1- Department of Plant Molecular Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran
2- Department of Cell & Molecular Biology, Faculty of Life Sciences and Biotechnology, Shahid Beheshti University, Tehran, Iran
2- Department of Cell & Molecular Biology, Faculty of Life Sciences and Biotechnology, Shahid Beheshti University, Tehran, Iran
Abstract: (447 Views)
Extended Abstract
Background: The safflower oilseed plant (Carthamus tinctorius L.) has favorable agronomic and yield characteristics that make it a suitable crop for various practical purposes and achieving sustainable development in the food, energy, and pharmaceutical industries. This plant has various medical, industrial, and nutritional applications. The high quality of the oil (more than 90% unsaturated fatty acids of oleic and linoleic acids), high tolerance to abiotic stresses (cold, salinity, and drought), and the wide range of cultivation have always been of interest. Knowledge of the expression atlas of this plant during its growth and development helps to modify agricultural characteristics, including improving yield, quality, nutritional, and health factors. Due to the good quality of the oil extracted from its seeds, the cultivation of this industrial plant has recently received attention in countries with hot and dry ecosystems. The lack of detailed knowledge about the genome structure and the function of genes is one of the limiting factors to accelerate safflower breeding programs to improve the quality and quantity of this plant. Most safflower NGS studies have been conducted to investigate genetic diversity and identify molecular markers. In this study, the molecular factors involved in the biosynthesis of fatty acids and tocopherol of safflower seeds are considered based on RNA-seq data.
Methods: In this research, the Goldasht variety was selected among the local and commercial safflower varieties available in Iran. This variety has outstanding features, such as high tolerance to salinity and drought, red flowers, spineless, medium height, and large capitula. The seeds were planted in the research farm of the National Institute of Genetic Engineering and Biotechnology in the 2018-2019 cultivation year. The plants were sampled with the daily monitoring of plant growth after the start of reproductive growth. Samples were collected from seed development stages, including seed formation (14 days after flowering), seed filling (28 days after flowering), and physiological ripening (35 days after flowering), to examine the transcriptome of safflower seed development. Each sample consisted of five sub-replicates and two replicates. Total RNA was extracted from the samples using a modified Trizol method. In this study, the BGISEQ-500 platform was used for sequencing and produced 100 bp pair-end reads for each library. FastQC and Trimmomatic software were used to evaluate the quality and clean up the raw reads of each library, respectively. Trinity software was used to assemble the filtered reads of all samples. Differentially Expressed Transcripts (DETs) and their clustering were obtained using the DESeq2 package in R. Functional annotation was performed using Trinotate, gene enrichment was performed using the goseq package in R, and the biochemical pathway was identified using the KEGG database.
Results: An average of 71 million reads was obtained from each library. As a result of reassembling and creating a reference transcriptome, the alignment rate of raw data against the final assembled file was 97.29%. After screening the data, 86,585 transcripts were categorized into 68,809 genes. A total of 16,755 DETs were identified in this analysis. The highest number of transcripts identified with differential expression in pairwise comparisons and during the seed development stages belonged to the transition phase from seed filling to seed maturation, with 10,198 transcripts. Three main patterns of gene expression were evident based on the results of seed transcriptome atlas data analysis. Most genes at 14 days after flowering (DAF) coincided with a rapid increase in oil content during seed development. Genes that were highly expressed at 14 and 28 DAF were related to the formation of metabolites and early stages of oil biosynthesis, including the formation of pyruvate and acetyl-CoA. Moreover, genes related to protein storage peaked at 35 DAF. Genes were identified using five public databases (NR, COG, Swiss-Prot, KEGG, and GO). In the KEGG database, all identified genes were classified into 398 biological pathways and eight functional categories, which were related to fatty acid and tocopherol biosynthesis, with the synthesis of lipid metabolites and ubiquinone during safflower seed growth and development.
Conclusion: The expression profile of the genes involved in the biosynthesis of unsaturated fatty acids showed a higher frequency of their expression in the first stage of seed development. Furthermore, this level of expression was at its lowest level during seed maturation. Studying the expression profile of the genes involved in the metabolic pathway of tocopherol biosynthesis showed a higher frequency of occurrence of these genes in the middle and final stages of seed development, which indicates a clear temporal relationship between oil accumulation and physiological changes in safflower seeds. This study shows that the increased expression of genes related to fatty acid biosynthesis occurs earlier than seed development. Besides, oil accumulation begins before the accumulation of proteins in safflower seeds. Identifying the molecular factors involved in improving plant yield and the identification of the interaction between them as well as with the environment can be a valuable tool for better crop management and breeding activities.
