Cold Spring Harbor Laboratory

Isolation of new alleles of anther ear and indeterminate

--Joseph Colasanti and Venkatesan Sundaresan

The genes for anther ear (an1), indeterminate (id) and bronze-2 (bz2) are known to lie within 1 or 2 map units of each other on the long arm of chromosome 1. We used the mutable bronze allele bz2-m2, which contains a Ds2 transposable element insertion in the Bz2 gene, in a series of crosses in the hopes of obtaining an insertion of the Ds2 element into the anther ear gene.

The first set of crosses, in the summer of 1989, involved a directed cross of bz2-m2/bz2-m2 plants carrying an active Ac element (a gift from Kelly Dawe, RK, U. C. Berkeley) as the female parent with plants homozygous for the an1 bz2 deletion (a gift from Virginia Walbot, V, U. Stanford) as the male parent. The an1 bz2 plants were semi-dwarf in stature and required exogenous application of gibberellic acid (GA).

Approximately 500 ears were isolated from this cross; most of the kernels had bz-mutable aleurones, but there were approximately 1 to 4 completely purple kernels per ear which corresponded to germinal excision of the Ds2 element from the bz2 gene. These germinal revertant kernels were selected to facilitate the screening of mutants by reducing the number of plants to screen. From 1000 purple kernels planted in the summer of 1990, one semi-dwarf plant was found (#853) and subsequently selfed and outcrossed. At harvest, the ears of plant #853 had anthers typical of a GA-deficient phenotype. All other non-dwarf plants were also selfed with the intent of screening for other Ds2-induced recessive mutations (see below).

The progeny from the self of plant #853 were crossed to a line carrying an an1 single gene mutation (from the Maize Coop) and also backcrossed to the an1 bz2 deletion line. Plant #853 was found to be allelic to an1 and the new allele tentatively designated an1-*. In addition, in all plants examined so far, the an1-* allele is closely linked to the Bz2 gene, which presumably resulted from germinal excision of Ds2 from the bz2-m2 gene.

For molecular analysis, a 100bp fragment of the Ds2 element was obtained from Sarah Hake, S (U.S.D.A., Albany) and used to screen the outcross progeny of plant #853 by Southern blotting. The use of this fragment reduces the number of bands that correspond to the Ds2 element family and that correspond to other Ds-like elements. The number of Ds2-hybridizing bands in these progeny varied from 4 to 12, with the greatest number of bands in the selfed progeny. One 4.3kb band from a BamHI digest was found to always associate with an1-* progeny and was always absent in an1 bz2 deletion strains. Experiments are in progress to further characterize this 4.3kb BamHI fragment and to determine the effect of the presence of an active Ac element on the expression of the an1-* phenotype.

In the summer of 1991 the selfed, normal progeny from the previous summer were screened for any interesting whole plant recessive mutations. In all of these families, purple and bronze kernels segregated 3:1, as expected. Bronze kernels corresponded to a homozygous an1 bz2 deletion genotype and therefore only purple kernels were selected. Twenty purple kernels from each of 600 families were screened. Several distinct phenotypes were found (mostly dwarfs and mini-plants) but the most prominent was family #62. Fifteen of 20 kernels from family #62 grew to adult plants; of these, all but 7 plants had completely shed out by the first week of August, as did all the other plants in the other families of the screen. These seven plants continued to grow taller and exhibited other indeterminate phenotypes, such as prop root formation up to the ninth node and an increased number of internodes. Eventually, 2 of the 7 plants had formed tassels and shed pollen by the end of August; these were outcrossed to various plants in the field which were delayed in planting and were flowering at this time (courtesy of Chang-deok Han, C-d). The other 5 "indeterminate" plants did not form tassels until the middle to end of September. The normal siblings of family #62 had between 9 and 12 nodes at maturity, whereas the 7 late flowering plants had between 17 and 25 nodes at season's end and none of these 7 plants formed viable ears. Interestingly, id/id plants from the Maize Coop (id-R) that were planted at the same time in another part of the field did not flower until the end of October.

In preliminary experiments, purple kernels from family #62 that were germinated and grown under short day conditions (16 hour nights/8 hour days) for 4 or 6 weeks continued to segregate late-flowering plants; i.e. this light regime had no effect on abrogating the indeterminate phenotype, as has been reported for other id alleles. Experiments are in progress to determine whether this is a new id allele and if it is the result of a Ds2 insertion.

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