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Genome walking (GW) refers to the capture and sequencing of unknown regions in a long DNA molecule that are adjacent to a region with a known sequence. A novel PCR-based method, palindromic sequence-targeted PCR (PST-PCR), was developed. PST-PCR is based on a distinctive design of walking primers and special thermal cycling conditions. The walking primers (PST primers) match palindromic sequences (PST sites) that are randomly distributed in natural DNA. The PST primers have palindromic sequences at their 3′-ends. Upstream of the palindromes there is a degenerate sequence (8–12 nucleotides long); defined adapters are present at the 5′-termini. The thermal cycling profile has a linear amplification phase and an exponential amplification phase differing in annealing temperature. Changing the annealing temperature to switch the amplification phases at a defined cycle controls the balance between sensitivity and specificity. In contrast to traditional genome walking methods, PST-PCR is rapid (two to three hours to produce GW fragments) as it uses only one or two PCR rounds. Using PST-PCR, previously unknown regions (the promoter and intron 1) of the VRN1 gene of Timothy-grass (Phleum pratense L.) were captured for sequencing. In our experience, PST-PCR had higher throughput and greater convenience in comparison to other GW methods.

Retrotransposable elements are widely distributed and diverse in eukaryotes. Their copy number increases through reverse-transcription-mediated propagation, while they can be lost through recombinational processes, generating genomic rearrangements. We previously identified extensive structurally uniform retrotransposon groups in which no member contains the gag, pol, or env internal domains. Because of the lack of protein-coding capacity, these groups are non-autonomous in replication, even if transcriptionally active. The Cassandra element belongs to the non-autonomous group called terminal-repeat retrotransposons in miniature (TRIM). It carries 5S RNA sequences with conserved RNA polymerase (pol) III promoters and terminators in its long terminal repeats (LTRs). Here, we identified multiple extended tandem arrays of Cassandra retrotransposons within different plant species, including ferns. At least 12 copies of repeated LTRs (as the tandem unit) and internal domain (as a spacer), giving a pattern that resembles the cellular 5S rRNA genes, were identified. A cytogenetic analysis revealed the specific chromosomal pattern of the Cassandra retrotransposon with prominent clustering at and around 5S rDNA loci. The secondary structure of the Cassandra retroelement RNA is predicted to form super-loops, in which the two LTRs are complementary to each other and can initiate local recombination, leading to the tandem arrays of Cassandra elements. The array structures are conserved for Cassandra retroelements of different species. We speculate that recombination events similar to those of 5S rRNA genes may explain the wide variation in Cassandra copy number. Likewise, the organization of 5S rRNA gene sequences is very variable in flowering plants, part of what is taken for 5S gene copy variation may be variation in Cassandra number. The role of the Cassandra 5S sequences remains to be established.

We evaluated copy number variation (CNV) for four genes in rat strains differing in nervous system excitability. rpl13a copy number is significantly reduced in hippocampus and bone marrow in rats with a high excitability threshold and stress. The observed phenomenon may be associated with a role for rpl13a in lipid metabolism.

Retrotransposon-based molecular markers were applied for the first time within the genera Xanthosoma and Colocasia to assess intraspecific variability among 27 accessions of cocoyam (Xanthosoma sagittifolium) and taro (Colocasia esulenta). Over 16 distinct retrotransposon fragments were isolated, sequenced, and LTR primers were designed to obtain Inter-Retrotransposon Amplified Polymorphism (IRAP) fingerprints. The set of six polymorphic LTR primers yielded 433 reproducible bands across a set of 20 X. sagittifolium samples. Out of the 433 bands, 400 fragments (92%) were polymorphic. In seven C. esculenta accessions, the six primers amplified a total of 354 reproducible, informative data points, of which 285 (80.5%) were polymorphic. Data concerning the number of polymorphic bands and Shannon’s index in X. sagittifolium accessions suggest that retrotransposon activity continued after Xanthosoma speciation. Cluster analysis placed all the accessions in two groups according to their species delimitation. The accessions of X. sagittifolium were further divided into two subgroups corresponding to their ploidy level. Moreover, our results showed that the genetic variability accessed by IRAP markers allows separation of X. sagittifolium and C. esculenta accessions according to their type and botanical variety respectively. These data provide a basis for better germplasm management, future systematic studies and genetic improvement, as well as for exploration of the role of retrotransposons in cocoyam and taro polyploid formation and genome dynamics.

