RFLP mapping of loci controlling self-incompatibility in <Emphasis Type="Italic">Brassica campestris</Emphasis> and their comparative mapping with <Emphasis Type="Italic">B. napus</Emphasis> and <Emphasis Type="Italic">B. oleracea</Emphasis>

RFLP analysis of a cDNA probe SLG6, governing self incompatibility (SI) in Brassica oleracea, using a recombinant inbred population of Brassica campestris followed by genetic linkage analysis led to the detection of two marker loci, SLG6a and SLG6b controlling SI. SLG6a was mapped in linkage group (LG) 9 and was flanked by the RFLP markers ec4f10 (6.4 cM) and wg5b9 (4.2 cM). SLG6b positioned in LG 2 and was flanked by the RFLP markers wg2d11 (9.9 cM) and ec4e7 (26.9 cM). These results indicated the scope of marker-aided introgression of these genes into self-compatible genotypes for production of SI lines suitable for hybridization in B. campestris. Comparative mapping of LG 9 containing SLG6b with corresponding linkage groups of B. oleracea (BO 2) and B. napus (BN 16) led to the detection of small homologous regions with SLG6 locus linked with another RFLP locus. This evidenced for homology of the SLG genes across Brassica species and possibility of using any single cloned SLG gene for development of SI lines in any Brassica species.     

Identification of <Emphasis Type="Italic">S</Emphasis> haplotypes in <Emphasis Type="Italic">Brassica</Emphasis> by dot-blot analysis of <Emphasis Type="Italic">SP11</Emphasis> alleles

A self-incompatibility system is used for F(1) hybrid breeding in Brassicaceae vegetables. The determinants of recognition specificity of self-incompatibility in Brassica are SRK in the stigma and SP11/SCR in the pollen. Nucleotide sequences of SP11 alleles are more highly variable than those of SRK. We analyzed the S haplotype specificity of SP11 DNA by Southern-blot analysis and dot-blot analysis using 16 S haplotypes in Brassica oleracea, and found that DNA fragments of a mature protein region of SP11 cDNA, SP11(m), of eight S haplotypes can detect only the SP11 alleles of the same S haplotypes. This specificity makes these methods useful for S haplotype identification. Therefore, we developed two methods of dot-blot analysis for SP11. One is dot blotting of DNA samples, i.e. plant genomic DNA probed with labeled SP11(m), and the other is dot blotting of SP11(m) DNA fragments probed with labeled DNA samples, i.e. the SP11 coding region labeled by PCR using a template of plant genomic DNA. The former is useful for testing many plant materials. The latter is suitable, if there is no previous information on the S haplotypes of plant materials.     

Phylogenetic analysis of <Emphasis Type="Italic">Brassiceae</Emphasis> based on the nucleotide sequences of the <Emphasis Type="Italic">S</Emphasis>-locus related gene, <Emphasis Type="Italic">SLR1</Emphasis>

Nucleotide sequences of orthologs of the S-locus related gene, SLR1, in 20 species of Brassicaceae were determined and compared with the previously reported SLR1 sequences of six species. Identities of deduced amino-acid sequences with Brassica oleracea SLR1 ranged from 66.0% to 97.6%, and those with B. oleracea SRK and SLR2 were less than 62% and 55%, respectively. In multiple alignment of deduced amino-acid sequences, the 180-190th amino-acid residues from the initial methionine were highly variable, this variable region corresponding to hypervariable region I of SLG and SRK. A phylogenetic tree based on the deduced amino-acid sequences showed a close relationship of SLR1 orthologs of species in the Brassicinae and Raphaninae. Brassica nigra SLR1 was found to belong to the same clade as Sinapis arvensis and Diplotaxis siifolia, while the sequences of the other Brassica species belonged to another clade together with B. oleracea and Brassica rapa. The phylogenetic tree was similar to previously reported trees constructed using the data of electrophoretic band patterns of chloroplast DNA, though minor differences were found. Based on synonymous substitution rates in SLR1, the diversification time of SLR1 orthologs between species in the Brassicinae was estimated. The evolution and function of SLR1 and the phylogenetic relationship of Brassiceae plants are discussed.     

