The cereal endosperm is a major organ of the seed and an important component of the world's food supply. To understand the development and physiology of the endosperm of cereal seeds, we focused on the identification of genes expressed at various times during maize endosperm development. We constructed several cDNA libraries to identify full-length clones and subjected them to a twofold enrichment. A total of 23,348 high-quality sequence-reads from 5'- and 3'-ends of cDNAs were generated and assembled into a unigene set representing 5326 genes with paired sequence-reads. Additional sequencing yielded a total of 3160 (59%) completely sequenced, full-length cDNAs. From 5326 unigenes, 4139 (78%) can be aligned with 5367 predicted rice genes and by taking only the "best hit" be mapped to 3108 positions on the rice genome. The 22% unigenes not present in rice indicate a rapid change of gene content between rice and maize in only 50 million years. Differences in rice and maize gene numbers also suggest that maize has lost a large number of duplicated genes following tetraploidization. The larger number of gene copies in rice suggests that as many as 30% of its genes arose from gene amplification, which would extrapolate to a significant proportion of the estimated 44,027 candidate genes of its entire genome. Functional classification of the maize endosperm unigene set indicated that more than a fourth of the novel functionally assignable genes found in this study are involved in carbohydrate metabolism, consistent with its role as a storage organ.

Different maize inbred lines are polymorphic for the presence or absence of genic sequences at various allelic chromosomal locations. In the bz genomic region, located in 9S, sequences homologous to four different genes from rice and Arabidopsis are present in line McC but absent from line B73. It is shown here that this apparent intraspecific violation of genetic colinearity arises from the movement of genes or gene fragments by Helitrons, a recently discovered class of eukaryotic transposons. Two Helitrons, HelA and HelB, account for all of the genic differences distinguishing the two bz locus haplotypes. HelA is 5.9 kb long and contains sequences for three of the four genes found only in the McC bz genomic region. A nearly identical copy of HelA was isolated from a 5S chromosomal location in B73. Both the 9S and 5S sites appear to be polymorphic in maize, suggesting that these Helitrons have been active recently. Helitrons lack the strong predictive terminal features of other transposons, so the definition of their ends is greatly facilitated by the identification of their vacant sites in Helitron-minus lines. The ends of the 2.7-kb HelB Helitron were discerned from a comparison of the McC haplotype sequence with that of yet a third line, Mo17, because the HelB vacant site is deleted in B73. Maize Helitrons resemble rice Pack-MULEs in their ability to capture genes or gene fragments from several loci and move them around the genome, features that confer on them a potential role in gene evolution.

Maize (Zea mays) has a large class of seed mutants with opaque or nonvitreous endosperms that could improve the nutritional quality of our food supply. The phenotype of some of them appears to be linked to the improper formation of protein bodies (PBs) where zein storage proteins are deposited. Although a number of genes affecting endosperm vitreousness have been isolated, it has been difficult to clone opaque7 (o7), mainly because of its low penetrance in many genetic backgrounds. The o7-reference (o7-ref) mutant arose spontaneously in a W22 inbred, but is poorly expressed in other lines. We report here the isolation of o7 with a combination of map-based cloning and transposon tagging. We first identified an o7 candidate gene by map-based cloning. The putative o7-ref allele has a 12-bp in-frame deletion of codons 350-353 in a 528-codon-long acyl-CoA synthetase-like gene (ACS). We then confirmed this candidate gene by generating another mutant allele from a transposon-tagging experiment using the Activator/Dissociation (Ac/Ds) system in a W22 background. The second allele, isolated from approximately 1 million gametes, presented a 2-kb Ds insertion that resembles the single Ds component of double-Ds, McClintock's original Dissociation element, at codon 496 of the ACS gene. PBs exhibited striking membrane invaginations in the o7-ref allele and a severe number reduction in the Ds-insertion mutant, respectively. We propose a model in which the ACS enzyme plays a key role in membrane biogenesis, by taking part in protein acylation, and that altered PBs render the seed nonvitreous.