Molecular markers are essential in plant and animal breeding and biodiversity applications, in human forensics, and for map-based cloning of genes. The long terminal repeat (LTR) retrotransposons are well suited as molecular markers. As dispersed and ubiquitous transposable elements, their "copy and paste" life cycle of replicative transposition leads to new genome insertions without excision of the original element. Both the overall structure of retrotransposons and the domains responsible for the various phases of their replication are highly conserved in all eukaryotes. Nevertheless, up to a year has been required to develop a retrotransposon marker system in a new species, involving cloning and sequencing steps as well as the development of custom primers. Here, we describe a novel PCR-based method useful both as a marker system in its own right and for the rapid isolation of retrotransposon termini and full-length elements, making it ideal for "orphan crops" and other species with underdeveloped marker systems. The method, iPBS amplification, is based on the virtually universal presence of a tRNA complement as a reverse transcriptase primer binding site (PBS) in LTR retrotransposons. The method differs from earlier retrotransposon isolation methods because it is applicable not only to endogenous retroviruses and retroviruses, but also to both Gypsy and Copia LTR retrotransposons, as well as to non-autonomous LARD and TRIM elements, throughout the plant kingdom and to animals. Furthermore, the inter-PBS amplification technique as such has proved to be a powerful DNA fingerprinting technology without the need for prior sequence knowledge.

The BARE retrotransposon family comprises more than 10(4) copies in the barley (Hordeum vulgare) genome. The element is bounded by long terminal repeats (LTRs, 1829 bp) containing promoters and RNA-processing motifs required for retrotransposon replication. Members of the BARE1 subfamily are transcribed, translated, and form virus-like particles. Very similar retrotransposons are expressed as RNA and protein in other cereals and grasses. The BARE2 subfamily is, however, non-autonomous because it cannot produce the GAG capsid protein. The pattern of plant development implies that inheritance of integrated copies should critically depend, in the first instance, on cell-specific and tissue-specific expression patterns. We examined transcription of BARE within different barley tissues and analyzed the promoter function of the BARE LTR. The two promoters of the LTR vary independently in activity by tissue. In embryos TATA1 was almost inactive, whereas transcription in callus appears to be less tightly regulated than in other tissues. Deletion analyses of the LTR uncovered strong positive and negative regulatory elements. The promoters produce multiple groups of transcripts that are distinct by their start and stop points, by their sequences, and by whether they are polyadenylated. Some of these groups do not share the common end structures needed for template switching during replication. Only about 15% of BARE transcripts are polyadenylated. The data suggest that distinct subfamilies of transcripts may play independent roles in providing the proteins and replication templates for the BARE retrotransposon life cycle.

To date, all linkage maps of hexaploid oat ( Avena sativa L.) have been constructed using recombinant inbred lines (RILs). Doubled haploids (DHs), however, have the advantage over RILs of their comprehensive homozygosity. DHs have been used for mapping in several cereal species, but in oats the production of large DH populations has only recently become an option. A linkage map of hexaploid oat was constructed using an anther culture–derived DH population (137 individuals) from the F1individuals of a cross between the Finnish cultivar ‘Aslak’ and the Swedish cultivar ‘Matilda’. The map is composed of 28 linkage groups containing 625 DNA markers: 375 AFLPs (amplified fragment length polymorphisms), 3 IRAPs (inter-retrotransposon amplified polymorphisms), 12 ISSRs (inter simple sequence repeats), 12 microsatellites, 57 RAPDs (random amplified polymorphic DNAs), 59 REMAPs (retrotransposon-microsatellite amplified polymorphisms), 105 SRAPs (sequence-related amplified polymorphisms), and 2 SNPs (single-nucleotide polymorphisms). The total map size is 1526 cM. Over half of the markers in the map showed distorted segregation, with alleles from ‘Aslak’ usually prevailing. This is explained by the better performance of ‘Aslak’ in anther culture. Quantitative trait loci affecting some important quality and agronomic traits are being localized on the map.