Sequence comparisons among dispersed members of the Brassica S multigene family in an S 9 genome

Self-incompatibility (SI) systems prevent self-pollination and promote outbreeding. In Brassica, the SI genes SLG (for S-locus glycoprotein) and SRK (for S-receptor kinase) are members of the S multigene family, which share the SLG-like domain (S domain), which encodes a putative receptor. We have cloned members of the S multigene family from the S9 haplotype of B. campestris (syn. rapa). In addition, eight distinct genomic regions harboring 10 SLG/SRK-like genes were characterized in the present study. Sequence analysis revealed two novel SRK-like genes, BcRK3 and BcRK6 (for B. campestris receptor kinases 3 and 6, respectively). Other genes that were characterized included SFR2 (for S gene family receptor 2), SLR2 (for S locus related gene 2), and a pseudogene. Based on phylogenetic analysis of the nucleotide sequences of the S domain regions, SLG and SRK appear to be distinct from other members of the S multigene family. Linkage analysis showed that most members of the S multigene family are dispersed in the Brassica genome, and that SLR1 (S locus related gene 1) is not linked to the SLR2 in B. campestris.     

Molecular mechanisms of self-incompatibility in Brassica

In Brassica species, self-incompatibility has been mapped genetically to a single chromosomal location. In this region several closely linked genes have been identified. One of them, S-locus receptor kinase (SRK), determines S haplotype specificity of the stigma and it's the key protein for SI reaction. The role of the S locus glycoprotein (SLG) gene remains unclear. In the last decade approximately 15 additional genes linked to S-locus have been found. Recently, a gene has been identified (SCR) that encodes a small cysteine-rich protein which is a candidate for the pollen ligand. In addition to S locus linked genes there are unlinked SLRgenes (S-locus related genes). In this review, we discuss the role of these genes and the current view on the self-incompatibility mechanism in Brassica.     

Molecular and genetic characterization of the <Emphasis Type="Italic">S</Emphasis> locus in <Emphasis Type="Italic">Hordeum bulbosum</Emphasis> L., a wild self-incompatible species related to cultivated barley

Gametophytic self-incompatibility (GSI) in the grasses is controlled by a distinct two-locus genetic system governed by the multiallelic loci S and Z. We have employed diploid Hordeum bulbosum as a model species for identifying the self-incompatibility (SI) genes and for elucidating the molecular mechanisms of the two-locus SI system in the grasses. In this study, we attempted to identify S haplotype-specific cDNAs expressed in pistils and anthers at the flowering stage in H. bulbosum, using the AFLP-based mRNA fingerprinting (AMF, also called cDNA-AFLP) technique. We used the AMF-derived DNA clones as markers for fine mapping of the S locus, and found that the locus resided in a chromosomal region displaying remarkable suppression of recombination, encompassing a large physical region. Furthermore, we identified three AMF-derived markers displaying complete linkage to the S locus, although they showed no significant homology with genes of known functions. Two of these markers showed expression patterns that were specific to the reproductive organs (pistil or anther), suggesting that they could be potential candidates for the S gene.     

Just how complex is the Brassica S-receptor complex?

Of the plant self-incompatibility (SI) systems investigated to date, that possessed by members of the Brassicaceae is currently the best understood. Whilst the recent demonstrations of interactions between the male determinant (S-locus cysteine rich protein, SCR) and the female determinant (S-locus receptor kinase, SRK) indicate the minimal requirement for SI in Brassica, no consensus exists as to the nature of these molecules in vivo and the potential involvement of accessory molecules in establishing the active S-receptor complex. Variation between S haplotypes appears to be present in the molecular composition of the receptor complex, the regulation of downstream signalling and the requirement for accessory molecules. This review discusses what constitutes an active receptor complex and highlights potential differences between haplotypes. The role of accessory molecules, in particular SLG (S-locus glycoprotein) and low molecular weight pollen coat proteins (PCPs), in pollination are discussed, as is the link between SI and unilateral incompatibility (UI).     

