Identification and characterization of a new member of the SINE Au retroposon family (GmAu1) in the soybean, Glycine max (L.) Merr., genome and its potential application

A plant short interspersed element (SINE) was identified in Glycine max after re-sequencing of the soybean sequence characterized amplified region (SCAR) markers. Detailed analysis revealed that this newly recognized SINE element consisted of a tRNA-related region, a tRNA non-related region, direct flanking repeat sequences, and a short stretch of Ts at the 3′-terminal region. These features are similar to previously characterized SINEs. To investigate the evolution of the SINE retroposon, BLASTN was used to search against genome sequences of other plants. Since it is homologous with the retroposon Au in Aegilops umbellulata (wheat) and its homology in soybean, the SINE is named as GmAu1. Genome analysis of the Glycine max var. Willimas 82 uncovered more than 847 copies of GmAu1 per haploid genome of soybean. Examination of the regions flanking the inserted GmAu1 sequences indicated a preference for introns over exons or other noncoding regions. Considering the flanking insertion sequences, 146 primers were designed in order to detect insertion mutations by a PCR-based method. Seventy-seven primers displayed polymorphism and were used to develop corresponding GmAu1-based SCAR markers. The retroposon GmAu1 and its related SCAR markers identified in this study will prove valuable to future investigations into the genetic mapping, phylogeny, and evolution of the Glycine genus.     

The Au family, a novel short interspersed element (SINE) from Aegilops umbellulata

A novel plant short interspersed nuclear element (SINE) was identified in the second intron of the acetyl CoA carboxylase gene of Aegilops umbellulata which has been designated ”Au”, for the host species in which it was discovered. Au elements have a tRNA-related region, direct flanking repeats, and a short stretch of T at the 3′ end, which are features common to Au and previously characterized SINEs. Au elements are detected in the genomes of several monocots and dicots by DNA dot hybridization and are also found in the tobacco genome by database searching. Au elements are present at an especially high copy number (approximately 10⁴ copies per haploid genome) in wheat and Ae. umbellulata. This suggests a recent amplification of Au in the Triticum and Aegilops species. In situ hybridization revealed a dispersed distribution of Au elements on wheat chromosomes. Au elements were amplified by PCR from monocot and dicot species and the phylogenetic relationships among Au elements were inferred. This phylogenetic analysis suggests amplification of Au elements in a manner consistent with the retrotransposon model for SINE dispersion. The high copy number of Au elements and their dispersed distribution in wheat are desirable characteristics for a molecular marker system in this important species. Received: 15 April 2000 / Accepted: 24 August 2000     

Genomic targeting and mapping of tiller inhibition gene (<Emphasis Type="Italic">tin3</Emphasis>) of wheat using ESTs and synteny with rice

Changes in plant architecture have been central to the domestication of wild species. Tillering or the degree of branching determines shoot architecture and is a key component of grain yield and/or biomass. Previously, a tiller inhibition mutant with monoculm phenotype was isolated and the mutant gene (tin3) was mapped in the distal region of chromosome arm 3A^mL of Triticum monococcum. As a first step towards isolating a candidate gene for tin3, the gene was mapped in relation to physically mapped expressed sequence tags (ESTs) and sequence tag site (STS) markers developed based on synteny with rice. In addition, we investigated the relationship of the wheat region containing tin3 with the corresponding region in rice by comparative genomic analysis. Wheat ESTs that had been previously mapped to deletion bins provided a useful framework to identify closely related rice sequences and to establish the most likely syntenous region in rice for the wheat tin3 region. The tin3 gene was mapped to a 324-kb region spanned by two overlapping bacterial artificial chromosomes (BACs) of rice chromosome arm 1L. Wheat–rice synteny was exceptionally high at the tin3 region despite being located in the high-recombination, gene-rich region of wheat. Identification of tightly linked flanking EST and STS markers to the tin3 gene and its localization to highly syntenic rice BACs will assist in the future development of a high-resolution map and map-based cloning of the tin3 gene. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Identification and verification of microRNA in wheat (Triticum aestivum)

MicroRNAs (miRNAs) are small, endogenous RNAs that regulate gene expression in both plants and animals. A large number of miRNAs has been identified from various animals and model plant species such as Arabidopsis thaliana and rice (Oryza sativa); however, characteristics of wheat (Triticum aestivum) miRNAs are poorly understood. Here, computational identification of miRNAs from wheat EST sequences was preformed by using the in-house program GenomicSVM, a prediction model for miRNAs. This study resulted in the discovery of 79 miRNA candidates. Nine out of 22 miRNA representatives randomly selected from the 79 candidates were experimentally validated with Northern blotting, indicating that prediction accuracy is about 40%. For the 9 validated miRNAs, 59 wheat ESTs were predicted as their putative targets. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Weibo Jin and Nannan Li contributed equally to the work.     

