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1.
Background
Molecular profiling of gene families is a versatile tool to study diversity between individual genomes in sexual crosses and germplasm. Nucleotide binding site (NBS) profiling, in particular, targets conserved nucleotide binding site-encoding sequences of resistance gene analogs (RGAs), and is widely used to identify molecular markers for disease resistance (R) genes.Results
In this study, we used NBS profiling to identify genome-wide locations of RGA clusters in the genome of potato clone RH. Positions of RGAs in the potato RH and DM genomes that were generated using profiling and genome sequencing, respectively, were compared. Largely overlapping results, but also interesting discrepancies, were found. Due to the clustering of RGAs, several parts of the genome are overexposed while others remain underexposed using NBS profiling. It is shown how the profiling of other gene families, i.e. protein kinases and different protein domain-coding sequences (i.e., TIR), can be used to achieve a better marker distribution. The power of profiling techniques is further illustrated using RGA cluster-directed profiling in a population of Solanum berthaultii. Multiple different paralogous RGAs within the Rpi-ber cluster could be genetically distinguished. Finally, an adaptation of the profiling protocol was made that allowed the parallel sequencing of profiling fragments using next generation sequencing. The types of RGAs that were tagged in this next-generation profiling approach largely overlapped with classical gel-based profiling. As a potential application of next-generation profiling, we showed how the R gene family associated with late blight resistance in the SH*RH population could be identified using a bulked segregant approach.Conclusions
In this study, we provide a comprehensive overview of previously described and novel profiling primers and their genomic targets in potato through genetic mapping and comparative genomics. Furthermore, it is shown how genome-wide or fine mapping can be pursued by choosing different sets of profiling primers. A protocol for next-generation profiling is provided and will form the basis for novel applications. Using the current overview of genomic targets, a rational choice can be made for profiling primers to be employed.2.
Background
In vivo detection of protein-bound genomic regions can be achieved by combining chromatin-immunoprecipitation with next-generation sequencing technology (ChIP-seq). The large amount of sequence data produced by this method needs to be analyzed in a statistically proper and computationally efficient manner. The generation of high copy numbers of DNA fragments as an artifact of the PCR step in ChIP-seq is an important source of bias of this methodology.Results
We present here an R package for the statistical analysis of ChIP-seq experiments. Taking the average size of DNA fragments subjected to sequencing into account, the software calculates single-nucleotide read-enrichment values. After normalization, sample and control are compared using a test based on the ratio test or the Poisson distribution. Test statistic thresholds to control the false discovery rate are obtained through random permutations. Computational efficiency is achieved by implementing the most time-consuming functions in C++ and integrating these in the R package. An analysis of simulated and experimental ChIP-seq data is presented to demonstrate the robustness of our method against PCR-artefacts and its adequate control of the error rate.Conclusions
The software ChIP-seq Analysis in R (CSAR) enables fast and accurate detection of protein-bound genomic regions through the analysis of ChIP-seq experiments. Compared to existing methods, we found that our package shows greater robustness against PCR-artefacts and better control of the error rate. 相似文献3.
4.
Background
Preparation of large quantity and high quality genomic DNA from a large number of plant samples is a major bottleneck for most genetic and genomic analyses, such as, genetic mapping, TILLING (Targeting Induced Local Lesion IN Genome), and next-generation sequencing directly from sheared genomic DNA. A variety of DNA preparation methods and commercial kits are available. However, they are either low throughput, low yield, or costly. Here, we describe a method for high throughput genomic DNA isolation from sorghum [Sorghum bicolor (L.) Moench] leaves and dry seeds with high yield, high quality, and affordable cost.Results
We developed a high throughput DNA isolation method by combining a high yield CTAB extraction method with an improved cleanup procedure based on MagAttract kit. The method yielded large quantity and high quality DNA from both lyophilized sorghum leaves and dry seeds. The DNA yield was improved by nearly 30 fold with 4 times less consumption of MagAttract beads. The method can also be used in other plant species, including cotton leaves and pine needles.Conclusion
A high throughput system for DNA extraction from sorghum leaves and seeds was developed and validated. The main advantages of the method are low cost, high yield, high quality, and high throughput. One person can process two 96-well plates in a working day at a cost of $0.10 per sample of magnetic beads plus other consumables that other methods will also need. 相似文献5.