Self-compatible peach (<Emphasis Type="Italic">Prunus persica</Emphasis>) has mutant versions of the <Emphasis Type="Italic">S</Emphasis> haplotypes found in self-incompatible <Emphasis Type="Italic">Prunus</Emphasis> species

This study demonstrates that self-compatible (SC) peach has mutant versions of S haplotypes that are present in self-incompatible (SI) Prunus species. All three peach S haplotypes, S ¹ , S ² , and S ^2m , found in this study encode mutated pollen determinants, SFB, while only S ^2m has a mutation that affects the function of the pistil determinant S-RNase. A cysteine residue in the C5 domain of the S ^2m -RNase is substituted by a tyrosine residue, thereby reducing RNase stability. The peach SFB mutations are similar to the SFB mutations found in SC haplotypes of sweet cherry (P. avium) and Japanese apricot (P. mume). SFB ¹ of the S ¹ haplotype, a mutant version of almond (P. dulcis) S ^k haplotype, encodes truncated SFB due to a 155 bp insertion. SFB ² of the S ² and S ^2m haplotypes, both of which are mutant versions of the S ^a haplotype in Japanese plum (P. salicina), encodes a truncated SFB due to a 5 bp insertion. Thus, regardless of the functionality of the pistil determinant, all three peach S haplotypes are SC haplotypes. Our finding that peach has mutant versions of S haplotypes that function in almond and Japanese plum, which are phylogenetically close and remote species, respectively, to peach in the subfamily Prunoideae of the Roasaceae, provides insight into the SC/SI evolution in Prunus. We discuss the significance of SC pollen part mutation in peach with special reference to possible differences in the SI mechanisms between Prunus and Solanaceae.     

Characterization of directly transformed weedy <Emphasis Type="Italic">Brassica rapa</Emphasis> and introgressed <Emphasis Type="Italic">B. rapa</Emphasis> with Bt <Emphasis Type="Italic">cry1Ac</Emphasis> and <Emphasis Type="Italic">gfp</Emphasis> genes

Crop to weed transgene flow, which could result in more competitive weed populations, is an agricultural biosafety concern. Crop Brassica napus to weedy Brassica rapa hybridization has been extensively characterized to better understand the transgene flow and its consequences. In this study, weedy accessions of B. rapa were transformed with Bacillus thuringiensis (Bt) cry1Ac- and green fluorescence protein (gfp)-coding transgenes using Agrobacterium to assess ecological performance of the wild biotype relative to introgressed hybrids in which the transgenic parent was the crop. Regenerated transgenic B. rapa events were characterized by progeny analysis, Bt protein enzyme-linked immunosorbent assay (ELISA), Southern blot analysis, and GFP expression assay. GFP expression level and Bt protein concentration were significantly different between independent transgenic B. rapa events. Similar reproductive productivity was observed in comparison between transgenic B. rapa events and B. rapa × B. napus introgressed hybrids in greenhouse and field experiments. In the greenhouse, Bt transgenic plants experienced significantly less herbivory damage from the diamondback moth (Plutella xylostella). No differences were found in the field experiment under ambient, low, herbivore pressure. Directly transformed transgenic B. rapa plants should be a helpful experimental control to better understand crop genetic load in introgressed transgenic weeds.     