The nuclear genome of Brachypodium distachyon: analysis of BAC end sequences

Due in part to its small genome (~350 Mb), Brachypodium distachyon is emerging as a model system for temperate grasses, including important crops like wheat and barley. We present the analysis of 10.9% of the Brachypodium genome based on 64,696 bacterial artificial chromosome (BAC) end sequences (BES). Analysis of repeat DNA content in BES revealed that approximately 11.0% of the genome consists of known repetitive DNA. The vast majority of the Brachypodium repetitive elements are LTR retrotransposons. While Bare-1 retrotransposons are common to wheat and barley, Brachypodium repetitive element sequence-1 (BRES-1), closely related to Bare-1, is also abundant in Brachypodium. Moreover, unique Brachypodium repetitive element sequences identified constitute approximately 7.4% of its genome. Simple sequence repeats from BES were analyzed, and flanking primer sequences for SSR detection potentially useful for genetic mapping are available at . Sequence analyses of BES indicated that approximately 21.2% of the Brachypodium genome represents coding sequence. Furthermore, Brachypodium BES have more significant matches to ESTs from wheat than rice or maize, although these species have similar sizes of EST collections. A phylogenetic analysis based on 335 sequences shared among seven grass species further revealed a closer relationship between Brachypodium and Triticeae than Brachypodium and rice or maize. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. N. Huo and G.R. Lazo contributed equally to this work.     

Integrating genes and phenotype: a wheat–Arabidopsis–rice glycosyltransferase database for candidate gene analyses

Glycosyltransferases (GTs) constitute a very large multi-gene superfamily, containing several thousand members identified in sequenced organisms especially in plants. GTs are key enzymes involved in various biological processes such as cell wall formation, storage polysaccharides biosynthesis, and glycosylation of various metabolites. GTs have been identified in rice (Oryza sativa) and Arabidopsis thaliana, but their precise function has been demonstrated biochemically for only a few. In this work we have established a repertoire of virtually all the wheat (Triticum aestivum) GT sequences, using the large publicly available banks of expressed sequences. Based on sequence similarity with Arabidopsis and rice GTs compiled in the carbohydrate active enzyme database (CAZY), we have identified and classified these wheat sequences. The results were used to feed a searchable database available on the web () that can be used for initiating an exhaustive candidate gene survey in wheat applied to a particular biological process. This is illustrated through the identification of GT families which are expressed during cell wall formation in wheat grain maturation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. This work was funded by a grant of the French ministry of research.     

Structure and dynamics of retrotransposons at wheat centromeres and pericentromeres

Little is known of the dynamics of centromeric DNA in polyploid plants. We report the sequences of two centromere-associated bacterial artificial chromosome clones from a Triticum boeoticum library. Both autonomous and non-autonomous wheat centromeric retrotransposons (CRWs) were identified, both being closely associated with the centromeres of wheat. Fiber-fluorescence in situ hybridization and chromatin immunoprecipitation analysis showed that wheat centromeric retrotransposons (CRWs) represent a dominant component of the wheat centromere and are associated with centromere function. CRW copy number showed variation among different genomes: the D genome chromosomes contained fewer copies than either the A or B genome chromosomes. The frequency of lengthy continuous CRW arrays was higher than that in either rice or maize. The dynamics of CRWs and other retrotransposons at centromeric and pericentromeric regions during diploid speciation and polyploidization of wheat and its related species are discussed. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Zhao Liu and Wei Yue made an equal contribution to this work.     

