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In Brassicaceae self‐incompatibility (SI), self‐pollen rejection is initiated by the S‐haplotype specific interactions between the pollen S cysteine‐rich/S‐locus protein 11 (SCR/SP11) ligands and the stigma S receptor kinases (SRK). In Brassica SI, a member of the Plant U‐Box (PUB) E3 ubiquitin ligases, ARM‐repeat containing 1 (ARC1), is then activated by SRK in this stigma and cellular events downstream of this cause SI pollen rejection by inhibiting pollen hydration and pollen tube growth. During the transition to selfing, Arabidopsis thaliana lost the SI components, SCR, SRK, and ARC1. However, this trait can be reintroduced into A. thaliana by adding back functional copies of these genes from closely related SI species. Both SCR and SRK are required for this, though the degree of SI pollen rejection varies between A. thaliana accessions, and ARC1 is not always needed to produce a strong SI response. For the A. thaliana C24 accession, only transforming with Arabidopsis lyrataSCR and SRK confers a strong SI trait (SI‐C24), and so here, we investigated if ARC1‐related PUBs were involved in the SI pathway in the transgenic A. thaliana SI‐C24 line. Two close ARC1 homologs, PUB17 and PUB16, were selected, and (Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR‐associated protein 9 (Cas9) technology was used to generate pub17 and pub16 mutations in the C24 accession. These mutants were then crossed into the transgenic A. thaliana SI‐C24 line and their potential impact on SI pollen rejection was investigated. Overall, we did not observe any significant differences in SI responses to implicate PUB17 and PUB16 functioning in the transgenic A. thaliana SI‐C24 stigma to reject SI pollen. [ABSTRACT FROM AUTHOR]

Brassica napus is an amphidiploid species with the A genome from Brassica rapa and the C genome from Brassica oleracea. Although B. rapa, B. oleracea and artificially synthesized amphidiploids with the AC genome are self-incompatible, B. napus is self-compatible. Six S genotypes were identified in B. napus, five of which had class I S haplotypes from one species and a class II S haplotype from the other species, and mutations causing self-compatibility were identified in three of these S genotypes. The most predominant S genotype ( BnS-1;BnS-6), which is that of cv. ‘Westar’, had a class I S haplotype similar to B. rapa S-47 ( BrS-47) and a class II S haplotype similar to B. oleracea S-15 ( BoS-15). The stigmas of ‘Westar’ rejected the pollen grains of both BrS-47 and BoS-15, while reciprocal crossings were compatible. Insertion of a DNA fragment of about 3.6 kb was found in the promoter region of the SP11/SCR allele of BnS-1, and transcripts of SP11/SCR were not detected in ‘Westar’. The nucleotide sequence of the SP11 genomic DNA of BnS-6 was 100% identical to that of SP11 of BoS-15. Class I SP11 alleles from one species showed dominance over class II SP11 alleles from the other species in artificially synthesized B. napus lines, suggesting that the non-functional dominant SP11 allele suppressed the expression of the recessive SP11 allele in ‘Westar’. Two other S genotypes in B. napus also had non-functional class I S haplotypes together with recessive BnS-6. These observations suggest independent origins of self-compatibility in B. napus. [ABSTRACT FROM AUTHOR]

SummaryThe determinants of recognition specificity of self-incompatibility in Brassica are SRK in the stigma and SP11/SCR in the pollen, respectively. In the pair of S haplotypes BrS 46 (S 46 in B. rapa ) and BoS 7 (S 7 in B. oleracea ), which have highly similar SRK alleles, the SP11 alleles were found to be similar, with 96.1% identity in the deduced amino acid sequence. Two other pairs of S haplotypes, BrS 47 and BoS 12 , and BrS 8 and BoS 32 , having highly similar SRK and SP11 alleles between the two species were also found. The haplotypes in each pair are considered to have been derived from a single S haplotype in the ancestral species. The allotetraploid produced by interspecific hybridization between homozygotes of BrS 46 and BoS 15 showed incompatibility with a BoS 7 homozygote and compatibility with other B. oleracea S haplotypes in reciprocal crossings. This result indicates that BrS 46 and BoS 7 have maintained the same recognition specificity after the divergence of the two species and that amino acid substitutions found in such cases in both SRK alleles and SP11 alleles do not alter the recognition specificity. DNA blot analysis of SRK, SP11, SLG and other S -locus genes showed different DNA fragment sizes between the interspecific pairs of S haplotypes. A much lower level of sequence similarity was observed outside the genes of SRK and SP11 between BrS 46 and BoS 7 . These results suggest that the DNA sequences of the regions intervening between the S -locus genes were diversified after or at the time of speciation. This is the first report demonstrating the presence of common S haplotypes in different plant species and presenting definite evidence of the trans-specific evolution of self-incompatibility genes. [ABSTRACT FROM AUTHOR]