Exploring valid reference genes for gene expression studies in <Emphasis Type="Italic">Brachypodium distachyon</Emphasis>by real-time PCR

Background  

The wild grass species Brachypodium distachyon (Brachypodium hereafter) is emerging as a new model system for grass crop genomics research and biofuel grass biology. A draft nuclear genome sequence is expected to be publicly available in the near future; an explosion of gene expression studies will undoubtedly follow. Therefore, stable reference genes are necessary to normalize the gene expression data.     

Validation of reference genes for real-time quantitative PCR studies in gene expression levels of <Emphasis Type="Italic">Lactobacillus casei</Emphasis> Zhang

Lactobacillus casei Zhang, a potential probiotic strain isolated from homemade koumiss in Inner Mongolia of China, has been sequenced and deposited in GenBank. Real-time quantitative PCR is one of the most widely used methods to study related gene expression levels of Lactobacillus casei Zhang. For accurate and reliable gene expression analysis, normalization of gene expression data using one or more appropriate reference genes is essential. We used three statistical methods (geNorm, NormFinder, and BestKeeper) to evaluate the expression levels of five candidate reference genes (GAPD, gyrB, LDH, 16s rRNA, and recA) under different culture conditions and different growth phases to find a suitable housekeeping gene which can be used as internal standard. The results showed that the best reference gene was GAPD, and a set of two genes, GAPD and gyrB (which were the most stable reference genes), is recommended for normalization of real-time quantitative PCR experiments under all the different experimental conditions tested. The systematic validation of candidate reference genes is important for obtaining reliable analysis results of real-time quantitative PCR studies in gene expression levels of Lactobacillus casei Zhang.     

Transient expression of <Emphasis Type="Italic">AtNCED3</Emphasis> and <Emphasis Type="Italic">AAO3</Emphasis> genes in guard cells causes stomatal closure in <Emphasis Type="Italic">Vicia faba</Emphasis>

Abscisic acid (ABA) regulates stomatal closure in response to water loss. Here, we examined the competence of guard cells to synthesize ABA, using two Arabidopsis ABA biosynthetic enzymes. 35S pro::AtNCED3-GFP and AAO3-GFP were introduced into guard cells of broad bean leaves. AtNCED3-GFP expression was detected at the chloroplasts, whereas green fluorescent protein (GFP) and AAO3-GFP were in the cytosol. The stomatal aperture was decreased in AtNCED3-GFP- and AAO3-GFP-transformed guard cells. This indicated that ABA biosynthesis is stimulated by heterologous expression of AtNCED3 and Arabidopsis aldehyde oxidase 3 (AAO3) proteins, which both seem to be regulatory enzymes for ABA biosynthesis in these cells. Furthermore, stomatal closure by the expression of AtNCED3 and AAO3 suggested that the substrates of the enzymes are present and native ABA-biosynthesis enzymes are active in guard cells. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. V. Melhorn and K. Matsumi contributed equally to this work.     

Selection of reference genes for quantitative real-time PCR in <Emphasis Type="Italic">Casuarina equisetifolia</Emphasis> under salt stress

Real time quantitative PCR (qPCR) is widely used in gene expression analysis for its accuracy and sensitivity. Reference genes serving as endogenous controls are necessary for gene normalization. In order to select an appropriate reference gene to normalize gene expression in Casuarina equisetifolia under salt stress, 10 potential reference genes were evaluated using real time qPCR in the leaves and roots of plants grown under different NaCl concentrations and treatment durations. GeNorm, NormFinder, and BestKeeper analyses reveal that elongation factor 1-alpha (EF1α) and ubiquitin-conjugating enzyme E2 (UBC) were the most appropriate reference genes for real time qPCR under salt stress. However, β-tubulin (βTUB) and actin 7, which were widely used as reference genes in other plant species, were not always stably expressed. The combination of EF1α, UBC, uncharacterized protein 2, DNAJ homolog subfamily A member 2, and glyceraldehyde-3-phosphate dehydrogenase should be ideal reference genes for normalizing gene expression data in all samples under salt stress. It indicates the need for reference gene selection for normalizing gene expression in C. equisetifolia. In addition, the suitability of reference genes selected was confirmed by validating the expression of WRKY29-like and expansin-like B1. The results enable analysis of salt response mechanism and gene expression in C. equisetifolia.     

Identification of plant architecture and yield-related QTL in <Emphasis Type="Italic">Vicia faba</Emphasis> L.

