Isolation of genomic DNA from defatted oil seed residue of rapeseed (Brassica napus)

A simple protocol for obtaining pure, restrictable and amplifiable megabase genomic DNA from oil-free seed residue of Brassica napus, an important oil seed plant, has been developed. Oil from the dry seeds was completely recovered in an organic solvent and quantified gravimetrically followed by processing of the residual biomass (defatted seed residue) for genomic DNA isolation. The isolated DNA can be cut by a range of restriction enzymes. The method enables simultaneous isolation and recovery of lipids and genomic DNA from the same test sample, thus allowing two independent analyses from a single sample. Multiple micro-scale oil extraction from the commercial seeds gave approximately 39% oil, which is close to the usual oil recovery from standard oil seed. Most of the amplified fragments were scored in the range of 2.5 to 0.5 kb, best suited for scoring as molecular diagnostics.     

Alternative approach for consistent yields of total genomic DNA from cotton (Gossypium hirsutum L.)

A persistent limitation to molecular biological research on cotton (Gossypium spp.) has been the difficulty in isolation of total genomic DNA from the plant tissue. This report describes a reliable strategy for isolation of genomic DNA from cotton. The mini-preparation procedure involves use of lyophilized, etiolated cotyledons and an anion exchange column kit. The isolated DNA had a molecular weight in excess of 50 kb with minimal degradation or shearing. Routine yields ranged from 5 to 7 μg DNA per etiolated cotyledon pair (corresponding to 100 ng/mg dry weight), in contrast to little or no DNA from equivalent amounts of either green cotyledons or mature leaf tissue. The decreased yields from the latter tissues appeared to be correlated with increased afmounts of flavonoid. The DNA was amenable to routine molecular applications as demonstrated by: digestibility with a number of restriction enzymes (Eco RI,HindIII,Sau 3A), and hybridization of a tomato genomic clone containing the gene for S-adenosylmethionine synthetase to a 13.3-kbEco RI fragment of cotton. Using DNA from an isoline immune to root-knot nematodes, we observed no impediment to genomic cloning.     

Rapid isolation of DNA from Dioscorea species suitable for PCR,restriction digestion and pathogen screening

The methods employed for DNA extraction from many plants is difficult because of the metabolites that interfere with DNA isolation procedures. We have developed a reliable and efficient method for isolating genomic DNA free from polysaccharide, polyphenols and protein contaminants from Dioscorea spp. The method involves inactivation of contaminant proteins by using CTAB/Proteinase K and precipitation of polysaccharides in the presence of high concentration of salt. The purity of genomic DNA was confirmed by A260/280 and A260/230 ratios calculated from the spectrophotometric readings and further by restriction analysis of the isolated DNA using restriction enzymes Eco RI. The total genomic DNA extracted by the new protocol was used for polymerase chain reaction amplification, RAPD analysis, restriction digestion and pathogen screening. The new protocol can be successfully used for both small- and large-scale preparation of genomic DNA from different tissues of Dioscorea spp. The quarantine of seed tubers and use of pathogen-free tubers for planting is a prerequisite for integrated disease management strategy. The protocol can be used for the isolation of genomic DNA from other crop plants too.     

RAPD analysis in flax: Optimization of yield and reproducibility using klenTaq 1 DNA polymerase,chelex 100, and gel purification of genomic DNA

We have developed an optimized RAPD analysis approach using the unusually heat-stable KlenTaq1 DNA polymerase. This enzyme is used in conjunction with a genomic DNA isolation method that includes a modified CTAB DNA isolation protocol, ethanol re-precipitation of resuspended nucleic acids from 2M NaCl, and Chelex 100 treatment. When needed, additional gel purification and isolation of high molecular weight DNA for use as a template in RAPD analysis is shown to remove amplification product ambiguity from within isolates of the same line as well as from between lines. This optimized RAPD analysis was used to define polymorphisms in lines of flax nearly isogenic for rust resistance at theL locus. It should also be useful for any plant species.     

