Comparative Studies on S-Glycoproteins Purified from Different S-Genotypes in Self-Incompatible BRASSICA Species I. Purification and Chemical Properties

S-glycoproteins, i.e. stigma glycoproteins that are heritable in correlation with S allele in self-incompatible Brassica species, were apparently purified for three S alleles in B. oleracea. From SDS gel electrophoresis, the estimated molecular weight for two of the S-glycoproteins was 57,000. The other S-glycoprotein was considered to be heterogeneous with molecular weights of 60,000 and 65,000. Distinct differences in amino acid content were found; in general, cysteine, methionine and histidine were low, and serine, glutamate, glycine, leucine, arginine and aspartate were high and variable between the S-glycoproteins. Differences in the isoelectric point were mainly attributed to the amino acid composition of each S-glycoprotein.     

Analysis of self-incompatibility interactions in 30 resynthesized Brassica napus lines. II. Expression of S-locus glycoproteins (SLGs)

Thirty resynthesized Brassica napus lines with defined S-allele constitution and the ancestral B. oleracea and B. campestris lines were used for the analysis of S- locus glycoproteins (SLGs). The aim of this study was to investigate (1) whether the S-specific glycoproteins of the diploid ancestor lines were also expressed in the amphidiploid hybrids and (2) whether the occurrence of SLG bands was correlated with the activity of the respective S-alleles, which had been tested by means of diallele pollination tests in a previous study. Stigma proteins were separated by isoelectric focusing (IEF)-gel electrophoresis, and glycoprotein bands were identified by Western blotting and Con-A/peroxidase reaction. The SLG bands of the B. campestris parent could be detected in all 30 resynthesized B. napus lines. In contrast, B. oleracea SLG bands could only be detected in 12 resynthesized B. napus lines. Only B. napus lines which carried the dominant B. oleracea S-alleles S₈ and S₂₉ showed respective SLG bands in all cases. Nine B. napus lines showed only glycoprotein bands of the B. campestris parent, although the biological functioning of the B. oleracea S-alleles was demonstrated by test-pollinations. New SLG bands different from those of the B. oleracea and B. campestris parents occurred in 16 B. napus lines. The expression level of the SLGs in B. napus was not correlated with the self-incompatibility phenotype, not only in the case of recessive S-alleles (S₂, S₁₅), but also for dominant alleles (e.g. S₁₄, S₃₂, S₄₅). Received: 22 January 1999 / Accepted: 30 January 1999     

Analysis of self-incompatibility interactions in 30 resynthesized Brassica napus lines. I. Fluorescence microscopic studies

Thirty Brassica napus lines have been developed through interspecific hybridization of B. oleracea and B. campestris lines with defined S-allele constitutions. These lines, which represent 29 different S-allele combinations, were tested in a diallel of test-pollinations to determine the activity of the introgressed S-alleles and intergenomic dominance relationships. Some consistent trends were observed: B. oleracea S-alleles high in the dominance series (e.g. S₈, S₁₄, S₂₉) were always active in the resynthesized B. napus lines, whereas recessive S-alleles (S₂, S₁₅) lost their activity in some test combinations. The B. campestris S-alleles were active in most cases, although 2 alleles were partially inactivated by the recessive B. oleracea allele S₁₅.     

Inheritance of isozyme and RFLP markers in Brassica campestris and comparison with B. oleracea

Summary Using primarily cDNA restriction fragment length polymorphism markers (RFLPs) previously located to Brassica oleracea (cabbage, 2n=18) chromosomes, we initiated a comparative RFLP map in an F₂ population of B. campestris (turnip x mock pak-choi, 2n=20). As with B. oleracea, the genome of B. campestris showed extensive gene duplication, and the majority of detected duplicated loci were unlinked. Only 6 of the 49 identified loci were represented as a single copy, and 3 of these 6 were clustered on a single linkage group showing a distorted segregation ratio. Comparison with B. Oleracea indicates this synteny is conserved between species. Two other linkage groups also appeared syntenic between B. oleracea and B. campestris. One single copy locus appears to have changed synteny between B. oleracea and B. campestris. These observations suggest that B. oleracea and B. campestris share a common ancestor, but that chromosome repatterning has occurred during or after speciation. Within B. campestris, 5 loci appeared duplicated in one parent or the other, and 2 of these were linked. Differentiation through subspecies-specific duplication or deletion events is suggested as one mechansim for the evolution of numerous morphotypes within each of these species.     

