A simple method for extraction of fungal genomic DNA

We have developed a new, simple and effective method for extraction of fungal genomic DNA. The initial steps involved suspension of freeze-dried mycelium in buffer containing sodium dodecyl sulphate, detachment of DNA from polysaccharides by mild shearing, NaCl precipitation of polysaccharides and protein, chloroform extraction and ethanol precipitation. The ethanol precipitate was then subjected to a second round of mild shearing, NaCl precipitation, chloroform extraction and ethanol precipitation. The procedure required approximately 1 h to perform. The method yielded 8-32 microg of high molecular weight DNA per 30 mg of freeze-dried mycelium when tested on six fungal species: Aspergillus niger, A. flavus, Fusarium graminarum, Neotyphodium lolii, Penicillium citrinum and Rhizopus nigricanes. The DNA was digestible with EcoRI, HindIII, SalI and BamHI. For the slow-growing N. lolii, a modification of the method was developed that removed the agar residue from colonies grown on agar plates by centrifugation at 13 000 rev min(-1) in the presence of CsCl. The modified method yielded 1.5-2 microg of high molecular weight DNA per colony.     

Different genomic relationship matrices for single-step analysis using phenotypic,pedigree and genomic information

Background

The incorporation of genomic coefficients into the numerator relationship matrix allows estimation of breeding values using all phenotypic, pedigree and genomic information simultaneously. In such a single-step procedure, genomic and pedigree-based relationships have to be compatible. As there are many options to create genomic relationships, there is a question of which is optimal and what the effects of deviations from optimality are.

Methods

Data of litter size (total number born per litter) for 338,346 sows were analyzed. Illumina PorcineSNP60 BeadChip genotypes were available for 1,989. Analyses were carried out with the complete data set and with a subset of genotyped animals and three generations pedigree (5,090 animals). A single-trait animal model was used to estimate variance components and breeding values. Genomic relationship matrices were constructed using allele frequencies equal to 0.5 (G05), equal to the average minor allele frequency (GMF), or equal to observed frequencies (GOF). A genomic matrix considering random ascertainment of allele frequencies was also used (GOF*). A normalized matrix (GN) was obtained to have average diagonal coefficients equal to 1. The genomic matrices were combined with the numerator relationship matrix creating H matrices.

Results

In G05 and GMF, both diagonal and off-diagonal elements were on average greater than the pedigree-based coefficients. In GOF and GOF*, the average diagonal elements were smaller than pedigree-based coefficients. The mean of off-diagonal coefficients was zero in GOF and GOF*. Choices of G with average diagonal coefficients different from 1 led to greater estimates of additive variance in the smaller data set. The correlation between EBV and genomic EBV (n = 1,989) were: 0.79 using G05, 0.79 using GMF, 0.78 using GOF, 0.79 using GOF*, and 0.78 using GN. Accuracies calculated by inversion increased with all genomic matrices. The accuracies of genomic-assisted EBV were inflated in all cases except when GN was used.

Conclusions

Parameter estimates may be biased if the genomic relationship coefficients are in a different scale than pedigree-based coefficients. A reasonable scaling may be obtained by using observed allele frequencies and re-scaling the genomic relationship matrix to obtain average diagonal elements of 1.     

A simple method to separate cellulose-binding domains of fungal cellulases after digestion by a protease

Core-binding domains of fungal cellulases from Trichoderma reesei were purified using a new and simple technique. Cellulases were hydrolysed with papain and the binding domains were then separated from the digested mixture by ultrafiltration. The enzymatic digestion process was monitored using capillary electrophoresis. This methodology produced a yield of 85% of binding domains.     

