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. 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. Our results showed that our Agrobacterium-mediated CRISPR/Cas9 system can be used for targeted mutations and facilitated wheat genetic improvement.

The CRISPR/Cas9 system has been successfully used in hexaploid wheat. Although it has been reported that the induced mutations can be passed to the next generation, gene editing and transmission patterns in later generations still need to be studied. In this study, we demonstrated that the CRISPR/Cas9 system could achieve efficient mutagenesis in five wheat genes via Agrobacterium-mediated transformation of an sgRNA targeting the D genome, an sgRNA targeting both the A and B homologues and three tri-genome guides targeting the editing of all three homologues. High mutation rates and putative homozygous or biallelic mutations were observed in the T0 plants. The targeted mutations could be stably inherited by the next generation, and the editing efficiency of each mutant line increased significantly across generations. The editing types and inheritance of targeted mutagenesis were similar, which were not related to the targeted subgenome number. The presence of Cas9/sgRNA could cause new mutations in subsequent generations, while mutated lines without Cas9/sgRNA could retain the mutation type. Additionally, off-target mutations were not found in sequences that were highly homologous to the selected sgRNA sequences. Overall, the results suggested that CRISPR/Cas9-induced gene editing via Agrobacterium-mediated transformation plays important roles in wheat genome engineering.

Disease resistance genes encoding proteins with nucleotide binding sites and Leucine-Rich Repeat (NB-LRR) domains include many members involved in the effector-triggered immunity pathway in plants. The transcript levels of these defense genes are negatively regulated by diverse microRNAs (miRNAs) in angiosperms and gymnosperms. In wheat, using small RNA expression datasets and degradome datasets, we identified five miRNA families targeting NB-LRR defense genes in monocots, some of which arose in the Triticeae species era. These miRNAs regulate different types of NB-LRR genes, most of them with coil-coiled domains, and trigger the generation of secondary small interfering RNAs (siRNA) as a phased pattern in the target site regions. In addition to acting in response to biotic stresses, they are also responsive to abiotic stresses such as heat, drought, salt, and light stress. Their copy number and expression variation in Triticeae suggest a rapid birth and death frequency. Altogether, non-conserved miRNAs as conserved transcriptional regulators in gymnosperms and angiosperms regulating the disease resistance genes displayed quick plasticity including the variations of sequences, gene copy number, functions, and expression level, which accompanied with NB-LRR genes may be tune-regulated to plants in natural environments with various biotic and abiotic stresses.

Background: Photoperiod and/or thermo-sensitive male sterility is an effective pollination control system in crop two-line hybrid breeding. We previously discovered the spontaneous mutation of a partially male sterile plant and developed a thermo-sensitive genic male sterile (TGMS) line 373S inBrassica napusL. The present study characterized this TGMS line through cytological observation, photoperiod/ temperature treatments, and genetic investigation.Results: Microscopic observation revealed that the condensed cytoplasm and irregular exine of microspores and the abnormal degradation of tapetum are related to pollen abortion. Different temperature and photoperiod treatments in field and growth cabinet conditions indicated that the fertility alteration of 373S was mainly caused by temperature changes. The effects of photoperiod and interaction between temperature and photoperiod were insignificant. The critical temperature leading to fertility alteration ranged from 10 °C (15 °C/5 °C) to 12 °C (17 °C/7 °C), and the temperature-responding stage was coincident with anther development from pollen mother cell formation to meiosis stages. Genetic analysis indicated that the TGMS trait in 373S was controlled by one pair of genes, with male sterility as the recessive. Multiplex PCR analysis revealed that the cytoplasm of 373S ispoltype.Conclusions: Our study suggested that the 373S line inB. napushas a novel thermo-sensitive geneBnms^t1inPolCMS cytoplasm background, and its fertility alteration is mainly caused by temperature changes. Our results will broaden the TGMS resources and lay the foundation for two-line hybrid breeding inB. napus.

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. 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. 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.