Exploitation And Identification Of Stay-green Mutations In Brassica Rapa

The stay-green trait is mostly characterized by leaves of plant that remain green during the period of senescence.Stay-green materials can increase the yield by extending the time of photosynthesis,and some of them also have the effect of delaying the senescence of plants.The stay-green mutants also have the stress tolerance and disease resistance.Leaf color is an important agronomic trait for Brassica rapa.The stay-green trait could not only improve the commodity quality but also prolong the shelf life of Brassica rapa.The aims of this research are to indentify the stay-green variation of Brassica rapa and assess the breeding potential for the variety improvement.In the first part,a natural stay-green mutant of pakchoi,nye,was identified.The pigment catabolism,the chloroplast structure stability and physiological photosynthetic characteristics of nye were analyzed,and the stay-green mutant gene Brnye1 was fine mapped by BSA-seq and the candidate gene was predicted,then the mutant was used to create diversity stay-green DH lines and cytoplasmic male sterile lines and prepared for variety improvement in Pakchoi;In the second part,a DH 'FT' of Chinese cabbage was used as the material,and the stay-green mutants of Chinese cabbage were created by EMS mutagenesis its germinating seeds.Using the above stay-green Chinese cabbage mutants,the Mut Map,KASP,heterogenic overexpression verification,spatiotemporal expression and subcellular localization methods were carried out to locate and clone the mutant gene,verify the gene function and explore the expression characteristics of stay-green gene.The main research results are as follows:1.Chlorophyll metabolism and physiological photosynthetic characteristics of the stay-green mutant nye in Pakchoi.During leaf senescence,the total chlorophyll,Chl a and Chl b of the stay-green mutant nye were significantly accumulated,the chloroplast structure was more compact,and the chloroplast degradation was delayed.Along with the increase of the degree of leaf senescence in nye,the activity of chlorophyll catabolism enzyme Chlase and PAO was increased and induced by senescence signal,while the Mg-dechelatase defects which can't catalyze the degradation of chlorophyll a,suggests that the lesion step of Chl degradation occurs at the beginning of Chl degradation in nye.No significant excessive accumulation of Chlide a,Pheophytin a,or Pheide a was observed in nye during leaf senescence,and there might be no dangerous phototoxic molecule affect mutant normal growth.The variation trend of net photosynthetic rate(Pn)and photochemical efficiency(Fv/Fm)of mutant leaves in different senescence degrees was not significantly different from that of '13A510'.The nye was a non-functional stay-green mutant.2.The stay-green mutant gene Brnye1 was fine mapped using molecular markers associated with linkage analysis and BSA-Seq.Genetic analysis showed that the stay-green phenotype was controlled by a single recessive nuclear gene.By using the BSA-seq method,the stay-green mutant gene Brnye1 was mapped on chromosome A03 23-16 Mb candidate region.Then 1,561 stay-green plants in an F2 population helped us localize Brnye1 to an 81.01-kb interval between SSR markers SSRWN27 and SSRWN30 via linkage analysis,with the genetic distances of 0.06 c M and 0.19 c M,respectively.We identified 12 genes in this region,based on the 1.5 version Brassica rapa database,an orthologous gene of the Arabidopsis gene At NYE1,Bra019346(Br SGR1),was identified as the potential candidate for Brnye1.Sequence analysis revealed a 40-bp insertion in the second exon of Bra019346 in nye,which generated the TAA stop codon.A candidate gene-specific Indel marker in 1,561 F2 individuals showed perfect cosegregation with Brnye1 in the nye mutant.3.Based on the stay-green mutant nye,320 diversity homozygous stay-green breeding materials were created,and 6 new cytoplasmic male sterile lines were created,and two excellent stay-green hybrid combinations were screened out.Using the stay-green nye and excellent lines of common pakchoi as the parent to prepare the hybrid combinations,320 homozygous stay-green lines(DH lines)were created by rapidly by isolated microspore culture.In order to solve the problem of hybrid seed production,6 cytoplasmic male sterile lines were created by the method of saturation backcross.The hybrid combination was prepared by using the newly created stay-green male sterile line and the stay-green DH lines,through the variety comparison test,two excellent hybrid combinations ZH02 and ZH03 were screened out.Storage experiments showed that ZH03 could prolong the shelf life and be shelf-stable.4.EMS mutagenesis was used to treat the germinating seeds of DH line of Chinese cabbage,and eight homozygous stay-green mutants of Chinese cabbage were created.The germinating seeds of 'FT' DH lines were treated with 0.8% EMS mutagenesis and 3,750 M1 lines were obtained.Totally eight stay-green mutant materials were screened out through the identification.Genetic analysis showed that the stay-green trait of eight stay-green mutants was controlled by a single recessive nuclear gene,involving three different allelic genes.5.The stay-green gene Brsgr1 was identified in stay-green mutant nym1 of Chinese cabbage by the Mut Map and KASP technologies and the heterologous overexpression was used to verify the gene function.The stay-green mutant nym1 of Chinese cabbage obtained by EMS mutagenesis was used as the material and the stay-green gene Brsgr1 was identified by Mut Map and KASP.Bra A03g050600.3C(Br SGR1)gene encodes an At NYE1/SGR1 protein,which is involved in chlorophyll degradation.Compared with the wild type,a non-synonymous SNP mutation occurred in the stay-green conserved domain of Brsgr1 gene,which leads to the substitution of amino acids from Leu to Phe.Therefore,the mutant has an extra benzene ring in the protein configuration of the mutation site.The Wild-type SGR(Br SGR1)gene restores the mutant nye1-1 stay-green phenotype of Arabidopsis thaliana with SGR defects.