Jianwei Gao

High-throughput sequencing discovery of conserved and novel microRNAs in Chinese cabbage (Brassica rapa L. ssp. pekinensis)

MicroRNAs (miRNAs) are a class of 21–24 nucleotide non-coding RNAs that down-regulate gene expression by cleaving or inhibiting the translation of target gene transcripts. miRNAs have been extensively analyzed in a few model plant species such as Arabidopsis, rice and Populus, and partially investigated in other non-model plant species. However, only a few conserved miRNAs have been identified in Chinese cabbage, a common and economically important crop in Asia. To identify novel and conserved miRNAs in Chinese cabbage (Brassica rapa L. ssp. pekinensis) we constructed a small RNA library. Using high-throughput Solexa sequencing to identify microRNAs we found 11,210 unique sequences belonging to 321 conserved miRNA families and 228 novel miRNAs. We ran a Blast search with these sequences against the Chinese cabbage mRNA database and found 2,308 and 736 potential target genes for 221 conserved and 125 novel miRNAs, respectively. The BlastX search against the Arabidopsis genome and GO analysis suggested most of the targets were involved in plant growth, metabolism, development and stress response. This study provides the first large scale-cloning and characterization of Chinese cabbage miRNAs and their potential targets. These miRNAs add to the growing database of new miRNAs, prompt further study on Chinese cabbage miRNA regulation mechanisms, and help toward a greater understanding of the important roles of miRNAs in Chinese cabbage.

Transcriptome analysis of rosette and folding leaves in Chinese cabbage using high-throughput RNA sequencing.

In this study, we report the first use of RNA-sequencing to gain insight into the wide range of transcriptional events that are associated with leafy head development in Chinese cabbage. We generated 53.5 million sequence reads (90 bp in length) from the rosette and heading leaves. The sequence reads were aligned to the recently sequenced Chiifu genome and were analyzed to measure the gene expression levels, to detect alternative splicing events and novel transcripts, to determine the expression of single nucleotide polymorphisms, and to refine the annotated gene structures. The analysis of the global gene expression pattern suggests two important concepts, which govern leafy head formation. Firstly, some stimuli, such as carbohydrate levels, light intensity and endogenous hormones might play a critical role in regulating the leafy head formation. Secondly, the regulation of transcription factors, protein kinases and calcium may also be involved in this developmental process.

Genome-wide identification and analysis of the growth-regulating factor family in Chinese cabbage (Brassica rapa L. ssp. pekinensis)

BackgroundGrowth regulating factors (GRFs) have been shown to play important roles in plant growth and development. GRF genes represent a large multigene family in plants. Recently, genome-wide structural and evolutionary analyses of the GRF gene families in Arabidopsis, rice, and maize have been reported. Chinese cabbage (Brassica rapa L. ssp. pekinensis) is one of the most important vegetables for agricultural production, and a full genome assembly for this plant has recently been released. However, to our knowledge, the GRF gene family from Chinese cabbage has not been characterized in detail.ResultsIn this study, genome-wide analysis was carried out to identify all the GRF genes in Chinese cabbage. Based on the complete Chinese cabbage genome sequence, 17 nonredundant GRF genes, named BrGRFs, were identified and classified into six groups. Phylogenetic analysis of the translated GRF protein sequences from Chinese cabbage, Arabidopsis, and rice indicated that the Chinese cabbage GRF proteins were more closely related to the GRF proteins of Arabidopsis than to those of rice. Expression profile analysis showed that the BrGRF genes had uneven transcript levels in different organs or tissues, and the transcription of most BrGRF genes was induced by gibberellic acid (GA3) treatment. Additionally, over-expression of BrGRF8 in transgenic Arabidopsis plants increased the sizes of the leaves and other organs by regulation of cell proliferation.ConclusionsThe data obtained from this investigation will contribute to a better understanding of the characteristics of the GRF gene family in Chinese cabbage, and provide a basis for further studies to investigate GRF protein function during development as well as for Chinese cabbage-breeding programs to improve yield and/or head size.

