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Transcriptome analyses of variegated leaf development in ornamental kale

ABSTRACT: Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of leaf color at different development stages. The goals of this study are to compare chlorophyll metabolism and chloroplast organization transcriptome profiling (RNA-seq) to microarray and quantitative reverse transcription polymerase chain reaction (qRT–PCR) methods and to evaluate protocols for optimal high-throughput data analysis Methods: leaf mRNA profiles of 12 RNA sequencing libraries (S1, S2, S3_S, and S3_C) were generated by deep sequencing, in triplicate, using an Illumina HiSeq 4000 system. After removing reads of low quality, those that remained were mapped to the reference genome (ftp://ftp.ensemblgenomes.org/pub/release-38/plants/genbank/brassica_oleracea/) using the HISAT package, allowing for a maximum of two mismatches and multiple alignments per read (up to 20 by default). qRT–PCR validation was performed using SYBR Green assays Results: Using an optimized data analysis workflow, we mapped about 571.74 million sequence reads per sample to the the reference genome (ftp://ftp.ensemblgenomes.org/pub/release-38/plants/genbank/brassica_oleracea/) and identified 1028, 4323, 428, and 1033 DEGs were detected in pairwise comparison (S2 vs. S1, S3_S vs. S2, S3_S vs. S2, and S3_S vs. S3_C, respectively). The DEGs were associated with ‘photosynthesis’, ‘carbon fixation in photosynthetic organisms’, ‘porphyrin and chlorophyll metabolism’ and other pathways in the Kyoto Encyclopedia of Genes and Genomes database; DEGs related to chloroplast organization were identified in the Gene Ontology analysis. The DEGs identified by RNA sequencing were confirmed by qRT-PCR analysis, indicating that the data were reliable. These findings provide information that can be useful for investigating the molecular basis for leaf variegation in ornamental kale and other plants. Conclusions: The results presented here reveal changes in the transcriptome profile of a variegated leaf kale. DEGs related to chlorophyll metabolism and chloroplast organization were detected. These results demonstrate that leaf color at different stages of development is influenced by chloroplast and pigment metabolism, providing a foundation for investigating the molecular basis for leaf variegation in ornamental kale and other plants.

ORGANISM(S): Brassica oleracea

PROVIDER: GSE112493 | GEO | 2021/03/27

REPOSITORIES: GEO

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Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of leaf color at different development stages. The goals of this study are to compare anthocyanin biosynthesis, chlorophyll metabolism and chloroplast organization transcriptome profiling (RNA-seq) to microarray and quantitative reverse transcription polymerase chain reaction (qRT–PCR) methods and to evaluate protocols for optimal high-throughput data analysis Methods: Leaf mRNA profiles of 12 RNA sequencing libraries (S1, S2, S3_S, and S3_C) were generated by deep sequencing, in triplicate, using an Illumina HiSeq 4000 system. After removing reads of low quality, those that remained were mapped to the reference genome (ftp://ftp.ensemblgenomes.org/pub/release-38/plants/genbank/brassica_oleracea/) using the HISAT package, allowing for a maximum of two mismatches and multiple alignments per read (up to 20 by default). qRT–PCR validation was performed using SYBR Green assays Results: Using an optimized data analysis workflow, we mapped about 571.74 million sequence reads per sample to the the reference genome (ftp://ftp.ensemblgenomes.org/pub/release-38/plants/genbank/brassica_oleracea/) and identified 99, 391, 74, and 543 DEGs were detected in pairwise comparison (S2 vs. S1, S3_S vs. S2, S3_C vs. S2, and S3_S vs. S3_C, respectively). The DEGs were associated with ‘photosynthesis’and other pathways in the Kyoto Encyclopedia of Genes and Genomes database; DEGs related to chloroplast organization were identified in the Gene Ontology analysis. The DEGs identified by RNA sequencing were confirmed by qRT-PCR analysis, indicating that the data were reliable. These findings provide information that can be useful for investigating the molecular basis for leaf variegation in ornamental kale and other plants. Conclusions: The results presented here reveal changes in the transcriptome profile of a bicolor leaf kale. DEGs related to anthocyanin biosynthesis, chlorophyll metabolism and chloroplast organization were detected. These results demonstrate that leaf color at different stages of development is influenced by anthocyanin biosynthesis, chloroplast and pigment metabolism, providing a foundation for investigating the molecular basis for bicolor leaf in ornamental kale and other plants.

