Meng-Yao Li

Identification of SSRs and differentially expressed genes in two cultivars of celery (Apium graveolens L.) by deep transcriptome sequencing.

Celery (Apium graveolens L.) is one of the most important and widely grown vegetables in the Apiaceae family. Due to the lack of comprehensive genomic resources, research on celery has mainly utilized physiological and biochemical approaches, rather than molecular biology, to study this crop. Transcriptome sequencing has become an efficient and economic technology for obtaining information on gene expression that can greatly facilitate molecular and genomic studies of species for which a sequenced genome is not available. In the present study, 15 893 516 and 19 818 161 high-quality sequences were obtained by RNA-seq from two celery varieties ‘Ventura’ and ‘Jinnan Shiqin’, respectively. The obtained reads were assembled into 39 584 and 41 740 unigenes with mean lengths of 683 bp and 690 bp, respectively. A total of 1939 simple sequence repeat (SSR) markers were identified in ‘Ventura’ and 2004 SSRs in ‘Jinnan Shiqin’. Di-nucleotide repeats were the most common repeat motif, accounting for 55.49% and 54.84% in ‘Ventura’ and ‘Jinnan Shiqin’, respectively. A comparison of expressed genes between the two libraries, identified 338 differentially expressed genes (DEGs). Three hundred and three of the DEGs were annotated based on a sequence similarity search utilizing eight public databases. Additionally, the expression profile of eight annotated DEGs was characterized in response to abiotic stresses. The collective data generated in the present research represent a valuable resource for further genetic and molecular studies in celery.

Genome-wide analysis of Dof family transcription factors and their responses to abiotic stresses in Chinese cabbage

BackgroundChinese cabbage is an important leaf vegetable that experienced long-term cultivation and artificial selection. Dof (DNA-binding One Zinc Finger) transcription factors, with a highly conserved Dof domain, are members of a major plant-specific transcription factor family that play important roles in many plant biological processes. The Dof family transcription factors, one of the most important families of transcriptional regulators in higher plants, are involved in massive aspects of plant growth, development, and response to abiotic stresses. Our study will supply resources for understanding how Dof transcription factors respond to abiotic stress and the interaction network of these genes in tolerance mechanism.ResultsIn this study, we performed a comprehensive analysis of Dof family factors in Chinese cabbage. In total, 76 genes encoding BraDof family transcription factor were identified from Chinese cabbage, and those BraDof factors were divided into nine classes. Fifteen motifs were found based on Dof amino acid sequence alignments. Chromosome locations and gene duplications of BraDof family genes were also analyzed. Ten duplicate events of BraDof genes were discovered in Chinese cabbage chromosomes. The uneven distribution of BraDof genes in Brassica chromosomes may cause the expansion of BraDof genes. In the Dof family, 37 and 7 orthologous genes were identified between Chinese cabbage and Arabidopsis and between Chinese cabbage and Oryza sativa, respectively. The interaction networks of Dof factors in Chinese cabbage were also constructed. Expression profiles of nine selected genes from different nine classes subjected to four abiotic stresses (cold, heat, salt and drought) were further investigated by quantitative real-time PCR to obtain a better understanding of the functions and regulation mechanisms of BraDof family transcription factors in two Chinese cabbage varieties, ‘Lubaisanhao’ and ‘Qingdao 87-114’.ConclusionsDof-family transcription factors were analyzed in genome of Chinese cabbage. Chromosomal locations showed that duplication might result in expansion. Response to abiotic stresses was elucidated in Chinese cabbage varieties. The results provide novel insights into the stress responses of BraDof genes and promote a better understanding of the construction and function of Dofs in Chinese cabbage.

