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Journal of Heredity Advance Access published online on October 30, 2008
Journal of Heredity, doi:10.1093/jhered/esn091
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Published by Oxford University Press 2008.

The Maize enr System of r1 Haplotype–Specific Aleurone Color Enhancement Factors

Philip S. Stinard, Jerry L. Kermicle, and Martin M. Sachs

From the United States Department of Agriculture/Agricultural Research Service, Soybean/Maize Germplasm, Pathology and Genetics Research Unit, Urbana, IL (Stinard and Sachs); Laboratory of Genetics, University of Wisconsin, Madison, WI (Kermicle); and Department of Crop Sciences, University of Illinois, Urbana, IL (Sachs)

Address correspondence to Martin M. Sachs, Maize Genetics Cooperation Stock Center, S-123 Turner Hall, 1102 S. Goodwin Avenue, Urbana, IL 61801, or e-mail: msachs{at}uiuc.edu.

We describe a family of 3 dominant r1 haplotype–specific enhancers of aleurone color in Zea mays. Stable alleles of the 3 enhancement of r1 loci (enr1, enr2, and enr3) intensify aleurone color conferred by certain pale and near-colorless r1 haplotypes. In addition, unstable alleles of enr1 act on the same set of r1 haplotypes, producing spotted kernels. Components of this instability cross react with the Fcu system of instability. Two of the enr loci are linked with one another but none of the 3 are linked with r1. The r1 haplotypes affected by enr alleles overlap those affected by the inr family of r1 haplotype–specific inhibitors of aleurone color, suggesting a possible interaction.

Key Words: dominant color enhancer • epistatic interactions • Fcu • r1 locus • transposable elements • zea mays


   Introduction
Top
 Introduction
Materials and Methods
 Results
 Discussion
 Funding
 References
 
Open-pollinated populations of maize are rife with phenotypic variegations—striped, spotted, and mottled kernels—and variegated plants. In an early genetic analysis, Emerson (1917) described variegated pericarp and cob conditioned by a mutable allele at the p1 locus, P1-vv, that frequently produced full-color germinal revertants. Rhoades (1935, 1938) later described mutability of an allele at the a1 aleurone color locus, a1-m, which was controlled by a second locus, Dt1. In the course of studying chromosome breakage, Barbara McClintock (1950) isolated many examples of mutable alleles and characterized them, providing the first comprehensive description of a transposable element system, Ac/Ds. This insight was promptly applied to P1-vv variegation (Brink and Nilan 1952). Since then, the basis of many naturally occurring instabilities in maize has been attributed to the action of various transposable element systems (En/Spm, Bg, Mutator, and others; for reviews, see Feschotte et al. 2002; Gierl et al. 1989).

In a classical transposable element system, there are 2 kinds of elements: 1) nonautonomous elements, which are not capable of transposition in the absence of an autonomous element and 2) autonomous elements that control the transposition of nonautonomous elements and are generally themselves capable of transposition (Fincham and Sastry 1974). In a typical mutable system, a gene's function is knocked out by the insertion of either an autonomous or a nonautonomous element. In the case of an autonomous element insert, gene function is restored upon the excision of the autonomous element. In the case of a nonautonomous element insert, gene function remains knocked out unless an autonomous element is present to allow excision of the nonautonomous element.

Fcu was identified by Gonella and Peterson (1977) as the factor responsible for aleurone color sectoring at the r1 locus in Cuna tribal maize from Colombia. The Fcu system was described as being comprised of 2 components: a responsive r1 haplotype, r1-cu, and the controlling element Fcu. The r1-cu haplotype produces a variable pale aleurone coloration in the absence of Fcu but produces sectors of full-color pigmentation on a pale background in the presence of Fcu. Two other r1 haplotypes, R1-r(sd2) (spotted dilute2; also referred to as R-r#2; Gonella and Peterson 1978) and R1-mo(cu) (Gonella and Peterson 1976) also responded to Fcu, producing sectors.

Gonella and Peterson (1977) proposed the presence of a nonautonomous element Icu in r1-cu, disrupting the S (seed color) expression of r1, that is either excised or turned off in the presence of the autonomous element Fcu (Figure 1A). They based this conclusion on the isolation of the responsive R1-mo(cu) haplotype, which arose spontaneously from an unresponsive full-color r1 haplotype.


Figure 1
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  		Figure 1. Former and revised models of the interaction between Fcu and r1-cu. (A) r1-cu is R1 with an Icu insert according to the model of Gonella and Peterson (1977). In the presence of transposase produced by Fcu, the Icu element excises from r1-cu, leaving a functional R1 allele. (B)The data presented here indicate that Fcu (now called enr1-Fcu) is a mutable enhancer of r1 aleurone color expression. The mutability could be due to the presence of an autonomous element at the enhancer (enr1) locus. Excision of the transposable element restores Enr1 expression resulting in a colored sector.

 
Fcu was tested against receptor alleles for Dt, En/Spm, and Ac transposable element systems and was found not to elicit a response (Gonella and Peterson 1977). However, when crossed to the receptor allele for the Spf system (Sastry and Kurmi 1970), R1-r(sd2), kernel sectoring was observed (Gonella and Peterson 1978). Subsequently, Spf was found to be a member of the En/Spm system of controlling elements and R1-r(sd2) a typical receptor allele (Stinard and Sachs 2005). On the other hand, Spf elicited no response in r1-cu. The response of R1-r(sd2) to 2 different autonomous elements is contrary to the specific relation of autonomous to receptor loci. The difference in response of r1-cu and R1-r(sd2) to Fcu and Spf was proposed to be due to the presence of an additional function in Fcu not present in Spf (Gonella and Peterson 1978).

