Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 29.500.212
Filtrar
1.
Methods Mol Biol ; 2072: 1-7, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31541433

RESUMEN

Many of the functional genomics methods require isolation of genomic DNA from large population of plants. The selection of DNA isolation protocols depends on several factors such as choice of starting material, ease of handling, time and labor required for isolation, the final quantity as well as the quality of genomic DNA. We outline here a high-throughput method of DNA extraction from different plant species including cereal crops. The protocol can be used for extraction of DNA in single tubes as well as for large formats in 96-well plates. The protocol includes steps for eliminating interfering secondary products such as phenolics. This protocol can be applied for high-throughput isolation of DNA for varied applications such as TILLING, mapping, fingerprinting, etc. as a cost-effective protocol compared to commercial kits.

2.
Methods Mol Biol ; 2072: 9-14, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31541434

RESUMEN

Cereal improvement is based upon effective utilization of genetic resources. These include germplasm and genomics data and tools. Cereal germplasm is available from major global seed banks. Wild material remains an additional less well utilized resource. Sourcing of germplasm requires protocols to ensure intellectual property matters are adequately addressed. Advances in genomics technology have made extensive data set available for the cereals. Reference genome sequences, transcriptome resources, and pan genomes are now available for the major cereal species. The use of genomic data is facilitated by the addition of user-friendly interfaces that allow breeders to access the information they need.

3.
Methods Mol Biol ; 2072: 15-25, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31541435

RESUMEN

Identification of genetic basis for important agronomic traits is essential for marker-assisted crop improvement. Linkage mapping is one of the most popular approaches utilized for identification of major quantitative trait loci (QTLs) governing important agronomic traits in cereals. However, the identified QTLs usually span large genomic intervals and very few of these are subsequently fine mapped to single major effect gene. This hinders application of these QTLs in marker-aided breeding and crop genetic enhancement. On the contrary, association mapping, another popular approach for identification of QTLs, provides very high resolution but suffers from high level of false positives. Joint linkage-association analysis provides a way to combine advantages and avoid the pitfalls associated with both these methods. In this context, we recently developed MetaQTL specific regional association analysis and demonstrated its utility to rapidly narrow down previously identified QTL intervals to few candidate genes. Here, we describe the detailed step-by-step guide for performing MetaQTL specific regional association analysis to identify important genomic regions and underlying potential major effect genes governing traits of agronomic importance in cereals.

4.
Methods Mol Biol ; 2072: 27-37, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31541436

RESUMEN

Next generation sequencing (NGS) and assembly are key pieces in the success of cereal crops genomes sequencing. One interesting strategy for reducing the complexity in large genomes, the case of several cereal crops, is the sequencing of individual chromosomes. This has been done with success by flow cytometric chromosome sorting followed by sequencing using available next generation (high throughput) sequencing platforms. In this chapter, methodologies for sequencing and assembly of flow sorted chromosomes and whole genomes in cereal crops, with special emphasis on the case of the International Wheat Genome Sequencing Consortium (IWGSC), are reviewed.

5.
Methods Mol Biol ; 2072: 39-50, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31541437

RESUMEN

Transposable elements can be highly mutagenic because when they transpose they can insert into genes and disrupt their function, a propensity which has been exploited in many organisms to generate tagged mutant alleles. The Mutator (Mu) family transposon is a family of DNA-type transposons in maize with a particularly high duplication frequency, which results in large numbers of new mutations in lineages that carry active Mu elements. Here we describe a rapid and cost-effective Miseq-based Mu transposon profiling pipeline. This method can also be used for identifying flanking sequences of other types of long insertions such as T-DNAs.

6.
Methods Mol Biol ; 2072: 51-63, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31541438

RESUMEN

Real-time PCR is a powerful technique used for quantification of defined nucleic acid sequences. Numerous applications of this method have been described including: gene expression studies, diagnosis of pathogens, and detection of genetically modified organisms or mutations. Here, we describe a simple and efficient protocol to determine gene expression in cereals, based on real-time PCR using SYBR® Green dye. This technique provide an inexpensive alternative, since no probes are required, allowing for the quantification of a high number of genes with reduced cost.

