The self-compatibility is acquired after polyploidization: a case study of Brassica napus self-incompatible trilinear hybrid breeding system.

Self-incompatibility plays a vital role in angiosperms, by preventing inbreeding depression and maintaining genetic diversity within populations. Following polyploidization, many angiosperm species transition from self-incompatibility to self-compatibility. Here, we investigated the S-locus in Brassicaceae and identified distinct origins for the sRNA loci, SMI and SMI2 (SCR Methylation Inducer 1 and 2), within the S-locus. The SMI loci were found to be widespread in Cruciferae, whereas the SMI2 loci were exclusive to Brassica species. Additionally, we discovered four major S-haplotypes (BnS-1, BnS-6, BnS-7, and BnS-1300) in rapeseed. Overexpression of BnSMI-1 in self-incompatible Brassica napus ('S-70S1300S6 ') resulted in a significant increase in DNA methylation in the promoter regions of BnSCR-6 and BnSCR-1300, leading to self-compatibility. Conversely, by overexpressing a point mutation of BnSmi-1 in the 'S-70S1300S6 ' line, we observed lower levels of DNA methylation in BnSCR-6 and BnSCR-1300 promoters. Furthermore, the overexpression of BnSMI2-1300 in the 'SI-326S7S6 ' line inhibited the expression of BnSCR-7 through transcriptional repression of the Smi2 sRNA from the BnS-1300 haplotype. Our study demonstrates that the self-compatibility of rapeseed is determined by S-locus sRNA-mediated silencing of SCR after polyploidization, which helps to further breed self-incompatible or self-compatible rapeseed lines, thereby facilitating the utilization of heterosis.

Two aspartic proteases, BnaAP36s and BnaAP39s, regulate pollen tube guidance in Brassica napus.

Pollen tube (PT) growth towards the micropyle is critical for successful double fertilization. However, the mechanism of micropyle-directed PT growth is still unclear in Brassica napus. In this study, two aspartate proteases, BnaAP36s and BnaAP39s, were identified in B. napus. BnaAP36s and BnaAP39s were localized to the plasma membrane. The homologues of BnaAP36 and BnaAP39 were highly expressed in flower organs, especially in the anther. Sextuple and double mutants of BnaAP36s and BnaAP39s were then generated using CRISPR/Cas9 technology. Compared to WT, the seed-set of cr-bnaap36 and cr-bnaap39 mutants was reduced by 50% and 60%, respectively. The reduction in seed-set was also found when cr-bnaap36 and cr-bnaap39 were used as the female parent in a reciprocal cross assay. Like WT, cr-bnaap36 and cr-bnaap39 pollen were able to germinate and the relative PTs were able to elongate in style. Approximately 36% and 33% of cr-bnaap36 and cr-bnaap39 PTs, respectively, failed to grow towards the micropyle, indicating that BnaAP36s and BnaAP39s are essential for micropyle-directed PT growth. Furthermore, Alexander's staining showed that 10% of cr-bnaap39 pollen grains were aborted, but not cr-bnaap36, suggesting that BnaAP39s may also affect microspore development. These results suggest that BnaAP36s and BnaAP39s play a critical role in the growth of micropyle-directed PTs in B. napus. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-023-01377-1.

Comparative phosphoproteomic analysis of compatible and incompatible pollination in Brassica napus L.

Self-incompatibility (SI) promotes outbreeding and prevents self-fertilization to promote genetic diversity in angiosperms. Several studies have been carried to investigate SI signaling in plants; however, protein phosphorylation and dephosphorylation in the fine-tuning of the SI response remain insufficiently understood. Here, we performed a phosphoproteomic analysis to identify the phosphoproteins in the stigma of self-compatible 'Westar' and self-incompatible 'W-3' Brassica napus lines. A total of 4109 phosphopeptides representing 1978 unique protein groups were identified. Moreover, 405 and 248 phosphoproteins were significantly changed in response to SI and self-compatibility, respectively. Casein kinase II (CK II) phosphorylation motifs were enriched in self-incompatible response and identified 127 times in 827 dominant SI phosphorylation residues. Functional annotation of the identified phosphoproteins revealed the major roles of these phosphoproteins in plant-pathogen interactions, cell wall modification, mRNA surveillance, RNA degradation, and plant hormone signal transduction. In particular, levels of homolog proteins ABF3, BKI1, BZR2/BSE1, and EIN2 were significantly increased in pistils pollinated with incompatible pollens. Abscisic acid and ethephon treatment partially inhibited seed set, while brassinolide promoted pollen germination and tube growth in SI response. Collectively, our results provided an overview of protein phosphorylation during compatible/incompatible pollination, which may be a potential component of B. napus SI responses.

Mechanism of Salt-Induced Self-Compatibility Dissected by Comparative Proteomic Analysis in Brassica napus L.

