Identification and distribution of S haplotypes in Brassica vegetables from China

The amplification efficiency and identification diversity with gene-specific primers derived from S locus glycoprotein gene (SLG) and S locus receptor kinase gene (SRK) were compared, and the geographical distribution for S haplotypes was investigated by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) in 72 genotypes of 5 Brassica vegetables from China. The amplification efficiency and identification diversity with class I SRK primers were generally higher than that with class I SLG in most crops tested. Class I alleles were found in total 66 genotypes and they were classified into 16, 10, 7 and 10 groups for Chinese cabbage, purple flowering stalk, cauliflower and cabbage respectively. The number of amplification accessions and identification diversity using the primers of class II SLG and SRK were quite similar. Class II alleles were detected in 55 genotypes and further grouped into one type in mustard and three in other crops. The nucleotide sequences showed high similarity between identical S haplotypes determined by reciprocal pollination and PCR-RFLP tests. It demonstrated that the PCR-RFLP analysis was feasible for identification of S alleles, and SRK should be considered as a better marker for the identification of S haplotypes than SLG. The types of S haplotypes are highly diverse in Brassica vegetables from China. Nevertheless, they were geographically limited in some Brassica vegetables, so the exchange of germplasm resources should be enhanced for breeding.     

Protocol: high throughput silica-based purification of RNA from Arabidopsis seedlings in a 96-well format

Background

The Arabidopsis thaliana-Pseudomonas syringae model pathosystem is one of the most widely used systems to understand the mechanisms of microbial pathogenesis and plant innate immunity. Several inoculation methods have been used to study plant-pathogen interactions in this model system. However, none of the methods reported to date are similar to those occurring in nature and amicable to large-scale mutant screens.

Results

In this study, we developed a rapid and reliable seedling flood-inoculation method based on young Arabidopsis seedlings grown on MS medium. This method has several advantages over conventional soil-grown plant inoculation assays, including a shorter growth and incubation period, ease of inoculation and handling, uniform infection and disease development, requires less growth chamber space and is suitable for high-throughput screens. In this study we demonstrated the efficacy of the Arabidopsis seedling assay to study 1) the virulence factors of P. syringae pv. tomato DC3000, including type III protein secretion system (TTSS) and phytotoxin coronatine (COR); 2) the effector-triggered immunity; and 3) Arabidopsis mutants affected in salicylic acid (SA)- and pathogen-associated molecular pattern (PAMPs)-mediated pathways. Furthermore, we applied this technique to study nonhost resistance (NHR) responses in Arabidopsis using nonhost pathogens, such as P. syringae pv. tabaci, pv. glycinea and pv. tomato T1, and confirmed the functional role of FLAGELLIN-SENSING 2 (FLS2) in NHR.

Conclusions

The Arabidopsis seedling flood-inoculation assay provides a rapid, efficient and economical method for studying Arabidopsis-Pseudomonas interactions with minimal growth chamber space and time. This assay could also provide an excellent system for investigating the virulence mechanisms of P. syringae. Using this method, we demonstrated that FLS2 plays a critical role in conferring NHR against nonhost pathovars of P. syringae, but not to Xanthomonas campestris pv. vesicatoria. This method is potentially ideal for high-throughput screening of both Arabidopsis and pathogen mutants.     

Cloning and characterization of 5 MADS-box cDNAs isolated from citrus fruit tissue

We isolated and characterized 5 MADS-box cDNA clones of CitMADS1, CitMADS3, CitMADS5, CitMADS6 and CitMADS8 from fruit tissues of Satsuma mandarin (Citrus unshiu Marc.). Sequence analysis revealed that they have high sequence identities with known MADS-box genes and that their predicted proteins possess the general structural features of the M domain and the K domain. In a phylogenetic tree with 24 known Arabidopsis MADS-box genes, 5 Citrus MADS-box genes were classified into 4 clades of the MADS-box gene with independent gene functions. Their broad expression profiles in the Citrus life cycle suggested that they play roles in flower and fruit development. Most of them, except CitMADS1 and CitMADS3, are expressed in seedlings before the phase transition from vegetative to reproductive growth. CitMADS1 and CitMADS3 are not expressed in vegetative organs and could serve as a molecular marker for reproductive development.     

Universal endogenous gene controls for bisulphite conversion in analysis of plant DNA methylation

Background

Protein extraction is a frequent procedure in biological research. For preparation of plant cell extracts, plant materials usually have to be ground and homogenized to physically break the robust cell wall, but this step is laborious and time-consuming when a large number of samples are handled at once.

