Expression of a self-incompatibility gene in a self-compatible line of Brassica oleracea.

In cruciferous plants, self-pollination is prevented by the action of genes situated at the self-incompatibility locus or S-locus. The self-incompatibility reaction is associated with expression of stigma glycoproteins encoded by the S-locus glycoprotein (SLG) gene. Only a few cases of self-compatible plants derived from self-incompatible lines in the crucifer Brassica have been reported. In these cases, self-compatibility was generally ascribed to the action of single genes unlinked to the S-locus. In contrast, we report here a line of Brassica oleracea var acephala with a self-compatible phenotype linked to the S-locus. By means of both biochemical and immunochemical analyses, we showed that this self-compatible (Sc) line nonetheless possesses stigmatic SLGs (SLG-Sc) that are expressed with a similar spatial and temporal pattern to that described for the SLGs of self-incompatible Brassica plants. Moreover, the SLG-Sc products segregate with the self-compatibility phenotype in F2 progeny, suggesting that changes at the S-locus may be responsible for the occurrence of the self-compatibility character. A cDNA clone encoding the SLG-Sc product was isolated, and the deduced amino acid sequence showed this glycoprotein to be highly homologous to the pollen recessive S2 allele glycoprotein. Hence, self-compatibility in this Brassica Sc line correlates with the expression of a pollen recessive-like S allele in the stigma.     

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     

Identification of an S-locus glycoprotein allele introgressed from B. napus ssp. rapifera to B. napus ssp. oleifera

Self-incompatible Brassica napus ssp. oleifera lines were generated by introgressing the S-locus from the self-incompatible B. napus ssp. rapifera Z line into the self-compatible cultivars, Topas and Regent, resulting in T2 and R2, respectively. Screening of a cDNA library made from R2 stigma RNA produced several candidate SLG (S-locus glycoprotein) cDNAs. One of the cDNAs, A14, was found to be represented in only the R2, T2 and Z lines. In addition, the corresponding A14 gene was demonstrated to segregate with the T2 self-incompatibility phenotype in an F2 population derived from a cross between T2 and Topas, and to exhibit high mRNA levels in the stigmas prior to anthesis. Sequence analysis of the A14 cDNA revealed close homology to B. oleracea SLG alleles associated with a Class I high activity self-incompatibility phenotype.     

The self-incompatibility (S) haplotypes of Brassica contain highly divergent and rearranged sequences of ancient origin.

In Brassica, the recognition of self-related pollen by the stigma is controlled by the highly polymorphic S locus that encodes several linked and coadapted genes and can span several hundred kilobases. We used pulsed-field gel electrophoresis to analyze the structure of different S haplotypes. We show that the S2 and S13 haplotypes of Brassica oleracea contain extensive sequence divergence and rearrangement relative to each other. In contrast, haplotypic configuration is more conserved between B. oleracea S13 and B. campestris S8, two haplotypes that have been proposed to be derived from a common ancestral haplotype based on sequence comparisons. These results support the view that extensive restructuring of the S locus preceded speciation in Brassica. This structural heteromorphism, together with haplotype-specific sequences, may suppress recombination within the S locus complex, potentially providing a mechanism for maintaining the linkage of coadapted allelic combinations of genes over time.     

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.     

Phylogenetic analysis of <Emphasis Type="Italic">Brassiceae</Emphasis> based on the nucleotide sequences of the <Emphasis Type="Italic">S</Emphasis>-locus related gene, <Emphasis Type="Italic">SLR1</Emphasis>

Nucleotide sequences of orthologs of the S-locus related gene, SLR1, in 20 species of Brassicaceae were determined and compared with the previously reported SLR1 sequences of six species. Identities of deduced amino-acid sequences with Brassica oleracea SLR1 ranged from 66.0% to 97.6%, and those with B. oleracea SRK and SLR2 were less than 62% and 55%, respectively. In multiple alignment of deduced amino-acid sequences, the 180-190th amino-acid residues from the initial methionine were highly variable, this variable region corresponding to hypervariable region I of SLG and SRK. A phylogenetic tree based on the deduced amino-acid sequences showed a close relationship of SLR1 orthologs of species in the Brassicinae and Raphaninae. Brassica nigra SLR1 was found to belong to the same clade as Sinapis arvensis and Diplotaxis siifolia, while the sequences of the other Brassica species belonged to another clade together with B. oleracea and Brassica rapa. The phylogenetic tree was similar to previously reported trees constructed using the data of electrophoretic band patterns of chloroplast DNA, though minor differences were found. Based on synonymous substitution rates in SLR1, the diversification time of SLR1 orthologs between species in the Brassicinae was estimated. The evolution and function of SLR1 and the phylogenetic relationship of Brassiceae plants are discussed.     

羽衣甘蓝柱头酵母双杂交cDNA文库的构建与分析

目的:构建应用于酵母双杂交系统的羽衣甘蓝柱头cDNA文库。方法:以羽衣甘蓝S13-bS13-b自交不亲和系为材料,提取柱头的总RNA,用亲和层析法分离纯化mRNA,利用CytoTrapXR建库试剂盒构建羽衣甘蓝柱头cDNA文库。结果:羽衣甘蓝柱头cDNA原始文库的库容量为2.5×105;扩增后文库的库容量约为4×108,重组率为96%,插入片段大小为0.4～3kb,平均长度在0.8kb左右。结论:构建了应用于酵母双杂交系统的羽衣甘蓝自交不亲和系柱头的cDNA文库,为探讨芸苔属植物自交不亲和的分子机理奠定了基础。     

Isolation of a Second S-Locus-Related Cdna from Brassica Oleracea: Genetic Relationships between the S Locus and Two Related Loci

Self-incompatibility in Brassica oleracea is controlled by the highly polymorphic S locus. Isolation and subsequent characterization of the S-locus-glycoprotein (SLG) gene, which encodes the S-locus-specific glycoprotein (SLSG), has revealed the presence of a self-incompatibility multigene family. One of these S-locus-related genes, SLR1, has been shown to be expressed. In this study we present the isolation and preliminary characterization of a second expressed S-locus-related sequence, SLR2. Through restriction fragment length polymorphism (RFLP) linkage analysis we demonstrate that the SLR1 and SLR2 loci reside approximately 18.5 map units apart in one linkage group that segregates independently of the S-locus. The identification of a second SLR gene expressed in stigmas suggests that loci unlinked to the S-locus may play a role in the self-incompatibility response, or in pollination in general.     

