Genetic Analysis Of Cotyledon Yellow To Lethal Mutant Ytl In Brassica Napus

In general,plants generate energy and nutrients via photosynthesis,which not only maintains their own growth activities,but also provides food for human beings and animals.Chloroplasts are the main organelles for photosynthesis in plants.The generation of leaf color mutants may be due to the mutant genes participate in many important pathways such as chlorophyll synthesis and degradation,cytoplasmic nuclear signal transduction,photomorphogenesis and chloroplast differentiation.Thus they are ideal materials for researching chloroplasts.In this research,a yellow to lethal mutant named ytl was found in the pedigree of one single plant from the Brassica napus recurrent selection population of restorer lines.The phenotype of the mutant was investigated,the genetic model was analyzed,and the fine location of the mutation site was completed.The results were as follows:1.The mutant ytl came from the pedigree of one single plant from the recurrent selection population.Compared with the wild type of the same family,the cotyledons of the mutant had been in the yellowing stage since emergence,and did not turn green after germinating,meanwhile the true leaves weren’t able to come out,then ytl died in 9-15days after sowing.The 7 days’old cotyledons of the wild type and mutant ytl were collected after sowing,then we measured the content of photosynthetic pigments and observed the chloroplast through transmission electron microscope.As shown by the results,the contents in the mutant ytl were significantly lower than wild type.Compared with the wild type plants,the content of fat granules in the mutant cells was sparse,the chloroplast dysplasia remained in the plastid stage.In addition,the thylakoid stacks were less stacked,no lamellar structure was formed,and the granawere sparse.2.Investigating the traits segregation ratio of selfing progeny of heterozygous individuals showed the ratio of the number of normal individuals to those of the mutated individuals was 275:104,which conformed to the segregation ratio of 3:1(χ²=1.20<χ²_0.05,1=3.84).Due to Brassica napus is double diploid,heterozygous individuals with the recessive loci were selected to cross with ZS11.There were 4 of 8 F₂ plants from the cross had some segregations,the segregation ratio of 3 lines was 15:1(χ²<χ²_0.05,1=3.84),and one line appeared partial segregation.It was suggested that this character was controlled by two pairs of recessive reduplicative nuclear genes.3.BSA and Brassica napus 60K IlluminaSNP array were used to analysis the initial mapping of the mutant.The results showed that the different SNPs between the mutant individuals and the wild type individuals were mainly concentrated on chromosomes C09and C02.According to the ZS11 reference genome sequence,the SSR markers were uniformly designed within difference segment,then the polymorphic markers were screened for the mixed pools of the mutant and wild type plants in the S1 population and the S2 population.Finally,two candidate loci were located on chromosomes C09 and C02,named BnaC09.YTL and BnaC02.YTL,respectively.4.BnaC09.YTL was identified by fining mapping with 777 yellow to lethal individuals from the S1 population.Eventually BnaC09.YTL was located in 198 kb DNA segment between the marker SSR-140 and PBZIN-1 compared to the ZS11 reference genome,with 10 annotated genes.At the same time,we analyzed the expressions of 10candidate genes between the mutant and wild type by q RT-PCR.According to the results,the 4th and 10th genes were not expressed at the cotyledon stage,and the expression levels of the other eight candidate genes were all down regulated in the mutant,while the2nd,3rd,5th,and 7th genes’expression were significantly different between the mutant and wild type.5.All candidate genes were amplified and sequence comparisons were performed in the mutant and wild type.As shown by the results,there were no sequence differences in the 4th to 10th genes between the wild type and the mutant.The candidate genes were focused on the first three genes.6.Brassica napus 60K IlluminaInfinium SNP array results indicated that some enrichment of the SNPs were on the chromosome C02,then 41 pairs of SSR markers were uniformly designed on 3 to 10 Mbp of chromosome C02,the polymorphism markers were screened by using the ytl bulk and the wild type bulk selected from the S2population,then easily identifiable markers were selected for small group verification.A total of 25 available polymorphic markers were gained from 41 pairs of designed primers,the polymorphism markers were located on the same side,corresponding to the position of 3 to 6 Mbp on the ZS11 chromosome C02.