Results

We developed a chemical method for lysing Arabidopsis cells without grinding. In this method, plants are boiled for just 10 minutes in a solution containing a Ca²⁺ chelator and detergent. Cell extracts prepared by this method were suitable for SDS-PAGE and immunoblot analysis. This method was also applicable to genomic DNA extraction for PCR analysis. Our method was applied to many other plant species, and worked well for some of them.

Conclusions

Our method is rapid and economical, and allows many samples to be prepared simultaneously for protein analysis. Our method is useful not only for Arabidopsis research but also research on certain other species.     

Exploration of plant genomes in the FLAGdb++ environment

Background

Efficient high throughput screening systems of useful mutants are prerequisite for study of plant functional genomics and lots of application fields. Advance in such screening tools, thanks to the development of analytic instruments. Direct analysis in real-time (DART)-mass spectrometry (MS) by ionization of complex materials at atmospheric pressure is a rapid, simple, high-resolution analytical technique. Here we describe a rapid, simple method for the genetic discrimination of intact Arabidopsis thaliana mutant seeds using metabolic profiling by DART-MS.

Results

To determine whether this DART-MS combined by multivariate analysis can perform genetic discrimination based on global metabolic profiling, intact Arabidopsis thaliana mutant seeds were subjected to DART-MS without any sample preparation. Partial least squares-discriminant analysis (PLS-DA) of DART-MS spectral data from intact seeds classified 14 different lines of seeds into two distinct groups: Columbia (Col-0) and Landsberg erecta (Ler) ecotype backgrounds. A hierarchical dendrogram based on partial least squares-discriminant analysis (PLS-DA) subdivided the Col-0 ecotype into two groups: mutant lines harboring defects in the phenylpropanoid biosynthetic pathway and mutants without these defects. These results indicated that metabolic profiling with DART-MS could discriminate intact Arabidopsis seeds at least ecotype level and metabolic pathway level within same ecotype.

Conclusion

The described DART-MS combined by multivariate analysis allows for rapid screening and metabolic characterization of lots of Arabidopsis mutant seeds without complex metabolic preparation steps. Moreover, potential novel metabolic markers can be detected and used to clarify the genetic relationship between Arabidopsis cultivars. Furthermore this technique can be applied to predict the novel gene function of metabolic mutants regardless of morphological phenotypes.     

Anaerobic Soil Disinfestation and Brassica Seed Meal Amendment Alter Soil Microbiology and System Resistance

ABSTRACT

Brassica seed meal amendments and anaerobic soil disinfestation (ASD) control a spectrum of soil-borne plant pathogens via a diversity of mechanisms. Transformations in microbial community structure and function in certain instances were determinants of disease control and enhanced plant performance. For instance, in strawberry field soils, increased strawberry yields in response to ASD were attained in a carbon-source input-dependent manner. ASD conducted with rice bran as the carbon input, but not molasses, resulted in significantly greater yields. Effective ASD treatments, but not ineffective treatments, resulted in increased abundance of bacteria in phylum Bacteroidetes in treated soils, specific genera of which are known to provide biological disease control. Brassica seed meal amendment resulted in development of a soil system suppressive towards disease incited by the root pathogen Macrophomina phaseolina. Brassica seed meal treated orchard soil systems exhibit resistance to re-infestation by soil-borne pathogens, including the plant parasitic nematode Pratylenchus penetrans. System resistance is associated with significant and prolonged changes in rhizosphere microbiology and specifically amplification of specific organisms with potential to parasitize P. penetrans.     

sunTILL: a TILLING resource for gene function analysis in sunflower

Background

Cultivated sunflower (Helianthus annus L.) is a globally important oilseed crop, subjected to intensive genetic and genomic studies. Although classical mutagenesis has successfully been applied to Helianthus genus in the past, we have developed the first sunflower TILLING resource.