A simple and rapid method for calculating identity-by-descent matrices using multiple markers

A fast, partly recursive deterministic method for calculating Identity-by-Descent (IBD) probabilities was developed with the objective of using IBD in Quantitative Trait Locus (QTL) mapping. The method combined a recursive method for a single marker locus with a method to estimate IBD between sibs using multiple markers. Simulated data was used to compare the deterministic method developed in the present paper with a stochastic method (LOKI) for precision in estimating IBD probabilities and performance in the task of QTL detection with the variance component approach. This comparison was made in a variety of situations by varying family size and degree of polymorphism among marker loci. The following were observed for the deterministic method relative to MCMC: (i) it was an order of magnitude faster; (ii) its estimates of IBD probabilities were found to agree closely, even though it does not extract information when haplotypes are not known with certainty; (iii) the shape of the profile for the QTL test statistic as a function of location was similar, although the magnitude of the test statistic was slightly smaller; and (iv) the estimates of QTL variance was similar. It was concluded that the method proposed provided a rapid means of calculating the IBD matrix with only a small loss in precision, making it an attractive alternative to the use of stochastic MCMC methods. Furthermore, developments in marker technology providing denser maps would enhance the relative advantage of this method.     

A simple and rapid method for preparing genomic DNA from gram-positive bacteria

Molecular epidemiologic and other studies may require preparation of genomic DNA from large numbers of bacteria in sufficiently pure form for restriction endonuclease digestion, cloning, RAPD-PCR, Southern hybridization, and so on.Staphylococcus and other Gram-positive bacteria have a rigid cell wall and can be difficult to lyse. Here, a simple and rapid method for the preparation of genomic DNA from multiple samples is reported. This method produces clean DNA for use in most molecular biology methods in <90 min.     

A simple and sensitive method for quantifying human genomic DNA in forensic specimen extracts

The analysis of DNA restriction fragment length polymorphisms by Southern blot hybridization requires that sufficient quantities of high molecular weight genomic DNA be extracted from biological specimens. Prior to analysis, it is necessary to determine the quantity and quality of the extracted DNA. For many applications, it is also desirable to determine the amount of DNA which is of human origin. In this report, we describe a simple and highly sensitive procedure for the specific quantification of human genomic DNA in forensic extracts or any biological sample. A small fraction of the extract is immobilized onto a nylon membrane and subsequently hybridized to p17H8 (D17Z1), a cloned probe which detects highly repetitive, primate-specific alpha satellite DNA. The procedure requires less than four hours to complete and can be used to quantify subnanogram amounts of hybridizable human genomic DNA.     

A simple method for enhancing hybridization efficiency in chromosome and array comparative genomic hybridization

Abstract

The accuracy of comparative genomic hybridization (CGH) analysis is affected by hybridization efficiency. We describe here a simple method for enhancing hybridization efficiency. The hybridization procedure is essentially the same as that of conventional methods. Hybridization solution containing denatured DNA probe mixture was applied to a metaphase chromosome slide or DNA chip slide and covered with a coverslip. In the new method, however, the slide was inverted by turning the coverslip downward prior to hybridization. We termed this method the inverted slide method. To estimate the efficiency of the new method, metaphase chromosome slides and DNA chip slides were treated by both the conventional and inverted slide methods and incubated in a moist chamber at 37°C for 12, 24, 48, and 72 h. Hybridization signals were approximately 1.5 to 2 times brighter on the slides using the inverted slide method than those using the conventional method after 48 and 72 h of incubation. Furthermore, topographical differences in fluorescence intensity were smaller in slides using the inverted-slide method than in those prepared by the conventional method. The inverted slide method is methodologically very simple and improves the resolution of CGH.     

Long-term response to genomic selection: effects of estimation method and reference population structure for different genetic architectures

Background

Genomic selection has become an important tool in the genetic improvement of animals and plants. The objective of this study was to investigate the impacts of breeding value estimation method, reference population structure, and trait genetic architecture, on long-term response to genomic selection without updating marker effects.