With a size approximating that of the human genome, the maize genome is about to become the largest plant genome yet sequenced. Contributing to that size are a whole-genome duplication event and a retrotransposition explosion that produced a large amount of repetitive DNA. This DNA is greatly under-represented in cDNA collections, so analysis of the maize transcriptome has been an expedient way of assessing the gene content of maize. Over 2 million maize cDNA sequences are now available, making maize the third most widely studied organism, behind mouse and man. To date, the sequencing of large-sized DNA clones has been largely driven by the genetic interests of different investigators. The recent construction of a physical map that is anchored to the genetic map will aid immensely in the maize genome-sequencing effort. However, studies showing that the repetitive DNA component is highly polymorphic among maize inbred lines point to the need to sample vertically a few specific regions of the genome to evaluate the extent and importance of this variability.

Domesticated maize and its wild ancestor (teosinte) differ strikingly in morphology and afford an opportunity to examine the connection between strong selection and diversity in a major crop species. The tb1 gene largely controls the increase in apical dominance in maize relative to teosinte, and a region of the tb1 locus 5' to the transcript sequence was a target of selection during maize domestication. To better characterize the impact of selection at a major "domestication" locus, we have sequenced the upstream tb1 genomic region and systematically sampled nucleotide diversity for sites located as far as 163 kb upstream to tb1. Our analyses define a selective sweep of approximately 60-90 kb 5' to the tb1 transcribed sequence. The selected region harbors a mixture of unique sequences and large repetitive elements, but it contains no predicted genes. Diversity at the nearest 5' gene to tb1 is typical of that for neutral maize loci, indicating that selection at tb1 has had a minimal impact on the surrounding chromosomal region. Our data also show low intergenic linkage disequilibrium in the region and suggest that selection has had a minor role in shaping the pattern of linkage disequilibrium that is observed. Finally, our data raise the possibility that maize-like tb1 haplotypes are present in extant teosinte populations, and our findings also suggest a model of tb1 gene regulation that differs from traditional views of how plant gene expression is controlled.

Maize (Zea mays L.) is one of the most important cereal crops and a model for the study of genetics, evolution, and domestication. To better understand maize genome organization and to build a framework for genome sequencing, we constructed a sequence-ready fingerprinted contig-based physical map that covers 93.5% of the genome, of which 86.1% is aligned to the genetic map. The fingerprinted contig map contains 25,908 genic markers that enabled us to align nearly 73% of the anchored maize genome to the rice genome. The distribution pattern of expressed sequence tags correlates to that of recombination. In collinear regions, 1 kb in rice corresponds to an average of 3.2 kb in maize, yet maize has a 6-fold genome size expansion. This can be explained by the fact that most rice regions correspond to two regions in maize as a result of its recent polyploid origin. Inversions account for the majority of chromosome structural variations during subsequent maize diploidization. We also find clear evidence of ancient genome duplication predating the divergence of the progenitors of maize and rice. Reconstructing the paleoethnobotany of the maize genome indicates that the progenitors of modern maize contained ten chromosomes.

BACKGROUND: Retrotransposons are commonly occurring eukaryotic transposable elements (TEs). Among these, long terminal repeat (LTR) retrotransposons are the most abundant TEs and can comprise 50-90% of the genome in higher plants. By comparing the orthologous chromosomal regions of closely related species, the effects of TEs on the evolution of plant genomes can be studied in detail. RESULTS: Here, we compared the composition and organization of TEs within five orthologous chromosomal regions among three grass species: maize, sorghum, and rice. We identified a total of 132 full or fragmented LTR retrotransposons in these regions. As a percentage of the total cumulative sequence in each species, LTR retrotransposons occupy 45.1% of the maize, 21.1% of the rice, and 3.7% of the sorghum regions. The most common elements in the maize retrotransposon-rich regions are the copia-like retrotransposons with 39% and the gypsy-like retrotransposons with 37%. Using the contiguous sequence of the orthologous regions, we detected 108 retrotransposons with intact target duplication sites and both LTR termini. Here, we show that 74% of these elements inserted into their host genome less than 1 million years ago and that many retroelements expanded in size by the insertion of other sequences. These inserts were predominantly other retroelements, however, several of them were also fragmented genes. Unforeseen was the finding of intact genes embedded within LTR retrotransposons. CONCLUSION: Although the abundance of retroelements between maize and rice is consistent with their different genome sizes of 2,364 and 389 Mb respectively, the content of retrotransposons in sorghum (790 Mb) is surprisingly low. In all three species, retrotransposition is a very recent activity relative to their speciation. While it was known that genes re-insert into non-orthologous positions of plant genomes, they appear to re-insert also within retrotransposons, potentially providing an important role for retrotransposons in the evolution of gene function.