Oleosins in the gametophytes of Pinus and Brassica and their phylogenetic relationship with those in the sporophytes of various species

Oleosins, which are structural proteins on the surface of intracellular oil bodies, have been found in the sporophytic seeds of angiosperms. Here, we report an oleosin from the female gametophyte of gymnosperm Pinus ponderosa Laws, seed and another oleosin from the male gametophyte of Brassica napus L. With the pine seed gametophyte, we identified two putative oleosins of 15 and 10 kDa, which are similar to the oleosins in angiosperm seeds in terms of their presence in the oil bodies in massive quantity. The complete sequence of the cDNA encoding the gametophytic 15-kDa oleosin was obtained, and it has a predicted amino-acid sequence similar to those of oleosins in angiosperm sporophytic seeds. A Brassica napus pollen cDNA sequence, which was reported earlier, would encode an amino-acid sequence somewhat similar to those of seed oleosins. We tested if the dissimilarity signifies a substantially different oleosin in the Brassica male gametophyte or an analytic error. By direct sequencing of a polymerase chain reaction (PCR)-amplified fragment of genomic DNA, we obtained evidence showing that this reported dissimilarity is likely to have arisen from a sequencing error. Our predicted sequence of the Brassica pollen oleosin has all the structural characteristics of seed oleosins. A phylogenic tree of 20 oleosins, including those from sporophytic and gametophytic tissues of angiosperm and gymnosperm, was constructed based on their amino-acid sequences. We discuss the evolution of oleosins, and conclude that oleosins are ancient proteins with multiple lineages whose root cannot be determined at this time.Abbreviations PCR polymerase chain reaction - TAG triacylglycerols This work was supported by USDA grant 91-01439 (AHCH). We thank Dr. Mike Lassner of Calgene, Inc., (Davis, Calif., USA) for providing us with the unpublished jojoba oleosin amino acid sequence.     

A rapid method for extraction of cotton (Gossypium spp.) genomic DNA suitable for RFLP or PCR analysis

Extraction of high-quality genomic DNA fromGossypium (cotton) species is difficult due to high levels of polysaccharide, oxidizable quinones, and other interfering substances. We describe a procedure that consistently permits isolation of cotton genomic DNA of satisfactory size and quality for RFLP and PCR analysis, as well as for most routine cloning applications. Several antioxidants, phenol-binding reagents, and phenol oxidase inhibitors are used throughout the procedure, and most polysaccharides are eliminated early in the procedure by isolation of nuclei.     

Early,non-destructive selection of microspore-derived embryo genotypes in oilseed rape (<Emphasis Type="Italic">Brassica napus</Emphasis> L.) by molecular markers and oil quality analysis

In oilseed rape (Brassica napus L.) breeding, microspore culture is frequently applied for the immediate regeneration of homozygous doubled haploid (DH) plants. From the regenerated microspore-derived embryos (MDEs), usually only a smaller subset of around 200 are used for plantlet regeneration and cultivation in the greenhouse until seed harvest, without there being any knowledge about their quality traits and agronomic performance. The random selection of MDEs implies that valuable rare recombinant genotypes may be discarded at an early stage of in vitro culture. We report here on the development of a simple protocol for simultaneously extracting lipids (for oil quality analysis) and the isolation of DNA (for marker-assisted selection) from single cotyledons dissected from MDEs under aseptic conditions, thus keeping the rest of the embryo in vitro for plantlet regeneration. Neither the fatty acid extraction nor the transmethylation with sodium methylate at high pH interfered with subsequent DNA isolation. The feasibility of the protocol was tested using MDEs from a cross segregating for two linked transgenes, fae1 and plsC, affecting the fatty acid composition. Multiplex PCR was performed with specific PCR primers for the plsC gene and with locus-specific primers for a resident single copy fad2 gene. The amplification of the fad2 gene provided a control for the presence of DNA in sufficient quantity and quality, whereas the amplification of the plsC gene showed a 1:1 segregation expected for a single copy transgene in a segregating DH population. The early identification of the 50% MDE genotypes carrying the desired transgenes, along with a high expression of the trait, allows their early selection for plantlet regeneration.     