Self-incompatibility (SI) in plants genetically prevents self-fertilization to promote outcrossing and genetic diversity. Its hybrids in Brassica have been widely cultivated due to the propagation of SI lines by spraying a salt solution. We demonstrated that suppression of Brassica napus SI from edible salt solution treatment was ascribed to sodium chloride and independent of S haplotypes, but it did not obviously change the expression of SI-related genes. Using the isobaric tags for relative and absolute quantitation (iTRAQ) technique, we identified 885 differentially accumulated proteins (DAPs) in Brassica napus stigmas of un-pollinated (UP), pollinated with compatible pollen (PC), pollinated with incompatible pollen (PI), and pollinated with incompatible pollen after edible salt solution treatment (NA). Of the 307 DAPs in NA/UP, 134 were unique and 94 were shared only with PC/UP. In PC and NA, some salt stress protein species, such as glyoxalase I, were induced, and these protein species were likely to participate in the self-compatibility (SC) pathway. Most of the identified protein species were related to metabolic pathways, biosynthesis of secondary metabolites, ribosome, and so on. A systematic analysis implied that salt treatment-overcoming SI in B.napus was likely conferred by at least five different physiological mechanisms: (i) the use of Ca2+ as signal molecule; (ii) loosening of the cell wall to allow pollen tube penetration; (iii) synthesis of compatibility factor protein species for pollen tube growth; (iv) depolymerization of microtubule networks to facilitate pollen tube movement; and (v) inhibition of protein degradation pathways to restrain the SI response.

Exo84c-regulated degradation is involved in the normal self-incompatible response in Brassicaceae.

The self-incompatibility system evolves in angiosperms to promote cross-pollination by rejecting self-pollination. Here, we show the involvement of Exo84c in the SI response of both Brassica napus and Arabidopsis. The expression of Exo84c is specifically elevated in stigma during the SI response. Knocking out Exo84c in B. napus and SI Arabidopsis partially breaks down the SI response. The SI response inhibits both the protein secretion in papillae and the recruitment of the exocyst complex to the pollen-pistil contact sites. Interestingly, these processes can be partially restored in exo84c SI Arabidopsis. After incompatible pollination, the turnover of the exocyst-labeled compartment is enhanced in papillae. However, this process is perturbed in exo84c SI Arabidopsis. Taken together, our results suggest that Exo84c regulates the exocyst complex vacuolar degradation during the SI response. This process is likely independent of the known SI pathway in Brassicaceae to secure the SI response.

Genetic and Molecular Characterization of a Self-Compatible Brassica rapa Line Possessing a New Class II S Haplotype.

Most flowering plants have evolved a self-incompatibility (SI) system to maintain genetic diversity by preventing self-pollination. The Brassica species possesses sporophytic self-incompatibility (SSI), which is controlled by the pollen- and stigma-determinant factors SP11/SCR and SRK. However, the mysterious molecular mechanism of SI remains largely unknown. Here, a new class II S haplotype, named BrS-325, was identified in a pak choi line '325', which was responsible for the completely self-compatible phenotype. To obtain the entire S locus sequences, a complete pak choi genome was gained through Nanopore sequencing and de novo assembly, which provided a good reference genome for breeding and molecular research in B. rapa. S locus comparative analysis showed that the closest relatives to BrS-325 was BrS-60, and high sequence polymorphism existed in the S locus. Meanwhile, two duplicated SRKs (BrSRK-325a and BrSRK-325b) were distributed in the BrS-325 locus with opposite transcription directions. BrSRK-325b and BrSCR-325 were expressed normally at the transcriptional level. The multiple sequence alignment of SCRs and SRKs in class II S haplotypes showed that a number of amino acid variations were present in the contact regions (CR II and CR III) of BrSCR-325 and the hypervariable regions (HV I and HV II) of BrSRK-325s, which may influence the binding and interaction between the ligand and the receptor. Thus, these results suggested that amino acid variations in contact sites may lead to the SI destruction of a new class II S haplotype BrS-325 in B. rapa. The complete SC phenotype of '325' showed the potential for practical breeding application value in B. rapa.

Joint genome-wide association and transcriptome sequencing reveals a complex polygenic network underlying hypocotyl elongation in rapeseed (Brassica napus L.).

Hypocotyl elongation is considered an important typical seedling trait contributing directly to an increase in and stabilization of the yield in Brassica napus, but its molecular genetic mechanism is poorly understood. In the present study, hypocotyl lengths of 210 lines were measured in an illuminated culture room. A genome-wide association study (GWAS) was performed with 23,435 single nucleotide polymorphisms (SNPs) for hypocotyl length. Three lines with long hypocotyl length and three lines with short hypocotyl length from one doubled haploid line (DH) population were used for transcriptome sequencing. A GWAS followed by transcriptome analysis identified 29 differentially expressed genes associated with significant SNPs in B. napus. These genes regulate hypocotyl elongation by mediating flowering morphogenesis, circadian clock, hormone biosynthesis, or important metabolic signaling pathways. Among these genes, BnaC07g46770D negatively regulates hypocotyl elongation directly, as well as flowering time. Our results indicate that a joint GWAS and transcriptome analysis has significant potential for identifying the genes responsible for hypocotyl elongation; The extension of hypocotyl is a complex biological process regulated by a polygenic network.