Project description:Camelina sativa (L.) Crantz an oilseed crop of the Brassicaceae family is gaining attention due to its potential as a source of high value oil for food, feed or fuel. The hexaploid domesticated C. sativa has limited genetic diversity, encouraging the exploration of related species for novel allelic variation for traits of interest. The current study utilized genotyping by sequencing to characterize 193 Camelina accessions belonging to seven different species collected primarily from the Ukrainian-Russian region and Eastern Europe. Population analyses among Camelina accessions with a 2n = 40 karyotype identified three subpopulations, two composed of domesticated C. sativa and one of C. microcarpa species. Winter type Camelina lines were identified as admixtures of C. sativa and C. microcarpa Eighteen genotypes of related C. microcarpa unexpectedly shared only two subgenomes with C. sativa, suggesting a novel or cryptic sub-species of C. microcarpa with 19 haploid chromosomes. One C. microcarpa accession (2n = 26) was found to comprise the first two subgenomes of C. sativa suggesting a tetraploid structure. The defined chromosome series among C. microcarpa germplasm, including the newly designated C. neglecta diploid née C. microcarpa, suggested an evolutionary trajectory for the formation of the C. sativa hexaploid genome and re-defined the underlying subgenome structure of the reference genome.
Project description:BACKGROUND: Lipids extracted from seeds of Camelina sativa have been successfully used as a reliable source of aviation biofuels. This biofuel is environmentally friendly because the drought resistance, frost tolerance and low fertilizer requirement of Camelina sativa allow it to grow on marginal lands. Improving the species growth and seed yield by genetic engineering is therefore a target for the biofuels industry. In Arabidopsis, overexpression of purple acid phosphatase 2 encoded by Arabidopsis (AtPAP2) promotes plant growth by modulating carbon metabolism. Overexpression lines bolt earlier and produce 50% more seeds per plant than wild type. In this study, we explored the effects of overexpressing AtPAP2 in Camelina sativa. RESULTS: Under controlled environmental conditions, overexpression of AtPAP2 in Camelina sativa resulted in longer hypocotyls, earlier flowering, faster growth rate, higher photosynthetic rate and stomatal conductance, increased seed yield and seed size in comparison with the wild-type line and null-lines. Similar to transgenic Arabidopsis, activity of sucrose phosphate synthase in leaves of transgenic Camelina was also significantly up-regulated. Sucrose produced in photosynthetic tissues supplies the building blocks for cellulose, starch and lipids for growth and fuel for anabolic metabolism. Changes in carbon flow and sink/source activities in transgenic lines may affect floral, architectural, and reproductive traits of plants. CONCLUSIONS: Lipids extracted from the seeds of Camelina sativa have been used as a major constituent of aviation biofuels. The improved growth rate and seed yield of transgenic Camelina under controlled environmental conditions have the potential to boost oil yield on an area basis in field conditions and thus make Camelina-based biofuels more environmentally friendly and economically attractive.
