Interspecific pairs of class II S haplotypes having different recognition specificities between Brassica oleracea and Brassica rapa

There are several pairs of similar class I S haplotypes between Brassica oleracea and Brassica rapa. The similar S halotypes in these interspecific pairs have been reported to have the same recognition specificities. In the present study, three interspecific pairs showing a high sequence similarity were found in class II S haplotypes, i.e. between BoS-2b (B. oleracea S-2b) and BrS-44 (B. rapa S-44), between BoS-5 and BrS-40, and between BoS-15 and BrS-60. By pollination tests using interspecific hybrids between B. oleracea and B. rapa, BoS-5 and BoS-2b were revealed to have slightly and completely different recognition specificities from those of BrS-40 and BrS-44, respectively. The recognition reaction between SP11 and SRK of BoS-15 was suggested to be incomplete. The regions of class II SP11 and SRK important for self-recognition specificity and the diversification of class II S haplotypes are discussed herein.     

The self-compatibility mechanism in <Emphasis Type="Italic">Brassica napus</Emphasis> L. is applicable to F<Subscript>1</Subscript> hybrid breeding

Brassica napus, an allopolyploid species having the A genome of B. rapa and the C genome of B. oleracea, is self-compatible, although both B. rapa and B. oleracea are self-incompatible. We have previously reported that SP11/SCR alleles are not expressed in anthers, while SRK alleles are functional in the stigma in B. napus cv. ‘Westar’, which has BnS-1 similar to B. rapa S-47 and BnS-6 similar to B. oleracea S-15. This genotype is the most frequent S genotype in B. napus, and we hypothesized that the loss of the function of SP11 is the primary cause of the self-compatibility of ‘Westar’. To verify this hypothesis, we transformed ‘Westar’ plants with the SP11 allele of B. rapa S-47. All the transgenic plants and their progeny were completely self-incompatible, demonstrating self-compatibility to be due to the S haplotype having the non-functional SP11 allele in the A genome, which suppresses a functional recessive SP11 allele in the C genome. An artificially synthesized B. napus line having two recessive SP11 alleles was developed by interspecific hybridization between B. rapa and B. oleracea. This line was self-incompatible, but F₁ hybrids between this line and ‘Westar’ were self-compatible. These results suggest that the self-compatibility mechanism of ‘Westar’ is applicable to F₁ seed production in B. napus.     

Distribution of <Emphasis Type="Italic">S</Emphasis> haplotypes and its relationship with restorer–maintainers of self-incompatibility in cultivated <Emphasis Type="Italic">Brassica napus</Emphasis>

Brassica napus (AACC, 2n = 38) is a self-compatible amphidiploid plant that arose from the interspecies hybridization of two self-incompatible species, B. rapa (AA, 2n = 20) and B. oleracea (CC, 2n = 18). Self-incompatibility (S) haplotypes in one self-incompatible line and 124 cultivated B. napus lines were detected using S-locus-specific primers, and their relationships with restorer-maintainers were investigated. Two class I (S-I ( SLG ) a and S-I ( SLG ) b) and four class II (S-II ( SLG ) a, S-II ( SLG ) b, S-II ( SP11 ) a and S-II ( SP11 ) b) S haplotypes were observed, of which S-II ( SP11 ) b was newly identified. The nucleotide sequence of SP11 showed little similarity to the reported SP11 alleles. The lines were found to express a total of eleven S genotypes. The self-incompatible line had a specific genotype consisting of S-II ( SP11 ) a, similar to B. rapa S-60, and S-II ( SLG ) a, similar to B. oleracea S-15. Restorers expressed six genotypes: the most common genotype contained S-I ( SLG ) a, similar to B. rapa S-47, and S-II ( SLG ) b, similar to B. oleracea S-15. Maintainers expressed nine genotypes: the predominant genotype was homozygous for two S haplotypes, S-II ( SLG ) a and S-II ( SP11 ) b. One genotype was specific to restorers and four genotypes were specific to maintainers, whereas five genotypes were expressed in both restorers and maintainers. This suggests that there is no definitive correlation between the distribution of S genotypes and restorer-maintainers of self-incompatibility. The finding that restorers and maintainers express unique genotypes, and share some common genotypes, would be valuable for detecting the interaction of S haplotypes in inter- or intra-genomes as well as for developing markers-assisted selection in self-incompatibility hybrid breeding.     