Results

To balance the maximum mutation density with an acceptable plant survival rate, a 'kill curve' analysis was first conducted with different ethylmethanesulfonate (EMS) dosages and different exposure times. According to the germination rate, a treatment with 0.7% EMS for 6 h was chosen. An M₂ progeny of 3,651 fertile plants was obtained. Totally, 4.79% of the whole population showed clear aberrant phenotypes. A microsatellite analysis on a representative sample of the original seed stock and mutant lines confirmed the uniformity of the genetic background of plant material. The TILLING procedure was successfully applied to sunflower genome, initially by a CelI-nuclease mismatch cleavage assay coupled with a DNA-pooling level test. To investigate the efficiency of the mutagenic treatment, a pilot screening was carried out on 1,152 M₂ lines focusing on four genes, three involved in the fatty acid biosynthetic pathway and one for downy mildew resistance. A total of 9 mutant lines were identified and confirmed by sequencing; thereby, the estimated overall mutation frequency for the pilot assay resulted to be 1/475 kb.

Conclusion

A first TILLING population for a high throughput identification of EMS-induced point mutations in sunflower genome has been successfully obtained. This represents a powerful tool to a better understanding of gene function in sunflower.     

Investigations of barley stripe mosaic virus as a gene silencing vector in barley roots and in <Emphasis Type="Italic">Brachypodium distachyon</Emphasis> and oat

Background  

Gene silencing vectors based on Barley stripe mosaic virus (BSMV) are used extensively in cereals to study gene function, but nearly all studies have been limited to genes expressed in leaves of barley and wheat. However since many important aspects of plant biology are based on root-expressed genes we wanted to explore the potential of BSMV for silencing genes in root tissues. Furthermore, the newly completed genome sequence of the emerging cereal model species Brachypodium distachyon as well as the increasing amount of EST sequence information available for oat (Avena species) have created a need for tools to study gene function in these species.     

Development of a pooled probe method for locating small gene families in a physical map of soybean using stress related paralogues and a BAC minimum tile path

Background  

Genome analysis of soybean (Glycine max L.) has been complicated by its paleo-autopolyploid nature and conserved homeologous regions. Landmarks of expressed sequence tags (ESTs) located within a minimum tile path (MTP) of contiguous (contig) bacterial artificial chromosome (BAC) clones or radiation hybrid set can identify stress and defense related gene rich regions in the genome. A physical map of about 2,800 contigs and MTPs of 8,064 BAC clones encompass the soybean genome. That genome is being sequenced by whole genome shotgun methods so that reliable estimates of gene family size and gene locations will provide a useful tool for finishing. The aims here were to develop methods to anchor plant defense- and stress-related gene paralogues on the MTP derived from the soybean physical map, to identify gene rich regions and to correlate those with QTL for disease resistance.     

The complete chloroplast genome sequence of the folk medicinal and vegetable plant purslane (Portulaca oleracea L.)

Portulaca oleracea is an important and widely distributed medicinal and edible plant that has great economic value in the medical and food industries. We sequenced the complete chloroplast (cp) genome of P. oleracea,whichis156,533 bp in length, including a pair of inverted repeats (IRs) of 25,501 bp separated by a large single copy (LSC) region of 87,436 bp and a small single copy (SSC) region of 18,095 bp. The genome contains 85 protein-coding genes, 37 transfer RNA (tRNA) genes, and 8 ribosomal RNA (rRNA) genes. There are 40 repeat units and 111 simple sequence repeats (SSRs) in the genome. We also carried out complete cp genome comparison with other Caryophyllales species. The size, gene content, and structural organisation of P. oleracea are relatively conserved, but the genes ycf1 and ycf2 have larger introns. The simple sequence repeats (SSR) in the introns of ycf3, ycf1, atpB, rrn23 and ndhH have significant polymorphism. Comparisons of IR boundaries among 8 Caryophyllales species showed contraction and expansion. The complete cp genome sequence will contribute to a better understanding of the evolutionary mode of the cp genome and to the development of new markers for Caryophyllales classification.     

Spacing Experiments on Vegetables: V. The Effect of Spacing and Manuring on the Composition of the Foliage of Shallots and Globe Beet,Considered in Relation to the Growth of the Plants