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. 相似文献6.
Robin A Atherton Bennet J McComish Lara D Shepherd Lorraine A Berry Nick W Albert Peter J Lockhart 《Plant methods》2010,6(1):22
Background
Complete chloroplast genome sequences provide a valuable source of molecular markers for studies in molecular ecology and evolution of plants. To obtain complete genome sequences, recent studies have made use of the polymerase chain reaction to amplify overlapping fragments from conserved gene loci. However, this approach is time consuming and can be more difficult to implement where gene organisation differs among plants. An alternative approach is to first isolate chloroplasts and then use the capacity of high-throughput sequencing to obtain complete genome sequences. We report our findings from studies of the latter approach, which used a simple chloroplast isolation procedure, multiply-primed rolling circle amplification of chloroplast DNA, Illumina Genome Analyzer II sequencing, and de novo assembly of paired-end sequence reads. 相似文献7.
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Background
Deep sequencing is a powerful tool for novel small RNA discovery. Illumina small RNA sequencing library preparation requires a pre-adenylated 3?? end adapter containing a 5??,5??-adenyl pyrophosphoryl moiety. In the absence of ATP, this adapter can be ligated to the 3?? hydroxyl group of small RNA, while RNA self-ligation and concatenation are repressed. Pre-adenylated adapters are one of the most essential and costly components required for library preparation, and few are commercially available.Results
We demonstrate that DNA oligo with 5?? phosphate and 3?? amine groups can be enzymatically adenylated by T4 RNA ligase 1 to generate customized pre-adenylated adapters. We have constructed and sequenced a small RNA library for tomato (Solanum lycopersicum) using the T4 RNA ligase 1 adenylated adapter.Conclusion
We provide an efficient and low-cost method for small RNA sequencing library preparation, which takes two days to complete and costs around $20 per library. This protocol has been tested in several plant species for small RNA sequencing including sweet potato, pepper, watermelon, and cowpea, and could be readily applied to any RNA samples. 相似文献9.
Background
CRISPR-Cas is a recent and powerful addition to the molecular toolbox which allows programmable genome editing. It has been used to modify genes in a wide variety of organisms, but only two alga to date. Here we present a methodology to edit the genome of Thalassiosira pseudonana, a model centric diatom with both ecological significance and high biotechnological potential, using CRISPR-Cas.Results
A single construct was assembled using Golden Gate cloning. Two sgRNAs were used to introduce a precise 37 nt deletion early in the coding region of the urease gene. A high percentage of bi-allelic mutations (≤61.5%) were observed in clones with the CRISPR-Cas construct. Growth of bi-allelic mutants in urea led to a significant reduction in growth rate and cell size compared to growth in nitrate.Conclusions
CRISPR-Cas can precisely and efficiently edit the genome of T. pseudonana. The use of Golden Gate cloning to assemble CRISPR-Cas constructs gives additional flexibility to the CRISPR-Cas method and facilitates modifications to target alternative genes or species.10.
Background
To identify plant genes involved in various key traits, QTL mapping is a powerful approach. This approach is based on the use of mapped molecular markers to identify genomic regions controlling quantitative traits followed by a fine mapping and eventually positional cloning of candidate genes. Mapping technologies using SNP markers are still rather expensive and not feasible in every laboratory. In contrast, microsatellite (also called SSR for Simple Sequence Repeat) markers are technologically less demanding and less costly for any laboratory interested in genetic mapping.Results
In this study, we present the development and the characterization of a panel of 96 highly polymorphic SSR markers along the Arabidopsis thaliana genome allowing QTL mapping among accessions of the Versailles 24 core collection that covers a high percentage of the A. thaliana genetic diversity. These markers can be used for any QTL mapping analysis involving any of these accessions. We optimized the use of these markers in order to reveal polymorphism using standard PCR conditions and agarose gel electrophoresis. In addition, we showed that the use of only three of these markers allows differentiating all 24 accessions which makes this set of markers a powerful tool to control accession identity or any cross between any of these accessions.Conclusion
The set of SSR markers developed in this study provides a simple and efficient tool for any laboratory focusing on QTL mapping in A. thaliana and a simple means to control seed stock or crosses between accessions. 相似文献11.
