Molecular Biology of <Emphasis Type="Italic">Plasmodiophora brassicae</Emphasis>

Initially, molecular techniques were used to detect and distinguish Plasmodiophora pathotypes in soil. Meanwhile, chromosomes from 2.2 Mb to 680 kb are characterized and the total genome size is estimated to be approximately 20 Mb. Furthermore, the genomic gene structure and the cDNA structure of several genes have been revealed, and the expression of those genes has been linked to development of clubroot to some extent. In addition, the sequence data have reinforced the inclusion of the plasmodiophorids within the Cercozoa. The recent successes in molecular biology have produced new approaches for clubroot research.     

Detection and Measurement of <Emphasis Type="Italic">Plasmodiophora brassicae</Emphasis>

Clubroot, caused by Plasmodiophora brassicae, is one of the most important diseases of brassicas. Management of clubroot is difficult, and the best means of avoiding the disease include planting in areas where P. brassicae is not present and using plants and growing media free from pathogen inoculum. As P. brassicae is not culturable, its detection has traditionally relied on plant bioassays, which are time-consuming and require large amounts of glasshouse space. More recently, fluorescence microscopy, serology, and DNA-based methods have all been used to test soil, water, or plant samples for clubroot. The use of fluorescence microscopy to detect and count pathogen spores in the soil requires significant operator skill and is unlikely to serve as the basis for a routine diagnostic test. By contrast, serologic assays are inexpensive and amenable to high-throughput screening but need to be based on monoclonal antibodies because polyclonal antisera cannot be reproduced and are therefore of limited quantity. Several polymerase chain reaction (PCR)-based assays have also been developed; these are highly specific for P. brassicae and have been well-correlated with disease severity. As such, PCR-based diagnostic tests have been adopted to varying extents in Canada and Australia, but wide implementation has been restricted by sample processing costs. Efforts are underway to develop inexpensive serologic on-farm diagnostic kits and to improve quantification of pathogen inoculum levels through real-time PCR. Proper detection and quantification of P. brassicae will likely play an increasingly important role in the development of effective clubroot management strategies.     

<Emphasis Type="Italic">WUS</Emphasis> and <Emphasis Type="Italic">STM</Emphasis>-based reporter genes for studying meristem development in poplar

We describe the development of a reporter system for monitoring meristem initiation in poplar using promoters of poplar homologs to the meristem-active regulatory genes WUSCHEL (WUS) and SHOOTMERISTEMLESS (STM). When ~3 kb of the 5′ flanking regions of close homologs were used to drive expression of the GUSPlus gene, 50–60% of the transgenic events showed expression in apical and axillary meristems. However, expression was also common in other organs, including in leaf veins (40 and 46% of WUS and STM transgenic events, respectively) and hydathodes (56% of WUS transgenic events). Histochemical GUS staining of explants during callogenesis and shoot regeneration using in vitro stems as explants showed that expression was detectable prior to visible shoot development, starting 3–15 days after explants were placed onto callus inducing medium. A minority of WUS and STM events also showed expression in the cambium, phloem, or xylem of regenerated, greenhouse grown plants undergoing secondary growth. Based on microarray gene expression data, a paralog of poplar WUS was detectably up-regulated during shoot initiation, but the other paralog was not. Both paralogs of poplar STM were down-regulated threefold to sixfold during early callus initiation. We identified 15–35 copies of cytokinin response regulator binding motifs (ARR1AT) and one copy of the auxin response element (AuxRE) in both promoters. Several of the events recovered may be useful for studying the process of primary and secondary meristem development, including treatments intended to stimulate meristem development to promote clonal propagation and genetic transformation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterization of expressed class II MHC sequences in the banner-tailed kangaroo rat (<Emphasis Type="Italic">Dipodomys spectabilis</Emphasis>) reveals multiple <Emphasis Type="Italic">DRB</Emphasis> loci

