Chuan Xia

Optimizing de novo common wheat transcriptome assembly using short-read RNA-Seq data

BackgroundRapid advances in next-generation sequencing methods have provided new opportunities for transcriptome sequencing (RNA-Seq). The unprecedented sequencing depth provided by RNA-Seq makes it a powerful and cost-efficient method for transcriptome study, and it has been widely used in model organisms and non-model organisms to identify and quantify RNA. For non-model organisms lacking well-defined genomes, de novo assembly is typically required for downstream RNA-Seq analyses, including SNP discovery and identification of genes differentially expressed by phenotypes. Although RNA-Seq has been successfully used to sequence many non-model organisms, the results of de novo assembly from short reads can still be improved by using recent bioinformatic developments.ResultsIn this study, we used 212.6 million pair-end reads, which accounted for 16.2 Gb, to assemble the hexaploid wheat transcriptome. Two state-of-the-art assemblers, Trinity and Trans-ABySS, which use the single and multiple k-mer methods, respectively, were used, and the whole de novo assembly process was divided into the following four steps: pre-assembly, merging different samples, removal of redundancy and scaffolding. We documented every detail of these steps and how these steps influenced assembly performance to gain insight into transcriptome assembly from short reads. After optimization, the assembled transcripts were comparable to Sanger-derived ESTs in terms of both continuity and accuracy. We also provided considerable new wheat transcript data to the community.ConclusionsIt is feasible to assemble the hexaploid wheat transcriptome from short reads. Special attention should be paid to dealing with multiple samples to balance the spectrum of expression levels and redundancy. To obtain an accurate overview of RNA profiling, removal of redundancy may be crucial in de novo assembly.

A wheat R2R3-MYB gene, TaMYB30-B, improves drought stress tolerance in transgenic Arabidopsis

The MYB-type proteins are involved in various processes of plant growth, development, and stress response. In a previous work, a polyethylene glycol (PEG) stress-induced gene, TaMYB30, which encodes a R2R3-type MYB protein was identified in wheat. In this study, the isolation and functional characterization of the TaMYB30 gene are reported. Three homologous sequences of TaMYB30 were isolated from hexaploid wheat and designated as TaMYB30-A, TaMYB30-B, and TaMYB30-D genes based on the localizations of these three sequences to chromosomes 2A, 2B, and 2D, respectively. The expression levels of these three genes were similar under PEG stress conditions, and TaMYB30-B was selected for further analysis. The TaMYB30-B protein was localized to the nucleus where it activated transcription. The detailed characterization of Arabidopsis transgenic plants that overexpress the TaMYB30-B gene revealed that the TaMYB30-B protein can improve drought stress tolerance during the germination and the seedling stages. It was also found that overexpression of TaMYB30-B resulted in altered expression levels of some drought stress-responsive genes and changes in several physiological indices, which allow plants to overcome adverse conditions. These results indicate that the TaMYB30-B protein plays important roles in plant stress tolerance, and modification of its expression may improve drought stress tolerance in crop plants.

A novel wheat bZIP transcription factor, TabZIP60, confers multiple abiotic stress tolerances in transgenic Arabidopsis.

The basic region/leucine zipper (bZIP) transcription factors (TFs) play vital roles in the response to abiotic stress. However, little is known about the function of bZIP genes in wheat abiotic stress. In this study, we report the isolation and functional characterization of the TabZIP60 gene. Three homologous genome sequences of TabZIP60 were isolated from hexaploid wheat and mapped to the wheat homoeologous group 6. A subcellular localization analysis indicated that TabZIP60 is a nuclear-localized protein that activates transcription. Furthermore, TabZIP60 gene transcripts were strongly induced by polyethylene glycol, salt, cold and exogenous abscisic acid (ABA) treatments. Further analysis showed that the overexpression of TabZIP60 in Arabidopsis resulted in significantly improved tolerances to drought, salt, freezing stresses and increased plant sensitivity to ABA in seedling growth. Meanwhile, the TabZIP60 was capable of binding ABA-responsive cis-elements that are present in promoters of many known ABA-responsive genes. A subsequent analysis showed that the overexpression of TabZIP60 led to enhanced expression levels of some stress-responsive genes and changes in several physiological parameters. Taken together, these results suggest that TabZIP60 enhances multiple abiotic stresses through the ABA signaling pathway and that modifications of its expression may improve multiple stress tolerances in crop plants.

Characterization of a Wheat R2R3-MYB Transcription Factor Gene, TaMYB19, Involved in Enhanced Abiotic Stresses in Arabidopsis

MYB-type proteins have been shown to participate in multiple stress responses. In the present study, we identified a gene in wheat induced by multiple abiotic stresses, TaMYB19, which encodes a R2R3-type MYB protein. Three highly homologous sequences of TaMYB19 were isolated from hexaploid wheat. Using the nulli-tetrasomic (NT) lines of Chinese Spring wheat, the three sequences were localized to chromosomes 1A, 1B and 1D and designated as TaMYB19-A, TaMYB19-B and TaMYB19-D, respectively. The expression patterns of these three genes were similar under different stress conditions. The TaMYB19-B sequence was selected for further analysis. The TaMYB19-B protein localized to the nucleus. A detailed characterization of Arabidopsis transgenic plants overexpressing the TaMYB19-B gene revealed that the TaMYB19-B protein could improve tolerance to multiple stresses during the seedling stage. We also found that the overexpression of TaMYB19-B resulted in changes in several physiological indices and altered the expression levels of a number of abiotic stress-related genes, allowing the plants to overcome adverse conditions. These results indicate that the TaMYB19 protein plays an important role in plant stress tolerance and that modification of the expression of this protein may improve abiotic stress tolerance in crop plants.

