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Essential amino acids like lysine and tryptophan are deficient in corn meal because of the abundance of zein storage proteins that lack these amino acids. A natural mutant, opaque 2 ( o2 ) causes reduction of zeins, an increase of nonzein proteins, and as a consequence, a doubling of lysine levels. However, o2 ’s soft inferior kernels precluded its commercial use. Breeders subsequently overcame kernel softness, selecting several quantitative loci (QTLs), called o2 modifiers, without losing the high-lysine trait. These maize lines are known as “quality protein maize” (QPM). One of the QTLs is linked to the 27-kDa γ-zein locus on chromosome 7S. Moreover, QPM lines have 2- to 3-fold higher levels of the 27-kDa γ-zein, but the physiological significance of this increase is not known. Because the 27- and 16-kDa γ-zein genes are highly conserved in DNA sequence, we introduced a dominant RNAi transgene into a QPM line (CM105 Mo2 ) to eliminate expression of them both. Elimination of γ-zeins disrupts endosperm modification by o2 modifiers, indicating their hypostatic action to γ-zeins. Abnormalities in protein body structure and their interaction with starch granules in the F1 with Mo2 /+; o2 / o2 ; γRNAi/+ genotype suggests that γ-zeins are essential for restoring protein body density and starch grain interaction in QPM. To eliminate pleiotropic effects caused by o2 , the 22-kDa α-zein, γ-zein, and β-zein RNAis were stacked, resulting in protein bodies forming as honeycomb-like structures. We are unique in presenting clear demonstration that γ-zeins play a mechanistic role in QPM, providing a previously unexplored rationale for molecular breeding.

Opaque or nonvitreous phenotypes relate to the seed architecture of maize (Zea mays) and are linked to loci that control the accumulation and proper deposition of storage proteins, called zeins, into specialized organelles in the endosperm, called protein bodies. However, in the absence of null mutants of each type of zein (i.e. alpha, beta, gamma, and delta), the molecular contribution of these proteins to seed architecture remains unclear. Here, a double null mutant for the delta-zeins, the 22-kD alpha-zein, the beta-zein, and the gamma-zein RNA interference (RNAi; designated as z1CRNAi, betaRNAi, and gammaRNAi, respectively) and their combinations have been examined. While the delta-zein double null mutant had negligible effects on protein body formation, the betaRNAi and gammaRNAi alone only cause slight changes. Substantial loss of the 22-kD alpha-zeins by z1CRNAi resulted in protein body budding structures, indicating that a sufficient amount of the 22-kD zeins is necessary for maintenance of a normal protein body shape. Among different mutant combinations, only the combined betaRNAi and gammaRNAi resulted in drastic morphological changes, while other combinations did not. Overexpression of alpha-kafirins, the homologues of the maize 22-kD alpha-zeins in sorghum (Sorghum bicolor), in the beta/gammaRNAi mutant failed to offset the morphological alterations, indicating that beta- and gamma-zeins have redundant and unique functions in the stabilization of protein bodies. Indeed, opacity of the beta/gammaRNAi mutant was caused by incomplete embedding of the starch granules rather than by reducing the vitreous zone.

Using an Affymetrix sugarcane genechip, we previously identified 154 genes differentially expressed between grain and sweet sorghum. Although many of these genes have functions related to sugar and cell wall metabolism, dissection of the trait requires genetic analysis. Therefore, it would be advantageous to use microarray data for generation of genetic markers, shown in other species as single-feature polymorphisms (SFPs). As a test case, we used the GeSNP software to screen for SFPs between grain and sweet sorghum. Based on this screen, out of 58 candidate genes, 30 had single-nucleotide polymorphisms (SNPs) from which 19 had validated SFPs. The degree of nucleotide polymorphism found between grain and sweet sorghum was in the order of one SNP per 248 base pairs, with chromosome 8 being highly polymorphic. Indeed, molecular markers could be developed for a third of the candidate genes, giving us a high rate of return by this method.

If one considers the Gordon Conference in Plant Genetics in 1997 as the origin of the international effort to sequence the rice ( Oryza sativa ) genome and the publication of the data in 2005 as the completion of production and analysis, it took about 8 years and 14 major laboratories from nine

Comparative analyses of the sequence of entire genomes have shown that gene duplications, chromosomal segmental duplications, or even whole genome duplications (WGD) have played prominent roles in the evolution of many eukaryotic species. Here, we used the ancient duplication of a well known transcription factor in maize, encoded by the Opaque-2 ( O2 ) locus, to examine the general features of divergences of chromosomal segmental duplications in a lineage-specific manner. We took advantage of contiguous chromosomal sequence information in rice ( Oryza sativa Nipponbare), sorghum ( Sorghum bicolor Btx623), and maize ( Zea mays B73) that were aligned by conserved gene order (synteny). This analysis showed that the maize O2 locus is contained within a 1.25 million base-pair (Mb) segment on chromosome 7, which was duplicated ≈56 million years ago (mya) before the split of rice and maize 50 mya. The duplicated region on chromosome 1 is only half the size and contains the maize OHP gene, which does not restore the o2 mutation although it encodes a protein with the same DNA and protein binding properties in endosperm. The segmental duplication is not only found in rice, but also in sorghum, which split from maize 11.9 mya. A detailed analysis of the duplicated regions provided examples for complex rearrangements including deletions, duplications, conversions, inversions, and translocations. Furthermore, the rice and sorghum genomes appeared to be more stable than the maize genome, probably because maize underwent allotetraploidization and then diploidization.

The Rice Annotation Project Database (RAP-DB) was created to provide the genome sequence assembly of the International Rice Genome Sequencing Project (IRGSP), manually curated annotation of the sequence, and other genomics information that could be useful for comprehensive understanding of the rice biology. Since the last publication of the RAP-DB, the IRGSP genome has been revised and reassembled. In addition, a large number of rice-expressed sequence tags have been released, and functional genomics resources have been produced worldwide. Thus, we have thoroughly updated our genome annotation by manual curation of all the functional descriptions of rice genes. The latest version of the RAP-DB contains a variety of annotation data as follows: clone positions, structures and functions of 31 439 genes validated by cDNAs, RNA genes detected by massively parallel signature sequencing (MPSS) technology and sequence similarity, flanking sequences of mutant lines, transposable elements, etc. Other annotation data such as Gnomon can be displayed along with those of RAP for comparison. We have also developed a new keyword search system to allow the user to access useful information. The RAP-DB is available at: http://rapdb.dna.affrc.go.jp/ and http://rapdb.lab.nig.ac.jp/.