Genome-Wide Identification and Analysis of the VQ Motif-Containing Protein Family in Chinese Cabbage (Brassica rapa L. ssp. Pekinensis)

Previous studies have showed that the VQ motif–containing proteins in Arabidopsis thaliana and Oryza sativa play an important role in plant growth, development, and stress responses. However, little is known about the functions of the VQ genes in Brassica rapa (Chinese cabbage). In this study, we performed genome-wide identification, characterization, and expression analysis of the VQ genes in Chinese cabbage, especially under adverse environment. We identified 57 VQ genes and classified them into seven subgroups (I–VII), which were dispersedly distributed on chromosomes 1 to 10. The expansion of these genes mainly contributed to segmental and tandem duplication. Fifty-four VQ genes contained no introns and 50 VQ proteins were less than 300 amino acids in length. Quantitative real-time PCR showed that the VQ genes were differentially expressed in various tissues and during different abiotic stresses and plant hormone treatments. This study provides a comprehensive overview of Chinese cabbage VQ genes and will benefit the molecular breeding for resistance to stresses and disease, as well as further studies on the biological functions of the VQ proteins.

MicroRNA expression analysis of rosette and folding leaves in Chinese cabbage using high-throughput Solexa sequencing.

In this study, a global analysis of miRNA expression from rosette leaves (RLs) and folding leaves (FLs) of Chinese cabbage (Brassica rapa L. ssp. pekinensis) was conducted using high-throughput Solexa sequencing. In total, over 12 million clean reads were obtained from each library. Sequence analysis identified 64 conserved miRNA families in each leaf type and 104 and 95 novel miRNAs from RLs and FLs, respectively. Among these, 61 conserved miRNAs and 61 novel miRNAs were detected in both types of leaves. Furthermore, six conserved and 21 novel miRNAs were differentially expressed between the two libraries. Target gene annotation suggested that these differentially expressed miRNAs targeted transcription factors, F-box proteins, auxin and Ca(2+) signaling pathway proteins, protein kinases and other proteins that may function in governing leafy head formation. This study advanced our understanding of the important roles of miRNAs in regulating leafy head development in Chinese cabbage.

Characterization and Development of EST-SSRs by Deep Transcriptome Sequencing in Chinese Cabbage (Brassica rapa L. ssp. pekinensis)

Simple sequence repeats (SSRs) are among the most important markers for population analysis and have been widely used in plant genetic mapping and molecular breeding. Expressed sequence tag-SSR (EST-SSR) markers, located in the coding regions, are potentially more efficient for QTL mapping, gene targeting, and marker-assisted breeding. In this study, we investigated 51,694 nonredundant unigenes, assembled from clean reads from deep transcriptome sequencing with a Solexa/Illumina platform, for identification and development of EST-SSRs in Chinese cabbage. In total, 10,420 EST-SSRs with over 12 bp were identified and characterized, among which 2744 EST-SSRs are new and 2317 are known ones showing polymorphism with previously reported SSRs. A total of 7877 PCR primer pairs for 1561 EST-SSR loci were designed, and primer pairs for twenty-four EST-SSRs were selected for primer evaluation. In nineteen EST-SSR loci (79.2%), amplicons were successfully generated with high quality. Seventeen (89.5%) showed polymorphism in twenty-four cultivars of Chinese cabbage. The polymorphic alleles of each polymorphic locus were sequenced, and the results showed that most polymorphisms were due to variations of SSR repeat motifs. The EST-SSRs identified and characterized in this study have important implications for developing new tools for genetics and molecular breeding in Chinese cabbage.

Integrative Analysis of mRNA and miRNA Expression Profiles of the Tuberous Root Development at Seedling Stages in Turnips

The tuberous root of Brassica rapa L. (turnip) is an important modified organ for nutrition storage. A better understanding of the molecular mechanisms involved in the process of tuberous root development is of great value in both economic and biological context. In this study, we analyzed the expression profiles of both mRNAs and miRNAs in tuberous roots at an early stage before cortex splitting (ES), cortex splitting stage (CSS), and secondary root thickening stage (RTS) in turnip based on high-throughput sequencing technology. A large number of differentially expressed genes (DEGs) and several differentially expressed miRNAs (DEMs) were identified. Based on the DEG analysis, we propose that metabolism is the dominant pathway in both tuberous root initiation and secondary thickening process. The plant hormone signal transduction pathway may play a predominant role in regulating tuberous root initiation, while the starch and sucrose metabolism may be more important for the secondary thickening process. These hypotheses were partially supported by sequential DEM analyses. Of all DEMs, miR156a, miR157a, and miR172a exhibited relatively high expression levels, and were differentially expressed in both tuberous root initiation and the secondary thickening process with the expression profiles negatively correlated with those of their target genes. Our results suggest that these miRNAs play important roles in tuberous root development in turnips.

