Related polymorphic F-box protein genes between haplotypes clustering in the BAC contig sequences around the S-RNase of Japanese pear.

Most fruit trees in the Rosaceae exhibit self-incompatibility, which is controlled by the pistil S gene, encoding a ribonuclease (S-RNase), and the pollen S gene at the S-locus. The pollen S in Prunus is an F-box protein gene (SLF/SFB) located near the S-RNase, but it has not been identified in Pyrus and Malus. In the Japanese pear, various F-box protein genes (PpSFBB(-α-γ)) linked to the S-RNase are proposed as the pollen S candidate. Two bacterial artificial chromosome (BAC) contigs around the S-RNase genes of Japanese pear were constructed, and 649 kb around S(4)-RNase and 378 kb around S(2)-RNase were sequenced. Six and 10 pollen-specific F-box protein genes (designated as PpSFBB(4-u1-u4, 4-d1-d2) and PpSFBB(2-u1-u5,) (2-d1-d5), respectively) were found, but PpSFBB(4-α-γ) and PpSFBB(2-γ) were absent. The PpSFBB(4) genes showed 66.2-93.1% amino acid identity with the PpSFBB(2) genes, which indicated clustering of related polymorphic F-box protein genes between haplotypes near the S-RNase of the Japanese pear. Phylogenetic analysis classified 36 F-box protein genes of Pyrus and Malus into two major groups (I and II), and also generated gene pairs of PpSFBB genes and PpSFBB/Malus F-box protein genes. Group I consisted of gene pairs with 76.3-94.9% identity, while group II consisted of gene pairs with higher identities (>92%) than group I. This grouping suggests that less polymorphic PpSFBB genes in group II are non-S pollen genes and that the pollen S candidates are included in the group I PpSFBB genes.

Deletion of a 236 kb region around S 4-RNase in a stylar-part mutant S 4sm-haplotype of Japanese pear.

Japanese pear (Pyrus pyrifolia Nakai) has a gametophytic self-incompatibility (GSI) mechanism controlled by a single S-locus with multiple S-haplotypes, each of which contains separate genes that determine the allelic identity of pistil and pollen. The pistil S gene is the S-ribonuclease (S-RNase) gene, whereas good candidates for the pollen S gene are the F-box protein genes. A self-compatible (SC) cultivar, 'Osa-Nijisseiki', which is a bud mutant of 'Nijisseiki' (S (2) S (4)), has a stylar-part mutant S(4)sm-haplotype, which lacks the S (4)-RNase gene but retains the pollen S gene. To delineate the deletion breakpoint in the S(4)sm-haplotype, we constructed a bacterial artificial chromosome (BAC) library from an S (4)-homozygote, and assembled a BAC contig of 570 kb around the S (4)-RNase. Genomic PCR of DNA from S (4)- and S(4)sm-homozygotes and the DNA sequence of the BAC contig allowed the identification of a deletion of 236 kb spanning from 48 kb upstream to 188 kb downstream of S (4)-RNase. The S(4)sm-haplotype lacks 34 predicted open reading frames (ORFs) including the S (4)-RNase and a pollen-specific F-box protein gene (termed as S (4) F-box0). Genomic PCR with a primer pair designed from the deletion junctions yielded a product specific for the S(4)sm-haplotype. The product could be useful as a maker for early selection of SC cultivars harboring the S(4)sm-haplotype.