A method is presented for the rapid isolation of high molecular weight plant DNA (50,000 base pairs or more in length) which is free of contaminants which interfere with complete digestion by restriction endonucleases. The procedure yields total cellular DNA (i.e. nuclear, chloroplast, and mitochondrial DNA). The technique is ideal for the rapid isolation of small amounts of DNA from many different species and is also useful for large scale isolations.

Rapid isolation of high molecular weight plant DNA

Spacer-length (sl) variation in ribosomal RNA gene clusters (rDNA) was surveyed in 502 individual barley plants, including samples from 50 accessions of cultivated barley, 25 accessions of its wild ancestor, and five generations of composite cross II (CCII), an experimental population of barley. In total, 17 rDNA sl phenotypes, made up of 15 different rDNA sl variants, were observed. The 15 rDNA sl variants comprise a complete ladder in which each variant differs in length from adjacent variants by approximately equal to 115 nucleotide pairs. Studies of four rDNA sl variants in an F2 population showed that these variants are located at two unlinked loci, Rrn1 and Rrn2, each with two codominant alleles. Using wheat-barley addition lines, we determined that Rrn1 and Rrn2 are located on chromosomes 6 and 7, respectively. The nonrandom distribution of sl variants between loci suggests that genetic exchange occurs much less frequently between than within the two loci, which demonstrates that Rrn1 and Rrn2 are useful as new genetic markers. Frequencies of rDNA sl phenotypes and variants were monitored over 54 generations in CCII. A phenotype that was originally infrequent in CCII ultimately became predominant, whereas the originally most frequent phenotype decreased drastically in frequency, and all other phenotypes originally present disappeared from the population. We conclude that the sl variants and/or associated loci are under selection in CCII.

https://www.pnas.org/content/pnas/81/24/8014.full.pdf

Ribosomal DNA spacer-length polymorphisms in barley: mendelian inheritance, chromosomal location, and population dynamics.

Tomato (Solanum lycopersicum) is a major crop plant and a model system for fruit development. Solanum is one of the largest angiosperm genera1 and includes annual and perennial plants from diverse habitats. Here we present a high-quality genome sequence of domesticated tomato, a draft sequence of its closest wild relative, Solanum pimpinellifolium2, and compare them to each other and to the potato genome (Solanum tuberosum). The two tomato genomes show only 0.6% nucleotide divergence and signs of recent admixture, but show more than 8% divergence from potato, with nine large and several smaller inversions. In contrast to Arabidopsis, but similar to soybean, tomato and potato small RNAs map predominantly to gene-rich chromosomal regions, including gene promoters. The Solanum lineage has experienced two consecutive genome triplications: one that is ancient and shared with rosids, and a more recent one. These triplications set the stage for the neofunctionalization of genes controlling fruit characteristics, such as colour and fleshiness.

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The tomato genome sequence provides insights into fleshy fruit evolution

A very simple, fast, universally applicable and reproducible method to extract high quality megabase genomic DNA from different organisms is described. We applied the same method to extract high quality complex genomic DNA from different tissues (wheat, barley, potato, beans, pear and almond leaves as well as fungi, insects and shrimps' fresh tissue) without any modification. The method does not require expensive and environmentally hazardous reagents and equipment. It can be performed even in low technology laboratories. The amount of tissue required by this method is approximately 50-100 mg. The quantity and the quality of the DNA extracted by this method is high enough to perform hundreds of PCR-based reactions and also to be used in other DNA manipulation techniques such as restriction digestion, Southern blot and cloning.

