Screening Of Mesocotyl Elongation Germplasm And Mapping Of Genes Regulating Mesocotyl Elongation In Rice

The rice mesocotyl is the part between the coleoptile node and the germination point of the radicle.Under the condition of direct seeding of rice,seedlings in low temperature and hypoxia environment,seedling emergence rate is easy to be affected,and lodging phenomenon is more likely in the middle and late stages.Mesocotyl elongated varieties can break water?soil?and emerge,relieve anaerobic stress,which is conducive to the survival of seedlings and improve the emergence rate.Screening mesocotyl elongation rice seed resources and discovering related genes are of great significance for breeding rice varieties suitable for direct seeding.In this study,rice germplasm screening,physiological and biochemical studies,genetic regulation and gene mapping of mesocotyl elongation rice were carried out,and the following results were obtained.?1?Screening and Cluster Analysis of Long mesocotyl Germplasm Resources of Rice Seedlings:375 germplasm resources with rich genetic background were treated by submergence stress,agronomic traits at seedling stage were investigated,and all varieties were screened and classified by correlation analysis and cluster analysis.The results showed that there was a significant positive correlation between mesocotyl length and bud length.375 experimental materials were clustered into 5 groups.Two typical mesocotyl elongation materials,Ezhong 4,DH4038,and two short mesocotyl materials,Huahang 31 and DH4040,were selected to carry out physiological,biochemical and gene mapping studies related to the characteristics of the mesocotyl.?2?Effects of Melatonin?MT?Seed Soaking on the growth of rice seedlings under submergence stress:The four seed materials were treated with different concentration gradients of melatonin soaking.Measuring agronomic traits such as Mesocotyl length,coleoptile length,shoot length,fresh weight,survival rate and the physiological and biochemical indexes such as enzyme activity of peroxidase?POD?,superoxide dismutase?SOD?,catalase?CAT?and malondialdehyde?MDA?content.The effects of MT soaking-seed on rice germination under submergence stress can be brought two results,low concentration MT alleviating stress and high concentration MT aggravating toxicity.MT soaking-seed could reduce MDA content,increased POD,SOD,CAT activity,promoted agronomic traits such as mesocotyl length,coleoptile length,shoot length and fresh weight,and 100?mol/L MT soaking-seed could also promote related agronomic traits.Soaking-seeds with appropriate concentration of melatonin can improve the vigor and emergence rate of rice seedlings under submergence stress,and significantly reduce the toxicity of submergence stress on rice seedlings.?3?Gene mapping of mesocotyl elongation in rice:F₂ mapping population was constructed by using long mesocotyl variety Ezhong 4 and short mesocotyl variety Huahang 31.1000 F₂ germinated seeds were treated by submergence and dark stress.The BSA mixing pool was separatelyconstructed by taking 30 mesocotyl materials with significant elongation and 30 materials non-elongation.Using BSA sequencing analysis to screen candidate genes for mesocotyl elongation.A total of 49.39G raw sequencing data were obtained,and the coverage rate was higher than 88%compared with the reference genome of rice.Among them,the sequence depth of parents was 25×and that of offspring mixed pools was 12×.The number of SNP markers was29677 by mutation locus detection.A significant single peak was found in the region of chromosome 5?25-30Mb?.A candidate gene LOC_Os05g44810 was screened and its function annotated as auxin response factor?OsIAA18?.?4?Cloning and bioinformatics analysis of OsIAA18 gene:OsIAA18 genome sequencing of Ezhong 4 and Huahang 31 showed that compared with Huahang 31,Ezhong 4 inserted a 27 BP sequence in OsIAA18 5'UTR region.Sequencing analysis of more varieties with different mesocotyl elongation showed that there was a high correlation between the mesocotyl elongation varieties and the 27 bp insertion sequence in 5'UTR region.Quantitative polymerase chain reaction?qPCR?showed that submergence stress could induce the expression of OsIAA18,which was higher in long mesocotyl varieties than in short mesocotyl varieties.Bioinformatics was used to analyze the promoter of OsIAA18 gene,the tertiary structure of OsIAA18 protein and the homology of rice AUX/IAA gene family.It was found that OsIAA18 promoter had a large number of response elements related to stress,OsIAA18 had the highest homology with OsIAA6,and OsIAA18 protein had a typical PB1 domain.These analyses indicate that OsIAA18 gene may be related to stress-induced mesocotyl elongation,which lays a foundation for further functional analysis of OsIAA18 gene.