Sorghum, an African grass related to sugar cane and maize, is grown for food, feed, fibre and fuel. We present an initial analysis of the approximately 730-megabase Sorghum bicolor (L.) Moench genome, placing approximately 98% of genes in their chromosomal context using whole-genome shotgun sequence validated by genetic, physical and syntenic information. Genetic recombination is largely confined to about one-third of the sorghum genome with gene order and density similar to those of rice. Retrotransposon accumulation in recombinationally recalcitrant heterochromatin explains the approximately 75% larger genome size of sorghum compared with rice. Although gene and repetitive DNA distributions have been preserved since palaeopolyploidization approximately 70 million years ago, most duplicated gene sets lost one member before the sorghum-rice divergence. Concerted evolution makes one duplicated chromosomal segment appear to be only a few million years old. About 24% of genes are grass-specific and 7% are sorghum-specific. Recent gene and microRNA duplications may contribute to sorghum's drought tolerance.

Maize (Zea mays or corn), both a major food source and an important cytogenetic model, evolved from a tetraploid that arose about 4.8 million years ago (Mya). As a result, maize has extensive duplicated regions within its genome. We have sequenced the two copies of one such region, generating 7.8 Mb of sequence spanning 17.4 cM of the short arm of chromosome 1 and 6.6 Mb (25.6 cM) from the long arm of chromosome 9. Rice, which did not undergo a similar whole genome duplication event, has only one orthologous region (4.9 Mb) on the short arm of chromosome 3, and can be used as reference for the maize homoeologous regions. Alignment of the three regions allowed identification of syntenic blocks, and indicated that the maize regions have undergone differential contraction in genic and intergenic regions and expansion by the insertion of retrotransposable elements. Approximately 9% of the predicted genes in each duplicated region are completely missing in the rice genome, and almost 20% have moved to other genomic locations. Predicted genes within these regions tend to be larger in maize than in rice, primarily because of the presence of predicted genes in maize with larger introns. Interestingly, the general gene methylation patterns in the maize homoeologous regions do not appear to have changed with contraction or expansion of their chromosomes. In addition, no differences in methylation of single genes and tandemly repeated gene copies have been detected. These results, therefore, provide new insights into the diploidization of polyploid species.

Two instances of genetic transmission of spontaneous epimutation of the maize P-rr gene were identified. Transmission gave rise to two similar, moderately stable alleles, designated P-pr-1 and P-pr-2, that exhibited Mendelian behavior. Both isolates of P-pr conditioned a variable and variegated phenotype, unlike the uniform pigmentation conditioned by P-rr. Extensive genomic analysis failed to reveal insertions, deletions or restriction site polymorphisms between the new allele and its progenitor. However, methylation of the P gene was increased in P-pr relative to P-rr, and was greatly reduced (though not lost) in a revertant to uniform pigmentation. Variability in pigmentation conditioned by P-pr correlated with variability in transcript levels of the P gene, and both correlated inversely with variability in its methylation. Part of the variability in methylation could be accounted for by a developmental decrease in methylation in all tissues of plants carrying P-pr. We hypothesize that the variegated phenotype results from a general epigenetic pathway which causes a progressive decrease in methylation and increase in expression potential of the P gene as a function of cell divisions in each meristem of the plant. This renders all tissues chimeric for a functional gene; chimerism is visualized as variegation only in pericarp due to the tissue specificity of P gene expression. Therefore, this allele that originates from epimutation may exemplify an epigenetic mechanism for variegation in maize.