7.
Methods Mol Biol ; 2072: 65-84, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31541439

RESUMEN

RNA sequencing (RNA-seq) coupled to DNA methylation strategies enables the detection and characterization of genes which expression levels might be mediated by DNA methylation. Here we describe a bioinformatics protocol to analyze gene expression levels using RNA-seq data that allow us to identify candidate genes to be tested by bisulfite assays. The candidate methylated genes are usually those that are low expressed in a particular condition or developmental stage.

8.
Methods Mol Biol ; 2072: 85-99, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31541440

RESUMEN

cis-regulatory DNA elements (CREs) are noncoding but functional DNA sequences. The binding of regulatory proteins into CRE regions leads to chromatin high sensitive to DNase I digestion, which are termed as DNase I hypersensitive sites (DHSs). These DHSs can be efficiently detected through DNase I digestion followed by high-throughput DNA sequencing (DNase-seq). Thus, DNase-seq has become a powerful technique for DHSs mapping at whole-genome level in both plants and animals. Here we describe a DNase-seq procedure modified and developed for crop plants. These plants usually contain large amounts of repetitive sequences and complex organic constituents. With the main improvement in nuclei isolation, this method has been successfully used in mapping DHSs in cotton and sugarcane.

9.
Methods Mol Biol ; 2072: 101-117, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31541441

RESUMEN

Chromatin immunoprecipitation coupled with sequencing (ChIP-seq) is a widely used method for mapping the genome-wide locations of chromatin-associated proteins. This protocol has been developed and utilized to perform ChIP on histone covalent modifications in various plant species including cereals. DNA and chromatin-associated proteins are crosslinked with formaldehyde. Chromatin is then isolated from nuclei and sheared via sonication. Antibodies targeting the histone modification of interest are incubated with the sheared chromatin and nonspecific interactions are washed away. DNA is purified via phenol-chloroform extraction, end-repaired, ligated to sequencing adapters, and PCR-amplified.

10.
Methods Mol Biol ; 2072: 119-128, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31541442

RESUMEN

Whole-genome bisulfite sequencing (WGBS) is a technique used for the analysis of genome-wide DNA methylation patterns (DNA methylomes) at a single-base resolution. Here, I describe a simple DNA extraction method from rice endosperm and the universal protocol of WGBS, MethylC-sequencing library preparation. The use of benzyl chloride allows for the extraction of high-quality genomic DNA from starchy endosperm, while sodium bisulfite converts unmethylated cytosine to uracil, whereas methylated cytosine is unchanged. The bisulfite conversion of whole genome sequencing libraries before the final amplification step allows for the discrimination of methylated from unmethylated cytosines in a genome-wide manner.

11.
Methods Mol Biol ; 2072: 129-139, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31541443

RESUMEN

Upon fertilization, normal endosperm and embryo development require the contribution of both the maternal and paternal genomes. However, certain genes are expressed in a parent-of-origin-dependent manner, an epigenetic phenomenon known as genomic imprinting. Despite the blast of new technologies and the crucial advances of the past decades in the epigenetics field, novel imprinted genes are yet to be discovered and thus key regulators of early seed development. Using rice plant as a model, we describe a method for the identification of imprinted genes based on an RNA-Seq approach, which allows the identification of maternal and paternal gene expression in a parent-of-origin-specific manner.

12.
Methods Mol Biol ; 2072: 141-156, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31541444

RESUMEN

The study of heritable genetic changes that do not implicate alterations in the DNA sequence-epigenetics-represents one of the most prolific and expanding fields in plant biology during the last two decades. With a focus on DNA methylation and histone modifications, recent advances also reported the identification of epigenetic regulatory mechanisms that control reproductive development in cereal crop plants. One of the most powerful methods to selectively study interactions between epigenetic factors or specific proteins bound to genomic DNA regions is called chromatin immunoprecipitation (ChIP). ChIP can be widely used to determine the presence of particular histones with posttranslational modifications at specific genomic regions or whether and where specific DNA-binding proteins including transcription factors interact with candidate target genes. ChIP is also an exciting tool to study and compare chromatin states under normal and stress conditions. Here, we present a detailed step-by-step ChIP assay to investigate epigenetic chromatin marks during vegetative and reproductive development in cereals. However, the method described here can be used for all plant tissues and plant species.