Cadmium (Cd) is a nonessential heavy metal that is highly toxic to living cells at very low concentrations. Most of the Cd in plants derives from soils. Owing to the large amounts consumed, cereals are the major source of dietary Cd, and Cd content in oat can exceed accepted limits. Plants have a set of mechanisms that control the uptake, accumulation, trafficking, and detoxification of Cd and other metals. Genetic factors affect the variation in Cd level between plant species and cultivars, and the development of cultivars that poorly accumulate Cd is a worthwhile goal. Because of the expense of Cd screening, the use of molecular markers linked to low Cd accumulation could be an alternative to phenotyping for selection. In this study, such markers were sought using bulked-segregant analysis in an F2 population from the cross between oat cultivars ‘Aslak’ and ‘Salo’, the second of which is known to be a high Cd accumulator. Four markers associated with grain Cd concentration were found: 2 RAPDs (random amplified polymorphic DNAs), 1 REMAP (retrotransposon-microsatellite amplified polymorphism), and 1 SRAP (sequence-related amplified polymorphism). The first 3 were converted into more reproducible SCAR (sequence-characterized amplified region) markers. The 4 markers were assigned to 1 linkage group that exhibited a QTL (quantitative trait locus) representing a major gene for grain Cd concentration.

Net blotch of barley ( Hordeum vulgare L.), caused by the fungal phytopathogen Pyrenophora teres Drechs. f. teres Smedeg., constitutes one of the most serious constraints to barley production worldwide. Two forms of the disease, the net form, caused by P. teres f. teres, and the spot form, caused by P. teres f. maculata, are differentiated by the type of symptoms on leaves. Several barley lines with major gene resistance to net blotch have been identified. Earlier, one of these was mapped in the Rolfi × CI 9819 cross to barley chromosome 6H, using a mixture of 4 Finnish isolates of P. teres f. teres. In this study, we used the same barley progeny to map resistance to 4 spot-type isolates and 4 net-type isolates of P. teres. With all net-type isolates, a major resistance gene was located on chromosome 6H, in the same position as described previously, explaining up to 88% of the phenotypic variation in infection response in the progeny. We designate this gene Rpt5. Several minor resistance genes were located on chromosomes 1H, 2H, 3H, 5H, and 7H. These minor genes were not genuinely isolate-specific, but their effect varied among isolates and experiments. When the spot-type isolates were used for infection, a major isolate-specific resistance gene was located on chromosome 5H, close to microsatellite marker HVLEU, explaining up to 84% of the phenotypic variation in infection response in the progeny. We designate this gene Rpt6. No minor gene effects were detected in spot-type isolates. The Ethiopian 2-rowed barley line CI 9819 thus carries at least 2 independent major genes for net-blotch resistance: Rpt5, active against net-type isolates; and Rpt6, active against specific spot-type isolates.

