Physical distances between S-locus genes in various S haplotypes of Brassica rapa and B. oleracea

In Brassica, self-incompatibility genes SLG (for S-locus glycoprotein) and SRK (for S-receptor kinase) are located in the S-locus complex region with several other S-linked genes. The S locus is a highly polymorphic region: polymorphism has been observed not only in sequences of SLG and SRK but also in the location of the S-locus genes. In order to compare the physical location of the S-locus genes in various S haplotypes, we used six class-I S haplotypes of B. rapa and seven class-I S haplotypes of B. oleracea in this study. DNA gel blot analysis using pulsed-field gel electrophoresis (PFGE) showed that the physical distances between SLG and SRK in B. rapa are significantly shorter than those in B. oleracea and that the sizes of MluI and BssHII fragments harboring SLG and SRK are less variable within B. rapa than within B. oleracea. We concluded that several large genomic fragments might have been inserted into the S-locus region of B. oleracea after allelic differentiation of S-locus genes. Received: 20 September 1999 / Accepted: 8 October 1999     

Sequence and expression of endogenous S-locus glycoprotein genes in self-compatible Brassica napus

Brassica napus is an amphidiploid plant which is self-compatible even though it is derived from hybridisation of the self-incompatible species B. oleracea and B. campestris. Experiments were undertaken to establish if S-locus glycoprotein (SLG) genes exist in B. napus and whether these are expressed as in self-incompatible Brassica species. Two different stigma-specific cDNA sequences homologous to SLG genes were obtained from the B. napus cultivar Westar. One of these sequences, SLG _WS1, displayed highest homology to class I SLG alleles, whereas the other, SLG _WS2, showed greatest homology to class II SLG genes. Both were expressed at high levels in Westar stigmas following a developmental pattern typical of SLG genes in the self-incompatible diploids. We infer that they represent the endogenous SLG genes at the two homoeologous S-loci. The occurrence of normally expressed SLG genes and its relevance to the self-compatible phenotype of B. napus is discussed.     

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.     

Introduction of SLG (S locus glycoprotein) alters the phenotype of endogenous S haplotype,but confers no new S haplotype specificity in Brassica rapa L.

Self-incompatibility (SI) in Brassicaceae is genetically controlled by the S locus complex in which S locus glycoprotein (SLG) and S receptor kinase (SRK) genes have been identified, and these two genes encoding stigma proteins are believed to play important roles in SI recognition reaction. Here we introduced the SLG⁴³ gene of Brassica rapa into a self-incompatible cultivar, Osome, of B. rapa, and examined the effect of this transgene on the SI behavior of the transgenic plants. Preliminary pollination experiments demonstrated that Osome carried S⁵² and S⁶⁰, and both were codominant in stigma, but S⁵² was dominant to S⁶⁰ in pollen. S⁴³ was found to be recessive to S⁵² and codominant with S⁶⁰ in stigma. The nucleotide sequence of SLG⁴³ was more similar to that of SLG⁵² (87.8% identity) than to that of SLG⁶⁰ (74.8% identity). Three of the ten primary transformants (designated No. 1 to No. 10) were either completely (No. 9) or partially (No. 6 and No. 7) self-compatible; the SI phenotype of the stigma was changed from S⁵²S⁶⁰ to S⁶⁰, but the SI phenotype of the pollen was not altered. In these three plants, the mRNA and protein levels of both SLG⁴³ and SLG⁵² were reduced, whereas those of SLG⁶⁰ were not. All the plants in the selfed progeny of No. 9 and No. 6 regained SI and they produced a normal level of SLG⁵². These results suggest that the alteration of the SI phenotype of the stigma in the transformants Nos. 6, 7, and 9 was the result of specific co-suppression between the SLG⁴³ transgene and the endogenous SLG⁵² gene. Three of the transformants (Nos. 5, 8 and 10) produced SLG⁴³ protein, but their SI phenotype was not altered. The S⁶⁰ homozygotes in the selfed progeny of No. 10 which produced the highest level of SLG⁴³ were studied because S⁴³ was codominant with S⁶⁰ in the stigma. They produced SLG⁴³ at approximately the same level as did S⁴³S⁶⁰ heterozygotes, but did not show S⁴³ haplotype specificity at the stigma side. We conclude that SLG is necessary for the expression of the S haplotype specificity in the stigma but the introduction of SLG alone is not sufficient for conferring a novel S haplotype specificity to the stigma.     

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’.     