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.     

Diversification and alteration of recognition specificity of the pollen ligand SP11/SCR in self-incompatibility of Brassica and Raphanus

The recognition specificity of the pollen ligand of self-incompatibility (SP11/SCR) was investigated using Brassica rapa transgenic plants expressing SP11 transgenes, and SP11 of Raphanus sativus S-21 was found to have the same recognition specificity as that of B. rapa S-9. In a set of three S haplotypes, whose sequence identities of SP11 and SRK are fairly high, R. sativus S-6 showed the same recognition specificity as Brassica oleracea S-18 and a slightly different specificity from B. rapa S-52. B. oleracea S-18, however, showed a different specificity from B. rapa S-52. Using these similar S haplotypes, chimeric SP11 proteins were produced by domain swapping. Bioassay using the chimeric SP11 proteins revealed that the incompatibility response induction activity was altered by the replacement of Region III and Region V. Pollen grains of Brassica transgenic plants expressing chimeric SP11 of the B. oleracea SP11-18 sequence with Region III and Region V from B. rapa SP11-52 (chimeric BoSP11-18[52]) were partially incompatible with the B. rapa S-52 stigmas, and those expressing the R. sativus SP11-6 sequence with Region III and Region V from B. rapa SP11-52 (chimeric RsSP11-6[52]) were completely incompatible with the stigmas having B. rapa S-52. However, the transgenic plant expressing chimeric RsSP11-6(52) also showed incompatibility with B. oleracea S-18 stigmas. These results suggest that Regions III and Region V of SP11 are important for determining the recognition specificity, but not the sole determinant. A possible process of the generation of a new S haplotype is herein discussed.     

Chromosomal localization and characterization of rDNA loci in the Brassica A and C genomes.

Using fluorescence in situ hybridization, we located ribosomal DNA loci on prometaphase chromosomes of the diploid species Brassica rapa and Brassica oleracea and their amphidiploid Brassica napus. Based on comparisons of chromosome morphology and hybridization patterns, we characterized the individual B. napus rDNA loci according to their presumed origins in the Brassica A and C genomes. As reported in other studies, the sum of rDNA loci observed on B. rapa (AA genome) and B. oleracea (CC genome) chromosomes was one greater than the total number of loci seen in their amphidiploid B. napus (AACC). Evidence is presented that this reduction in B. napus rDNA locus number results from the loss of the smallest A genome rDNA site in the amphidiploid.     

Effects of recombination on hitchhiking diversity in the Brassica self-incompatibility locus complex

In self-incompatibility, a number of S haplotypes are maintained by frequency-dependent selection, which results in trans-specific S haplotypes. The region of several kilobases (approximately 40-60 kb) from SP6 to SP2, including self-incompatibility-related genes and some adjacent genes in Brassica rapa, has high nucleotide diversity due to the hitchhiking effect, and therefore we call this region the "S-locus complex." Recombination in the S-locus complex is considered to be suppressed. We sequenced regions of >50 kb of the S-locus complex of three S haplotypes in B. rapa and found higher nucleotide diversity in intergenic regions than in coding regions. Two highly similar regions of >10 kb were found between BrS-8 and BrS-46. Phylogenetic analysis using trans-specific S haplotypes (called interspecific pairs) of B. rapa and B. oleracea suggested that recombination reduced the nucleotide diversity in these two regions and that the genes not involved in self-incompatibility in the S-locus complex and the kinase domain, but not the S domain, of SRK have also experienced recombination. Recombination may reduce hitchhiking diversity in the S-locus complex, whereas the region from the S domain to SP11 would disfavor recombination.     