[EN]: Grande is an abundant gypsy-like retrotransposon present in the genera Zea and Tripsacum. Related retro transposon families can be found in sorghum, rice, and barley, but not in wheat or rye. We have amplified and sequenced several copies of part of the integrase domain derived from the Zea mays, Zea diploperennis, and Tripsacum dactyloides genomes. There are no significant differences in divergence or clustering between the integrase sequences of these species. The substitution rate for synonimous sites was found to be higher than those of non-synomymous sites; this indicates that Grande integrase has been under purifying selection for function. Grande is transcribed in leaves. The transcripts show sequence diversity similar to that of genomic sequences, but belong to restricted clades; this indicates that only some evolutionary branches of Grande have retained transcriptional competence.Key words: maize, plant genome, retrotransposon, Zea diploperennis, Zea genus., [FR]: L'élément Grande est un rétrotransposon de type gypsy qui est abondant chez les genres Zea et Tripsacum. Des familles apparentées de rétrotransposons sont présentes chez le sorgho, le riz et l'orge, mais pas chez le blé ou le seigle. Les auteurs ont amplifié et séquencé plusieurs copies d'une partie du domaine intégrase chez les génomes du Zea mays, du Zea diploperennis et du Tripsacum dactyloides. Il n'y a pas de différences significatives quant à la divergence ou au regroupement entre les séquences d'intégrase chez ces espèces. Le taux moyen de substitutions synonymes plus élevé que celui de substitutions non-synonymes indique que l'intégrase de Grande a subi une sélection purificatrice pour sa fonction. L'élément Grande est transcrit dans les feuilles. Les transcrits présentent une diversité nucléotidique semblable à celle observée chez les séquences génomiques, mais appartiennent à des clades restreints, ce qui indique que seules certaines branches évolutives de l'élément Grande ont conservé la capacité d'être transcrites.Mots clés : maïs, génome de plante, rétrotransposon, Zea diploperennis, genre Zea.

Short straw is a desired trait in oat germplasm (Avena sativa L.). Marker-assisted selection, a key tool for achieving this objective, is limited by the presence and number of available markers. Here, we have attempted to develop markers sufficiently linked to a gene specifying short straw so that marker-assisted selection could be applied. Bulked-segregant analysis was used to identify anonymous PCR-based markers associated with the dwarfing gene Dw6 in an F2 population from the cross between A. sativa 'Aslak' and A. sativa 'Kontant'. One random amplified polymorphic DNA (RAPD) and 1 retrotransposon-microsatellite amplified polymorphism (REMAP) marker were found to be associated with height. These were converted into codominant single-nucleotide polymorphism (SNP) markers. The SNP–REMAP and the SNP–RAPD markers were located 5.2 and 12.6 cM from Dw6, respectively. They can be used in future efforts both to enhance oat germplasm by application of molecular markers and to determine the nature of the gene through positional cloning.Key words: Avena sativa, short straw, marker-assisted selection, RAPD, REMAP, SNP.

Retrotransposon markers have been demonstrated to be powerful tools for investigating linkage, evolution and genetics diversity in plants. In the present study, we identified and cloned three full-size TRIM (terminal-repeat retrotransposon in miniature) group retrotransposon elements from apple (Malus domestica) cv. 'Antonovka', the first from the Rosaceae. To investigate their utility as markers, we designed primers to match the long terminal repeats (LTRs) of the apple TRIM sequences. We found that PCR reactions with even a single primer produced multiple bands, suggesting that the copy number of these TRIM elements is relatively high, and that they may be locally clustered or nested in the genome. Furthermore, the apple TRIM primers employed in IRAP (inter-retrotransposon amplified polymorphism) or REMAP (retrotransposon-microsatellite amplified polymorphism) analyses produced unique, reproducible profiles for 12 standard apple cultivars. On the other hand, all seven of the sport mutations in this study were identical to their mother cultivar. Genetic similarity values calculated from the IRAP/REMAP analyses or the STMS (sequence tagged microsatellite sites) analysis were generally comparable. PAUP cluster analysis based on IRAP and REMAP markers in apple and Japanese quince generated an NJ tree that is in good accordance with both a tree based on SMTS markers and the origin of the studied samples. Our results demonstrate that, although they do not encode the proteins necessary to carry out a life cycle and are thereby non-autonomous, TRIMs are at least as polymorphic in their insertion patterns as conventional complete retrotransposons.