High throughput sequencing of two celery varieties small RNAs identifies microRNAs involved in temperature stress response

BackgroundMicroRNAs (miRNAs) are small, non-coding RNAs of 20 to 24 nucleotides that regulate gene expression and responses to biotic and abiotic stress. Till now, no reports have previously been published concerning miRNAs in celery.ResultsTwo small RNAs libraries were constructed from two celery varieties, ‘Jinnan Shiqin’ and ‘Ventura’, and characterized by deep sequencing. A total of 431 (418 known and 13 novel) and 346 (341 known and five novel) miRNAs were identified in celery varieties ‘Jinnan Shiqin’ and ‘Ventura’, respectively. Potential miRNA-target genes were predicted and annotated by screening diverse protein databases, including Gene Ontology, Cluster of Orthologous Groups and Kyoto Encyclopedia of Genes and Genomes. Significant differential expression between the two varieties was seen for 221 miRNAs. qRT-PCR was used to analyze the abundance of six miRNAs under cold and heat stress conditions. The results showed that miRNAs may have important functions in controlling temperature stress in celery.ConclusionA large number of miRNAs were identified in celery, and their target genes, functional annotations, and gene expression patterns have been explored.These findings provide the first information on celery miRNAs and enhance understanding of celery miRNA regulatory mechanisms under extreme temperature stress.

Selection of suitable reference genes for qPCR normalization under abiotic stresses in Oenanthe javanica (BI.) DC.

Accurate normalization of gene expression data is an absolute prerequisite to obtain reliable results in qPCR analysis. Oenanthe javanica, an aquatic perennial herb, belongs to the Oenanthe genus in Apiaceae family, with known medicinal properties. In the current study, O. javanica was subjected to hormone stimuli (gibberellin, salicylic acid, methyl jasmonate, and abscisic acid) and abiotic stresses (heat, cold, salt, and drought), and the expression of nine candidate reference genes (eIF-4α, ACT7, TIP41, GAPDH, SAND, EF-1α, PP2A, TBP, and TUB) was evaluated. Stability of the genes was assessed using geNorm, NormFinder and BestKeeper. All the genes presented distinct expression profiles under the experimental conditions analyzed. Under abiotic stress conditions, ACT7 and PP2A genes displayed the maximum stability; PP2A and SAND were the most stable genes under hormone stimuli. Even though PP2A gene was most stable across all the samples, individual analysis revealed changes in expression profile. To further validate the suitability of the reference genes identified in this study, the expression level of M6PR gene under salt treatment was studied. Based on our data, we propose that it is essential to normalize the target gene expression with specific reference genes under different experimental conditions for most accurate results. To our knowledge, this is the first systematic analysis for reference genes under abiotic stress and hormone stimuli conditions in O. javanica. This will be beneficial for future studies on O. javanica and other plants in Apiaceae family at molecular level.

Validation and Comparison of Reference Genes for qPCR Normalization of Celery (Apium graveolens) at Different Development Stages

A suitable reference gene is an important prerequisite for guarantying accurate and reliable results in qPCR analysis. Celery is one of the representative vegetable in Apiaceae and is widely cultivated and consumed in the world. However, no reports have been previously published concerning reference genes in celery. In this study, the expression stabilities of nine candidate reference genes in leaf blade and petiole at different development stages were evaluated using three statistics algorithms geNorm, NormFinder, and BestKeeper. Our results showed that TUB-B, TUB-A, and UBC were the most reference genes among all tested samples. GAPDH represented the maximum stability for most individual sample, while the UBQ displayed the minimum stability. To further validate the stability of reference genes, the expression pattern of AgAP2-2 was calculated by using the selected genes for normalization. In addition, the expression patterns of several development-related genes were studied using the selected reference gene. Our results will be beneficial for further studies on gene transcription in celery.

Genome-wide Analysis of the Distribution of AP2/ERF Transcription Factors Reveals Duplication and Elucidates their Potential Function in Chinese Cabbage ( Brassica rapa ssp. pekinensis )

The AP2/ERF family is one of the largest groups of transcription factors in plants. Members of this family are involved in various biological processes, such as plant development, pathogen defense and stress responses. Here, we identified a total of 289 AP2/ERF transcription factors from Chinese cabbage based on the genome sequence. According to sequence similarity and the number of AP2/ERF domains, the 289 AP2/ERF proteins were classified into five groups (DREB, ERF, AP2, RAV and Soloist). The amino acid sequence, composition, physical and chemical characterization, phylogenetic tree and conserved domain were predicted and analyzed. The analysis results showed that AP2/ERF transcription factors from Chinese cabbage and Arabidopsis share high similarity. The sequences of 286 AP2/ERF genes were distributed unevenly among the ten chromosomes of the Chinese cabbage genome. Three genes could not be assigned to any specific chromosome, and were located on scaffold sequences. Gene duplication events of AP2/ERF family genes exist on Chinese cabbage chromosomes. Eight AP2/ERF genes, chosen from five subfamilies, respectively, were further investigated for their stress response by quantitative RT-PCR analysis. In total, eight genes were expressed differentially during cold or heat stress conditions.

