STRUCTURAL-ANALYSIS OF THE RDNA INTERGENIC SPACER OF BRASSICA-NIGRA -EVOLUTIONARY DIVERGENCE OF THE SPACERS OF THE 3 DIPLOID BRASSICA SPECIES

EcoRI restriction of the B. nigra rDNA recombinants, isolated from a l ambda genomic library, showed that the 3.9-kb fragment corresponded to the Intergenic Spacer (IGS), which was sequenced and found to be 3,92 8 bp in size. Sequence and dot-matrix analyses showed that the organiz ation of the B. nigra rDNA IGS was typical of most rDNA spacers, consi sting of a central repetitive region and flanking unique sequences on either side, The repetitive region was composed of two repeat families -RE 'A' and RF 'B.' The B. nigra RF 'A' consisted of a tandem array of three full-length copies of a 106-bp sequence element. RF 'B' was com posed of 66 tandemly repeated elements. Each 'B' element was only 21-b p in size and this is the smallest repeat unit identified in plant rDN A to date. The putative transcription initiation site (TIS) was identi fied as nucleotide position 3,110. Based on the sequence analysis it w as suggested that the present organization of the repeal families was generated by successive cycles of deletions and amplifications and was being maintained by homogenization processes such as gene conversion and crossing-over. A detailed comparison of the rDNA IGS sequences of the three diploid Brassica species-namely, B. nigra, B. campestris, an d B. oleracea-was carried out. First, comparisons revealed that B. cam pestris and B. oleracea were close to each other as the repeat familie s in both showed high sequence homology between each other. Second, th e repeat elements in both the species were organized in an intersperse d manner. Third, a 52-bp sequence, present just downstream of the repe ats in B. campestris, was found to be identical to the B. oleracea rep eats, thereby suggesting a common progenitor. On the other hand, in B. nigra no interspersion pattern of organization of repeats was observe d. Further, the B. nigra RF 'A' was identified as distinct from the re peat families of B. campestris and B. oleracea. Based on this analysis , it was suggested that during speciation B. campestris and B. olerace a evolved in one lineage whereas B. nigra diverged into a separate lin eage. The comparative analysis of the ICS helped in identifying not on ly conserved ancestral sequence motifs of possible functional signific ance such as promoters and enhancers, but also sequences which showed variation between the three diploid species and were therefore identif ied as species-specific sequences.