Recognition specificity of self-incompatibility maintained after the divergence of Brassica oleracea and Brassica rapa

The determinants of recognition specificity of self-incompatibility in Brassica are SRK in the stigma and SP11/SCR in the pollen, respectively. In the pair of S haplotypes BrS46 (S46 in B. rapa) and BoS7 (S7 in B. oleracea), which have highly similar SRK alleles, the SP11 alleles were found to be similar, with 96.1% identity in the deduced amino acid sequence. Two other pairs of S haplotypes, BrS47 and BoS12, and BrS8 and BoS32, having highly similar SRK and SP11 alleles between the two species were also found. The haplotypes in each pair are considered to have been derived from a single S haplotype in the ancestral species. The allotetraploid produced by interspecific hybridization between homozygotes of BrS46 and BoS15 showed incompatibility with a BoS7 homozygote and compatibility with other B. oleracea S haplotypes in reciprocal crossings. This result indicates that BrS46 and BoS7 have maintained the same recognition specificity after the divergence of the two species and that amino acid substitutions found in such cases in both SRK alleles and SP11 alleles do not alter the recognition specificity. DNA blot analysis of SRK, SP11, SLG and other S-locus genes showed different DNA fragment sizes between the interspecific pairs of S haplotypes. A much lower level of sequence similarity was observed outside the genes of SRK and SP11 between BrS46 and BoS7. These results suggest that the DNA sequences of the regions intervening between the S-locus genes were diversified after or at the time of speciation. This is the first report demonstrating the presence of common S haplotypes in different plant species and presenting definite evidence of the trans-specific evolution of self-incompatibility genes.     

Conservation of the microstructure of genome segments in Brassica napus and its diploid relatives

The cultivated Brassica species are the group of crops most closely related to Arabidopsis thaliana (Arabidopsis). They represent models for the application in crops of genomic information gained in Arabidopsis and provide an opportunity for the investigation of polyploid genome formation and evolution. The scientific literature contains contradictory evidence for the dynamics of the evolution of polyploid genomes. We aimed at overcoming the inherent complexity of Brassica genomes and clarify the effects of polyploidy on the evolution of genome microstructure in specific segments of the genome. To do this, we have constructed bacterial artificial chromosome (BAC) libraries from genomic DNA of B. rapa subspecies trilocularis (JBr) and B. napus var Tapidor (JBnB) to supplement an existing BAC library from B. oleracea. These allowed us to analyse both recent polyploidization (under 10,000 years in B. napus) and more ancient polyploidization events (ca. 20 Myr for B. rapa and B. oleracea relative to Arabidopsis), with an analysis of the events occurring on an intermediate time scale (over the ca. 4 Myr since the divergence of the B. rapa and B. oleracea lineages). Using the Arabidopsis genome sequence and clones from the JBr library, we have analysed aspects of gene conservation and microsynteny between six regions of the genome of B. rapa with the homoeologous regions of the genomes of B. oleracea and Arabidopsis. Extensive divergence of gene content was observed between the B. rapa paralogous segments and their homoeologous segments within the genome of Arabidopsis. A pattern of interspersed gene loss was identified that is similar, but not identical, to that observed in B. oleracea. The conserved genes show highly conserved collinearity with their orthologues across genomes, but a small number of species-specific rearrangements were identified. Thus the evolution of genome microstructure is an ongoing process. Brassica napus is a recently formed polyploid resulting from the hybridization of B. rapa (containing the Brassica A genome) and B. oleracea (containing the Brassica C genome). Using clones from the JBnB library, we have analysed the microstructure of the corresponding segments of the B. napus genome. The results show that there has been little or no change to the microstructure of the analysed segments of the Brassica A and C genomes as a consequence of the hybridization event forming natural B. napus. The observations indicate that, upon polyploid formation, these segments of the genome did not undergo a burst of evolution discernible at the scale of microstructure.     