Universal and rapid salt-extraction of high quality genomic DNA for PCR-based techniques

The Wealth of India. Raw Materials

The reassociation kinetics of pea (Pisum sativum L.) DNA fragments (300 nucleotides) were measured with hydroxylapatite. The most slowly reassociating fragments do so with a rate constant of 2 X 10(-4) L mol-1s-1, as determined from experiments with total DNA as well as with a tracer enriched for slowly renaturing sequences. This rate is about 1000 times slower than that observed for Escherichia coli DNA included as an internal kinetic standard, indicating a kinetic complexity of 4.5 X 10(9) nucleotide pairs or 4.6 pg of DNA per haploid nucleus. This estimate is in good agreement with previous chemical and cytophotometric measurements. The majority (85%) of the 300 nucleotide fragments contain repetitive sequences. While the reassociation of repetitive DNA could be modeled with two theoretical second-order components, the data did not specify a unique solution. The reassociation kinetics of isolated high- and low-frequency fractions indicate that repetitive sequence families in pea DNA probably cover a broad range of frequencies ranging from 100 to 10 000 or more copies per haploid genome. Single-copy sequences account for about 30% of the DNA, but because of extensive interpersion of repetitive sequences only about 15% of 300 nucleotide fragments reassociate with single-copy kinetics. From studies of hydroxylapatite binding as a function of fragment length, we conclude that the major class of single-copy sequences has a modal length of about 300 nucleotides. Long tracer reassociation kinetics indicate that sequences with an apparent repetition frequency of about 10 000 copies are interspersed at intervals of less than 1300 nucleotides throughout 75% of the genome. At a detection limit of about 3%, we find no single-copy sequences longer than 1000 nucleotides.

DNA sequence organization in the pea genome.

Essentially all of the sequences in the pea (Pisum sativum) genome which reassociate with single copy kinetics at standard (Tm -25°C) criterion follow repetitive kinetics at lower temperatures (about Tm-35°C). Analysis of thermal stability profiles for presumptive single copy duplexes show that they contain substantial mismatch even when formed at standard criterion. Thus most of the sequences in the pea genome which are conventionally defined as “single copy” are actually “fossil repeats” — that is, they are members of extensively diverged (mutuated) and thus presumably ancient families of repeated sequences. Coding sequences as represented by a cDNA probe prepared from poly-somal poly(A) + mRNA reassociate with single copy kinetics regardless of criterion and do not form mismatched duplexes. The coding regions thus appear to be composed of true single copy sequences but they cannot represent more than a few percent of the pea genome. Ancient diverged repeats are present, but not a prominent feature of the smaller mung bean (Vigna radiata) genome. An extension of a simple evolutionary model is proposed in which these and other differences in genome organization are considered to reflect different rates of sequence amplification or genome turnover during evolution. The model accounts for some of the differences between typical plant and animal genomes.

Ancient repeated sequences in the pea and mung bean genomes and implications for genome evolution

We have investigated the DNase I sensitivity of transcriptionally active DNA sequences in intact nuclei and isolated chromatin from embryos of wheat (Triticum aestivum L.). Nuclei or isolated chromatin was incubated with DNase I, and the extent of DNA digestion was monitored as percentage acid solubility. The resistant DNA and DNA from sham-digested controls were used to drive reassociation reactions with cDNA populations corresponding to either total poly(A)+RNA from unimbibed wheat embryos or polysomal poly(A)+RNA from embryos that had imbibed for 3 hr. Sequences complementary to either probe were depleted in DNase I-resistant DNA from nuclei and from chromatin isolated under low-ionic-strength conditions. This indicates that transcriptionally active sequences are preferentially DNase I sensitive in plants. In chromatin isolated at higher ionic strength, cDNA complementary sequences were not preferentially depleted by DNase I treatment. Therefore, the chromatin structure that confers preferential DNase I sensitivity to transcriptionally active genes appears to be lost when the higher-ionic-strength method of preparation is used. Treatment of wheat nuclei with DNase I causes the release of four prominent nonhistone chromosomal proteins that comigrate with wheat high mobility group proteins on NaDodSO4 gels.

https://www.pnas.org/content/pnas/80/3/815.full.pdf

Michael G. Murray

Papers

Rapid isolation of high molecular weight plant DNA

DNA sequence organization in the pea genome.

Ancient repeated sequences in the pea and mung bean genomes and implications for genome evolution

DNase I sensitivity of transcriptionally active genes in intact nuclei and isolated chromatin of plants

Altered chromatin conformation of the higher plant gene phaseolin