SUMMARYWe explored genetic variation by sequencing a selection of 84 tomato accessions and related wild species representative of the Lycopersicon, Arcanum, Eriopersicon and Neolycopersicon groups, which has yielded a huge amount of precious data on sequence diversity in the tomato clade. Three new reference genomes were reconstructed to support our comparative genome analyses. Comparative sequence alignment revealed group-, species-and accession-specific polymorphisms, explaining characteristic fruit traits and growth habits in the various cultivars. Using gene models from the annotated Heinz 1706 reference genome, we observed differences in the ratio between non-synonymous and synonymous SNPs (dN/dS) in fruit diversification and plant growth genes compared to a random set of genes, indicating positive selection and differences in selection pressure between crop accessions and wild species. In wild species, the number of single-nucleotide polymorphisms (SNPs) exceeds 10 million, i.e. 20-fold higher than found in most of the crop accessions, indicating dramatic genetic erosion of crop and heirloom tomatoes. In addition, the highest levels of heterozygosity were found for allogamous self-incompatible wild species, while facultative and autogamous self-compatible species display a lower heterozygosity level. Using whole-genome SNP information for maximum-likelihood analysis, we achieved complete tree resolution, whereas maximum-likelihood trees based on SNPs from ten fruit and growth genes show incomplete resolution for the crop accessions, partly due to the effect of heterozygous SNPs. Finally, results suggest that phylogenetic relationships are correlated with habitat, indicating the occurrence of geographical races within these groups, which is of practical importance for Solanum genome evolution studies.

The ability of the nitrogen-fixing bacterial endophyte Acetobacter diazotrophicus strain PAl5 to enhance the growth of sugarcane SP70-1143 was evaluated in the growth chamber, greenhouse, and field by comparing plants inoculated with wild-type and Nif mutant MAd3A in two independent experiments. The wild-type and Nif mutant strains colonized sugarcane plants equally and persisted in mature plants. In N-deficient conditions, sugarcane plants inoculated with A. diazotrophicus PAl5 generally grew better and had a higher total N content 60 days after planting than did plants inoculated with mutant MAd3A or uninoculated plants. These results indicate that the transfer of fixed N from A. diazotrophicus to sugarcane might be a significant mechanism for plant growth promotion in this association. When N was not limiting, growth enhancement was observed in plants inoculated with either wild-type or Nif- mutants, suggesting the additional effect of a plant growth promoting factor provided by A. diazotrophicus. A 15N2 incorporation experiment demonstrated that A. diazotrophicus wild-type strains actively fixed N2 inside sugarcane plants, whereas the Nif- mutants did not.

A major 30.5-kb cluster of nif and associated genes of Acetobacter diazotrophicus (syn. Gluconacetobacter diazotrophicus), a nitrogen-fixing endophyte of sugarcane, was sequenced and analyzed. This cluster represents the largest assembly of contiguous nif-fix and associated genes so far characterized in any diazotrophic bacterial species. Northern blots and promoter sequence analysis indicated that the genes are organized into eight transcriptional units. The overall arrangement of genes is most like that of the nif-fix cluster in Azospirillum brasilense, while the individual gene products are more similar to those in species of Rhizobiaceae or in Rhodobacter capsulatus.Biological nitrogen fixation occurs in species of more than 100 genera distributed among several of the major phylogenetic divisions of prokaryotes (Eubacteria and Archaea) (25). Sequence and mutational analyses of the genes necessary for nitrogen fixation (nif) in many diazotrophs indicate that their products have common structures and functions, while the degree of linkage and arrangement of specific nif and associated genes vary considerably (5,8,17). In addition, nif genes and genes involved in plant invasion and nitrogen fixation effectiveness, such as nod and fix in species of Rhizobiaceae, are often linked.The identification of nitrogen-fixing bacteria with endophytic habitats raises the possibility of a new classification of symbiosis (3). The relationship of a proteobacterial ␣ group member, Acetobacter diazotrophicus (syn. Gluconacetobacter diazotrophicus), with sugarcane represents a promising model system for the study of an association between a monocot and an endophytic nitrogen-fixing bacterium (13,22). The ability of A. diazotrophicus to enhance sugarcane growth has been documented, and while the benefit to plant growth might be due at least in part to the transfer of bacterially fixed N, another plant growth-stimulating factor(s) is indicated, possibly auxin production by A. diazotrophicus (22). Because of its potential agronomic use and unique status as the only diazotrophic species of Acetobacter so far identified, it was of interest to isolate and characterize genes that are involved in nitrogen fixation and regulation.Identification of a major cluster of nif and associated genes. A genomic library of A. diazotrophicus constructed in the widehost-range cosmid pLAFR3 (Tet r ) (4) was transferred by conjugation from Escherichia coli to several different nif mutants of Azotobacter vinelandii (12). Two mutants that yielded Nif ϩ Tet r transconjugants were DJ71 (nifV) and DJ35 (nifE), and several transconjugants of both strains carried the same cosmid, pAD71, the 22-kb insert fragment of which carries nifD through mcpA (Fig.

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