Maize Genetics Cooperation Stock Center
The Spotted-dilute controlling element system revisited
Sastry and Kurmi (MNL44:101–105, 1970) describe a mutable system at the r1 locus involving three elements: the receptor haplotype R1-r(sd2) (spotted dilute2), the autonomous controlling element Spf (spotting factor), which elicits aleurone color mutability at R1-r(sd2), and Dil (diluting factor), which suppresses background aleurone color of R1-r(sd2). In the absence of Spf and Dil, kernels bearing the R1-r(sd2) haplotype are near full colored; in the presence of Dil, R1-r(sd2) kernels have pale aleurone color; in the presence of Spf, R1-r(sd2) kernels are dark with barely distinguishable darker sectors; and in the presence of both Spf and Dil, R1-r(sd2) kernels have dark sectors on a pale background. Full colored revertants of R1-r(sd2) can be isolated from mutable lines. The apparent progenitor haplotype of R1-r(sd2) is R1-r(Cornell) (Stadler and Emmerling, MNL 30:61–63, 1956), which may be identical to R1-r(standard). Sastry and Kurmi performed tests indicating that Spf elicits mutability at a2-m1::dSpm. We performed additional tests (described elsewhere in this newsletter) that demonstrate that Spf and En/Spm are functionally equivalent at eliciting mutability at both R1-r(sd2) and various dSpm/I receptor alleles. We also demonstrated that Dil is very tightly linked, if not identical to, the r1 haplotype-specific aleurone color inhibitor Inr1 (also described elsewhere in this newsletter).
What sets the Spotted-dilute system apart from other controlling element systems are certain peculiar behaviors described by Sastry and Kurmi. They indicate that after Spf and Dil have segregated away from R1-r(sd2), R1-r(sd2) is not capable of responding to either Spf or Dil once these factors have been reintroduced. However, as long as at least one of the two factors is present, R1-r(sd2) will also show a response to the other factor once it is reintroduced. They also indicate that R1-ch (most likely the R1-ch(Stadler) haplotype) responds to Spf and Dil regardless of whether either factor was previously present in the R1-ch line. We performed several experiments in order to investigate these unusual behaviors. We obtained two R1-r(sd2) Dil Spf lines from Jerry Kermicle, one with R1-r(sd2) linked to K10, and one with R1-r(sd2) residing on a normal chromosome 10. Both lines segregated for R1-r(sd2) (the other r1 haplotype present was r1-r on N10), Spf, and Dil. From these lines, we isolated three homozygous r1-r N10 lines: one homozygous for both Spf and Dil, one segregating for Spf without Dil, and one homozygous for Dil and segregating for Spf. A W22 r1-g(Stadler) line without Spf and Dil was used as a control. We also developed the following R1-r(sd2) testers: R1-r(sd2) N10 with Dil, R1-r(sd2) N10 without Dil, and R1-r(sd2) K10 without Dil. Crosses were made of the r1-r lines onto the R1-r(sd2) testers and onto R1-ch(Stadler) in various combinations, and the data obtained are summarized in Table 1.
Some of our results conflict with those of Sastry and Kurmi. Crosses of r1 Dil (no Spf) onto the R1-r(sd2) N10 tester without Dil yielded pale colored kernels. Crosses of r1 Spf (no Dil) onto the R1-r(sd2) N10 tester without Dil yielded colored kernels with darker sectors. Finally, crosses of r1 Dil Spf onto the R1-r(sd2) N10 tester without Dil yielded pale colored kernels with sectors. In other words, R1-r(sd2) that has been separated from Spf and Dil does respond to both Spf and Dil separately or in combination when these factors are reintroduced, providing that R1-r(sd2) is carried on an N10 chromosome.