Here, we report 2 categories of independently collected variation that prove to be related to the Fcu system. First, 2 sources of instability, initially called arv-m (mutable amplifiers of aleurone color in certain R1-Venezuela haplotypes), elicit aleurone color sectoring in crosses to certain pale Venezuelan r1 haplotypes (Kermicle 2003). Stable aleurone color enhancement factors termed Arv also are present in certain Venezuelan accessions. The locus affected by these factors (r1), similar sectored kernel phenotypes, and their northern South American origin are suggestive of a relationship with Fcu.

A second category of factors is known to enhance aleurone color in crosses to certain near-colorless derivatives of R1-st, such as r1-sc:m6. These stable modifiers, E(Nc) (enhancers of near colorless), have been isolated from open-pollinated maize land races collected from locations as diverse as Turkey, Ethiopia, and Mexico. Although these factors were originally identified as affecting r1 haplotypes unrelated to the Venezuelan accessions, we evaluated these pigment enhancers for behavior parallel to Fcu and Arv in crosses to responsive haplotypes.

In addition to characterizing these new sources of variation and their relation to Fcu, we extend the analysis of Fcu and reinterpret the relationship between its components.

Observations on these diverse materials reveal a family of loci that enhance aleurone color of specific r1 haplotypes, hence the designation enr (enhancement of r1). Under this system, Fcu becomes enr1-Fcu and previously reported arv alleles become enr1 alleles. Factors that map to other chromosomal locations are designated enr2 and enr3, respectively. This report of kernel color enhancement factors is complementary to a previous report of r1 haplotype–specific inhibitors of aleurone color (Stinard and Sachs 2002).


   Materials and Methods
Top
 Introduction
 Materials and Methods
Results
 Discussion
 Funding
 References
 
Genetic Stocks
All stocks are homozygous for alleles at the anthocyanin loci necessary for purple aleurone color (A1 A2 C1 C2 R1) unless otherwise indicated. All stocks have been deposited with the Maize Genetics Cooperation Stock Center, Urbana, IL.

r1 haplotype lines (all in W22-inbred background unless otherwise indicated):

R1-ch(Stadler)—aleurone layer of the kernel mottled to full purple, variable red plant color; confers strong purple pericarp color upon exposure to sunlight or in the presence of the plant color allele Pl1 (Sastry 1970)
R1-d(Catspaw)—purple aleurone, dilute plant color (Stadler 1948; Walker and Panavas 2001)
R1-Randolph—purple aleurone, variable red plant color (Stinard and Sachs 2002). Pale aleurone lines of R1-Randolph carrying the inhibitors Inr1-JD, Inr1-ref, or Inr2-JD in W23-inbred background were also used in this study.
R1-r(Venezuela412-PI302347), R1-r(Venezuela559-PI302355), R1-r(Venezuela594-PI302363), and R1-r(Venezuela 694#16037)—pale aleurone, red plant color (Van Der Walt and Brink 1969)
R1-r(spotted dilute2) [R1-r(sd2)] with Inr1-Dil—purple aleurone in absence of the color inhibitor Inr1-Dil; pale aleurone in presence of Inr1-Dil; red plant color (Sastry and Kurmi 1970; Stinard and Sachs 2005)
R1-sc:124—full-color derivative of R1-st; purple aleurone, green plant color (McWhirter and Brink 1962; Eggleston et al. 1995)
R1-r(Std)—purple aleurone, red plant color (Bray and Brink 1966; Walker et al. 1995)
r1-cu—variable pale aleurone, red plant color (Gonella and Peterson 1977)
r1-sc:m6—a near-colorless aleurone derivative of R1-sc:134 that carries a single functional Nc (Near colorless) gene and has its main seed color component, Sc, inactivated by a Ds transposable element insertion (Eggleston et al. 1995)
r1-g(Stadler)—colorless aleurone, green plant color (Kermicle 1984)
r1-g(Nc)3-5—a near-colorless aleurone derivative of R1-st that carries an Sc component inactivated by a transposition-defective I-R element and likely carries 3 Nc genes (Kermicle 1984; Eggleston et al. 1995)
R1-g:8:pale—a pale derivative of R1-g:8, which in turn is a green plant color derivative of R1-r(Std) (Bray and Brink 1966; Kermicle 1984)

Aleurone color enhancement lines (all in W22-inbred background except for enr1-Fcu and its revertants):

enr1-Fcu—a mutable enhancement factor (isolate PAP74-1033-8) received from Peter Peterson of Iowa State University (Gonella and Peterson 1977)
Enr1-Fcu-R2003-2653-2, Enr1-Fcu-R2003-2653-6, Enr1-Fcu-R2004-947-5, and Enr1-Fcu-R2004-947-10—full-color revertants of enr1-Fcu isolated as described below
enr1-m594—a mutable enhancement factor isolated from Van Der Walt and Brink's (1969) R1-r(Venezuela594-PI302363) line
Enr1-m594-R5874, Enr1-m594-R6117b, Enr1-m594-R6117c, and Enr1-m594-R6117d—full-color revertants of enr1-m594 isolated as described below
enr1-m694—a mutable enhancement factor isolated from Van Der Walt and Brink's (1969) R1-r(Venezuela694#16037) line
Enr1-m694-R6118a—full-color revertant of enr1-m694 isolated as described below
Enr1-628—a full-color enhancement factor isolated from Van Der Walt and Brink's (1969) R1-r(Venezuela628#16038) line
Enr1-ZC—a full-color enhancement factor isolated as an enhancer of Nc aleurone color from the land race Zapalote Chico
Enr2-6117a—a near full-color enhancement factor linked to but distinct from enr1, isolated from an enr1-m594 line as described below
Enr2-694—a full-color enhancement factor isolated from Van Der Walt and Brink's (1969) R1-r(Venezuela694#16037) line
Enr3-594—a full-color enhancement factor unlinked to enr1 and enr2, isolated from Van Der Walt and Brink's (1969) R1-r(Venezuela594-PI302363) line

Linkage stocks (heterogeneous background):

wx1-marked translocation lines—obtained from the Maize Genetics Stock Center. These lines are generally colorless aleurone (c1 r1).
R1-r(sd2) Inr1-Dil wx1—tester for wx1-marked translocation linkage tests that carries an enr1-responsive r1 haplotype (R1-r(sd2)), a background color suppressor (Inr1-Dil), and a recessive wx1 allele.
fl1 v4 w3 Ch1—chromosome 2 linkage stock from Maize Genetics Stock Center.

r1 Haplotype Response Tests
Tests of response of r1 haplotypes to unstable enhancement factors were made by crossing homozygous r1 haplotype lines as females by enhancement factor lines homozygous for the colorless aleurone variant r1-g. The resulting F1 ears were scored for the presence of kernel sectoring.