SLR1 function is dispensable for both self-incompatible rejection and self-compatible pollination processes inBrassica

TheSLR1 gene inBrassica is related both in DNA sequence and in pattern of expression to theS-locus glycoprotein (SLG) gene involved in the self-incompatibility mechanism which recognises and arrests the germination of self pollen. However,SLR1 shows minimal allelic variation and is expressed in both self-incompatible and compatibleBrassica lines and in related, self-compatible cruciferous plants. The function of the SLR1 protein is unknown. TheSLR1 gene was specifically ablated in self-incompatible and self-compatibleBrassica plants byAgrobacterium-mediated transformation with an antisense construct. Primary transformants and homozygous T2 progeny of both self-incompatibleB. oleracea and self-compatibleB. napus recipients were found to exhibit normal pollination responses despite having no detectable SLR1 glycoprotein. This shows that the high, wild-type level of SLR1 protein is not required to sustain the self-incompatibility reaction, nor is it necessary for successful intra-specific cross-pollination between compatible lines.     

Isolation of S-alleles from a wild population of Brassica campestris L. at Balcesme,Turkey and their characterization by S-glycoproteins

Summary S-alleles of self-incompatibility were isolated from a wild population of Brassica campestris growing at Balcesme, Turkey. Out of 88 plants observed, 73 were self-incompatible and 4 were self-compatible. In certain families, selfed progenies from a self-incompatible plant segregated into fewer than three incompatibility classes, which is consistent with a one-locus sporophytic genetic control of self-incompatibility. Out of 25 combinations of S-alleles tested, dominance interactions were observed in 6 of them on the pollen side and on 5 of them on the stigma side. The 35 S-homozygotes thus isolated consisted of 18 independent S-alleles. The number of S-alleles in this population was estimated to be more than 30. The S-locus glycoproteins (SLGs) corresponding to the respective S-alleles were identified by iso-electric focusing (IEF)-gel immunoblotting with a polyclonal antiserum against SLG⁸. SLGs in a stigma were generally composed of several bands, one major and a few minor ones, whose molecular weight was similar to each other, and the major and minor bands were heritable in correlation with each other. SDS-PAGE analysis of SLGs differentiated a few juxtaposed bands between 50 and 60 kDa, and the variations in these bands were considered to be due to differences in the number of polysaccharide residues. General features of the variation of S-genes and their SLGs between the populations in Balcesme, Turkey and Oguni, Japan, were comparatively similar to one another, despite the different surroundings and history of these populations.     

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.     

Polymorphism of the S -locus glycoprotein gene ( SLG ) and the S -locus related gene ( SLR1 ) in Raphanus sativus L. and self-incompatible ornamental plants in the Brassicaceae

The S-locus glycoprotein gene, SLG, which participates in the pollen-stigma interaction of self-incompatibility, and its unlinked homologue, SLR1, were analyzed in Raphanus sativus and three self-incompatible ornamental plants in the Brassicaceae. Among twenty-nine inbred lines of R. sativus, eighteen S haplotypes were identified on the basis of DNA polymorphisms detected by genomic Southern analysis using Brassica SLG probes. DNA fragments of SLG alleles specifically amplified from eight S haplotypes by PCR with class I SLG-specific primers showed different profiles following polyacrylamide gel electrophoresis, after digestion with a restriction endonuclease. The nucleotide sequences of the DNA fragments of these eight R. sativus SLG alleles were determined. Degrees of similarity of the nucleotide sequences to a Brassica SLG (S  ⁶ SLG) ranged from 85.6% to 91.9%. Amino acid sequences deduced from these had the twelve conserved cysteine residues and the three hypervariable regions characteristic of Brassica SLGs. Phylogenetic analysis of the SLG sequences from Raphanus and Brassica revealed that the Raphanus SLGs did not form an independent cluster, but were dispersed in the tree, clustering together with Brassica SLGs. These results suggest that diversification of the SLG alleles of Raphanus and Brassica occurred before differentiation of these genera. Although SLR1 sequences from Orychophragmus violaceus were shown to be relatively closely related to Brassica and Raphanus SLR1 sequences, DNA fragments that are highly homologous to the Brassica SLG were not detected in this species. Two other ornamental plants in the Brassicaceae, which are related more distantly to Brassica than Orychophragmus, also lacked sequences highly homologous to Brassica SLG genes. The evolution of self-incompatibility in the Brassicaceae is discussed. Received: 9 October 1997 / Accepted: 27 January 1998     

Characterization of S tester lines in Brassica oleracea: polymorphism of restriction fragment length of SLG homologues and isoelectric points of S-locus glycoproteins