Molecular peculiarities of the chitin-binding peroxidases of plants

The chitin-binding ability of isoperoxidases isolated from 23 plants of different species was studied. The activation of peroxidases in a protein extract in the presence of this polysaccharide was found for 14 of the studied plants. Anionic isoperoxidases were shown to be sorbed on chitin and eluted from them with 1M NaCl for 16 of the plant species. Cationic isoforms of the peroxidases of some species of the Fabaceae and Cucurbitaceae plant families also bound to chitin. An immunochemical similarity was found between the chitin-binding isoperoxidases of taxonomically distant plant species (the Pomaceous, Fabaceae, and Cucurbitaceae). Moreover, a high homology of the molecular structures of the polysaccharide-binding sites was revealed for the anionic peroxidases of rice, wheat, oat, zucchini, cucumber, and radish. We propose the existence of a special class of plant peroxidases that bind with polysaccharides (chitin) and participate in the protective reactions of plants against pathogens.     

<Emphasis Type="Italic">hATpin</Emphasis>, a Family of MITE-like <Emphasis Type="Italic">hAT</Emphasis> Mobile Elements Conserved in Diverse Plant Species that Forms Highly Stable Secondary Structures

We identified a 178 bp mobile DNA element in lettuce with characteristic CGAGC/GCTCG repeats in the subterminal regions. This element has terminal inverted repeats and 8-bp target site duplications typical of the hAT superfamily of class II mobile elements, but its small size and potential to form a single-stranded stable hairpin-like secondary structure suggest that it is related to MITE elements. In silico searches for related elements identified 252 plant sequences with 8-bp target site duplications and sequence similarity in their terminal and subterminal regions. Some of these sequences were predicted to encode transposases and may be autonomous elements; these constituted a separate clade within the phylogram of hAT transposases. We demonstrate that the CGAGC/GCTCG pentamer maximizes the hairpin stability compared to any other pentamer with the same C + G content, and the secondary structures of these elements are more stable than for most MITEs. We named these elements collectively as hATpin elements because of the hAT similarity and their hairpin structures. The nearly complete rice genome sequence and the highly advanced genome annotation allowed us to localize most rice elements and to deduce insertion preferences. hATpin elements are distributed on all chromosomes, but with significant bias for chromosomes 1 and 10 and in regions of moderate gene density. This family of class II mobile elements is found primarily in monocot species, but is also present in dicot species. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Microsatellites and microsynteny in the chloroplast genomes of Oryza and eight other Gramineae species

Primer pairs flanking ten chloroplast microsatellite loci, originally identified in Oryza sativa cv Nipponbare, were evaluated for amplification and allelic diversity using a panel of 13 diverse cultivars of rice (O. sativa), 19 accessions of wild rice (three O. officinalis, five O. latifolia, five O. minuta, four O. australiensis, one O. brachyantha and one O. ridleyi) and eight other Gramineae species (maize, teosinte, wheat, oat, barley, pearl millet, sorghum and sugarcane). Amplified products were obtained for all samples at nine out of ten loci. Among the rice cultivars, the number of alleles per locus ranged from one to four, with monomorphic patterns observed at five loci. The average polymorphism information content (PIC) value at the other five (polymorphic) loci was 0.54 among the 13 cultivars. When wild rice and the other Gramineae species were compared based on the proportion of shared alleles, their phylogenetic relationships were in agreement with previous studies using different types of markers; however, the magnitude of the differences based on chloroplast microsatellites underestimated the genetic distance separating these divergent species and genera. A sequence-based comparison of homologous regions of the rice and maize chloroplast genomes revealed that, while a high level of microsynteny is evident, the occurrence of actively evolving microsatellite motifs in specific regions of the rice chloroplast genome appears to be mainly a species or genome-specific phenomenon. Thus the chloroplast primer pairs used in this study bracketed mutationally active microsatellite motifs in rice but degenerate, interrupted motifs or highly conserved, mutationally inert motifs in distantly related genera. Received: 17 March 1999 / Accepted: 11 November 1999     

Cladosporium sphaerospermum as a new plant growth-promoting endophyte from the roots of Glycine max (L.) Merr.

