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Title: Sequence analysis of the Petunia inflata S ‐locus region containing 17 S‐Locus F‐Box genes and the S‐RNase gene involved in self‐incompatibility

Self‐incompatibility inPetuniais controlled by the polymorphicS‐locus, which containsS‐RNaseencoding the pistil determinant and 16–20S‐locus F‐box(SLF) genes collectively encoding the pollen determinant. Here we sequenced and assembled approximately 3.1 Mb of theS2‐haplotype of theS‐locus inPetunia inflatausing bacterial artificial chromosome clones collectively containing all 17SLFgenes,SLFLike1, andS‐RNase. TwoSLFpseudogenes and 28 potential protein‐coding genes were identified, 20 of which were also found at theS‐loci of both theS6a‐haplotype ofP. inflataand theSN‐haplotype of self‐compatiblePetunia axillaris, but not in theS‐locus remnants of self‐compatible potato (Solanum tuberosum) and tomato (Solanum lycopersicum). Comparative analyses ofS‐locus sequences of these threeS‐haplotypes revealed potential genetic exchange in the flanking regions ofSLFgenes, resulting in highly similar flanking regions between different types ofSLFand between alleles of the same type ofSLFof differentS‐haplotypes. The high degree of sequence similarity in the flanking regions could often be explained by the presence of similar long terminal repeat retroelements, which were enriched at theS‐loci of all threeS‐haplotypes and in the flanking regions of allS‐locus genes examined. We also found evidence of the association of transposable elements withSLFpseudogenes. Based on the hypothesis thatSLFgenes were derived by retrotransposition, we identified 10F‐boxgenes as putativeSLFparent genes. Our results shed light on the importance of non‐coding sequences in the evolution of theS‐locus, and on possible evolutionary mechanisms of generation, proliferation, and deletion ofSLFgenes.

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Journal Name:
The Plant Journal
Page Range / eLocation ID:
p. 1348-1368
Medium: X
Sponsoring Org:
National Science Foundation
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  1. Self-incompatibility (SI), an inbreeding-preventing mechanism, is regulated in Petunia inflata by the polymorphic S-locus, which houses multiple pollen-specific S-locus F-box (SLF) genes and a single pistil-specific S-RNase gene. S2-haplotype and S3-haplotype possess the same 17 polymorphic SLF genes (named SLF1 to SLF17), and each SLF protein produced in pollen is assembled into an SCF (Skp1–Cullin1– F-box) E3 ubiquitin ligase complex. A complete suite of SLF proteins is thought to collectively interact with all non-self S-RNases to mediate their ubiquitination and degradation by the 26S proteasome, allowing cross-compatible pollination. For each SCFSLF complex, the Cullin1 subunit (named PiCUL1-P) and Skp1 subunit (named PiSSK1), like the F-box protein subunits (SLFs), are pollen-specific, raising the possibility that they also evolved specifically to function in SI. Here we used CRISPR/Cas9-meditated genome editing to generate frame-shift indel mutations in PiSSK1, and examined the SI behavior of a T0 plant (S2S3) with biallelic mutations in the pollen genome and two progeny plants (S2S2) each homozygous for one of the indel alleles and not carrying the Cas9-containing T-DNA. Their pollen was completely incompatible with pistils of seven otherwise compatible S-genotypes, but fully compatible with pistils of an S3S3 transgenic plant in which production of S3-RNase was completely suppressed by an antisense S3-RNase gene, and with pistils of immature flower buds, which produce little S-RNase. These results suggest that PiSSK1 specifically functions in SI, and support the hypothesis that SLF-containing SCF complexes are essential for compatible pollination. 
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  2. Summary

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