Gene expression is regulated at many different levels during the life cycle of all plant species. Recent investigations have taken advantage of next-generation sequencing to study the relevance of DNA methylation and sRNAs in controlling tissue-specific gene expression in maize at the genome-wide level. Here, we profiled H3K27ac in maize, which has one of the largest sequenced plant genomes due to the amplification of retrotransposons. Because transcribed genes represent only a small proportion of its genome, gene-specific epigenetic modifications are concentrated in a relatively small percentage of the genome. Indeed, H3K27ac marks are mostly in gene-rich, in contrast to gene-poor regions. A large proportion of those marks are located in transcribed regions of genes, including 111 out of 458 known genetic loci. Moreover, increased transcription correlates with the presence of H3K27ac modification in gene bodies. Using maize as an example, we suggest that H3K27ac marks actively transcribed genes in plants.

Maize (Zea mays or corn) plays many varied and important roles in society. It is not only an important experimental model plant, but also a major livestock feed crop and a significant source of industrial products such as sweeteners and ethanol. In this study we report the systematic analysis of contiguous sequences of the maize genome. We selected 100 random regions averaging 144 kb in size, representing about 0.6% of the genome, and generated a high-quality dataset for sequence analysis. This sampling contains 330 annotated genes, 91% of which are supported by expressed sequence tag data from maize and other cereal species. Genes averaged 4 kb in size with five exons, although the largest was over 59 kb with 31 exons. Gene density varied over a wide range from 0.5 to 10.7 genes per 100 kb and genes did not appear to cluster significantly. The total repetitive element content we observed (66%) was slightly higher than previous whole-genome estimates (58%–63%) and consisted almost exclusively of retroelements. The vast majority of genes can be aligned to at least one sequence read derived from gene-enrichment procedures, but only about 30% are fully covered. Our results indicate that much of the increase in genome size of maize relative to rice (Oryza sativa) and Arabidopsis (Arabidopsis thaliana) is attributable to an increase in number of both repetitive elements and genes.

Rice, one of the world's most important food plants, has important syntenic relationships with the other cereal species and is a model plant for the grasses. Here we present a map-based, finished quality sequence that covers 95% of the 389 Mb genome, including virtually all of the euchromatin and two complete centromeres. A total of 37,544 non-transposable-element-related protein-coding genes were identified, of which 71% had a putative homologue in Arabidopsis. In a reciprocal analysis, 90% of the Arabidopsis proteins had a putative homologue in the predicted rice proteome. Twenty-nine per cent of the 37,544 predicted genes appear in clustered gene families. The number and classes of transposable elements found in the rice genome are consistent with the expansion of syntenic regions in the maize and sorghum genomes. We find evidence for widespread and recurrent gene transfer from the organelles to the nuclear chromosomes. The map-based sequence has proven useful for the identification of genes underlying agronomic traits. The additional single-nucleotide polymorphisms and simple sequence repeats identified in our study should accelerate improvements in rice production.

It is generally believed that maize (Zea maysL. ssp.mays) arose as a tetraploid; however, the two progenitor genomes cannot be unequivocally traced within the genome of modern maize. We have taken a new approach to investigate the origin of the maize genome. We isolated and sequenced large genomic fragments from the regions surrounding five duplicated loci from the maize genome and their orthologous loci in sorghum, and then we compared these sequences with the orthologous regions in the rice genome. Within the studied segments, we identified 11 genes that were conserved in location, order, and orientation. We performed phylogenetic and distance analyses and examined the patterns of estimated times of divergence for sorghum and maize gene orthologs and also the time of divergence for maize orthologs. Our results support a tetraploid origin of maize. This analysis also indicates contemporaneous divergence of the ancestral sorghum genome and the two maize progenitor genomes about 11.9 million years ago (Mya). On the basis of a putative conversion event detected for one of the genes, tetraploidization must have occurred before 4.8 Mya, and therefore, preceded the major maize genome expansion by gene amplification and retrotransposition.

PubMed ID: 15466290, Maize (Zea mays L. ssp. mays), one of the most important agricultural crops in the world, originated by hybridization of two closely related progenitors. To investigate the fate of its genes after tetraploidization, we analyzed the sequence of five duplicated regions from different chromosomal locations. We also compared corresponding regions from sorghum and rice, two important crops that have largely collinear maps with maize. The split of sorghum and maize progenitors was recently estimated to be 11.9 Mya, whereas rice diverged from the common ancestor of maize and sorghum ~50 Mya. A data set of roughly 4 Mb yielded 206 predicted genes from the three species, excluding any transposon-related genes, but including eight gene remnants. On average, 14% of the genes within the aligned regions are noncollinear between any two species. However, scoring each maize region separately, the set of noncollinear genes between all four regions jumps to 68%. This is largely because at least 50% of the duplicated genes from the two progenitors of maize have been lost over a very short period of time, possibly as short as 5 million years. Using the nearly completed rice sequence, we found noncollinear genes in other chromosomal positions, frequently in more than one. This demonstrates that many genes in these species have moved to new chromosomal locations in the last 50 million years or less, most as single gene events that did not dramatically alter gene structure. © 2004 by Cold Spring Harbor Laboratory Press.

Zea mays L. ssp. mays , or corn, one of the most important crops and a model for plant genetics, has a genome ≈80% the size of the human genome. To gain global insight into the organization of its genome, we have sequenced the ends of large insert clones, yielding a cumulative length of one-eighth of the genome with a DNA sequence read every 6.2 kb, thereby describing a large percentage of the genes and transposable elements of maize in an unbiased approach. Based on the accumulative 307 Mb of sequence, repeat sequences occupy 58% and genic regions occupy 7.5%. A conservative estimate predicts ≈59,000 genes, which is higher than in any other organism sequenced so far. Because the sequences are derived from bacterial artificial chromosome clones, which are ordered in overlapping bins, tagged genes are also ordered along continuous chromosomal segments. Based on this positional information, roughly one-third of the genes appear to consist of tandemly arrayed gene families. Although the ancestor of maize arose by tetraploidization, fewer than half of the genes appear to be present in two orthologous copies, indicating that the maize genome has undergone significant gene loss since the duplication event.

