Functional Genomics of Gametogenesis ( NSF Genome project website )
There is a discrepancy between the number of identified gametophyte mutants and the number expected based on the number of genes expressed in gametophytes. One explanation for this phenomenon is that many such mutants are gametophyte lethal. Gametophyte lethals are quickly removed new mutations from the test population because they produce no seed. If the gametophytes fail prior to competent gamete formation, mutant heterozygotes either are never produced or cannot be maintained. However, if the new mutation causes 100% gametophyte lethality, a heterozygous seed is never produced. Similarly, if a mutation is 90% lethal it is ten times less likely to pass to the next generation than a mutation with no effect. Consequently, we are taking advantage of maize genetics to identify mutations in essential genes by generating them in the presence of a genetic duplication. We are taking advantage of the translocations of arms from the 10 normal A chromosomes to the supernumerary B chromosome. This system can be used to generate tertiary trisomic stocks in which the chromosome arm in question is linked to the B centromere and inherits independently of the A chromosome, frequently producing gametophytes with two copies of a chromosome arm. Mutagenesis of the normal chromosome with the regionally biased Activator transposable element can be used to generate lethal mutants that are covered by the duplication in some gametophytes and therefore viable. Activator elements distributed throughout the genome by the laboratory of Thomas Brutnell are being used for this mutagenesis.
We are part of a collaborative project funded by the National Science Foundation Plant Genome Research Program to identify genes important for gametophyte function. These genes are to be identified in two ways: by screening for mutants with reduced transmission through the gametophytes, as described above, and by characterizing the transcriptome of expressed genes of the male and female gametophytes using next generation sequencing. This project is a collaboration between our lab and John Fowler and Scott Givan at Oregon State University, Erik Vollbrecht at Iowa State University, and Don Auger at South Dakota State University. As part of this project we are also collaborating with Kelly Beck at the Science in Service program of the Haas Center for Public Service of Stanford University to develop and deliver a plant biology curriculum to high school age students in local Boys & Girls clubs.
Figure 1. Screen for gametophyte lethal mutants. The long arm of chromosome 5 is shown for example. B chromosome sequences are shown in blue. pr1=purple aleurone1 locus. fgl=female gametophyte lethal. Ac=Activator transposable element. Tertiary trisomic stocks carrying an Ac element linked to a recessive marker and covered by a tertiary trisome have been made. Seeds are selected that carry the trisome and a new Ac element (based on increased Ac dosage). Plants are grown and tested for inheritance of the Ac element. If the Ac is inserted into a gametophyte lethal locus, it can only be transmitted in the presence of the trisome, which provides wild type gene function.
Figure 2. Ear of a maize plant with kernels homozygous for bz1-m2(DI)::Ds (except for a germinal revertant pictured) and segregating two different Ac elements and also segregating for a chromosome 5L trisome marked by the wild-type allele of the pr1 locus, which conditions purple aleurone (or brown in combination with bz1), while the normal chromosomes carry mutant pr1, which conditions red aleurone (or orange in combination with bz1). If Ac is also present in a kernel, then bz1-m2(DI)::Ds is unstable and produces somatic wild-type revertant sectors (spots) on a mutant background. When there are two Ac’s present in the genome, the revertant sectors are less frequent and smaller than they are with only one Ac. In this way, the presence of the trisome and the number of active Ac’s can be determined by endosperm phenotype.