Molecular genetic maps are commonly constructed by analyzing the segregation of restriction fragment length polymorphisms (RFLPs) among the progeny of a sexual cross. Here we describe a new DNA polymorphism assay based on the amplification of random DNA segments with single primers of arbitrary nucleotide sequence. These polymorphisms, simply detected as DNA segments which amplify from one parent but not the other, are inherited in a Mendelian fashion and can be used to construct genetic maps in a variety of species. We suggest that these polymorphisms be called RAPD markers, after Random Amplified Polymorphic DNA.

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The 5.67-megabase genome of the plant pathogen Agrobacterium tumefaciens C58 consists of a circular chromosome, a linear chromosome, and two plasmids. Extensive orthology and nucleotide colinearity between the genomes of A. tumefaciens and the plant symbiont Sinorhizobium meliloti suggest a recent evolutionary divergence. Their similarities include metabolic, transport, and regulatory systems that promote survival in the highly competitive rhizosphere; differences are apparent in their genome structure and virulence gene complement. Availability of the A. tumefaciens sequence will facilitate investigations into the molecular basis of pathogenesis and the evolutionary divergence of pathogenic and symbiotic lifestyles.

Central America (1), natural genetic variations in flowering time enabled early Native Americans to select maize adapted to a range of latitudes and lengths of growing seasons, including the very short summer season typical of the eastern Canadian region of Quebec. Under such conditions, early flowering allows seed to mature before the onset of frost. Flowering time is also a key trait of improved drought tolerance. Indeed, it has been shown that a single day of drought during flowering can decrease yield by as much as 8% (2). One way to address such losses is to develop and grow cultivars characterized by a short cycle and able to flower before predictable drought episodes.The genetic variability available for maize breeding is essentially quantitative; i.e., it involves allelic variation at different quantitative trait loci (QTLs), which are influenced by environmental effects. Although a large body of mapping information on QTLs is available for flowering time (3), relatively little is known about the molecular basis of QTLs, with only one gene, Dwarf8, correlated thus far with quantitative effects (4, 5). Furthermore, a few mutants for flowering time have been described (6, 7), two of which, id1 (8) and dlf1 (9), have been cloned. Our results (i) show that the allelic variation responsible for the major flowering-time QTL, Vegetative to generative transition 1 (Vgt1) (10, 11) on chromosome 8, is confined to an Ϸ2-kb intergenic region upstream of an Ap2-like flowering-time gene, (ii) identify maize-sorghum-rice evolutionarily conserved noncoding sequences (CNSs) within Vgt1, and (iii) support a cisacting transcription-regulatory role for Vgt1. ResultsPositional Cloning of Vgt1. Previous work (12) mapped Vgt1 to a 1.3-cM region (Fig. 1A) on bin 8.05, based on a mapping population derived from the cross N28 ϫ C22-4. The strain C22-4 is nearly isogenic to N28 and carries the early Vgt1 allele in an Ϸ7-cM introgression originating from the early maize variety Gaspé Flint. By using standard positional cloning, Vgt1 was confined to an Ϸ2-kb region (Fig. 1 B-D). Sequence annotation of the original BAC clone and the corresponding sequences derived from N28 and Gaspé Flint genetic backgrounds showed that Vgt1 is apparently noncoding and is located Ϸ70 kb (61-76 kb, depending on the genetic background) upstream of an Ap2-like gene identified here as ZmRap2.7. This gene is orthologous to Rap2.7 (also known as TOE1), a transcription factor that regulates flowering time in Arabidopsis (13,14). No other genes were annotated between Vgt1 and ZmRap2.7. Pseudogenes due to transduplication events mediated by nonautonomous helitron elements (15) were observed in N28 and other genetic backgrounds but not in Gaspé Flint (data not shown). Within the Vgt1 region, the contrasting QTL alleles showed 29 SNPs and insertion/deletion-type polymorphisms (Indels) and one 143-bp insertion into the Gaspé Flint allele of a Mite transposon belonging to the Tourist (16) family [ Fig. 4 Lower and supporting information (SI) Fig. 5].Association M...

The maize genome, with its large complement of transposons and repeats, is a paradigm for the study of epigenetic mechanisms such as paramutation and imprinting. Here, we present the genome-wide map of cytosine methylation for two maize inbred lines, B73 and Mo17. CG (65%) and CHG (50%) methylation (where H = A, C, or T) is highest in transposons, while CHH (5%) methylation is likely guided by 24-nt, but not 21-nt, small interfering RNAs (siRNAs). Correlations with methylation patterns suggest that CG methylation in exons (8%) may deter insertion of Mutator transposon insertion, while CHG methylation at splice acceptor sites may inhibit RNA splicing. Using the methylation map as a guide, we used low-coverage sequencing to show that parental methylation differences are inherited by recombinant inbred lines. However, frequent methylation switches, guided by siRNA, persist for up to eight generations, suggesting that epigenetic inheritance resembling paramutation is much more common than previously supposed. The methylation map will provide an invaluable resource for epigenetic studies in maize.

