Variation and phylogenetic utility of the Arabidopsis thaliana Rps2 homolog in various species of the tribe Brassiceae

Our objective was to analyze the evolutionary paths of cultivated diploid Brassica species and a few related wild species (tribe Brassiceae) in relation to Arabidopsis thaliana (tribe Arabidae), using the Rps2 sequence. Rps2 confers resistance to Pseudomonas syringae in A. thaliana. We found that similar to Arabidopsis, the Rps2 homolog in Brassica species is present in a single copy. Primers based in the Rps2 sequence amplified Rps2 homologs from the other species. Maximum-parsimony analysis based on number of nucleotide substitutions yielded a single tree, grouping the species as expected from other evolutionary inferences. Age of divergence between the two tribes was within the range of previous estimates. Indels in the different sequences were also useful for distinguishing some of the species. The Rps2 gene is a useful phylogenetic tool for more comprehensive studies of the species of Brasicaceae.     

Assessment of the degree of restriction fragment length polymorphism in Brassica

Summary The feasibility of creating a restriction fragment length polymorphism (RFLP) linkage map in Brassica species was assessed by screening EcoRI-, HindIII-, or EcoRV-digested total genomic DNA from several accessions of B. campestris, B. oleracea, and B. napus using random genomic DNA clones from three Brassica libraries as hybridization probes. Differences in restriction fragment hybridization patterns occurred at frequencies of 95% for comparisons of accessions among species, 79% for comparisons of accessions among subspecies within species, and 70% for comparisons among accessions within subspecies. In addition, species differences in the level of hybridization were noted for some clones. The high degree of polymorphism found even among closely related Brassica accessions indicates that RFLP analysis will be a very useful tool in genetic, taxonomic, and evolutionary studies of the Brassica genus. Development of RFLP linkage maps is now in progress.     

Map-based cloning and characterization of a gene controlling hairiness and seed coat color traits in <Emphasis Type="Italic">Brassica rapa</Emphasis>

A glabrous, yellow-seeded doubled haploid (DH) line and a hairy, black-seeded DH line in Chinese cabbage (B. rapa) were used as parents to develop a DH line population that segregated for both hairiness and seed coat color traits. The data showed that both traits completely co-segregated each other, suggesting that one Mendelian locus controlled both hairiness and seed coat color in this population. A fine genetic map was constructed and a SNP marker that was located inside a Brassica ortholog of TRANSPARENT TESTA GLABRA 1 (TTG1) in Arabidopsis showed complete linkage to both the hairiness and seed coat color gene, suggesting that the Brassica TTG1 ortholog shared the same gene function as its Arabidopsis counterpart. Further sequence analysis of the alleles from hairless, yellow-seeded and hairy, black-seeded DH lines in B. rapa showed that a 94-base deletion was found in the hairless, yellow-seeded DH lines. A nonfunctional truncated protein in the hairless, yellow-seeded DH lines in B. rapa was suggested by the coding sequence of the TTG1 ortholog. Both of the TTG1 homologs from the black and yellow seeded B. rapa lines were used to transform an Arabidopsis ttg1 mutant and the results showed that the TTG1 homolog from the black seeded B. rapa recovered the Arabidopsis ttg1 mutant, while the yellow seeded homolog did not, suggesting that the deletion in the Brassica TTG1 homolog had led to the yellow seeded natural mutant. This was the first identified gene in Brassica species that simultaneously controlled both hairiness and seed coat color traits.     

On the origin of the woody buckwheat Fagopyrum tibeticum (=Parapteropyrum tibeticum) in the Qinghai-Tibetan Plateau

Here we tested whether ‘insular woodiness’, a striking evolutionary pattern that commonly occurs on islands, has also appeared in QTP continental endemics. Parapteropyrum, a monotypic shrubby genus occurring in the central QTP, has been previously placed in the tribe Atraphaxideae of the family Polygonaceae, while all the other woody species of this tribe mainly occur in western and central Asia. We studied sequence variations of nuclear ITS (internal transcribed spacer) and cp (chloroplast) DNA (rbcL and accD) of this genus and the other ten genera. The constructed phylogenies based on ITS, cpDNA or a combination of both datasets, suggest that the woody Parapteropyrum is nested within and most likely evolved from the herbaceous Fagopyrum. We propose that the large-scale uplift of the QTP not only promoted continental species radiation, but also the secondary feature of woodiness in a few herbaceous lineages in response to strong selection pressures, similar to those acting on island flora. In addition, the confirmation of Parapteropyrum within Fagopyrum highlights its potential use as a new, perennial source of buckwheat.     

