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.     

Characterization of the S locus genes, SLG and SRK, of the Brassica S3 haplotype: identification of a membrane-localized protein encoded by the S locus receptor kinase gene

The S locus, which controls the self-incompatibility response in Brassica, has been shown to contain at least two genes. SLG encodes a secreted S locus glycoprotein whilst SRK encodes a putative S locus receptor kinase. SRK has been shown potentially to encode a functional kinase and genetic evidence indicates that this gene is essential for the self-incompatibility response. Here the characterization of the SRK and SLG genes of a Brassica line homozygous for the S₃ haplotype is described. A 120 kDa glycoprotein was identified in stigmas and several lines of evidence indicated that this protein is encoded by the SRK₃ gene. First, the 120 kDa glycoprotein was recognized by antibodies raised against peptides based on the SRK₃ gene sequence. Secondly, this protein is polymorphic and, in an F₂ population segregating for the S₃ haplotype, was expressed only in plants possessing the S₃ haplotype. Thirdly, the 120 kDa protein was expressed specifically in stigmas. Finally, the 120 kDa protein was only extracted from stigmas in the presence of detergent indicating that it is anchored in the membrane. SRK has been predicted to encode a transmembrane glycoprotein based on the deduced amino acid sequence. Located on the membrane, SRK is in a position to interface between an extracellular recognition event between pollen and pistil and an intracellular signal transduction pathway which initiates the self-incompatibility response.     

Genomic organization of the S locus: Identification and characterization of genes in SLG/SRK region of S(9) haplotype of Brassica campestris (syn. rapa).

In Brassica, two self-incompatibility genes, encoding SLG (S locus glycoprotein) and SRK (S-receptor kinase), are located at the S locus and expressed in the stigma. Recent molecular analysis has revealed that the S locus is highly polymorphic and contains several genes, i.e., SLG, SRK, the as-yet-unidentified pollen S gene(s), and other linked genes. In the present study, we searched for expressed sequences in a 76-kb SLG/SRK region of the S(9) haplotype of Brassica campestris (syn. rapa) and identified 10 genes in addition to the four previously identified (SLG(9), SRK(9), SAE1, and SLL2) in this haplotype. This gene density (1 gene/5.4 kb) suggests that the S locus is embedded in a gene-rich region of the genome. The average G + C content in this region is 32.6%. An En/Spm-type transposon-like element was found downstream of SLG(9). Among the genes we identified that had not previously been found to be linked to the S locus were genes encoding a small cysteine-rich protein, a J-domain protein, and an antisilencing protein (ASF1) homologue. The small cysteine-rich protein was similar to a pollen coat protein, named PCP-A1, which had previously been shown to bind SLG.     

Polymorphism of the kinase domain of the S-locus receptor kinase gene (SRK) in Brassica oleracea L.

 DNA polymorphism of the S-locus receptor kinase gene (SRK) participating in self-incompatibility in Brassica was analyzed by PCR-RFLP and nucleotide sequencing. In the screening of primers for specific amplification of polymorphic DNA fragments of SRK, the best combination was that of a forward primer (PK1) having the nucleotide sequence of the second exon of S6 SRK and a reverse primer (PK4) having the complementary nucleotide sequence of the fifth exon of S6 SRK. PCR using this primer pair amplified DNA fragments of 0.9–1.0 kb from 36 S haplotypes out of 42 tested. These DNA fragments showed high polymorphism in polyacrylamide-gel electrophoresis after digestion with restriction endonuclease(s): 25 types were found in a double digestion with MboI and AfaI. Nucleotide sequencing of the DNA fragments amplified from five S haplotypes showed that the third, fourth, and fifth exons of SRK are highly conserved, and that there are high variations of the second and third introns of SRK, which produced polymorphism of the band pattern in PCR-RFLPs. Another forward primer (PK5) having the nucleotide sequence of the second exon, which is derived from S2 SRK, amplified DNA fragments of almost the same region of SRK from 27 S haplotypes in combination with PK4. Although SRK alleles of the class-II S haplotypes were not amplified, all of the class-I S-haplotypes were amplified with a primer mixture of PK1, PK4 and PK5. The DNA fragments of both SRK alleles in S heterozygotes, or a 1 : 1 mixture of the genomic DNA of different S homozygotes, were amplified without exception, suggesting the usefulness of these primers for the identification of S heterozygotes. The DNA fragment sizes obtained by digestion with restriction endonucleases served as markers for the identification of S haplotypes. Received: 15 December 1996 / Accepted: 14 February 1997     

SRK, the stigma-specific S locus receptor kinase of Brassica, is targeted to the plasma membrane in transgenic tobacco.