The construction and characteristics of a BAC library for <Emphasis Type="Italic">Cucumis sativus</Emphasis> L. ‘B10’

Cloning using bacterial artificial chromosomes (BACs) can yield high quality genomic libraries, which are used for the physical mapping, identification and isolation of genes, and for gene sequencing. A BAC genomic library was constructed from high molecular weight DNA (HMW DNA) obtained from nuclei of the cucumber (Cucumis sativus L. cv. Borszczagowski; B10 line). The DNA was digested with the HindIII restriction enzyme and ligated into the pCC1BAC vector. The library consists of 34,560 BAC clones with an average insert size of 135 kb, and 12.7x genome coverage. Screening the library for chloroplast and mitochondrial DNA content indicated an exceptionally low 0.26% contamination with chloroplast DNA and 0.3% with mitochondrial DNA.     

A rapid method for tissue collection and high-throughput isolation of genomic DNA from mature trees

Collection of tissue and subsequent isolation of genomic DNA from mature tree species often proves difficult. DNA extraction from needles, leaves, or buds is recommended in many protocols. Collecting these tissues from mature trees generally requires the use of firearms or climbing if sampling is to be nondestructive. As a result, sample collection is a major expense of many tree-based projects. Tree (and plant) tissues generally contain large amounts of polysaccharides and phenolic compounds that are difficult to separate from DNA. Many methods aim to overcom these problems, with most involving extraction in buffers containing the nonionic detergent cetyltrimethyl-ammonium bromide (CTAB), followed by numerous steps to clean contaminants from the DNA, using organic solvents and differential salt precipitation. These steps are time-consuming, such that isolation of DNA becomes the bottleneck in many molecular studies. This paper presents a new, efficient, cambium collection method for tree species and a DNA extraction protocol based on that of Doyle and Doyle (1987), with follow-up purification using the Wizard nuclei lysis and protein precipitation solutions (Promega). Results show a significant improvement in yield and DNA purity compared with other published methods, with consistently high yields of pure genomic DNA and high sample throughput. The relatively low cost per extraction, no requirement for use of liquid nitrogen, no requirement for freezer storage, and long-term sample stability after collection are important additional benefits.     

Cloning genomic sequences using long-range PCR

Protocols are presented for preparing DNA from a genomic library in λ phage and for synthesizing genomic fragments using PCR with nested vector- and gene-specific primers and linker-primers. Library DNA, isolated fromE. coli liquid lysates by a simple protocol, is used as template in PCR following a commercial protocol. The method produces library DNA sufficient for several hundred PCRs, incorporates nested primers to reduce nonspecific product formation, and enables the synthesis of linker-containing DNA fragments containing selected restriction sites to simplify subsequent cloning. The isolation of 5′ upstream sequences of three different arabidopsis genes by this methodod is described.     

Two high linolenic mutants of Arabidopsis thaliana contain megabase-scale genome duplications encompassing the FAD3 locus

Understanding the quantitative control of fatty acid desaturation during the biosynthesis of seed storage oil has become a priority area for research, as a consequence of its importance for both human health and the substitution of mineral oil for industrial applications. We have analysed the genome structure of two mutants in Arabidopsis thaliana that show substantially elevated content of the omega‐3 polyunsaturated fatty acid linolenic acid in their seed oil. In one, rfc4, sequences totalling approximately 2 Mb from chromosome 2 have been duplicated and inserted into chromosome 3. In the other mutant, ife, chromosome 2 sequences totalling approximately 1.4 Mb have been duplicated and inserted into a linked position. In both cases, the duplications encompass the FAD3 locus, which encodes the linoleate desaturase responsible for the biosynthesis of linolenic acid for accumulation in seed storage oil. The results show that mutagens such as fast neutrons (used for the induction of rfc4) and T‐DNA (used for the induction of ife, which is not linked to the T‐DNA present in the line) can result in the duplication of very large genome segments. They also show that increasing the dosage of the FAD3‐containing genomic region results in an increase in the linolenic acid content of seed oil. Consequently, screening methods for duplication of FAD3 orthologues in oil crops may be an appropriate approach for the identification of germplasm for breeding varieties with increased proportions of linolenic acid in the oil that they produce.     