We have exploited the repetitive and dispersed nature of many long terminal repeat (LTR)-retrotransposon families for characterizing genome constitutions and classifying cultivars of the genusMusa. Insertional polymorphisms of the elements were studied using seven published and two newly designed primers facing outwards from the LTRs and reverse transcriptase (RT) domain of the retrotransposon. The primers generated specific amplification patterns showing the universal applicability of this marker type. The Inter-Retrotransposon Amplified Polymorphism (IRAP) markers distinguished the A and B genomes of the banana species (Musa acuminata Col la andMusa balbisiana Colla) and between banana cultivars. The IRAP markers enabled phylogenetic analysis of 16 Malaysian banana cultivars and determination of the genome constitution of hybrid banana (AAB, ABB, AABB, and AAAB), and gave information about ancestral genotypes of the hybrids. In addition, the IRAP detected new retrotransposon insertions into the genome of tissue culture regenerants. This PCR-based IRAP assay is amenable to large-scale throughput demands in screening breeding populations and is applicable for any crop.

Cellular genes comprise at most 5% of the barley genome; the rest is occupied primarily by retrotransposons. Retrotransposons move intracellularly by a replicative mechanism similar to that of retroviruses. We describe the major classes of retrotransposons in barley, including the two nonautonomous groups that were recently identified, and detail the evidence supporting our current understanding of their life cycle. Data from analyses of long contiguous segments of the barley genome, as well as surveys of the prevalence of full-length retrotransposons and their solo LTR derivatives in the genus Hordeum, indicate that integration and recombinational loss of retrotransposons are major factors shaping the genome. The sequence conservation and integrative capacity of barley retrotransposons have made them excellent sources for development of molecular marker systems.

Retroviruses and LTR retrotransposons comprise two long-terminal repeats (LTRs) bounding a central domain that encodes the products needed for reverse transcription, packaging, and integration into the genome. We describe a group of retrotransposons in 13 species and four genera of the grass tribe Triticeae, including barley, with long, ∼4.4-kb LTRs formerly called Sukkula elements. The ∼3.5-kb central domains include reverse transcriptase priming sites and are conserved in sequence but contain no open reading frames encoding typical retrotransposon proteins. However, they specify well-conserved RNA secondary structures. These features describe a novel group of elements, called LARDs or large retrotransposon derivatives (LARDs). These appear to be members of the gypsy class of LTR retrotransposons. Although apparently nonautonomous, LARDs appear to be transcribed and can be recombinationally mapped due to the polymorphism of their insertion sites. They are dispersed throughout the genome in an estimated 1.3 × 103 full-length copies and 1.16 × 104 solo LTRs, indicating frequent recombinational loss of internal domains as demonstrated also for the BARE-1 barley retrotransposon., This study was supported by the European Union Research Directorate (contract no. QLK5-CT-1999-01499) and by the Academy of Finland (project 44404).

This paper reviews marker methods based on retrotransposons and illustrates examples from oil palm. Prior to this study, very little had been known about the repetitive DNA present in oil palm and no marker systems based on retrotransposons had been developed. Firstly,copialike retrotransposons ofElaeis oleifera, Elaeis guineensisandCocos nuciferawere characterized by performing phylogenetic analyses on a portion of the reverse transcriptase (RT) gene ofcopia-like retrotransposons. The results identified three classes ofcopia-like retrotransposons in the three species studied. While the C-class RT sequences seemed to have amplified preferentially only inElaeis guineensis, the classes A and B were present inElaeis guineensisandCocos nuciferaas well. Secondly, long terminal repeats (LTRs) from these retrotransposon classes were isolated as a prelude to developing a marker system in palm based on retrotransposons. The B- and C-class LTRs were isolated by primer walking from the RT region and the B-class fromElaeis oleiferaandCocos nuciferawith the use of oil palm-specific RnaseH primers. The method has been developed and applied for breeding purposes in oil palm, hybrids betweenE. oleiferaandE. guineensisand within the tribe Cocoeae. There is scope for the method to be used in the isolation of new retrotransposon families endogenous for each palm species and as an alternative to amplified fragment length polymorphism (AFLP) in diversity studies within the Palmae.

