Identification and characterization of a putative light-harvesting chlorophyll a/b-binding protein gene encoded at a fertility restorer locus for the Ogura CMS in Brassica napus L.

Abstract 

The nuclear-encoded fertility restorer locus for Ogura radish cytoplasmic male sterility (CMS) was previously transferred from radish into Brassica napus through intergeneric crosses, with the putative restorer gene(s) being expressed in leaves and flower buds. To identify transcripts corresponding to this restorer locus, we performed a PCR-selected cDNA subtraction using leaf mRNA from the restorer line and its recurrent parent. The resulting library was differentially screened using the subtracted cDNAs for the library itself and the cDNAs from the reverse subtraction (recurrent parent subtracted against the restorer line). Forty-eight cDNA clones were isolated based on their specific hybridization with cDNAs for the library construction. Sequence analyses revealed that these 48 clones correspond to only three different genes: two light-harvesting chlorophyll a/b-binding protein (Cab) genes and one chloroplast-encoded gene (a chloroplast 50S ribosomal protein gene). Of the two putative Cab genes, one showed polymorphism on the Southern blot with the EcoRI-digested genomic DNA from the restorer line and its recurrent parent. Segregation analyses confirmed that this putative Cab gene comes from radish DNA and co-segregates with the restorer locus. A sequence comparison of this putative Cab gene with its homeolog in Brassica napus revealed that (1) their coding regions share a 94% similarity, (2) they differ in the size of the intron, and (3) they are identical in the deduced amino acid sequences, except that the one encoded at the restorer locus contained an additional alanine at position 34.