Methods

Three methods were used to estimate genomic breeding values: a BLUP method with relationships estimated from genome-wide markers (GBLUP), a Bayesian method, and a partial least squares regression method (PLSR). A shallow (individuals from one generation) or deep reference population (individuals from five generations) was used with each method. The effects of the different selection approaches were compared under four different genetic architectures for the trait under selection. Selection was based on one of the three genomic breeding values, on pedigree BLUP breeding values, or performed at random. Selection continued for ten generations.

Results

Differences in long-term selection response were small. For a genetic architecture with a very small number of three to four quantitative trait loci (QTL), the Bayesian method achieved a response that was 0.05 to 0.1 genetic standard deviation higher than other methods in generation 10. For genetic architectures with approximately 30 to 300 QTL, PLSR (shallow reference) or GBLUP (deep reference) had an average advantage of 0.2 genetic standard deviation over the Bayesian method in generation 10. GBLUP resulted in 0.6% and 0.9% less inbreeding than PLSR and BM and on average a one third smaller reduction of genetic variance. Responses in early generations were greater with the shallow reference population while long-term response was not affected by reference population structure.

Conclusions

The ranking of estimation methods was different with than without selection. Under selection, applying GBLUP led to lower inbreeding and a smaller reduction of genetic variance while a similar response to selection was achieved. The reference population structure had a limited effect on long-term accuracy and response. Use of a shallow reference population, most closely related to the selection candidates, gave early benefits while in later generations, when marker effects were not updated, the estimation of marker effects based on a deeper reference population did not pay off.     

Single-step genomic prediction of Eucalyptus dunnii using different identity-by-descent and identity-by-state relationship matrices

Genomic selection based on the single-step genomic best linear unbiased prediction (ssGBLUP) approach is becoming an important tool in forest tree breeding. The quality of the variance components and the predictive ability of the estimated breeding values (GEBV) depends on how well marker-based genomic relationships describe the actual genetic relationships at unobserved causal loci. We investigated the performance of GEBV obtained when fitting models with genomic covariance matrices based on two identity-by-descent (IBD) and two identity-by-state (IBS) relationship measures. Multiple-trait multiple-site ssGBLUP models were fitted to diameter and stem straightness in five open-pollinated progeny trials of Eucalyptus dunnii, genotyped using the EUChip60K. We also fitted the conventional ABLUP model with a pedigree-based covariance matrix. Estimated relationships from the IBD estimators displayed consistently lower standard deviations than those from the IBS approaches. Although ssGBLUP based in IBS estimators resulted in higher trait-site heritabilities, the gain in accuracy of the relationships using IBD estimators has resulted in higher predictive ability and lower bias of GEBV, especially for low-heritability trait-site. ssGBLUP based on IBS and IBD approaches performed considerably better than the traditional ABLUP. In summary, our results advocate the use of the ssGBLUP approach jointly with the IBD relationship matrix in open-pollinated forest tree evaluation.Subject terms: Plant breeding, Genomics     

A simple method for detecting single base substitutions and its application to HLA-DPB1 typing.

We have developed a simple and reliable method, PCR-PHFA (polymerase chain reaction dependent preferential homoduplex formation assay), for detection of single base substitutions within PCR amplicons. This technique is based upon strand competition during hybridization between a double labeled amplicon, prepared from biotin and DNP labeled primers, and an unlabeled amplicon. Under the precisely controlled temperature gradient, the preferential formation of a homoduplex over a heteroduplex occurs. After annealing, the identical sequence of the double labeled and unlabeled amplicon resulted in a low population of regenerated double labeled dsDNA due to strand exchange between them. Even when the two differed by only a single base substitution, double labeled molecule was regenerated efficiently because of preferential homoduplex formation. The regenerated double labeled molecule was captured onto a streptavidin coated microtiter plate and quantified enzymatically with a chromogenic substrate. The technique has been successfully applied in HLA-DPB1 typing. Furthermore, we detected a mutated gene even in the presence of a large excess of the corresponding normal gene.     

