Wusirika Ramakrishna

Transposable elements, genes and recombination in a 215-kb contig from wheat chromosome 5Am

Sequencing of a contiguous 215-kb interval of Triticum monococcum showed the presence of five genes in the same order as in previously sequenced colinear barley and rice BACs. Gene 2 was in the same orientation in wheat and rice but inverted in barley. Gene density in this region was 1xa0gene per 43xa0kb and the ratio of physical to genetic distance was estimated to be 2,700xa0kb cM–1. Twenty more-or-less intact retrotransposons were found in the intergenic regions, covering at least 70% of the sequenced region. The insertion times of 11 retrotransposons were less than 5xa0millionxa0years ago and were consistent with their nested structure. Five new families of retroelements and the first full-length elements for two additional retrotransposon families were discovered in this region. Significantly higher values of GC content were observed for Triticeae BACs compared with rice BACs. Relative enrichment or depletion of certain dinucleotides was observed in the comparison of introns, exons and retrotransposons. A higher proportion of transitions in CG and CNG sites that are targets for cytosine methylation was observed in retrotransposons (76%) than in introns (37%). These results showed that the wheat genome is a complex mixture of different sequence elements, but with general patterns of content and interspersion that are similar to those seen in maize and barley.

Numerous small rearrangements of gene content, order and orientation differentiate grass genomes

Comparative genetic mapping has indicated that the grass family (Poaceae) exhibits extensive chromosomal collinearity. In order to investigate microcollinearity in these genomes, several laboratories have begun to undertake comparative DNA sequence analyses of orthologous chromosome segments from various grass species. Five different regions have now been investigated in detail, with four regions sequenced for maize, rice and sorghum, plus two for wheat and one for barley. In all five of these segments, gene rearrangements were observed in at least one of the comparisons. Most of the detected rearrangements are small, involving the inversion, duplication, translocation or deletion of DNA segments that contain only 1-3 genes. Even closely related species, like barley and wheat or maize and sorghum, exhibit approximately 20% alterations in gene content or orientation. These results indicate that thousands of small genetic rearrangements have occurred in several grass lineages since their divergence from common ancestors. These rearrangements have largely been missed by genetic mapping and will both complicate and enrich the use of comparative genetics in the grasses.

Differential Expansion and Expression of α- and β-Tubulin Gene Families in Populus

Microtubule organization is intimately associated with cellulose microfibril deposition, central to plant secondary cell wall development. We have determined that a relatively large suite of eight α-TUBULIN (TUA) and 20 β-TUBULIN (TUB) genes is expressed in the woody perennial Populus. A number of features, including gene number, α:β gene representation, amino acid changes at the C terminus, and transcript abundance in wood-forming tissue, distinguish the Populus tubulin suite from that of Arabidopsis thaliana. Five of the eight Populus TUAs are unusual in that they contain a C-terminal methionine, glutamic acid, or glutamine, instead of the more typical, and potentially regulatory, C-terminal tyrosine. Both C-terminal Y-type (TUA1) and M-type (TUA5) TUAs were highly expressed in wood-forming tissues and pollen, while the Y-type TUA6 and TUA8 were abundant only in pollen. Transcripts of the disproportionately expanded TUB family were present at comparatively low levels, with phylogenetically distinct classes predominating in xylem and pollen. When tension wood induction was used as a model system to examine changes in tubulin gene expression under conditions of augmented cellulose deposition, xylem-abundant TUA and TUB genes were up-regulated. Immunolocalization of TUA and TUB in xylem and phloem fibers of stems further supported the notion of heavy microtubule involvement during cellulose microfibril deposition in secondary walls. The high degree of sequence diversity, differential expansion, and differential regulation of Populus TUA and TUB families may confer flexibility in cell wall formation that is of adaptive significance to the woody perennial growth habit.

Genomic sequencing reveals gene content, genomic organization, and recombination relationships in barley

Barley (Hordeum vulgare L.) is one of the most important large-genome cereals with extensive genetic resources available in the public sector. Studies of genome organization in barley have been limited primarily to genetic markers and sparse sequence data. Here we report sequence analysis of 417.5xa0kb DNA from four BAC clones from different genomic locations. Sequences were analyzed with respect to gene content, the arrangement of repetitive sequences and the relationship of gene density to recombination frequencies. Gene densities ranged from 1xa0gene per 12xa0kb to 1xa0gene per 103xa0kb with an average of 1xa0gene per 21xa0kb. In general, genes were organized into islands separated by large blocks of nested retrotransposons. Single genes in apparent isolation were also found. Genes occupied 11% of the total sequence, LTR retrotransposons and other repeated elements accounted for 51.9% and the remaining 37.1% could not be annotated.

Structural Analysis of the Maize Rp1 Complex Reveals Numerous Sites and Unexpected Mechanisms of Local Rearrangement

Rp1 is a complex disease resistance locus in maize that is exceptional in both allelic variability and meiotic instability. Genomic sequence analysis of three maize BACs from the Rp1 region of the B73 inbred line revealed 4 Rp1 homologs and 18 other gene-homologous sequences, of which at least 16 are truncated. Thirteen of the truncated genes are found in three clusters, suggesting that they arose from multiple illegitimate break repairs at the same sites or from complex repairs of each of these sites with multiple unlinked DNA templates. A 43-kb region that contains an Rp1 homolog, six truncated genes, and three Opie retrotransposons was found to be duplicated in this region. This duplication is relatively recent, occurring after the insertion of the three Opie elements. The breakpoints of the duplication are outside of any genes or identified repeat sequence, suggesting a duplication mechanism that did not involve unequal recombination. A physical map and partial sequencing of the Rp1 complex indicate the presence of 15 Rp1 homologs in regions of ∼250 and 300 kb in the B73 inbred line. Comparison of fully sequenced Rp1-homologous sequences in the region demonstrates a history of unequal recombination and diversifying selection within the Leu-rich repeat 2 region, resulting in chimeric gene structures.