Summary

A leaf curl disease was observed on croton (Codiaeum variegatum L.), a popular ornamental plant in botanical, home, and office gardens in and around Bengaluru, South India. Diseased plants showed typical symptoms of vein thickening, severe inward curling and a reduction in leaf size, and stunting. The pathogen responsible was transmitted to healthy croton plants by grafting of infected scions, and through the whitefly vector, Bemisia tabaci, suggesting that the disease was caused by a begomovirus. The association of a begomovirus with the disease was further confirmed by the amplification of viral DNA fragments of ca. 520 bp and 575 bp derived from the coat protein (CP) gene of DNA-A using degenerate primers and total DNA extracted from infected, but not from healthy croton plants. The 575 bp fragment corresponding to the core region of the CP gene was cloned and sequenced. Phylogenetic analysis of the core CP sequence grouped the croton-infecting begomovirus, which we tentatively called croton leaf curl virus (CrLCuV), with Ageratum yellow vein virus (AJ810825), with which it shared the highest nucleotide identity (95%). The core CP sequence was similar (90 – 95%) to many other begomoviruses from the Indian sub-continent that infect tomato, tobacco, cotton, and papaya. Thus, its precise taxonomic denomination will require sequencing of the complete ssDNA viral genome.     

Effect of glucose on glucosinolates,antioxidants and metabolic enzymes in Brassica sprouts

The purpose of this study was to investigate the effect of glucose on glucosinolates, antioxidants and metabolic enzymes in Brassica sprouts. After glucose treatment, total glucosinolates, phenolics and anthocyanin contents and antioxidant activity were significantly enhanced in Chinese kale and pak choi sprouts, while only the anthocyanin and ascorbic acid contents were increased in radish sprouts. These results indicate that glucose treatment has selectively improved the nutritional compounds in different Brassica sprouts.     

Photosynthetic and growth responses of juvenile Chinese kale (Brassica oleracea var. alboglabra) and Caisin (Brassica rapa subsp. parachinensis) to waterlogging and water deficit

Chinese kale (Brassica oleracea var. alboglabra) and Caisin (Brassica rapa subsp. parachinensis) are leafy vegetable crops grown in south-east Asian countries where rainfall varies dramatically from excess to deficit within and between seasons. We investigated the physiological and growth responses of these plants to waterlogging and water deficit in a controlled experiment in a glasshouse. Juvenile plants were subjected to waterlogging or water deficit for 19 days in case of Chinese kale and 14 days in case of Caisin and compared with well-watered controls. Caisin tolerated waterlogging better than Chinese kale because it produced hypocotyl roots and gas spaces developed at the stem base. In Chinese kale, waterlogging reduced plant fresh weight (90%), leaf area (86%), dry weight (80%) and leaf number (38%). In contrast, waterlogging had no impact on leaf number in Caisin and reduced plant fresh and dry weights and leaf area by 60–70%. Water deficit reduced leaf area, fresh weight and dry weight of both species by more than half. Leaf number in Chinese kale was reduced by 38% but no effect occurred in Caisin. Water deficit increased the concentration of nitrogen in the leaf dry matter by more than 60% in both species and the leaf colour of water deficient plants was dark green compared with the leaf colour of well-watered plants. Soil water deficit delayed flowering of Caisin while waterlogging accelerated it. Thickening and whitening of the cuticle on the leaves of Chinese kale probably increased its ability to retain water under drought while Caisin adjusted osmotically and Chinese kale did not. Waterlogging and water deficit had strong effects on leaf gas exchange of both Brassica species. Water deficit closed the stomata in both species and this was associated with a leaf water content of 9 g g⁻¹ DW. In contrast, waterlogging reduced conductance from 1.0 to 0.1 mol H₂O m⁻² s⁻¹ in direct proportion to changes in leaf water content, which fell from 11 to 5 g g⁻¹ DW. This separation of the effects of water deficit and waterlogging on conductance was reflected in transpiration, internal CO₂ concentration and net photosynthesis. In conclusion, Chinese kale and Caisin showed rather different adaptations in response to waterlogging and water deficit. Caisin was more tolerant of waterlogging than Chinese kale and also showed evidence of tolerance of drought. There is genetic variation to waterlogging within the Brassica genus among the leafy vegetables that could be used for cultivar improvement.     

Targeted identification of genomic regions using TAGdb

Background

The introduction of second generation sequencing technology has enabled the cost effective sequencing of genomes and the identification of large numbers of genes and gene promoters. However, the assembly of DNA sequences to create a representation of the complete genome sequence remains costly, especially for the larger and more complex plant genomes.

Results

We have developed an online database, TAGdb, that enables researchers to identify paired read sequences that share identity with a submitted query sequence. These tags can be used to design oligonucleotide primers for the PCR amplification of the region in the target genome.

Conclusions

The ability to produce large numbers of paired read genome tags using second generation sequencing provides a cost effective method for the identification of genes and promoters in large, complex or orphan species without the need for whole genome assembly.