BackgroundOne of the goals of livestock genomics research is to identify the genetic differences responsible for variation in phenotypic traits, particularly those of economic importance. Characterizing the genetic variation in livestock species is an important step towards linking genes or genomic regions with phenotypes. The completion of the bovine genome sequence and recent advances in DNA sequencing technology allow for in-depth characterization of the genetic variations present in cattle. Here we describe the whole-genome resequencing of two Bos taurus bulls from distinct breeds for the purpose of identifying and annotating novel forms of genetic variation in cattle.ResultsThe genomes of a Black Angus bull and a Holstein bull were sequenced to 22-fold and 19-fold coverage, respectively, using the ABI SOLiD system. Comparisons of the sequences with the Btau4.0 reference assembly yielded 7 million single nucleotide polymorphisms (SNPs), 24% of which were identified in both animals. Of the total SNPs found in Holstein, Black Angus, and in both animals, 81%, 81%, and 75% respectively are novel. In-depth annotations of the data identified more than 16 thousand distinct non-synonymous SNPs (85% novel) between the two datasets. Alignments between the SNP-altered proteins and orthologues from numerous species indicate that many of the SNPs alter well-conserved amino acids. Several SNPs predicted to create or remove stop codons were also found. A comparison between the sequencing SNPs and genotyping results from the BovineHD high-density genotyping chip indicates a detection rate of 91% for homozygous SNPs and 81% for heterozygous SNPs. The false positive rate is estimated to be about 2% for both the Black Angus and Holstein SNP sets, based on follow-up genotyping of 422 and 427 SNPs, respectively. Comparisons of read depth between the two bulls along the reference assembly identified 790 putative copy-number variations (CNVs). Ten randomly selected CNVs, five genic and five non-genic, were successfully validated using quantitative real-time PCR. The CNVs are enriched for immune system genes and include genes that may contribute to lactation capacity. The majority of the CNVs (69%) were detected as regions with higher abundance in the Holstein bull.ConclusionsSubstantial genetic differences exist between the Black Angus and Holstein animals sequenced in this work and the Hereford reference sequence, and some of this variation is predicted to affect evolutionarily conserved amino acids or gene copy number. The deeply annotated SNPs and CNVs identified in this resequencing study can serve as useful genetic tools, and as candidates in searches for phenotype-altering DNA differences.

Aluminum (Al) toxicity is a major constraint for crop production in acid soils, although crop cultivars vary in their tolerance to Al. We have investigated the potential role of citrate in mediating Al tolerance in Al-sensitive yeast (Saccharomyces cerevisiae; MMYO11) and canola (Brassica napus cv Westar). Yeast disruption mutants defective in genes encoding tricarboxylic acid cycle enzymes, both upstream (citrate synthase [CS]) and downstream (aconitase [ACO] and isocitrate dehydrogenase [IDH]) of citrate, showed altered levels of Al tolerance. A triple mutant of CS (⌬cit123) showed lower levels of citrate accumulation and reduced Al tolerance, whereas ⌬aco1-and ⌬idh12-deficient mutants showed higher accumulation of citrate and increased levels of Al tolerance. Overexpression of a mitochondrial CS (CIT1) in MMYO11 resulted in a 2-to 3-fold increase in citrate levels, and the transformants showed enhanced Al tolerance. A gene for Arabidopsis mitochondrial CS was overexpressed in canola using an Agrobacterium tumefaciens-mediated system. Increased levels of CS gene expression and enhanced CS activity were observed in transgenic lines compared with the wild type. Root growth experiments revealed that transgenic lines have enhanced levels of Al tolerance. The transgenic lines showed enhanced levels of cellular shoot citrate and a 2-fold increase in citrate exudation when exposed to 150 m Al. Our work with yeast and transgenic canola clearly suggest that modulation of different enzymes involved in citrate synthesis and turnover (malate dehydrogenase, CS, ACO, and IDH) could be considered as potential targets of gene manipulation to understand the role of citrate metabolism in mediating Al tolerance.Aluminum toxicity is one of the major factors limiting crop productivity in acid soils. The root apex is considered the primary site of Al-induced injury, and inhibition of root elongation is one of the most visible symptoms of Al toxicity. Although most plants are sensitive to Al, several crop species exhibit genetic variation in their ability to tolerate Al. One possible mechanism of Al tolerance is the chelation of Al by organic anions within root cells or in the rhizosphere (Taylor, 1991). Exudation of a variety of low M r organic anions such as citrate, malate, or oxalate has been reported in several crop species upon exposure to Al (Miyasaka et al., 1991; Basu et al., 1994;Pellet et al., 1995;Larsen et al., 1998). Although a rapid release of organic anions (malate 2Ϫ ) was observed in near isogenic, Al-tolerant wheat (Triticum aestivum) lines (Delhaize et al., 1993), several lines of evidence suggest that a lag phase may also exist between exposure of roots to Al and excretion of organic anions. Citrate exudation from roots of rye was observed only after 120 h of exposure (Li et al., 2000). Similarly, an Alinduced, de novo synthesis of malate leading to enhanced malate efflux 24 h after exposure was demonstrated in Al-tolerant wheat cv Katepwa (Basu et al., 1994). Delayed exudation of organic anions could possibly ...