A newly-developed community microarray resource for transcriptome profiling in Brassica species enables the confirmation of Brassica-specific expressed sequences

Background  

The Brassica species include an important group of crops and provide opportunities for studying the evolutionary consequences of polyploidy. They are related to Arabidopsis thaliana, for which the first complete plant genome sequence was obtained and their genomes show extensive, although imperfect, conserved synteny with that of A. thaliana. A large number of EST sequences, derived from a range of different Brassica species, are available in the public database, but no public microarray resource has so far been developed for these species.     

A cDNA clone encoding an IgE-binding protein from Brassica anther has significant sequence similarity to Ca2+-binding proteins

Thirteen cDNA clones encoding IgE-binding proteins were isolated from expression libraries of anthers of Brassica rapa L. and B. napus L. using serum IgE from a patient who was specifically allergic to Brassica pollen. These clones were divided into two groups, I and II, based on the sequence similarity. All the group I cDNAs predicted the same protein of 79 amino acids, while the group II predicted a protein of 83 amino acids with microheterogeneity. Both of the deduced amino acid sequences contained two regions with sequence similarity to Ca²⁺-binding sites of Ca²⁺-binding proteins such as calmodulin. However flanking sequences were distinct from that of calmodulin or other Ca²⁺-binding proteins. RNA-gel blot analysis showed the genes of group I and II were preferentially expressed in anthers at the later developmental stage and in mature pollen. The recombinant proteins produced in Escherichia coli was recognized in immunoblot analysis by the IgE of a Brassica pollen allergic patient, but not by the IgE of a non-allergic patient. The cDNA clones reported here, therefore, represent pollen allergens of Brassica species.     

Brassica taxonomy based on nuclear restriction fragment length polymorphisms (RFLPs)

Summary Restriction fragment length polymorphisms (RFLPs) of nuclear DNAs have been used to explore the origin and evolution of the six cultivated Brassica species. Extensive RFLP variation was found at the species, subspecies and variety levels. Based on RFLP data from Brassica and related genera, a detailed phylogenetic tree was generated using the PAUP microcomputer program, which permits a quantitative analysis of the interrelationships among Brassica species. The results suggested that 1) B. nigra originated from one evolutionary pathway with Sinapis arvensis or a close relative as the likely progenitor, whereas B. campestris and B. oleracea came from another pathway with a possible common ancestor in wild B. oleracea or a closely related nine chromosome species; 2) the amphidiploid species B. napus and B. juncea have evolved through different combinations of the diploid morphotypes and thus polyphyletic origins may be a common mechanism for the natural occurrence of amphidiploids in Brassica; 3) the cytoplasm has played an important role in the nuclear genome evolution of amphidiploid species when the parental diploid species contain highly differentiated cytoplasms. A scheme for the origins of diploid and amphidiploid species is depicted based on evidence gathered from nuclear RFLP analysis, cpDNA RFLP analysis, cytogenetic studies and classical taxonomy.     

Chemical composition of the lipopolysaccharides of Rhodobacter sulfidophilus,Rhodopseudomonas acidophila,and Rhodopseudomonas blastica

The lipopolysaccharides of Rhodobacter sulfidophilus and the two budding species Rhodopseudomonas acidophila and Rhodopseudomonas blastica were isolated and chemically analyzed. The all have a lipid A backbone structure with glucosamine as the only amino sugar. The lipid A's of Rb. sulfidophilus and Rps. blastica contain phosphate, their fatty acids are characterized by ester-linked, unsubstituted 3-OH-10:0 and amide-linked 3-OH-14:0 (Rb. sulfidophilus) or 3-oxo-14:0 (Rps. blastica). Lipid A of Rps. acidophila is free of phosphate and contains the rare 3-OH-16:0 fatty acid in amide linkage.The lipopolysaccharides of all three species contain 2-keto-3-deoxy-octonate (KDO) but are devoid of heptoses. Neutral sugars with the exception of glucose are lacking in the lipopolysaccharide of Rb. sulfidophilus. This shows a high galacturonic acid content. The lipopolysaccharides of Rps. acidophila and Rps. blastica have neutral sugar spectra indicative for typical O-chains (rhamnose, mannose, galactose, glucose in both species, and in Rps. blastica additionally 2-O-methyl-6-deoxy-hexose). The taxonomic value of the data is discussed.This paper is dedicated to Prof. Dr. Norbert Pfennig on the occasion of his 60th birthday     