Genes of the major histocompatibility complex (MHC) are exceptionally polymorphic due to the combined effects of natural and sexual selection. Most research in wild populations has focused on the second exon of a single class II locus (DRB), but complete gene sequences can provide an illuminating backdrop for studies of intragenic selection, recombination, and organization. To this end, we characterized class II loci in the banner-tailed kangaroo rat (Dipodomys spectabilis). Seven DRB-like sequences (provisionally named MhcDisp-DRB*01 through *07) were isolated from spleen cDNA and most likely comprise ≥5 loci; this multiformity is quite unlike the situation in muroid rodents such as Mus, Rattus, and Peromyscus. In silico translation revealed the presence of important structural residues for glycosylation sites, salt bonds, and CD4+ T-cell recognition. Amino-acid distances varied widely among the seven sequences (2–34%). Nuclear DNA sequences from the Disp-DRB*07 locus (∼10 kb) revealed a conventional exon/intron structure as well as a number of microsatellites and short interspersed nuclear elements (B4, Alu, and IDL-Geo subfamilies). Rates of nucleotide substitution at Disp-DRB*07 are similar in both exons and introns (π = 0.015 and 0.012, respectively), which suggests relaxed selection and may indicate that this locus is an expressed pseudogene. Finally, we performed BLASTn searches against Dipodomys ordii genomic sequences (unassembled reads) and find 90–97% nucleotide similarity between the two kangaroo rat species. Collectively, these data suggest that class II diversity in heteromyid rodents is based on polylocism and departs from the muroid architecture. 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 EU817477–EU817485.     

Identification of two loci for resistance to clubroot (<Emphasis Type="Italic">Plasmodiophora brassicae</Emphasis> Woronin) in <Emphasis Type="Italic">Brassica rapa</Emphasis> L.

In an analysis of 114 F₂ individuals from a cross between clubroot-resistant and susceptible lines of Brassica rapa L., 'G004' and 'Hakusai Chukanbohon Nou 7' (A9709), respectively, we identified two loci, Crr1 and Crr2, for clubroot (caused by Plasmodiophora brassicae Woronin) resistance. Each locus segregated independently among the F₂ population, indicating that the loci reside on a different region of chromosomes or on different chromosomes. Genetic analysis showed that each locus had little effect on clubroot resistance by itself, indicating that these two loci are complementary for clubroot resistance. The resistance to clubroot was much stronger when both loci were homozygous for resistant alleles than when they were heterozygous. These results indicate that clubroot resistance in B. rapa is under oligogenic control and at least two loci are necessary for resistance.Communicated by H.C. Becker     

Genomewide analysis of <Emphasis Type="Italic">ABCB</Emphasis>s with a focus on <Emphasis Type="Italic">ABCB1</Emphasis> and <Emphasis Type="Italic">ABCB19</Emphasis> in <Emphasis Type="Italic">Malus domestica</Emphasis>

The B subfamily of ATP-binding cassette (ABC) proteins (ABCB) plays a vital role in auxin efflux. However, no systematic study has been done in apple. In this study, we performed genomewide identification and expression analyses of the ABCB family in Malus domestica for the first time. We identified a total of 25 apple ABCBs that were divided into three clusters based on the phylogenetic analysis. Most ABCBs within the same cluster demonstrated a similar exon–intron organization. Additionally, the digital expression profiles of ABCB genes shed light on their functional divergence. ABCB1 and ABCB19 are two well-studied auxin efflux carrier genes, and we found that their expression levels are higher in young shoots of M106 than in young shoots of M9. Since young shoots are the main source of auxin synthesis and auxin efflux involves in tree height control. This suggests that ABCB1 and ABCB19 may also take a part in the auxin efflux and tree height control in apple.     

Genetics of Clubroot Resistance in <Emphasis Type="Italic">Brassica</Emphasis> Species

Clubroot disease, caused by the obligate plant pathogen Plasmodiophora brassicae Wor., is one of the most economically important diseases affecting Brassica crops in the world. The genetic basis of clubroot resistance (CR) has been well studied in three economically important Brassica species: B. rapa, B. oleracea, and B. napus. In B. rapa, mainly in Chinese cabbage, one minor and seven major CR genes introduced from European fodder turnips have been identified. Mapping of these CR genes localized Crr1 on R8, Crr2 on R1, CRc on R2, and Crr4 on R6 linkage groups of Chinese cabbage. Genes Crr3, CRa, CRb, and CRk mapped to R3, but at two separate loci, CRa and CRb are independent of Crr3 and CRk, which are closely linked. Further analysis suggested that Crr1, Crr2, and CRb have similar origins in the ancestral genome as in chromosome 4 of Arabidopsis thaliana. Genetic analysis of clubroot resistance genes in B. oleracea suggests that they are quantitative traits. Twenty-two quantitative trait loci (QTLs) were mapped in different linkage groups of B. oleracea. In B. napus, genetic analysis of clubroot resistance was found to be governed by one or two dominant genes, whereas resistance conferred by two recessive genes is reported. The quantitative analysis approach, however, proved that they are polygenic. In total, at least 16 QTLs have been detected on eight chromosomes of B. napus, N02, N03, N08, N09, N13, N15, N16, and N19. The chromosomal location of the other six QTLs is not clear. Cloning of any of these QTLs or resistance loci was not, however, possible until recently. Progress in genomics, particularly the techniques of comparative mapping and genome sequencing, supplements cloning and allows improved characterization of CR genes. Further development of DNA markers linked to CR genes will in turn hasten the breeding of clubroot-resistant Brassica cultivars.     

