Isolation and identification of the pollen-expressed polymorphic F-box genes linked to the S-locus in apple (Malus × domestica)

Abstract

Using degenerate primers designed based on the conserved regions of the reported SLF/SFB genes, more than a dozen of different cDNA clones were isolated from pollen of three different apple S-genotypes by reverse transcription (RT)-PCR. The 5¢-and 3¢-rapid amplification of cDNA end in combination with RT-PCR allowed isolation of two fulllength cDNAs, whose corresponding genes (RJ28-2 and Mg15, later renamed SLF 1 and SLF 2 ) were specifically expressed in pollen in an S-haplotype-specific manner. PCR analysis of seven different cultivars further showed the linkage between SLF 1 and S 1 -RNase and between SLF 2 and S 2 -RNase. The predicted ORFs of the two genes encode two F-box proteins of 393 amino acids in length with 70% amino acid identity. These features are consistent with the supposition that SLF 1 and SLF 2 are good candidates for pollen S-genes. Phylogenetic trees for the SLF/SFB and S-RNase proteins reported in the Solanaceae, Scrophulariaceae and Rosaceae (including apple) were constructed. It was found that the SLFs and S-RNases from apple were more closely related to those from Petunia (a genus of the Solanaceae) and Antirrhinum (a genus of the Scrophulariaceae) than to those from Prunus (a genus of the Rosaceae), implying a potential co-evolution between SLF/SFB and S-RNase. Furthermore, six SLF-like genes that shared a high level of sequence similarity (amino acid identity from 68 to 72%) to SLFs were also isolated. Among them, RJ28, Jt24 and Jt24-4 were confirmed to be expressed specifically in pollen in an S-haplotype-unspecific manner. These results are discussed in relation to the possible evolution of the SLF/SFB in S-RNase-based self-incompatible species.

Molecular Basis of Self-(in)compatibility and Current Status of S-genotyping in Rosaceous Fruit Trees

“…Similarly, a pollen-S candidate has been identified by sequencing a 317-kb region of the S 9 -locus in apple and PCR with degenerate primers derived from the conserved regions of Prunus pollen-S (Cheng et al, 2006). Although Cheng et al (2006) isolated a single Slocus-linked F-box gene from different S-alleles, Sassa et al (2007) isolated multiple F-box genes (SFBBs) in a genome region that encodes a certain S-haplotype. SFBBs are specifically expressed in pollen, and variable regions of SFBBs are under positive selection.…”

Identification and characterization of S-RNase genes and S-genotypes in Prunus and Malus species

“…However, genes for the pollen S-determinant in Malus species, belonging to the Maloideae subfamily, have not been functionally characterized (Cheng et al 2006;Kakui et al 2007Kakui et al , 2011Okada et al 2008Okada et al , 2011Sassa et al 2007Sassa et al , 2010.…”

Apple Pollination Biology for Stable and Novel Fruit Production: Search System for Apple Cultivar Combination Showing Incompatibility, Semicompatibility, and Full-Compatibility Based on theS-RNaseAllele Database

“…The gametophytic self-incompatibility (GSI) system in apple is controlled by a multiallelic single locus, called Slocus [13,14]. The genes responsible for apple GSI, (S-RNase as the pistil factor and SFB/SLF as the pollen factor) have been cloned [15,16]. Both, S-RNase and SFB/SLF are located at the S-locus, and their combined alleles are called S-haplotype for they are tightly linked.…”

“…In the case of commercial cultivars, the central flower opened first followed by the laterals [8]. I observed that the opening of the central flower of "Fuji" was between half a day and one day earlier than that of the laterals at Nagoya, Japan, in 2013 (the temperature during the flowering period at day time was [13][14][15][16][17][18][19][20][21][22][23] ∘ C). Also in the case of "Maypole, " the central flower opened first at all 5 to 7 flower clusters investigated.…”