Sequence variability and gene structure at the self-incompatibility locus of Solanum tuberosum

Summary Allelic complexity is a key feature of self-incompatibility (S) loci in gametophytic plants. We describe in this report the allelic diversity and gene structure of the S locus in Solanum tuberosum revealed by the isolation and characterization of genomic and cDNA clones encoding S-associated major pistil proteins from three alleles (S ₁, S _r1, S ₂). Genomic clones encoding the S₁ and S₂ proteins provide evidence for a simple gene structure: Two exons are separated by a small intron of 113 (S ₁) and 117 by (S ₂). Protein sequences deduced from cDNA clones encoding S₁ and S_r1 proteins show 95% homology. 15 of the 25 residues that differ between these S ₁and S _r1alleles are clustered in a short hypervariable protein segment (amino acid positions 44–68), which corresponds in the genomic clones to DNA sequences flanking the single intron. In contrast, these alleles are only 66% homologous to the S ₂allele, with the residues that differ between the alleles being scattered throughout the sequence. DNA crosshybridization experiments identify a minimum of three classes of potato S alleles: one class contains the alleles S ₁, S _r1and S ₃, the second class S ₂and an allele of the cultivar Roxy, and the third class contains at present only S ₄. It is proposed that these classes reflect the origin of the S alleles from a few ancestral S sequence types.     

A cDNA encoding an S-locus specific glycoprotein from Brassica oleracea plants containing the S5 self-incompatibility allele

Summary A cDNA sequence homologous to the Brassica self-incompatibility locus specific glycoprotein (SLSG) sequence was isolated from stigmas of B. oleracea plants homozygous for the S₅ allele. The nucleotide sequence of this cDNA was obtained and compared with the S₆ allelic form of the SLSG. Evidence is presented which indicates that this sequence does not specify the self-incompatibility response of pollen.Abbreviations SDS sodium dodecyl sulphate - PVP polyvinylpyrrolidone - BSA bovine serum albumin - SLSG self-incompatibility locus specific glycoprotein     

Physical linkage of the SLG and SRK genes at the self-incompatibility locus of Brassica oleracea

Summary In Brassica oleracea, the pollen-stigma interaction of self-incompatibility is controlled by a single genetically defined locus designated S. Molecular studies have identified two genes that are tightly linked to the classically defined S locus: The S-Locus Glycoprotein (SLG) gene and the S-Receptor Kinase (SRK) gene. In previous RFLP linkage analyses with probes specific for SLG and SRK, we were unable to identify any recombination events between SLG, SRK, and self-incompatibility phenotype. In this paper, we use pulsed-field gel electrophoresis (PFGE) in conjunction with DNA blot analysis to characterize the S-locus region from two highly divergent self-incompatibility genotypes, S ₂ and S ₆. We establish the physical linkage of SLG and SRK in each genotype, and demonstrate that the two genes are separated by a maximum distance of 220 kb in the S ₆ genotype and 350 kb in the S ₂ genotype. Furthermore, a comparison of the data from the two genotypes reveals that a high level of polymorphism exists across the entire S-locus region.     

Transformation of Brassica oleracea with an S-locus gene from B. campestris changes the self-incompatibility phenotype

Summary An SLG gene derived from the S-locus and encoding and S-locus-specific glycoprotein of Brassica campestris L. was introduced via Agrobacterium-mediated transformation into B. oleracea L. A self-incompatible hybrid and another with partial self-compatibility were used as recipients. The transgenic plants were altered in their pollen-stigma interaction and were fully compatible upon self-pollination. Reciprocal crosses between the transgenic plants and untransformed control plants indicated that the stigma reaction was changed in one recipient strain while the pollen reaction was altered in the other. Due to interspecific incompatibility, we could not demonstrate whether or not the introduced SLG gene confers a new allelic specificity in the transgenic plants. Our results show that the introduced SLG gene perturbs the self-incompatibility phenotype of stigma and pollen.     