Mapping enr1-Fcu and enr1-m594 Using wx1-Marked Reciprocal A–A Translocations
Mapping through the use of wx1 marked reciprocal A-A translocations was performed as described by Patterson (1994), with the following modifications: Lines carrying enr1-Fcu and enr1-m594 were crossed to a series of wx1 marked A-A translocations homozygous for r1 and lacking color modifiers, and plants grown from the resulting F1s were backcrossed as females to a line homozygous for R1-r(sd2), Inr1-Dil, and wx1. R1-r(sd2) is a reporter allele for the status of enr1, and Inr1-Dil suppresses background aleurone color in R1-r(sd2), allowing enr1 sectoring to be visualized more readily. The resulting kernels were scored for the presence/absence of the "waxy" trait and for aleurone color sectoring. The results were tabulated, and linkage values were calculated according to Coe (1994).

Five-Point Linkage Test of enr1-Fcu with fl1, v4, w3, and Ch1
A line homozygous for enr1-Fcu was crossed by a line homozygous for fl1, v4, and Ch1 and segregating for the recessive lethal w3. Progeny plants were self-pollinated and outcrossed to the enr1-responsive r1 haplotype R1-r(Venezuela559-PI302355). Only those outcrosses of plants carrying the factor w3 were advanced to the next generation. Kernels from outcross ears were separated into spotted (enr1-Fcu) and nonspotted (enr1) classes, planted in the field, and mature plants were self-pollinated. The resulting ears were scored for the presence of fl1, w3, and Ch1, and the presence/absence of aleurone color sectoring was used to confirm enr1 status. Samples from each ear consisting of fifty kernels were planted in sand benches under cool conditions (Demerec 1924) and the resulting seedlings were scored for the presence of v4. The results were tabulated and linkage values were calculated according to Coe (1994).

Four-Point Linkage Test of enr1-m594 and Enr2–6117a with fl1 and v4
A line carrying enr1-m594 and Enr2-6117a linked in coupling was crossed by a line homozygous for fl1, v4, and the responsive r1 haplotype R1-r(Venezuela559-PI302355). Plants grown from full-colored (Enr2) kernels of this cross were backcrossed by the homozygous fl1 v4 R1-r(Venezuela559-PI302355) line. Kernels from backcross ears carrying enr1-m594 and Enr2-6117a were separated into full-color (Enr2), spotted (enr1-m), and pale (enr1 enr2) classes, and kernels within each class were separated into floury (fl) and nonfloury (Fl) classes. All kernels from each class were planted in sand benches under cool conditions and the resulting seedlings were scored for virescent (v) versus green (V). Only partial classification was possible within the Enr2 class because intense coloration obscured scoring for enr1 versus enr1-m594. The results were tabulated, and linkage values were calculated according to Coe (1994).

Direct Pairwise Linkage Tests between Individual Factors
To directly map enr factors with respect to each other, we made F1s between lines homozygous for the individual factors and then crossed the F1s as male onto the responsive haplotype R1-r(Venezuela559-PI302355) unless otherwise noted. In the case of 2 mutable factors, both parental classes and the double-recombinant class would be expected to produce sectored kernels; in the case of 2 stable factors, both parental classes and the double-recombinant class would be expected to produced full-colored kernels; and in the case of one mutable and one stable factor, one parental class would produce sectored kernels, one parental class would produce full-colored kernels, and the double-recombinant class would produce full-colored kernels. However, in all 3 cases, the recombinant class lacking both factors would be expected to be stable (nonsectored) pale. Because the class lacking both factors is the only recombinant class that can be identified phenotypically, this class was used in calculating linkage values.

The kernels on the backcross ears were scored for presence or absence of aleurone color sectoring. Exceptional stable pale kernels were planted the following season, and the resulting plants self-pollinated and outcrossed to R1-r(Venezuela559-PI302355) testers to confirm genotypes. The number of confirmed stable pale kernels was doubled to account for the undetected 2-factor crossover class. Population sizes were reduced by the proportion of exceptional kernels that could not be tested in order to arrive at an effective population size for the purpose of linkage calculations. Linkage values were calculated according to Coe (1994).

Isolation of Germinal Revertants
No germinal revertants from the enr1-Fcu system have been reported (Gonella and Peterson 1978). In order to isolate enr1-Fcu revertants for further study, we made crosses of an r1-g enr1-Fcu line onto the responsive pale r1 haplotype, R1-r(Venezuela559-PI302355). All full-colored putative revertant kernels were planted, and the resulting plants were self-pollinated and outcrossed to R1-r(Venezuela559-PI302355) testers in order to test for heritability and verify the presence of contamination markers. Two heritable revertants were tested for linkage with enr1-Fcu, and effective population sizes and linkage values were calculated as described above for direct pairwise linkage tests between individual factors.

Germinal revertants of enr1-m594 were isolated in the following manner: Plants homozygous for enr1-m594 and the responsive haplotype R1-r(Venezuela 594-PI302363) were crossed as females by a homozygous r1-g(Stadler) tester. Full-colored putative germinal revertants were tested for heritability and for linkage with enr1-m594.

Similarly, germinal revertants of enr1-m694 were isolated by crossing a line homozygous for enr1-m694 and the responsive haplotype R1-r(Venezuela 694#16037) by a homozygous r1-g(Stadler) tester. Full-colored putative germinal revertants were tested for heritability and for linkage with an enr1-m594 revertant.