The Sequence-Specific Amplification Polymorphism (S-SAP) method, and the related molecular marker techniques IRAP (inter-retrotransposon amplified polymorphism) and REMAP (retrotransposon-microsatellite amplified polymorphism), are based on retrotransposon activity, and are increasingly widely used. However, there have been no systematic analyses of the parameters of these methods or of the utility of different retrotransposon families in producing polymorphic, scorable fingerprints. We have generated S-SAP, IRAP, and REMAP data for three barley ( Hordeum vulgare L.) varieties using primers based on sequences from six retrotransposon families ( BARE -1, BAGY-1, BAGY-2, Sabrina, Nikita and Sukkula). The effect of the number of selective bases on the S-SAP profiles has been examined and the profiles obtained with eight MseI+3 selective primers compared for all the elements. Polymorphisms detected in the insertion pattern of all the families show that each can be used for S-SAP. The uniqueness of each transposition event and differences in the historic activity of each family suggest that the use of multiple retrotransposon families for genetic analysis will find applications in mapping, fingerprinting, and marker-assisted selection and evolutionary studies, not only in barley and other Hordeum species and related taxa, but also more generally.

F2 progeny segregating for linolenic acid content were used to identify genes and develop markers for linolenic acid in spring turnip rape (Brassica rapa ssp. oleifera). A candidate gene approach applying the rapeseed fad3 gene and bulked segregant analysis with RAPD markers was used. A total of 27 markers were distributed in three linkage groups which each exhibited a QTL for linolenic acid. Jointly the three QTLs accounted for 73.5% of the variation in linolenic acid level in this population. The fad3 gene was mapped near one QTL controlling 23.5% of the variation. Allele-specific markers were developed for fad3 and can be used for marker-assisted selection in future spring turnip rape breeding programmes.

α-Amylolysis of native granular starch represents a biochemical process, in which an enzyme in solution solubilises a highly organised, semicrystalline substrate. To study this process, which is very slow compared to the hydrolysis of gelatinised starch [1], it is necessary to use high concentrations of enzyme or very long incubation times. The solubilised dextrins are therefore easily hydrolysed further into small fragments, preventing a study of the products originally released from the granules. A method that enables a collection of the dextrins solubilised from starch granules by α-amylase was developed [1] and has made it possible to study the composition of the products at early stages of the process. In this method, the starch granules are packed as a thin layer in a two-compartment column [2] and a diluted enzyme solution is pumped through the starch. Before a secondary hydrolysis occurs, the enzyme is bound to an anionexchange matrix (DEAE-Sepharose) in the upper compartment of the column, whereas the solubilised dextrins are collected into fractions representing successive stages of the

The BARE-1 retrotransposon is an active, dispersed, and highly abundant component of the genome of barley (Hordeum vulgare) and other species in its genus. Like all retrotransposons of its kind, BARE-1 is bounded by long terminal repeats (LTRs). We have developed two amplification-based marker methods based on the position of given LTRs within the genome. The IRAP (Inter-Retrotransposon Amplified Polymorphism) markers are generated by the proximity of two LTRs using outward-facing primers annealing to LTR target sequences. In REMAP (REtrotransposon-Microsatellite Amplified Polymorphism), amplification between LTRs proximal to simple sequence repeats such as constitute microsatellites produces markers. The methods can distinguish between barley varieties and produce fingerprint patterns for species across the genus. The patterns indicate that although the BARE-1 family of retrotransposons is disperse, these elements are locally clustered or nested and often found near tandem arrays of a simple sequence repeat.

Propagation of long terminal repeat (LTR)-bearing retrotransposons and retroviruses requires integrase (IN, EC 2.7.7.-), encoded by the retroelements themselves, which mediates the insertion of cDNA copies back into the genome. An active retrotransposon family, BARE-1, comprises approximately 7% of the barley (Hordeum vulgare subsp. vulgare) genome. We have generated models for the secondary and tertiary structure of BARE-1 IN and demonstrate their similarity to structures for human immunodeficiency virus 1 and avian sarcoma virus INs. The IN core domains were compared for 80 clones from 28 Hordeum accessions representative of the diversity of the genus. Based on the structural model, variations in the predicted, aligned translations from these clones would have minimal structural and functional effects on the encoded enzymes. This indicates that Hordeum retrotransposon IN has been under purifying selection to maintain a structure typical of retroviral INs. These represent the first such analyses for plant INs.