Structural and functional comparative mapping between the Brassica A genomes in allotetraploid Brassica napus and diploid Brassica rapa

Brassica napus (AACC genome) is an important oilseed crop that was formed by the fusion of the diploids B. rapa (AA) and B. oleracea (CC). The complete genomic sequence of the Brassica A genome will be available soon from the B. rapa genome sequencing project, but it is not clear how informative the A genome sequence in B. rapa (A(r)) will be for predicting the structure and function of the A subgenome in the allotetraploid Brassica species B. napus (A(n)). In this paper, we report the results of structural and functional comparative mapping between the A subgenomes of B. napus and B. rapa based on genetic maps that were anchored with bacterial artificial chromosomes (BACs)-sequence of B. rapa. We identified segmental conservation that represented by syntenic blocks in over one third of the A genome; meanwhile, comparative mapping of quantitative trait loci for seed quality traits identified a dozen homologous regions with conserved function in the A genome of the two species. However, several genomic rearrangement events, such as inversions, intra- and inter-chromosomal translocations, were also observed, covering totally at least 5% of the A genome, between allotetraploid B. napus and diploid B. rapa. Based on these results, the A genomes of B. rapa and B. napus are mostly functionally conserved, but caution will be necessary in applying the full sequence data from B. rapa to the B. napus as a result of genomic rearrangements in the A genome between the two species.     

Molecular characterisation and chromosomal localisation of a telomere-like repetitive DNA sequence highly enriched in the C genome of Brassica

The aim of this work was to find C genome specific repetitive DNA sequences able to differentiate the homeologous A (B. rapa) and C (B. oleracea) genomes of Brassica, in order to assist in the physical identification of B. napus chromosomes. A repetitive sequence (pBo1.6) highly enriched in the C genome of Brassica was cloned from B. oleracea and its chromosomal organisation was investigated through fluorescent in situ hybridisation (FISH) in B. oleracea (2n = 18, CC), B. rapa (2n = 20, AA) and B. napus (2n = 38, AACC) genomes. The sequence was 203 bp long with a GC content of 48.3%. It showed up to 89% sequence identity with telomere-like DNA from many plant species. This repeat was clearly underrepresented in the A genome and the in situ hybridisation showed its B. oleracea specificity at the chromosomal level. Sequence pBo1.6 was localised at interstitial and/or telomeric/subtelomeric regions of all chromosomes from B. oleracea, whereas in B. rapa no signal was detected in most of the cells. In B. napus 18 to 24 chromosomes hybridised with pBo1.6. The discovery of a sequence highly enriched in the C genome of Brassica opens the opportunity for detailed studies regarding the subsequent evolution of DNA sequences in polyploid genomes. Moreover, pBo1.6 may be useful for the determination of the chromosomal location of transgenic DNA in genetically modified oilseed rape.     

Identification of the A and C genomes of amphidiploid Brassica napus (oilseed rape).

A genetic linkage map consisting of 399 RFLP-defined loci was generated from a cross between resynthesized Brassica napus (an interspecific B. rapa x B. oleracea hybrid) and "natural" oilseed rape. The majority of loci exhibited disomic inheritance of parental alleles demonstrating that B. rapa chromosomes were each pairing exclusively with recognisable A-genome homologues in B. napus and that B. oleracea chromosomes were pairing similarly with C-genome homologues. This behaviour identified the 10 A genome and 9 C genome linkage groups of B. napus and demonstrated that the nuclear genomes of B. napus, B. rapa, and B. oleracea have remained essentially unaltered since the formation of the amphidiploid species, B. napus. A range of unusual marker patterns, which could be explained by aneuploidy and nonreciprocal translocations, were observed in the mapping population. These chromosome abnormalities were probably caused by associations between homoeologous chromosomes at meiosis in the resynthesized parent and the F1 plant leading to nondisjunction and homoeologous recombination.     