Purification and N-terminal sequencing of style glycoproteins associated with self-incompatibility in Petunia hybrida

We report isolation and N-terminal amino acid sequencing of three style glycoproteins, which segregate with three S (self-incompatibility) alleles of Petunia hybrida. The S-glycoproteins were expressed mainly in the upper part of the pistil and showed an increasing concentration during flower development. The glycoproteins were purified by a combination of ConA-Sepharose and cation exchange fast protein liquid chromatography. The amount of S-glycoproteins recovered from style extracts varied from 0.5 to 1.6 g per style, which was 40–60% of the amount recovered by a simplified analytical method. N-terminal amino acid sequences of S₁-, S₂- and S₃-glycoprotein showed homology within the fifteen amino terminal residues. These amino acid sequences were compared with the previously published sequences of S-glycoproteins from Nicotiana alata and Lycopersicon peruvianum.     

Genetic variation within and among different cultivars and landraces of Brassica campestris L. and B. oleracea L. based on isozymes

Genetic variation based on isozymes was studied in 43 landraces and cultivars of Brassica campestris from China, 4 cultivars of B. campestris from Sweden and 1 from India, and 5 cultivars of B. oleracea from Sweden and 1 from China (B. alboglabra). A total of 17 isozyme loci was studied, 10 of these were polymorphic in B. campestris and 6 were polymorphic in B. oleracea. The level of heterozygosity seemed to be reduced in the Swedish cultivars compared to the Chinese landraces and cultivars of B. campestris. The level of heterozygosity in B. oleracea was even lower than that in the Swedish cultivars of B. campestris. A phylogeny of the cultivars and landraces of B. campestris showed that the B. campestris var yellow sarson cultivar, originating from India, deviated significantly from the other cultivars of B. campestris. A phylogeny of the cultivars of B. oleracea confirmed the expectations that the cultivar B. alboglabra was not closely related to the cultivated forms of B. oleracea.     

Variations in and Inheritance of NS-Glycoprotein in Self-Incompatible Brassica campestris L.

The stigma of Brassica species contain NS-glycoproteins thatexhibit a high degree of structural homology to the S-glycoproteinsof self-incompatibility. Inheritance of and variations in theNS-glycoprotein were studied with reference to self-incompatibility.The detection of NS-glycoproteins was performed by cross-reactionwith an antiserum raised against a purified NS-glycoprotein.In B. campestris, four isoforms of the NS-glycoprotein weredifferentiated by their pI values, but their molecular weightswere identical to one another. The genes for these isoformsof NS-glycoprotein were controlled by alleles at a single locus,tentatively named the NS allele, which was independent of Salleles at both the protein and the DNA level. Segregation ofF₂ plants with respect to the self-incompatibility behaviorof pollen tubes can be explained by the S allele model, butit appears not to be affected by the NS alleles. NS-glycoproteinswere found in all 21 species of Brassica and its allies examinedto date. The pI values of these glycoproteins varied among differentspecies. In addition to the isoforms of the NS alleles, maturestigmas contained other groups of proteins that reacted weaklywith the antiserum against the NS-glycoprotein. (Received July 30, 1991; Accepted February 21, 1992)     

Intergeneric and interspecific hybrids through in vitro ovule culture in the Cruciferae

A procedure to obtain interspecific and intergeneric hybrids has been developed through culture in vitro of ovules of cruciferous crops using Brassica oleracea as the female plant. A modified medium of Murashige and Skoog supplemented with 10% (v/v) coconut milk, 300 mg/l casein hydrolysate, 0.1 mg/l 1-napthaleneacetic acid (NAA) and 0.1 mg/l kinein were effective for culturing hybrid ovules. All the interspecific and intergeneric hybrids between B. oleracea and Brassica campestris; and between B. oleracea and Raphanus sativus had an intermediate plant morphology in combination with their parental traits. Hybrids of cross combinations between B. oleracea and B. campestris, and between B. olecacea and R. sativus were amphihaploids. This was revealed through chromosome and isozyme analyses.     

