ENOD12, AN EARLY NODULIN GENE, IS NOT REQUIRED FOR NODULE FORMATION AND EFFICIENT NITROGEN-FIXATION IN ALFALFA

To demonstrate the importance of an extensively studied early nodulin gene ENOD12 in symbiotic nodule development, plants of different Medic ago sativa subspecies were tested for the presence or absence of ENOD1 2 alleles. In M. s. ssp coerulea w2 (Mcw2), two ENOD12 genes were dete cted, whereas in M. s. ssp quasifalcata k93 (Mqk93) only one gene was present. In both plants, the ENOD12 genes were expressed in nodules in duced by Rhizobium meliloti. The nucleotide sequence of the ENOD12 gen es showed that the two Mcw2-specific genes were similar to the ENOD12A and ENOD12B genes of the tetraploid M. s. ssp sativa. ENOD12 from Mqk 93 was similar to the corresponding gene found in M. truncatula. From the aligned ENOD12 sequences, an evolutionary tree was constructed. Ge netic analysis of the progenies of a cross between Mqk93 and Mcw2 show ed that several offspring in F1 carried a null allele originating from Mcw2, and among the F2 progenies, plants with the null allele only la cking the ENOD12 gene appeared. Surprisingly, the ENOD12-deficient pla nts were similar to their wild-type parents in viability, nodule devel opment, nodule structure, and nitrogen fixation efficiency. Therefore, we concluded that in Medicago the ENOD12 gene is not required for sym biotic nitrogen fixation. Furthermore, we proposed that the heterozygo us nature of these legumes can be exploited for the identification of mutated alleles of other known nodulin genes; this will permit the con struction of plant mutants deficient in these genes.