Genomic changes in resynthesized Brassica napus and their effect on gene expression and phenotype

Many previous studies have provided evidence for genome changes in polyploids, but there are little data on the overall population dynamics of genome change and whether it causes phenotypic variability. We analyzed genetic, epigenetic, gene expression, and phenotypic changes in approximately 50 resynthesized Brassica napus lines independently derived by hybridizing double haploids of Brassica oleracea and Brassica rapa. A previous analysis of the first generation (S0) found that genetic changes were rare, and cytosine methylation changes were frequent. Our analysis of a later generation found that most S0 methylation changes remained fixed in their S5 progeny, although there were some reversions and new methylation changes. Genetic changes were much more frequent in the S5 generation, occurring in every line with lines normally distributed for number of changes. Genetic changes were detected on 36 of the 38 chromosomes of the S5 allopolyploids and were not random across the genome. DNA fragment losses within lines often occurred at linked marker loci, and most fragment losses co-occurred with intensification of signal from homoeologous markers, indicating that the changes were due to homoeologous nonreciprocal transpositions (HNRTs). HNRTs between chromosomes A1 and C1 initiated in early generations, occurred in successive generations, and segregated, consistent with a recombination mechanism. HNRTs and deletions were correlated with qualitative changes in the expression of specific homoeologous genes and anonymous cDNA amplified fragment length polymorphisms and with phenotypic variation among S5 polyploids. Our data indicate that exchanges among homoeologous chromosomes are a major mechanism creating novel allele combinations and phenotypic variation in newly formed B. napus polyploids.     

A highly conserved Brassica gene with homology to the S-locus-specific glycoprotein structural gene.

The S-locus-specific glycoprotein of Brassica and the gene encoding it (the SLG gene) are thought to be involved in determining self-incompatibility phenotype in this genus. It has been shown that the Brassica genome contains multiple SLG-related sequences. We report here the cloning and characterization of a Brassica oleracea gene, SLR1, which corresponds to one of these SLG-related sequences. Like the SLG gene, SLR1 is developmentally regulated. It is maximally expressed in the papillar cells of the stigma at the same stage of flower development as the onset of the incompatibility response. Unlike SLG, the SLR1 genes isolated from different S-allele homozygotes are highly conserved, and this gene, which appears to be ubiquitous in crucifers, is expressed in self-compatible strains as well as self-incompatible strains. Most importantly, we show that the SLR1 gene is not linked to the S-locus and therefore cannot be a determinant of S-allele specificity in Brassica.     

The gene for ornithine decarboxylase is co-amplified in hydroxyurea-resistant hamster cells

We have constructed a cDNA library from the highly hydroxyurea-resistant hamster cell line 600H in which the activity of ribonucleotide reductase is elevated more than 80-fold. Using the technique of differential hybridization, we have isolated a number of cDNA clones from this library which are homologous to genomic DNA sequences amplified in the 600H cell line compared to the V79 parental line. One of these cDNA clones by sequence analysis was found to code for ornithine decarboxylase. This was confirmed by in vitro translation of poly(A+) RNA isolated by hybridization-selection followed by immunoprecipitation with antiserum specific for mouse ornithine decarboxylase. Genomic sequences homologous to the cDNA clone were shown to be sequentially amplified 6-20-fold in hamster cell lines selected stepwise for resistance to increasing concentrations of hydroxyurea. Genomic sequences homologous to a cDNA for the M2 subunit of ribonucleotide reductase were also amplified in these cell lines, and the degree of M2 sequence amplification corresponded to the degree of amplification of ornithine decarboxylase sequences, suggesting that the two genes had been co-amplified during the selection of the hydroxyurea-resistant phenotype.     

The self-incompatibility phenotype in brassica is altered by the transformation of a mutant S locus receptor kinase

The self-incompatible (SI) Brassica napus line W1, which carries the 910 S allele, was transformed with an inactive copy of the 910 S locus receptor kinase (SRK) gene. Two transformed lines were analyzed based on their heritable ability to set self-seed. The first line was virtually completely self-compatible (SC), and reciprocal pollinations with the original W1 line demonstrated that only the stigma side of the SI phenotype was altered. An analysis of the expression of endogenous SRK-910 demonstrated that the mechanism of transgene action is via gene suppression. Furthermore, the expression of the S locus glycoprotein gene present in the 910 allele (SLG-910), SLG-A10, which is derived from a nonfunctional S allele, and an S locus-related gene were also suppressed. When the transgene was crossed into another SI line carrying the A14 S allele, it was also capable of suppressing the expression of the endogenous genes and of making this line SC. The second transgenic line studied was only partly SC. In this case as well, only the stigma phenotype was affected, although no gene suppression was detected for endogenous SRK-910 or SLG-910. In this line, the expression of the transgene most likely was causing the change in phenotype, and no effect was observed when this transgene was crossed into the other SI line. Therefore, this work reinforces the hypothesis that the SRK gene is required, but only for the stigma side of the SI phenotype, and that a single transgene can alter the SI phenotype of more than one S allele.