   Results
Top
 Introduction
 Materials and Methods
 Results
Discussion
 Funding
 References
 
r1 Haplotype Response Patterns
Crosses of enr1-Fcu to the enr1-reporter haplotype r1-cu elicit in F1 kernels a small number of dark, irregularly shaped, small to medium sized sectors on a pale background (Gonella and Peterson 1977). The enr1-Fcu stock elicited a similar pattern of sectoring when crossed as male onto the following full aleurone color r1 haplotypes: R1-ch(Stadler) (illustrated in Figure 2), R1-d(Catspaw), and R1-Randolph. enr1-Fcu also induced sectoring in crosses to pale aleurone color haplotypes R1-r(Venezuela412-PI302347) and R1-r(Venezuela559-PI302355) as well as in crosses to R1-Randolph lines that have pale or colorless aleurone due to the presence of the r1 haplotype–specific aleurone color inhibitor alleles Inr1-ref, Inr1-JD, or Inr2-JD; and R1-r(sd2) in the presence of the aleurone color inhibitor Inr1-Dil.


Figure 2
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  		Figure 2. Cross of r1-g enr1-Fcu Inr1-Fcu onto R1-ch(Stadler).

 
The sectoring in crosses to the full aleurone color haplotypes R1-ch(Stadler), R1-d(Catspaw), and R1-Randolph appeared on a background of paler aleurone color. It was subsequently determined that the enr1-Fcu line obtained from P.A. Peterson carries a dominant inhibitory allele at the inr1 locus, Inr1-Fcu, that suppresses color in these particular haplotypes (Stinard 2004). The inhibitor lightens background color, allowing Enr somatic sectors to appear with greater contrast against a pale background. Although sectoring can still be observed, it is much more difficult to visualize in crosses to these same haplotypes without inhibitors present.

The haplotype r1-sc:m6 produced pale sectors on a near-colorless background in response to enr1-Fcu.

The following haplotypes gave no response to enr1-Fcu: R1-r(Std), R1-sc:124, r1-g(Nc)3-5, R1-g:8:pale, and r1-g(Stadler) (a colorless control).

enr1-m594 gave the same response pattern as enr1-Fcu in crosses to all of the above r1 haplotypes, except for R1-d(Catspaw), which was not tested. enr1-m694 elicited sectoring of R1-r(Venezuela559-PI302355), R1-r(Venezuela 594-PI302363), and R1-d(Catspaw), but was not tested against other haplotypes. Thus, none of the r1 haplotypes tested differentiated between enr1-Fcu, enr1-m594, and enr1-m694.

Mapping Studies
wx1-Marked Translocation Linkage Data
To further characterize the similarities and differences between enr1-Fcu and enr1-m factors, enr1-Fcu and enr1-m594 were mapped to chromosome region using a series of wx1 marked A-A translocations. Mapping crosses involving the translocations T1-9c, T1-9(4995), T1-9(8389), T3-9(8447), T3-9(8562), T4-9e, T4-9(5657), T5-9c, T5-9a, T6-9b, T7-9(4363), T7-9a, T8-9d, T9-10b, and T9-10(8630) failed to show linkage between wx1 and enr1-Fcu (data not shown). Mapping crosses involving the translocations T1-9c, T1-9(5622), T1-9(8389), T3-9(8447), T3-9(8562), T4-9e, T4-9(5657), T5-9(022-11), T5-9a, T6-9e, T7-9(4363), T7-9a, T8-9d, T9-10(059-10), and T9-10b failed to show linkage between wx1 and enr1-m594 (data not shown). However, mapping crosses involving T2-9c, T2-9b, and T2-9d showed linkage of wx1 with both enr1-Fcu (Table 1) and enr1-m594 (Table 2).


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  		Table 1. Two-point linkage data for wx1 and enr1-Fcu in crosses involving T2-9 translocations

 


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  		Table 2. Two-point linkage data for wx1 and enr1-m594 in crosses involving T2-9 translocations

 
Both enr1-Fcu and enr1-m594 show the same general pattern of linkage with wx1 in the T2-9 translocations (Tables 1 and 2). It should be noted that the T2-9c data show distortions in the ratios of waxy to nonwaxy kernels indicative of the transmission of duplicate-deficient chromosomal segments through the female. Linkage was tightest with T2-9d (2L.83; 9L.27; 13.6; and 9.4 cM, respectively) and weakest with T2-9c (2S.49; 9S.33; 39.3; and 30.8 cM, respectively), indicating that both spotting factors probably reside on the long arm of chromosome 2. However, the linkage values differ for the 2 factors, all linkage values lying well outside each other's standard errors in side-by-side comparisons. Because these factors were subsequently found to map to the same chromosomal location, the differences are most likely due to differences in genetic background between the lines, which can affect linkage values (Patterson 1952).

Mapping data for wx1 T2-9d with respect to enr1-m694 (16.0 ± 1.5 cM, n = 587), the enr1-Fcu revertant Enr1-Fcu-R2003-2653-6 (11.5 ± 1.7 cM, n = 348), and the full-color Venezuelan enhancement factor Enr1-628 (15.0 ± 1.2 cM, n = 842) were also collected (data not shown) and are consistent with placement of these factors on 2L.

Five-Point Linkage Data for enr1-Fcu with Respect to the Chromosome 2 Markers fl1, v4, w3, and Ch1
In order to further refine the position of enr1 on 2L, we conducted a 5-point linkage test of enr1-Fcu with the chromosome 2 markers fl1, v4, w3, and Ch1. The linkage testcross and the results are presented in Table 3. The following linkage order and distances (in centiMorgans) were established:

Formula


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  		Table 3. Five-point linkage data for fl1 v4 enr1 w3 ch1

 
The linkage values for fl1, v4, w3, and ch1 are close to those previously reported on the 1993 genetic map of chromosome 2 (Neuffer et al. 1997), fl1 – 15 – v4 – 28 – w3 – 44 – ch1, there being a slight discrepancy in the v4 – w3 distance (22.5 cM, our data, vs. 28 cM, genetic map). However, individually reported linkage data from Robertson (1961; 22.7 cM, n = 304; and 23.9, n = 376) and Patterson et al. (1968; 24 cM, n = 71) are in close agreement with our data.

