| Helianthus annuus L.is an important oil crop in the world.The main characteristic of sunflower oil is rich in unsaturated fatty acids,among which monounsaturated fatty acid oleic acid has health care effect on human body,and can also reduce oxidation and rancidity thus prolong the shelf life of oil.However,the proportion of polyunsaturated fatty acid linoleic acid in unsaturated fatty acid of sunflower seed is large,while the proportion of monounsaturated fatty acid oleic acid is small.Improving the content of oleic acid in sunflower seed will greatly improve its edible nutritional value and storage stability,which has become an important goal of sunflower breeding.In this study,a near-infrared spectroscopic(NIRS)model was developed for quantitative analysis of sunflower seed fat and four major fatty acids,which could quickly and nondestructively detect the quality of sunflower and provided technical support for the selection of sunflower germplasm resources and quality breeding.The high-density genetic map of sunflower was constructed by SLAF-seq technology,and the QTL of oleic acid content was mapping,which could lay the foundation for fine mapping of QTL and molecular marker-assisted breeding.The transcriptome sequencing of high oleic acid and low oleic acid sunflower seeds was carried out to analyze the differentially expressed genes related to fatty acid metabolism,and the key enzyme genes that may affect the oleic acid content were mined.Gene cloning and expression pattern analysis were performed to provide genetic resources and theoretical basis for improving sunflower quality by molecular means.The main results are as follows:1.Four spectral pretreatment methods were used to establish a NIRS model for quantitative analysis of fat and four main fatty acids(linoleic acid,oleic acid,stearic acid and palmitic acid)in sunflower seed,and the best spectral pretreatment method was determined.The correlation coefficients of calibration and validation of fat,linoleic acid and oleic acid content models were all greater than 0.96,and the values of correction for root mean square error(RMSEC)and prediction for root mean square error(RMSEP)were all less than 0.99,which could be used for the rapid determination of component content in samples.For stearic acid and palmitic acid models,the correlation coefficients of calibration were 0.92 and 0.82,respectively,and the correlation coefficients of validation were 0.83 and 0.74,respectively.The correlation between near-infrared scanning spectroscopy and chemical determination results of the models was relatively low,but the values of RMSEC and RMSEP were low,which could be used for rough estimation of component content in samples.2.The F2 population was constructed by crossing the high oleic acid maintainer line "L-1-OL-1”and low oleic acid maintainer line "86-1".The high-density genetic map of sunflower including 17 linkage groups was constructed by SLAF-seq technology.There were 6136 markers in the map.The total distance of the map was 2221.86 cM,and the average distance of the map was 0.36 cM.Compared with the high-density genetic map of sunflower previously reported,the total distance of this map is longer and contains more markers.3.Based on the high density genetic map of sunflower,3 QTLs controlling oleic acid content,2 QTLs controlling plant height,2 QTLs controlling diameter of flower disc and 1 QTL controlling stem diameter were detected by composite interval mapping,which were distributed in linkage groups LG5,LG6,LG7,LG9,LG 16 and LG 17,explaining 5.18%-15.65%of phenotypic variation.The phenotypic contribution rate of 4 QTLs were greater than 10.4.Transcriptome sequencing was performed on seeds of sunflower high oleic acid maintainer "L-1-OL-1" and low oleic acid maintainer "86-1" in two developmental stages.A total of 86.68gb clean data was obtained.With screening standard of Fold Change?2 and FDR<0.01,5971,4906,12761 and 9883 differentially expressed genes(DEGs)were obtained from D8617dvsD8627d,D8617dvsL17d,D8627dvsL27d and L17dvsL27d,respectively.They were enriched in 122,120,125 and 124 metabolic pathways in KEGG database respectively.In order to find out the genes related to oleic acid content,DEGs of D8617dvsL17d and D8627dvsL27d in six metabolic pathways that may affect oleic acid synthesis were analyzed,including unsaturated fatty acid biosynthesis,fatty acid biosynthesis,fatty acid prolongation,fatty acid degradation,carbon metabolism and starch and sucrose metabolism.It was found that D8627dvsL27d group contained more DEGs in these metabolic pathways and most of them were down regulated.The expression trend of 14 genes selected in fatty acid metabolism pathway were all consistent by qRT-PCR and transcriptome sequencing,which indicated that the result of transcriptome sequencing was reliable.5.Seven key enzyme genes in fatty acid metabolism pathway which may affect the oleic acid content of sunflower seeds were screened out from transcriptome data:FAD2,FAB2,fab2,ACSL,KAR,FATE and fabF.FAD2 and FAB2 are two key genes directly related to oleic acid synthesis.?9-stearoyl-ACP dehydrogenase FAB2 dehydrogenates stearic acid to form oleic acid,and ?-12-fatty-acid desaturase FAD2 catalyzes oleic acid to form linoleic acid.The open reading frame(ORF)sequences of sunflower FAD2 and FAB2 were cloned,and the nucleotide sequence length was 1137 bp and 1191 bp,respectively.Phylogenetic analysis showed that FAD2 and FAB2 genes were most closely related to the evolution of Dimorphotheca sinuata and Mikania micrantha,respectively.Bioinformatics analysis revealed that the FAD2 encoding protein was an alkaline protein,the FAB2 encoding protein was an acidic protein,and the two gene encoding proteins were both a-helix and hydrophilic proteins.The expression patterns of 7 genes in seeds at different development stages and in roots,stems,leaves,tubular florets and ray florets were analyzed by qRT-PCR.It was found that only FAD2 was particularly highly expressed in seeds.The expression of FAD2 in low oleic acid seeds was significantly higher than that in high oleic acid seeds,and the accumulation of more linoleic acid resulted in the low oleic acid content,which proved that FAD2 was an important gene controlling the oleic acid content in sunflower.The expression patterns of FAB2,fabZ,ACSL,KAR,FATB and fabF were different in seeds with high and low oleic acid content,which indicated that these genes might have different expression regulation mechanisms in seeds with different oleic acid content. |
PDF Full Text Request