A Genetic Change Associated with the R Locus in Maize Which Is Directed and Potentially Reversible*
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A GENETIC CHANGE ASSOCIATED WITH THE R LOCUS IN MAIZE WHICH IS DIRECTED AND POTENTIALLY REVERSIBLE* R. ALEXANDER BRINK Department of Genetics, University of Wisconsin, Madison, Wisconsin Received July 12, 1956 NTEGRITY of the gene in heterozygotes, first inferred from MENDEL'S(1865) I Pisum data and repeatedly corhrmed since in a wide variety of organisms, is re- garded as a universal principle in Mendelian heredity. Departures from the law have been reported from time to time, only to prove unfounded, in most instances at least, on close analysis. The present study is concerned with a seeming exception to the rule at the well-known R locus in maize conditioning aleurone and plant color. All the RI c? gametes (self-colored aleurone) produced by RrRStplants (heterozy- gous stippled aleurone) were found to give a different phenotype in testcrosses on rrrr 9 9 (colorless aleurone) than the R' 8 gametes formed by R'R' sibs. The altered form of R' in the Rrrr testcross progeny is transmitted through the succeeding sporo- phyte generation and, therefore, is heritable. The change in Rr is potentially reversible with equal regularity. R' gametes formed by all RrRI plants derived from RrRst heterozygotes by seliing were found to have regained the standard (or near-standard) level of pigment-producing action in test- crosses with rrrr 9 9. The present communication is a preliminary account of this unusual phenomenon. MATERIALS AND METHODS The R' gene used conditions self-colored (purple or red) aleurone when homozygous. The superscript in the symbol (EMERSON,ET AL. 1935) denotes that the allele also produces anthocyanin pigment in vegetative tissues of the plant. The other allele is Rat (stippled), which gives an irregular pattern of mostly small colored spots with sharply defined boundaries in the aleurone, the outer layer of endosperm cells. Aleurone color alone could be scored in the present experiment (see later). The plant color action of the RT and Ratgenes was utilized only to identify R'R', &Rat, and RstRatplants in segregating families at the seedling and flowering stages. RrRr plants have red coleoptiles and anthers, whereas these tissues are green in RatRat individuals. The anthers in the heterozygote, RTRat,are light red, although there is overlap with the R'R' class. In order to improve readability of the text the plant color superscripts will be omitted from the gene symbols except as the complete designations are necessary to make the meaning clear. R, therefore, will be substituted for P,and r' will be represented by r. The R gene in question has been in the writer's cultures for ten years or more, without having disclosed any unusual properties. It is evident from STADLER'S(1951) * Paper No. 630 from the Department of Genetics, College of Agriculture, University of Wis- consin. DIRECTED GENETIC CHANGE IN MAIZE 873 extensive work on this locus, however, that many R alleles of this class are only moderately stable as compared, for example, with most of the commonly occurring representatives of the A1 and C loci, also concerned with anthocyanin pigmentation in maize. The particular R gene used is probably typical in this respect. Adequate tests of stability have not yet been made, but examination of about 14,000 kernels from a population of RR plants of this stock grown in isolation for another purpose revealed seven mottled kernels, indicative of mutations to or toward r, transmitted through the macrospores. These are not definitive data on mutation rate but they demonstrate, at least, that this R allele is not in the highly mutable category. The Rst gene, on the other hand, is relatively unstable, mutating to self-color as frequently, perhaps, as 1 per 100 kernels in the present stock. Here again adequate data are not at hand. The figure given is based on kernel phenotype in the few stippled ears currently available to the writer. Most Rr alleles, including the one in question, give an irregularly broken pattern of aleurone color when present with two r (colorless) genes in the triploid endosperm nuclei. This well-known phenomenon is termed “R-mottling”. The R-mottling in the stock here used is much lighter in RrrCcc than in RrrCCC endosperms. That is to say, the dosage of C affects R-mottling, a fact of obvious importance in scoring kernels for intensity of pigmentation which seems not to have been recorded in theliterature. The dosage effects of R alleles in general on aleurone color have not been studied in detail, but it is apparent that the action of the R gene here used is not simply additive in heterozygotes with r;RRr(Ccc) is much more than twice as heavily pigmented as Rrr(Ccc). Seemingly, therefore, each of the R genes in