Abstract
Tobacco (Nicotiana tabacum L.) is a complex allotetraploid species with a large 4.5-Gb genome that carries duplicated gene copies. In this study, we describe the development of a whole-exome sequencing (WES) procedure in tobacco and its application to characterize a test population of ethyl methanesulfonate (EMS)-induced mutations. A probe set covering 50.3-Mb protein coding regions was designed from a reference tobacco genome. The EMS-induced mutations in 19 individual M2 lines were analyzed using our mutation analysis pipeline optimized to minimize false positives/negatives. In the target regions, the on-target rate of WES was approximately 75%, and 61,146 mutations were detected in the 19 M2 lines. Most of the mutations (98.8%) were single nucleotide variants, and 95.6% of them were C/G to T/A transitions. The number of mutations detected in the target coding sequences by WES was 93.5% of the mutations detected by whole-genome sequencing (WGS). The amount of sequencing data necessary for efficient mutation detection was significantly lower in WES (11.2 Gb), which is only 6.2% of the required amount in WGS (180 Gb). Thus, WES was almost comparable to WGS in performance but is more cost effective. Therefore, the developed target exome sequencing, which could become a fundamental tool in high-throughput mutation identification, renders the genome-wide analysis of tobacco highly efficient.
Highlights
Tobacco (Nicotiana tabacum) is one of the most widely cultivated non-food crops and is cultivated in more than 125 countries (Kakar et al, 2020)
The quality of the Nitab-v4.5 genome was a significant improvement over the previous K326 genome, more than one third (1.68 Gb) of the scaffold sequences were still not incorporated into the pseudo-chromosomes
These results indicated that the gene structure and their sequences that were predicted using the previous genome sequences were good enough to be transferred to the new release for better sequence accuracy and contiguity
Summary
Tobacco (Nicotiana tabacum) is one of the most widely cultivated non-food crops and is cultivated in more than 125 countries (Kakar et al, 2020). As comprehensive reverse genetic resources, chemically or physically induced mutant libraries are extremely valuable tools for studying gene function as well as for increasing the number of useful alleles in plant breeding (Suprasanna et al, 2015). Tobacco carries a pair of duplicated genes (referred to as homeologs) from the S- and T-genomes. Due to this redundancy, loss-of-function mutations in any single homeolog are typically masked by the correspondent, limiting the use of forward genetic phenotypic screens. Loss-of-function mutations in any single homeolog are typically masked by the correspondent, limiting the use of forward genetic phenotypic screens Notwithstanding, such a characteristic allows tobacco to tolerate high densities of induced mutations, they are hidden though functional ones
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