Phylogenetic Analysis of S-Locus Genes Reveals the Complicated Evolution Relationship of S Haplotypes in Brassica

Self-incompatibility (SI) is reported to play a key role in the evolution of species as it promotes their outcrossing through the recognition and rejection of self-pollen grains. In Brassica, two S-locus genes expressed in the stigma, S-locus glycoprotein (SLG) gene and S-locus receptor kinase (SRK) gene, and one expressed in the pollen, S-locus protein 11 (SP11) gene, were linked as an S haplotype. In order to analyze the evolutionary relationships of S haplotypes in Brassica, a total of 39 SRK, 37 SLG, and 58 SP11 sequences of Brassica oleracea, Brassica rapa and Brassica napus were aligned. Two phylogenetic trees with similar pattern were constructed based on the nucleotide sequences of SRK/SLG and SP11, respectively. Class I and class II alleles were clustered into two distinct groups, and alleles from different species, including all the interspecific pairs of S haplotypes, were closely related to each other. The S-locus genes identified in B. napus were intermingled in phylogenetic trees. All these observations showed that class I and class II S haplotypes diverged ahead of the species differentiation in Brassica. The evolution and the genetic diversity of S haplotypes in Brassica were discussed. Moreover, the relationships between S haplotypes and SI phenotypes in Brassica, especially in B. napus, were also discussed.     

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.     

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’.     

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.     

Single-seed PCR-RFLP analysis for the identification of S haplotypes in commercial F1 hybrid cultivars of broccoli and cabbage

 Several simple methods of DNA preparation from plant tissues were evaluated for PCR-RFLP analyses of SLG and SRK alleles, which can be used for the identification of S haplotypes of breeding lines in broccoli and cabbage (Brassica oleracea L.) and in purity tests of F₁ hybrid seeds. On the five methods tested, the NaI method was found to be the most suitable for the amplification of the SLG and SRK alleles. This method enables the use of a single seed as testing material. Using this method, we identified S haplotypes of 31 broccoli and 31 cabbage cultivars. Ninety-four percent of the cultivars of broccoli and 97% of those of cabbage were-single cross F₁ hybrids. Nine and 15 S haplotypes were found in broccoli and cabbage, respectively. The small number of S haplotypes in broccoli suggests the importance of incorporating new S haplotypes in the breeding program. Received: 18 February 1999 / Revision received: 4 May 1999 / Accepted: 14 May 1999     

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     

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     

Does the genome of Corylus avellana L. contain sequences homologous to the self-incompatibility gene of Brassica?

Self-incompatibility is a genetic mechanism enforcing cross-pollination in plants. Hazelnut (Corylus avellana L.) expresses the sporophytic type of self-incompatibility, for which the molecular genetic basis is characterized only in Brassica. The hypothesis that the hazelnut genome contains homologs of Brassica self-incompatibility genes was tested. The S-locus glycoprotein gene (SLG) and the kinase-encoding domain of the S-receptor kinase (SRK) gene of B. oleracea L. were used to probe blots of genomic DNA from six genotypes of hazelnut. Weak hybridization with the SLG probe was detected for all hazelnut genotypes tested; however, no hybridization was detected with PCR-generated probes corresponding to two conserved regions of the SLG gene. One of these PCR probes included the region of SLG encoding the 11 invariant cysteine residues that are an important structural feature of all S-family genes. The present evidence suggests that hazelnut DNA hybridizing to SLG differs significantly from the Brassica gene, and that the S-genes cloned from Brassica will not be useful for exploring self-incompatibility in hazelnut.     

