BICD 123 Spring 2007 Yanofsky Arabidopsis Performing crosses and testing for allelism Overview Arabidopsis has become a model system for experiments in plant molecular genetics Among the many features that make Arabidopsis an ideal organism for studies of plant development are the rapid generation time 5 6 weeks ease of performing genetic crosses and the fact that the complete genome sequence is now available Other resources include the availability of a whole genome microarray chip for genome wide analysis of gene expression and insertional mutant lines that can be used in reverse genetics studies This laboratory will be used to demonstrate 1 how genetic crosses are performed in plants such as Arabidopsis and 2 how tests for allelism are performed Introduction The Arabidopsis genome has been subjected to extensive mutagenesis and a wide variety of mutants affecting floral morphogenesis have been isolated and characterized Flowers of Arabidopsis and many other plant species consist of four concentric rings or whorls of organs For Review see Coen and Meyerowitz The War of the Whorls Nature 353 31 1991 These whorls refer to a geographical location within the developing flower In wildtype flowers the first or outermost whorl consists of four green sepals Interior to the sepals are the second whorl organs which consist of four white petals Six stamens make up the third whorl each consisting of a long filament capped by a pollen bearing anther The fourth and innermost whorl is occupied by two fused carpels In order to further characterize the genetic interaction between different mutants it is necessary to perform crosses One particular class of mutants causes homeotic conversions of petals into sepals and of stamens into carpels Extensive studies have demonstrated that this class of mutants is represented by mutations in two different genes called APETALA3 AP3 and PISTILLATA PI One of the goals of this lab session will be to demonstrate how independently isolated mutants with the same or similar phenotype are determined to be either a allelic to one another or b mutations in different genes For example ap3 and pi mutants look the same Thus if you were to isolate a new mutant that had the ap3 pi mutant phenotype how would you determine if your new mutant has a mutation in the AP3 gene the PI gene or perhaps a completely different gene In addition to working with floral homeotic mutants we will also be taking advantage of Arabidopsis trichome mutants Trichomes in Arabidopsis are readily visible on the surfaces of leaves and form a branched structure We will be working with several distinct trichome mutants each of which contains a mutation in a different gene These genes are called ANGUSTIFOLIA AN Embo J 21 1280 ZWICHEL ZWI PNAS 94 6261 as well as FURCA1 and FURCA3 FUR Development 126 5547 All of the alleles that we will be working with are recessive QUESTION What do we expect the phenotype to be of the F1 progeny if we cross plants that are homozygous for the an mutation to plants homozygous for the zwi mutation QUESTION What do we expect the phenotypes to be of the F1 progeny if we cross a plant that is heterozygous for a mutation in AP3 AP3 ap3 to a plant that that is homozygous for a mutation in AP3 ap3 ap3 QUESTION What do we expect the phenotypes to be of the F1 progeny if we cross a plant that is heterozygous for a mutation in AP3 AP3 ap3 to a plant that that is homozygous for a mutation in PI pi pi The objectives of these experiments include 1 learning how to perform crosses between different mutants and 2 learning how to determine if a newly identified mutant is allelic to a previously described 2 mutant These experiments will be carried out over the entire length of the lab course and during the final session the results will be obtained and analyzed Day 1 Each group will be provided with one known and one unknown The known plants will segregate for the ap3 mutant In other words the parents of these plants were heterozygous for a mutation in the AP3 gene These parents self pollinated and gave rise to the plants that you have in your pots in the following ratios 25 AP3 AP3 50 AP3 ap3 25 ap3 ap3 The unknown plants will segregate for either a mutation in the AP3 gene or a mutation in the PI gene By crossing your unknown to your known plants you will be able to determine the genotype of your unknown plants In other words you will be able to determine if they have a mutation in the AP3 or the PI gene What exactly is in these unknown pots The A and B pots should have plants that segregate for the ap3 or pi mutant phenotypes That is approximately 25 of them have the mutant phenotype Keep in mind that these mutants completely lack stamens and without stamens there can be no pollen and without pollen these mutant plants are not self fertile However because these mutations are recessive the mutants can be maintained in the heterozygous condition Thus the parents for these plants were heterozygous for the mutation but they had a wild type phenotype Self pollination of these parents X gave rise to the 25 mutants that you see in your unknown pots 3 Question What fraction of the plants in the A and B pots are heterozygous for the mutation Examine the phenotypes of these mutants Pollinate ap3 mutant plants with pollen from each of the unknowns Experimental Plan Pollinate ap3 mutant flowers using pollen from your unknown plants Note that it is not possible to obtain pollen from the mutant plants so you must use pollen from plants that appear wild type but in fact are segregating for the mutation You should perform at least three separate pollinations using pollen from at least three different donor plants Tie a small piece of thread or use a small piece of tape just below the pollinated flowers so that you will know that it is those flowers above the thread that you will later collect seeds from Be sure to label plants carefully so that you know which plant you used for each pollination Keep in mind that you will be collecting seed not only from the crosses you perform but also from the plants used to obtain pollen Question Why is it important to collect seeds from the plants used to obtain pollen Day 2 Lab period date to be determined Keep an eye on developing siliques When they turn brown in about 2 weeks they are ready to harvest Use care as it is easy to lose all seeds at this stage Collect seeds from crosses by opening siliques over a piece of paper and pour seeds into a 1 5 ml