Analysis of S-locus and expression of S-alleles of self-compatible rapid-cycling Brassica oleracea ‘TO1000DH3’

Brassica oleracea is a strictly self-incompatible (SI) plant, but rapid-cycling B. oleracea ‘TO1000DH3’ is self-compatible (SC). Self-incompatibility in Brassicaceae is controlled by multiple alleles of the S-locus. Three S-locus genes, S-locus glycoprotein (SLG), S-locus receptor kinase (SRK) and S-locus protein 11 or S-locus cysteine-rich (SP11/SCR), have been reported to date, all of which are classified into class I and II. In this study, we investigated the molecular mechanism behind alterations of SI to SC in rapid-cycling B. olerace ‘TO1000DH3’. Class I SRK were identified by genomic DNA PCR and PCR-RFLP analysis using SRK specific markers and found to be homozygous. Cloning and sequencing of class I SRK revealed a normal kinase domain without any S-domain/transmembrane domain. Moreover, S-locus sequencing analysis revealed only an SLG sequence, but no SP11/SCR. Expression analysis showed no SRK expression in the stigma, although other genes involved in the SI recognition reaction (SLG, MLPK, ARC1, THL) were found to have normal expression in the stigma. Taken together, the above results suggest that structural aberrations such as deletion of the SI recognition genes may be responsible for the breakdown of SI in rapid-cycling B. oleracea ‘TO1000DH3’.     

Conservation of S-locus for self-incompatibility in Brassica napus (L.) and Brassica oleracea (L.)

 Self-incompatibility (SI) in Brassica is a sporophytic system, genetically determined by alleles at the S-locus, which prevents self-fertilization and encourages outbreeding. This system occurs naturally in diploid Brassica species but is introduced into amphidiploid Brassica species by interspecific breeding, so that in both cases there is a potential for yield increase due to heterosis and the combination of desirable characteristics from both parental lines. Using a polymerase chain reaction (PCR) based analysis specific for the alleles of the SLG (S-locus glycoprotein gene) located on the S-locus, we genetically mapped the S-locus of B. oleracea for SI using a F₂ population from a cross between a rapid-cycling B. oleracea line (CrGC-85) and a cabbage line (86-16-5). The linkage map contained both RFLP (restriction fragment length polymorphism) and RAPD (random amplified polymorphic DNA) markers. Similarly, the S-loci were mapped in B. napus using two different crosses (91-SN-5263×87-DHS-002; 90-DHW-1855-4×87-DHS-002) where the common male parent was self-compatible, while the S-alleles introgressed in the two different SI female parents had not been characterized. The linkage group with the S-locus in B. oleracea showed remarkable homology to the corresponding linkage group in B. napus except that in the latter there was an additional locus present, which might have been introgressed from B. rapa. The S-allele in the rapid-cycling Brassica was identified as the S₂₉ allele, the S-allele of the cabbage was the S ₅ allele. These same alleles were present in our two B. napus SI lines, but there was evidence that it might not be the active or major SI allele that caused self-incompatibility in these two B. napus crosses. Received: 7 June 1996/Accepted: 6 September 1996     

