Genetic Analysis On Phytate Content In Seeds Of Oilseed Rape(Brassica Napus L.)

Phosphorus(P)is an essential macroelement for higher plant growth and development.Phytate(inositol hexaphosphate)is the storage form of P in seeds and plays vitally important roles in P sensing and Pi homeostasis during seed development.However,phytic acid is highly negatively charged,which chelates positive ions of essential minerals.Phytate can not be absorbed and used by human and monogastric animals without phytases in the digestive system,which results in low bioavailability of mineral elements in human being and monogastric animals.Moreover,phytate in animal wastes is excreted into rivers,lakes and oceans,which contributes to a waste of P resource and the eutrophication of water.Oilseed rape is one of the main oil crops in China,and soil available phosphorus in its main producing areas is relatively low.Besides,rapeseed cakes are commonly used as feed.Hence,the study of the genetic mechanism of seed phytate accumulation in Brassica napus is of great significance for the research on P efficiency in oilseed rape and the breeding of low-phytate crops.An association panel of Brassica napus has been genotyped using the Brassica 60 K Illumina SNP array in our lab.In this study,seed phytate concentration and content of 390 cultivars in the association panel were determined by iron precipitation method,and 19397 high-quality single nucleotide polymorphisms(SNPs)were used to performe the genome-wide association study(GWAS)on seed phytate traits.Additionally,seed phytate concentration and content of 182 lines of Bna TNDH population as well as two parents ‘Tapidor' and ‘Ningyou 7' under low P(LP,9 P2O5 kg/ha)and optimal P(HP,90 P2O5 kg/ha)conditions were also determined.Quantitative traits loci(QTLs)affecting seed phytate conentration and content were detected.Correlation analysis between seed phytate traits and root morphology traits of the association panel and Bna TNDH population at seedling stage were performed.The main results were as follows.1.Seed phytate concentration,content and seed weight of 390 cultivars in the association panel showed high genetic diversity,continuous variation and an approximate normal distribution.All the seed phytate raits were used to perform association analysis with GLM and MLM independently to determine the optimal model for association mapping in this study by TASSEL5.0.In total,35 single-nucleotide polymorphisms were significantly associated with seed phytate concentration and content of Brassica napus,which explained the phenotypic variation from 5.33% to 7.29%.These SNPs were located on chromosomes A2,A3,A4,A5,A6,A9,A10,C1,C2,C3,C4,C7 and C8 in B.napus.The low phenotypic variation explained by single SNP showed that seed phytate concentration and content might be contributed by numerous minor genes.The SNP marker Bn-Scaffold000321-p23083 was the repeatedly identified peak SNP on A9.Four candidate genes located in the LD decay region were involoved in phosphate uptake and transport,phytate biosynthesis and transport.Five closely adjacent significant SNPs formed eight haplotypes,named ‘Bn A09 Hap haplotypes'.Among them,haplotype E(AAYNA)had higher seed phyate content.Whereas,haplotype C(AANAA)showed lower seed phytate content,compared to other haplotypes.2.Seed phytate concentration and content of 182 double haploid lines of Bna TNDH population at low P and high P were determined in this study.A total of six QTLs,one at LP and five at HP,were detected for seed phytate concentration or content with Bna TNDH 2041 map.Most of the QTLs were located in linkage groups A03,A09 and C06 in B.napus.The genome of Brassica napus cultivar ‘Darmor-bzh' was used as a reference,and GO enrich analysis of target genes in the QTL intervals for seed phytate traits were performed.Five candidate genes were identified in these QTL regions on the basis of the gene annotation.These genes were involved in phytate biosynthesis,sulfate tranporter or plant responses to P deficiency.3.In Bna TNDH population,seed phytate concentration negatively correlated with shoot fresh weight and shoot dry weight at LP.In GWAS population,seed phytate content positively correlated with shoot dry weight,root dry weight,primary root length and total root length at LP.Due to the low correlation coefficient,it is only statistically significant and makes no sense in biology.The correlation between seed phytate concentration,content and root morphology traits at seedling stage should be further confirmed by more experiments.