PCR detection of transcripts homologous to the self-incompatibility gene in anthers ofBrassica

The polymerase chain reaction (PCR) is particularly well suited for the detection of rare sequences. Taking advantage of the recent isolation of sequences associated with stigma self-incompatibility inBrassica oleracea, we used PCR amplifications with primers synthesized to the S6 cDNA sequence, to demonstrate the presence of mRNA homologous to stigmaS-locus gene (SLG) in anthers during early microsporogenesis. In addition, otherS-locus-related (SLR) sequences were shown to be transcribed in sexual as well as in vegetative tissues (roots, leaves), suggesting that the SLG family might be involved not only in pollen-stigma recognition, but more generally in various forms of plant cell signalling processes. This information corroborates the recent discovery of a cDNA-deduced protein kinase from maize roots, whose extracellular receptor displays high homology withBrassica S-locus-specific glycoproteins.Communicated by H.F. Linskens     

The molecular biology of self-incompatibility systems in flowering plants

Self-incompatibility is a common mechanism by which flowering plants can exert some control over the process of fertilization. Typically, the self-incompatibility response involves the recognition and rejection of self-incompatible pollen which leads to a block in self-fertilization and, as a consequence, promotes outcrossing. In recent years, considerable progress has been made in the molecular understanding of several self-incompatibility systems. Interestingly, a common mechanism for self-incompatibility is not employed by all flowering plants, but in fact quite diverse mechanisms have been recruited for the rejection of self-incompatible pollen. In this review, the recent advances in the self-incompatibility systems of the Solanaceae, Papaveraceae, and Brassicaceae will be described as well as some of the molecular work that is emerging for the Poaceae and the heteromorphic self-incompatibility systems.     

Sequence complexity of the S receptor kinase gene family in Brassica

A genomic library from an S ₂₉/S ₂₉ self-incompatible genotype of Brassica oleracea was screened with a probe carrying part of the catalytic domain of a Brassica S-receptor kinase (SRK)-like gene. Six positive phage clones with varying hybridisation intensities (K1 to K6) were purified and characterised. A 650–700 by region corresponding to the probe was excised from each clone and sequenced. DNA and predicted protein sequence comparisons based on a multiple alignment identified K5 as a pseudogene, whereas the others could encode functional proteins. K3 was found to have lost an intron from its genomic sequence. The six genes display different degrees of sequence similarity and form two distinct clusters in a dendrogram. The 98% similarity between K4 and K6, which extends across intron sequences, suggests that these might be very recently diverged alleles or daughters of a duplication. In addition, K2 showed a comparably high similarity to the probe. Clones K1, K3 and K5 cross-hybridised with an SLG ₂₉ cDNA probe, indicating the presence of upstream receptor domains homologous to the Brassica SLG gene. This suggests that the previously reported S sequence complexity may be ascribed to a large receptor kinase gene family.     

Effect of hexane and humidity on self-incompatibility in Brassica oleracea

Summary The effects of hexane, high humidity, flower age and temperature in overcoming the self-incompatibility of Brassica oleracea were studied using three plants, each of which was homozygous for a different dominant S-allele. Hexane had a significant effect in all cases, but the size of the effect varied considerably. In one plant there was a marked interaction between the effect of hexane, humidity and flower age, but temperature had relatively little effect. In another plant high humidity alone gave a very much greater response than hexane alone. This plant gave as many self-seeds from the high humidity treatment as from bud selfing, indicating that the incompatibility reaction was almost completely overcome by the high humidity. The results are discussed in the light of current views of the mechanism of incompatibility in Brassica.     

Use of the polymerase chain reaction to isolate an S-locus glycoprotein cDNA introgressed from Brassica campestris into B. napus ssp. oleifera.

Summary A self-incompatible canola-quality Brassica napus ssp. oleifera line (W1) was generated by introgressing the S-locus from a self-incompatible B. campestris plant into the Westar cultivar. Using the polymerase chain reaction (PCR) with primers derived from conserved regions in S-locus glycoprotein (SLG) alleles, the central region of the active SLG gene (910) was obtained. The remaining portions of the cDNA for this 910 gene were subsequently cloned using the PCR-rapid amplification of cDNA ends (RACE) procedure. Sequence analysis revealed that the 910 cDNA show a high degree of sequence similarity to SLG alleles associated with Class I self-incompatible lines. The 910 gene was found to be absent in the original self-compatible cv. Westar (B. napus) and segregated with self-incompatibility in a mixed population generated from a cross between self-incompatible W1 and self-compatible Westar. RNA blot analysis indicated that high levels of 910 mRNAs were present in the stigma as buds approached anthesis. Thus, the SLG allele of W1 transferred from B. campestris via backcrosses to a line of cv. Westar has been identified.     