High Temperature Stimulates <Emphasis Type="Italic">DWARF4 (DWF4)</Emphasis> Expression to Increase Hypocotyl Elongation in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Hypocotyl growth occurs as a result of an interaction between environmental factors and endogenous phytohormones. In Arabidopsis, high temperature promotes auxin synthesis to increase hypocotyl growth. We previously showed that exogenously provided auxin stimulates expression of the brassinosteroid (BR) biosynthetic gene DWARF4. To determine whether temperature-induced hypocotyl elongation depends on BR biosynthesis, we examined the morphological responses to high temperature and the expression pattern of DWF4pro:GUS in different genetic backgrounds, which are as follows: Ws-2 wild-type, iaa19/msg2, bri1-5, and dwf7-1. In contrast to the wild-type, growth of the three genotypes at 29°C did not significantly increase hypocotyl length; whereas, with the exception of iaa19/msg2, the roots were elongated. These results confirm that BR biosynthesis and signaling pathways are required for hypocotyl growth at high temperature. Furthermore, a GUS histochemical assay revealed that a temperature of 29°C greatly increased DWF4pro:GUS expression in the shoot and root tips compared to a temperature of 22°C. Quantitative measurements of GUS activity in DWF4pro:GUS revealed that growth at 29°C is similar to the level of growth after addition of 100 nM IAA to the medium. Our results suggest that temperature-dependent synthesis of free auxin stimulates BR biosynthesis, particularly via the key biosynthetic gene DWF4, and that the BRs thus synthesized are involved in hypocotyl growth at high temperature.     

Predation capacity and prey preference of <Emphasis Type="Italic">Chrysoperla carnea</Emphasis> on <Emphasis Type="Italic">Pieris brassicae</Emphasis>

The predation capacity and prey preference of larvae of Chrysoperla carnea (Stephens) (Neuroptera: Chrysopidae) on eggs or larvae of Pieris brassicae (Linnaeus) (Lepidoptera: Pieridae) in the absence and presence of cabbage aphids as an alternative prey were evaluated in laboratory experiments at 25°C. Both instars preyed upon butterfly eggs and larvae as well as on cabbage aphids with the third instar being the most voracious. The lacewings had a strong preference for caterpillars to butterfly eggs. In the presence of the aphids the predation on P. brassicae eggs or larvae was either completely abandoned or reduced by about 70%, respectively, by second instar lacewings and either reduced by about 80% or maintained, respectively, by third instar lacewings. Both instars thus had a clear preference for aphids compared to eggs of P. brassicae. However, second instar lacewings preferred aphids to caterpillars whereas the opposite was the case for third instar lacewings. The results indicate that 3rd instar C. carnea has a potential as biocontrol agent against P. brassicae.     

Induction and origin of adventitious roots from chimeras of <Emphasis Type="Italic">Brassica juncea</Emphasis> and <Emphasis Type="Italic">Brassica oleracea</Emphasis>

Adventitious roots were induced from shoots and leaves of the chimera plant TCC (LI-LII-LIII = TCC; T = Tuber mustard, C = Red Cabbage), previously developed by in vitro grafting of tuber mustard (Brassica juncea) and red cabbage (B. oleracea). The regeneration frequency of adventitious roots from TCC shoots and leaf sections was markedly higher than that obtained from the parents TTT (tuber mustard) and CCC (red cabbage). Moreover, levels of α-naphthaleneacetic acid in the culture medium had lower effects on rooting efficiency of TCC chimeras compared to those of TTT and CCC. The number and fresh weight of adventitious roots per TCC shoot, 13.11 roots and 0.274 g, respectively, were also significantly higher than those of the parents. This demonstrated that replacing the histogenic LI layer (the outermost apical cell layer) with a different genotype might improve adventitious root induction capability of these vegetative tissues due to likely synergistic effects between LI and the other two histogenic layers, LII and LIII. Following polymerase chain reaction analysis and histological investigation, it was found that these adventitious roots originated from the LIII histogenic layer.     