Endophytic fungi are plant symbionts that produce a variety of beneficial metabolites for plant growth and protection against herbivory and pathogens. Fourteen fungal samples were isolated from the roots of soybean cultivar Daemangkong and screened on waito-c rice for their plant growth-promoting capacity. Twelve of the fungal isolates promoted plant growth, while two inhibited it. The fungal isolate DK-1-1 induced maximum plant growth in both waito-c rice and soybean. The plant growth promotion capacity of DK-1-1 was higher than the wild type Gibberella fujikuroi. Gibberellin (GA) analysis of culture filtrate of DK-1-1 showed the presence of higher amounts of bioactive GA₃, GA₄, and GA₇ (6.62, 2.1 and 1.26 ng/mL, respectively) along with physiologically inactive GA₅, GA₁₅, GA₁₉, and GA₂₄. Phylogenetic analysis of 18S rDNA sequence identified the fungal isolate as a new strain of Cladosporium sphaerospermum. Gibberellin production and plant growth-promoting ability of genus Cladosporium are reported for the first time in the present study. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Comparison of the starch synthesis genes between maize and rice: copies, chromosome location and expression divergence

Gene duplication and divergence are important evolutionary processes. It has been suggested that a whole genome duplication (WGD) event occurred in the Gramineae, predating its divergence, and a second WGD occurred in maize during its evolution. In this study we compared the fate of the genes involved in the core pathway of starch biosynthesis following the ancient and second WGDs in maize and rice. In total, thirty starch synthesis genes were detected in the maize genome, which covered all the starch synthesis gene families encoded by 27 genes in rice. All of these genes, except ZmGBSSIIb and ZmBEIII, are anchored within large-scale synteny blocks of rice and maize chromosomes. Previous findings and our results indicate that two of the current copies of many starch synthesis genes (including AGPL, AGPS, GBSS, SSII, SSIII, and BEII) probably arose from the ancient WGD in the Gramineae and are still present in the maize and rice genome. Furthermore, two copies of at least six genes (AGPS1, SSIIb, SSIIIb, GBSSII, BEI, and ISA3) appear to have been retained in the maize genome after its second WGD, although complete coding regions were only detected among the duplicate sets of AGPS1, SSIIb, and SSIIIb. The expression patterns of the remaining duplicate sets of starch synthesis genes (AGPL1/2, AGPS1/2, SSIIa/b, SSIIIa/b, GBSSI/II, and BEIIa/b) differ in their expression and could be classified into two groups in maize. The first group is mainly expressed in the endosperm, whereas the second is expressed in other organs and the early endosperm development. The four duplicate sets of ZmGBSSII, ZmSSIIb, ZmSSIIIb and AGPS1, which arose from the second WGD diverged in gene structure and/or expression patterns in maize. These results indicated that some duplicated starch synthesis genes were remained, whereas others diverged in gene structure and/or expression pattern in maize. For most of the duplicated genes, one of the copies has disappeared in the maize genome after the WGD and the subsequent “diploidization”. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Moths that Vector a Plant Pathogen also Transport Endophytic Fungi and Mycoparasitic Antagonists

Claviceps paspali, a common fungal pathogen of Paspalum grasses, attracts moth vectors by producing sugary exudates in the grass florets it infects. These exudates also support mycoparasitic Fusarium species that may negatively influence C. paspali fitness. We examined the potential for moths on which C. paspali depends to also transmit mycoparasitic Fusarium and fungal endophytes, which inhabit asymptomatic plant tissue and may influence host susceptibility to pathogens. We quantified infections by C. paspali, Fusarium spp., and endophytic fungi associated with Paspalum spp. at focal sites in the southeastern USA and used data from the nuclear internal transcribed spacer (ITS rDNA) to compare communities of plant-associated and moth-borne fungi. ITS sequences of moth-borne fungi were identical to reference sequences of mycoparasitic Fusarium heterosporum and to three distinct endophytic fungi isolated from Paspalum species. Our results demonstrate an unexpected overlap of fungal communities between disparate locations and among plant species and plant tissues, and suggest an unexpected role of moths, which vector a plant pathogen, to transmit other guilds of fungi. In turn, the potential for insects to transmit plant pathogens as well as mycoparasites and endophytic fungi suggests complex interactions underlying a commonly observed grass–pathogen system. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A one-time inducible transposon for creating knockout mutants