Project description:Plants produce diverse secondary metabolites. Although each metabolite is made through its respective biosynthetic pathway, plants coordinate multiple biosynthetic pathways simultaneously. One example is an interaction between glucosinolate and phenylpropanoid pathways in Arabidopsis thaliana. Glucosinolates are defense compounds made primarily from methionine and tryptophan, while phenylpropanoids are made from phenylalanine. Recent studies have shown that the accumulation of glucosinolate intermediate such as indole-3-acetaldoxime (IAOx) or its derivatives represses phenylpropanoid production via the degradation of phenylalanine ammonia lyase (PAL) functioning at the entry point of the phenylpropanoid pathway. Given that IAOx is a precursor of other bioactive compounds other than glucosinolates and that the phenylpropanoid pathway is present in most plants, we hypothesized that this interaction is relevant to other species. Camelina sativa is an oil crop and produces camalexin from IAOx. We enhanced IAOx production in Camelina by overexpressing Arabidopsis CYP79B2 which encodes an IAOx-producing enzyme. The overexpression of AtCYP79B2 results in increased auxin content and its associated morphological phenotypes in Camelina but indole glucosinolates were not detected in Camelina wild type as well as the overexpression lines. However, phenylpropanoid contents were reduced in AtCYP79B2 overexpression lines suggesting a link between aldoxime metabolism and phenylpropanoid production. Interestingly, the expression of PALs was not affected in the overexpression lines although PAL activity was reduced. To test if PAL degradation is involved in the crosstalk, we identified F-box genes functioning in PAL degradation through a phylogenetic study. A total of 459 transcript models encoding kelch-motifs were identified from the Camelina sativa database. Among them, the expression of CsKFBs involved in PAL degradation is up-regulated in the transgenic lines. Our results suggest a link between aldoxime metabolism and phenylpropanoid production in Camelina and that the molecular mechanism behind the crosstalk is conserved in Arabidopsis and Camelina.
Project description:BACKGROUND: Camelina sativa, an oilseed crop in the Brassicaceae family, has inspired renewed interest due to its potential for biofuels applications. Little is understood of the nature of the C. sativa genome, however. A study was undertaken to characterize two genes in the fatty acid biosynthesis pathway, fatty acid desaturase (FAD) 2 and fatty acid elongase (FAE) 1, which revealed unexpected complexity in the C. sativa genome. RESULTS: In C. sativa, Southern analysis indicates the presence of three copies of both FAD2 and FAE1 as well as LFY, a known single copy gene in other species. All three copies of both CsFAD2 and CsFAE1 are expressed in developing seeds, and sequence alignments show that previously described conserved sites are present, suggesting that all three copies of both genes could be functional. The regions downstream of CsFAD2 and upstream of CsFAE1 demonstrate co-linearity with the Arabidopsis genome. In addition, three expressed haplotypes were observed for six predicted single-copy genes in 454 sequencing analysis and results from flow cytometry indicate that the DNA content of C. sativa is approximately three-fold that of diploid Camelina relatives. Phylogenetic analyses further support a history of duplication and indicate that C. sativa and C. microcarpa might share a parental genome. CONCLUSIONS: There is compelling evidence for triplication of the C. sativa genome, including a larger chromosome number and three-fold larger measured genome size than other Camelina relatives, three isolated copies of FAD2, FAE1, and the KCS17-FAE1 intergenic region, and three expressed haplotypes observed for six predicted single-copy genes. Based on these results, we propose that C. sativa be considered an allohexaploid. The characterization of fatty acid synthesis pathway genes will allow for the future manipulation of oil composition of this emerging biofuel crop; however, targeted manipulations of oil composition and general development of C. sativa should consider and, when possible take advantage of, the implications of polyploidy.
