Background
Mutagenesis has been an important part of both basic and applied genetic research. It is a fundamental tool for gene discovery as well as for understanding individual gene function and interaction between genes. By 2004, worldwide more than 2250 cultivars have been released that have been derived either as direct mutants or from their progenies (Ahloowalia et al., 2004). An allelic series for a locus is extremely powerful when analyzed together to understand gene function and to maximize the range of phenotypes that can be observed. The depth and breadth of such an allelic series will depend on mutation spectrum, mutation density and the number of individuals that can be practically screened. In maize and many other plant species (Weil and Monde, 2009; Sikora et al., 2011), EMS (ethyl methanesulfonate) has been the chemical of choice for generating mutagenized populations. However, in species such as maize and Arabidopsis, the mutation spectrum of EMS is limited, with essentially all sequenced EMS-induced mutations being G:C to A:T transitions (Till et al., 2004; Greene et al., 2003). Of the 420 possible amino acid and nonsense interconversions (21 coding/noncoding options x 20 alternatives), 170 can be achieved by single nucleotide changes, and of these, only 29 (17%) are possible through EMS mutagenesis (Perry et al., 2009; Figure 1). Thus, only a fraction of the possible mutational spectrum is surveyed by EMS mutagenesis (Figure 1).

If only amorphic (null) or hypomorphic mutations are of interest, then this limited spectrum is likely not a concern. However, rarer types of mutations such as dominant gain of function (hypermorphic: an increase in normal gene function; antimorphic: acting in opposition to normal gene function; and neomorphic: gaining a new function) may require more specific amino acid substitutions. For example, Yu et al. (2007) listed 8 amino acid changes within the plastidic acetyl-coenzyme A carboxylase (ACCase) that were associated with ACCase-inhibiting herbicide resistant Lolium rigidum populations; none of the eight changes could have been produced by the G:C to A:T change caused by EMS. In order to broaden the induced mutation spectrum in maize, a number of alternative mutagens were tested on seeds, including ENU (N-ethyl-N-nitrosourea), sodium azide and gamma ray irradiation. An assay based on leaf sectors indicating a change in the phenotype at the oil yellow1 (oy1) locus was utilized to produce sequences for mutation spectrum analysis (Figure 2).

 
1 Results
In preliminary experiments with a large number of putative mutagenic agents, three agents (in addition to EMS): ENU, sodium azide and gamma ray irradiation produced leaf sectors with reduced (pale-yellow) or no (white) chlorophyll indicative of mutations produced in the oy1 gene whereas none were observed in the control (no mutagen) plants (Figure 2; Section 3.2). Most of the putative mutagenic agents failed under our conditions to induce leaf sectors (Table 1).

A larger experiment was subsequently conducted with these three agents which included sequence analysis of the oy1 gene. Although the results are informative, resources to complete additional sequencing and thus a more reliable estimate of the mutation spectrum for each mutagen were unavailable.

1.1 EMS
A limited number of EMS-induced sectors (n=10) from the preliminary experiments were sequenced as it has been well-established that its mutation spectrum in maize is almost exclusively G:C to A:T (Till et al., 2004). All ten of the sequenced EMS-induced oy1 sectors contained G:C to A:T mutations (Figure 3).

 
1.2 Sodium Azide
Five percent of plants derived from seeds treated with sodium azide produced sectors (Table 1). Germination was 60% of the control. Sugihara et al. (2013) recently confirmed sodium azide as an effective mutagen in maize; however, no information on the mutation spectrum was presented. In barley (Olsen et al., 1993), sodium azide was shown to produce A:T to G:C (62%), G:C to A:T (24%) and A:T to T:A (14%) changes. Our sequencing results (n=35) in maize are 86% (n=30) G:C to A:T;  11% (n=4) G:C to T:A;  and 3% (n=1) A:T to G:C (Figure 3). Thus in maize, sodium azide does not appear to broaden the mutation spectrum very much relative to EMS. In addition to the single nucleotide substitutions, a 2 bp deletion was observed in one case.

1.3 ENU
ENU has also been shown to induce mutations in maize (Schy and Plewa, 1985), but no sequence information was provided. Although in this experiment ENU had a sectoring frequency about half that of sodium azide and gamma-irradiation (Table 2), in other experiments with different inbred lines, ENU was found to be highly mutagenic, with up to 40% of M₃ families producing visible seedling mutants (unpublished results).

Table 2 Germination and sector rates after mutagen treatment of maize seeds

Given the high germination rate (99% of control), it appears that the DPF inbred background used (Section 3.2) can tolerate a higher concentration of ENU. ENU has been used in many other organisms, with a varying but generally broad mutation spectrum (De Stasio and Dorman, 2001; Weinholds et al., 2003; Van Boxtel et al., 2008). Sequencing results (n=104) in maize show that ENU produced 5 out of the 6 possible types of single base changes (Figure 3); however, the A:T to T:A change predominated (55%; n=57). In contrast to EMS (100%), G:C to A:T changes composed only 8.5% (n=9) of the total. From a total of 104 sequence changes identified, only the G:C to C:G change was not observed.