The Novel Wheat Transcription Factor TaNAC47 Enhances Multiple Abiotic Stress Tolerances in Transgenic Plants

NAC transcription factors play diverse roles in plant development and responses to abiotic stresses. However, the biological roles of NAC family members in wheat are not well understood. Here, we reported the isolation and functional characterization of a novel wheat TaNAC47 gene. TaNAC47 encoded protein, localizing in the nucleus, is able to bind to the ABRE cis-element and transactivate transcription in yeast, suggesting that it likely functions as a transcriptional activator. We also showed that TaNAC47 is differentially expressed in different tissues, and its expression was induced by the stress treatments of salt, cold, polyethylene glycol and exogenous abscisic acid. Furthermore, overexpression of TaNAC47 in Arabidopsis resulted in ABA hypersensitivity and enhancing tolerance of transgenic plants to drought, salt, and freezing stresses. Strikingly, overexpression of TaNAC47 was found to activate the expression of downstream genes and change several physiological indices that may enable transgenic plants to overcome unfavorable environments. Taken together, these results uncovered an important role of wheat TaNAC47 gene in response to ABA and abiotic stresses.

Overexpression of a wheat MYB transcription factor gene, TaMYB56-B, enhances tolerances to freezing and salt stresses in transgenic Arabidopsis.

The MYB proteins play central roles in the stress response in plants. Our previous works identified a cold stress-related gene, TaMYB56, which encodes a MYB protein in wheat. In this study, we isolated the sequences of TaMYB56 genes, and mapped them to the wheat chromosomes 3B and 3D. The expression levels of TaMYB56-B and TaMYB56-D were strongly induced by cold stress, but slightly induced by salt stress in wheat. The detailed characterization of the Arabidopsis transgenic plants that overexpress TaMYB56-B revealed that TaMYB56-B is possibly involved in the responses of plant to freezing and salt stresses. The expression of some cold stress-responsive genes, such as DREB1A/CBF3 and COR15a, were found to be elevated in the TaMYB56-B-overexpressing Arabidopsis plants compared to wild-type. These results indicate that TaMYB56-B may act as a regulator in plant stress response.

Characterization and mapping of novel chlorophyll deficient mutant genes in durum wheat

The yellow-green leaf mutant has a non-lethal chlorophyll-deficient mutation that can be exploited in photosynthesis and plant development research. A novel yellow-green mutant derived from Triticum durum var. Cappelli displays a yellow-green leaf color from the seedling stage to the mature stage. Examination of the mutant chloroplasts with transmission electron microscopy revealed that the shape of chloroplast changed, grana stacks in the stroma were highly variable in size and disorganized. The pigment content, including chlorophyll a, chlorophyll b, total chlorophyll and carotene, was decreased in the mutant. In contrast, the chla/chlb ratio of the mutants was increased in comparison with the normal green leaves. We also found a reduction in the photosynthetic rate, fluorescence kinetic parameters and yield-related agronomic traits of the mutant. A genetic analysis revealed that two nuclear recessive genes controlled the expression of this trait. The genes were designated ygld1 and ygld2. Two molecular markers co-segregated with these genes. ygld 1 co-segregated with the SSR marker wmc110 on chromosome 5AL and ygld 2 co-segregated with the SSR marker wmc28 on chromosome 5BL. These results will contribute to the gene cloning and the understanding of the mechanisms underlying chlorophyll metabolism and chloroplast development in wheat.

The wheat transcription factor, TabHLH39, improves tolerance to multiple abiotic stressors in transgenic plants

Although bHLH transcription factors play important roles regulating plant development and abiotic stress response and tolerance, few functional studies have been performed in wheat. In this study, we isolated and characterized a bHLH gene, TabHLH39, from wheat. The TabHLH39 gene is located on wheat chromosome 5DL, and the protein localized to the nucleus and activated transcription. TabHLH39 showed variable expression in roots, stems, leaves, glumes, pistils and stamens and was induced by polyethylene glycol, salt and cold treatments. Further analysis revealed that TabHLH39 overexpression in Arabidopsis significantly enhanced tolerance to drought, salt and freezing stress during the seedling stage, which was also demonstrated by enhanced abiotic stress-response gene expression and changes to several physiological indices. Therefore, TabHLH39 has potential in transgenic breeding applications to improve abiotic stress tolerance in crops.

A TRIM insertion in the promoter of Ms2 causes male sterility in wheat

The male-sterile ms2 mutant has been known for 40 years and has become extremely important in the commercial production of wheat. However, the gene responsible for this phenotype has remained unknown. Here we report the map-based cloning of the Ms2 gene. The Ms2 locus is remarkable in several ways that have implications in basic biology. Beyond having no functional annotation, barely detectable transcription in fertile wild-type wheat plants, and accumulated destructive mutations in Ms2 orthologs, the Ms2 allele in the ms2 mutant has acquired a terminal-repeat retrotransposon in miniature (TRIM) element in its promoter. This TRIM element is responsible for the anther-specific Ms2 activation that confers male sterility. The identification of Ms2 not only unravels the genetic basis of a historically important breeding trait, but also shows an example of how a TRIM element insertion near a gene can contribute to genetic novelty and phenotypic plasticity.

The wheat MYB‐related transcription factor TaMYB72 promotes flowering in rice

Through large-scale transformation analyses, TaMYB72 was identified as a flowering time regulator in wheat. TaMYB72 is a MYB family transcription factor localized to the nucleus. Three TaMYB72 homologs, TaMYB72-A, TaMYB72-B and TaMYB72-D, cloned from hexaploid wheat were mapped to the short arm of the group 6 chromosomes. Under the long-day conditions, over-expression of the TaMYB72 in rice shortened the flowering time by approximately 12 d. Expression analyses suggest that TaMYB72 may function through up-regulation of florigen genes Hd3a and RFT1.