Crosses of Dil and Spf on to the R1-r(sd2) K10 tester without Dil gave results that are tantalizing but incomplete. All crosses intoducing Dil resulted in pale aleurone color, indicating that the presence of K10 has no effect on the response of R1-r(sd2) to Dil. However, none of the crosses introducing Spf resulted in sectored kernels. Control crosses of most of the plants used in the K10 crosses to an N10 version of R1-r(sd2) were not made because the K10 result was unanticipated. Thus, all of the putative r1 Dil Spf parents crossed to the K10 line were not independently verified to carry Spf, although it is likely that they do since the line was homozygous for Dil and Spf when tested in the previous generation. However, all r1 Spf (no Dil) parents were independently tested, and all of those known to carry Spf did not induce mutability when crossed to the K10 line.
We tentatively conclude that R1-r(sd2) that has been separated from Spf and Dil does not respond to Spf when it is reintroduced either alone or in combination with Dil, when R1-r(sd2) resides on a K10 chromosome. However, R1-r(sd2) does respond to Dil regardless of whether R1-r(sd2) resides on an N10 or K10 chromosome. These results will have to be tested more thoroughly because there exists the possibility that the R1-r(sd2) K10 haplotype that we tested has lost its ability to respond to Spf for reasons other than the presence of K10. We derived our R1-r(sd2) K10 line from a single stable pale kernel segregant from an ear segregating for Spf and Dil. It is possible that this kernel was stable not because it lacked Spf, but rather because a mutation event or an imprecise excision event resulted in the formation of a nonresponsive R1-r(sd2) derivative. We plan to test other, independent R1-r(sd2) K10 derivatives, as well as create new R1-r(sd2) K10 derivatives by crossing over using the N10 source of R1-r(sd2) that is known to respond to Spf and an r1 K10 line. We also plan to separate the nonresponsive R1-r(sd2) from K10 by crossing over to determine whether it can regain its ability to respond to Spf when carried on an N10 chromosome.
K10 is known to affect meiotic drive (Hiatt and Dawe, Genetics 164:699–709, 2003) and paramutation at the r1 locus (Brink, Mutation Res 8:285–302, 1969). Our results suggest another possible effect of K10, the repression of transposable element excision. Whether this effect is confined to R1-r(sd2) or whether it could affect other mutable r1 haplotypes or other linked or unlinked receptor loci is not known at this time. Clearly, the presence of a linked K10 does not automatically shut down transposable element excision events at r1. Descendants of the original R1-r(sd2) K10 mutational event retain mutability at R1-r(sd2) in the presence of Spf until Spf has segregated away. K10 also has no appreciable effect on mutability of R1-st (Brink, 1969). If the K10 effect is real, then apparently the mutable R1-r(sd2) on K10 is in a certain “state” in the presence of Spf; this state is reset once Spf has segregated away, and is not regained when Spf is reintroduced. This “state” could be a particular chromatin structure maintained by an Spf product such as a transposase that is irreversibly lost when the product is no longer present. Much remains to be learned about this system.
The Spotted-dilute system arose in a line in which R1-r(Cornell) was carried on a K10 chromosome (Stadler and Emmerling, MNL 30:61–63, 1956). If the K10 effect is real, its presence could be the reason that Sastry and Kurmi found that Spf did not induce mutability in R1-r(sd2) after Spf had been separated from it and then reintroduced. However, we found that the presence of K10 has no effect on the response of R1-r(sd2) to Dil, and this result conflicts with that of Sastry and Kurmi. Finally, we found that Dil suppresses aleurone color in crosses to R1-ch(Stadler), but no combination of Spf with or without Dil induced mutability of R1-ch(Stadler), and this also conflicts with Sastry and Kurmi. However, since Sastry and Kurmi did not indicate their source of R1-ch, it is possible that they used a different r1 haplotype that happens to respond to Spf.