Direct Mapping between Factors
To determine whether enr1-Fcu, enr1-m594, and other related factors reside at the same chromosome 2 locus, the stocks were intercrossed pairwise and the F1 plants testcrossed to an r1-responsive enr strain. Heritably pale progeny kernels indicate recombination between the 2 parental factors. The results are summarized in Table 4.


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  		Table 4. Results of mapping among mutable and stable factors

 
enr1-Fcu with enr1-m594
Thirteen stable pale putative crossover kernels were recovered from a population of 3223 kernels. Subsequent testing revealed that these kernels were parental types. Thus, there were no crossovers in a population of 3223 kernels, indicating a separation of less than 0.06 ± 0.04 cM between enr1-Fcu and enr1-m594.

enr1-Fcu with enr1-m694
Eight stable pale kernels were recovered from a population of 2372 kernels. Upon further testing, 7 of the stable kernels proved not to be crossovers and 1 did not survive to pollination and could not be tested. Based on our inability to recover crossovers from an effective population of 2076 kernels, we calculate that these 2 factors are separated by less than 0.10 ± 0.07 cM.

enr1-m594 with enr1-m694
Four stable pale kernels were recovered from a population of 1620 kernels. Upon further testing, 2 of the stable kernels proved to be truly stable (not carrying a mutable factor). Thus, we calculate a map distance of 0.25 ± 0.12 cM between these 2 factors. We could not rule out these stable kernels being the result of self-contamination by the R1-r(Venezuela559-PI302355) tester used as the female parent in the testcrosses, but the plants grown from the exceptional kernels appeared to be more vigorous than the tester, which is highly inbred. We also cannot rule out the possibility that the exceptional kernels represent stable derivatives of the mutable factors and not crossovers.

From a testcross population of 2958 kernels in which the F1 was crossed as a female, one stable pale kernel was recovered. It and its progeny were very weakly pigmented, indicating a change at r1 rather than at enr1. This test reveals a separation of less than 0.07 ± 0.05 cM.

enr1-m594 with Enr1-628
Thirty-seven pale stable kernels were recovered from a population of 7475 kernels. Upon further testing, 6 proved to carry Enr1-628 and 21 carried enr1-m594 and were thus members of parental classes and not recombinants. Ten kernels did not germinate, giving an effective population of 5455 kernels. Based on our failure to recover recombinants, we calculate that these factors are separated by less than 0.04 ± 0.03 cM.

Enhancement of Nc Expression by enr1-Fcu and Enr1 Sources
Some derivatives of the R1-st complex carry genes designated Nc that consist of coding sequences carrying Doppia sequences in their promoter region (Matzke et al. 1996). In order to test enr1 for ability to enhance aleurone color expression of Nc genes, a line homozygous for the stable enhancement factor Enr1-628 and the colorless aleurone r1 haplotype r1-g(Stadler) was crossed to a line homozygous for r1-sc:m6, a derivative of R1-sc:134 that carries a single functional Nc gene (Eggleston et al. 1995) and has its main seed color component, Sc, inactivated by a Ds transposable element insertion. Kernels from this cross had a lightly mottled phenotype, whereas the r1-sc:m6 parental line without enhancement factors had virtually colorless aleurone. (Ac is not present in these lines.) Thus, Enr1-628 appears to enhance Nc expression. Lines homozygous for both Enr1-628 and r1-sc:m6 produce heavily mottled kernels indicating a positive dosage effect (Figure 3).


Figure 3
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  		Figure 3. Nc-enhancing effect of Enr1 alleles. Top row: Enr1-ZC with r1-sc:m6. Second row: Enr1-628 with r1-sc:m6. Third row: enr1 with r1-sc:m6 (control). All kernels are homozygous for their respective factors.

 
Crosses of Enr1-628 were also made to the Nc line carrying the haplotype r1-g(Nc)3-5, which is a derivative of R1-st that carries an Sc component inactivated by a transposition-defective I-R element (Kermicle 1984), and likely retains the 3 Nc genes of its progenitor, R1-st (Eggleston et al. 1995). All kernels from such crosses had virtually colorless aleurone, but such a result is not unexpected because multiple copies of Nc genes in cis have a negative dosage effect on Nc expression (Eggleston et al. 1995), and therefore, any enhancing effect of Enr1-628 on Nc might go undetected.

Crosses of enr1-Fcu and enr1-m594 to r1-sc:m6 produced sectors of mottling on a near-colorless background. Crosses of these 2 factors to r1-g(Nc)3-5 produced virtually colorless kernels. These results follow the response pattern for Enr1-628 described above.

Factors known to enhance Nc expression are present in some open-pollinated populations. A stock homozygous for one such Nc enhancer isolated from the land race Zapalote Chico and homozygous for r1-g(Stadler) was outcrossed to r1-sc:m6, r1-g(Nc)3-5, and to R1-r(Venezuela559-PI302355). Crosses to r1-sc:m6 produced a light mottled phenotype, and crosses to R1-r(Venezuela559-PI302355) produced full-colored kernels. On the other hand, crosses to r1-g(Nc)3-5 produced virtually colorless kernels. Thus, the Nc enhancer behaved similarly to Enr1-628 in these tests.

Direct mapping of this Nc enhancer with respect to Enr1-628 suggests that these factors are also very tightly linked and likely allelic (Table 4). Six stable pale kernels and 13 colorless kernels were recovered from a population of 4754 kernels. Upon further testing, 5 of the stable kernels and 12 of the colorless kernels proved not to be crossovers. The remaining 2 exceptions did not survive to pollination and could not be tested. Based on our inability to recover crossovers from an effective population of 4254 kernels, we calculate that these 2 factors are separated by less than 0.05 ± 0.03 cM. This Nc enhancer is now called Enr1-ZC.

