Sodium azide mutagenesis induces a unique pattern of mutations

Abstract

The nature and effect of mutations are of fundamental importance to the evolutionary process. The generation of mutations with mutagens has also played important roles in genetics. Applications of mutagens include dissecting the genetic basis of trait variation, inducing desirable traits in crops, and understanding the nature of genetic load. Previous studies of sodium azide-induced mutations have reported single nucleotide variants (SNVs) found in individual genes. To characterize the nature of mutations induced by sodium azide, we analyze whole-genome sequencing (WGS) of 11 barley lines derived from sodium azide mutagenesis, where all lines were selected for diminution of plant fitness owing to induced mutations. We contrast observed mutagen-induced variants with those found in standing variation in WGS of 13 barley landraces. Here, we report indels that are two orders of magnitude more abundant than expected based on nominal mutation rates. We found induced SNVs are very specific, with C → T changes occurring in a context followed by another C on the same strand (or the reverse complement). The codons most affected by the mutagen include the sodium azide-specific CC motif (or the reverse complement), resulting in a handful of amino acid changes and few stop codons. The specific nature of induced mutations suggests that mutagens could be chosen based on experimental goals. Sodium azide would not be ideal for gene knockouts but will create many missense mutations with more subtle effects on protein function.

Fig 1. A) The number and percentage of SNVs and indels in mutagenized, untreated rare, and untreated common categories. Private is defined as variants in mutated lines found in a single sample. Untreated are naturally occurring variants with rare variants defined as those with a non-reference allele count ≤ 2, while untreated common variants have alternate allele count ≥ 3. B) The number of SNVs and indels identified in each mutagenized line. Colors indicate whether 10X Genomics linked read technology was utilized for the sample. Dashed lines indicate the expected number of SNPs and indels based on average mutation rates and accounting for experimental design.

Fig 2. The mutational spectrum of mutagenized, untreated rare, and untreated common SNPs.

Each bin includes the reverse complement. For example, the C → T* bin also includes G → A changes.

Fig 3. The distribution of insertion and deletion lengths for sodium azide-treated lines versus rare and common variant categories.

Insertions are shown as positive values and deletions are shown as negative values. Only variants with lengths <20 bp are shown here.

Fig 4. The nucleotide sequence context for C → T transitions relative to the reference genome in the mutated (N = 10,048), untreated rare (N = 439,563), and untreated common (N = 732,565) SNVs.

Position 0 indicates where the C → T change occurred. The relative height of the letters indicates their relative entropy (RE), with a higher RE indicating a position has a greater influence on the mutation. Upright letters indicate overrepresented bases, whereas upside-down letters indicate underrepresented bases at positions neighboring position 0. The null expectation (RE of zero) is based on randomly sampling a nearby location with the same starting base (e.g., for a C → T mutation, a random choice of a position with a C is selected.

Fig 5. Grain yield for 25 mutated lines, the Morex W2017 parent, and 8 check lines.

The box plots are sorted by the median for each line, and the bars in the box plot indicate the mean. Sodium azide-treated lines are represented by red outlines. Red shaded boxes indicate mutated lines that were sequenced in this study.