The CACTA transposon Bot1 played a major role in Brassica genome divergence and gene proliferation

We isolated and characterized a Brassica C genome-specific CACTA element, which was designated Bot1 (Brassica oleracea transposon 1). After analysing phylogenetic relationships, copy numbers and sequence similarity of Bot1 and Bot1 analogues in B. oleracea (C genome) versus Brassica rapa (A genome), we concluded that Bot1 has encountered several rounds of amplification in the oleracea genome only, and has played a major role in the recent rapa and oleracea genome divergence. We performed in silico analyses of the genomic organization and internal structure of Bot1, and established which segment of Bot1 is C-genome specific. Our work reports a fully characterized Brassica repetitive sequence that can distinguish the Brassica A and C chromosomes in the allotetraploid Brassica napus, by fluorescent in situ hybridization. We demonstrated that Bot1 carries a host S locus-associated SLL3 gene copy. We speculate that Bot1 was involved in the proliferation of SLL3 around the Brassica genome. The present study reinforces the assumption that transposons are a major driver of genome and gene evolution in higher plants.     

Resistance of some cultivated Brassicaceae to infestations by Plutella xylostella (Lepidoptera: Plutellidae)

Selecting insect-resistant plant varieties is a key component of integrated management programs of oligophagous pests such as diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), but rigorous research on important life history parameters of P. xylostella in relation to host plant resistance is rare. We evaluated six conventional brassicaceous species, namely, Brassica napus L. 'Q2', B. rapa L., B. juncea (L.) Czern., B. carinata L., B. oleracea L., and Sinapis alba L., and two herbicide-tolerant cultivars, namely, B. napus 'Liberty' and B. napus 'Conquest' for their resistance against P. xylostella. Brassicaceae species and cultivars varied considerably in their susceptibilities as hosts for P. xylostella. Sinapis alba and B. rapa plants were highly preferred by ovipositing females and trichome density on adaxial and abaxial leaf surfaces had nonsignificant effects on P. xylostella oviposition. Larval survival was similar on the genotypes we tested, but host plants significantly affected larval and pupal developmental time, herbivory, pupal weight, silk weight, adult body weight, forewing area and longevity (without food) of both male and female P. xylostella. Larval and pupal development of females was fastest on B. juncea and S. alba, respectively. Specimens reared on B. napus Liberty and B. oleracea, respectively, produced the lightest female and male pupae. Defoliation by both female and male larvae was highest on B. rapa, whereas least herbivory occurred on S. alba. Females reared on S. alba were heavier and lived longer in the absence of food than their counterparts raised on other tested host plants. Brassica oleracea could not compensate for larval feeding to the level of the other species we evaluated. B. napus Conquest, B. napus Q2, B. carinata, B. rapa, and S. alba produced, respectively, 1.6-, 1.8-, 1.8-, 3.9-, and 5.5-fold heavier root systems when infested than their uninfested counterparts, suggesting that these species were better able to tolerate P. xylostella infestations.     

Conserved patterns of chromosome pairing and recombination in Brassica napus crosses.

The patterns of chromosome pairing and recombination in two contrasting Brassica napus F1 hybrids were deduced. One hybrid was from a winter oilseed rape (WOSR) x spring oilseed rape cross, the other from a resynthesized B. napus x WOSR cross. Segregation at 211 equivalent loci assayed in the population derived from each hybrid produced two collinear genetic maps. Alignment of the maps indicated that B. napus chromosomes behaved reproducibly as 19 homologous pairs and that the 19 distinct chromosomes of B. napus each recombined with unique chromosomes from the interspecific hybrid between Brassica rapa and Brassica oleracea. This result indicated that the genomes of the diploid progenitors of amphidiploid B. napus have remained essentially unaltered since the formation of the species and that the progenitor genomes were similar to those of modern-day B. rapa and B. oleracea. The frequency and distribution of crossovers were almost indistinguishable in the two populations, suggesting that the recombination machinery of B. napus could cope easily with different degrees of genetic divergence between homologous chromosomes. Efficient recombination in wide crosses will facilitate the introgression of novel alleles into oilseed rape from B. rapa and B. oleracea (via resynthesized B. napus) and reduce linkage drag.     

