Cloning and Characterizing Two PpSFBB-Alpha Genes In Chinese Sand Pear (Pyrus pyrifolia Nakai.)

Gametophytic self-incompatibility (GSI) is genetically controlled mechanism to prevent inbreeding and promote out-crossing. It is controlled by a single multi-allelic S-locus, which contains two separate genes (a stylar S-RNase gene and a pollen S-gene). In gametophytic self-incompatible Japanese pear (Pyrus pyrifolia Nakai.) and apple (Malus domestica), three types of SFB genes (SFBB-alpha, SFBB-beta, and SFBB-gamma) have been isolated of which the SFBB-alpha genes were identified as the best candidates for pollen S-genes. To investigate sequence characteristics of the possible pollen S-gene and elucidate possible GSI mechanism of Chinese sand pear (P. pyrifolia), SFBB-alpha homologies in Chinese sand pear cultivars were cloned and characterized by degenerate primers, PSFBA-F and PSFBA-R, that were designed based on Japanese pear and apple SFBB-alpha sequence information. Results showed that the PSFBA-F and PSFBA-R primers successfully amplified a fragment of approximately 1,300 bp from ‘Maogong’ (S₁₂S₁₃), ‘Hongsucui’ (S₄S₁₂), ‘Tianchengzi’ (S₇S₁₂), and ‘Mantianhong’ (S₄S₁₂), corresponding to pear SFBB-alpha gene. A total of two distinct sequences derived from the 1,300 bp product were identified that were named PpSFBB12-alpha (P. pyrifolia SFBB12-alpha) and PpSFBB13-alpha, respectively. RT-PCR revealed that both PpSFBB-alpha genes were expressed specifically in the pollen grains. The coding region of PpSFBB12-alpha was 1,194 bp in length encoding 397 amino acids with predicted molecular weight of 45.8 kDa and isoeletric point of 5.03. The coding region of PpSFBB13-alpha was 1,179 bp in length encoding 392 amino acids with predicted molecular weight of 45.4 kDa and isoeletric point of 4.92. Both genes displayed normal structural characteristics of SFB/SLF genes, i.e. an F-box motif and four variable regions. At the deduced amino acid level, they shared 18.6% to 97.7% similarities with other SFB/SLFs of rosaceous plants. These characteristics of the two PpSFBB-alpha genes fully demonstrated that they are good candidates of pollen S-gene. Phylogenetic analysis revealed that 34 rosaceous SFB/SLFs were dived into two subfamily groups, but did not further form species subgroup. The evolutionary pattern of SFB/SLFs concurred with that of rosaceous S-RNases, suggesting that SFB/SLFs occurred after divergence of subfamily, but before the divergence of species as S-RNases in Rosaceae. The two PpSFBB-alpha sequence data should be useful for studying the interaction between SFBs and S-RNases and clarifying the mechanism of GSI at the molecular level in Chinese sand pear.