Department of Crop and Soils Sciences, North Carolina State University, Raleigh, NC, 27695, USA.
Yunnan Academy of Tobacco Agricultural Sciences, No. 33 Yuantong St., Kunming, 650021, People's Republic of China.
BMC Genomics. 2018 Jun 20;19(1):484. doi: 10.1186/s12864-018-4839-y.
Advances in genomics technologies are making it increasingly feasible to characterize breeding lines that carry traits of agronomic interest. Tobacco germplasm lines that carry loci designated VAM and va have been extensively investigated due to their association with potyvirus resistance (both VAM and va) and defects in leaf surface compounds originating from glandular trichomes (VAM only). Molecular studies and classical genetic analyses are consistent with the model that VAM and va represent deletion mutations in the same chromosomal region. In this study, we used RNA-seq analysis, together with emerging tobacco reference genome sequence information to characterize the genomic regions deleted in tobacco lines containing VAM and va.
Tobacco genotypes TI 1406 (VAM), K326-va and K326 (wild type) were analyzed using RNA-seq to generate a list of genes differentially expressed in TI 1406 and K326-va, versus the K326 control. Candidate genes were localized onto tobacco genome scaffolds and validated as being absent in only VAM, or missing in both VAM and va, through PCR analysis. These results enabled the construction of a map that predicted the relative extent of the VAM and va mutations on the distal end of chromosome 21. The RNA-seq analyses lead to the discovery that members of the cembratrienol synthase gene family are deleted in TI 1406. Transformation of TI 1406 with a cembratrienol synthase cDNA, however, did not recover the leaf chemistry phenotype. Common to both TI 1406 and K326-va was the absence of a gene encoding a specific isoform of a eukaryotic translation initiation factor (eiF4E1.S). Transformation experiments showed that ectopic expression of eiF4E1.S is sufficient to restore potyvirus susceptibility in plants possessing either the va or VAM mutant loci.
We have demonstrated the feasibility of using RNA-seq and emerging whole genome sequence resources in tobacco to characterize the VAM and va deletion mutants. These results lead to the discovery of genes underlying some of the phenotypic traits associated with these historically important loci. Additionally, initial size estimations were made for the deleted regions, and dominant markers were developed that are very close to one of the deletion junctions that defines va.
基因组学技术的进步使得对具有农艺学兴趣的性状的育种系进行特征描述变得越来越可行。由于与马铃薯 Y 病毒抗性（VAM 和 va 均有）和来源于腺毛的叶表面化合物缺陷有关，携带 VAM 和 va 位点的烟草种质系已得到广泛研究（仅 VAM）。分子研究和经典遗传分析与 VAM 和 va 代表同一染色体区域缺失突变的模型一致。在这项研究中，我们使用 RNA-seq 分析，以及新兴的烟草参考基因组序列信息，来描述携带 VAM 和 va 的烟草品系中缺失的基因组区域。
使用 RNA-seq 对 TI 1406（VAM）、K326-va 和 K326（野生型）烟草基因型进行分析，生成 TI 1406 和 K326-va 与 K326 对照相比差异表达的基因列表。通过 PCR 分析将候选基因定位到烟草基因组支架上，并验证仅在 VAM 中缺失，或在 VAM 和 va 中均缺失。这些结果使得构建一个预测 VAM 和 va 突变在 21 号染色体远端相对程度的图谱成为可能。RNA-seq 分析导致发现萜烯合酶基因家族的成员在 TI 1406 中缺失。然而，用萜烯合酶 cDNA 转化 TI 1406 并没有恢复叶化学表型。TI 1406 和 K326-va 共有的是缺失编码真核翻译起始因子（eiF4E1.S）特定同工型的基因。转化实验表明，在外源表达 eiF4E1.S 足以恢复具有 va 或 VAM 突变基因座的植物对马铃薯 Y 病毒的敏感性。
我们已经证明了使用 RNA-seq 和新兴的烟草全基因组序列资源来描述 VAM 和 va 缺失突变体的可行性。这些结果导致发现了与这些历史重要基因座相关的一些表型性状的基因。此外，对缺失区域进行了初步大小估计，并开发了非常接近定义 va 的缺失连接点之一的显性标记。