Chloroplast DNA phylogeography of <Emphasis Type="Italic">Pedicularis</Emphasis> ser. <Emphasis Type="Italic">Gloriosae</Emphasis> (Orobanchaceae) in Japan

Phylogeographic analyses using chloroplast DNA (cpDNA) variation were performed for Pedicularis ser. Gloriosae (Orobanchaceae). Eighty-one plants of 18 populations of 6 species (P. gloriosa, P. iwatensis, P. nipponica, P. ochiaiana, P. sceptrum-carolinum and P. grandiflora) were analyzed. Fifteen distinct haplotypes were identified based on six cpDNA regions: the intergenic spacer between the trnT and trnL 3′exon, trnL 3′exon-trnF, atpB-rbcL, accD–psaI, the rpl16 intron and the trnK region (including the matK gene). Via phylogenetic analyses of the haplotypes, two continental species, P. sceptrum-carolinum and P. grandiflora, were placed at the most ancestral position in the trees. The former species is widely distributed in the Eurasian continent, and the latter is distributed in Far East Asia. Two robust major cpDNA clades (clades I and II) were revealed in the Japanese archipelago, although the statistical values of monophyly of these clades were weak. Clade I included the haplotypes (A-1, A-2, B-1, B-2 and J) of three species (P. gloriosa, P. iwatensis and P. ochiaiana), and Clade II included seven haplotypes (C-D, E-1, E-2 and F-H) of P. nipponica. These results suggest that this series originated on the Eurasian continent and that subsequently populations at the eastern edge of the continent differentiated into the two Japanese lineages. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Association between <Emphasis Type="Italic">GSTM1</Emphasis>, <Emphasis Type="Italic">GSTT1</Emphasis>, and <Emphasis Type="Italic">GSTP1</Emphasis> polymorphisms and lung cancer risk in a Turkish population

Several studies focused on investigating genetic polymorphisms in order to estimate genetic contribution to lung cancer often showed conflicting results. In this study, we investigated the role of GSTM1, GSTT1, GSTP1 exon 5 and exon 6 polymorphisms on developing lung cancer and histological subtypes in 213 lung cancer patients and 231 controls. GSTM1 null, GSTT1 null, and GSTP1 exon 5 variant genotypes did not show a significant risk for developing lung cancer overall. Significant association was noted between GSTP1 exon 6 variant genotypes and overall lung cancer risk (OR 2.17, 95% CI 1.25–3.78; P = 0.006). These results show that GSTP1 exon 6 polymorphism might be an important factor in determining lung cancer susceptibility in a Turkish population.     

In vitro regeneration of <Emphasis Type="Italic">Carlina acaulis</Emphasis> subsp. <Emphasis Type="Italic">simplex</Emphasis> from seedling explants

The aim of the study was to obtain an efficient system for Carlina acaulis subsp. simplex propagation. The experimental materials were shoot tips, fragments of hipocotyls, cotyledons and roots isolated from 10-day-old seedlings. The explants were transferred to the proliferation medium supplemented with different types of cytokinin: BA (13.3 μM), kinetin (13.9 μM) and zeatin (13.7 μM) in combination with NAA (0.54 μM). The best morphogenetic response was observed when explants were cultured on the BA supplemented medium. The maximum shoot organogenesis frequency was observed for shoot tip (nearly 94%). On average 8.6 axillary shoots were induced per explant. Multiplication rate increased during the first three subcultures. The shoots revealed a wide range of morphogenetic responses. Differences were observed in the presence or absence of hair on the surface of lamina. These changes had epigenetic character and were the effect of changes in DNA methylation, which is shown by differences in methylation pattern between 18S rRNA and 25S rRNA genes in the analyzed regenerated plants. Nearly 94% of plantlets were rooted on auxin lacking medium. Addition of auxin (NAA or IAA) increased both the rooting percentage (100%) and the number of roots per shoot, but their growth was inhibited. Shortening of the auxin exposition time reduced the number of roots. Moreover, high efficiency (90%) was observed for ex vitro rooting. Plantlets with a large number of roots survived better than the ones with only a few roots. Plants were able to flower and gave viable seeds.