Finally, some comments should be made about what structural features of R1-r(sd2) could account for the phenotypes produced by its interactions with Spf and Dil. First, Dil seems to be similar, if not identical to Inr1. Inr1 seems to work by suppressing S (seed color) subcomponents of certain r1 haplotypes (Stinard and Sachs, J Hered 93:421–428, 2002). It is particularly effective at suppressing S subcomponents of inverted repeat haplotypes that have a single functional S subcomponent such as R1-ch(Stadler) and R1-d(Catspaw). It does not, however, significantly reduce aleurone color when crossed to R1-r(standard), a haplotype with two functional S subcomponents. In the case of R1-r(standard), it could be that Inr1 suppresses one S subcomponent, but not the other. If the lesion in R1-r(sd2) is a defective transposable element insertion into a nonsuppressible S subcomponent that knocks out its expression, then in the absence of Inr1/Dil and the autonomous element Spf, the suppressible S subcomponent would be expressed and give rise to near full color aleurone. In the presence of Inr1/Dil, the suppressible S subcomponent would be suppressed, giving rise to weak or no aleurone color. However, in the presence of both Inr1/Dil and Spf, excision of the defective transposable element inserted at the nonsuppressible S subcomponent would give rise to purple tissue sectors carrying a functional S subcomponent on a pale suppressed S background. This model will be further tested.
Table 1. Kernel segregations on ears of crosses of r1 with various combinations of Dil and Spf to various R1-r(sd2) testers and R1-ch(Stadler).
|male parent||male genotype1||kernel segregations2 on crosses to R1-r(sd2) N10 / r1-g N10 tester||kernel segregations on crosses to R1-r(sd2) K10 / r1-g N10 tester||kernel segregations on crosses to R1-r(sd2) N10 Dil / r1-g N10 dil tester||kernel seg-regations on crosses to R1-ch (Stadler) tester|
|2003-962-2||r1 Dil ?Spf||Pale Cl|
|2003-962-3||r1 Dil ?Spf||Pale Cl, cl; K10 ratio||Pale Cl|
|2003-962-4||r1 Dil ?Spf||Pale Cl, cl; K10 ratio||Pale Cl|
|2003-962-5||r1 Dil Spf||Pale Cl sec, cl||Pale Cl|
|2003-962-6||r1 Dil ?Spf||Pale Cl, cl; K10 ratio||Pale Cl|
|2003-962-9||r1 Dil ?Spf||Pale Cl, cl; K10 ratio||Pale Cl|
|2003-962-10||r1 Dil ?Spf||Pale Cl, cl; K10 ratio||Pale Cl|
|2003-962-11||r1 Dil ?Spf||Pale Cl, cl; K10 ratio||Pale Cl|
|2003-963-3||r1 Dil spf||Pale Cl, cl||Pale Cl|
|2003-963-7||r1 Dil Spf/spf||Pale Cl, Pale Cl sec, cl||Pale Cl|
|2003-963-9||r1 Dil spf||Pale Cl, cl||Pale Cl|
|2003-963-10||r1 Dil Spf/spf||Pale Cl, Pale Cl sec, cl||Pale Cl|
|2003-963-11||r1 Dil Spf/spf||Pale Cl, Pale Cl sec, cl|
|2003-964-1||r1 dil Spf/spf||Cl, sec base, cl||Cl, sec base, Pale Cl, sec, cl||Cl|
|2003-964-2||r1 dil Spf||Cl, sec base, cl||Cl, sec base, Pale Cl sec, cl||Cl|
|2003-964-4||r1 dil Spf||Cl, cl; K10 ratio||Cl, sec base, Pale Cl sec, cl||Cl|
|2003-964-7||r1 dil Spf/spf||Cl, cl; K10 ratio||Cl, sec base, Pale Cl, sec. cl||Cl|
|2003-964-8||r1 dil spf||Cl, cl||Cl, Pale Cl, cl||Cl|
|2003-911-3||r1 dil spf||Cl, cl||Cl, Pale Cl, cl||Cl|
|2003-911-9||r1 dil spf||Cl, cl 1:1 ratio = N10 crossover||Cl, Pale Cl, cl||Cl|
1Male genotypes are based solely upon the results of test crosses made this generation. In the previous generation, family 2003-962 tested homozygous for both Spf and Dil, 2003-963 tested homozygous for Dil and segregating for Spf, and 2003-964 tested no Dil and segregating for Spf. 2003-911 is the W22 r1-g(Stadler) tester (no Dil or Spf).
2Pale Cl = pale aleurone color
cl = colorless aleurone
Cl = colored aleurone
sec = sectored aleurone
sec base = colored aleurone with visible darker sectors at kernel base