The maize transposon Ac can move to a new location within the genome to create knockout mutants in transgenic plants. In rice, Ac transposon is very active but sometimes undergoes further transposition and leaves an empty mutated gene. Therefore, we developed a one-time transposon system by locating one end of the transposon in the intron of the Ac transposase gene, which is under the control of the inducible promoter (PR-1a). Treatment with salicylic acid induced transposition of this transposon, COYA, leading to transposase gene breakage in exons. The progeny plants inheriting the transposition events become stable knockout mutants, because no functional transposase could be yielded. The behavior of COYA was analyzed in single-copy transgenic rice plants. We determined the expression of the modified transposase gene and its ability to trigger transposition events in transgenic rice plants. The COYA element thus exhibits potential for development of an inducible transposon system suitable for gene isolation in heterologous plant species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Germin-like proteins (GLPs) in cereal genomes: gene clustering and dynamic roles in plant defence

The recent release of the genome sequences of a number of crop and model plant species has made it possible to define the genome organisation and functional characteristics of specific genes and gene families of agronomic importance. For instance, Sorghum bicolor, maize (Zea mays) and Brachypodium distachyon genome sequences along with the model grass species rice (Oryza sativa) enable the comparative analysis of genes involved in plant defence. Germin-like proteins (GLPs) are a small, functionally and taxonomically diverse class of cupin-domain containing proteins that have recently been shown to cluster in an area of rice chromosome 8. The genomic location of this gene cluster overlaps with a disease resistance QTL that provides defence against two rice fungal pathogens (Magnaporthe oryzae and Rhizoctonia solani). Studies showing the involvement of GLPs in basal host resistance against powdery mildew (Blumeria graminis ssp.) have also been reported in barley and wheat. In this mini-review, we compare the close proximity of GLPs in publicly available cereal crop genomes and discuss the contribution that these proteins, and their genome sequence organisation, play in plant defence.     

The PIP and TIP aquaporins in wheat form a large and diverse family with unique gene structures and functionally important features

Aquaporins, members of major intrinsic proteins (MIPs), transport water across cellular membranes and play vital roles in all organisms. Adversities such as drought, salinity, or chilling affect water uptake and transport, and numerous plant MIPs are reported to be differentially regulated under such stresses. However, MIP genes have been not yet been characterized in wheat, the largest cereal crop. We have identified 24 PIP and 11 TIP aquaporin genes from wheat by gene isolation and database searches. They vary extensively in lengths, numbers, and sequences of exons and introns, and sequences and cellular locations of predicted proteins, but the intron positions (if present) are characteristic. The putative PIP proteins show a high degree of conservation of signature sequences or residues for membrane integration, water transport, and regulation. The TIPs are more diverse, some with potential for water transport and others with various selectivity filters including a new combination. Most genes appear to be expressed as expressed sequence tags, while two are likely pseudogenes. Many of the genes are highly identical to rice but some are unique, and many correspond to genes that show differential expression under salinity and/or drought. The results provide extensive information for functional studies and developing markers for stress tolerance. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Evolutionary modes of emergence of short interspersed nuclear element (SINE) families in grasses

Short interspersed nuclear elements (SINEs) are non‐autonomous transposable elements which are propagated by retrotransposition and constitute an inherent part of the genome of most eukaryotic species. Knowledge of heterogeneous and highly abundant SINEs is crucial for de novo (or improvement of) annotation of whole genome sequences. We scanned Poaceae genome sequences of six important cereals (Oryza sativa, Triticum aestivum, Hordeum vulgare, Panicum virgatum, Sorghum bicolor, Zea mays) and Brachypodium distachyon to examine the diversity and evolution of SINE populations. We comparatively analyzed the structural features, distribution, evolutionary relation and abundance of 32 SINE families and subfamilies within grasses, comprising 11 052 individual copies. The investigation of activity profiles within the Poaceae provides insights into their species‐specific diversification and amplification. We found that Poaceae SINEs (PoaS) fall into two length categories: simple SINEs of up to 180 bp and dimeric SINEs larger than 240 bp. Detailed analysis at the nucleotide level revealed that multimerization of related and unrelated SINE copies is an important evolutionary mechanism of SINE formation. We conclude that PoaS families diversify by massive reshuffling between SINE families, likely caused by insertion of truncated copies, and provide a model for this evolutionary scenario. Twenty‐eight of 32 PoaS families and subfamilies show significant conservation, in particular either in the 5′ or 3′ regions, across Poaceae species and share large sequence stretches with one or more other PoaS families.