A simple method for genomic selection of moderately sized dairy cattle populations

An efficient algorithm for genomic selection of moderately sized populations based on single nucleotide polymorphism chip technology is described. A total of 995 Israeli Holstein bulls with genetic evaluations based on daughter records were genotyped for either the BovineSNP50 BeadChip or the BovineSNP50 v2 BeadChip. Milk, fat, protein, somatic cell score, female fertility, milk production persistency and herd-life were analyzed. The 400 markers with the greatest effects on each trait were first selected based on individual analysis of each marker with the genetic evaluations of the bulls as the dependent variable. The effects of all 400 markers were estimated jointly using a 'cow model,' estimated from the data truncated to exclude lactations with freshening dates after September 2006. Genotype probabilities for each locus were computed for all animals with missing genotypes. In Method I, genetic evaluations were computed by analysis of the truncated data set with the sum of the marker effects subtracted from each record. Genomic estimated breeding values for the young bulls with genotypes, but without daughter records, were then computed as their parent averages combined with the sum of each animal's marker effects. Method II genomic breeding values were computed based on regressions of estimated breeding values of bulls with daughter record on their parent averages, sum of marker effects and birth year. Method II correlations of the current breeding values of young bulls without daughter records in the truncated data set were higher than the correlations of the current breeding values with the parent averages for fat and protein production, persistency and herd-life. Bias of evaluations, estimated as a difference between the mean of current breeding values of the young bulls and their genomic evaluations, was reduced for milk production traits, persistency and herd-life. Bias for milk production traits was slightly negative, as opposed to the positive bias of parent averages. Correlations of Method II with the means of daughter records adjusted for fixed effects were higher than parent averages for fat, protein, fertility, persistency and herd-life. Reducing the number of markers included in the analysis from 400 to 300 did not reduce correlations of genomic breeding values for protein with current breeding values, but did slightly reduce correlations with means of daughter records. Method II has the advantages as compared with the method of VanRaden in that genotypes of cows can be readily incorporated into the Method II analysis, and it is more effective for moderately sized populations.     

Rapid amplification of genomic ends (RAGE) as a simple method to clone flanking genomic DNA

This report describes the amplification of upstream genomic sequences using the polymerase chain reaction (PCR) based solely on downstream DNA information from a cDNA clone. In this novel and rapid technique, genomic DNA (gDNA) is first incubated with a restriction enzyme that recognizes a site within the 5′ end of a gene, followed by denaturation and polyadenylation of its free 3′ ends with terminal transferase. The modified gDNA is then used as template for PCR using a gene-specific primer complementary to a sequence in the 3′ end of its cDNA and an anchored deoxyoligothymidine primer. A second round of PCR is then performed with a second, nested gene-specific primer and the anchor sequence primer. The resulting PCR product is cloned and its sequence determined. Three independent plant genomic clones were isolated using this method that exhibited complete sequence identity to their cDNAs and to the primers used in the amplification.     

A simple enzymic method to separate [3H]inositol 1,4,5- and 1,3,4-trisphosphate isomers in tissue extracts.

A novel method to separate [3H]Ins(1,4,5)P3 and [3H]Ins(1,3,4)P3 in tissue extracts is described. It is based on the selective metabolism of Ins(1,3,4)P3 by a crude cerebral supernatant in a Mg2+-free buffer followed by separation of [3H]inositol trisphosphates using conventional anion-exchange chromatography. Evaluation of the assay was performed using [3H]Ins(1,3,4)P3 standards and tissue extracts containing different proportions of [3H]Ins(1,4,5)P3 and [3H]Ins(1,3,4)P3. Parallel h.p.l.c. separations of extracts established the selective and complete metabolism of [3H]Ins(1,3,4)P3 under the above conditions and demonstrated that the enzymic method provides an accurate estimate of the trisphosphate isomers in rat cerebral cortex, parotid gland and bovine tracheal smooth muscle.     