Characterization of proteins associated with self-incompatibility in Solanum tuberosum

Summary The gametophytic self-incompatibility system of Solarium tuberosum is controlled by a single locus, designated as the S-locus. Protein extracts from potato styles of defined S-genotypes have been analysed by two-dimensional gel electrophoresis, and found to contain a group of basic glycoproteins. Each genetically determined allele S ₁ to S ₄ was associated with the presence of one of a number of these polypeptides differing slightly in isoelectric points (in the range 8.3–>9.1) and/or apparent molecular weight (ranging from 23,000 to 29,000). Two abundant basic polypeptides, one of which is apparently not glycosylated, were present in all genotypes examined. Amino-terminal protein sequence determinations revealed homologies of the S. tuberosum stylar proteins S₂, S₃ and S₄ with SI-associated polypeptides from Nicotiana alata and Lycopersicon peruvianum. With an oligonucleotide generated to the potato-S₂ N-terminal protein sequence, it was possible to detect a style-specific RNA species of 920 nucleotides. The oligonucleotide also behaved as an allele-specific probe when hybridized to total RNA of different S-genotypes.     

A glycoprotein associated with the acquisition of the self-incompatibility system by maturing stigmas of Brassica oleracea

Iso-electric focusing of extracts derived from stigmatic homogenates of Brassica oleracea reveals that the mature stigma possesses large quantities of a glycoprotein not present in earlier stages of development in the bud. Pollen germination experiments carried out in parallel with the biochemical tests suggest that the appearance of this glycoprotein, which has an isoelectric point of pH 5.8, is coincident with the development of the self-incompatibility response. The site of this protein, and the role it may play in pollen-stigma interactions are discussed in terms of current models of the self-incompatibility system in Brassica.Abbreviations PAS periodic acid-schiffs - PEG polyethylene glycol - SI system self-incompatibility system     

Towards an in vitro bioassay for the self-incompatibility response in Brassica oleracea

Summary Eluates of stigmas of Brassica oleracea that were known to contain S locus-specific glycoproteins (SLSG) discriminated between self and cross pollen in vitro in three different media. Discrimination was equally evident in experiments that were the in vitro equivalents of reciprocal pollinations. In a TAPS-buffered medium, self eluates depressed pollen germination in a dose-dependent manner. TAPS medium allowed a bioassay of the effects of SLSG in eluates because it optimized germination in a way that eliminated the complicating features of the stimulatory substances in the eluates. Stigma eluates affected percentage pollen germination and optimum calcium concentrations in vitro whether or not SLSG were present in the eluates, but differently in different media, and depending on whether the eluates were cross or self with respect to the pollen tested. Thus, the effect of stigma eluates on the in vitro germination of pollen in Brassica depends on the balance of stimulatory versus inhibitory substances in the eluates.     

Two large Arabidopsis thaliana gene families are homologous to the Brassica gene superfamily that encodes pollen coat proteins and the male component of the self-incompatibility response

The male component of the self-incompatibility response in Brassica has recently been shown to be encoded by the S locus cysteine-rich gene (SCR). SCR is related, at the sequence level, to the pollen coat protein (PCP) gene family whose members encode small, cysteine-rich proteins located in the proteo-lipidic surface layer (tryphine) of Brassica pollen grains. Here we show that the Arabidopsis genome includes two large gene families with homology to SCR and to the PCP gene family, respectively. These genes are poorly predicted by gene-identification algorithms and, with few exceptions, have been missed in previous annotations. Based on sequence comparison and an analysis of the expression patterns of several members of each family, we discuss the possible functions of these genes. In particular, we consider the possibility that SCR-related genes in Arabidopsis may encode ligands for the S gene family of receptor-like kinases in this species.     

A modifier locus affecting the expression of the S-RNase gene could be the cause of breakdown of self-incompatibility in almond

Self-compatibility has become the primary objective of most almond (Prunus amygdalus Batsch) breeding programmes in order to avoid the problems related to the gametophytic self-incompatibility system present in almond. The progeny of the cross ‘Vivot’ (S ₂₃ S _fa) × ‘Blanquerna’ (S ₈ S _fi) was studied because both cultivars share the same S _f allele but have a different phenotypic expression: active (S _fa) in ‘Vivot’ and inactive (S _fi) in ‘Blanquerna’. In addition, the microscopic observation of pollen tube growth after self-pollination over several years showed an unexpected self-incompatible behaviour in most seedlings of this cross. The genotypes of this progeny showed that the S _fi pollen from ‘Blanquerna’ was not able to grow down the pistils of ‘Vivot’ harbouring the S _fa allele, confirming the active function of this allele against the inactive form of the same allele, S _fi. As self-compatibility was observed in some S ₈ S ₂₃ and S ₈ S _fa individuals of this progeny, the S _f haplotype may not always be linked to the expression and transmission of self-compatibility in almond, suggesting that a modifier locus may be involved in the mechanism of self-incompatibility in plants.