A Novel Small Heat Shock Protein Gene, vis1, Contributes to Pectin Depolymerization and Juice Viscosity in Tomato Fruit

We have characterized a novel small heat shock protein gene,viscosity 1 (vis1) from tomato (Lycopersicon esculentum) and provide evidence that it plays a role in pectin depolymerization and juice viscosity in ripening fruits. Expression of vis1 is negatively associated with juice viscosity in diverse tomato genotypes. vis1exhibits DNA polymorphism among tomato genotypes, and the alleles vis1-hta (high-transcript accumulator; accession no. AY128101) andvis1-lta (low transcript accumulator; accession no. AY128102) are associated with thinner and thicker juice, respectively. Segregation of tomato lines heterogeneous forvis1 alleles indicates that vis1influences pectin depolymerization and juice viscosity in ripening fruits. vis1 is regulated by fruit ripening and high temperature and exhibits a typical heat shock protein chaperone function when expressed in bacterial cells. We propose that VIS1 contributes to physiochemical properties of juice, including pectin depolymerization, by reducing thermal denaturation of depolymerizing enzymes during daytime elevated temperatures.

On the Tetraploid Origin of the Maize 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. Swigonova et 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.

Comparative Analysis of Divergent and Convergent Gene Pairs and Their Expression Patterns in Rice, Arabidopsis, and Populus

Comparative analysis of the organization and expression patterns of divergent and convergent gene pairs in multiple plant genomes can identify patterns that are shared by more than one species or are unique to a particular species. Here, we study the coexpression and interspecies conservation of divergent and convergent gene pairs in three plant species: rice (Oryza sativa), Arabidopsis (Arabidopsis thaliana), and black cottonwood (Populus trichocarpa). Strongly correlated expression levels between divergent and convergent genes were found to be quite common in all three species, and the frequency of strong correlation appears to be independent of intergenic distance. Conservation of divergent or convergent arrangement among these species appears to be quite rare. However, conserved arrangement is significantly more frequent when the genes display strongly correlated expression levels or have one or more Gene Ontology (GO) classes in common. A correlation between intergenic distance in divergent and convergent gene pairs and shared GO classes was observed, in varying degrees, in rice and Populus but not in Arabidopsis. Furthermore, multiple GO classes were either overrepresented or underrepresented in Arabidopsis and Populus gene pairs, while only two GO classes were underrepresented in rice divergent gene pairs. Three cis-regulatory elements common to both Arabidopsis and rice were overrepresented in the intergenic regions of strongly correlated divergent gene pairs compared to those of noncorrelated pairs. Our results suggest that shared as well as unique mechanisms operate in shaping the organization and function of divergent and convergent gene pairs in different plant species.

Comparative Sequence Analysis of the Sorghum Rph Region and the Maize Rp1 Resistance Gene Complex

A 268-kb chromosomal segment containing sorghum (Sorghum bicolor) genes that are orthologous to the maize (Zea mays) Rp1 disease resistance (R) gene complex was sequenced. A region of approximately 27 kb in sorghum was found to contain five Rp1 homologs, but most have structures indicating that they are not functional. In contrast, maize inbred B73 has 15 Rp1 homologs in two nearby clusters of 250 and 300 kb. As at maize Rp1, the cluster of R gene homologs is interrupted by the presence of several genes that appear to have no resistance role, but these genes were different from the ones found within the maize Rp1complex. More than 200 kb of DNA downstream from the sorghumRp1-orthologous R gene cluster was sequenced and found to contain many duplicated and/or truncated genes. None of the duplications currently exist as simple tandem events, suggesting that numerous rearrangements were required to generate the current genomic structure. Four truncated genes were observed, including one gene that appears to have both 5′ and 3′ deletions. The maize Rp1region is also unusually enriched in truncated genes. Hence, the orthologous maize and sorghum regions share numerous structural features, but all involve events that occurred independently in each species. The data suggest that complex R gene clusters are unusually prone to frequent internal and adjacent chromosomal rearrangements of several types.

Identification of minor DNA variations in rice somaclonal variants

Abstract Some somaclonal variants derived from a landrace rice variety, Indrayani, were shown to be high yielding and resistant to multiple diseases in previous analysis carried out in our laboratory. An attempt was made to assess the effect of culturing and regeneration of rice plants on DNA variation at microsatellite loci in R2 progeny of callus-derived rice plants. Different somaclones of the rice line Indrayani differing in yield and disease response (high, low and no change in yield, as compared to the original genotype) were used as genetic material for these analyses. Analysis of microsatellite loci was accomplished by digesting DNA from regenerated rice somaclones and assaying for polymorphisms at microsatellite loci by in-gel hybridization with synthetic oligonucleotide probes such as (GATA)4, (CAC)5 and (TG)10. Specific variation at a PCR-amplified locus containing three internal microsatellite repeats (1E6) using restriction site fingerprinting was also investigated. The locus-specific amplification of a sequence-tagged microsatellite marker followed by digestion with HinfI and Sau3AI restriction endonucleases showed differences in some somaclonal variants. The technique used in this study enables monitoring of DNA changes in successive generations of somaclonal variants as a measure of DNA variability and possibly to identify the regions which are responsible for specific traits.