Effects Of Mutation Of Bnamiks On Seed Phytate Concentration And Transcriptome-wide Association Study On Seed Phytate Concentration In Brassica Napus

Phosphorus(P)is one of the essential macronutrients for plant growth and development.Phytate is the main storage form of P in plant seeds.Although phytate is crucial for seed germination and plant development,seed derived phytate functions as an antinutrient(substances that have adverse effects on the digestion,absorption,and utilization of nutrients)when consumed in human and animal diets since phytate is not efficiently utilized by monogastric animals.In addition,large amounts of excreted phytate in animal production caused environmental pollution.Oilseed rape(Brassica napus.L)is the largest oil crop in China,and the concentration of seed phytate is very high.In order to reduce the negative effects of phytate,breeding low phytate(lpa)oilseed rape is necessary.In plants,phytate is produced by two pathways: lipidindependent and-dependent.Lipid-independent pathway is considered to be the main pathway of phytate synthesis in seeds.Myo-inositol kinase(MIK)is involved in the first step of phosphorylation reaction of lipid-independent pathway,which promotes the conversion from inositol phosphorylation to inositol-3-phosphate.In this study,all the members of BnaMIKs in Brassica napus were identified and their expression pattern and subcellular localization were analyzed.bnac09.mik and bnaa10.mik mutants were created by CRISPR-Cas9 technology to study the effects of BnaMIKs mutation on seed phytate synthesis and agronomic traits.Furthermore,three lpa haplotypes were screened through transcriptome-wide association study(TWAS)and candidate gene association study.The gene BnaA06g08470 that may affect phytate synthesis of seeds was identified,which will provide a basis for lpa breeding in Brassica napus.The main results are as follows:(1)Gene structure characterization of BnaMIKs and effects of mutation of BnaMIKs on seed phytate concentration in Brassica napusThere were two copies of MIK gene in Brassica napus,namely Bna C09.MIK and BnaA10.MIK.Evolutionary analysis showed that the amino acid sequence similarity between Bna C09.MIK and BnaA10.MIK was as high as 96%.Their amino acid sequence similarity with Arabidopsis At MIK was more than 80%.The three amino acid sites that maintain the activity of MIK kinase were very conservative,The results of q PCR,transcriptome and GUS staining experiments all showed that the expression of Bna C09.MIK and BnaA10.MIK gradually increased in the seeds during 15-30 days after flowering(15-30 DAF),and the expression of 30-40 DAF gradually decreased.The change of seed phytate concentration was similar to the change of gene expression of both genes during the seed development.At the same time,the expression of Bna C09.MIK is always slightly higher than that of BnaA10.MIK.The results of subcellular localization showed that both Bna C09.MIK and BnaA10.MIK were located in the cytoplasm.The seed phytate concentration of their mutants is significantly reduced by about 15%.However,seed total P concentration,seed size and 1000-seed weight of these two mutants were not changed significantly.(2)Transcriptome-wide association study of seed phytate concentration in Brassica napusThe transcriptome sequencing data of seeds of 20 DAF of 309 cultivars and seeds of 40 DAF of 274 cultivars in the natural population were employed to carry out TWAS.TWAS were performed with seed phytate concentration,and 181 and nine genes were identified in the seeds of 20 DAF and 40 DAF,respectively.GO enrichment indicated that these genes were mainly involved in processes such as photosynthesis,glucose-6-phosphate metabolism,and lipid biosynthesis.Three genes including BnaA06g08470 D,BnaA06g09130 D and BnaA09g40530 D were identified simultaneously by the phytate concentration in the seed 20 DAF and in the seed 40 DAF,respectively.Association analysis of candidate genes showed that these three genes contained six,seven and three SNPs(single nucleotide polymorphism)respectively,which were significantly correlated with seed phytate concentration.Among them,six SNPs of BnaA06g08470 D had two haplotypes,Hap1(CACCGA)and Hap2(TTTGAG);seven SNPs of BnaA06g09130 D had two haplotypes,Hap1(CCTTGAG)and Hap2(TTCCCGA);and three SNPs of BnaA09g40530 D had two haplotypes,Hap1(CAA)and Hap2(GGT).The varieties with Hap2 showed a lpa phenotype.Field experiments showed that there was no significant difference in plant height,branch number and 1000-seed weight between the varieties with three lpa haplotypes and the varieties with three high phytate haplotypes.In addition,the seed phytate concentration of the T-DNA insertion mutant of AtRPF5(the homologous gene of BnaA06g08470 D in Arabidopsis thaliana)significantly reduced by 20%.As compared with the wild type(WT),the seed total P concentration was also significantly decreased,but the growth was not significantly affected.In the atrpf5 mutant,the expression of genes involved in phytate synthesis pathway,such as At MIPS1,At MIK,At ITPK4,At IPK1 and At MRP5,was significantly reduced,indicating that AtRPF5 can positively regulate the synthesis of seed phytate.Therefore,we speculate that BnaA06g08470 D could also positively regulate the synthesis of seed phytate in Brassica napus.Taken together,two MIK genes Bna C09.MIK and BnaA10.MIK,were idenfified to be involved in phytate synthesis in Brassica napus.Their mutations significantly reduced the seed phytate concentration by 15%,however,the diameter size and 1000-seed weight of the mutant seeds were not affected significantly.In addition,three lpa haplotypes and one gene BnaA06g08470 D that may affect seed phytate synthesis in the panel of Brassica napus by TWAS and association analysis of candidate genes.These results provide elite germplasm and gene resources for lpa breeding in Brassica napus.