Except for the initial studies on the effects of barley stripe mosaic virus (BSMV) in maize in which mutation rates were analyzed (Sprague et al., Science 141:1052-1053, 1963), reports involving putative genetic effects of the virus have dealt with distorted Mendelian ratios. Attempts to formulate genetic mechanisms for which the virus may be responsible from these kinds of data suffer because when off ratios occur, they could be caused by factors which do not directly affect the gene(s) being followed. As discussed in the previous report, elements modifying the transmission rates of whole chromosomes could be responsible. One way to surmount this problem is to screen for mutations at particular loci in the F1 generation of virus-infected plants.
One locus ideally suited for this kind of analysis is Adh. The gene products are identifiable electrophoretically, several alleles exist with different migration rates and, as reported by Schwartz and Osterman (Genetics 85:63-65, 1976) and Freeling and Cheng (Genet. Res. 31:107-130, 1978) Adh-negative mutants and mutants with low levels of Adh expression can be selected by allyl alcohol treatment of pollen.
Attempts to recover virus-induced mutants of Adh were begun in the summer of 1980 in Berkeley. Plants of the stock Adh1-S Adh2-P (also c c, r r, pr pr) from Freeling's lab proved to be quite susceptible to infection by BSMV, strain ND-18. When grown in 6" pots under reduced sunlight in a lathhouse and in the relatively cool temperatures (70-80 F days, 60-65 F nights) of the Berkeley summer, a substantial number of plants expressed viral symptoms in all leaves and shed adequate amounts of pollen.
Pollen from 36 of these plants was treated with allyl alcohol at concentrations which would allow the survival of grains possessing low levels of ADH, and applied to silks of plants homozygous for Adh1-F Adh2-N C R and pr. The Adh2-P, c, r and pr alleles of the male parent served as contamination markers in these crosses as no other plants of that genetic makeup were grown at Berkeley in 1980. To reduce the possibility of stray pollen alighting on silks of the female parent, all pollinations were conducted in an isolation field. Tassels of the virus-treated plants were bagged in the afternoon, placed in a room devoid of other corn and transported to the field the following morning.
Approximately 300 pollinations were made, and it is estimated that about 2,000 grains were applied in each cross. Of 309 kernels on the resulting ears, three were identified as putative mutants. In electrophoretograms of scutellar extracts, two exhibited no Adh1-S band and one showed a low level of S expression. All three, in subsequent analyses with root tissue, proved to contain Adh2-P and also carried the c, r and pr alleles, thus confirming the virus-treated plants as their source. These mutants have been designated Adh1-S-v1, Adh1-S-v2 and Adhl-S-v3. When treated with an ADH-specific stain, the pollen of plants grown from the three mutant kernels all segregated approximately 1:1 for purple and white grains, confirming the presence of the mutant alleles. Samples collected from non-mutant plants of the same cross stained all purple.
Adh1-s-v1, in addition to the Adh null phenotype, also expressed knotted in the F1 plant. In some allelic combinations Kn is less than 0.1 units away from the Adh locus (Freeling, personal communication). Although Kn produces a dominant phenotype, the mutant allele may in fact be a null and the knots due to a dosage effect of the kn gene product. If this is the case, then the Adh1-s-v1 mutant is either a deletion of the two loci or both markers are suppressed.
Of 54 F2 scutella subjected to electrophoretic analysis, 6 showed no ADH band and thus were probably S-v1 null homozygotes. Of the remaining 48, all exhibited only the F band. In an F2 population of 54, 13.5 S-v1 homozygotes are expected. Hence, the deficiency (P = 0.02) may indicate a reduced transmission of this mutant through the gametophyte stage, but more data are needed to confirm this.
Adh1-S-v2 expressed a low level of ADH in the F1 scutellum. Although pollen of the plant from this mutant kernel segregated approximately 1:1 purple and white* grains when stained for ADH activity, expression of the S allele in F2 scutella appeared normal in a population of 83 heterozygotes. The nature of this mutant, whether it be unstable, expressed at different levels in various tissues, or what, awaits further analyses.
In a small sample of F2 kernels, Adh1-S-v3 has exhibited only a null expression. The frequency of S-v3 homozygotes in the F2 is as expected; hence its transmission through the gametophyte stage appears normal.
Freeling (Nature 267:154-156 1977) reported that the spontaneous forward mutation rate for Adh is less than 10-7. Hence it appears safe to conclude that these mutants are of viral origin. Further studies with these variants at both the genetic and molecular level should shed substantial light on the manner in which BSMV is interacting with the maize genome.
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