Search

Abstract Background PhasiRNAs (phased secondary siRNAs) play important regulatory roles in the development processes and biotic or abiotic stresses in plants. Some of phasiRNAs involve in the reproductive development in grasses, which include two categories, 21-nt (nucleotide) and 24-nt phasiRNAs. They are triggered by miR2118 and miR2275 respectively, in premeiotic and meiotic anthers of rice, maize and other grass species. Wheat (Triticum aestivum) with three closely related subgenomes (subA, subB and subD), is a model of allopolyploid in plants. Knowledge about the role of phasiRNAs in the inflorescence development of wheat is absent until now, and the evolution of PHAS loci in polyploid plants is also unavailable. Results Using 261 small RNA expression datasets from various tissues, a batch of PHAS (phasiRNA precursors) loci were identified in the young spike of wheat, most of which were regulated by miR2118 and miR2275 in their target site regions. Dissection of PHAS and their trigger miRNAs among the diploid (AA and DD), tetraploid (AABB) and hexaploid (AABBDD) genomes of Triticum indicated that distribution of PHAS loci were dominant randomly in local chromosomes, while miR2118 was dominant only in the subB genome. The diversity of PHAS loci in the three subgenomes of wheat and their progenitor genomes (AA, DD and AABB) suggested that they originated or diverged at least before the occurrence of the tetraploid AABB genome. The positive correlation between the PHAS loci or the trigger miRNAs and the ploidy of genome indicated the expansion of genome was the major drive force for the increase of PHAS loci and their trigger miRNAs in Triticum. In addition, the expression profiles of the PHAS transcripts suggested they responded to abiotic stresses such as cold stress in wheat. Conclusions Altogether, non-coding phasiRNAs are conserved transcriptional regulators that display quick plasticity in Triticum genome. They may be involved in reproductive development and abiotic stress in wheat. It could be referred to molecular research on male reproductive development in Triticum.

Abstract Background Hexaploid bread wheat (Triticum aestivum L) arose by two polyploidisation events from three diploid species with homoeologous genomes. Nullisomic-tetrasomic (nulli-tetra or NT) lines are aneuploid wheat plants lacking two and adding two of six homoeologous chromosomes. These plants can grow normally, but with significantly morphological variations because the adding two chromosomes or the remaining four chromosomes compensate for those absent. Despite these interesting phenomena, detailed molecular mechanisms underlying dosage deletion and compensation in these useful genetic materials have not been determined. Results By sequencing the transcriptomes of leaves in two-week-old seedlings, we showed that the profiles of differentially expressed genes between NT stocks for homoeologous group 7 and the parent hexaploid Chinese Spring (CS) occurred throughout the whole genome with a subgenome and chromosome preference. The deletion effect of nulli-chromosomes was compensated partly by the tetra-chromosomes via the dose level of expressed genes, according to the types of homoeologous genes. The functions of differentially regulated genes primarily focused on carbon metabolic process, photosynthesis process, hormone metabolism, and responding to stimulus, and etc., which might be related to the defective phenotypes that included reductions in plant height, flag leaf length, spikelet number, and kernels per spike. Conclusions The perturbation of the expression levels of transcriptional genes among the NT stocks for homoeologous group 7 demonstrated the gene dosage effect of the subgenome at the genome-wide level. The gene dosage deletion and compensation can be used as a model to elucidate the functions of the subgenomes in modern polyploid plants.

Abstract Background Recently, the CRISPR/Cas9 system has been widely used to precisely edit plant genomes. Due to the difficulty in Agrobacterium-mediated genetic transformation of wheat, the reported applications in CRISPR/Cas9 system were all based on the biolistic transformation. Results In the present study, we efficiently applied targeted mutagenesis in common wheat (Triticum aestivum L.) protoplasts and transgenic T0 plants using the CRISPR/Cas9 system delivered via Agrobacterium tumefaciens. Seven target sites in three genes (Pinb, waxy and DA1) were selected to construct individual expression vectors. The activities of the sgRNAs were evaluated by transforming the constructed vectors into wheat protoplasts. Mutations in the targets were detected by Illumina sequencing. Genome editing, including insertions or deletions at the target sites, was found in the wheat protoplast cells. The highest mutation efficiency was 6.8% in the DA1 gene. The CRISPR/Cas9 binary vector targeting the DA1 gene was then transformed into common wheat plants by Agrobacterium tumefaciens-mediated transformation, resulting in efficient target gene editing in the T0 generation. Thirteen mutant lines were generated, and the mutation efficiency was 54.17%. Mutations were found in the A and B genomes of the transgenic plants but not in the D genome. In addition, off-target mutations were not detected in regions that were highly homologous to the sgRNA sequences. Conclusions Our results showed that our Agrobacterium-mediated CRISPR/Cas9 system can be used for targeted mutations and facilitated wheat genetic improvement.

Abstract Background This study aimed to validate the function of CKX gene on grain numbers in wheat. Methods we constructed and transformed a RNA interference expression vector of TaCKX2.4 in bread wheat line NB1. Southern blotting analysis was used to select transgenic plants with single copy. The expression of TaCKX2.4 gene was estimated by Quantitative real-time PCR (qRT-PCR) analysis. Finally, the relation between expression of TaCKX2.4 gene and grain numbers was validated. Results Totally, 20 positive independent events were obtained. Homozygous lines from 5 events with a single copy of transformed gene each were selected to evaluate the expression of TaCKX2.4 and grain numbers per spike in T3 generation. Compared with the control NB1, the average grain numbers per spike significantly increased by 12.6%, 8.3%, 6.5% and 5.8% in the T3 lines JW39-3A, JW1-2B, JW1-1A and JW5-1A, respectively. Conclusion Our study indicated that the expression level of TaCKX2.4 was negatively correlated with the grain number per spike, indicating that the reduced expression of TaCKX2.4 increased grain numbers per spike in wheat.