1.4 Gamma ray irradiation
Although frequently used to induce large deletions or chromosome breaks, gamma ray irradiation, particularly if the dose is kept relatively low (Schwartz et al., 2000), can cause single base changes in plants (Sato et al., 2006; Cho et al., 2010). Nearly six percent of plants derived from seeds treated with gamma ray irradiation produced sectors (Table 2). Given the high germination rate (95% of control), a higher dosage could be considered, although this must be balanced with the expected increase in large deletions (Schwartz et al., 2000). Although only a very limited set was able to be sequenced, 2 of 7 (28%) single base changes identified were G:C to C:G changes, a class which was not observed in the other 3 mutagens tested. In addition to the single nucleotide substitutions, a tandem mutation (CC to AT) was observed in one case.

None of the mutagens tested at the depth we were able to complete produced all six of the possible single nucleotide changes. Consequently, in maize, an optimal mutagenized population may be derived from a treatment consisting of a mixture of mutagens, or a population consisting of sub-populations treated with different mutagens individually.

2 Discussion
As stated previously, the depth and breadth of an allelic series created by mutagenesis will depend on mutation spectrum, mutation density and the number of families that can be practically screened. Tsai et al. (2013) found that in Arabidopsis, autotetraploid Col-0 demonstrated higher tolerance to EMS treatment than its isogenic diploid, resulting in a 4-fold increase in mutation density. Another possibility to increase mutation density is to mutagenize in a background deficient in DNA repair genes (Greene et al., 2003) or mutagen metabolism. The use of next-generation sequencing technology to identify mutations has greatly increased screening throughput (Rigola et al., 2009; Tsai et al., 2011).

It has been shown here that all of the six possible single nucleotide changes can be produced by alternatives to EMS, which greatly expands the possible mutation spectrum in maize. However, this is still only 40% (170/420; Figure 1) of the possible amino acid and nonsense interconversions. Is it possible to expand beyond this? The probability of inducing two or three independent single nucleotide mutations in the same 3 base codon would be extremely rare, and beyond practical use. To break through the ceiling of 170 interconversions possible with single nucleotide changes (Figure 1), mutagens which are potential intra-strand crosslinkers may be useful. DNA intra-strand crosslinkers can induce tandem mutations. The CC to TT (GG to AA on the opposite strand) tandem mutation is induced by UV radiation; moreover, the frequency of this tandem mutation is increased in human cells deficient in DNA repair genes (Shin-Darlak et al., 2005). Although the DNA was exposed to the mutagen in vitro, in acetaldehyde-treated plasmids that were transfected into human cell cultures it was found that 63 percent of the mutations found in the selection gene (supF/nalidixic acid resistance) were tandem (Matsuda et al., 1998). Although these were primarily GG to TT (61%; CC to AA on the opposite strand) changes due to G-G crosslinking, nine other types of tandem mutations were observed at frequencies ranging from 2 to 11 percent. Similar to the results above for UV radiation, the mutation frequency was five times higher in cells which were DNA excision repair-deficient.

It has been stated that “Plant biotechnology is now entering a new phase where random mutagenesis methods, such as EMS mutagenesis and γ-radiation, are being superseded by genome editing technologies that enable precise manipulation of specific genomic sequences (Belhaj et al., 2015).” This is certainly true when it is known what specific gene needs to be mutated, and especially if the specific nucleotide change(s) desired are known. However, these advances in genome engineering technologies do not abolish the need for random mutagenesis as the current knowledge of gene and protein function is far from absolute. Random mutagenesis, and improvements of it, will remain a useful tool going forward.

3 Materials and Methods
3.1 Mutagenesis
EMS (ethyl methanesulfonate; CAS 62-50-0; Sigma-Ald-rich M0880): Dry seeds were soaked for 8 h at room temperature with 0.075 M EMS in water as described in Neuffer (1994).

Sodium azide (CAS 26628-22-8; Sigma-Aldrich S2002): Dry seeds were soaked for 24 h at room temperature with 2 mM sodium azide in 100 mM pH 6.0 sodium phosphate buffer (Teknova #P2060) followed by two rinses with water. This protocol was developed based on Sugihara et al. (2013) and Prina and Favret (1993). ENU (N-ethyl-N-nitrosourea; CAS 759-73-9; Sigma-Aldrich 1 g Isopac N3385): Dry seeds were soaked for 8 h at room temperature with 12.2 mM ENU in 100 mM pH 6.0 sodium phosphate buffer (Teknova #P2060). To avoid potential exposure to ENU powder, 10 mL ethanol was injected into the sealed Isopac with a syringe. Once dissolved, the ethanol-ENU solution was added to 1 L of the buffer. After the treatment, the seeds were rinsed twice with water, and for safer handling, the seeds were subsequently soaked for an additional 2 h in 100 mM pH 8.0 sodium phosphate buffer. The increase in pH from 6.0 to 8.0 dramatically reduces the half-life of ENU (at 25ºC, from 280 min to 7 min; deKok et al., 1983; Tosato et al., 1987; Dennis and Rosencrance, 1995).