Synthesis of high erucic acid rapeseed (Brassica napus L.) somatic hybrids with improved agronomic characters

Novel Brassica napus somatic hybrids have been created through protoplast fusion of B. oleracea var. botrytis and B. rapa var. oleifera genotypes selected for high erucic acid (22:1) content in the seed oil. Fifty amphidiploids (aacc) and one putative hexaploid (aacccc) hybrid were recovered in one fusion experiment. Conversely, only one amphidiploid and numerous regenerates with higher DNA contents were produced in a similar fusion using a different B. rapa partner. Hybridity was confirmed by morphology, isozyme expression, flow cytometry, and DNA hybridization. Analysis of organellar DNA revealed a distinct bias toward the inheritance of chloroplasts from the B. rapa (aa) genome. All amphidiploids set self-pollinated seed. A erucic acid content as high as 57.4% was found in the seed oil of one regenerated plant. Fatty acid composition was stable in the R₁ generation and was coupled with increased female fertility. Other novel agronomic characters in the hybrids recovered include large seed size, lodging resistance, and non-shattering seed pods.     

Identification of quantitative trait loci controlling sucrose content in soybean (Glycine max)

Sucrose is a primary constituent of soybean (Glycine max) seed; however, little information concerning the inheritance of seed sucrose in soybean is available. The objective of this research was to use molecular markers to identify genomic regions significantly associated with quantitative trait loci (QTL) controlling sucrose content in a segregating F₂ population. DNA samples from 149 F₂ individuals were analyzed with 178 polymorphic genetic markers, including RFLPs, SSRs, and RAPDs. Sucrose content was measured on seed harvested from each of 149 F_2:3 lines from replicated field experiments in 1993 and 1995. Seventeen marker loci, mapping to seven different genomic regions, were significantly associated with sucrose variation at P<0.01. Individually, these markers explained from 6.1% to 12.4% of the total phenotypic variation for sucrose content in this population. In a combined analysis these genomic regions; explained 53% of total variation for sucrose content. No significant evidence of epistasis among QTLs was observed. Comparison of our QTL mapping results for sucrose content and those previously reported for protein and oil content (the other major seed constituents in soybean), suggests that seed quality traits are inherited as clusters of linked loci or that `major' QTLs with pleiotropic effects may control all three traits. Of the seven genomic regions having significant effects on sucrose content, three were associated with significant variation for protein content and three were significantly associated with oil content.     

A Novel Approach to Isolation and Functional Characterization of Genomic DNA Sequences from the Methylotrophic Yeast Hansenula polymorpha

A novel approach to isolation and functional characterization of the Hansenula polymorpha genes basing on the use of two strains of different origin is described. One of these strains is better suited for the isolation of genomic DNA fragments, while the other is preferable for their functional analysis. Thirty three genomic sequences governing expression of a reporter protein have been isolated. Analysis of the sequence encoding a homolog of the Saccharomyces cerevisiae cofilin revealed two introns. Another isolated DNA fragment encoded a homolog of the S. cerevisiae Vps10p. Disruption of the corresponding gene resulted in secretion of a vacuolar protein, carboxypeptidase Y, into the culture medium.     

