Breakthrough Technologies Positional Cloning in Arabidopsis Why It Feels Good to Have a Genome Initiative Working for You1 Wolfgang Lukowitz C Stewart Gillmor and Wolf Ru diger Scheible Department of Plant Biology Carnegie Institution of Washington 260 Panama Street Stanford California 94305 W L C S G W R S and Department of Biological Sciences Stanford University Gilbert Hall Stanford California 94305 C S G Positional or map based cloning techniques are widely used to identify the protein products of genes defined by mutation In Arabidopsis the information generated by the Genome Initiative is giving this approach a decisive boost A wealth of sequence polymorphisms and molecular markers is now available and can be exploited for fine mapping with technically simple and robust polymerase chain reaction based methods As a result it has become possible to complete positional cloning projects in a short time and with relatively little effort Over the past decade the use of Arabidopsis genetics has become increasingly popular for the study of plant biochemistry physiology pathology and developmental biology for review see Meinke et al 1998 Much information can be gathered from analysis of genetic variation without knowing anything about the underlying molecular causes However most researchers would ultimately like to know which genes have been altered to produce a given phenotype If a mutation is caused by the insertion of a T DNA or transposon the inserted sequence provides a tag pointing directly to the gene Most genetic variability however is not of this kind Chemical agents and radiation are widely used to induce mutations for a number of good reasons Ethyl methanesulfonate for example is easy to use more effective than many other mutagens and since it typically causes single basepair exchanges more likely to create mutations with special properties such as weak dominant or conditional alleles Apart from induced mutations there is a wealth of naturally occurring genetic variability in traits like resistance to pathogens flowering time seed size and many others This variability is likely to reflect rather subtle changes in the DNA sequence In all of these cases there is little alternative but to isolate the genes involved based on their map position a process that until recently has been considered time consuming and expensive With public accessibility of the complete and annotated sequence of the Arabidopsis genome anticipated in only a few months this situation 1 W L was partially supported by a postdoctoral fellowship LT 594 96 from the Human Frontier Science Program Organization and by a fellowship from the Carnegie Institution of Washington C S G was supported by a U S Department of Energy National Science Foundation U S Department of Agriculture TRI Agency Training Grant and W R S was supported by a postdoctoral fellowship Sche 548 1 1 from the Deutsche Forschungsgemeinschaft Corresponding author e mail lubo andrew2 stanford edu fax 650 325 6857 has radically changed Chromosome walks formerly the most tedious and technically problematic steps in the process are a thing of the past Molecular mapping can be vastly enhanced by systematically exploiting the available sequence information As a consequence it now takes just a few rather basic molecular biology routines and as little as a few months to isolate almost any mutation that can be mapped In this paper we discuss how to approach a positional cloning project in Arabidopsis with an emphasis on techniques that have been found to be reliable simple and inexpensive Protocols for many of the procedures mentioned have been deposited on an accompanying Web page Table I Our hope is to convince researchers who have little or no experience in positional cloning that now is a good time to capitalize on the achievements of the Arabidopsis Genome Initiative and find out what your favorite mutant is all about MAPPING RESOLUTION In contrast to gene tagging positional or mapbased cloning is an essentially indirect approach mapping will narrow down the genetic interval containing a mutation by successively excluding all other parts of the genome Once an interval is defined other criteria have to be employed to find out which of the genes within the interval is mutated Naturally this is easier if the interval is small or the mapping resolution is high For the purpose of this paper we will refer to the mapping resolution as the average distance between two recombination breakpoints in a given mapping population In Arabidopsis a genetic distance of 1 recombination corresponds on average to a physical distance of about 250 kb However the ratio between genetic and physical distance is by no means constant and it varies with respect to position on the chromosome as well Plant Physiology July 2000 Vol 123 pp 795 805 www plantphysiol org 2000 American Society of Plant Physiologists 795 Lukowitz et al Table I Internet resources Web Site Supplemental material for this paper Nottingham Stock Centre U K Recombinant Inbred map Ohio Stock Center U S A TAIR databasea homepage Recombinant Inbred map mirror site CAPS markers Sequence table SNP collection CEREON collection of polymorphisms SSLP markers TIGR genome annotations Database of Ler sequences Kasuza DNA Research Institute genome annotations MIPS genome annotations SINS database of transposon insertions Internet Address http carnegiedpb stanford edu methods ppsuppl html http nasc nott ac uk http nasc nott ac uk new ri map html http aims cps msu edu aims http www arabidopsis org http www arabidopsis org cgi bin maps Riintromap http www arabidopsis org aboutcaps html http www arabidopsis org cgi bin maps Seqtable pl http www arabidopsis org SNPs html http www arabidopsis org cereon http genome bio upenn edu SSLP info SSLP html http www tigr org tdb athl htmls index html http www tigr org tdb atgenome Ler html http www kazusa or jp kaos http websvr mips biochem mpg de proj thal http www jic bbsrc ac uk sainsbury lab jonathan jones jjhome htm a The Arabidopsis Information Resource TAIR monitors a large variety of information relevant for researchers working with Arabidopsis and provides links to most of the web sites listed here TAIR plans to incorporate all available information about polymorphic sequences and molecular markers at one central location in the future as with respect to different mapping populations For example a substantial difference was observed in two mapping populations involving