Gamma-irradiation: Dry seeds were exposed to a dose of 150 Gy from a cobalt-60 source. Seeds were irradiated at the Radiation Science & Engineering Center, Breazeale Nuclear Reactor, Pennsylvania State University, University Park, PA 16802.

3.2 Oy1 Mutation Assay
To determine the mutagenicity of chemicals and gamma ray irradiation, and to produce sequences for mutation spectrum analysis, an assay was developed based on Becraft et al. (2001) to identify mutations in a specific maize gene, oil yellow1 (oy1; Sawers et al., 2006; Figure 2). Oil yellow1 (Oy1) mutants of maize are deficient in the conversion of protoporphin IX to magnesium protoporphin IX, the first committed step of chlorophyll biosynthesis. Plants heterozygous (Figure 2B: seedling a) for semi-dominant alleles of Oy1 are viable but accumulate a reduced level of chlorophyll and exhibit a yellow-green phenotype intermediate to that of wildtype (Figure 2B: seedling b) and pale-yellow homozygous mutants (Figure 2B: seedling c). If the wildtype allele is disrupted in a yellow-green heterozygote, an albino or pale-yellow sector results (Figure 2C). The semi-dominant allele Oy1-N700 was obtained from the Maize Genetics Cooperation Stock Center (#X27G), and maintained and increased by backcrossing 4 times to Pioneer inbred line DPF. Such seed would segregate 1:1 for heterozygous (Oy1-N700/Oy1) and wildtype (Oy1/Oy1). Batches of this seed (Figure 2A) were treated with various potential mutagens and grown. The wildtype (green) plants were discarded and the heterozygous (yellow-green) plants were observed at flowering for the presence of albino or pale-yellow leaf sectors (Figure 2C).

3.3 Sequencing
Leaf punches of albino or pale-yellow leaf sectors were taken, lyophilized and DNA extracted (Gentra Systems Puregene® DNA Isolation Kit). The wildtype Oy1 allele in inbred DPF does not contain the XmaI/SmaI restriction site (nucleotides (nt) 377-382 Gramene ID: GRMZM2G419806; Figure 2) present in B73-Oy1, W22-Oy1 and Oy1-N700 (Figure 2D). Digestion with XmaI (Thermofisher) was conducted for 16 h at 30ºC prior to PCR amplification in order to specifically amplify the DPF allele and avoid the production of recombinant PCR fragments (Lahr and Katz, 2009). The Oy1 gene was amplified using forward primer 5’-CTCCCCGTCATGGCTTCCACCTT-3’ (nt 324-346; the start codon of Oy1 is underlined) and reverse primer 5’-GAACAAGAGGGTTCCCGC-3’ (nt 1824-1841; 39 bp 3’ of the TAG stop codon), and high-fidelity DNA polymerase Phusion Hotstart II (Thermofisher) for 35 cycles with an annealing temperature of 67.5ºC; other conditions were according to the manufacturer’s recommendation. The resulting  1508 bp fragment was gel purified (Qiagen QIAquick Gel Extraction Kit), ligated into the EcoRV-digested plasmid pLITMUS28i (New England Biolabs), transformed into One Shot™ TOP10 E. coli cells (Invitrogen) and white colonies were selected on LB plates containing 50 μg/mL X–gal and 100 μg/mL ampicillin. Plasmid DNA was isolated (Qiagen QIAprep Spin Miniprep Kit) and inserts were confirmed by EcoRI-HindIII double digests; the specific DPF fragment was confirmed by the lack of XmaI digestion. Inserts were sequenced using four primers: standard M13 forward and reverse primers present on pLITMUS28i, and 2 Oy1-specific primers, Oy1r 5’-GAGCCGATGAGGATGAAGCG-3’ (nt 1290-1309) and Oy1f 5’-ATCCCGCAGCCCGCAGGAAC-3’ (nt 645-665). Three plasmids were sequenced for each entry. The set of sequences produced for each entry were assembled and analyzed in Sequencher® (GeneCodes). Mutations were only counted if present in all three plasmids.

Acknowledgement
The author would like to thank DuPont Pioneer colleagues Stephen Sowinski and Mildred Widmann for lab and field assistance, respectively. In addition, Candace Davison , Gamma Irradiation Outreach Coordinator at the Radiation Science & Engineering Center at Penn State University and the Maize Genetics Cooperation Stock Center for Stock #X27G.

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