Registration of S alleles in Brassica campestris L by the restriction fragment sizes of SLGs

Polymorphism of SLG (the S-locus glycoprotein gene) in Brassica campestris was analyzed by PCR-RFLP using SLG-specific primers. Nucleotide sequences of PCR products from 15 S genotypes were determined in order to characterise the exact DNA fragment sizes detected in the PCR-RFLP analysis. Forty-seven lines homozygous for 27 S-alleles were used as plant material. One combination of primers, PS5 + PS 15, which had a nucleotide sequence specific to a class-I SLG, gave amplification of a single DNA fragment of approximately 1.3kb from the genomic DNA of 15 S genotypes. All the DNA fragments showed different electrophroetic profiles from each other after digestion with MboI or MspI. Different lines having the same S genotype had an identical electrophoretic profile even between the lines collected in Turkey and in Japan. Another class-I SLG-specific primer, PS 18, gave amplification of a 1.3-kb DNA fragment from three other S genotypes in combination with PS 15, and the PCR product also showed polymorphism after cleavage with the restriction endonucleases. Genetic analysis, Southern-hybridization analysis, and determination of the nucleotide sequences of the PCR products suggested that the DNA fragments amplified with these combinations of primers are class-I SLGs. Expected DNA fragment sizes in the present PCR-RFLP condition were calculated from the determined nucleotide sequence of SLG PCR products. A single DNA fragment was also amplified from six S genotypes by PCR with a combination of primers, PS3 + PS21, having a nucleotide sequence specific to a class-II SLG. The amplified DNA showed polymorphisnm after cleavage with restriction endonucleases. The cleaved fragments were detected by Southern-hybridization analysis using a probe of S ⁵ SLG cDNA, a class-IISLG. Partial sequencing revealed a marked similarity of these amplified DNA fragments to a class-II SLG, demonstrating the presence of class-I and class-II S alleles also in B. campestris. The high SLG polymorphism detected by the present investigation suggests the usefulness of the PCR-RFLP method for the identification of S alleles in breeding lines and for listing S alleles in B. campestris.     

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.     

Distribution of similar self-incompatibility (<Emphasis Type="Italic">S</Emphasis>) haplotypes in different genera,<Emphasis Type="Italic"> Raphanus</Emphasis> and<Emphasis Type="Italic"> Brassica</Emphasis>

The nucleotide sequences of ten SP11 and nine SRK alleles in Raphanus sativus were determined, and deduced amino acid sequences were compared with those of Brassica SP11 and SRK. The amino acid sequence identity of class-I SP11s in R. sativus was about 30% on average, the highest being 52.2%, while that of the S domain of class-I SRK was 77.0% on average and ranged from 70.8% to 83.9%. These values were comparable to those of SP11 and SRK in Brassica oleracea and B. rapa. SP11 of R. sativus S-21 was found to be highly similar to SP11 of B. rapa S-9 (89.5% amino acid identity), and SRK of R. sativus S-21 was similar to SRK of B. rapa S-9 (91.0%). SP11 and SRK of R. sativus S-19 were also similar to SP11 and SRK of B. oleracea S-20, respectively. These similarities of both SP11 and SRK alleles between R. sativus and Brassica suggest that these S haplotype pairs originated from the same ancestral S haplotypes.     

Characterization of self-incompatibility genes in the intergeneric hybrid xBrassicoraphanus

Brassica rapa and Raphanus sativus are strictly self-incompatible (SI) plants; however, xBrassicoraphanus (baemoochae) is an intergeneric hybrid synthesized following hybridization of B. rapa and R. sativus that is self-compatible (SC). Self-incompatibility in Brassicaceae is controlled by multiple alleles of the S-locus. Two S-locus genes, SRK (S-locus receptor kinase) and SP11/SCR (S-locus protein 11 or S-locus cysteine-rich), have been reported to date, both of which are classified into class I and II. In this study, we investigate if there is an alteration in the structure or the expression in SRK or SP11 genes behind the alteration of SI to SC in baemoochae. Class I and II SRK were identified by PCR of the genomic DNA of baemoochae using SRK-specific universal primers. Cloning and sequencing of both classes of SRK was conducted and compared with SRK genes of parental species. Comparison analysis showed no genomic alterations and both of them showed expression in the stigma. Similarly, SP11 genes also showed no genomic alterations and normally are expressed in the anther. Other SI-related genes (MLPK, ARC1, THL) also showed normal expression in the stigma and anther. Taken together, these results revealed that no structural/gene expression change in these genes was responsible for the breakdown of SI in baemoochae. Rather, the transformation from SI parents to SC descendants could be responding to epigenetic mechanisms.     

