Genetic Analysis And Positional Cloning Of Leaf Color,Flower Color And Plant Height Mutants In Brassica Napus L.

Brassica napus L is an important oil crop.Variation of important traits,gene mapping and cloning are important tasks in rapeseed genetics and breeding.In this study,genetic analysis,gene mapping and cloning of yellow leaf,yellowish-white flower,and semi-dwarf mutants produced by EMS mutagenesis were conducted.Physiological and biochemical assays and transcriptome analysis were used to explore mechanisms of trait formation.The main results are as follows:1. The research of yellow leaf mutantThe original material of EMS mutagenesis was zhongshuang 9?ZS9?.The yellow leaf mutant has been selfing for many years and has a stable phenotype.?1?Compared with the original material ZS9,the mutant showed that the newly heart leaves were yellow,and the yellow leaves was relieved with the growth and development of the plant,and gradually changed to virescent from cotyledon to mature.During the whole growth period,the leaves of mutant showed the development trend of yellow first and then virescent.?2?The mutant and the original material ZS9,both in the seven-leaf stage,were selected as the research objects.The chlorophyll content of the younger leaf in the mutant was significantly lower than that of ZS9.With the growth and development,the chlorophyll of the older leaf in the mutant was increased,but it was still significantly lower than that of ZS9.The result of transmission electron microscopy showed that the chloroplast development of yvl was stagnant and slow.So the mutant was named yvl.Interestingly,there was no significant difference in photosynthetic rate of younger and older leaves between ZS9 and yvl.?3?Genetic analysis indicated that the leaf color of yvl was controlled by a single recessive nuclear gene.The target gene was initially mapped to 1.0-4.0 Mb region on A03chromosome by BSA-seq.Further fine mapping using the BC₂F₂population,the candidate gene yvl was narrowed in the 70 kb region.Sequence alignment of 14 genes in the candidate region revealed that only a C to T substitution occurred in the fifth exon of the gene Bna A03g04440D,leading to premature termination,which encodes the magnesium chelate enzyme large subunit H?CHLH?in the chlorophyll synthesis pathway and involves retrograde signaling to regulate chloroplast development.The expression pattern analysis of Bna A03.CHLH was found to be expressed in all tissues,especially in leaves.Evolutionary analysis revealed the presence of another CHLH gene on C03chromosome in B.napus.In yvl,it was found that the up-regulation of Bna C03.CHLH expression significantly compensated the mutant Bna A03.CHLH.?4?Differential expression analysis of younger and older leaves in ZS9 and yvl was performed by RNA-seq technique.Transcriptional activity of plastid-encoded RNA polymerase?PEP?in yvl younger leaf was severely suppressed.The expression of photosynthesis-related electron transport chains,redox reaction and photosynthesis device genes were not affected in yvl.2. The research of yellowish-white flower mutantThe original material of EMS mutagenesis was zhongshuang 9?ZS9?.The yellowish-white flower mutant has been selfing for many years and has a stable phenotype.?1?Compared with the original material ZS9,the petals of mutant were milky white,between yellow and white.There was no significant difference in the length of the main inflorescence,the silique number of main inflorescences and 1000-seed weight between ZS9 and mutant,but at the plant height and the initial branch heigh were reduced to different extents in the mutant.?2?The petals of ywf and ZS9 in the blooming stage were selected as the research objects.In ywf,the content of carotenoids and the components was significantly lower than ZS9.The result of transmission electron microscopy showed that the number of plastid in ywf petals was significantly reduced and the shape was not full.So the mutant was named ywf.?3?Genetic analysis showed that the petal color of ywf was controlled by a single recessive nuclear gene.The target gene was initially mapped to 11.0-17.0 Mb region on A08 chromosome by BSA-seq.Further fine mapping using the F₂population,the candidate gene ywf was narrowed in the 857 kb region.In this region,there was only one"C"to"T"mutation site,which was located at the first exon of the gene Bna A08g17170D,leading to the premature termination.The gene encodes phytoene desaturase 3?PDS3?and involved in the carotenoid biosynthetic pathway.?4?The expression pattern analysis of Bna A08.PDS3 was found to be expressed in all tissues,especially in petals.Evolutionary and synteny analysis revealed that the PDS3gene on the A08 chromosome was broken,forming two PDS3 genes,Bna A08g17160D?Bna A08.PDS3;1?and Bna A08g17170D?Bna A08.PDS3;2?.?5?The transcriptome analysis of ZS9 and ywf petals revealed that KEGG was significantly enriched in the carotenoid biosynthesis pathway.The expression analysis of genes related to the carotenoids biosynthetic pathway revealed that the candidate gene Bna A08.PDS3;2 was interrupted the biosynthesis of carotenoids,resulting in the lack of carotenoids content.?6?Metabolome analysis of ZS9 and ywf petals showed that a total of 59 differentially significant metabolites,including flavonoids,flavanones and flavonols were all up-regulated in ywf petals.The integrated analysis of transcriptome and metabolome showed that differentially expressed genes and differential metabolites were significantly enriched in flavonoid biosynthesis,which may be involved in the disease resistance and photoprotection of rapeseed.3. The research of semi-dwarf mutantThe original material of EMS mutagenesis was zhongshuang 9?ZS9?.The semi-dwarf mutant has been selfing for many years and has a stable phenotype.Because the plant height of mutant was 110-120 cm,it was named semi-dwarf mutant?sdw?.?1?The sdw was developed slowly from the seedling stage.Compaired with ZS9,the leaf color of sdw was greenish and the leaves were bent downward and slightly shrinking.At maturity the plant height of sdw was about 110-120 cm and 30%lower than ZS9.?2?Genetic analysis showed that the plant height of sdw mutant was controlled by two recessive nuclear genes,named sdw1 and sdw2.To efficiently locate these two genes,sdw1 and sdw2 were isolated and constructed into two independent segregated population.The candidate genes sdw1 and sdw2 were respectively mapped to the 647 kb region on the C03 chromosome and the 400 kb region of the A02 chromosome by BSA-seq and fine mapping.In the 647 kb region of sdw1,a substitution from"C"to"T"occurred in the seventh exon of Bna C03g62810D,leading to the change from proline to leucine.Bna C03g62810D encodes shaggy-like protein kinase BIN2,which involves in brassinolide and auxin signal transduction pathways.In the 400 kb region of sdw2,the expression level of Bna A02g17120D,which encodes BZR1,a positive regulator in the signal transduction pathway of brassinolide,was significantly reduced in the sdw stem.Therefore,We predicted that the two candidate genes controlling the plant height of sdw are Bna C03.BIN2 and Bna A02.BZR1,respectively.?3?RNA-seq analysis of shoot apical meristem and stem in squaring stage of ZS9 and sdw showed that genes related to plant hormone signal transduction pathway were significantly changed.Determination of gibberellins?GAs?,auxin?IAA?and brassinolide?BR?of ZS9 and sdw stems in squaring stage showed no significant difference in GA,while BR and IAA were significant accumulation in sdw stem.Therefore,it is concluded that sdw may be abnormal in the BR and IAA signal transduction pathways.