Project description:BACKGROUND: Biofuels extracted from the seeds of Camelina sativa have recently been used successfully as environmentally friendly jet-fuel to reduce greenhouse gas emissions. Camelina sativa is genetically very close to Arabidopsis thaliana, and both are members of the Brassicaceae. Although public databases are currently available for some members of the Brassicaceae, such as A. thaliana, A. lyrata, Brassica napus, B. juncea and B. rapa, there are no public Expressed Sequence Tags (EST) or genomic data for Camelina sativa. In this study, a high-throughput, large-scale RNA sequencing (RNA-seq) of the Camelina sativa transcriptome was carried out to generate a database that will be useful for further functional analyses. RESULTS: Approximately 27 million clean "reads" filtered from raw reads by removal of adaptors, ambiguous reads and low-quality reads (2.42 gigabase pairs) were generated by Illumina paired-end RNA-seq technology. All of these clean reads were assembled de novo into 83,493 unigenes and 103,196 transcripts using SOAPdenovo and Trinity, respectively. The average length of the transcripts generated by Trinity was 697 bp (N50 = 976), which was longer than the average length of unigenes (319 bp, N50 = 346 bp). Nonetheless, the assembly generated by SOAPdenovo produced similar number of non-redundant hits (22,435) with that of Trinity (22,433) in BLASTN searches of the Arabidopsis thaliana CDS sequence database (TAIR). Four public databases, the Kyoto Encyclopedia of Genes and Genomes (KEGG), Swiss-prot, NCBI non-redundant protein (NR), and the Cluster of Orthologous Groups (COG), were used for unigene annotation; 67,791 of 83,493 unigenes (81.2%) were finally annotated with gene descriptions or conserved protein domains that were mapped to 25,329 non-redundant protein sequences. We mapped 27,042 of 83,493 unigenes (32.4%) to 119 KEGG metabolic pathways. CONCLUSIONS: This is the first report of a transcriptome database for Camelina sativa, an environmentally important member of the Brassicaceae. We showed that C. savita is closely related to Arabidopsis spp. and more distantly related to Brassica spp. Although the majority of annotated genes had high sequence identity to those of A. thaliana, a substantial proportion of disease-resistance genes (NBS-encoding LRR genes) were instead more closely similar to the genes of other Brassicaceae; these genes included BrCN, BrCNL, BrNL, BrTN, BrTNL in B. rapa. As plant genomes are under long-term selection pressure from environmental stressors, conservation of these disease-resistance genes in C. sativa and B. rapa genomes implies that they are exposed to the threats from closely-related pathogens in their natural habitats.
Project description:Hybridization between crops and their wild relatives has the potential to introduce novel variation into wild populations. Camelina (Camelina sativa) is a promising oilseed and cultivars with modified seed characteristics and herbicide resistance are in development, prompting a need to evaluate the potential for novel trait introgression into weedy relatives. Little-podded false flax (littlepod; Camelina microcarpa) is a naturalized weed in Canada and the USA. Here we evaluated the hybridization rate between the three cytotypes of littlepod (?) and camelina (?), assessed characteristics of hybrids, and evaluated the fitness of hexaploid littlepod and camelina hybrids in the glasshouse and field. In total we conducted, 1,005 manual crosses with diploid littlepod, 1, 172 crosses with tetraploid littlepod, and 896 crosses with hexaploid littlepod. Hybrids were not produced by the diploids, but were produced by the tetraploids and hexaploids at rates of one hybrid for 2,000 ovules pollinated and 24 hybrids for 25 ovules pollinated, respectively. Hybrids between tetraploid littlepod and camelina showed low pollen fertility and produced a small number of seeds. In the glasshouse, hybrids between hexaploid littlepod and camelina also showed significantly lower pollen fertility and seed production than parental lines, but their seeds showed high viability. A similar pattern was observed in field trials, with hybrids showing earlier flowering, reduced biomass, seed production and seed weight. However, seed produced by the hybrids showed greater viability than that produced by hexaploid littlepod and is potentially the result of a shortened lifecycle. The introgression of lifecycle traits into littlepod populations may facilitate range expansion and contribute to crop gene persistence. Consequently, future work should evaluate the hybridization rate in the field, the fitness of advanced generation backcrosses, and the role of time to maturity in limiting hexaploid littlepod's distribution.
Project description:<i>Camelina sativa</i>, a largely relict crop, has recently returned to interest due to its potential as an industrial oilseed. Molecular markers are key tools that will allow <i>C. sativa</i> to benefit from modern breeding approaches. Two complementary methodologies, capture of 3' cDNA tags and genomic reduced-representation libraries, both of which exploited second generation sequencing platforms, were used to develop a low density (768) Illumina GoldenGate single nucleotide polymorphism (SNP) array. The array allowed 533 SNP loci to be genetically mapped in a recombinant inbred population of <i>C. sativa</i>. Alignment of the SNP loci to the <i>C. sativa</i> genome identified the underlying sequenced regions that would delimit potential candidate genes in any mapping project. In addition, the SNP array was used to assess genetic variation among a collection of 175 accessions of <i>C. sativa</i>, identifying two sub-populations, yet low overall gene diversity. The SNP loci will provide useful tools for future crop improvement of <i>C. sativa</i>.