Long terminal repeat (LTR) retrotransposons have been shown to make up much of the maize genome. Although these elements are known to be prevalent in plant genomes of a middle-to-large size, little information is available on the relative proportions composed by specific families of elements in a single genome. We sequenced a library of randomly sheared genomic DNA from maize to characterize this genome. BLAST analysis of these sequences demonstrated that the maize genome is composed of diverse sequences that represent numerous families of retrotransposons. The largest families contain the previously described elements Huck, Ji, and Opie. Approximately 5% of the sequences are predicted to encode proteins. The genomic abundance of 16 families of elements was estimated by hybridization to an array of 10,752 maize bacterial artificial chromosome (BAC) clones. Comparisons of the number of elements present on individual BACs indicated that retrotransposons are in general randomly distributed across the maize genome. A second library was constructed that was selected to contain sequences hypomethylated in the maize genome. Sequence analysis of this library indicated that retroelements abundant in the genome are poorly represented in hypomethylated regions. Fifty-six retroelement sequences corresponding to the integrase and reverse transcriptase domains were isolated from ∼407,000 maize expressed sequence tags (ESTs). Phylogenetic analysis of these and the genomic retroelement sequences indicated that elements most abundant in the genome are less abundant at the transcript level than are more rare retrotransposons. Additional phylogenies also demonstrated that rice and maize retrotransposon families are frequently more closely related to each other than to families within the same species. An analysis of the GC content of the maize genomic library and that of maize ESTs did not support recently published data that the gene space in maize is found within a narrow GC range, but does indicate that genic sequences have a higher GC content than intergenic sequences (52% vs. 47% GC).

The phytohormone abscisic acid (ABA) plays important regulatory roles in many plant developmental processes including seed dormancy, germination,growth, and stomatal movements. These physiological responses to ABA are in large part brought about by changes in gene expression. To study genome-wide ABA-responsive gene expression we applied massively parallel signature sequencing (MPSS) to samples from Arabidopsis thaliana wildtype (WT)and abi1-1 mutant seedlings. We identified 1354 genes that are either up- or downregulated following ABA treatment of WT seedlings. Among these ABA-responsive genes, many encode signal transduction components. In addition,we identified novel ABA-responsive gene families including those encoding ribosomal proteins and proteins involved in regulated proteolysis. In the ABA-insensitive mutant abi1-1, ABA regulation of about 84.5% and 6.9%of the identified genes was impaired or strongly diminished, respectively;however, 8.6% of the genes remained appropriately regulated. Compared to other methods of gene expression analysis, the high sensitivity and specificity of MPSS allowed us to identify a large number of ABA-responsive genes in WT Arabidopsis thaliana. The database given in our supplementary materialprovides researchers with the opportunity to rapidly assess whether genes of interest may be regulated by ABA. Regulation of the majority of the genes by ABA was impaired in the ABA-insensitive mutant abi1-1. However, a subset of genes continued to be appropriately regulated by ABA, which suggests the presence of at least two ABA signaling pathways, only one of which is blocked in abi1-1.

Allelic chromosomal regions totaling more than 2.8 Mb and located on maize (Zea mays) chromosomes 1L, 2S, 7L, and 9S have been sequenced and compared over distances of 100 to 350 kb between the two maize inbred lines Mo17 and B73. The alleles contain extended regions of nonhomology. On average, more than 50% of the compared sequence is noncolinear, mainly because of the insertion of large numbers of long terminal repeat (LTR)-retrotransposons. Only 27 LTR-retroelements are shared between alleles, whereas 62 are allele specific. The insertion of LTR-retrotransposons into the maize genome is statistically more recent for nonshared than shared ones. Most surprisingly, more than one-third of the genes (27/72) are absent in one of the inbreds at the loci examined. Such nonshared genes usually appear to be truncated and form clusters in which they are oriented in the same direction. However, the nonshared genome segments are gene-poor, relative to regions shared by both inbreds, with up to 12-fold difference in gene density. By contrast, miniature inverted terminal repeats (MITEs) occur at a similar frequency in the shared and nonshared fractions. Many times, MITES are present in an identical position in both LTRs of a retroelement, indicating that their insertion occurred before the replication of the retroelement in question. Maize ESTs and/or maize massively parallel signature sequencing tags were identified for the majority of the nonshared genes or homologs of them. In contrast with shared genes, which are usually conserved in gene order and location relative to rice (Oryza sativa), nonshared genes violate the maize colinearity with rice. Based on this, insertion by a yet unknown mechanism, rather than deletion events, seems to be the origin of the nonshared genes. The intergenic space between conserved genes is enlarged up to sixfold in maize compared with rice. Frequently, retroelement insertions create a different sequence environment adjacent to conserved genes.