Project description:The leaf of Chinese cabbage is the major place of photosynthesis, the mutation of leaf may directly affect the rate of plant growth and development and the formation of leafy head, and ultimately influence the yield and quality of Chinese cabbage. We identified a developmentally retarded mutant (drm) exhibiting stable inheritance, which was derived from Chinese cabbage DH line âFTâ using a combination of isolated microspore culture and radiation treatment (60Co Î³-rays). The drm exhibited slow growth and development at the seedling and heading stages, leading to the production of a tiny, leafy head, as well as chlorophyll-deficient leaves, especially in seedlings. Genetic analysis indicated that the phenotype of drm was controlled by a single recessive nuclear gene. Compared with wild-type line âFTâ, the drmâs chlorophyll content was significantly reduced and its chloroplast structure was abnormal. Moreover, the photosynthetic efficiency and chlorophyll fluorescence parameters were significantly decreased. The changes in leaf color, combined with these altered physiological characters may influence the growth and development of plant, ultimately resulting in the developmentally retarded phenotype of drm. To further understand the molecular regulatory mechanisms of phenotypic differences between âFTâ and drm, comparative transcriptome analysis were performed using RNA-Seq, a total of 338 differentially expressed genes (DEGs) were detected between âFTâ and drm. According to GO and KEGG pathway analysis, a number of DEGs which involved in the chlorophyll degradation and photosynthesis were identified, such as chlorophyllase and ribulose-1,5-bisphosphate carboxylase/oxygenase. In addition, the expression patterns of 12 DEGs, including three chlorophyll degradation- and photosynthesis-related genes and nine randomly selected genes, were confirmed by qRT-PCR. Numerous single nucleotide polymorphisms were also identified, providing a valuable resource for research and molecular marker-assistant breeding in Chinese cabbage. These results contribute to our understanding of the molecular regulatory mechanisms underlying growth and development and lay the foundation for future genetic and functional genomics studies in Chinese cabbage. The RNA from the third true leaves (day 15 to day 24 after the appearance of the third true leaves) of a developmentally retarded mutant (drm) and its wild type âFTâ in Chinese cabbage were sequenced by RNA-Seq, in triplicate.

Project description:The leaf of Chinese cabbage is the major place of photosynthesis, the mutation of leaf may directly affect the rate of plant growth and development and the formation of leafy head, and ultimately influence the yield and quality of Chinese cabbage. We identified a developmentally retarded mutant (drm) exhibiting stable inheritance, which was derived from Chinese cabbage DH line ‘FT’ using a combination of isolated microspore culture and radiation treatment (60Co γ-rays). The drm exhibited slow growth and development at the seedling and heading stages, leading to the production of a tiny, leafy head, as well as chlorophyll-deficient leaves, especially in seedlings. Genetic analysis indicated that the phenotype of drm was controlled by a single recessive nuclear gene. Compared with wild-type line ‘FT’, the drm’s chlorophyll content was significantly reduced and its chloroplast structure was abnormal. Moreover, the photosynthetic efficiency and chlorophyll fluorescence parameters were significantly decreased. The changes in leaf color, combined with these altered physiological characters may influence the growth and development of plant, ultimately resulting in the developmentally retarded phenotype of drm. To further understand the molecular regulatory mechanisms of phenotypic differences between ‘FT’ and drm, comparative transcriptome analysis were performed using RNA-Seq, a total of 338 differentially expressed genes (DEGs) were detected between ‘FT’ and drm. According to GO and KEGG pathway analysis, a number of DEGs which involved in the chlorophyll degradation and photosynthesis were identified, such as chlorophyllase and ribulose-1,5-bisphosphate carboxylase/oxygenase. In addition, the expression patterns of 12 DEGs, including three chlorophyll degradation- and photosynthesis-related genes and nine randomly selected genes, were confirmed by qRT-PCR. Numerous single nucleotide polymorphisms were also identified, providing a valuable resource for research and molecular marker-assistant breeding in Chinese cabbage. These results contribute to our understanding of the molecular regulatory mechanisms underlying growth and development and lay the foundation for future genetic and functional genomics studies in Chinese cabbage.

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Transcriptome analyses of variegated leaf development in ornamental kale

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