Mechanism Of Yellowing And Delayed Ripening And Key Gene Slym1 Identification Of Tomato Yellow Leaf Mutant Ym

As an important vegetable crop and model research plant in the world,tomato has the characteristics of self pollination and heterosis.Therefore,hybrid seed production is widely used in tomato breeding.However,in the process of hybrid seed production,the offspring will have character separation,which makes a large number of hybrid plants appear in the hybrid progeny.It needs to be screened by multiple generations to obtain the pure and stable materials.Leaf color,as an extremely obvious agronomic trait,can be used as a special auxiliary marker for the selection of hybrid offspring in crop hybridization breeding.This method could shorten the screening time of characters,simplify the selection of hybrid offspring,and improve the efficiency of hybrid seed production.Nowdays,using leaf color as marker to screen hybrid progeny has been widely used in rice breeding.However few studies on leaf color mutant of tomato have been carried out.If the leaf color mutant is used as a marker in hybrid production of tomato,the screening efficiency of characters could be improved.In conclusion,the study of leaf color mutant of tomato can provide an important theoretical basis for in-depth analysis of the mechanism of photosynthesis,and has important application significance for the selection of tomato characters too.In the early study stages of our group,one plant with natural mutation of yellow leaves had been found in the field.The stable yellow leaves tomato line was obtained after breeding for generations.We named it tomato yellowing mutant.As a non-lethal leaf color mutant,yellow leaf color mutant is mainly the color of the yellow leaf color mutant,meanwhile shown the characterized by the delayed ripening of the fruit.Therefore,the study on this mutant not only could provide provides theoretical basis and plant materials for in-depth analysis of the mechanism of photosynthesis,but also provide the new ideas of the characteristics storage and transportation resistance of tomato fruit.The physiological mechanism of leaf yellow mutant is studied by physiological index determination,chloroplast ultrastructural observation and photosynthetic pigment content determination.High throughput sequencing BSA-seq and GBTS genotyping are used to locate and identify the candidate genes regulate these characteristics.Bioinformatics screening,physiological and biochemical experiments,subcellular localization and tissue-specific analysis are combined to found the candidate genes' function.Genes' s silencing and over-expression,yeast two hybrid,two molecule fluorescence complementation experiments and comparative transcriptome analysis are used to study on the mechanism of leaf discoloration and fruit ripening.The main results obtained in this study are as follows:(1)The yield and biomass of mutant ym are not significantly different from those of control zs4.The result of physiological and biochemical indexes showed that the excessive accumulation of hydrogen peroxide,the ability to clear free oxygen decreased and the dynamic balance of ROS destroyed in mutant ym plants,lead to premature senescence and oxygen poisoning of mutant ym's cells.In mutant ym,the chlorophyll content is decreased significantly,and the chloroplast structure is destroyed.The net photosynthetic rate,intercellular carbon dioxide content,stomatal conductance and cell activity of mutant ym are decreased significantly.The photosynthetic capacity of mutant ym is decreased significantly.These results lead the mutant ym's leaf color turned yellow;(2)The mutant and the control plant are hybridified to obtain the isolated population.Genetic analysis showed that the yellow trait is controlled by a single gene.A candidate interval of 1.88 Mb was screened by using BSA genome localization and sequencing.In this interval,a candidate gene Slym1 is identified.The Slym1 encodes F-box family proteins and plays an important role in nucleus.In the mutant ym,a non-synonymous SNP mutation occurs at the 704 bp position of the coding region,and nucleotide is mutated from G to A;(3)Over-expression of Slym1 significantly decreased photosynthetic,photoresponsive,photoreceptor proteins and photosynthetic pigment synthesis genes' expression level in tomato plants.Silencing Slym1 showed the opposite result of over-expression.Slym1 plays a negative role in plant photosynthesis;(4)Yeast two hybrid and bimolecular fluorescence complementation experiments showed that the protein Sl YM1 indirectly interact with the key transcription factor HY5 in plant's photomorphogenesis.A competitive relationship,may affect the dynamic balance between PIF3 and HY5,and further affecting a series of reactions such as plant photosynthesis;(5)The result of fruit color change,internal structure change and soluble solids content change in the process of fruit mature and mature fruit weight of mutant ym and control zs4 show that the fruit of mutant ym delay a period of mature compare with control zs4.The mutant ym's fruit characterized slow accumulation of pigment,the sepals color change from green to yellow;(6)The leaves of mutant ym and control zs4 are analyzed by transcriptome.The transcriptome analysis showed that Solyc01g110340.3,Solyc04g081300.3,Solyc05g005080.3,Solyc08g082250.3 and Solyc09g075360.3 are down regulated in the mutant lines,which may lead the cellulose in the mutant fruit slow degradation then cause late ripening in mutant fruit;(7)The GBTS genotyping is used to shorten the length of candidate interval.Except Slym1,other two genes are obtained after screening and identification.Solyc08g006690.1.1 and Solyc08g006720.2.1.Enrichment analysis showed that these two genes are involved in leaf yellowing and fruit late ripening,respectively.