Abstract
In Malus × domestica (Rosaceae) the product of each SFBB gene (the pollen component of the gametophytic self-incompatibility (GSI) system) of a S-haplotype (the combination of pistil and pollen genes that are linked) interacts with a sub-set of non-self S-RNases (the pistil component), but not with the self S-RNase. To understand how the Malus GSI system works, we identified 24 SFBB genes expressed in anthers, and determined their gene sequence in nine M. domestica cultivars. Expression of these SFBBs was not detected in the petal, sepal, filament, receptacle, style, stigma, ovary or young leaf. For all SFBBs (except SFBB15), identical sequences were obtained only in cultivars having the same S-RNase. Linkage with a particular S-RNase was further established using the progeny of three crosses. Such data is needed to understand how other genes not involved in GSI are affected by the S-locus region. To classify SFBBs specificity, the amino acids under positive selection obtained when performing intra-haplotypic analyses were used. Using this information and the previously identified S-RNase positively selected amino acid sites, inferences are made on the S-RNase amino acid properties (hydrophobicity, aromatic, aliphatic, polarity, and size), at these positions, that are critical features for GSI specificity determination.
Highlights
Gametophytic self-incompatibility (GSI), the most common reproductive system in flowering plants, is a pre-zygotic genetic mechanism that prevents self-fertilization and promotes out-crossing, by enabling the pistil to reject pollen from genetically related individuals[2]
Because the main goal of this work was to identify as many as possible candidate SFBB genes involved in pollen specificity through transcriptome sequencing of anthers, we first assessed the coverage of the transcriptomes used (Supplementary Table S1)
According to the accumulation curve obtained for the nine anthers transcriptomes, the number of expressed Malus CDS detected in the sample increases at a slower rate after 6000000 paired reads (Supplementary Fig. S1), suggesting that the sampling is sufficient for the discovery of new SFBB genes
Summary
Gametophytic self-incompatibility (GSI), the most common reproductive system in flowering plants (see Fig. 1 in Igic et al.1), is a pre-zygotic genetic mechanism that prevents self-fertilization and promotes out-crossing, by enabling the pistil to reject pollen from genetically related individuals[2]. Using the predicted tertiary structure of S-RNases and SFBBs and their binding energies, based on the Wilcoxon rank-sum test, when the hypervariable region of the S-RNase is considered, it has been shown that SFBBs of a S-haplotype interact more strongly with non-self than with self S-RNases[42] It seems that in Malus the GSI system works in a similar way to that of Petunia. Furtermore, in Petunia, the study of 12 homozygous plants, using a combination of next-generation sequencing (from mature pollen and unopened mature anthers) and PCR techniques, revealed that the number of SLF genes per S-haplotype varies from 16 to 2020 These genes define 18 specificity types, and within each type, variation in terms of copy number and amino acid sequence polymorphism was found.
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