The DNA sequence of 106 BAC/PAC clones in the minimum tiling path (MTP) of the long arm of rice chromosome 11, between map positions 57.3 and 116.2 cM, has been assembled to phase 2 or PLN level. This region has been sequenced to 10x redundancy by the Indian Initiative for Rice Genome Sequencing (IIRGS) and is now publicly available in GenBank. The region, excluding overlaps, has been predicted to contain 2,932 genes using different software. A gene-by-gene BLASTN search of the NCBI wheat EST database of over 420,000 cDNA sequences revealed that 1,143 of the predicted rice genes (38.9%) have significant homology to wheat ESTs (bit score >/= 100). Further BLASTN search of these 1,143 rice genes with the GrainGenes database of sequence contigs containing bin-mapped wheat ESTs allowed 113 of the genes to be placed in bins located on wheat chromosomes of different homoeologous groups. The largest number of genes, about one-third, mapped to the homoeologous group 4 chromosomes of wheat, suggesting a common evolutionary origin. The remaining genes were located on wheat chromosomes of different groups with significantly higher numbers for groups 3 and 5. Location of bin-mapped wheat contigs to chromosomes of all the seven homoeologous groups can be ascribed to movement of genes (transpositions) or chromosome segments (translocations) within rice or the hexaploid wheat genomes. Alternatively, it could be due to ancient duplications in the common ancestral genome of wheat and rice followed by selective elimination of genes in the wheat and rice genomes. While there exists definite conservation of gene sequences and the ancestral chromosomal identity between rice and wheat, there is no obvious conservation of the gene order at this level of resolution. Lack of extensive colinearity between rice and wheat genomes suggests that there have been many insertions, deletions, duplications and translocations that make the synteny comparisons much more complicated than earlier thought. However, enhanced resolution of comparative sequence analysis may reveal smaller conserved regions of colinearity, which will facilitate selection of markers for saturation mapping and sequencing of the gene-rich regions of the wheat genome.

Data from cytological and genetic mapping studies suggest that maize arose as a tetraploid. Two previous studies investigating the most likely mode of maize origin arrived at different conclusions. Gaut and Doebley [7] proposed a segmental allotetraploid origin of the maize genome and estimated that the two maize progenitors diverged at 20.5 million years ago (mya). In a similar study, using larger data set, Brendel and colleagues (quoted in [8]) suggested a single genome duplication at 16 mya. One of the key components of such analyses is to examine sequence divergence among strictly orthologous genes. In order to identify such genes, Lai and colleagues [10] sequenced five duplicated chromosomal regions from the maize genome and the orthologous counterparts from the sorghum genome. They also identified the orthologous regions in rice. Using positional information of genetic components, they identified 11 orthologous genes across the two duplicated regions of maize, and the sorghum and rice regions. Swigonovaet al. [12] analyzed the 11 orthologues, and showed that all five maize chromosomal regions duplicated at the same time, supporting a tetraploid origin of maize, and that the two maize progenitors diverged from each other at about the same time as each of them diverged from sorghum, about 11.9 mya.

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 ≈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.

Prolamine and glutelin RNAs are localized to two subdomains of the cortical endoplasmic reticulum (ER), the protein body ER and the cisternal ER, in developing rice seeds. The addition of nearly full-length prolamine sequences at the 3 � untranslated region of a reporter RNA redirects its localization from the cisternal ER to the protein body ER. Deletion analysis of prolamine RNA sequences indicates the presence of two partially redundant cis elements required for protein body ER targeting. The addition of glutelin 3 � untranslated region to protein body ER cis sequences, however, redirects RNA localization to the cisternal ER. These results indicate that there are at least two regulated RNA transport pathways as well as a constitutive pathway to the cortical ER.

The chapter begins by describing celiac disease (CD), its symptoms, typical ages of presentation and etiology, before discussing gluten toxicity and the classification of the relevant cereal proteins. The pathogenesis of CD and the adaptive and innate immune responses are then presented. In vitro and in vivo testing for gluten toxicity are briefly presented before the chapter proceeds to discuss EU labelling and codex guidelines and methods of measuring gluten contamination in foods such as malted cereals, communion wafers and oats. Finally, some pointers are given to future methods for diagnosing CD and to possible research priorities.

RNA editing is the post-transcriptional conversion from C to U before translation, providing a unique feature in the regulation of gene expression. Here, we used a robust and efficient method based on RNA-seq from non-ribosomal total RNA to simultaneously measure chloroplast-gene expression and RNA editing efficiency in the Greater Duckweed, Spirodela polyrhiza, a species that provides a new reference for the phylogenetic studies of monocotyledonous plants. We identified 66 editing sites at the genome-wide level, with an average editing efficiency of 76%. We found that the expression levels of chloroplast genes were relatively constant, but 11 RNA editing sites show significant changes in editing efficiency, when fronds turn into turions. Thus, RNA editing efficiency contributes more to the yield of translatable transcripts than steady state mRNA levels. Comparison of RNA editing sites in coconut, Spirodela, maize, and rice suggests that RNA editing originated from a common ancestor.

A new approach has been undertaken to analyze the sequences and linear organization of the 19-kD zein genes in maize (Zea mays). A high-coverage, large-insert genomic library of the inbred line B73 based on bacterial artificial chromosomes was used to isolate a redundant set of clones containing members of the 19-kD zein gene family, which previously had been estimated to consist of 50 members. The redundant set of clones was used to create bins of overlapping clones that represented five distinct genomic regions. Representative clones containing the entire set of 19-kD zein genes were chosen from each region and sequenced. Seven bacterial artificial chromosome clones yielded 1,160 kb of genomic DNA. Three of them formed a contiguous sequence of 478 kb, the longest contiguous sequenced region of the maize genome. Altogether, these DNA sequences provide the linear organization of 25 19-kD zein genes, one-half the number previously estimated. It is suggested that the difference is because of haplotypes exhibiting different degrees of gene amplification in the zein multigene family. About one-half the genes present in B73 appear to be expressed. Because some active genes have only been duplicated recently, they are so conserved in their sequence that previous cDNA sequence analysis resulted in “unigenes” that were actually derived from different gene copies. This analysis also shows that the 22- and 19-kD zein gene families shared a common ancestor. Although both ancestral genes had the same incremental gene amplification, the 19-kD zein branch exhibited a greater degree of far-distance gene translocations than the 22-kD zein gene family.

