Differences In Phytate Accumulation In Seeds Between High And Low Phytate Brassica Napus Cultivars And Identification Of Candidate Genes For Phytate Synthesis

Oilseed rape?Brassica napus.L?is an important oilseed crops in China as well as in the world,which needs more phosphorus?P?and show sensitive to P deficiency.P deficiency occurred in large areas of the main Brassica napus production in China and application of phosphate fertilizer is one of the important strategies to keep the high and stable seed yield of Brassica napus.Phytate?inositol hexaphosphate?is the storage form of P in the seed and plays vitally important roles in P sensing and inorganic phosphorus?Pi?homeostasis during seed development.During seed germination,mineral nutrients are released by hydrolysis to provide nutrients for seed germination.At present,very few study are involved in the the effect of phytate in the seedon the seed germination and seedling growth in Brassica napus,and the difference in the dynamic change of phytate accumulation and the gene expression of phytate synthesis genes during the seed development between the cultivars with high?HPA?and low?LPA?phytate in the seed.In addition,the progress on the study of genetic mechanism of phytate accumulation in the seed of Brassica napus is limited.Hence,it is of great significance to study the the difference in the phytate synthesis mechanism in the seed between HPA and LPA Brassica napus cultivars,map and identify the genetic loci for the phytate in the seed,and isolate and clone the genes that could reduce the phytate content in the seed,but do not affect the germination and seed yield.The phytate concentration and phytate content in the seed of an association panel of Brassica napus?404 cultivars?were measured by iron precipitation method in our group.In this study,HPA and LPA Brassica napus cultivars were screened to study the difference between them in the seed germination,shoot and root growth at the seedling stage as well as seed yield and yield related traits at the mature stage under P deficient and sufficient conditions.The difference in the dynamic change of phytate content,and in the gene expression profile of phytate synthesis gene expression during seed developmentbetween HPA and LPA cultivars were also investigated,and the key genes decided the phytate in the seed were predicted.Finally,genome-wide association study?GWAS?were conducted to identify the significant SNP associated with the phytate concentration and content in seeds.The main results were as follows:?1?Forty eight cultivars were randomly selected from an association panel of Brassica napus to conduct seed germination experiment.The results showed that the average germination rate of seeds with high phytate concentration?41.14 mg/g-23.17 mg/g?and content?1.09 mg-0.55 mg?was 10%and 6%higher than that of seeds with low phytate concentration?22.91 mg/g-8.69 mg/g?and content?0.54 mg-0.04 mg?,respectively.However,some LPA cultivars showed a high germination rate of 80%-100%Moreover,there is no significant correlation between the germination rate and the phytate concentration and content in the seed.These indicated that the low phytate in the seed did not affect the germination in this study.The mineral elements in the seeds of 8 HPA cultivars and 7 LPA cultivars were determined.The mineral nutrients in the seed were mainly P,calcium?Ca?,potassium?K?and magnesium?Mg?,and minute quantities of iron?Fe?,manganese?Mn?,zinc?Zn?and copper?Cu?.However,there were no significant difference in the above nutrients between HPA and LPA Brassica napus cultivars.The biomass of shoot and root at the seedling stage,and the seed yield of LPA cultivars showed slightly higher than that of the HPA cultivars under P deficient conditions.?2?The biomass,P concentration and content of root,branches and the shell of pod of branches of HPA381 were significantly higher than that of LPA10,but there were no significant difference in biomass,P concentration and content of main inflorenscence and the shell of pod of branches between HPA381 and LPA10.59.64%of the P was distributed in the seed in HPA381;whereas 75%of the P was in the seed in LPA10 when the plants were harvested.On the 35^th day after flowering?DAF?,the concentration of Pi in the seed of LPA10 was significantly higher than that HPA381.The phytate concentration of HPA381 was higher than that LPA10 during the whole period of seed development,and the difference between HPA 381 and LPA10 was the most significant on 30 DAF.Thus,30 DAF could be the key time point for the difference of phytate synthesis in the seed between HPA381 and LPA10.q RT-PCR results showed that,the expression of manys genes involved the phytate synthesis showed up-regulated in HPA381 and LPA 10 in 30 DAF compared with 15-25 DAF,and the expression of six genes of them in HPA381 showed significantly different from LPA10.?3?The phytate concentration and content in the seed in the association panel showed an approximate normal distribution and had high genetic diversity.A total of 129 significant SNP loci associated with the phytate concentration and content in the seed of the association panel of Brassica napus in 2013 and 2015 by genome wide association study?GWAS?.Among them,22 SNPs associated with phytate concentration and 12 SNPs associated with phytate content were located on A09.The physical distance from the candidate genes of ABC transporter family gene Bna A09g10230D and Bna A09g10220D to the lead SNP of chr A09?5198034 were 40.422 kb and 46.191 kb,respectively.Association analysis of candidate genes showed that four SNPs of Bna A09g10230D had two haplotypes including Hap1?TCAA?and Hap2?GTCC?,and five SNPs of Bna A09g10220D had two haplotypes including Hap1?TCAGA?and Hap2?GTCCC?.The varieties with Hap1 haplotypes showed low phytate in the seed,which indicated that Hap1 haplotypes would be benefit to low phytate crop breeding.A total of 43 and 13significant SNPs were identified associated with phytate concentration and content of the association panel of Brassica napus under P sufficient and deficient conditions,respectively,in 2018.A total of 14 candidate genes were predicted,including five candidate genes under P sufficient condition and nine candidate genes under P deficient condition.The expression amount of three phospholipase genes in the seed of LPA10 was significantly lower than that of HPA381 during the seed development?15 DAF-40 DAF?under P sufficient condition.?4?The crosses between two groups of HPA and LPA Brassica napus cultivars had been finished.A total of 400 and 300 seedlings regenerated from the pollens?microspore?culture of of F₁ hybrid derived the cross between LPA10 and HPA381,and of F₁ hybrid derived the cross between LPA52 and HPA352,respectively.