MOESM2 of SNP-based genetic linkage map of tobacco (Nicotiana tabacum L.) using next-generation RAD sequencing

Additional file 2. A flowchart for bioinformatic analysis procedure in this study

Additional file 1: of Characterization of Nicotiana tabacum genotypes possessing deletion mutations that affect potyvirus resistance and the production of trichome exudates

Table S1. Primer sequences. (DOCX 551 kb

Additional file 4: of Characterization of Nicotiana tabacum genotypes possessing deletion mutations that affect potyvirus resistance and the production of trichome exudates

Figure S1. Typical examples of potyvirus infection of TI 1406 and K326-va plants transformed with 35S::eiF4E1.S construct or vector control (VC). Pictures were taken 14 days post-infection with PVYNN or TEV. (DOCX 54 kb

Additional file 2: of Characterization of Nicotiana tabacum genotypes possessing deletion mutations that affect potyvirus resistance and the production of trichome exudates

Table S2. Annotations of genes within scaffolds expected to be part of VAM. (XLSX 10 kb

Additional file 4: Table S3. of Transcriptomic profile of tobacco in response to Tomato zonate spot orthotospovirus infection

Significantly enriched GO terms. (XLSX 80 kb

Additional file 1: Table S1. of Transcriptomic profile of tobacco in response to Tomato zonate spot orthotospovirus infection

Primers used for the validation of DEGs. (DOCX 17 kb

Image_3_Establishment and application of Agrobacterium-delivered CRISPR/Cas9 system for wild tobacco (Nicotiana alata) genome editing.jpeg

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR-Cas9) system has been widely applied in cultivated crops, but limited in their wild relatives. Nicotiana alata is a typical wild species of genus Nicotiana that is globally distributed as a horticultural plant and well-studied as a self-incompatibility model. It also has valuable genes for disease resistance and ornamental traits. However, it lacks an efficient genetic transformation and genome editing system, which hampers its gene function and breeding research. In this study, we developed an optimized hypocotyl-mediated transformation method for CRISPR-Cas9 delivery. The genetic transformation efficiency was significantly improved from approximately 1% to over 80%. We also applied the CRISPR-Cas9 system to target the phytoene desaturase (NalaPDS) gene in N. alata and obtained edited plants with PDS mutations with over 50% editing efficiency. To generate self-compatible N. alata lines, a polycistronic tRNA-gRNA (PTG) strategy was used to target exonic regions of allelic S-RNase genes and generate targeted knockouts simultaneously. We demonstrated that our system is feasible, stable, and high-efficiency for N. alata genome editing. Our study provides a powerful tool for basic research and genetic improvement of N. alata and an example for other wild tobacco species.</p

Table_1_Establishment and application of Agrobacterium-delivered CRISPR/Cas9 system for wild tobacco (Nicotiana alata) genome editing.docx

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR-Cas9) system has been widely applied in cultivated crops, but limited in their wild relatives. Nicotiana alata is a typical wild species of genus Nicotiana that is globally distributed as a horticultural plant and well-studied as a self-incompatibility model. It also has valuable genes for disease resistance and ornamental traits. However, it lacks an efficient genetic transformation and genome editing system, which hampers its gene function and breeding research. In this study, we developed an optimized hypocotyl-mediated transformation method for CRISPR-Cas9 delivery. The genetic transformation efficiency was significantly improved from approximately 1% to over 80%. We also applied the CRISPR-Cas9 system to target the phytoene desaturase (NalaPDS) gene in N. alata and obtained edited plants with PDS mutations with over 50% editing efficiency. To generate self-compatible N. alata lines, a polycistronic tRNA-gRNA (PTG) strategy was used to target exonic regions of allelic S-RNase genes and generate targeted knockouts simultaneously. We demonstrated that our system is feasible, stable, and high-efficiency for N. alata genome editing. Our study provides a powerful tool for basic research and genetic improvement of N. alata and an example for other wild tobacco species.</p

Image_2_Establishment and application of Agrobacterium-delivered CRISPR/Cas9 system for wild tobacco (Nicotiana alata) genome editing.jpeg

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR-Cas9) system has been widely applied in cultivated crops, but limited in their wild relatives. Nicotiana alata is a typical wild species of genus Nicotiana that is globally distributed as a horticultural plant and well-studied as a self-incompatibility model. It also has valuable genes for disease resistance and ornamental traits. However, it lacks an efficient genetic transformation and genome editing system, which hampers its gene function and breeding research. In this study, we developed an optimized hypocotyl-mediated transformation method for CRISPR-Cas9 delivery. The genetic transformation efficiency was significantly improved from approximately 1% to over 80%. We also applied the CRISPR-Cas9 system to target the phytoene desaturase (NalaPDS) gene in N. alata and obtained edited plants with PDS mutations with over 50% editing efficiency. To generate self-compatible N. alata lines, a polycistronic tRNA-gRNA (PTG) strategy was used to target exonic regions of allelic S-RNase genes and generate targeted knockouts simultaneously. We demonstrated that our system is feasible, stable, and high-efficiency for N. alata genome editing. Our study provides a powerful tool for basic research and genetic improvement of N. alata and an example for other wild tobacco species.</p

Image_1_Establishment and application of Agrobacterium-delivered CRISPR/Cas9 system for wild tobacco (Nicotiana alata) genome editing.jpeg

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR-Cas9) system has been widely applied in cultivated crops, but limited in their wild relatives. Nicotiana alata is a typical wild species of genus Nicotiana that is globally distributed as a horticultural plant and well-studied as a self-incompatibility model. It also has valuable genes for disease resistance and ornamental traits. However, it lacks an efficient genetic transformation and genome editing system, which hampers its gene function and breeding research. In this study, we developed an optimized hypocotyl-mediated transformation method for CRISPR-Cas9 delivery. The genetic transformation efficiency was significantly improved from approximately 1% to over 80%. We also applied the CRISPR-Cas9 system to target the phytoene desaturase (NalaPDS) gene in N. alata and obtained edited plants with PDS mutations with over 50% editing efficiency. To generate self-compatible N. alata lines, a polycistronic tRNA-gRNA (PTG) strategy was used to target exonic regions of allelic S-RNase genes and generate targeted knockouts simultaneously. We demonstrated that our system is feasible, stable, and high-efficiency for N. alata genome editing. Our study provides a powerful tool for basic research and genetic improvement of N. alata and an example for other wild tobacco species.</p