Regulation of ascorbic acid biosynthesis and recycling during root development in carrot (Daucus carota L.).

Ascorbic acid (AsA), also known as vitamin C, is an essential nutrient in fruits and vegetables. The fleshy root of carrot (Daucus carota L.) is a good source of AsA for humans. However, the metabolic pathways and molecular mechanisms involved in the control of AsA content during root development in carrot have not been elucidated. To gain insights into the regulation of AsA accumulation and to identify the key genes involved in the AsA metabolism, we cloned and analyzed the expression of 21 related genes during carrot root development. The results indicate that AsA accumulation in the carrot root is regulated by intricate pathways, of which the l-galactose pathway may be the major pathway for AsA biosynthesis. Transcript levels of the genes encoding l-galactose-1-phosphate phosphatase and l-galactono-1,4-lactone dehydrogenase were strongly correlated with AsA levels during root development. Data from this research may be used to assist breeding for improved nutrition, quality, and stress tolerance in carrots.

Heat shock factors in carrot: genome-wide identification, classification, and expression profiles response to abiotic stress

Heat shock factors (HSFs) play key roles in the response to abiotic stress in eukaryotes. In this study, 35 DcHSFs were identified from carrot (Daucus carota L.) based on the carrot genome database. All 35 DcHSFs were divided into three classes (A, B, and C) according to the structure and phylogenetic relationships of four different plants, namely, Arabidopsis thaliana, Vitis vinifera, Brassica rapa, and Oryza sativa. Comparative analysis of algae, gymnosperms, and angiosperms indicated that the numbers of HSF transcription factors were related to the plant’s evolution. The expression profiles of five DcHsf genes (DcHsf 01, DcHsf 02, DcHsf 09, DcHsf 10, and DcHsf 16), which selected from each subfamily (A, B, and C), were detected by quantitative real-time PCR under abiotic stresses (cold, heat, high salinity, and drought) in two carrot cultivars, D. carota L. cvs. Kurodagosun and Junchuanhong. The expression levels of DcHsfs were markedly increased by heat stress, except that of DcHsf 10, which was down regulated. The expression profiles of different DcHsfs in the same class also differed under various stress treatments. The expression profiles of these DcHsfs were also different in tissues of two carrot cultivars. This study is the first to identify and characterize the DcHSF family transcription factors in plants of Apiaceae using whole-genome analysis. The results of this study provide an in-depth understanding of the DcHSF family transcription factors’ structure, function, and evolution in carrot.

Genomic identification of WRKY transcription factors in carrot (Daucus carota) and analysis of evolution and homologous groups for plants.

WRKY transcription factors belong to one of the largest transcription factor families. These factors possess functions in plant growth and development, signal transduction, and stress response. Here, we identified 95 DcWRKY genes in carrot based on the carrot genomic and transcriptomic data, and divided them into three groups. Phylogenetic analysis of WRKY proteins from carrot and Arabidopsis divided these proteins into seven subgroups. To elucidate the evolution and distribution of WRKY transcription factors in different species, we constructed a schematic of the phylogenetic tree and compared the WRKY family factors among 22 species, which including plants, slime mold and protozoan. An in-depth study was performed to clarify the homologous factor groups of nine divergent taxa in lower and higher plants. Based on the orthologous factors between carrot and Arabidopsis, 38 DcWRKY proteins were calculated to interact with other proteins in the carrot genome. Yeast two-hybrid assay showed that DcWRKY20 can interact with DcMAPK1 and DcMAPK4. The expression patterns of the selected DcWRKY genes based on transcriptome data and qRT-PCR suggested that those selected DcWRKY genes are involved in root development, biotic and abiotic stress response. This comprehensive analysis provides a basis for investigating the evolution and function of WRKY genes.

AP2/ERF Transcription Factors Involved in Response to Tomato Yellow Leaf Curly Virus in Tomato

SlAP2/ERF factors respond to the TYLCV infection. GCC‐box binding ability of SlERF factors was different in tomato cultivars. SlERFs interact with other proteins in tomato. The defense mechanism to TYLCV is a complicated network.