 Forty three S tester lines of Brassica oleracea were characterized using DNA and protein gel-blotting analyses. DNA gel-blot analysis of HindIII-digested genomic DNA with class-I and class-II SLG probes revealed that 40 lines could be classified as class-I S haplotypes while three lines could be classified as class-II S haplotypes. The band patterns in the S tester lines were highly polymorphic. Although the S tester lines typically showed two bands corresponding to SLG and SRK in the analysis with the class-I SLG probe, only one band was observed in the S ²⁴ homozygote. This band was identified as SRK, suggesting that this haplotype has no class-I SLG band. In the analysis using the class-II SLG probe, one plant yielded a different band pattern from the known class-II haplotypes, S ² , S ⁵ and S ¹⁵ . Unexpectedly, this plant was reciprocally cross-incompatible with the S ² haplotype. Therefore, it was designated as S ^2-b . We found an S ¹³ haplotype having a restriction fragment length polymorphism different from that of the S ¹³ homozygotes of the S tester line. These findings indicate that S homozygous lines with the same S specificity do not necessarily show the same band pattern in the DNA gel-blot analysis. Soluble stigma proteins of 32 S homozygotes were separated by isoelectric focusing and detected using anti-S ²² SLG antiserum. S haplotype-specific bands were detected in 27 S homozygotes but not in five S homozygotes, including the S ²⁴ homozygote. This is consistent with the observation that the S ²⁴ haplotype had no SLG band. Received: 13 July 1998 / Accepted: 29 September 1998     

Polymorphism of the S -locus glycoprotein gene (SLG ) and the S -locus related gene (SLR1 ) in Raphanus sativus L. and self-incompatible ornamental plants in the Brassicaceae

The S-locus glycoprotein gene, SLG, which participates in the pollen-stigma interaction of self-incompatibility, and its unlinked homologue, SLR1, were analyzed in Raphanus sativus and three self-incompatible ornamental plants in the Brassicaceae. Among twenty-nine inbred lines of R. sativus, eighteen S haplotypes were identified on the basis of DNA polymorphisms detected by genomic Southern analysis using Brassica SLG probes. DNA fragments of SLG alleles specifically amplified from eight S haplotypes by PCR with class I SLG-specific primers showed different profiles following polyacrylamide gel electrophoresis, after digestion with a restriction endonuclease. The nucleotide sequences of the DNA fragments of these eight R. sativus SLG alleles were determined. Degrees of similarity of the nucleotide sequences to a Brassica SLG (S? ⁶ SLG) ranged from 85.6% to 91.9%. Amino acid sequences deduced from these had the twelve conserved cysteine residues and the three hypervariable regions characteristic of Brassica SLGs. Phylogenetic analysis of the SLG sequences from Raphanus and Brassica revealed that the Raphanus SLGs did not form an independent cluster, but were dispersed in the tree, clustering together with Brassica SLGs. These results suggest that diversification of the SLG alleles of Raphanus and Brassica occurred before differentiation of these genera. Although SLR1 sequences from Orychophragmus violaceus were shown to be relatively closely related to Brassica and Raphanus SLR1 sequences, DNA fragments that are highly homologous to the Brassica SLG were not detected in this species. Two other ornamental plants in the Brassicaceae, which are related more distantly to Brassica than Orychophragmus, also lacked sequences highly homologous to Brassica SLG genes. The evolution of self-incompatibility in the Brassicaceae is discussed.     

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.     

Development of SCAR markers linked to self-incompatibility in <Emphasis Type="Italic">Brassica napus</Emphasis> L.

‘SI1300’ is a self-incompatible Brassica napus line generated by introgressing an S haplotype from B. rapa ‘Xishuibai’ into a rapeseed cultivar ‘Huayou No. 1’. Five S-locus specific primer pairs were employed to develop cleaved amplified polymorphic sequences (CAPS) markers linked the S haplotype of ‘SI1300’. Two segregating populations (F₂ and BC₁) from the cross between ‘SI1300’ and self-compatible European spring cultivar ‘Defender’, were generated to verify the molecular markers. CAPS analysis revealed no desirable polymorphism between self-incompatible and self-compatible plants. Twenty primer pairs were designed based on the homology-based candidate gene method, and six dominant sequence characterized amplified region (SCAR) markers linked with the S-locus were developed. Of the six markers, three were derived from the SRK and SP11 alleles of class II B. rapa S haplotypes and linked with S haplotype of ‘SI1300’. The other three markers were designed from the SLG-A10 and co-segregated with S haplotype of ‘Defender’. We successfully combined two pairs of them and characterized two multiplex PCR markers which could discriminate the homozygous and heterozygous genotypes. These markers were further validated in 24 F₃ and 22 BC₁F₂ lines of ‘SI1300 × Defender’ and another two segregating populations from the cross ‘SI1300 × Yu No. 9’. Nucleotide sequences of fragments linked with S-locus of ‘SI1300’ showed 99% identity to B. rapa class II S-60 haplotype, and fragments from ‘Defender’ were 97% and 94% identical to SLG and SRK of B. rapa class I S-47 haplotype, respectively. ‘SI1300’ was considered to carry two class II S haplotypes and the S haplotype on the A-genome derived from B. rapa ‘Xishuibai’ determines the SI phenotype, while ‘Defender’ carry a class I S haplotype derived from B. rapa and a class II S haplotype from B. oleracea. SCAR markers developed in this study will be helpful for improving SI lines and accelerating marker-assisted selection process in rapeseed SI hybrid breeding program.