Rice Genetics IV - IRRI books - International Rice Research Institute

Although these mutants are useful for identifying single-gene defects, their diversegenetic backgrounds make them less suitable for the systematic comparison of biologicalvariation. Thus, a comprehensive mutant population in an identical geneticbackground is needed for phenotyping and functional assignment of rice sequences.To begin the mutant project, we evaluated several mutagens that would give sizabledeletions that could be physically detected by reverse genetics (Aguirrezabalagaet al 1995, Yandell et al 1994). We selected diepoxybutane (DEB), fast neutron, andgamma ray to build the mutation collection based on such properties (Bruggemann etal 1996, Reardon et al 1987, Shirley et al 1992). For instance, DEB is known toproduce kilobase range deletions in plants and Drosophila, whereas fast neutron isexpected to produce larger deletions and translocations. The requirement to producelarge deletions, however, is no longer necessary with the development of a highthroughputtechnique to detect single-base changes (McCallum et al 2000a; see discussionbelow). Our goal is to develop a collection of mutants with a range of deletionsizes and point mutations that are suitable for both forward and reverse genetics.Prior to the production of IR64 mutants, several mutant collections were made toidentify specific genes. G. Khush and D. Brar (unpublished) produced an ethylmethanesulfonate-induced mutant population of IR36 in an attempt to select for mutationsrelated to apomixis. P. Ronald, University of California-Davis, made anothercollection in IRBB21 using DEB and fast neutron. The IRBB21 mutants carry Xa21in an IR24 background, providing the materials for investigating genes involved inthe Xa21 gene-mediated resistance pathway (see Wang et al, this volume).We have used the IRBB21 mutant collection to establish the parameters in screeningfor disease-response mutants and to determine the molecular events associated withthe mutations. Approximately 4,000 IRBB21 M 2 lines were screened for a changefrom resistance to susceptibility against the bacterial pathogen strain PXO99 (avirulentto Xa21). We recovered 31 mutants that have become fully susceptible (10 mutants)or partially susceptible (21 mutants) to nine races of the bacterial blight pathogen.Southern and polymerase chain reaction (PCR) analyses of the molecular changesat the Xa21 locus revealed two classes of fully susceptible mutants: those with a lossof both the kinase domain and leucine-rich repeats and those with rearrangements inthe kinase domain. Based on the presence or absence of a marker flanking Xa21, weestimated that some deletions are >100 kb whereas others are under 10 kb, consistentwith those reported in mutagenesis of other eukaryotes.For the production of IR64 mutants, our target is to produce 40,000 independentlines to give a high probability of inducing a mutation in most genes (except homozygouslethals). Following the calculation of Krysan et al (1996) for T-DNA tagging inArabidopsis, the probability of inducing a mutation in a gene in a given populationsize can be estimated. Assuming that the average size of rice genes is approximately3.2 kb (as in Arabidopsis), the rice genome of 430,000 kb could be imagined as 134,375targets (430,000/3.2). The mutation rate of 1/1,000 observed in the rice mutants suggeststhat each mutant probably harbors 10–20 mutated sites. Thus, estimating conservatively,10,000 mutant lines would carry 10 × 10,000 mutated sites. We can applythe formula of P = 1 – (1 – f) N to determine P, the probability of any gene beingDeletion mutants for functional genomics: . . . 241