Three maize (Zea mays) bacterial artificial chromosome (BAC) libraries were constructed from inbred line B73. High-density filter sets from all three libraries, made using different restriction enzymes (HindIII,EcoRI, and MboI, respectively), were evaluated with a set of complex probes including the185-bp knob repeat, ribosomal DNA, two telomere-associated repeat sequences, four centromere repeats, the mitochondrial genome, a multifragment chloroplast DNA probe, and bacteriophage λ. The results indicate that the libraries are of high quality with low contamination by organellar and λ-sequences. The use of libraries from multiple enzymes increased the chance of recovering each region of the genome. Ninety maize restriction fragment-length polymorphism core markers were hybridized to filters of the HindIII library, representing 6× coverage of the genome, to initiate development of a framework for anchoring BAC contigs to the intermated B73 × Mo17 genetic map and to mark the bin boundaries on the physical map. All of the clones used as hybridization probes detected at least three BACs. Twenty-two single-copy number core markers identified an average of 7.4 ± 3.3 positive clones, consistent with the expectation of six clones. This information is integrated into fingerprinting data generated by the Arizona Genomics Institute to assemble the BAC contigs using fingerprint contig and contributed to the process of physical map construction.

Background: In metazoans, microRNAs, or miRNAs, constitute a growing family of small regulatory RNAs that are usually 19–25 nucleotides in length. They are processed from longer precursor RNAs that fold into stem-loop structures by the ribonuclease Dicer and are thought to regulate gene expression by base pairing with RNAs of protein-coding genes. In Arabidopsis thaliana , mutations in CARPEL FACTORY ( CAF ), a Dicer homolog, and those in a novel gene, HEN1 , result in similar, multifaceted developmental defects, suggesting a similar function of the two genes, possibly in miRNA metabolism. Results: To investigate the potential functions of CAF and HEN1 in miRNA metabolism, we aimed to isolate miRNAs from Arabidopsis and examine their accumulation during plant development in wild-type plants and in hen1-1 and caf-1 mutant plants. We have isolated 11 miRNAs, some of which have potential homologs in tobacco, rice, and maize. The putative precursors of these miRNAs have the capacity to form stable stem-loop structures. The accumulation of these miRNAs appears to be spatially or temporally controlled in plant development, and their abundance is greatly reduced in caf-1 and hen1-1 mutants. HEN1 homologs are found in bacterial, fungal, and metazoan genomes. Conclusions: miRNAs are present in both plant and animal kingdoms. An evolutionarily conserved mechanism involving a protein, known as Dicer in animals and CAF in Arabidopsis , operates in miRNA metabolism. HEN1 is a new player in miRNA accumulation in Arabidopsis , and HEN1 homologs in metazoans may have a similar function. The developmental defects associated with caf-1 and hen1-1 mutations and the patterns of miRNA accumulation suggest that miRNAs play fundamental roles in plant development.

Summary The amino acid methionine is a common protein building block that is also important in other cellular processes. Plants, unlike animals, synthesize methionine de novo and are thus a dietary source of this nutrient. A new approach for using maize as a source of nutrient methionine is described. Maize seeds, a major component of animal feeds, have variable levels of protein-bound methionine. This variability is a result of post-transcriptional regulation of the Dzs10 gene, which encodes a seed-specific high-methionine storage protein. Here we eliminate methionine variability by identifying and replacing the cis-acting site for Dzs10 regulation using transgenic seeds. Interestingly, two different mechanisms affect mRNA accumulation, one dependent on and the other independent of the untranslated regions (UTRs) of Dzs10 RNA. Accumulation of chimeric Dzs10 mRNA was not reduced in hybrid crosses and was uncoupled from genomic imprinting by Dzr1, a regulator of Dzs10. Uniform high levels of Dzs10 protein were maintained over five backcross generations of the transgene. The increased level of methionine in these transgenic seeds allowed the formulation of a useful animal feed ration without the addition of synthetic methionine.

Most flowering plant seeds are composed of the embryo and endosperm, which are surrounded by maternal tissue, in particular the seed coat. Whereas the embryo is the dormant progeny, the endosperm is a terminal organ for storage of sugars and amino acids in proteins and carbohydrates, respectively. Produced in maternal leaves during photosynthesis, sugars, and amino acids are transported to developing seeds after flowering, and during germination they nourish early seedlings growth. Maize endosperm usually contains around 10% protein and 70% starch, and their composition ratio is rather stable, because it is strictly regulated through a pre-set genetic program that is woven by networks of many interacting or counteracting genes and pathways. Endosperm protein, however, is of low nutritional value due mainly to the high expression of the α-zein gene family, which encodes lysine-free proteins. Reduced levels of these proteins in the opaque 2 (o2) mutant and α-zein RNAi (RNA interference) transgenic seed is compensated by an increase of non-zein proteins, leading to the rebalancing of the nitrogen sink and producing more or less constant levels of total proteins in the seed. The same rebalancing of zeins and non-zeins has been observed for maize seeds bred for 30% protein. In contrast to the nitrogen sink, storage of sulfur is controlled through the accumulation of specialized sulfur-rich proteins in maize endosperm. Silencing the synthesis of α-zeins through RNAi fails to raise sulfur-rich proteins. Although overexpression of the methionine-rich δ-zein can increase the methionine level in seeds, it occurs at least in part at the expense of the cysteine-rich β- and γ-zeins, demonstrating a balance between cysteine and methionine in sulfur storage. Therefore, we propose that the throttle for the flow of sulfur is placed before the synthesis of sulfur amino acids when sulfur is taken up and reduced during photosynthesis.

The subfamily of the Lemnoideae belongs to a different order than other monocotyledonous species that have been sequenced and comprises aquatic plants that grow rapidly on the water surface. Here we select Spirodela polyrhiza for whole-genome sequencing. We show that Spirodela has a genome with no signs of recent retrotranspositions but signatures of two ancient whole-genome duplications, possibly 95 million years ago (mya), older than those in Arabidopsis and rice. Its genome has only 19,623 predicted protein-coding genes, which is 28% less than the dicotyledonous Arabidopsis thaliana and 50% less than monocotyledonous rice. We propose that at least in part, the neotenous reduction of these aquatic plants is based on readjusted copy numbers of promoters and repressors of the juvenile-to-adult transition. The Spirodela genome, along with its unique biology and physiology, will stimulate new insights into environmental adaptation, ecology, evolution and plant development, and will be instrumental for future bioenergy applications., Spirodela, or duckweed, is a basal monocotyledonous plant with both pharmaceutical and commercial value. Here, the authors sequence the genome of Spirodela polyrhiza, suggesting its genome has evolved by neotenous reduction and clonal propagation, and provide a platform for future comparative genomic studies in angiosperms.

