage,and it was found that the elongation of sterile lemma at lsl59appeared at SP7 stage of spikelet development.After observing spikelets of different lengths with a posture microscope（Leica S8APO）,it was found that with the growth of the young spikelets,the growth of lsl59 sterile lemma was also accompanied by the growth of the young spikelets,and finally reached maturity.In conclusion,LSL59 gene affects the growth and elongation of sterile lemma,but does not affect the development of flower organs.4.Genetic analysis showed that the mutant character of lsl59 was regulated by a single recessive gene.Candidate genes were located between Indel marker LYX-205 and LYX-216 on chromosome 12.Two genes with Index value of 1 were found in the localization interval by Mutmap.Os12g0161700 was completely isolated from the mutant phenotype.By sequencing,we found that the mutation site was at the 307^th base of CDS region,and the cytosine（C）was substitute to thymine（T）,leading to the coded amino change of 103^th proline（Pro）into serine（Ser）.Therefore,the candidate gene was identified as Os12g0161700.Finally,the gene Os12g0161700 was edited in the WT by using CRISPR/CAS9 system and the transgenic plants displayed long sterile lemma at T0 generation.The results indicated that Os12g0161700 regulated the long sterile lemma in lsl59.Up to now there is no report describing the gene in rice,so Os12g0161700 is named LSL59（Long sterile lemma59）.5.LSL59 gene is expressed to some extent in various organs,which is a constitutive expression.The expression was highest in palea/lemma and young spikelets.In addition,it was also highly expressed in pistil,but at a lower level during differentiation and development in spikelets.6.Through the analysis of the expressions of other genes related to the development of rice sterile lemma,it was found that in lsl59,OsMADS34 was significantly down-regulated,FZP and G1 were significantly up-regulated,while LF1 showed no significant changes comparing with YXB.These results suggest that the abnormal sterile lemma in lsl59 mutant may be related to the change of transcription level of sterile lemma genes.In conclusion,there has been no report on the regulation of LSL59 gene in rice to control sterile lemma development.Therefore,the study of this gene provides material basis for the mechanism of flower orange development.Ⅱ.The results of pt63 are as follows:1.According to the observation of agronomic characters,the biggest difference between pt63 plants and wild-type plants is that the panicle is irregularly curved,and the seed setting rate decreases by about 70%,and the 1000-grain weight decreases by about10%,resulting in low grain yield.2.Hormone assay showed that the content of IAA and ABA in the panicle of the mutant pt63 and the wild type were basically the same before curling,but the content of IAA in the panicle was significantly increased after twisted,while the content of ABA was decreased compared with the wild type.It is speculated that endogenous high IAA content may lead to uneven cell growth and distortion during the development of tissues and organs.3.Genetic analysis showed that the mutant character of pt63 was regulated by a single recessive nuclear gene.The target gene was located between Indel3-2 and Indel3-3on chromosome 3,with a physical distance of 36.899 Kb.Among them,6 annotated genes were sequenced,and only LOC_Os03g24910 gene was found a base substitutive change from G to A at the location of 66bp upstream of ATG.Therefore,LOC_Os03g24910 was selected as the candidate gene affecting panicle distortion for follow-up study,and transgenic function verification is in progress.