Reversion Studies
enr1-Fcu Revertants
Sixty-six full-colored putative enr1-Fcu revertants were isolated from a population of 23 430 testcross kernels. Of these, 2 proved to be contaminants, lacking the y1 and wx1 markers from the enr1-Fcu parent; 61 proved not to be heritable, segregating for enr1-Fcu sectored kernels in both selfs and outcrosses; and 3 proved to be heritable germinal revertants, segregating for full-colored stable kernels as well as the pollen contamination markers, in both selfs and outcrosses. These enr1-Fcu revertants are designated Enr1-Fcu-R2003-2653-2, Enr1-Fcu-R2004-947-5, and Enr1-Fcu-R2004-947-10. The frequency of enr1-Fcu reversion from this test based on the 3 heritable germinal revertants is 1.3 x 10–4.

One of the enr1-Fcu parents (2003-2653-6) used in the reversion study was found to be heterozygous for a reversion event that must have arisen unnoticed in the previous generation. Outcrosses of this particular parent to the R1-r(Venezuela559-PI302355) tester resulted in 1:1 segregation for full-colored and sectored kernels (data not shown). The self-pollinated ear from the male parent plant was homozygous for the y1 and wx1 pollen contamination markers, ruling out contamination of the enr1-Fcu parent line. The reproducibility of the 1:1 segregation in 4 separate outcrosses rules out contamination arising during pollination as well as contamination occurring in the R1-r(Venezuela559-PI302355) parent line. This revertant is designated Enr1-Fcu-R2003-2653-6.

Two of the enr1-Fcu revertants, Enr1-Fcu-R2003-2653-2 and Enr1-Fcu-R2003-2653-6, were mapped with respect to enr1-Fcu in order to test whether they map to the same location and thus represent changes at enr1 rather than another locus. Revertant mapping data are summarized in Table 5. We obtained no heritable pale stable crossovers between Enr1-Fcu-R2003-2653-2 and enr1-Fcu from a population of 3936 testcross kernels, indicating a separation of less than 0.05 ± 0.04 cM. Likewise, we obtained no heritable pale stable crossovers between Enr1-Fcu-R2003-2653-6 and enr1-Fcu from an effective population of 1632 testcross kernels, indicating a separation of less than 0.12 ± 0.09 cM. We conclude that these 2 variants represent reversions at the enr1 locus.


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  		Table 5. Results of mapping of enr1 revertants

 
enr1-m594 Revertants
Large-scale testcrosses of a pure breeding enr1-m594 stock uncovered 4 full-color and 1 nearly full-color heritable variants among 102 700 kernels. In progeny analyses, the 4 full-color variants assorted independently of R1-r(Venezuela 594-PI302363) and spotted kernels did not reappear. These variants are designated Enr1-m594-R5874, Enr1-m594-R6117b, Enr1-m594-R6117c, and Enr1-m594-R6117d. To determine whether they were derivatives of enr1-m594 and therefore allelic to it, Enr1-m594-R5874 was crossed with enr1-m594 and testcrossed. Each of 1060 progeny was either full color (536) or spotted (524), indicating a separation of less than 0.19 ± 0.13 cM. Using Enr1-m594-R5874 as a standard, the remaining 3 full-color variants produced no recombinants, that is, pale kernels, in testcrosses of F1 hybrids. The data are as follows: Enr1-m594-R6117b (n = 3920; separation less than 0.05 ± 0.04 cM), Enr1-m594-R6117c (n = 5370; separation less than 0.04 ± 0.03 cM), and Enr1-m594-R6117d (n = 4180; separation less than 0.05 ± 0.03 cM).

The nearly full-color variant, Enr2-6117a, behaved uniquely. Although it too assorted independently of R1-r(Venezuela 594-PI302363) in progeny generations, a low frequency of spotted kernels resembling parental enr1-m594 occurred. Testcrosses of the F1 combination with Enr1-m594-R5874 produced 203 pale kernels to 1533 full- and nearly full-color kernels, indicating a separation of 23.4 ± 1.0 cM between these 2 factors. (The pale kernel recombinant class is doubled to account for the undetected full-color, double-factor recombinant class.) A spotted stock extracted from this source, tested similarly against Enr1-m594-R5874, produced 968 full-color and 971 spotted kernels, with no stable pale kernels, 1 of the 2 classes expected from recombination. This outcome indicates that enr1-m594 was unchanged but that a new Enr mutation had occurred at a linked site. Thus, Enr2-6117a does not represent a true germinal revertant of enr1-m594 but rather an independent mutation at a linked site. The germinal reversion rate for this population based on the 4 germinal revertants recovered is 3.9 x 10–5.

When Enr2-6117a was tested for linkage with a stable Enr allele (Enr3-594) present in the original enr1-m594 accession, testcrosses indicated independent assortment (data not shown). However, testcrosses of Enr2-6117a with a stable Enr allele (Enr2-694) present in the enr1-m694 accession produced only full-color and near full-color kernels among 664 progeny, indicating a separation of less than 0.30 ± 0.21 cM. Thus, Enr2-6117a and Enr2-694 define a second enr locus on chromosome 2. We designate this second locus enr2. The unlinked factor, Enr3-594, represents a third locus, enr3.

enr1-m694 Revertants
Large-scale testcrosses of enr1-m694 conducted in parallel to those of enr1-m594 produced 1 heritable full-color variant, Enr1-m694-R6118a, among 41 600 progeny for a reversion rate of 2.4 x 10–5. Tests of Enr1-m694-R6118a for linkage against Enr1-m594-R5874 produced only full-color kernels among 2210 testcross progeny, indicating a separation of less than 0.09 ± 0.06 cM.

