Genetic Mapping And Candidate Gene Analysis Of Cotyledon Yellow To Lethal Mutatant Ytl In Brassica Napus

Plants use photosynthesis to maintain their own growth and development.Chloroplast is the main organelle for photosynthesis in plants.Mutations in genes involved in multiple important pathways such as chlorophyll synthesis and degradation,chloroplast development,etc.may lead to the production of leaf color mutants.Therefore,leaf color mutants are an ideal material for studying chloroplasts.In the single selfing offspring of the restorer line recurrent selection population,a yellowing lethal mutant was found and named ytl（yellow to lethal）.This project aims to map the phenotype of mutant yellowing death and predict candidate genes.The results are as follows:1.Compared with the wild type,the cotyledons of ytl have been in the yellowing stage since sowing,and die after 9-15 days.There is a significant difference in the length of the aboveground and underground parts between the two after 7 days of sowing.2.Hybridized single plant crosses with ZS11,F₁ leaf color is normal,4 out of 8 F₂lines show trait segregation,3 lines have a segregation ratio of 15:1,and 1 line shows partial segregation.It is preliminarily determined that this trait is a recessive trait controlled by two nuclear genes.In the offspring of a population with a segregation ratio of 15:1,molecular markers were used to select recessive homozygous individuals at the Bna C09.YTL locus,but with normal phenotypes,and bagged selfing was used to obtain offspring for trait segregation ratio testing.The Chi square test results showed that the segregation ratio of the yellowing lethal phenotype was 3:1,and the segregation ratio of the hybrid single plant offspring of this strain was also 3:1.It is preliminarily determined that this locus is controlled by a pair of recessive nuclear genes and can be used as a unit point segregation population for localization analysis.The gene is named Bna C02.YTL.3.Using 4803 F₃ and F₄ individual plants,locate Bna C02.YTL within the 418kb genome interval of the ZS11 reference genome,which contains 84 annotation genes.4.Combined with the information on the website of the transcriptome of Brassica napus and the annotation of gene function,11 candidate genes were identified.q RT-PCR showed that Bna C02G0055300ZS,Bna C02G0055700ZS,Bna C02G0056300ZS,Bna C02G0057300ZS,Bna C02G0057300ZS,Bna C02G0058000ZS,Bna C02G0059500ZS,and Bna C02G0061100ZS were downregulated in the mutant compared to the wild-type,while Bna C02G0060000ZS was upregulated in the mutant compared to the wild-type.Eight differentially expressed genes are expressed in various tissues of rapeseed,and all have the highest expression levels in leaves.Bna C02G0055300ZS,Bna C02G0055700ZS,Bna C02G0057300ZS,Bna C02G0057300ZS,and Bna C02G0061100ZS also have high expression levels in cotyledons,and the expression levels are relatively low in other tissue parts.Bna C02G0059500ZS is only highly expressed in leaves,and the expression levels are relatively low in other tissue parts.Although Bna C02G0056300ZS,Bna C02G0058000ZS,and Bna C02G0060000600ZS have the highest expression levels in leaves,their expression levels are similar in other tissue parts.5.Using mutants,wild-type,and ZS11 as templates for comparative sequencing,amplify the g DNA sequence of the candidate gene Bna C02G0055700ZS.Amplify without bands in mutant ytl,and amplify normally in wild-type and ZS11,with the sequence consistent with the reference genome sequence.Therefore,it is speculated that the gene may undergo insertion or deletion of large fragments in the ytl mutant,and this gene is the most likely candidate gene.6.Using CRISPR/Cas9 technology,T₀ hybrid mutants with Bna C09G029430ZS,Bna C09G0294400ZS,and Bna C09G0294500ZS edited were obtained in the Bna C09.YTL unit population.After further self pollination and isolation,T₁ mutant materials with homozygous editing of Bna C09G0294400ZS and Bna C09G0294500ZS were obtained,and their phenotypes were investigated.The related mutants did not exhibit a yellowing lethal phenotype during the cotyledon period.