Structure and expression of AtS1, an Arabidopsis thaliana gene homologous to the S-locus related genes of Brassica

Summary Genetic and molecular analysis of the self-incompatibility locus (S-locus) of the crucifer Brassica has led to the characterization of a multigene family involved in pollen-stigma interactions. While the crucifer Arabidopsis thaliana does not have a self-incompatibility system, S-related sequences were detected in this species by cross-hybridization with Brassica DNA probes. In this paper, we show that an A. thaliana S-related sequence, designated AtS1, is expressed specifically in flower buds. Sequence analysis suggests that AtS1 encodes a secreted glycoprotein that is most similar to the Brassica S-locus related protein SLR1. As has been proposed for SLR1, this gene may be involved in determining some fundamental aspect of pollen-stigma interactions during pollination. The molecular and genetic advantages of the Arabidopsis system will provide many avenues for testing this hypothesis.     

Genome-wide identification of NBS-encoding resistance genes in <Emphasis Type="Italic">Brassica rapa</Emphasis>

Nucleotide-binding site (NBS)-encoding resistance genes are key plant disease-resistance genes and are abundant in plant genomes, comprising up to 2% of all genes. The availability of genome sequences from several plant models enables the identification and cloning of NBS-encoding genes from closely related species based on a comparative genomics approach. In this study, we used the genome sequence of Brassica rapa to identify NBS-encoding genes in the Brassica genome. We identified 92 non-redundant NBS-encoding genes [30 CC-NBS-LRR (CNL) and 62 TIR-NBS-LRR (TNL) genes] in approximately 100 Mbp of B. rapa euchromatic genome sequence. Despite the fact that B. rapa has a significantly larger genome than Arabidopsis thaliana due to a recent whole genome triplication event after speciation, B. rapa contains relatively small number of NBS-encoding genes compared to A. thaliana, presumably because of deletion of redundant genes related to genome diploidization. Phylogenetic and evolutionary analyses suggest that relatively higher relaxation of selective constraints on the TNL group after the old duplication event resulted in greater accumulation of TNLs than CNLs in both Arabidopsis and Brassica genomes. Recent tandem duplication and ectopic deletion are likely to have played a role in the generation of novel Brassica lineage-specific resistance genes.     

Members of the<Emphasis Type="Italic"> S</Emphasis>-receptor kinase multigene family in<Emphasis Type="Italic"> Senecio squalidus</Emphasis> L. (Asteraceae), a species with sporophytic self-incompatibility

While the molecular basis of sporophytic self-incompatibility (SSI) has been investigated extensively in the Brassicaceae, almost nothing is known about the molecular regulation of SSI in other families, such as the Asteraceae. In species of Brassica and in Arabidopsis lyrata, a stigma-specific serine-threonine receptor kinase (SRK) and its cognate ligand, a pollen coating-borne cysteine-rich protein (SCR/SP11), determine the female and male sides of the SSI response, respectively. Here we have used RT-PCR with degenerate primers to conserved regions of SRK to amplify three SRK-like gene fragments expressed in stigmas of Senecio squalidus (Asteraceae). The Senecio S-receptor-like kinase (SSRLK) sequences share ~43% amino acid sequence identity with Brassica SRK₃ but higher amino acid sequence identity (~50%) with two Solanum bulbocastanum receptor-like kinase genes of unknown function. Despite expression in stigmas, all three SSRLKs were expressed at varying levels in floral and vegetative tissues. No allelic polymorphism was detected for the three SSRLKs in two S homozygous lines of S. squalidus or three other lines of S. squalidus carrying different S alleles. A full-length cDNA clone was obtained for SSRLK1 and its predicted amino acid sequence revealed significant structural differences to Brassica SRKs, most notably a major N-terminal truncation of 169 amino acids and the presence of just 8 conserved cysteine residues within the putative receptor domain instead of 12. Together, the sequence characteristics and expression characteristics of SSRLKs suggest that they are unlikely to be directly involved in the SSI response of S. squalidus. These findings are discussed in terms of the evolution of the SRK multigene family and the molecular basis of SSI in S. squalidus and the Asteraceae.     