The accessibility of DNA during fundamental processes, such as transcription, replication and DNA repair, is tightly modulated through a dynamic chromatin structure. Differences in large-scale chromatin structure at the microscopic level can be observed as euchromatic and heterochromatic domains in interphase nuclei. Here, key epigenetic marks, including histone H3 methylation and 5-methylcytosine (5-mC) as a DNA modification, were studied cytologically to describe the chromatin organisation of representative species of the five duckweed genera in the context of their nuclear DNA content, which ranged from 158 to 1881 Mbp. All studied duckweeds, including Spirodela polyrhiza with a genome size and repeat proportion similar to that of Arabidopsis thaliana, showed dispersed distribution of heterochromatin signatures (5mC, H3K9me2 and H3K27me1). This immunolabelling pattern resembles that of early developmental stages of Arabidopsis nuclei, with less pronounced heterochromatin chromocenters and heterochromatic marks weakly dispersed throughout the nucleus.

A common cis-acting element in the promoter region of many genes expressed during endosperm development of cereal seeds, the prolamine-box or P-box, is only 20 bp upstream of the α-class 22-kDa zein gene-specific cis element, the O2-box, which is recognized by the b-ZIP transcription factor, Opaque-2 (O2). The proximity of these two boxes has prompted a study of how two DNA-binding proteins of a different hierarchy might be involved in the activation and modulation of the 22-kDa zein-encoding genes. This was accomplished by utilizing a highly purified P-box-binding-factor-1 (PBF-1) and a bacterially expressed truncated form of the O2 protein. After adding the recombinant O2 to the purified fraction of PBF-1, binding studies were performed with a series of DNA probes combining the P- and O2-boxes from zein promoters. These studies have revealed an interesting inhibitory effect of PBF-1 over O2 function dependent on their ratio, consistent with its in-vivo properties and the developmental expression profiles of zein genes. We also could show that the P-box is specifically recognized by topoisomerase II and single-strand DNA-binding proteins, indicating a possible additional linkage between P-box and the scaffold-attachment-region (SAR).

A maize prolamin box (P-box)-binding factor (PBF-1) has been purified and characterized from immature endosperm tissue. PBF-1 has a molecular weight of 38kDa. It is detected only in endosperm, but not in root or leaf tissues, consistent with its tissue-specific function. Site-directed mutagenesis experiments reveal that both the P-box and its flanking sequences are important for PBF-1 DNA binding. Developmental studies show that PBF-1 accumulates in the endosperm from 8 to at least 30days after pollination (DAP). From 16 to 24DAP, however, multiple shifted bands of protein(s)-DNA complexes can be observed, which correlate with an increase in zein gene expression. PBF-1 can also bind to the P-box from '22-kDa' and '19-kDa' zein promoters, but at a lower affinity than to the '27-kDa' zein promoter. The effects of protein dephosphorylation and zinc ion chelators on PBF-1 DNA binding activity are also shown. A model is proposed where PBF-1 serves as a 'recruiter' of class-specific transcription factors like Opaque2 (O2).

Progress in agricultural and environmental technologies is hampered by a slower rate of gene discovery in plants than animals. The vast pool of genes in plants, however, will be an important resource for insertion of genes, via biotechnological procedures, into an array of plants, generating unique germ plasms not achievable by conventional breeding. It just became clear that genomes of grasses have evolved in a manner analogous to Lego blocks. Large chromosome segments have been reshuffled and stuffer pieces added between genes. Although some genomes have become very large, the genome with the fewest stuffer pieces, the rice genome, is the Rosetta Stone of all the bigger grass genomes. This means that sequencing the rice genome as anchor genome of the grasses will provide instantaneous access to the same genes in the same relative physical position in other grasses (e.g., corn and wheat), without the need to sequence each of these genomes independently. ( i ) The sequencing of the entire genome of rice as anchor genome for the grasses will accelerate plant gene discovery in many important crops (e.g., corn, wheat, and rice) by several orders of magnitudes and reduce research and development costs for government and industry at a faster pace. ( ii ) Costs for sequencing entire genomes have come down significantly. Because of its size, rice is only 12% of the human or the corn genome, and technology improvements by the human genome project are completely transferable, translating in another 50% reduction of the costs. ( iii ) The physical mapping of the rice genome by a group of Japanese researchers provides a jump start for sequencing the genome and forming an international consortium. Otherwise, other countries would do it alone and own proprietary positions.

Background Higher plants exhibit a remarkable phenotypic plasticity to adapt to adverse environmental changes. The Greater Duckweed Spirodela, as an aquatic plant, presents exceptional tolerance to cold winters through its dormant structure of turions in place of seeds. Abundant starch in turions permits them to sink and escape the freezing surface of waters. Due to their clonal propagation, they are the fastest growing biomass on earth, providing yet an untapped source for industrial applications. Results We used next generation sequencing technology to examine the transcriptome of turion development triggered by exogenous ABA. A total of 208 genes showed more than a 4-fold increase compared with 154 down-regulated genes in developing turions. The analysis of up-regulated differential expressed genes in response to dormancy exposed an enriched interplay among various pathways: signal transduction, seed dehydration, carbohydrate and secondary metabolism, and senescence. On the other side, the genes responsible for rapid growth and biomass accumulation through DNA assembly, protein synthesis and carbon fixation are repressed. Noticeably, three members of late embryogenesis abundant protein family are exclusively expressed during turion formation. High expression level of key genes in starch synthesis are APS1, APL3 and GBSSI, which could artificially be reduced for re-directing carbon flow from photosynthesis to create a higher energy biomass. Conclusions The identification and functional annotation of differentially expressed genes open a major step towards understanding the molecular network underlying vegetative frond dormancy. Moreover, genes have been identified that could be engineered in duckweeds for practical applications easing agricultural production of food crops.

