Genomic editing of intronic enhancers unveils their role in fine-tuning tissue-specific gene expression in Arabidopsis thaliana

Enhancers located in introns are abundant and play a major role in the regulation of gene expression in mammalian species. By contrast, the functions of intronic enhancers in plants have largely been unexplored and only a handful of plant intronic enhancers have been reported. We performed a genome-wide prediction of intronic enhancers in Arabidopsis thaliana using open chromatin signatures based on DNase I sequencing. We identified 941 candidate intronic enhancers associated with 806 genes in seedling tissue and 1,271 intronic enhancers associated with 1,069 genes in floral tissue. We validated the function of 15 of 21 (71%) of the predicted intronic enhancers in transgenic assays using a reporter gene. We also created deletion lines of three intronic enhancers associated with two different genes using CRISPR/Cas. Deletion of these enhancers, which span key transcription factor binding sites, did not abolish gene expression but caused varying levels of transcriptional repression of their cognate genes. Remarkably, the transcriptional repression of the deletion lines occurred at specific developmental stages and resulted in distinct phenotypic effects on plant morphology and development. Clearly, these three intronic enhancers are important in fine-tuning tissue- and development-specific expression of their cognate genes.

Heat-Shock-Induced Removal of Transgenes Using the Gene-Deletor System in Hybrid Aspen (Populus tremula × P. tremuloides)

To evaluate the efficacy of the gene-deletor system in aspen, we evaluated the system for foreign gene removal in a hybrid aspen clone, INRA 353-53 (Populus tremula × P. tremuloides). The recombinase flipping DNA (FLP) gene was under the control of the heat-inducible promoter of Gmhsp17.6-L, and the β-glucuronidase (gusA) gene which was under the control of the 35S promoter and were constructed using the gene-deletor system in the pCaLFGmFNLFG vector. Six transgenic plants and their sublines were heated at 42 °C for 8 h and gene deletion was verified by polymerase chain reaction (PCR). Three lines exhibited partial transgene deletion while the remaining three lines did not delete. Transgenic lines were evaluated by Southern-blot analyses, verifying that the six transgenic plant lines all had a single copy of transfer DNA (t-DNA). Two partial-deletion lines and two non-deletion lines were analysed for methylation and expression of promoter and recombinase. Hardly any methylation was detected in the Gmhsp17.6-L promoter or recombinase FLP gene sequences, however, the expression of the promoter and recombinase was increased significantly in the partial-deletion compared with the non-deletion line after heat-shock treatment. These results suggest that the excision efficiency had no direct relationship with methylation status of the Gmhsp17.6-L promoter and FLP recombinase, yet was affected by the expression of the Gmhsp17.6-L and FLP after heat-shock treatment.

Production and identification of wheat – Agropyron cristatum (1·4P) alien translocation lines

The P genome of Agropyron Gaertn., a wild relative of wheat, contains an abundance of desirable genes that can be utilized as genetic resources to improve wheat. In this study, wheat – Aegilops cylindrica Host gametocidal chromosome 2C addition lines were crossed with wheat – Agropyron cristatum (L.) Gaertn. disomic addition line accession II-21 with alien recombinant chromosome (1·4)P. We successfully induced wheat – A. cristatum alien chromosomal translocations for the first time. The frequency of translocation in the progeny was 3.75%, which was detected by molecular markers and genomic in situ hybridization (GISH). The translocation chromosomes were identified by dual-color GISH /fluorescence in situ hybridization (FISH). The P genomic DNA was used as probe to detect the (1·4)P chromosome fragment, and pHvG39, pAs1, or pSc119.2 repeated sequences were used as probes to identify wheat translocated chromosomes. The results showed that six types of translocations were identified in the three wheat – A. cristatum alien translocation lines, including the whole arm or terminal portion of a (1·4)P chromosome. The (1·4)P chromosome fragments were translocated to wheat chromosomes 1B, 2B, 5B, and 3D. The breakpoints were located at the centromeres of 1B and 2B, the pericentric locations of 5BS, and the terminals of 5BL and 3DS. In addition, we obtained 12 addition–deletion lines that contained alien A. cristatum chromosome (1·4)P in wheat background. All of these wheat – A. cristatum alien translocation lines and addition–deletion lines would be valuable for identifying A. cristatum chromosome (1·4)P-related genes and providing genetic resources and new germplasm accessions for the genetic improvement of wheat. The specific molecular markers of A. cristatum (1·4)P chromosome have been developed and used to track the (1·4)P chromatin.

Mapping barley Ds insertions using wheat deletion lines reveals high insertion frequencies in gene-rich regions with high to moderate recombination rates

Gene distribution is highly uneven in the large genomes of barley and wheat; however, location, order, and gene density of gene-containing regions are very similar between the two genomes. Flanking sequences from 35 unique, single-copy, barley Ds insertion events were physically mapped using wheat nullisomic-tetrasomic, ditelosomic, and deletion lines. Of the 35 sequences, 23 (66%) detected 34 loci mapping on all 7 homoeologous wheat groups. Seven sequences were not mapped owing to lack of polymorphism and the remaining 5 (14%) were barley-specific. All 34 loci physically mapped to the previously identified gene-rich regions (GRRs) of wheat, making the contained genes candidates for targeted mutagenesis by remobilization. Transpositions occurred preferentially into GRRs with higher recombination rates. The GRRs containing 17 of the 23 Ds insertions accounted for 60%–89% of the respective arm’s recombination. The remaining 6 (17%) insertions mapped to GRRs with <15% of the arm’s recombination. Overall, kb/cM estimates for the Ds-containing GRRs were twofold higher than those for regions without insertions. These results suggest that all genes may be targeted by transposon-based gene cloning, although the transposition frequency for genes present in recombination-poor regions is significantly less than that present in highly recombinogenic regions.