Organogenesis and <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of <Emphasis Type="Italic">Eucalyptus saligna</Emphasis> with <Emphasis Type="Italic">P5CS</Emphasis> gene

The purpose of this research was Eucalyptus saligna in vitro regeneration and transformation with P5CSF129A gene, which encodes Δ¹-pyrroline-5-carboxylate synthetase (P5CS), the key enzyme in proline biosynthesis. After selection of the most responsive genotype, shoot organogenesis was induced on leaf explants cultured on a callus induction medium (CI) followed by subculture on a shoot induction medium (SI). Shoots were subsequently cultured on an elongation medium (BE), then transferred to a rooting medium and finally transplanted to pots and acclimatized in a greenhouse. For genetic transformation, a binary vector carrying P5CSF129A and uidA genes, both under control of the 35SCaMV promoter, was used. Leaves were co-cultured with Agrobacterium tumefaciens in the dark on CI medium for 5 d. The explants were transferred to the selective callogenesis inducing medium (SCI) containing kanamycin and cefotaxime. Calli developed shoots that were cultured on an elongation medium for 14 d and finally multiplied. The presence of the transgene in the plant genome was demonstrated by PCR and confirmed by Southern blot analysis. Proline content in the leaves was four times higher in transformed than in untransformed plants while the proline content in the roots was similar in both types of plants.     

芸薹属自交不亲和基因的分子生物学及进化模式

芸薹属的自交不亲和性是受单基因座、复等位基因控制的孢子体控制型。自交不亲和基因座位（S-locus）是由多个基因组成的复杂区域,称之为S多基因家族,其大多数成员分布于芸薹属的整个染色体组。目前已鉴定出100多个S等位基因,它们的起源分化始于一千万年前。S-座位上存在的多基因有3种：SRK,SLG和SCR/SP11;SRK和SLG在柱头中表达,SCR/SP11在雄蕊中表达。SRK蛋白在识别同类花粉的过程中起主要作用,而SLG蛋白增强了这种自交不亲和反应。SLG与SRK基因中编码S-结构域的核苷酸序列相似性程度高达85％～98%。基因转换可能是SLG和SRK的高度同源性能够得以保持的原因。SRK,SLG和SCR基因紧密相连,并表现出高水平的序列多样性。SRK与SLG基因间的距离很近,在20～25 kb之间。在柱头和花粉中,自交不亲和等位基因之间的共显性关系要比显性和隐性关系更加普遍,这是芸薹属自交不亲和性的一大特点。自交不亲和基因的进化模式存在两种假说：双基因进化模式和中性变异体进化模式;可能存在几种不同的进化方式,它们共同在自然群体中新的S等位基因进化过程中起作用。     

Genetic relationships of Chukchi charr <Emphasis Type="Italic">Salvelinus andriashevi</Emphasis> and Taranetz charr <Emphasis Type="Italic">Salvelinus taranetzi</Emphasis>

On the basis of sequence analysis of the mitochondrial DNA (mtDNA) control region (CR), cytochrome b (Cytb), and cytochrome oxidase-1 (CoI) genes, the relationships of endemic species Salvelinus andriashevi Berg, 1948, represented by the only population from Lake Estikhed (Chukotka), were estimated. The data on the genealogical analysis of mtDNA haplotypes supported phylogenetic closeness of S. andriashevi and S. taranetzi. It was also demonstrated that the specimens of Chukchi charr, along with Salvelinus sp. 4 (Lake Nachikinskoe), S. krogiusae (Lake Dal’nee), S. boganidae and S. elgyticus (Lake El’gygytgyn), and S. a. erythrinus from Canada’s Northwest Territories (NWT) belonged to the Arctic group of Taranetz charr. The problem of coincidence of taxonomic differentiation of charrs of the genus Salvelinus based on morphological and genetic analyses is discussed.     