Retrotransposons comprise the bulk of large plant genomes, replicating via an RNA intermediate whereby the original, integrated element remains in place. Of the two main orders, the LTR retrotransposons considerably outnumber the LINEs. LINEs integrate into target sites simultaneously with the RNA transcript being copied into cDNA by target-primed reverse transcription. LTR retrotransposon replication is basically equivalent to the intracellular phase of retroviral life cycles. The envelope gene giving extracellular mobility to retroviruses is in fact widespread in plants and their retrotransposons. Evolutionary analyses of the retrotransposons and retroviruses suggest that both form an ancient monophyletic group. The particular adaptations of LTR retrotransposons to plant life cycles enabling their success remain to be clarified.

The shx mutant of barley (Hordeum vulgare L. cv. Bomi) produces grains greatly decreased in starch content and containing smaller A-starch granules together with normal B-granules. Soluble starch synthase (SSS) and ADP-glucose pyrophosphorylase (AGP) are reduced to about 20% normal activity. To help determine the position of the block to starch synthesis in shx, we measured enzyme activities and metabolite levels in developing grains of normal and mutant genotypes in a cv. Bomi background. We demonstrate that sucrose, free hexose, hexose phosphates and, critically, ADP-Glc accumulate as a result of the mutation. In addition to AGP and SSS, several other enzyme activities are affected in the shx mutant, most of them showing activities 50-80% of normal. Northern blots showed that transcripts for the AGP small subunit are much less abundant, but of normal size, in shx. These and earlier results together indicate that the metabolic block is at the end of the starch synthetic pathway, with a primary effect on a type I, primer-independent soluble starch synthase.

Soluble starch synthase (SSS, EC 2.4.1.21) catalyzes formation of the α-1,4 bonds of amylopectin. It occurs in multiple isozymes which are either type I, primer-independent in the presence of citrate, or type II. always primer-dependent. To analyze the enzyme. a sensitive native gel assay was developed, monitoring ADP-[14C]glucose incorporation into insoluble α-glucan in the presence of either sodium citrate or glycogen primer or both. Using this system, we observed multiple type I and type II forms in developing grains of barley (Hordeum vulgare L.) cv. Bomi, the relative activities of which vary with seed development. At least one form comigrates in native gels with starch branching enzyme. Assays of the shx mutant, which is severely reduced in starch accumulation and in type I SSS activity, indicate that one type I isozyme becomes primer-dependent.

Retroviruses and retrotransposons make up the broad class of retroelements replicating and transposing via reverse transcriptase. Retroelements have recently been found to be ubiquitous in the plants. We report here the isolation, sequence and analysis of a retroelement from barley (Hordeum vulgare L.) with all the features of a copia-like retrotransposon. This is named BARE-1 (for BArley RetroElement 1), the first such element described for barley. BARE-1 is 12,088 bp, with long terminal repeats (LTRs) of 1829 bp containing perfect 6 bp inverted repeats at their ends and flanked by 4 bp direct repeats in the host DNA. Between the long terminal repeats is an internal domain with a derived amino acid sequence of 1285 residues, bearing homology to the gag, pro, int and rt domains of retroviruses and both plant and non-plant copia-like retrotransposons. Cultivated barley contains about 5000 elements in the genome similar to the BARE-1 putative gag domain, but ten-fold more hybridizing to rt or LTR probes. The particular BARE-1 element reported here appears to be inactive, as the putative protein-coding domain is interrupted by four stop codons and a frameshift. In addition, the 3' LTR is 4% divergent from the 5' LTR and contains a 3135 bp insertion. Nevertheless, we have recently detected transcripts hybridizing to BARE-1 on northern blots, presumably from active copies. Analysis of BARE-1 expression and function in barley is currently underway.