Four-Point Linkage Data for enr1-m594 and Enr2-6117a with Respect to the Chromosome 2 Markers fl1 and v4
Once it was established that enr1 and enr2 are linked on chromosome 2, we conducted a 4-point linkage test of enr1-m594 and Enr2-6117a with respect to the chromosome 2 markers fl1 and v4. The linkage testcross and the results are presented in Table 6. The following linkage order and distances (in centiMorgans) were established:

Formula


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  		Table 6. Four-point linkage data for fl1 enr2 v4 enr1

 
These linkage values are consistent with the data for fl1 v4 enr1 w3 ch1 presented above with respect to the fl1 v4 linkage distance and fl1 v4 enr1 gene order, although the linkage value obtained for the v4 enr1 interval (10.3 cM) is less than the value obtained in the former experiment (15.9 cM). The linkage value for the enr2 enr1 interval (18.1 cM) is also less than the value obtained from direct linkage between these 2 factors reported in Table 5 (23.4 cM). Nevertheless, the gene order for these factors is clearly established on chromosome 2.


   Discussion
Top
 Introduction
 Materials and Methods
 Results
 Discussion
Funding
 References
 
Equivalence of enr1-Fcu, enr1-m594, and enr1-m694 Instability Factors
Linkage test results indicate that enr1-Fcu and the 2 mutable enhancement factors isolated from Venezuelan lines, enr1-m594 and enr1-m694, map to the same location on 2L. Furthermore, 2 enr1-Fcu revertants, 4 enr1-m594 revertants, 1 enr1-m694 revertant, and the stable Venezuelan enhancement factor Enr1-628 map to this same location. This linkage relationship, the identical r1 haplotype response patterns of enr1-Fcu and enr1-m594, and the observation that the phenotypes of the mutable enhancement factors and stable enhancement factors are virtually indistinguishable within their groups, suggest that these factors are closely related and probably represent a single locus. enr1-Fcu was isolated from Cuna tribal maize collected in Unguia, Colombia (Gonella and Peterson 1977); enr1-m594 derives from maize collected in Tinaquillo, Cojedes, Venezuela (Van Der Walt and Brink 1969); the exact origin of enr1-m694 is unknown, although it is from a Venezuelan accession. All 3 lines came to the United States through the Tulio Ospina Research Station, Instituto Colombiano Agropecuario, Medellín, Colombia (Gonella and Peterson 1977; Kermicle JL, unpublished). Whether the mutable enhancement factors are independent alleles or whether they represent the isolation of the same allele from different sources remains to be determined.

Mutability in enr1-Fcu/enr1-m System Reflects Changes at the Color Enhancement Locus, not at r1
Gonella and Peterson (1977) proposed Fcu as a classical 2-element controlling element system with an autonomous element, Fcu, and a receptor element, Icu, inserted at the r1 locus in the r1-cu haplotype. According to this view, r1-cu produces stable pale color in the absence of Fcu, but in its presence, Icu either excises or undergoes some other modification resulting in the production of revertant sectors of full color in the aleurone (Figure 1A). Subsequent observations (Gonella and Peterson 1978) supported this interpretation which, however, was made without the benefit of germinal revertants that would have enabled a more complete understanding.

We report the isolation of revertants to full color from large-scale testcrosses of the enr1-Fcu and enr1-m systems. These revertants were found to segregate independently of r1, and 2 revertants from enr1-Fcu as well as 5 revertants from enr1-m sources were subsequently mapped to the same location as their mutable progenitors on chromosome 2. These findings reveal that the somatic instability observed in crosses of mutable color enhancement factors to r1-cu and other reporter haplotypes is due to changes at the color-enhancing locus rather than at r1 (see Figure 1B for one possible model).

This interpretation is consistent with the observation by Gonella and Peterson (1977) that the number of revertant sectors on Fcu-responsive kernels does not vary with the dosage of the responsive r1 haplotype but varies only with the dosage of Fcu. In contrast, other 2-element transposable element systems commonly give a marked dosage effect when the number of copies of the reporter allele is varied (e.g., a1-m with Dt1; Rhoades 1938). With increasing numbers of copies of a reporter allele present, there is a greater opportunity for sectoring to be observed because copies of the reporter allele revert independently of each other. Copies of r1-reporter haplotypes for Fcu, on the other hand, reflect Fcu activity in a particular cell lineage but do not undergo reversion themselves.

Gonella and Peterson (1978) present the paradox of a nonreciprocal relationship between 2 different controlling element systems. They reported that when Fcu was crossed to the reporter allele for the Spf (En/Spm) system (Sastry and Kurmi 1970; Stinard and Sachs 2005), R1-r(sd2), kernel sectoring was observed. On the other hand, Spf elicited no response in r1-cu. The response of R1-r(sd2) to 2 different autonomous elements is contrary to the specific relation of autonomous to receptor loci. The difference in response of r1-cu and R1-r(sd2) to Fcu and Spf was attributed to the presence of an additional function in Fcu not present in Spf. However, our data suggest that R1-r(sd2) is responding to Spf and enr1-Fcu in fundamentally different ways. The response of R1-r(sd2) to Spf is that of a traditional 2-element transposable element system. R1-r(sd2) contains a defective nonautonomous element that excises in the presence of the autonomous element Spf; reversion occurs at the r1 locus. On the other hand, R1-r(sd2) is simply another indicator haplotype for enr1-Fcu like r1-cu, reflecting changes occurring at the enhancement locus.

