Evaluation Of Folate Determination In Cereals And The Genetic Basis Of Folate Metabolism In Rice (Oryza Sative L.)

Folates are water-soluble vitamin B,and are important cofactors essential for normal metabolic activities.Folate deficiency causes several human diseases.Folate biofortification by development of high-folate cultivars of rice（Oryza sativa L.）and other staple crops is an important tool to combat folate malnutrition.In this study,the natural variation of folate content in brown rice of 524 different accessions was determined and the genome-wide association studies（GWAS）was performed to explore the molecular mechanism of regulating folate accumulation in rice seeds.The results were as follows:1.For folate determination,the extraction and quantification of eight monoglutamate folate derivatives by high performance liquid chromatography-tandem mass spectrometry（HPLC-MS/MS）in cereals（maize,rice,and wheat）were optimized and validated.Di-enzyme treatment withα-amylase and rat deconjugase was proved ideal for folate extraction from the cereal grains.The HPLC–MS/MS method was validated based on its matrix effect,linearity,and sensitivity,recovery,inter-day and intra-day precision.The limits of detection and quantification of folate derivatives ranged from 0.03-1.0μg/100 g among the three cereal samples.The absolute recoveries of most folate derivatives including 10-formyl-folic acid（10-CHO-Pte Glu）,5,10-methenyl-tetrahydrofolate（5,10-CH=H₄Pte Glu）,5-formyl-tetrahydrofolate（5-CHO-H₄Pte Glu）,5-methyl-tetrahydrofolate（5-CH₃-H₄Pte Glu）,and oxidated folates（Mefox）were 72–96%for these cereal samples,with the exception of dihydrofolate（H₂Pte Glu）,tetrahydrofolate（H₄Pte Glu）,and folic acid（Pte Glu）,ranging from 44–65%.The inter-day and intra-day precisions were<12%.This validated method is an efficient approach for folate determination in cereal crops.2.To investigate the natural variation of folates among rice cultivars and identify high folate materials for breeding,we studied the folate contents of 524 brown rice samples of two different seasons and geographical locations by using the HPLC–MS/MS method.The total folate content ranged from 3.54 to90.41μg per 100?g grains,showing a remarkable variation.5-Formyl-tetrahydrate and 5-methyl-tetrahydrate were found to be the two major folate derivatives in rice germplasms.The results also revealed that folate derivatives were significantly correlated,and the highest correlation coefficient（r=0.91**）was found between 5-CH₃-H₄Pte Glu and total folates.In addition,we found that environment factor also had significant effect on folate production.Ten rice cultivars were identified as rich in folates（>66.72μg per 100?g）.3.Genome-wide association studies were performed to uncover the genotypic and phenotypic variations using a mixed linear model with GAPPAT R Package based on folates traits including 5-CH₃-H₄Pte Glu,5-CHO-H₄Pte Glu,Mefox and total folates in the 524 different rice accessions of two various agro-climatic conditions and geographical locations.The 16 SNP loci were identified.For 5-formyl-tetrahydrofolate trait,4 SNP locis were identified at-log₁₀ P value≥6.0;for 5-methyl-tetrahydrofolate trait,8 significant SNPs were selected at-log₁₀ P value≥6.5;for the total folates,4 significant SNPs were detected at-log₁₀ P value≥5.0;for Mefox,4 significant SNPs were identified at-log₁₀ P value≥7.0 and 16 candidate genes were identified based on these SNPs.This study optimized the extraction and quantification methods of folate analysis in cereal grains,which provides a fundamental technique for accurate folate detection;the folate levels of 524 rice cultivars were analyzed and 10 rice accessions rich in folates were screened,which are elite resources,could be used in rice breeding for folate improvement;through GWAS analysis,16 folate-associated candidate genes were identified,which are beneficial for molecular marker-assisted breeding for folate enhancement in rice.