| UV induced anthocyanin synthesis,as a kind of photomorphogenesis,is regulated by light receptors and related signal pathway.Our previous studies showed that anthocyanin synthesis is induced not only by the known light receptor pathway,but also by UV-A and synergy of blue+UV-B signaling in turnip;however,the light receptors and signal transductors involved in the response are not well addressed.To investigate the UV-A and blue+UV-B signal pathways in non-model turnip,preliminary screening,identification and genetic analysis of a series of light-insensitive anthocyanin synthesis mutants were performed.The anthocyanin synthesis related genes expressed in the swollen storage roots of target mutants under sunlight condition were identified by real-time RT-qPCR;meanwhile,population genetic analysis was performed for F2 populations of mutants with stable heredity phenotype.Finally,the high-throughput sequencing integrated MutMap analysis was performed for identifing the casual genes of anthocyanin-deficient mutant g56w,g97w and gl42w.The main results were presented as follows:1.Genetic analysis and RT-qPCR analysis for light-insensitive anthocyanin-synthesis related mutants.The anthocyanin contents of 8 stable mutant lines and WT were determined.The results showed that:for WT,the pigmentation in the storage root peels showed a light-sensitive model that significant difference was observed in the contents of anthocyanin between the above-ground parts exposed to sunlight and the under-the WT,anthocyanin content significantly decreased in above-ground part of the storage roots in the anthocyanin-deficient mutants(g56w;g83w,g97w,gl40w,g141),g142w and g143w),ground parts in dark condition.In contrast to and anthocyanin content significantly increased in anthocyanin-rich mutant g19r.The mutants g56w,g83w,g97h,g140w,g141w,g142w and g143w are anthocyanin-deficient phenotype under light condition,without anthocyanin synthesis be observed.After several generation self crossing for the mutants,the mutant traits were stably inherited,and then the test cross for anthocyanin synthesis related mutants was performed.The results showed that their mutation phenotypes of anthocyanin-deficient mutants were induced by different gene mutations.Genetic analysis showed that,in the F2 population of the mutants(g56w,g83w,g97w,g142w and g143w)backcrossed with WT,the proportions of phenotype between wild-type and mutants are 3:1 following the segregation law of Mendelian heredity.Therefore,we speculated that the mutated trait of each mutant is controlled by a single recessive gene.Whereas,the other 2 mutants,g140w and g141w are not following the Mendelian inheritance,and anthocyanin-rich mutant g19r is also controlled by a single dominant gene.With RT-qPCR the expression profiles of anthocyanin biosynthetic genes for anthocyanin-deficient mutants suggest that the expression levels of structure genes(BrCHS,BrCHI,BrANS and BrDFR)and regulatory genes(BrTT8 and BrPAPl)in the anthocyanin biosynthesis pathway of all these five stable mutants(i.e.g56w,g83w,g97w,g142w,g143w)in this study were significantly decreased compared with WT,and for anthocyanin-rich mutant g19r,transcript levels of all the structural and regulatory genes were much higher for gl9r than for the WT in the soil.The systemic expression changes of anthocyanin synthesis related gene in these mutants further indicated that the mutated genes of g56w,g83h,g97w,g142w and g143w could likely impact on upstream components of anthocyanin biosynthesis to repress light-induced anthocyanin accumulation.2.Preliminarily gene mapping for anthocyanin-deficient mutants g56w and g97w.The candidate genes of g56w and g97w were preliminarily mapped by the resequencing technology combined with MutMap;meanwhile,we further verify the candidate SNP sites by high resolution melting curve(HRM)technology.The results indicated that the mutant interval was mapped on A07 chromosome in 576kb intervals between 16,825,203bp and 17,401,586bp for g56w,and in 119kb intervals between 17,221,786 bp and 17,341,239 bp for g97w,in which the candidate genes mainly include methyl transferases,transmembrane receptor proteins,nucleotide binding proteins,protein kinases,zinc-finger proteins,protein phosphoralyation and protein ser/thr kinase related functional genes.3.Gene mapping for anthocyanin-deficient mutant bg142w.The seedlings of WT and mutant g142w were treated with different short-wave length light quality,and the different anthocyanin accumulation models in the hypcotyls of WT and g142w were observed,from which we prudently speculated that g142w was our mutant object.The anthocyanin components was determined by HPLC for the WT and g142w,indicating that the content of anthocyanin,but not the synthesis and metabolism pathway in g142w changed.Resequencing and MutMap analysis indicated that the candidate genes of g142w were mapped on A07 chromosome in the intervals between 16.0 Mb and 18.0 Mb;meanwhile,HRM technology was performed for exploring whether the candidate SNPs were correlated with phenotype or not.The results indicated that the mutant interval of g142w was located in 159 kb intervals between 16,902,778 bp and 17,061,871 bp on A07 chromosome,in which 8 candidate genes are included,including acyl carrier protein,polygalacturonase,auxin response factor,calmodulin binding protein,and so on.Transcriptome data from RNA-seq and RT-qPCR showed that Bra004127,Bra004129 and Bra004136 might be the candidate genes controlling mutated traits.Our study provides a series of stable,homozygous and valuable mutant resources for investigation of UV-A and blue+UV-B induced anthocyanin biosynthesis.The obtained mutants g56w,g97w and g142w were of the characteristics of anthocyanin synthesis-insensitivity,of which the candidate genes of g56w,g97w and g142w may be valuable for further elucidating the underlying mechanism of UV-A and blue+UV-B induced anthocyanin biosynthesis in higher plants. |
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