A simple and economic preservation method for genomic bacterial DNA from clinically significant pathogens

Bacterial culture was allowed to dry to completeness on Columbia agar base with defibrinated horse blood. Following 6 months storage at room temperature, microbial DNA was extracted and successfully amplified by PCR. This storage technique has the advantage over other methods of not requiring (i) a DNA extraction protocol prior to storage and (ii) refrigeration and/or freezing. This technique maybe useful in the transportation of bacterial genomic DNA in nonviable cells as well as reliable method for the storage of DNA in underdeveloped countries.     

A simple method to enrich an Agrobacterium-transformed population for plants containing only T-DNA sequences

A simple modification to standard binary vector design has been utilized to enrich an Agrobacterium-transformed population for plants containing only T-DNA sequences. A lethal gene was incorporated into the non-T-DNA portion of a binary vector, along with a screenable marker. The resulting class of vectors is designated as NTL T-DNA vectors (non-T-DNA lethal gene-containing T-DNA vectors). The lethal gene used here is a CaMV 35S-barnase gene with an intron in the coding sequence (barnase-INT); the screenable marker is a pMAS-luciferase gene with an intron in the coding sequence (LUC-int). To evaluate the utility of this vector design, tobacco plants were transformed with either the NTL T-DNA vector or a control vector from which most of the barnase-INT gene was deleted. Populations of 50 transgenic plants were scored for LUC expression. The results indicated a dramatic reduction in the presence of non-T-DNA sequences in the transgenic population using the NTL T-DNA vector. Only one transgenic plant was found to be LUC+ using the NTL vector, compared with 42 of 50 plants using the control vector. Importantly, the efficiency with which transformed tobacco plants was obtained was reduced by no more than 30%. The reduction in LUC+ transgenics was partially reversed when a barstar-expressing tobacco line was transformed, indicating that barnase expression was responsible for the reduced frequency of incorporating non-T-DNA sequences. Similar transformation results were obtained with tomato and grape. The incorporation of a barnase-INT gene outside the left border appears to provide a generally applicable tool for enriching an Agrobacterium-transformed population for plants containing only T-DNA sequences.     

A simple in situ method for estimating fungal population size in the rumen

Sediment and the overlying water samples were collected from 67 fresh and 38 salt water sites in the UK. Only 13% (five) of the samples taken from salt water sites were negative for magnetotactic bacteria compared with 47·7% (32) of the fresh water ones. Of the seven types of salt water habitats examined, saltmarsh ponds were found to be most suited to magnetotactic bacteria, 96% (25/26) of sites being positive.     

A simple method for generating full length cDNA from low abundance partial genomic clones

Background  

PCR amplification of target molecules involves sequence specific primers that flank the region to be amplified. While this technique is generally routine, its applicability may not be sufficient to generate a desired target molecule from two separate regions involving intron /exon boundaries. For these situations, the generation of full-length complementary DNAs from two partial genomic clones becomes necessary for the family of low abundance genes.     

A simple method of genomic DNA extraction suitable for analysis of bulk fungal strains

Aims: A simple and rapid method (designated thermolysis) for extracting genomic DNA from bulk fungal strains was described. Methods and Results: In the thermolysis method, a few mycelia or yeast cells were first rinsed with pure water to remove potential PCR inhibitors and then incubated in a lysis buffer at 85°C to break down cell walls and membranes. This method was used to extract genomic DNA from large numbers of fungal strains (more than 92 species, 35 genera of three phyla) isolated from different sections of natural Ophiocordyceps sinensis specimens. Regions of interest from high as well as single‐copy number genes were successfully amplified from the extracted DNA samples. The DNA samples obtained by this method can be stored at ?20°C for over 1 year. Conclusions: The method was effective, easy and fast and allowed batch DNA extraction from multiple fungal isolates. Significance and Impact of Study: Use of the thermolysis method will allow researchers to obtain DNA from fungi quickly for use in molecular assays. This method requires only minute quantities of starting material and is suitable for diverse fungal species.