Low and unstable yields across seasons and environments are among the main reasons which make profitability for farmers too low. Along with resistance/tolerance to biotic and abiotic stresses, plant architecture and yield-related traits are the main determinants of yield stability. The current study was conducted to identify and validate quantitative trait loci (QTL) for plant architecture and yield-related traits in faba bean; our results provide novel information about the genetics of plant architecture traits in this crop. An equina × paucijuga recombinant inbred line population was derived and submitted to field experiments at Córdoba (Spain) over a period of four seasons. Stable QTL were identified for eight of the traits evaluated. QTL clusters were identified on almost each chromosome. The high inter-trait correlations between some of the traits controlled by a cluster of QTL might reflect either a set of closely linked loci or, more likely, pleiotropic effects. The stability of many of these major QTL in different years offers the possibility of exploiting them via marker-assisted selection. Further fine mapping of these target regions will help to identify potential candidate genes using synteny.     

Reference genes selection for quantitative gene expression studies in <Emphasis Type="Italic">Pinus massoniana</Emphasis> L.

Key message

Evaluation and selection of reference genes in Pinus massoniana L. (PM) for gene expression studies of various tissues, floral organ development, and abiotic stress.

Abstract

An important prerequisite for obtaining accurate gene expression results using quantitative real-time PCR is the selection of a reference gene or a group of genes having a highly stable level of expression. Pinus massoniana L. (PM) is the predominant fast-growing timber forest tree species in southern China. In this study of PM, we evaluated various tissues, flowers in different developmental phases, leaves from a cultivar with insect resistance, and leaves from plants under several types of abiotic stresses. Comprehensive Analysis was performed using BestKeeper, Normfinder, geNorm, and RefFinder software to select the most stable reference gene or gene group from among 25 candidate genes in these samples. The results showed that different experimental conditions require the use of different reference genes: ACT1 could be used as a reference gene for all samples in this study; UBI4 was the best gene for various tissues and zinc stress; CYP was the most stable gene for leaves from insect-resistant materials and Pb stress; Fbox and UBI11 were the best reference genes for salt stress; Fbox + RRP8, ARF + TUBA, and EF1B + IDH were the best reference groups for drought stress, low temperature stress, and flowers in different developmental phases, respectively. This study presents a reliable selection of reference genes for Masson pine, and the conclusions are meaningful for improving the accuracy of expression analyses in future molecular biology studies.

     

Ectopic expression of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) <Emphasis Type="Italic">CsTIR</Emphasis>/<Emphasis Type="Italic">AFB</Emphasis> genes enhance salt tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

Auxin receptors TIR1/AFBs play an essential role in a series of signaling network cascades. These F-box proteins have also been identified to participate in different stress responses via the auxin signaling pathway in Arabidopsis. Cucumber (Cucumis sativus L.) is one of the most important crops worldwide, which is also a model plant for research. In the study herein, two cucumber homologous auxin receptor F-box genes CsTIR and CsAFB were cloned and studied for the first time. The deduced amino acid sequences showed a 78% identity between CsTIR and AtTIR1 and 76% between CsAFB and AtAFB2. All these proteins share similar characteristics of an F-box domain near the N-terminus, and several Leucine-rich repeat regions in the middle. Arabidopsis plants ectopically overexpressing CsTIR or CsAFB were obtained and verified. Shorter primary roots and more lateral roots were found in these transgenic lines with auxin signaling amplified. Results showed that expression of CsTIR/AFB genes in Arabidopsis could lead to higher seeds germination rates and plant survival rates than wild-type under salt stress. The enhanced salt tolerance in transgenic plants is probably caused by maintaining root growth and controlling water loss in seedlings, and by stabilizing life-sustaining substances as well as accumulating endogenous osmoregulation substances. We proposed that CsTIR/AFB-involved auxin signal regulation might trigger auxin mediated stress adaptation response and enhance the plant salt stress resistance by osmoregulation.     

Genetic variability in Tunisian populations of faba bean (<Emphasis Type="Italic">Vicia faba</Emphasis> L. var. <Emphasis Type="Italic">major</Emphasis>) assessed by morphological and SSR markers