Transformation of Brassica napus canola cultivars with Arabidopsis thaliana acetohydroxyacid synthase genes and analysis of herbicide resistance

Summary A survey of selected crop species and weeds was conducted to evaluate the inhibition of the enzyme acetohydroxyacid synthase (AHAS) and seedling growth in vitro by the sulfonylurea herbicides chlorsulfuron, DPX A7881, DPX L5300, DPX M6316 and the imidazolinone herbicides AC243,997, AC263,499, AC252,214. Particular attention was given to the Brassica species including canola cultivars and cruciferous weeds such as B. kaber (wild mustard) and Thlaspi arvense (stinkweed). Transgenic lines of B. napus cultivars Westar and Profit, which express the Arabidopsis thaliana wild-type AHAS gene or the mutant gene csr1-1 at levels similar to the resident AHAS genes, were generated and compared. The mutant gene was essential for resistance to the sulfonylurea chlorsulfuron but not to DPX A7881, which appeared to be tolerated by certain Brassica species. Cross-resistance to the imidazolinones did not occur. The level of resistance to chlorsulfuron in transgenic canola greatly exceeded the levels that were toxic to the Brassica species or cruciferous weeds. Direct selection of transgenic lines with chlorsulfuron sprayed at field levels under greenhouse conditions was achieved.     

In vitro pollen germination of species and two interspecific hybrids from the genus Brassica

In vitro pollen germination of five species and two interspecific hybrids from the genus Brassica was tested in four media. Genetically fixed differences in the demands for optimal pollen germination among species were found. The experiments were designed to define optimal content of mineral salts, sugar, and PEG for every investigated species or hybrids. The differences found among species are discussed in relation to the evolutionary trend.     

Differences in the control of bacteriochlorophyll formation by light and oxygen

Control of bacteriochlorophyll formation was studied with continuous cultures of Rhodospirillum rubrum, Rhodopseudomonas sphaeroides, and Rhodopseudomonas capsulata. Oxygen controlled specific bacteriochlorophyll contents of the three species in a hyperbolical fashion irrespective of the presence of light. In Rps. sphaeroides, this applied to oxygen concentrations above 16% air saturation of the medium while at lower oxygen concentrations control followed a kinetics with negative cooperativity. Cell protein formation of R. rubrum and Rsp. sphaeroides was independent of oxygen concentrations while protein formation of Rps. capsulata increased at lower concentrations. Light controlled bacteriochlorphyll contents of R. rubrum and Rps. sphaeroides in a sigmoidal fashion. When growing at a constant low oxygen concentration cell protein formation increased with light energy flux in Rps. sphaeroides but remained unaffected in R. rubrum. Protein formation of R. rubrum increased with light energy flux only under anaerobic conditions. Two factor analyses were performed with R. rubrum and Rps. sphaeroides to study the combined effects of light and oxygen on bacteriochlorophyll formation. The results showed that both factors act independent of each other.Abbreviations ALA 5-aminolevulinic acid - R Rhodospirillum - Rsp. Rhodopseudomonas     

Molecular and cytological characterization of repetitive DNA sequences in Brassica

Summary We isolated three different repetitive DNA sequences from B. campestris and determined their nucleotide sequences. In order to analyze organization of these repetitive sequences in Brassica, Southern blot hybridization and in situ hybridization with metaphase chromosomes were performed. The sequence cloned in the plasmid pCS1 represented a middle repetitive sequence present only in B. campestris and not detected in closely related B. Oleracea. This sequence was localized at centromeric regions of six specific chromosomes of B. campestris. The second plasmid, pBT4, contained a part of the 25S ribosomal RNA gene, and its copy number was estimated to be 1,590 and 1,300 per haploid genome for B. campestris and B. oleracea, respectively. In situ hybridization with this sequence showed a clear signal at the NOR region found in the second largest chromosome of B. Campestris. The third plasmid, pBT11, contained a 175-bp insert that belongs to a major family of tandem repeats found in all the Brassica species. This sequence was detected at centromeric regions of all the B. campestris chromosomes. Our study indicates that in situ hybridization with various types of repetitive sequences should give important information on the evolution of repetitive DNA in Brassica species.     

