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

We have developed a DNA extraction procedure for milligram amounts of plant tissue. Yields ranged from 0.3-200 nanograms of DNA per milligram of tissue. The factors affecting yield are discussed. Fresh tissue, as well as herbarium specimens (22-118 years old) and mummified seeds and embryos (500 to greater than 44 600 years old) were used. All tissues attempted (57 types from 29 species) yielded measurable amounts of DNA. In no case tested was inhibition observed for restriction enzymes BamHI or EcoRI.

Extraction of DNA from milligram amounts of fresh, herbarium and mummified plant tissues

The number of angiosperm species for which nuclear DNA amount estimates have been made has nearly trebled since the last collected lists of such values were published, and therefore, publication of a more comprehensive list is overdue. This paper lists absolute nuclear DNA amounts for 753 angiosperm species. The data were assembled primarily for reference purposes, and so the species are listed in alphabetical order, as this was felt to be more helpful to cyto- and biochemists whom, it is anticipated, will be among its major users. The paper also reviews aspects of the history, nomenclature, methods, accuracy and problems of nuclear DNA estimation in angiosperms. No attempt is made to reconsider those aspects of nuclear DNA estimation which have been fully revised previously, although the bibliography of such aspects is given. Instead, the paper is intended as a source of basic information regarding the terminology, practice and limitations of nuclear DNA estimation, especially by Feulgen microdensitometry, as currently practiced.

Nuclear DNA Amounts in Angiosperms

Plant cell walls are the starting materials for many commercial products, from lumber, paper and textiles to thickeners, films and explosives. The cell wall is secreted by each cell in the plant body, forming a thin fibreglass-like network with remarkable strength and flexibility. During growth, plant cells secrete a protein called expansin, which unlocks the network of wall polysaccharides, permitting turgor-driven cell enlargement. Germinating grass pollen also secretes an unusual expansin that loosens maternal cell walls to aid penetration of the stigma by the pollen tube. Expansin's action has puzzling implications for plant cell-wall structure. The recent explosion of gene sequences and expression data has given new hints of additional biological functions for expansins.

Loosening of plant cell walls by expansins

Genome sequences reveal that a deluge of DNA from organelles has constantly been bombarding the nucleus since the origin of organelles. Recent experiments have shown that DNA is transferred from organelles to the nucleus at frequencies that were previously unimaginable. Endosymbiotic gene transfer is a ubiquitous, continuing and natural process that pervades nuclear DNA dynamics. This relentless influx of organelle DNA has abolished organelle autonomy and increased nuclear complexity.

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Endosymbiotic Gene Transfer: Organelle Genomes Forge Eukaryotic Chromosomes

Gene transfer from the chloroplast to the nucleus has occurred over evolutionary time1. Functional gene establishment in the nucleus is rare, but DNA transfer without functionality is presumably more frequent. Here, we measured directly the transfer rate of chloroplast DNA (cpDNA) into the nucleus of tobacco plants (Nicotiana tabacum). To visualize this process, a nucleus-specific neomycin phosphotransferase gene (neoSTLS2) was integrated into the chloroplast genome, and the transfer of cpDNA to the nucleus was detected by screening for kanamycin-resistant seedlings in progeny. A screen for kanamycin-resistant seedlings was conducted with about 250,000 progeny produced by fertilization of wild-type females with pollen from plants containing cp-neoSTLS2. Sixteen plants of independent origin were identified and their progenies showed stable inheritance of neoSTLS2, characteristic of nuclear genes. Thus, we provide a quantitative estimate of one transposition event in about 16,000 pollen grains for the frequency of transfer of cpDNA to the nucleus. In addition to its evident role in organellar evolution, transposition of cpDNA to the nucleus in tobacco occurs at a rate that must have significant consequences for existing nuclear genes.

Direct measurement of the transfer rate of chloroplast DNA into the nucleus

The buoyant density of ribosomal DNA is similar in species with or without satellite DNA, and in all species examined was distinguishable from that of the satellite DNA. In melon tissues (Cucumus melo) the percentage satellite DNA is not correlated with the percentage hybridization to ribosomal RNA. Satellite DNA sequences do not appear to be dispersed between those coding for ribosomal RNA. There is no correlation between the presence of satellite DNA and high ribosomal RNA gene redundancy, but there is a correlation between satellite DNA and small genome size, which results in a correlation between satellite DNA and a high percentage hybridization to ribosomal RNA. Satellite DNAs are defined as minor components after CsCI centrifugation.

The Relationship between Satellite Deoxyribonucleic Acid, Ribosomal Ribonucleic Acid Gene Redundancy, and Genome Size in Plants.

During evolution, the genomes of eukaryotic cells have undergone major restructuring to meet the new regulatory challenges associated with compartmentalization of the genetic material in the nucleus and the organelles acquired by endosymbiosis (mitochondria and plastids). Restructuring involved the loss of dispensable or redundant genes and the massive translocation of genes from the ancestral organelles to the nucleus. Genomics and bioinformatic data suggest that the process of DNA transfer from organelles to the nucleus still continues, providing raw material for evolutionary tinkering in the nuclear genome. Recent reconstruction of these events in the laboratory has provided a unique tool to observe genome evolution in real time and to study the molecular mechanisms by which plastid genes are converted into functional nuclear genes. Here, we summarize current knowledge about plastid-to-nuclear gene transfer in the context of genome evolution and discuss new insights gained from experiments that recapitulate endosymbiotic gene transfer in the laboratory.

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Reconstructing evolution: gene transfer from plastids to the nucleus.

Relatively few chloroplast (ct) or mitochondrial (mt) proteins are encoded and synthesized within the organelles, the majority being made on cytoplasmic ribosomes from messenger RNAs transcribed from nuclear genes1,2. How this interaction of genetic systems evolved is poorly understood. Whether the chloroplast or mitochondrial specific genes now residing in the nucleus arose de novo, or by genetic exchange between the nucleus and a symbiotic autonomous prokaryote, is an intriguing unanswered question, although that such genetic exchange is possible is strongly suggested by recent reports of homology between maize mitochondrial and chloroplast genomes3 and the presence of mitochondrial sequences in yeast nuclei4. We now present evidence that the spinach nucleus contains integrated sequences that are homologous to ctDNA sequences and that these sequences are incorporated at specific sites within the genome. The experiments also show a region of homology between ctDNA and mtDNA.

Jeremy N. Timmis

Papers

Endosymbiotic Gene Transfer: Organelle Genomes Forge Eukaryotic Chromosomes

Direct measurement of the transfer rate of chloroplast DNA into the nucleus

The Relationship between Satellite Deoxyribonucleic Acid, Ribosomal Ribonucleic Acid Gene Redundancy, and Genome Size in Plants.

Reconstructing evolution: gene transfer from plastids to the nucleus.

Sequence homology between spinach nuclear and chloroplast genomes