Background: The safflower oilseed plant (Carthamus tinctorius L.) has favorable agronomic and yield characteristics that make it a suitable crop for various practical purposes and achieving sustainable development in the food, energy, and pharmaceutical industries. This plant has various medical, industrial, and nutritional applications. The high quality of the oil (more than 90% unsaturated fatty acids of oleic and linoleic acids), high tolerance to abiotic stresses (cold, salinity, and drought), and the wide range of cultivation have always been of interest. Knowledge of the expression atlas of this plant during its growth and development helps to modify agricultural characteristics, including improving yield, quality, nutritional, and health factors. Due to the good quality of the oil extracted from its seeds, the cultivation of this industrial plant has recently received attention in countries with hot and dry ecosystems. The lack of detailed knowledge about the genome structure and the function of genes is one of the limiting factors to accelerate safflower breeding programs to improve the quality and quantity of this plant. Most safflower NGS studies have been conducted to investigate genetic diversity and identify molecular markers. In this study, the molecular factors involved in the biosynthesis of fatty acids and tocopherol of safflower seeds are considered based on RNA-seq data.
Methods: In this research, the Goldasht variety was selected among the local and commercial safflower varieties available in Iran. This variety has outstanding features, such as high tolerance to salinity and drought, red flowers, spineless, medium height, and large capitula. The seeds were planted in the research farm of the National Institute of Genetic Engineering and Biotechnology in the 2018-2019 cultivation year. The plants were sampled with the daily monitoring of plant growth after the start of reproductive growth. Samples were collected from seed development stages, including seed formation (14 days after flowering), seed filling (28 days after flowering), and physiological ripening (35 days after flowering), to examine the transcriptome of safflower seed development. Each sample consisted of five sub-replicates and two replicates. Total RNA was extracted from the samples using a modified Trizol method. In this study, the BGISEQ-500 platform was used for sequencing and produced 100 bp pair-end reads for each library. FastQC and Trimmomatic software were used to evaluate the quality and clean up the raw reads of each library, respectively. Trinity software was used to assemble the filtered reads of all samples. Differentially Expressed Transcripts (DETs) and their clustering were obtained using the DESeq2 package in R. Functional annotation was performed using Trinotate, gene enrichment was performed using the goseq package in R, and the biochemical pathway was identified using the KEGG database.
Results: An average of 71 million reads was obtained from each library. As a result of reassembling and creating a reference transcriptome, the alignment rate of raw data against the final assembled file was 97.29%. After screening the data, 86,585 transcripts were categorized into 68,809 genes. A total of 16,755 DETs were identified in this analysis. The highest number of transcripts identified with differential expression in pairwise comparisons and during the seed development stages belonged to the transition phase from seed filling to seed maturation, with 10,198 transcripts. Three main patterns of gene expression were evident based on the results of seed transcriptome atlas data analysis. Most genes at 14 days after flowering (DAF) coincided with a rapid increase in oil content during seed development. Genes that were highly expressed at 14 and 28 DAF were related to the formation of metabolites and early stages of oil biosynthesis, including the formation of pyruvate and acetyl-CoA. Moreover, genes related to protein storage peaked at 35 DAF. Genes were identified using five public databases (NR, COG, Swiss-Prot, KEGG, and GO). In the KEGG database, all identified genes were classified into 398 biological pathways and eight functional categories, which were related to fatty acid and tocopherol biosynthesis, with the synthesis of lipid metabolites and ubiquinone during safflower seed growth and development.
Conclusion: The expression profile of the genes involved in the biosynthesis of unsaturated fatty acids showed a higher frequency of their expression in the first stage of seed development. Furthermore, this level of expression was at its lowest level during seed maturation. Studying the expression profile of the genes involved in the metabolic pathway of tocopherol biosynthesis showed a higher frequency of occurrence of these genes in the middle and final stages of seed development, which indicates a clear temporal relationship between oil accumulation and physiological changes in safflower seeds. This study shows that the increased expression of genes related to fatty acid biosynthesis occurs earlier than seed development. Besides, oil accumulation begins before the accumulation of proteins in safflower seeds. Identifying the molecular factors involved in improving plant yield and the identification of the interaction between them as well as with the environment can be a valuable tool for better crop management and breeding activities.
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