13.
Methods Mol Biol ; 2072: 157-163, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31541445

RESUMEN

Circular RNAs (circRNAs) are a widespread class of endogenous noncoding RNAs and they have been studied in the past few years, implying important biological functions in all kingdoms of life. Recently, circRNAs have been identified in many plant species, including cereal crops, showing differential expression during stress response and developmental programs, which suggests their role in these process. In the following years, it is expected that insights into the functional roles of circRNAs can be used by cereal scientists and molecular breeders with the aim to develop new strategies for crop improvement. Here, we briefly outline the current knowledge about circRNAs in plants and we also outline available computational resources for their validation and analysis in cereal species.

14.
Methods Mol Biol ; 2072: 165-181, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31541446

RESUMEN

Recent advances in genome engineering are revolutionizing crop research and plant breeding. The ability to make specific modifications to a plant's genetic material creates opportunities for rapid development of elite cultivars with desired traits. The plant genome can be altered in several ways, including targeted introduction of nucleotide changes, deleting DNA segments, introducing exogenous DNA fragments and epigenetic modifications. Targeted changes are mediated by sequence specific nucleases (SSNs), such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR (clustered regularly interspersed short palindromic repeats)-Cas (CRISPR associated protein) systems. Recent advances in engineering chimeric Cas nucleases fused to base editing enzymes permit for even greater precision in base editing and control over gene expression. In addition to gene editing technologies, improvement in delivery systems of exogenous DNA into plant cells have increased the rate of successful gene editing events. Regeneration of fertile plants containing the desired edits remains challenging; however, manipulation of embryogenesis-related genes such as BABY BOOM (BBM) has been shown to facilitate regeneration through tissue culture, often a major hurdle in recalcitrant cultivars. Epigenome reprogramming for improved crop performance is another possibility for future breeders, with recent studies on MutS HOMOLOG 1 (MSH1) demonstrating epigenetic-dependent hybrid vigor in several crops. While these technologies offer plant breeders new tools in creating high yielding, better adapted crop varieties, constantly evolving government policy regarding the cultivation of plants containing transgenes may impede the widespread adoption of some of these techniques. This chapter summarizes advances in genome editing tools and discusses the future of these techniques for crop improvement.

15.
Methods Mol Biol ; 2072: 183-198, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31541447

RESUMEN

CRISPR/Cas9-based genome editing technology has the potential to revolutionize agriculture, but many plant species and/or genotypes are recalcitrant to conventional transformation methods. Additionally, the long generation time of crop plants poses a significant obstacle to effective application of gene editing technology, as it takes a long time to produce modified homozygous genotypes. The haploid single-celled microspores are an attractive target for gene editing experiments, as they enable generation of homozygous doubled haploid mutants in one generation. Here, we describe optimized methods for genome editing of haploid wheat microspores and production of doubled haploid plants by microspore culture.

16.
Methods Mol Biol ; 2072: 199-205, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31541448

RESUMEN

Transient expression of inserted recombinant DNA in plant protoplasts is a widely used tool for functional genomics research. Recently it has been utilized to screen potential sgRNA guides for CRISPR-mediated genome editing. However, little research has been conducted into the use of transient expression of protoplasts in Lolium perenne (a globally important pasture, hay, and amenity grass), and no studies have been conducted into Lolium temulentum (a weed in cereal crops but a potentially useful model species for Lolium research). In this chapter, we describe a methodology of protoplast extraction and transformation from 14-day-old leaf mesophyll cells from L. perenne and L. temulentum. We believe this is the first report of a procedure for obtaining high density, viable protoplasts from L. temulentum. The method of polyethylene glycol (PEG)-mediated transformation is also described to achieve genetic transformation of protoplasts.

17.
Methods Mol Biol ; 2072: 207-216, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31541449

RESUMEN

Modification of the rice genome by Agrobacterium-mediated transformation is a general technique that can be easily performed today. Successful methods were established by vigorous studies on the culture system and the elucidation of Agrobacterium transformation mechanisms. This section provides a detailed description of routine and efficient rice transformation protocols by Agrobacterium. This method uses mature seeds as a material and can be applied to many japonica and some other varieties of rice. According to this method, it is even possible for beginners to obtain rice transformants.