Commonality of self-recognition specificity of S haplotypes between Brassica oleracea and Brassica rapa

We have identified several interspecific pairs of S haplotypes having highly similar SRK and SP11/SCR sequences between Brassica oleracea and Brassica rapa. The recognition specificities of S haplotypes in these pairs were examined with three different methods. Stigmas of interspecific hybrids between an S-32 homozygote in B. oleracea and an S-60 homozygote in B. rapa, which were produced to avoid the interspecific incompatibility between the two species, showed incompatibility to the pollen of an S-8 homozygote in B. rapa and to the pollen of an S-15 homozygote in B. oleracea, while it showed compatibility to the pollen of other S haplotypes, suggesting B. oleracea S-32 and B. rapa S-60 have the same recognition specificity as B. rapa S-8 and B. oleracea S-15. Pollen grains of transgenic S-60 homozygous plants in B. rapa carrying a transgene of SP11-24 from B. oleracea were incompatible to B. rapa S-36 stigma, indicating that B. oleracea S-24 and B. rapa S-36 have the same recognition specificity. Application of the SP11 protein of B. rapa S-41 and S-47 onto the surface of B. oleracea S-64 stigmas and S-12 stigmas, respectively, resulted in the incompatibility reaction to pollen grains of another S haplotype, but application onto the stigmas of other S haplotypes did not, suggesting that B. oleracea S-64 stigmas and S-12 stigmas recognized the B. rapa SP11-41 and SP11-47 proteins as self SP11 proteins, respectively. Besides having evolutionary implications, finding of many interspecific pairs of S haplotypes can provide insight into the molecular mechanism of self-recognition. Comparing deduced amino-acid sequences of SP11 proteins and SRK proteins in the pairs, regions of SP11 and SRK important for self-recognition are discussed.     

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.     

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     

Three members of the S multigene family are linked to the S locus of Brassica

Two self-incompatibility genes in Brassica, SLG and SRK (SLG encodes a glycoprotein; SRK encodes a receptor-like kinase), are included in the S multigene family. Products of members of the S multigene family have an SLG-like domain (S domain) in common, which may function as a receptor. In this study, three clustered members of the S multigene family, BcRK1, BcRL1 and BcSL1, were characterized. BcRK1 is a putative functional receptor kinase gene expressed in leaves, flower buds and stigmas, while BcRL1 and BcSL1 are considered to be pseudogenes because deletions causing frameshifts were identified in these sequences. Sequence and expression pattern of BcRK1 were most similar to those of the Arabidopsis receptor-like kinase gene ARK1, indicating that BcRK1 might have a function similar to that of ARK1, in processes such as cell expansion or plant growth. Interestingly, the region containing BcRK1, BcRL1 and BcSL1 is genetically linked to the S locus and the physical distance between SLG, SRK and the three S-related genes was estimated to be less than 610 kb. Thus the genes associated with self-incompatibility exist within a cluster of S-like genes in the genome of Brassica. Received: 15 April 1997 / Accepted: 13 June 1997     

Simple and efficient methods for <Emphasis Type="Italic">S</Emphasis> genotyping and <Emphasis Type="Italic">S</Emphasis> screening in genus <Emphasis Type="Italic">Brassica</Emphasis> by dot-blot analysis

In F₁ hybrid breeding of Brassica vegetables utilizing the self-incompatibility system, identification of S genotypes in breeding lines is required. In the present study, we developed S-tester lines of 87 S haplotypes, i.e., 42 S haplotypes in B. rapa and 45 S haplotypes in B. oleracea. With these materials, we established a simple, efficient, and reliable dot-blot technique for S genotyping for 40 S haplotypes of B. rapa and and 33 of B. oleracea using allele-specific oligonucleotide probes and allele-specific primer pairs designed from sequences of each SP11 allele. In this method, DNA fragments amplified using multiplex primer pairs with digoxigenin-dUTP were hybridized with dot-blotted allele-specific oligonucleotide probes with distinct signals. In addition, we developed a screening method for identification of plants harboring a particular S haplotype using a labeled allele-specific oligonucleotide probe. This method is considered to be useful for purity testing of F₁ hybrid seeds.