Brachypodium distachyon is small annual grass that has been adopted as a model for the grasses. Its small genome, high-quality reference genome, large germplasm collection, and selfing nature make it an excellent subject for studies of natural variation. We sequenced six divergent lines to identify a comprehensive set of polymorphisms and analyze their distribution and concordance with gene expression. Multiple methods and controls were utilized to identify polymorphisms and validate their quality. mRNA-Seq experiments under control and simulated drought-stress conditions, identified 300 genes with a genotype-dependent treatment response. We showed that large-scale sequence variants had extremely high concordance with altered expression of hundreds of genes, including many with genotype-dependent treatment responses. We generated a deep mRNA-Seq dataset for the most divergent line and created a de novo transcriptome assembly. This led to the discovery of >2400 previously unannotated transcripts and hundreds of genes not present in the reference genome. We built a public database for visualization and investigation of sequence variants among these widely used inbred lines. Office of Science (BER), US Department of Energy, USDA NIFA; USDA CRISUnited States Department of Agriculture (USDA) [5325-21000-017-00]; Direct For Biological SciencesNational Science Foundation (NSF)NSF - Directorate for Biological Sciences (BIO) [0922457] Funding Source: National Science Foundation We thank Uffe Hellsten for help with population genetics. Genomic DNA sequencing was performed at the US Department of Energy Joint Genome Institute through the Community Sequencing Program. Deep sequencing of the Bd1-1 transcriptome was performed at the Center of Genome Research and Biocomputing, Oregon State University, Corvallis, OR. This work was supported by the Office of Science (BER), US Department of Energy, USDA NIFA, and by the USDA CRIS project 5325-21000-017-00.

Geminiviruses (Geminiviridae) are a diverse group of plant viruses differing from other known plant viruses in possessing circular, single-stranded DNA. Current classification divides the family into three subgroups, defined in part by genome organization, insect vector, and plant host range. Previous phylogenetic assessments of geminiviruses have used DNA and/or amino acid sequences from the replication-associated and coat protein genes and have relied predominantly on distance analyses. We used amino acid and DNA sequence data from the replication-associated and coat protein genes from 22 geminivirus types in distance and parsimony analyses. Although the results of our analyses largely agree with those reported previously, we could not always predict viral relationships based on genome organization, plant host, or insect vector. Loss of correlation of these traits with phylogeny is likely due to improved sampling of geminivirus types. Unrooted parsimony trees suggest multiple independent origins for the monopartite genome. genome organization is therefore a dynamic character. Estimates of nonsynonymous and synonymous nucleotide substitutions for extant and inferred ancestral sequences were used to evaluate hypotheses that the replication-associated and coat protein sequences evolve to accommodate plant host and insect vector specificities, respectively. Results suggest that plant host specificity does not solely direct replication-associated protein-evolution but that coat protein sequence does evolve in response to insect vector specificity. Genome organization and, possibly, plant host specificity are not reliable taxonomic characters.

Celiac disease (CD) is an enteropathy-induced immune response that occurs on exposure to toxic gluten in the diet and is reversible in most cases once gluten is withdrawn. A gluten-free diet (GFD) is the preferred treatment for CD and leads to reversal of villous atrophy. Individuals with untreated CD may experience significant health complications. Managing CD can potentially prevent or cure some of the associated conditions, such as neurologic complications, nutritional deficiencies, and osteoporosis. Counseling, nutritional support, and follow-up are vital aspects in CD management. GFD remains the mainstay of treatment in CD. Adhering to a strict GFD remains challenging for some individuals, and it has its limitations and other alternatives are called for. There are new and novel treatments emerging, which will be discussed here.

Maize is one of the most important food crops and a key model for genetics and developmental biology. A genetically anchored and high-quality draft genome sequence of maize inbred B73 has been obtained to serve as a reference sequence. To facilitate evolutionary studies in maize and its close relatives, much like the Oryza Map Alignment Project (OMAP) (www.OMAP.org) bacterial artificial chromosome (BAC) resource did for the rice community, we constructed BAC libraries for maize inbred lines Zheng58, Chang7-2, and Mo17 and maize wild relatives Zea mays ssp. parviglumis and Tripsacum dactyloides. Furthermore, to extend functional genomic studies to maize and sorghum, we also constructed binary BAC (BIBAC) libraries for the maize inbred B73 and the sorghum landrace Nengsi-1. The BAC/BIBAC vectors facilitate transfer of large intact DNA inserts from BAC clones to the BIBAC vector and functional complementation of large DNA fragments. These seven Zea Map Alignment Project (ZMAP) BAC/BIBAC libraries have average insert sizes ranging from 92 to 148 kb, organellar DNA from 0.17 to 2.3%, empty vector rates between 0.35 and 5.56%, and genome equivalents of 4.7- to 8.4-fold. The usefulness of the Parviglumis and Tripsacum BAC libraries was demonstrated by mapping clones to the reference genome. Novel genes and alleles present in these ZMAP libraries can now be used for functional complementation studies and positional or homology-based cloning of genes for translational genomics.

Sustainable food production for the earth's fast-growing population is a major challenge for breeding new high-yielding crops, but enhancing the nutritional quality of staple crops can potentially offset limitations associated with yield increases. Sorghum has immense value as a staple food item for humans in Africa, but it is poorly digested. Although a mutant exhibiting high-protein digestibility and lysine content has market potential, the molecular nature of the mutation is previously unknown. Here, building on knowledge from maize mutants, we take a direct approach and find that the high-digestible sorghum phenotype is tightly linked to a single-point mutation, rendering the signal peptide of a seed storage protein kafirin resistant to processing, indirectly reducing lysine-poor kafirins and thereby increasing lysine-rich proteins in the seeds. These findings indicate that a molecular marker can be used to accelerate introduction of this high nutrition and digestibility trait into different sorghum varieties.

Corn is the highest yielding crop on earth and probably the most valuable agricultural product of the United States. Because it converts sun energy through photosynthesis into starch and proteins, we addressed energy savings by focusing on protein quality. People and animals require essential amino acids derived from the digestion of proteins. If proteins are relatively low in certain essential amino acids, the crop becomes nutritionally defective and has to be supplemented. Such deficiency affects meat and fish production and countries where corn is a staple. Because corn seed proteins have relatively low levels of lysine and methionine, a diet has to be supplemented with soybeans for the missing lysine and with chemically synthesized methionine. We therefore have studied genes expressed during maize seed development and their chromosomal organization. A critical technical requirement for the understanding of the molecular structure of genes and their positional information was DNA sequencing. Because of the length of sequences, DNA sequencing methods themselves were insufficient for this type of analysis. We therefore developed the so-called “DNA shotgun sequencing” strategy, where overlapping DNA fragments were sequenced in parallel and used to reconstruct large DNA molecules via overlaps. Our publications became the most frequently cited onesmore » during the decade of 1981-1990 and former Associate Director of Science for the Office of Basic Energy Sciences Patricia M. Dehmer presented our work as one of the great successes of this program. A major component of the sequencing strategy was the development of bacterial strains and vectors, which were also used to develop the first biotechnology crops. These crops possessed new traits thanks to the expression of foreign genes in plants. To enable such expression, chimeric genes had to be constructed using our materials and methods by the industry. Because we made our materials and methods freely available to academia and industry, progress in plant research and new crop development could accelerate and benefit the public.« less