18.
Methods Mol Biol ; 2072: 217-240, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31541450

RESUMEN

Phosphoenolpyruvate carboxylases (PEPCs), mostly known as the enzymes responsible for the initial CO2 fixation during C4 photosynthesis, are regulated by reversible phosphorylation in vascular plants. The phosphorylation site on a PEPC molecule is conserved not only among isoforms but also across plant species. An anti-phosphopeptide antibody is a common and powerful tool for detecting phosphorylated target proteins with high specificity. We generated two antibodies, one against a peptide containing a phosphoserine (phosphopeptide) and the other against a peptide containing a phosphoserine mimetic, (S)-2-amino-4-phosphonobutyric acid (phosphonopeptide). The amino acid sequence of the peptide was taken from the site around the phosphorylation site near the N-terminal region of the maize C4-isoform of PEPC. The former antibodies detected almost specifically the phosphorylated C4-isoform of PEPC, whereas the latter antibodies had a broader specificity for the phosphorylated PEPC in various plant species. The following procedures are described herein: (1) preparation of the phosphopeptide and phosphonopeptide; (2) preparation and purification of rabbit antibodies; (3) preparation of cell extracts from leaves for analyses of PEPC phosphorylation with antibodies; and (4) characterization of the obtained antibodies. Finally, (5) two cases involving the application of these antibodies are presented.

19.
J Affect Disord ; 260: 131-139, 2020 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-31494365

RESUMEN

OBJECTIVE: To examine the rate and time to relapse for remitters and responders to ketamine in treatment-resistant depression (TRD). METHODS: Subjects with TRD were randomized to a single infusion of one of several doses of intravenous ketamine, or midazolam. Using Kaplan-Meier survival function, the current report examines the rate and time to relapse, defined as MADRS ≥ 22, over a period of 30 days, in subjects who achieved remission (MADRS ≤ 10) or response (≥ 50% reduction in MADRS) on day three post-infusion of intravenous ketamine 0.1, 0.5, or 1.0 mg/kg. RESULTS: Of the 60 randomized participants who received a single ketamine (0.1, 0.5, or 1.0 mg/kg) infusion, 19 (34%) met criteria for remission and 27 (48%) for response, on day 3 post-infusion. A numerical dose-response relationship was observed, with remitters/responders on ketamine 1.0 mg/kg having the lowest relapse rate, followed by ketamine 0.5 mg/kg and 0.1 mg/kg, respectively (% of remitters who relapsed by day 14: 38% with 1.0 mg/kg, 50% with 0.5 mg/kg, 100% with 0.1 mg/kg;% of responders who relapsed by day 14: 30% with 1.0 mg/kg, 50% with 0.5 mg/kg, 80% with 0.1 mg/kg). LIMITATIONS: The sample size was small. No MADRS measurements at day one post-infusion. The study was not powered to assess differences in relapse prevention between different doses of ketamine. CONCLUSION: Time to relapse after successful treatment with a single infusion of ketamine appears to follow a dose-response relationship, where higher dosage leads to increased time to relapse.

20.
J Affect Disord ; 260: 183-186, 2020 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-31499373

RESUMEN

BACKGROUND: Disturbed sleep is a core symptom of major depressive disorder (MDD), with nearly 90% of those with MDD reporting disturbed sleep. However, combining insomnia and hypersomnia into a single diagnostic domain ignores distinct biological differences between those symptom presentations. To better understand depression it may be necessary to explore these symptoms independently, beginning with the more prevalent insomnia. METHOD: The present study evaluated global insomnia symptom severity in a broad sample of MDD outpatients from the Sequenced Treatment Alternatives to Relieve Depression (STAR*D) trial, excluding patients who reported hypersomnia symptoms. The three insomnia-related symptoms from the 16-item Quick Inventory of Depressive Symptomatology- clinician rated (QIDS-C) were combined to create a global insomnia score to classify baseline insomnia severity. A modified depression severity score was then used to assess depression severity (mQIDS-C), excluding sleep-related items. RESULTS: A repeated measures ANCOVA revealed a significant improvement in insomnia score over the acute phase treatment (F = 33.1, d.f. = 6, 9897, p < 0.0001). Improvement in insomnia score over the acute phase treatment remained statistically significant even after controlling for change in depression severity (p = 0.0004). Participants with one point higher insomnia score at baseline were significantly less likely to remit at study exit (odds ratio = 0.88, 95% confidence interval = 0.85, 0.92, p < 0.0001) even after controlling for baseline depression severity. LIMITATIONS: Objective confirmation of sleep profiles was not available. CONCLUSION: Greater severity of insomnia reduces likelihood of MDD remission, and insomnia symptoms improved independent of depression remission.

SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA