Rare germinal unequal crossing-over leading to recombinant gene formation and gene duplication in Arabidopsis thaliana

Small, multigene families organized in a tandem array can facilitate the ra pid evolution of the gene cluster by a process of meiotic unequal crossing- over. To study this process in a multicellular organism, we created a synth etic RBCSB gene cluster in Arabidopsis thaliana and used this to measure di rectly the Frequency of meiotic, intergenic unequal crossing-over between s ister chromatids, The synthetic RBCSB gene cluster was composed of a silent Delta RBCS1B::LUC chimeric gene fusion, lacking all 5' transcription and t ranslation signals, followed by RBCS2B and RBC3B genomic DNA. Expression of luciferase activity (luc(+)) required a homologous recombination event bet ween the Delta RBCS1B::LUC and the RBCS3B genes, yielding a novel recombina nt RBCS3B/1B:LUC chimeric gene whose expression was driven by RBCS3B 5' tra nscription and translation signals. Using sensitive, single-photon-imaging equipment, three luc(+) seedlings were identified in more than 1 million F2 seedlings derived from self-fertilized F1 plants hemizygous for the synthe tic RBCSB gene cluster. The F2 luc(+) seedlings were isolated, and molecula r and genetic analysis indicated that the luc(+) trait was caused by the Fo rmation of a recombinant chimeric RBCS3B/1B::LUC gene. A predicted duplicat ion of the RBCS2B gene also was present. The recombination resolution break points mapped adjacent to a region of intron I at which a disjunction in s equence similarity between RBCS1B and RBCS3B occurs; this provided evidence supporting models of gene cluster evolution by exon-shuffling processes. I n contrast to most measures of meiotic unequal crossing-over that require t he deletion of a gene in a gene cluster, these results directly measured th e frequency of meiotic unequal crossing over (approximate to 3 x 10(-6)), l eading to the expansion of the gene cluster and the formation of a novel re combinant gene.