Characterization of a novel satellite DNA sequence from Flying Dragon (Poncirus trifoliata)

Repetitive sequences constitute a significant component of most eukaryotic genomes, and the isolation and characterization of repetitive DNA sequences provide an insight into the organization of the genome of interest. Here, we report the isolation and the molecular analysis and methylation status of a novel tandemly organized repetitive DNA sequence from the genome of Poncirus trifoliata. Digestion of P. trifoliata DNA with Afa I produced a prominent fragment of approximately 400 bp. Southern blotting analysis of genomic DNA digested with the same enzyme revealed a ladder composed of DNA fragments that are multimers of the 400-bp Afa I band, indicating that the repetitive DNA is arrayed in tandem. This suggests that Afa I isolated a novel satellite that we have called Poncirus trifoliata satellite DNA 400 (PN400). This satellite composes 25% of the genome and it is also present in lemon, sour orange and kumquat. Analysis of the methylation status demonstrated that the cytosines in CCGG sequences in this satellite were methylated.     

The Homeobox Gene GLABRA2 Affects Seed Oil Content in Arabidopsis

Despite a good understanding of genes involved in oil biosynthesis in seed, the mechanism(s) that controls oil accumulation is still not known. To identify genes that control oil accumulation in seed, we have developed a simple screening method to isolate Arabidopsis seed oil mutants. The method includes an initial screen for seed density followed by a seed oil screen using an automated Nuclear Magnetic Resonance (NMR). Using this method, we isolated ten low oil mutants and one high oil mutant. The high oil mutant, p777, accumulated 8% more oil in seed than did wild type, but it showed no differences in seed size, plant growth or development. The high-oil phenotype is caused by the disruption of the GLABRA2 gene, a previously identified gene that encodes a homeobox protein required for normal trichome and root hair development. Knockout of GLABRA2 did not affect LEAFY COTYLEDON 1 and PICKLE expression in developing embryo. The result indicates that in addition to its known function in trichome and root hair development, GLABRA2 is involved in the control of seed oil accumulation.     

Isolation of HMW DNA from sunflower (Helianthus annuus L.) for BAC cloning

The cultivated sunflower (Helianthus annuus L.) is one of the most important oil crops in the world. The importance of sunflower oil in human nutrition and in the chemical industry makes the sunflower a major research interest. An essential element for genomic libraries is the preparation of high molecular weight (HMW) DNA. We developed 2 methods for isolating HMW sunflower DNA. We prepared the DNA from nuclei and from protoplasts isolated from mesophyll tissue with the enzymes cellulase RS and pectolyase Y23. The HMW DNA was digested with restriction endonucleases. The ethidium bromide-stained gel suggested the DNA to be completely digested. These results were confirmed by Southern analysis using a radiolabeled RFLP marker. Both methods made it possible to generate sufficient quantities of megabase-size sunflower DNA suitable for bacterial artificial chromosome (BAC) cloning.     

Rapid extraction of genomic DNA from leaves and bracts of dove tree (Davidia involucrata)

Large amounts of polyphenolics in dove tree leaves make it difficult to obtain high-quality genomic DNA during extraction. A rapid DNA minipreparation method was developed for dove tree (Davidia involucrata) and yields 40–50 μg genomic DNA from 0.1 g fresh matured and young leaves and bracts. The yield and quality of the resulting DNA is satisfactory, and the protocol can be scaled up according to sample size. The obtained DNA is suitable for PCR and the restriction enzyme digestion needed for Southern blotting.     

Nuclear DNA content inCelosia (Amaranthaceae)

Nuclear DNA amount of five species ofCelosia ranging from 2x to 12x varies from 3.26 (2x) to 9.70pg (12x). The diploidC. trigyna has twice as much DNA/basic genome as other taxa, which is commensurate with its taxonomic position and genetic isolation. There is insignificant variation in DNA/basic genome among 4x, 8x, and 12x taxa. Therefore, DNA/nucleus shows a strong positive correlation with ploidy level. The different accessions of 4x taxa show constancy of DNA amounts. There is no correlation of seed weight with DNA amount.