Stable chloroplast transformation in cabbage (<Emphasis Type="Italic">Brassica oleracea </Emphasis>L. var. <Emphasis Type="Italic">capitata </Emphasis>L.) by particle bombardment

The objectives of this research were first to isolate plastid gene sequences from cabbage (Brassica oleracea L. var. capitata L.), and to establish the chloroplast transformation technology of Brassica. A universal transformation vector (pASCC201) for Brassica chloroplast was constructed with trnV–rrn16S (left) and trnI–trnA–rrn23S (right) of the IR_A region as a recombination site for the transformed gene. In transforming plasmid pASCC201, a chimeric aadA gene was cloned between the rrn16S and rrn23S plastid gene borders. Expression of aadA confers resistance to spectinomycin and streptomycin antibiotics. The uidA gene was also inserted into the pASCC201 and transferred into the leaf cells of cabbage via particle gun mediated transformation. Regenerated plantlets were selected by 200 mg/l spectinomycin and streptomycin. After antibiotic selection, the regeneration percentage of the two cabbage cultivars was about 2.7–3.3%. The results of PCR testing and Southern blot analysis confirmed that the uidA and aadA genes were present in the chloroplast genome via homologously recombined. Northern blot hybridizations, immunoblotting and GUS histochemical assays indicated that the uidA gene were stable integrated into the chloroplast genome. Foreign protein was accumulated at 3.2–5.2% of the total soluble protein in transgenic mature leaves. These results suggest that the expression of a variety of foreign genes in the chloroplast genome will be a powerful tool for use in future studies.     

Evolution of the self-incompatibility system in the Brassicaceae: identification of S-locus receptor kinase (SRK) in self-incompatible Capsella grandiflora

Self-incompatibility (SI) has been well studied in the genera Brassica and Arabidopsis, which have become models for investigation into the SI system. To understand the evolution of the SI system in the Brassicaceae, comparative analyses of the S-locus in genera other than Brassica and Arabidopsis are necessary. We report the identification of six putative S-locus receptor kinase genes (SRK) in natural populations of Capsella grandiflora, an SI species from a genus which is closely related to Arabidopsis. These S-alleles display striking similarities to the Arabidopsis lyrata SRK alleles in sequence and structure. Our phylogenetic analysis supports the scenario of differing SI evolution along the two lineages (The Brassica lineage and Arabidopsis/Capsella lineage). Our results also argue that the ancestral S-locus lacked the SLG gene (S-locus glycoprotein) and that the diversification of S-alleles predates the separation of Arabidopsis and Capsella.     

<Emphasis Type="Italic">S</Emphasis> locus-linked F-box genes expressed in anthers of <Emphasis Type="Italic">Hordeum bulbosum</Emphasis>

Diploid Hordeum bulbosum (a wild relative of cultivated barley) exhibits a two-locus self-incompatibility (SI) system gametophytically controlled by the unlinked multiallelic loci S and Z. This unique SI system is observed in the grasses (Poaceae) including the tribe Triticeae. This paper describes the identification and characterization of two F-box genes cosegregating with the S locus in H. bulbosum, named Hordeum S locus-linked F-box 1 (HSLF1) and HSLF2, which were derived from an S ₃ haplotype-specific clone (HAS175) obtained by previous AMF (AFLP-based mRNA fingerprinting) analysis. Sequence analysis showed that both genes encode similar F-box proteins with a C-terminal leucine-rich repeat (LRR) domain, which are distinct from S locus (or S haplotype-specific) F-box protein (SLF/SFB), a class of F-box proteins identified as the pollen S determinant in S-RNase-based gametophytic SI systems. A number of homologous F-box genes with an LRR domain were found in the rice genome, although the functions of the gene family are unknown. One allele of the HSLF1 gene (HSLF1-S ₃) was expressed specifically in mature anthers, whereas no expression was detected from the other two alleles examined. Although the degree of sequence polymorphism among the three HSLF1 alleles was low, a frameshift mutation was found in one of the unexpressed alleles. The HSLF2 gene showed a low level of expression with no tissue specificity as well as little sequence polymorphism among the three alleles. The multiplicity of S locus-linked F-box genes is discussed in comparison with those found in the S-RNase-based SI system. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB511822–AB511825 and AB511859–AB511862.