The genetic diversity of 21 faba bean populations was examined using morphological and molecular markers. DNA was extracted from 189 individuals and 8 microsatellite markers were genotyped individually in these 21 populations. A total of 53 alleles were obtained in all populations, with an average of 6.62 alleles per locus. The expected and observed heterozygosity was 0.38 and 0.62 respectively. The average polymorphism index content of SSR markers was 0.61, ranging from 0.31 to 0.81. The unweighted pair group method with arithmetic mean dendrogram clustered all the populations into two groups, each for them subdivided into 3 sub-groups according to geographical origin. Morphological variation showed that the populations were not grouped according to their geographical origin. Therefore, patterns of differentiation of morphological traits did not coincide with molecular differentiation, indicating that morphological variation does not reflect genetic subdivision in studied faba bean populations. Analysis of molecular variance revealed high levels of genetic variation (83%) within population and provides a good base for designing genetic improvement programs. The result of Principal Component Analysis (PCA) revealed that three dimensional principal components (PC1, PC2 and PC3) contributed 40.56% of the total variability and accounted with values of 20.64, 11.22 and 8.70%, respectively. Cluster analysis based on PCA indicated three separate groups of populations. The genetic relationships found between the 21 populations samples were the same in both the PCA and STRUCTURE analysis which support the results observed. These data may serve as a foundation for the development of faba bean breeding programs.     

Characterization of Two Novel Biovar of <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> Isolated from Root Nodules of <Emphasis Type="Italic">Vicia faba</Emphasis>

A total of eight strains of bacteria were isolated from the root nodule of Vicia faba on the selective media of Rhizobium. Two of these strains produced phenotypically distinct mucoid colonies (one slow growing and the other fast growing) and were examined using a polyphasic approach for taxonomic identification. The two strains (MTCC 7405 and MTCC 7406) turned out to be new strains of biovar 1 Agrobacterium rather than Rhizobium, as they showed growth on alkaline medium as well as on 2% NaCl and neither catabolized lactose as the carbon source nor oxidized Tween-80. The distinctness between the two strains was marked with respect to their growth on dextrose and the production of lysine dihydrolase, ornithine decarboxylase and DNA G + C content. 16S rDNA sequencing and their comparison with the 16S rDNA sequences of previously described agrobacteria as well as rhizobia strains confirmed the novelty of the two strains. Both of the strains clustered with strains of Agrobacterium tumefaciens in the 16S rDNA-based phylogenetic tree. The phenotypic and biochemical properties of the two strains differed from those of the recognized biovar of A. tumefaciens. It is proposed that the strains MTCC 7405 and MTCC 7406 be classified as novel biovar of the species A. tumefaciens (Type strains MTCC 7405 = DQ383275 and MTCC 7406 = DQ383276).

Genetic Diversity of a Natural Population of <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> bv. <Emphasis Type="Italic">viciae</Emphasis> Nodulating Plants of <Emphasis Type="Italic">Vicia faba</Emphasis> in the Vesuvian Area

A total of 98 rhizobial strains, isolated during the winter of the years 2003 (35 isolates), 2004 (33 isolates), and 2005 (30 isolates) were analyzed to determine the genetic diversity of the natural population nodulating Vicia faba plants and to identify dominant genotypes. All isolates were identified as Rhizobium leguminosarum bv. viciae by biovar-specific polymerase chain reaction amplification of the nodC gene. Intraspecific DNA polymorphism was evaluated through the restriction endonucleases analysis combined with pulsed-field gel electrophoresis. Four genotypes characterized 53% of the isolates, showing a high occurrence; moreover, they were recovered over the 3 years, thus showing a lasting persistence in the soil, which could mean a high degree of saprophytic competitiveness. The richness, diversity, and dominance indexes of genotypes were calculated to monitor the evolution of the rhizobial population during the 3 years. The genetic diversity of the analyzed strains decreased along the 3 years. In fact, the biodiversity index H′ decreased from 2.6 in the first and second year to 1.9 in the third year; probably, as a result of bean monocropping, specific genotypes of Rh. leguminosarum bv. viciae were naturally selected.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

In planta transformation of <Emphasis Type="Italic">Notocactus scopa</Emphasis> cv. <Emphasis Type="Italic">Soonjung</Emphasis> by <Emphasis Type="Italic">Agrobacterium </Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>

Notocactus scopa cv. Soonjung was subjected to in planta Agrobacterium tumefaciens-mediated transformation with vacuum infiltration, pin-pricking, and a combination of the two methods. The pin-pricking combined with vacuum infiltration (20-30 cmHg for 15 min) resulted in a transformation efficiency of 67-100%, and the expression of the uidA and nptII genes was detected in transformed cactus. The established in planta transformation technique generated a transgenic cactus with higher transformation efficiency, shortened selection process, and stable gene expression via asexual reproduction. All of the results showed that the in planta transformation method utilized in the current study provided an efficient and time-saving procedure for the delivery of genes into the cactus genome, and that this technique can be applied to other asexually reproducing succulent plant species.