Quantitative cytology of the sperm cells of Brassica campestris and B. oleracea

Pollen grains of Brassica campestris L. var. acephala DC and B. oleracea L. were serially sectioned and examined using transmission electron microscopy to determine the three-dimensional organization of sperm cells within the microgametophyte and the quantity of membrane-bound organelles occurring within each cell. Sperm cells occur in pairs within each pollen grain, but are dimorphic, differing in size, morphology and mitochondrial content. The larger of the two sperm cells (S_vn) is distinguished by the presence of a blunt evagination, which in B. oleracea wraps around and lies within shallow furrows on the vegetative nucleus and in B. campestris can penetrate through internal enclaves of the vegetative nucleus. This sperm cell contains more mitochondria in both species than the second sperm cell (S_ua). This latter cell is linked to the first by a common cell junction with the S _vn, but is not associated with the vegetative nucleus and lacks a cellular evagination. Such differences are indicative of a system of cytoplasmic heterospermy in which sperm cells possess significantly different quantities of mitochondria.Abbreviations mtDNA mitochondrial DNA - S_ua sperm cell unassociated with the vegetative nucleus - S_vn Sperm cell physically associated with the vegetative nucleus     

Analysis of organelle genomes in a somatic hybrid derived from cytoplasmic male-sterile Brassica oleracea and atrazine-resistant B. campestris

Summary An atrazine-resistant, male-fertile Brassica napus plant was synthesized by fusion of protoplasts from the diploid species B. oleracea and B. campestris. Leaf protoplasts from B. oleracea var. italica carrying the Ogura male-sterile cytoplasm derived from Raphanus sativus were fused with etiolated hypocotyl protoplasts of atrazine-resistant B. campestris. The selection procedure was based on the inability of B. campestris protoplasts to regenerate in the media used, and the reduction of light-induced growth of B. oleracea tissue by atrazine. A somatic hybrid plant that differed in morphology from both B. oleracea and B. campestris was regenerated on medium containing 50 M atrazine. Its chromosome number was 36–38, approximately that of B. napus. Furthermore, nuclear ribosomal DNA from this hybrid was a mixture of both parental rDNAs. Southern blot analyses of chloroplast DNA and an assay involving tetrazolium blue indicated that the hybrid contained atrazine-resistant B. campestris chloroplasts. The hybrid's mitochondrial genome was recombinant, containing fragments unique to each parent, as well as novel fragments carrying putative crossover points. Although the plant was female-sterile, it was successfully used to pollinate B. napus.     

Identification, isolation, and N-terminal sequencing of style glycoproteins associated with self-incompatibility in Nicotiana alata.

S-Gene-associated glycoproteins (S-glycoproteins) from styles of Nicotiana alata, identified by non-equilibrium two-dimensional electrophoresis, were purified by cation exchange fast protein liquid chromatography with yields of 0.5 to 8 micrograms of protein per style, depending on the S-genotype of the plant. The method relies on the highly basic nature of the S-glycoproteins. The elution profiles of the different S-glycoproteins from the fast protein liquid chromatography column were characteristic of each S-glycoprotein, and could be used to establish the S-genotype of plants in outbreeding populations. In all cases, the S-genotype predicted from the style protein profile corresponded to that predicted from DNA gel blot analysis using S-allele-specific DNA probes and to that established by conventional breeding tests. Amino-terminal sequences of five purified S-glycoproteins showed a high degree of homology with the previously published sequences of N. alata and Lycopersicon esculentum S-glycoproteins.     

Establishment of Isolated Root Cultures of Brassica Species and Regeneration from Cultured-root Segments of Brassica oleracea var. italica

Isolated root cultures which could be maintained over severalmonths by serial subculture were established from Brassica oleraceavar. italica cv. Green Comet F₁. A modified White's medium wasfound to be the best of several salt compositions tested. Theeffects on isolated root growth of the following were also examined;nutritional components, culture vessel type and closure, pHof the medium and auxin type and concentration. Using a mediumdevised for Green Comet, root cultures were established fromsix other B. oleracea, B. napus and B. campestris cultivars. It was possible to regenerate shoots from segments of culturedroots by incubation on agar-solidified media containing cytokininand auxin. The effects on regeneration of various auxins andcytokinins were investigated; the combination of Picloram withKN gave the highest frequency of shoot formation. It was demonstratedthat isolated roots retained their regenerative ability overa period of 5 months in culture. Brassica oleracea var. italica, Brassica napus, Brassica campestris, isolated root culture, shoot regeneration, organ culture     