Characterization and comparative analysis of sequence-specific amplified polymorphisms based on two subfamilies of IRRE retrotransposons in <Emphasis Type="Italic">Iris missouriensis</Emphasis> (Iridaceae)

DNA markers based on transposable-element polymorphisms are potentially useful alternatives to anonymous fragment-length polymorphisms (AFLPs). We developed the retrotransposon sequence-specific amplified polymorphism (retrotransposon SSAP) technique for the angiosperm Iris missouriensis (Iridaceae) in order to evaluate its use in generating population-genetic markers. Our cloning strategy identified two groups of long-terminal repeat retrotransposons of the IRRE family. Primers homologous to conserved regions of these elements generated repeatable and polymorphic markers. In comparison, the AFLP protocol failed to produce useful markers in our hands in this species. To investigate the distribution and evolutionary tempo of the two retrotransposons, we developed a phylogeny of representative species of subgenus Limniris based on chloroplast sequence. Sequences of both groups of retrotransposons were widespread in Limniris, but we also found evidence of substantial sequence and copy-number evolution since the divergence of I. missouriensis from other Limniris species. We corroborated these results with quantitative real-time PCR estimates of relative copy number. Importantly, the geographic structure of retrotransposon SSAP was strikingly different for the two groups of retrotransposons, indicating that different mutational dynamics and/or selective pressures govern their distribution. Although these primers should be useful for population-genetic studies of Iris missouriensis and other Limniris species, our findings reinforce the need for caution in evaluating transposable-element markers used to analyze the relatedness of populations or cultivars, as very different conclusions may be reached depending on the sequence amplified.     

Phylogenetic relationships within Pappophoreae s.l. (Poaceae: Chloridoideae): Additional evidence based on ITS and trnL-F sequence data

Historically, Pappophoreae included the genera Cottea, Enneapogon, Kaokochloa, Pappophorum and Schmidtia. Some authors consider this tribe as a well-supported monophyletic group; while other evidences reveals Pappophoreae as polyphyletic, with Pappophorum separated from the rest of the tribe. When the latter happens, it can form a clade with Tridens flavus. Molecular phylogenetic analyses of the subfamily Chloridoideae have included few species of Pappophoreae; therefore, further research involving more representatives of this tribe is needed. With the aim of providing new evidence to help clarify the phylogenetic position of Pappophorum and its relationships with other genera of the tribe and the subfamily Chloridoideae, eight new sequences of ITS and trnL-F regions of Pappophoreae species were generated. These sequences were analyzed together with other available sequence data obtained from GenBank, using maximum parsimony and Bayesian inference, for individual (trnL-F or ITS) or combined trnL-F/ITS data sets. All analyses reveal that Pappophoreae is polyphyletic, with Pappophorum separated from the rest of the tribe forming a well-supported clade sister to Tridens flavus.     

Occurrence and evolutionary significance of two sulfate assimilation pathways in the rhodospirillaceae

The ability to use adenosine 5-phosphosulfate (APS) or 3-phosphoadenosine 5-phosphosulfate (PAPS) as the substrate for the initial reductive step in sulfate assimilation has been tested in most of the known Rhodospirillaceae species and in some chemotrophic bacteria. Improved and optimized methods for the synthesis and purification of the sulfonucleotides APS and PAPS are described. The production of acid volatile radioactivity from ³⁵S-APS and ³⁵S-PAPS was measured under various conditions in the presence and absence of non-labeled sulfate. Specific differences in the ability to reduce APS or PAPS were observed among the Rhodospirillaceae species and also the chemotrophic bacteria. APS was found to be the substrate of the thiolsulfotransferase in Rps. acidophila, Rps. globiformis, Rm. vannielii, Rc. purpureus, R. tenue, Rps. gelatinosa, in Alcaligenes eutrophus and Pseudomonas aeruginosa. PAPS was the substrate in Rps. capsulata, Rps. sphaeroides, Rps. sulfidophila, Rps. palustris, Rps. viridis, R. rubrum, R. fulvum, in Paracoccus denitrificans and in several Enterobacteriaceae. The presence of different enzymatic systems for sulfate reduction in the Rhodospirillaceae family is compared with their taxonomical grouping and their possible phylogenetic relatedness.Nonstandard Abbreviations APS adenosine 5-phosphosulfate - PAPS 3-phosphate adenosine 5-phosphosulfate - DTE dithioerythrol - Rc. Rhodocyclus - R. Rhodospirillum - Rm. Rhodomicrobium - Rps. Rhodopseudomonas