The enr Loci Represent r1 Haplotype–Specific Enhancers of Aleurone Color
Given that the sectoring observed in crosses of enr1-Fcu and enr1-m lines to responsive r1 haplotypes is due to events taking place at the enr1 locus rather than at r1; what is responsible for the sectoring associated with enr1? Based on the enhanced color response of specific pale r1 haplotypes to revertants of enr1-Fcu and enr1-m and to full-colored Enr1 variants, enr1-Fcu and enr1-m are viewed as unstable enhancers of r1 seed color. The instability could be due to the insertion of an autonomous transposable element insert within the enhancement factor, knocking out its function. Excision of the transposable element would restore the enhancement factor's function, giving rise to enhanced (full color) expression in revertant sectors. Another possibility is that sectors could reflect epigenetic changes (Bestor et al. 1994) in the enhancement factor occurring during endosperm development. Under this model, the initial state of enr1-Fcu during endosperm development would be "off." During the course of kernel development, enr1-Fcu is turned on in some cell lineages, giving rise to enhanced purple sectors. Germinal revertants would represent heritable derivatives in which enr1-Fcu is in the "on" state throughout kernel development.

Aleurone color enhancement could come about by a first-order interaction of enr loci with r1, perhaps through transcriptional activation, or it could represent a higher order interaction with other factors that regulate r1 seed color expression in a positive or negative way. Such factors have been reported elsewhere (Hernandez et al. 2007). One intriguing possibility is that enr genes might be acting to suppress inhibitors of aleurone color. Evidence for this comes from the presence of colored sectors in crosses of enr1-Fcu to suppressible r1 haplotypes carrying inhibitors (R1-ch(Stadler), R1-Randolph, and R1-d(Catspaw) with inhibitory alleles of inr1 or inr2; and R1-r(sd2) with Inr1-Dil; Stinard and Sachs 2002, 2005). In these crosses, perhaps, the colored sectors represent inactivation of the inhibitors. It is also possible that the colored sectors in such crosses are due to an enhancement of r1 seed color expression that is able to counteract the effect of inhibitors.

The Spm/Spotted dilute system (Stinard and Sachs 2005) provides a potential means of dissecting the interaction between the enr and inr loci and the r1 locus. Gonella and Peterson (1978) observed that kernels bearing R1-r(sd2) produce aleurone color sectors in the presence of either Spf (later characterized to be an autonomous Spm element; Stinard and Sachs 2005) or Enr1-Fcu. In the absence of the color inhibitor Inr1-Dil, the sectoring is barely distinguishable from the near full-color background, but in the presence of Inr1-Dil, background color is suppressed, allowing sectors to be clearly distinguished from the pale background. R1-r(sd2) was derived from R1-r(Cornell) (Stadler and Emmerling 1956), which exhibits the same recombinational behavior as, and may be identical to, R1-r(Std) (Dooner and Kermicle 1971). R1-r(Std) carries duplicate genes, S1 and S2, that encode myc-like transcriptional activators regulating the anthocyanin biosynthetic pathway in the aleurone arranged as inverted repeats flanking a promoter region (Walker et al. 1995). Both S1 and S2 are expressed in the aleurone, so a mutation in one of the S genes would not be expected to give a visible phenotype. However, if one of the S genes is inhibited by Inr1-Dil, a mutation in the other S gene would be readily detectable. We propose that this is the case with the Spotted dilute system. One possible model is presented in Figure 4A. In the model illustrated, S2 bears a dSpm insert and S1 is inhibited by Inr1-Dil, but the reverse could also be the case. Molecular analysis would be needed to resolve this with certainty.


Figure 4
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  		Figure 4. Proposed model of the interaction of Enr1-Fcu, Inr1-Dil, and Spm with R1-r(sd2). (A) R1-r(sd2) is a compound haplotype carrying 2 seed color genes, S1 and S2, arranged as inverted repeats flanking a central promoter region. One of the S genes (S2 in this illustration) carries a dSpm insert; the other S gene is inhibited by Inr1-Dil. Inr1-Dil suppresses background coloration arising from S1 gene expression. In the presence of Spm, the dSpm excises from S2, giving rise to full-color revertant sectors. (B) In this model, Enr1-Fcu bears an autonomous transposable element blocking expression. Background color is suppressed due to the action of Inr1-Dil on S1 and the dSpm insert in S2. Excision of the transposable element from Enr1-Fcu restores Enr1 resulting in a colored sector due to enhancement of S1 expression, either through interaction with Inr1-Dil or through direct interaction with S1.

 
Because Enr1-Fcu shows no direct interaction with the components of the Spm transposable element system (Gonella and Peterson 1977), it is likely that sectoring elicited by Enr1-Fcu in crosses to R1-r(sd2) in the presence of Inr1-Dil are due to an interaction of Enr1-Fcu with either Inr1-Dil or the S component inhibited by Inr1-Dil rather than with the S component bearing the dSpm insert. This is illustrated in Figure 4B. In the model illustrated, mutability is due to excision of an autonomous transposable element from Enr1–Fcu resulting in sectors bearing a functional enhancer. This is only one possible model for the epistatic interaction between the enr and inr loci.

Relationship of Stable and Mutable Enhancement Factors
The inseparable map locations of stable and unstable alleles of enr1 raises an interesting question: Did the mutable factors result from the insertion of a transposable element in one of the naturally occurring stable factors or did the stable factors arise as a result of reversion or change of state of one of the mutable factors? Molecular analysis may resolve which of the stable alleles are ancestral and which are derived. To this end, an Ac element located on the long arm of chromosome 2 linked to enr1 (Brutnell and Conrad 2003) is being combined with Enr1-628 in order to recover insertions of Ac into enr1, enabling cloning and characterization of this stable allele and ultimately the unstable alleles.


   Funding
Top
 Introduction
 Materials and Methods
 Results
 Discussion
 Funding
References
 
United States Department of Agriculture/Agricultural Research Service Project Number (3611-21000-022-00 to M.M.S.); United States Department of Agriculture/National Research Initiative Award (35301-13314 to J.L.K.).


   Acknowledgments
 
We thank Hugo Dooner for his suggestions for improving the manuscript, particularly in regard to nomenclature issues.


   Footnotes
 
Corresponding Editor: Susan Gabay-Laughnan

Received June 9, 2008
Revised September 5, 2008
Accepted September 22, 2008


   References
Top
 Introduction
 Materials and Methods
 Results
 Discussion
 Funding
 References
 

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