Testing Local Host Adaptation and Phenotypic Plasticity in a Herbivore When Alternative Related Host Plants Occur Sympatrically

Host race formation in phytophagous insects can be an early stage of adaptive speciation. However, the evolution of phenotypic plasticity in host use is another possible outcome. Using a reciprocal transplant experiment we tested the hypothesis of local adaptation in the aphid Brevicoryne brassicae. Aphid genotypes derived from two sympatric host plants, Brassica oleracea and B. campestris, were assessed in order to measure the extent of phenotypic plasticity in morphological and life history traits in relation to the host plants. We obtained an index of phenotypic plasticity for each genotype. Morphological variation of aphids was summarized by principal components analysis. Significant effects of recipient host on morphological variation and life history traits (establishment, age at first reproduction, number of nymphs, and intrinsic growth rate) were detected. We did not detected genotype × host plant interaction; in general the genotypes developed better on B. campestris, independent of the host plant species from which they were collected. Therefore, there was no evidence to suggest local adaptation. Regarding plasticity, significant differences among genotypes in the index of plasticity were detected. Furthermore, significant selection on PC1 (general aphid body size) on B. campestris, and on PC1 and PC2 (body length relative to body size) on B. oleracea was detected. The elevation of the reaction norm of PC1 and the slope of the reaction norm for PC2 (i.e., plasticity) were under directional selection. Thus, host plant species constitute distinct selective environments for B. brassicae. Aphid genotypes expressed different phenotypes in response to the host plant with low or nil fitness costs. Phenotypic plasticity and gene flow limits natural selection for host specialization promoting the maintenance of genetic variation in host exploitation.     

Expression of S-locus glycoprotein genes from Brassica oleracea and B. campestris in transgenic plants of self-compatible B. napus cv Westar

Summary Self-compatible Brassica napus var Westar was transformed with SLG, the S-locus-derived gene that encodes S-locus-specific glycoproteins (SLSG). Four allelic variants of SLG isolated from self-incompatible B. oleracea and B. campestris strains homozygous for different S alleles were used. We show that the transgenic plants synthesized SLSG with the same apparent charge, molecular weight, and antigenic properties as that produced by the corresponding self-incompatible strains from which the cloned SLG genes were isolated. In addition, transgene-encoded SLSG was detected specifically in the papillar cells of the stigma, and was correctly targeted to the papillar cell wall. However, SLSG was produced at reduced levels in transgenic plants relative to self-incompatible strains. The introduction of the SLG genes did not confer a self-incompatibility phenotype on the Westar cultivar.     

N-Linked Glycan Chains on S-Allele-Associated Glycoproteins from Nicotiana alata

The products of the self-incompatibility locus of flowering plants are glycoproteins. The specificity of different alleles at this locus might be expressed through differences in either amino acid sequences or by the glycan substituents. We have investigated the numbers of N-linked glycan chains on the S-glycoproteins and obtained information on their structure by enzymic cleavage with N-glycanase and endo-[beta]-N-acetylglucosaminidase H. In addition to there being variation in the numbers of chains on the S-glycoproteins, each glycoprotein appears to consist of a spectrum of "glycoforms" bearing chains of differing type and fine structure. This microheterogeneity in N-linked glycan chains may be functionally significant.     

Inheritance of isozymes in Brassica campestris L. and genetic divergence among different species of Brassiceae

Electrophoretic investigations of Brassica campestris revealed 8 polymorphic loci that had not been described earlier. The inheritance of 7 of them was determined by crosses between different accessions of B. campestris. A phylogeny of B. campestris, B. nigra, B. alboglabra, Sinapis arvensis and S. alba was estimated from the analysis of 15 isozyme loci. The phylogeny showed a close relationship between B. nigra and S. arvensis and a less close relationship between B. campestris and B. oleracea. S. alba was remotely-related to all of the other species.