Genetic map lengths do not correlate directly with genome size, suggesting that meiotic recombination is not uniform throughout the genome. Further, the abundance of repeated sequences in plant genomes requires that crossing over is restricted to particular genomic regions. We used a physical mapping approach to identify these regions without the bias introduced by phenotypic selection. This approach is based on the detection of nonparental polymorphisms formed by recombination between polymorphic alleles. In an F2 population of 48 maize plants, we identified a crossover at two of the seven restriction fragment length polymorphism loci tested. Characterization of one recombination event revealed that the crossover mapped within a 534bp region of perfect homology between the parental alleles embedded in a 2773-bp unique sequence. No transcripts from this region could be detected. Sequences immediately surrounding the crossover site were not detectably methylated, except for an SstI site probably methylated via non-CpG or CpXpG cytosine methylation. Parental methylation patterns at this SstI site and at the flanking repetitive sequences were faithfully inherited by the recombinant allele. Our observations suggest that meiotic recombination in maize occurs between perfectly homologous sequences, within unmethylated, nonrepetitive regions of the genome.

Expansion and contraction of microRNA (miRNA) families can be studied in sequenced plant genomes through sequence alignments. Here, we focused on miR169 in sorghum because of its implications in drought tolerance and stem-sugar content. We were able to discover many miR169 copies that have escaped standard genome annotation methods. A new miR169 cluster was found on sorghum chromosome 1. This cluster is composed of the previously annotated sbi-MIR169o together with two newly found MIR169 copies, named sbi-MIR169t and sbi-MIR169u. We also found that a miR169 cluster on sorghum chr7 consisting of sbi-MIR169l, sbi-MIR169m, and sbi-MIR169n is contained within a chromosomal inversion of at least 500 kb that occurred in sorghum relative to Brachypodium, rice, foxtail millet, and maize. Surprisingly, synteny of chromosomal segments containing MIR169 copies with linked bHLH and CONSTANS-LIKE genes extended from Brachypodium to dictotyledonous species such as grapevine, soybean, and cassava, indicating a strong conservation of linkages of certain flowering and/or plant height genes and microRNAs, which may explain linkage drag of drought and flowering traits and would have consequences for breeding new varieties. Furthermore, alignment of rice and sorghum orthologous regions revealed the presence of two additional miR169 gene copies (miR169r and miR169s) on sorghum chr7 that formed an antisense miRNA gene pair. Both copies are expressed and target different set of genes. Synteny-based analysis of microRNAs among different plant species should lead to the discovery of new microRNAs in general and contribute to our understanding of their evolution.

We have investigated the methylation status of the alpha-tubulin genes, and the degree of accumulation of their mRNAs in endosperm, embryo and seedling tissues of Zea mays L. We have found that many of the alpha-tubulin genes are differentially demethylated in the endosperm relative to the embryo and seedling. However, only for tub alpha 2 and tub alpha 4 could a correlation between DNA demethylation and increased RNA accumulation be detected. By analyzing the inbred lines W64A and A69Y and their reciprocal crosses, we have also identified in the endosperm two alpha-tubulin genes, tub alpha 3 and tub alpha 4, that are differentially demethylated if transmitted by the maternal germline, but that remain hypermethylated when transmitted by the paternal germline.

A map has been developed of nuclease-hypersensitive sites of P-rr, the standard allele of the P-locus of Zea mays L. Using a traditional DNase I assay, eight such sites have been found that are specific for the expressing tissue and span a region of more than 25 kb of the P-locus, making it one of the largest plant genes yet described. The maps of the standard allele have also been compared with the recently described moderately stable P-pr allele, which arose from epimutation. Six of the eight sites exhibit the same tissue-specificity in P-pr plants, while two stay repressed as in non-expressing tissues of plants with the standard allele. Interestingly, the two repressed sites coincide with two hypermethylated restriction sites that have previously been correlated with the expression potential of the P-pr allele. On the other hand, four of the DNase I sites, coinciding with CpG islands that were not hypermethylated by the epimutation, also showed no differences in their sensitivity to DNase I between the standard allele and the P-pr allele. This suggests that the epimutation affects both site-specific methylation changes and a specific local chromatin structure of the P gene involved in its regulation.

Cereal grains are an important nutritional source of amino acids for humans and livestock worldwide. Wheat, barley, and oats belong to a different subfamily of the grasses than rice and in addition to maize, millets, sugarcane, and sorghum. All their seeds, however, are largely devoid of free amino acids because they are stored during dormancy in specialized storage proteins. Prolamins, the major class of storage proteins in cereals with preponderance of proline and glutamine, are synthesized at the endoplasmic reticulum during seed development and deposited into subcellular structures of the immature endosperm, the protein bodies. Prolamins have diverged during the evolution of the grass family in their structure and their properties. Here, we used the expression of wheat glutenin-Dx5 in maize to examine its interaction with maize prolamins during endosperm development. Ectopic expression of Dx5 alters protein body morphology in a way that resembles non-vitreous kernel phenotypes, although Dx5 alone does not cause an opaque phenotype. However, if we lower the amount of γ-zeins in Dx5 maize through RNAi, a non-vitreous phenotype emerges and the deformation on the surface of protein bodies is enhanced, indicating that Dx5 requires γ-zeins for its proper subcellular organization in maize.

Seeds have evolved to accommodate complicated processes like senescence, dormancy, and germination. Central to these is the storage of carbohydrates and proteins derived from sugars and amino acids synthesized during photosynthesis. In the grasses, the bulk of amino acids is stored in the prolamin superfamily that specifically accumulates in seed endosperm during senescence. Their promoters contain a conserved cis-element, called prolamin-box (P-box), recognized by the trans-activator P-box binding factor (PBF). Because of the lack of null mutants in all grass species, its physiological role in storage–protein gene expression has been elusive. In contrast, a null mutant of another endosperm-specific trans-activator Opaque2 (O2) has been shown to be required for the transcriptional activation of subsets of this superfamily by binding to the O2 box. Here, we used RNAi to knockdown Pbf expression and found that only 27-kDa γ- and 22-kDa α-zein gene expression were affected, whereas the level of other zeins remained unchanged. Still, transgenic seeds had an opaque seed phenotype. Combination of PbfRNAi and o2 resulted in further reduction of α-zein expression. We also tested the interaction of promoters and constitutively expressed PBF and O2. Whereas transgenic promoters could be activated, endogenous promoters appeared to be not accessible to transcriptional activation, presumably due to differential chromatin states. Although analysis of the methylation of binding sites of PBF and O2 correlated with the expression of endogenous 22-kDa α-zein promoters, a different mechanism seems to apply to the 27-kDa γ-zein promoter, which does not undergo methylation changes.