The S locus receptor kinase (SRK) gene is one of two S locus genes required for the self-incompatibility response in Brassica. We have identified the product of the SRK6 gene in B. oleracea stigmas and have shown that it has characteristics of an integral membrane protein. When expressed in transgenic tobacco, SRK6 is glycosylated and targeted to the plasma membrane. These results provide definitive biochemical evidence for the existence in plants of a plasma membrane-localized transmembrane protein kinase with a known cell-cell recognition function. The timing of SRK expression in stigmas follows a time course similar to that previously described for another S locus-linked gene, the S locus glycoprotein (SLG) gene, and correlates with the ability of stigmas to mount a self-incompatibility response. Based on SRK6 promoter studies, the site of gene expression overlaps with that of SLG and exhibits predominant expression in the stigmatic papillar cells. Although reporter gene studies indicated that the SRK promoter was active in pollen, SRK protein was not detected in pollen, suggesting that SRK functions as a cell surface receptor exclusively in the papillar cells of the stigma.     

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.     

Identification of a gene linked to the Brassica S (self-incompatibility) locus by differential display

Self-incompatibility in Brassica is controlled by a complex locus, the S locus, that includes several expressed genes. Two S locus genes, SLG and SRK, are expressed in the stigma and have been implicated in self-pollen recognition. The male component of this recognition system is also predicted to be encoded by a gene at the S locus but this gene has not been identified to date. In this study, we have used differential display to screen for polymorphic, S-locus-linked genes that are expressed in anthers. This approach has allowed the identification of a gene, named S5J, which was shown to segregate completely with the S locus. We discuss the possible role of this gene in the self-incompatibility response and evaluate the utility of differential display for the identification of genes at specific genetic loci.     

Comparison of the genome structure of the self-incompatibility (S) locus in interspecific pairs of S haplotypes

The determinants of recognition specificity of self-incompatibility in Brassica are SRK in the stigma and SP11/SCR in the pollen, both of which are encoded in the S locus. The nucleotide sequence analyses of many SRK and SP11/SCR alleles have identified several interspecific pairs of S haplotypes having highly similar sequences between B. oleracea and B. rapa. These interspecific pairs of S haplotypes are considered to be derived from common ancestors and to have maintained the same recognition specificity after speciation. In this study, the genome structures of three interspecific pairs of S haplotypes were compared by sequencing SRK, SP11/SCR, and their flanking regions. Regions between SRK and SP11/SCR in B. oleracea were demonstrated to be much longer than those of B. rapa and several retrotransposon-like sequences were identified in the S locus in B. oleracea. Among the seven retrotransposon-like sequences, six sequences were found to belong to the ty3 gypsy group. The gag sequences of the retrotransposon-like sequences were phylogenetically different from each other. In Southern blot analysis using retrotransposon-like sequences as probes, the B. oleracea genome showed more signals than the B. rapa genome did. These findings suggest a role for the S locus and genome evolution in self-incompatible plant species.     

Growth of radish (Raphanus sativus) seedlings in magnetic fields

Radish seedlings were grown in asymmetric magnetic fields. The number of seeds germinated and the dry weight of the plants were the two criteria by which possible effects were examined. Two experiments were done; in the first the plants were grown for an average of 7.7 days and in the second for 14.1 days. A statistical analysis of the results failed to reveal any significant difference between control plants grown in dummy magnets and those subjected to the magnetic influence.     

Identification and expression of the kosena radish (Raphanus sativus cv. Kosena) homologue of the ogura radish CMS-associated gene,orf138

A CMS-associated gene, orf125, present in the Japanese radish cultivar Kosena, has a sequence homologous to that of the ogura CMS-associated gene, orf138, except for two amino acid substitutions and a 39 bp deletion in the orf138 coding region. In Kosena radish, orf125 is linked with orfB, whereas the orf125 locus differs in a Brassica napus CMS cybrid derived from protoplast fusion between Kosena radish and B. napus. A novel mtDNA sequence is present in the 3-flanking region of orf125 in the B. napus kosena CMS cybrid. The orf125 is expressed both in the radish and the B. napus kosena CMS cybrid. Its accumulation is strongly associated with the CMS phenotype in B. napus. Fertility restoration was accompanied by a decrease in the amount of ORF125 in B. napus.     