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Low temperature is one of the most common environmental stresses that seriously affect the growth and development of plants. However, plants have the plasticity in their defence mechanisms enabling them to tolerate and, sometimes, even survive adverse environmental conditions. MicroRNAs (miRNAs) are small non-coding RNAs, approximately 18-24 nucleotides in length, and are being increasingly recognized as regulators of gene expression at the post-transcriptional level and have the ability to influence a broad range of biological processes. There is growing evidence in the literature that reprogramming of gene expression mediated through miRNAs is a major defence mechanism in plants enabling them to respond to stresses. To date, numerous studies have established the importance of miRNA-based regulation of gene expression under low temperature stress. Individual miRNAs can modulate the expression of multiple mRNA targets, and, therefore, the manipulation of a single miRNA has the potential to affect multiple biological processes. Numerous functional studies have attempted to identify the miRNA-target interactions and have elaborated the role of several miRNAs in cold-stress regulation. This review summarizes the current understanding of miRNA-mediated modulation of the expression of key genes as well as genetic and regulatory pathways, involved in low temperature stress responses in plants.

An important principle of the functional organization of plant cells is the targeting of proteins to specific subcellular locations. The physical location of proteins within the apoplasm/ rhizosphere at the root-soil interface positions them to play a strategic role in plant response to biotic and abiotic stress. We previously demonstrated that roots of Triticum aestivum and Brassica napus exude a large suite of proteins to the apoplasm/rhizosphere [Basu et al. (1994) Plant Physiol 106:151-158; Basu et al. (1999) Physiol Plant 106:53-61]. This study is a first step to identify low abundance extracytosolic proteins from Arabidopsis thaliana and Brassica napus roots using recent advances in the field of proteomics. A total of 16 extracytosolic proteins were identified from B. napus using two-dimensional gel electrophoresis, tandem mass spectrometry (LC-MS/MS) and de novo sequencing. Another high-throughput proteomics approach, Multidimensional Protein Identification Technology (Mud PIT) was used to identify 52 extracytosolic proteins from A. thaliana. Signal peptide cleavage sites, the presence/absence of transmembrane domains and GPI modification were determined for these proteins. Functional classification grouped the extracellular proteins into different families including glycoside hydrolases, trypsin/ protease inhibitors, plastocyanin-like domains, copper-zinc superoxide dismutases, gamma-thioinins, thaumatins, ubiquitins, protease inhibitor/ seed storage/lipid transfer proteins, transcription factors, class III peroxidase, and plant basic secretory proteins (BSP). We have also developed an on-line, Extracytosolic Plant Proteins Database (EPPdb, http://eppdb.biology.ualberta.ca) to provide information about these extracytosolic proteins.

We investigated the role that manganese superoxide dismutase (MnSOD, EC 1·15·1·1), an important enzyme of the antioxidant pathway, may play in aluminium (Al) toxicity/ resistance. A wheat ( Triticum aestivum ) cDNA ( WMn-SOD ) was cloned and up-regulation of the transcript was observed in root tips after 24 h exposure to 100 µ µ µ µ M Al. The WMnSOD1 under the control of the CaMV 35S promoter was expressed in canola ( Brassica napus ). Transgenic plants were phenotypically normal. Northern analyses showed enhanced levels of the WMnSOD1 and WMnSOD1-GUS transcripts and total SOD activity was 1·5-to 2·5-fold greater in transgenic plants than in wild type (WT) plants. Transgenic (T 1 ) leaf discs showed increased retention of chlorophyll and reduced electrolyte leakage when exposed to methyl viologen (MV) as compared with WT leaf discs, suggesting that transgenic plants were more resistant to oxidative stress. When WT canola plants were exposed to aluminium (0-200 µ µ µ µ M ), inhibition of root growth, higher SOD activity and increased levels of malondialdehyde (MDA, an indicator of lipid peroxidation) were observed in roots. Aluminium-induced inhibition of root growth and accumulation of MDA was lower in homozygous transgenic plants (T 2 ) compared with WT plants. Transgenic lines also showed lower synthesis of 1,3-β β β βglucans (callose, a sensitive marker for Al injury) as compared with WT. These data suggest that resistance to Al toxicity can be improved by overexpressing WMnSOD1 .

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Sclerotinia stem rot caused by Sclerotinia sclerotiorum affects canola production worldwide. Emerging evidence suggests that long non-coding RNAs (lncRNAs) play important roles in the regulation of gene expression in plants, in response to both abiotic and biotic stress. So far, identification of lncRNAs has been limited to a few model plant species, and their roles in mediating responses to biotic stresses are yet to be characterized in Brassica napus. The present study reports the identification of novel lncRNAs responsive to S. sclerotiorum infection in B. napus at two time points after infection (24 hpi and 48 hpi) using a stranded RNA-Sequencing technique and a detection pipeline for lncRNAs. Of the total 3,181 lncRNA candidates, 2,821 lncRNAs were intergenic, 111 were natural antisense transcripts, 76 possessed exonic overlap with the reference coding transcripts while the remaining 173 represented novel lnc- isoforms. Forty one lncRNAs were identified as the precursors for microRNAs (miRNAs) including miR156, miR169 and miR394, with significant roles in mediating plant responses to fungal phytopathogens. A total of 931 differentially expressed lncRNAs were identified in response to S. sclerotiorum infection and the expression of 12 such lncRNAs was further validated using qRT-PCR. B. napus antisense lncRNA, TCONS_00000966, having 90% overlap with a plant defensin gene, showed significant induction at both infection stages, suggesting its involvement in the transcriptional regulation of defense responsive genes under S. sclerotiorum infection. Additionally, nine lncRNAs showed overlap with cis-regulatory regions of differentially expressed genes of B. napus. Quantitative RT-PCR verification of a set of S. sclerotiorum responsive sense/antisense transcript pairs revealed contrasting expression patterns, supporting the hypothesis that steric clashes of transcriptional machinery may lead to inactivation of sense promoter. Our findings highlight the potential contributions of lncRNAs in regulating expression of plant genes that respond to biotic stress.