Complex silencing mechanisms in plants and other kingdoms target transposons, repeat sequences, invasive viral nucleic acids and transgenes, but also endogenous genes and genes involved in paramutation. Paramutation occurs in a heterozygote when a transcriptionally active allele heritably adopts the epigenetic state of a transcriptionally and/or post-transcriptionally repressed allele. P1-rr and its silenced epiallele P1-pr, which encode a Myb-like transcription factor mediating pigmentation in floral organs of Zea mays, differ in their cytosine methylation pattern and chromatin structure at a complex enhancer site. Here, we tested whether P1-pr is able to heritably silence its transcriptionally active P1-rr allele in a heterozygote and whether DNA methylation is associated with the establishment and maintenance of P1-rr silencing. We found that P1-pr participates in paramutation as the repressing allele and P1-rr as the sensitive allele. Silencing of P1-rr is highly variable compared to the inducing P1-pr resulting in a wide range of gene expression. Whereas cytosine methylation at P1-rr is negatively correlated with transcription and pigment levels after segregation of P1-pr, methylation lags behind the establishment of the repressed p1 gene expression. We propose a model in which P1-pr paramutation is triggered by changing epigenetic states of transposons immediately adjacent to a P1-rr enhancer sequence. Considering the vast amount of transposable elements in the maize genome close to regulatory elements of genes, numerous loci could undergo paramutation-induced allele silencing, which could also have a significant impact on breeding agronomically important traits.

Background A balanced composition of amino acids in seed flour is critical because of the demand on essential amino acids for nutrition. However, seed proteins in cereals like maize, the crop with the highest yield, are low in lysine, tryptophan, and methionine. Although supplementation with legumes like soybean can compensate lysine deficiency, both crops are also relatively low in methionine. Therefore, understanding the mechanism of methionine accumulation in the seed could be a basis for breeding cultivars with superior nutritional quality. Results In maize (Zea mays), the 22- and 19-kDa α-zeins are the most prominent storage proteins, nearly devoid of lysine and methionine. Although silencing synthesis of these proteins through RNA interference (RNAi) raises lysine levels in the seed, it fails to do so for methionine. Computational analysis of annotated gene models suggests that about 57% of all proteins exhibit a lysine content of more than 4%, whereas the percentage of proteins with methionine above 4% is only around 8%. To compensate for this low representation, maize seeds produce specialized storage proteins, the 15-kDa β-, 18-kDa and 10-kDa δ-zeins, rich in methionine. However, they are expressed at variant levels in different inbred lines. A654, an inbred with null δ-zein alleles, methionine levels are significantly lower than when the two intact δ-zein alleles are introgressed. Further silencing of β-zein results in dramatic reduction in methionine levels, indicating that β- and δ-zeins are the main sink of methionine in maize seed. Overexpression of the 10-kDa δ-zein can increase the methionine level, but protein analysis by SDS-PAGE shows that the increased methionine levels occur at least in part at the expense of cysteines present in β- and γ-zeins. The reverse is true when β- and γ-zein expression is silenced through RNAi, then 10-kDa δ-zein accumulates to higher levels. Conclusions Because methionine receives the sulfur moiety from cysteine, it appears that when seed protein synthesis of cysteine-rich proteins is blocked, the synthesis of methionine-rich seed proteins is induced, probably at the translational level. The same is true, when methionine-rich proteins are overexpressed, synthesis of cysteine-rich proteins is reduced, probably also at the translational level. Although we only hypothesize a translational control of protein synthesis at this time, there are well known paradigms of how amino acid concentration can play a role in differential gene expression. The latter we think is largely controlled by the flux of reduced sulfur during plant growth.

Bioenergy is the reduction of carbon via photosynthesis. Currently, this energy is harvested as liquid fuel through fermentation. A major concern, however, is input cost, in particular use of excess water and nitrogen, derived from an energy-negative process, the Haber–Bosch method. Furthermore, the shortage of arable land creates competition between uses for food and fuel, resulting in increased living expenses. This review seeks to summarize recent knowledge in genetics, genomics, and gene expression of a rising model species for bioenergy applications, sorghum. Its diploid genome has been sequenced, it has favorable low-input cost traits, and genetic crosses between different cultivars can be used to study allelic variations of genes involved in stem sugar metabolism and incremental biomass.

A salient feature of genomes of higher organisms is the birth and death of gene copies. An example is the alpha prolamin genes, which encode seed storage proteins in grasses (Poaceae) and represent a medium-size gene family. To better understand the mechanism, extent, and pace of gene amplification, we compared prolamin gene copies in the genomes of two different tribes in the Panicoideae, the Paniceae and the Andropogoneae. We identified alpha prolamin (setarin) gene copies in the diploid foxtail millet (Paniceae) genome (490 Mb) and compared them with orthologous regions in diploid sorghum (730 Mb) and ancient allotetraploid maize (2,300 Mb) (Andropogoneae). Because sequenced genomes of other subfamilies of Poaceae like rice (389 Mb) (Ehrhartoideae) and Brachypodium (272 Mb) (Pooideae) do not have alpha prolamin genes, their collinear regions can serve as "empty" reference sites. A pattern emerged, where genes were copied and inserted into other chromosomal locations followed by additional tandem duplications (clusters). We observed both recent (species-specific) insertion events and older ones that are shared by these tribes. Many older copies were deleted by unequal crossing over of flanking sequences or damaged by truncations. However, some remain intact with active and inactive alleles. These results indicate that genomes reflect only a snapshot of the gene content of a species and are far less static than conventional genetics has suggested. Nucleotide substitution rates for active alpha prolamins genes were twice as high as for low copy number beta, gamma, and delta prolamin genes, suggesting that gene amplification accelerates the pace of divergence.