Project description:Camelina sativa is an annual oilseed crop that is under intensive development for renewable resources of biofuels and industrial oils. MicroRNAs, or miRNAs, are endogenously encoded small RNAs that play key roles in diverse plant biological processes. Here, we conducted deep sequencing on small RNA libraries prepared from camelina leaves, flower buds and two stages of developing seeds corresponding to initial and peak storage products accumulation. Computational analyses identified 207 known miRNAs belonging to 63 families, as well as 5 novel miRNAs. These miRNAs, especially members of the miRNA families, varied greatly in different tissues and developmental stages. The predicted miRNA target genes are involved in a broad range of physiological functions including lipid metabolism. This report is the first step toward elucidating roles of miRNAs in C. sativa and will provide additional tools to improve this oilseed crop for biofuels and biomaterials.
Project description:Camelina sativa is an oilseed with desirable agronomic and oil-quality attributes for a viable industrial oil platform crop. Here we generate the first chromosome-scale high-quality reference genome sequence for C. sativa and annotated 89,418 protein-coding genes, representing a whole-genome triplication event relative to the crucifer model Arabidopsis thaliana. C. sativa represents the first crop species to be sequenced from lineage I of the Brassicaceae. The well-preserved hexaploid genome structure of C. sativa surprisingly mirrors those of economically important amphidiploid Brassica crop species from lineage II as well as wheat and cotton. The three genomes of C. sativa show no evidence of fractionation bias and limited expression-level bias, both characteristics commonly associated with polyploid evolution. The highly undifferentiated polyploid genome of C. sativa presents significant consequences for breeding and genetic manipulation of this industrial oil crop.
Project description:As an important oilseed worldwide, Camelina sativa is being increasingly explored for its use in production of food, feed, biofuel and industrial chemicals. However, detailed mechanisms of camelina oil biosynthesis and accumulation, particularly in vegetative tissues, are understood to a very small extent. Here, we present genome-wide identification, cloning and functional analysis of phospholipid diacylglycerol acyltransferase (PDAT) in C. sativa, which catalyses the final acylation step in triacylglycerol (TAG) biosynthesis by transferring a fatty acyl moiety from a phospholipid to diacylglycerol (DAG). We identified five genes (namely CsPDAT1-A, B, and C and CsPDAT2-A and B) encoding PDATs from the camelina genome. CsPDAT1-A is mainly expressed in seeds, whereas CsPDAT1-C preferentially accumulates in flower and leaf tissues. High expression of CsPDAT2-A and CsPDAT2-B was detected in stem and root tissues, respectively. Cold stress induced upregulation of CsPDAT1-A and CsPDAT1-C expression by 3.5- and 2.5-fold, respectively, compared to the control. Salt stress led to an increase in CsPDAT2-B transcripts by 5.1-fold. Drought treatment resulted in an enhancement of CsPDAT2-A mRNAs by twofold and a reduction of CsPDAT2-B expression. Osmotic stress upregulated the expression of CsPDAT1-C by 3.3-fold. Furthermore, the cDNA clones of these CsPDAT genes were isolated for transient expression in tobacco leaves. All five genes showed PDAT enzymatic activity and substantially increased TAG accumulation in the leaves, with CsPDAT1-A showing a higher preference for ?-linolenic acid (18:3 ?-3). Overall, this study demonstrated that different members of CsPDAT family contribute to TAG synthesis in different tissues. More importantly, they are involved in different types of stress responses in camelina seedlings, providing new evidence of their roles in oil biosynthesis and regulation in camelina vegetative tissue. The identified CsPDATs may have practical applications in increasing oil accumulation and enhancing stress tolerance in other plants as well.