High Resolution Melt (HRM) analysis is an efficient tool to genotype EMS mutants in complex crop genomes 总被引:1,自引:0,他引:1
Lochlainn SO Amoah S Graham NS Alamer K Rios JJ Kurup S Stoute A Hammond JP Ostergaard L King GJ White PJ Broadley MR 《Plant methods》2011,7(1):43-9
Background
Targeted Induced Loci Lesions IN Genomes (TILLING) is increasingly being used to generate and identify mutations in target genes of crop genomes. TILLING populations of several thousand lines have been generated in a number of crop species including Brassica rapa. Genetic analysis of mutants identified by TILLING requires an efficient, high-throughput and cost effective genotyping method to track the mutations through numerous generations. High resolution melt (HRM) analysis has been used in a number of systems to identify single nucleotide polymorphisms (SNPs) and insertion/deletions (IN/DELs) enabling the genotyping of different types of samples. HRM is ideally suited to high-throughput genotyping of multiple TILLING mutants in complex crop genomes. To date it has been used to identify mutants and genotype single mutations. The aim of this study was to determine if HRM can facilitate downstream analysis of multiple mutant lines identified by TILLING in order to characterise allelic series of EMS induced mutations in target genes across a number of generations in complex crop genomes.Results
We demonstrate that HRM can be used to genotype allelic series of mutations in two genes, BraA.CAX1a and BraA.MET1.a in Brassica rapa. We analysed 12 mutations in BraA.CAX1.a and five in BraA.MET1.a over two generations including a back-cross to the wild-type. Using a commercially available HRM kit and the Lightscanner? system we were able to detect mutations in heterozygous and homozygous states for both genes.Conclusions
Using HRM genotyping on TILLING derived mutants, it is possible to generate an allelic series of mutations within multiple target genes rapidly. Lines suitable for phenotypic analysis can be isolated approximately 8-9 months (3 generations) from receiving M3 seed of Brassica rapa from the RevGenUK TILLING service. 相似文献12.
Background
Rapid improvements in DNA synthesis technology are revolutionizing gene cloning and the characterization of their encoded proteins. Xenopus laevis oocytes are a commonly used heterologous system for the expression and functional characterization of membrane proteins. For many plant proteins, particularly transporters, low levels of expression can limit functional activity in these cells making it difficult to characterize the protein. Improvements in synthetic DNA technology now make it quick, easy and relatively cheap to optimize the codon usage of plant cDNAs for Xenopus. We have tested if this optimization process can improve the functional activity of a two-component plant nitrate transporter assayed in oocytes.Results
We used the generally available software (http://www.kazusa.or.jp/codon/; http://genomes.urv.es/OPTIMIZER/) to predict a DNA sequence for the plant gene that is better suited for Xenopus laevis. Rice OsNAR2.1 and OsNRT2.3a DNA optimized sequences were commercially synthesized for Xenopus expression. The template DNA was used to synthesize cRNA using a commercially available kit. Oocytes were injected with cRNA mixture of optimized and original OsNAR2.1 and OsNRT2.3a. Oocytes injected with cRNA obtained from using the optimized DNA template could accumulate significantly more NO3- than the original genes after 16 h incubation in 0.5 mM Na15NO3. Two-electrode voltage clamp analysis of the oocytes confirmed that the codon optimized template resulted in significantly larger currents when compared with the original rice cDNA.Conclusion
The functional activity of a rice high affinity nitrate transporter in oocytes was improved by DNA codon optimization of the genes. This methodology offers the prospect for improved expression and better subsequent functional characterization of plant proteins in the Xenopus oocyte system.14.