Physiological and Transcriptomic Responses of Chinese Cabbage (Brassica rapa L. ssp. Pekinensis) to Salt Stress

Salt stress is one of the major abiotic stresses that severely impact plant growth and development. In this study, we investigated the physiological and transcriptomic responses of Chinese cabbage “Qingmaye” to salt stress, a main variety in North China. Our results showed that the growth and photosynthesis of Chinese cabbage were significantly inhibited by salt treatment. However, as a glycophyte, Chinese cabbage could cope with high salinity; it could complete an entire life cycle at 100 mM NaCl. The high salt tolerance of Chinese cabbage was achieved by accumulating osmoprotectants and by maintaining higher activity of antioxidant enzymes. Transcriptomic responses were analyzed using the digital gene expression profiling (DGE) technique after 12 h of treatment by 200 mM NaCl. A total of 1235 differentially expressed genes (DEGs) including 740 up- and 495 down-regulated genes were identified. Functional annotation analyses showed that the DEGs were related to signal transduction, osmolyte synthesis, transcription factors, and antioxidant proteins. Taken together, this study contributes to our understanding of the mechanism of salt tolerance in Chinese cabbage and provides valuable information for further improvement of salt tolerance in Chinese cabbage breeding programs.

Comparative Transcriptome Analysis Reveals Effects of Exogenous Hematin on Anthocyanin Biosynthesis during Strawberry Fruit Ripening

Anthocyanin in strawberries has a positive effect on fruit coloration. In this study, the role of exogenous hematin on anthocyanin biosynthesis was investigated. Our result showed that the white stage of strawberries treated with exogenous hematin had higher anthocyanin content, compared to the control group. Among all treatments, 5 μM of hematin was the optimal condition to promote color development. In order to explore the molecular mechanism of fruit coloring regulated by hematin, transcriptomes in the hematin- and non-hematin-treated fruit were analyzed. A large number of differentially expressed genes (DEGs) were identified in regulating anthocyanin synthesis, including the DEGs involved in anthocyanin biosynthesis, hormone signaling transduction, phytochrome signaling, starch and sucrose degradation, and transcriptional pathways. These regulatory networks may play an important role in regulating the color process of strawberries treated with hematin. In summary, exogenous hematin could promote fruit coloring by increasing anthocyanin content in the white stage of strawberries. Furthermore, transcriptome analysis suggests that hematin-promoted fruit coloring occurs through multiple related metabolic pathways, which provides valuable information for regulating fruit color via anthocyanin biosynthesis in strawberries.

Transcriptome Analysis of Orange Head Chinese Cabbage (Brassica rapa L. ssp. pekinensis) and Molecular Marker Development

Due to the visual appearance and high carotenoid content, orange inner leaves are a desirable trait for the Chinese cabbage. To understand the molecular mechanism underlying the formation of orange inner leaves, the BrCRTISO (Bra031539) gene, as the Br-or candidate gene, was analyzed among the white and orange varieties, and 7 single nucleotide polymorphisms (SNPs) were identified. However, only one SNP (C952 to T952) altered the amino acid sequence, resulting in a mutation from Leu318 to Phe318 in the orange varieties. Additionally, we analyzed differentially expressed genes (DEGs) between the orange and white F2 individuals (14-401 × 14-490) and found four downregulated genes were involved in the carotenoid biosynthesis pathway, which may lead to the accumulation of prolycopene and other carotenoid pigments in the orange inner leaves. In addition, we developed a novel InDel marker in the first intron, which cosegregates with the phenotypes of orange color inner leaves. In conclusion, these findings enhance our understanding of the underlying mechanism of pigment accumulation in the inner leaves of the Chinese cabbage. Additionally, the SNP (C952 to T952) and the InDel marker will facilitate the marker-assisted selection during Chinese cabbage breeding.