an RRr(Ccc) aleurone nucleus augments the pigment-producing action of the other. In view of the statement above concerning the modifying action of C on R-mottling, however, this non-linear effect may not be readily discernible in plants segregating both R and C. The R and Rststocks employed were modified forms of a relatively vigorous, fertile, and uniform dent inbred line, known commercially as W22. The initial outcross of W22 (Alcr) to the A1CR source was followed by five backcrosses to W22, before isolating the homozygous self-colored genotype, AlCR, by selfing. The Rs‘gene then was substituted for R in a parallel sub-line. Three backcrosses to the recurrent, near- homozygous, self-colored parent were made in this case. The genetic backgrounds of the R and RSfstrains, therefore, were both closely comparable and relatively homozygous. A simple mating plan was followed. A single RRst plant (Bk 701-1) in the third backcross generation of RRst to W22 A1CR was self-pollinated. The resulting 50 % of self-colored kernels were planted under the family number 45-5, and consisted of the genotypes RR (endosperm, RRR) and RRst (endosperm, RRR“). The mottled kernels were given the pedigree number 45-6, and comprised the genotypes RRst (endosperm, RRstRst)and RstRst(endosperm, PtRstPt). Self-pollinated ears from RtRSt,RRst, and RR plants in the converted W22 inbred stock are illustrated in figure 1. Several plants in each of the three classes of offspring (identified by anther color) were selfed, and pollen of each individual also was applied in turn to three Alcr 874 R. ALEXANDER BRINK .. ._.. FIGUREI.-Self-pollinated ears of homozygous self-colorecl (right), homozygous stippled (left), and the FI RrRnfheterozygote (middle) in the W22 stock. inbred lines. A few additional matings were made by placing pollen from RR and mf plants on REtRnLsibs. Classification of RR and RR", and RslRnLplants, inferred in the field from anther color, was confirmed from the selfed ears obtained in the two families, 45-5 and 4.5-6, also used as the pollen source in the testcrosses on YY. Three unrelated Alcr inbred strains of dent corn, W8, W22, and Ko~.?,were used as pistillate parents in the testcrosses with the W22 RR, RRI, and RafRnfsibs. Com- parable results were obtained in all three sets of matings. Detailed analysis was limited, however, to the -IC063 ears because they gave the fullest complement of seed and the sharpest differentiation of aleurone color. The 4C063 stock was highly inbred, and was supplied originally by DR.G. F. SPRAGUE,Iowa State College, Ames. The use of Y'Y' plants, which have colorless kernels but which form anthocyanin pigment in various vegetative tissues, as pistillate parents in the testcrosses limited observations in this experiment to effects on aleurone phenotype. Another series of testcrosses, using Y"Y" (colorless aleurone, green plant), rather than YY individuals as females, is necessary to determine whether the differential effect of Rr alleles emerg- ing from RrRr and RRnfsibs on aleurone color, which is the subject of this report, extends to plant pigmentation also. DIRECTED GENETIC CHANGE IN MAIZE 87.5 Samples of kernels from a few of the rr Q X RR 8, rr Q X RRst 8, and rr Q X RstRst8 testcross ears then were scored for amount of aleurone pigmentation. A dissecting microscope was used for this purpose which was fitted with a 20 X 20 reticule covering about 12 mm2 at 27X magnification. The kernels were placed on the stage so that the silk scar occupied a given position just outside the edge of the reticule scale. Each of the 400 squares into which the field was thus divided then was scored as "+" if it contained any pigmented aleurone cells and "-"if it was entirely colorless. The sum of the positive scores, divided by 4, was taken as the color index for a kernel. Occasionally the control point on the kernel was shifted slightly to complete the field, and size of field varied somewhat depending upon curvature of the kernel surface. Neither of these factors, however, is a source of bias in the results. This scoring method is tedious, but it provides definitive quantitative data on the question at issue. Progeny tests were necessary to identify the two overlapping classes of kernels resulting from the rr X RRst matings. A few kernels from the testcross ears were chosen whose color indexes approximated the two respective modal values. The plants grown from these kernels were either selfed or pollinated with inbred 4Co63, and pollen from them was applied to the 4Co63 stock (A~cr).Parallel control matings were based on random kernels from the rr X RR and rr X RStPttestcross ears, respectively. EXPERIMENTAL RESULTS Distribution of the kernels in terms of color index from the testcrosses on 4co63 Alcr females of the RR, RRst,and RstRRstsibs in families 45-5 and 45-6 are shown in table 1.