Polymorphic DNA haplotypes at the human low-density lipoprotein receptor gene locus in Koreans

Many low-density lipoprotein (LDL) receptor mutations have been identified and characterized, demonstrating a high degree of allelic heterogeneity at this locus. The ability to identify mutant LDL-receptor genes for prenatal diagnosis of familial hypercholesterolemia (FH) or to study the role of the LDL-receptor gene in polygenic hypercholesterolemia requires the use of closely linked restriction fragment lenghth polymorphisms (RFLPs). In the present study nine different RFLPs (TaqI, StuI, HincII, BstEII, AvaII, PvuII, MspIA, MspIB, and NcoI) and a sequence variation at Arg450 were used to clarify the characteristics of the LDL-receptor gene in Koreans. A total of 978 LDL-receptor alleles from 244 members of 43 different pedigrees (15 normal and 28 FH pedigrees) and 245 individuals (187 normal and 58 FH) were analyzed. Frequencies of these polymorphisms did not differ significantly between controls and FH patients. Individually, seven sites--TaqI, BstEII, AvaII, MspIA, MspIB, NcoI and Arg450--had heterozygosity indices ranging from 0.3610 to 0.4601, whereas the PvuII site displayed low levels of polymorphism and StuI was monomorphic. Haplotypes were constructed for 215 individuals of 13 normal and 24 FH pedigrees using the nine polymorphisms. Of 512 (= 2(9)) possible combinations for the nine polymorphic sites, 39 unique haplotypes were detected. The frequency distribution of individual haplotypes ranged from 1/155 (0.65%) to 40/155 (25.8%). The four most common haplotypes accounted for 59.4% of those sampled. Statistical analysis of the haplotypes indicated marked linkage disequilibrium for these 10 sites and throughout the region containing the LDL-receptor gene. Owing to the high degree of linkage disequilibrium over the entire locus, not all RFLPs were informative. We rank each RFLP according to its informativeness and present a strategy for the optimal selection of RFLPs for pedigree analysis.     

Effects of ethylene on tuberization in radish (Raphanus sativus)

The effects of ethylene on the elongation of radish hypocotyls and on dry matter partitioning between tubers and shoots were analysed in order to gain insight into the possible role of ethylene in the regulation of tuberization. Treatment of very young seedlings with ethylene results in heavier tubers (Vreugdenhil et al. 1984). Here we report that addition of ethylene or ethephon two days after germination inhibited the elongation of the hypocotyl; trapping of endogenously produced ethylene had a stimulative effect on elongation. Ethephon, sprayed at a later stage, changed the partitioning of assimilates between tubers and shoots, resulting in lower tuber weights. It is concluded that ethylene had a dual effect on tuberization in radish: at a very early stage of development it inhibited elongation of the hypocotyl, resulting in earlier tuber formation and heavier tubers. At a later stage, it had a negative effect on tuber weight by changing dry matter partitioning.     

Isolation and characterization of a novel PHGPx gene in Raphanus sativus

A full-length cDNA encoding putative phospholipid hydroperoxide glutathione peroxidase (PHGPx) was cloned from Raphanus sativus. The cDNA, designated RsPHGPx, includes an open reading frame which encodes 197 amino acid residues. The alignment of amino acid sequences showed that RsPHGPx had the highest sequence homology to plant PHGPx and contained an N-terminal extension characteristic of a mitochondrial targeting peptide. Northern blot analysis indicated that RsPHGPx was constitutively and ubiquitously expressed during radish development, and its expression was differently regulated by various stress conditions. The expression of RsPHGPx in a yeast PHGPx-deletion mutant significantly rescued the mutant sensitivity to oxidation-sensitive linolenic acid, just as the yeast PHGPx3 gene did. This suggested that RsPHGPx encodes a functional PHGPx protein.     

Identification and characterization of a polymorphic receptor kinase gene linked to the self-incompatibility locus of Arabidopsis lyrata

We study the segregation of variants of a putative self-incompatibility gene in Arabidopsis lyrata. This gene encodes a sequence that is homologous to the protein encoded by the SRK gene involved in self-incompatibility in Brassica species. We show by diallel pollinations of plants in several full-sib families that seven different sequences of the gene in A. lyrata are linked to different S-alleles, and segregation analysis in further sibships shows that four other sequences behave as allelic to these. The family data on incompatibility provide evidence for dominance classes among the S-alleles, as expected for a sporophytic SI system. We observe no division into pollen-dominant and pollen-recessive classes of alleles as has been found in Brassica, but our alleles fall into at least three dominance classes in both pollen and stigma expression. The diversity among sequences of the A. lyrata putative S-alleles is greater than among the published Brassica SRK sequences, and, unlike Brassica, the alleles do not cluster into groups with similar dominance.