III-4. Radiation Chemistry & Biology

RIKEN Accel. Prog. Rep. 55 (2022)

RIKEN Accel. Prog. Rep. 55 (2022)

Highly eﬃcient and comprehensive identiﬁcation of ethyl
methanesulfonate-induced mutations in Nicotiana tabacum L. bywhole-genome and whole-exome sequencing†
H. Ichida,∗1 H. Udagawa,∗2 T. Takeuchi,∗2 T. Abe,∗1 and Y. Takakura∗2Tobacco (Nicotiana tabacum L.) is one of the mostwidely cultivated non-food crops. It is a complex allotetraploid (2n = 4x = 48) species with a large genomeof 4.5 Gb, which possesses a high repetitive elementcontent.1) Tobacco carries a pair of duplicated genes(referred to as homeologs) from the S- and T-genomes,
and owing to this redundancy, loss-of-function mutations in any single homeolog are typically masked by
the correspondent, thereby limiting the use of forward
genetic phenotypic screens. Owing to its large genome
size, whole-genome sequencing is not readily applicable, as it requires a large amount of sequencing reads
and computational power and storage. Whole-exome
sequencing (WES) is an approach to primarily sequenceon genomic regions that encode proteins. Because theprotein-coding sequences only considers approximately
1.3% of the tobacco genome, this technique is particularly eﬀective in organisms with large genomes. In
the present study, we developed a set of whole-exon
capturing probes for tobacco and tested the set by analyzing 19 independent mutant lines produced by ethyl
methanesulfonate (EMS) mutagenesis.2,3) We also investigated the optimum conditions to detect mutations
in tobacco.
We employed 19 individual EMS-treated tobaccolines, designated NtEMS-01–19, and a technical replicate of NtEMS-19, called NtEMS-19-rep2, for the sequencing analyses. A commercial target enrichment
platform (SureSelect XT Custom kit, Agilent Technologies) was used to establish a whole-exon enrichment
method for tobacco. The custom-designed capturing kit
contained 517,835 oligonucleotide probes of 120 bases,
and it was expected to capture coding sequences with asize of 50.3 Mb spanning 41,038 genes that had at least
one coding sequence (CDS) deﬁned in the target genes.
After we produced the whole-exome capturing probes,
a new version of the K326 reference genome sequence,
Nitab-v4.5, which signiﬁcantly improves coverage andcontiguity, was published.4) Therefore, we translated
the target locations to the Nitab-v4.5 sequences andperformed all subsequent analysis based on the new reference genome sequence.
The number of read bases varied from 11.6 to 18.9 Gb
per sample, which corresponded to 165.3× to 270.8× of
the total CDS length and indicated that, as expected,
suﬃcient sequencing data were obtained. More than
98% of the reads were mapped to the reference se†∗1∗2quences, resulting in an average read depth of 93.7×
in the CDS regions that commonly existed in the newand previous reference genome assemblies. An averageof 97.1% of the target CDS bases in the 19 NtEMSlines were covered by at least 30 reads. Among all themapped read bases, approximately 75% were locatedon or were adjacent to the target regions, indicatingthat the whole-exome capturing was successfully accomplished here. In the variant calling, we used twodiﬀerent programs, GATK and BcfTools. Each program detected 60,884 and 50,260 mutations and resulted in a total of 61,146 non-redundant mutations.
Almost all (98.8%) of the detected mutations in the19 NtEMS lines were single-nucleotide variations, while
95.6% of them were C/G to T/A transitions, which isconsistent with the known properties of EMS mutagenesis.The numbers of detected mutations varied from 1987
to 4966 in the 19 NtEMS lines. The average numbersof detected mutations in each line were 2715.7 (1987–3857) and 3511.3 (2415–4966) in 0.6% and 0.8% EMS
treatments, respectively. The mutation density waslargely proportional to the target CDS density in thegenome, indicating that the EMS-induced mutations
were suﬃciently random, at least practically, despitebase preferences resulting from the chemical nature ofEMS mutagenesis. A computational experiment using
down-sampled sequencing reads demonstrated that approximately 80% of the mutations located in the target
regions could be detected with 60× sequencing reads.
At 160× of the total CDS length equivalent, a total of 20,960 out of the 35,667 genes located within thetargets harbored at least one mutation in the CDS regions of the 19 NtEMS lines deﬁned by WES. This indicates that an average of 1103 genes could be expected
to have mutated within their CDS regions in the mutagenized NtEMS population that was partly analyzed in
the present study. Based on the probability formula,5)
one can expect at least one mutation from 95 lines (at95% conﬁdence level) in the NtEMS library, for all of
the 35,667 genes in the target regions.
References
1) N. Sierro et al., Nat. Commun. 5, 3833 (2014).
2) T. Tajima et al., Ann. Phytopathol. Soc. Jpn. 77, 258(2011).
3) Y. Takakura et al., Mol. Plant Pathol. 19, 2124 (2018).
4) K. D. Edwards et al., BMC Genom. 18, 448 (2017).
5) L. Clarke, J. Carbon, Cell 9, 91 (1976).

Condensed from the article in Front. Plant Sci. 12, 671598(2021)RIKEN Nishina Center
Leaf Tobacco Research Center, Japan Tobacco Inc.

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