Background
Protein phosphorylation is one of the most important post-translational modifications catalyzed by protein kinases in living organisms. The advance of genome sequencing provided the information of protein kinase families in many organisms, including both model and non-model plants. The development of proteomics technologies also enabled scientists to efficiently reveal a large number of protein phosphorylations of an organism. However, kinases and phosphorylation targets are still to be connected to illustrate the complicated network in life.Results
Here we adapted Pro-Q® Diamond (Pro-Q® Diamond Phosphoprotein Gel Stain), a widely used phosphoprotein gel-staining fluorescence dye, to establish a rapid, economical and non-radioactive fluorescence detection in tube (FDIT) method to analyze phosphorylated proteins. Taking advantages of high sensitivity and specificity of Pro-Q® diamond, the FDIT method is also demonstrated to be rapid and reliable, with a suitable linear range for in vitro protein phosphorylation. A significant and satisfactory protein kinase reaction was detected as fast as 15 min from Wheat Kinase START 1.1 (WKS1.1) on a thylakoid ascorbate peroxidase (tAPX), an established phosphorylation target in our earlier study.Conclusion
The FDIT method saves up to 95% of the dye consumed in a gel staining method. The FDIT method is remarkably quick, highly reproducible, unambiguous and capable to be scaled up to dozens of samples. The FDIT method could serve as a simple and sensitive alternative procedure to determine protein kinase reactions with zero radiation exposure, as a supplementation to other widely used radioactive and in-gel assays.15.
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17.
Jing Wang Chongnan Wang Yan Long Clare Hopkins Smita Kurup Kede Liu Graham J King Jinling Meng 《Plant methods》2011,7(1):1-7
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. 相似文献18.
Javan M. Bauder David R. Breininger M. Rebecca Bolt Michael L. Legare Christopher L. Jenkins Betsie B. Rothermel Kevin McGarigal 《Landscape Ecology》2018,33(5):743-763
Context
Although multi-scale approaches are commonly used to assess wildlife-habitat relationships, few studies have examined selection at multiple spatial scales within different hierarchical levels/orders of selection [sensu Johnson’s (1980) orders of selection]. Failure to account for multi-scale relationships within a single level of selection may lead to misleading inferences and predictions.Objectives
We examined habitat selection of the federally threatened eastern indigo snake (Drymarchon couperi) in peninsular Florida at the level of the home range (Level II selection) and individual telemetry location (Level III selection) to identify influential habitat covariates and predict relative probability of selection.Methods
Within each level, we identified the characteristic scale for each habitat covariate to create multi-scale resource selection functions. We used home range selection functions to model Level II selection and paired logistic regression to model Level III selection.Results
At both levels, EIS selected undeveloped upland land covers and habitat edges while avoiding urban land covers. Selection was generally strongest at the finest scales with the exception of Level II urban edge which was avoided at a broad scale indicating avoidance of urbanized land covers rather than urban edge per se.Conclusions
Our study illustrates how characteristic scales may vary within a single level of selection and demonstrates the utility of multi-level, scale-optimized habitat selection analyses. We emphasize the importance of maintaining large mosaics of natural habitats for eastern indigo snake conservation.19.
Using step and path selection functions for estimating resistance to movement: pumas as a case study
Katherine A. Zeller Kevin McGarigal Samuel A. Cushman Paul Beier T. Winston Vickers Walter M. Boyce 《Landscape Ecology》2016,31(6):1319-1335
Context
GPS telemetry collars and their ability to acquire accurate and consistently frequent locations have increased the use of step selection functions (SSFs) and path selection functions (PathSFs) for studying animal movement and estimating resistance. However, previously published SSFs and PathSFs often do not accommodate multiple scales or multi-scale modeling.Objectives
We present a method that allows multiple scales to be analyzed with SSF and PathSF models. We also explore the sensitivity of model results and resistance surfaces to whether SSFs or PathSFs are used, scale, prediction framework, and GPS collar sampling interval.Methods
We use 5-min GPS collar data from pumas (Puma concolor) in southern California to model SSFs and PathSFs at multiple scales, to predict resistance using two prediction frameworks (paired and unpaired), and to explore potential bias from GPS collar sampling intervals.Results
Regression coefficients were extremely sensitive to scale and pumas exhibited multiple scales of selection during movement. We found PathSFs produced stronger regression coefficients, larger resistance values, and superior model performance than SSFs. We observed more heterogeneous surfaces when resistance was predicted in a paired framework compared with an unpaired framework. Lastly, we observed bias in habitat use and resistance results when using a GPS collar sampling interval longer than 5 min.Conclusions
The methods presented provide a novel way to model multi-scale habitat selection and resistance from movement data. Due to the sensitivity of resistance surfaces to method, scale, and GPS schedule, care should be used when modeling corridors for conservation purposes using these methods.20.