A pollen coat protein, SP11/SCR, determines the pollen S-specificity in the self-incompatibility of Brassica species

Many flowering plants have evolved self-incompatibility (SI) systems to prevent inbreeding. In the Brassicaceae, SI is genetically controlled by a single polymorphic locus, termed the S-locus. Pollen rejection occurs when stigma and pollen share the same S-haplotype. Recognition of S-haplotype specificity has recently been shown to involve at least two S-locus genes, S-receptor kinase (SRK) and S-locus protein 11 or S-locus Cys-rich (SP11/SCR). SRK encodes a polymorphic membrane-spanning protein kinase, which is the sole female determinant of the S-haplotype specificity. SP11/SCR encodes a highly polymorphic Cys-rich small basic protein specifically expressed in the anther tapetum and in pollen. In cauliflower (B. oleracea), the gain-of-function approach has demonstrated that an allele of SP11/SCR encodes the male determinant of S-specificity. Here we examined the function of two alleles of SP11/SCR of B. rapa by the same approach and further established that SP11/SCR is the sole male determinant of SI in the genus Brassica sp. Our results also suggested that the 522-bp 5'-upstream region of the S9-SP11 gene used to drive the transgene contained all the regulatory elements required for the unique sporophytic/gametophytic expression observed for the native SP11 gene. Promoter deletion analyses suggested that the highly conserved 192-bp upstream region was sufficient for driving this unique expression. Furthermore, immunohistochemical analyses revealed that the protein product of the SP11 transgene was present in the tapetum and pollen, and that in pollen of late developmental stages, the SP11 protein was mainly localized in the pollen coat, a finding consistent with its expected biological role.     

Yield reduction in Brassica napus, B. rapa, B. juncea, and Sinapis alba caused by flea beetle (Phyllotreta cruciferae (Goeze) (Coleoptera: Chrysomelidae)) infestation in northern Idaho

Phyllotreta cruciferae is an important insect pest of spring-planted Brassica crops, especially during the seedling stage. To determine the effect of early season P. cruciferae infestation on seed yield, 10 genotypes from each of two canola species (Brassica napus L. and Brassica rapa L.) and two mustard species (Brassica juncea L. and Sinapis alba L.) were grown in 2 yr under three different P. cruciferae treatments: (1) no insecticide control; (2) foliar applications of endosulfan; and (3) carbofuran with seed at planting plus foliar application of carbaryl. Averaged over 10 genotypes, B. rapa showed most visible P. cruciferae injury and showed greatest yield reduction without insecticide application. Mustard species (S. alba and B. juncea) showed least visible injury and higher yield without insecticide compared with canola species (B. napus and B. rapa). Indeed, average seed yield of S. alba without insecticide was higher than either B. napus or B. rapa with most effective P. cruciferae control. Significant variation occurred within each species. A number of lines from B. napus, B. juncea, anid S. alba showed less feeding injury and yield reduction as a result of P. cruciferae infestation compared with other lines from the same species examined, thus having potential genetic background for developing resistant cultivars.     

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.     

Karyotype and identification of all homoeologous chromosomes of allopolyploid Brassica napus and its diploid progenitors

Investigating recombination of homoeologous chromosomes in allopolyploid species is central to understanding plant breeding and evolution. However, examining chromosome pairing in the allotetraploid Brassica napus has been hampered by the lack of chromosome-specific molecular probes. In this study, we establish the identification of all homoeologous chromosomes of allopolyploid B. napus by using robust molecular cytogenetic karyotypes developed for the progenitor species Brassica rapa (A genome) and Brassica oleracea (C genome). The identification of every chromosome among these three Brassica species utilized genetically mapped bacterial artificial chromosomes (BACs) from B. rapa as probes for fluorescent in situ hybridization (FISH). With this BAC-FISH data, a second karyotype was developed using two BACs that contained repetitive DNA sequences and the ubiquitous ribosomal and pericentromere repeats. Using this diagnostic probe mix and a BAC that contained a C-genome repeat in two successive hybridizations allowed for routine identification of the corresponding homoeologous chromosomes between the A and C genomes of B. napus. When applied to the B. napus cultivar Stellar, we detected one chromosomal rearrangement relative to the parental karyotypes. This robust novel chromosomal painting technique will have biological applications for the understanding of chromosome pairing, homoeologous recombination, and genome evolution in the genus Brassica and will facilitate new applied breeding technologies that rely upon identification of chromosomes.