Although mutations affecting several enzymes of the starch synthetic pathway in developing cereal endosperm have been isolated, none has a major effect on soluble starch synthase We report a new recessive shrunken endosperm mutant in barley (Hordeum vulgare L. cv. Bomi-like), shx, which has 25% of normal starch content. We have assayed the activity of sucrose synthase (EC 2.4.1.13), ADP and UDP-glucose pyrophosphorylases (EC 2.7.7.27 and 2.7.7.9), branching enzyme (EC.2.4.1.18), and granule-bound and soluble starch synthase (EC 2.4.1.21) in shx. Sucrose synthase activity is reduced by 49% and UDP-glucose pyrrphosphorylase is 80% of the normal level. Branching enzyme and starch-bound starch synthase activities are normal, but ADP-glucose pyrophosphorylase activity is reduced by 72%. The soluble starch synthase that is primer-independent in the presence of sodium citrate shows 14% of normal activity in shx. whereas the primer-dependent form is unaffected. This lower starch synthase activity in shx cannot be explained by inhibition, substrate destruction or lack of primer. Although several starch-synthetic enzymes are affected, it is suggested that the primer independent from of soluble starch synthase may be the primary-site of the mutation in shx.

LTR (long terminal repeat) retrotransposons are the main components of higher plant genomic DNA. They have shaped their host genomes through insertional mutagenesis and by effects on genome size, gene expression and recombination. These Class I transposable elements are closely related to retroviruses such as the HIV by their structure and presumptive life cycle. However, the retrotransposon life cycle has been closely investigated in few systems. For retroviruses and retrotransposons, individual defective copies can parasitize the activity of functional ones. However, some LTR retrotransposon groups as a whole, such as large retrotransposon derivatives and terminal repeats in miniature, are non-autonomous even though their genomic insertion patterns remain polymorphic between organismal accessions. Here, we examine what is known of the retrotransposon life cycle in plants, and in that context discuss the role of parasitism and complementation between and within retrotransposon groups.

Aegilops tauschii (Coss.) Schmal. (2n = 2x = 14, DD) (syn. A. squarrosa L.; Triticum tauschii) is well known as the D-genome donor of bread wheat (T. aestivum, 2n = 6x = 42, AABBDD). Because of conserved synteny, a high-density map of the A. tauschii genome will be useful for breeding and genetics within the tribe Triticeae which besides bread wheat also includes barley and rye. We have placed 249 new loci onto a high-density integrated cytological and genetic map of A. tauschii for a total of 732 loci making it one of the most extensive maps produced to date for the Triticeae species. Of the mapped loci, 160 are defense-related genes. The retrotransposon marker system recently developed for cultivated barley (Hordeum vulgare L.) was successfully applied to A. tauschii with the placement of 80 retrotransposon loci onto the map. A total of 50 microsatellite and ISSR loci were also added. Most of the retrotransposon loci, resistance (R), and defense-response (DR) genes are organized into clusters: retrotransposon clusters in the pericentromeric regions, R and DR gene clusters in distal/telomeric regions. Markers are non-randomly distributed with low density in the pericentromeric regions and marker clusters in the distal regions. A significant correlation between the physical density of markers (number of markers mapped to the chromosome segment/physical length of the same segment in microm) and recombination rate (genetic length of a chromosome segment/physical length of the same segment in microm) was demonstrated. Discrete regions of negative or positive interference (an excess or deficiency of crossovers in adjacent intervals relative to the expected rates on the assumption of no interference) was observed in most of the chromosomes. Surprisingly, pericentromeric regions showed negative interference. Islands with negative, positive and/or no interference were present in interstitial and distal regions. Most of the positive interference was restricted to the long arms. The model of chromosome structure and function in cereals with large genomes that emerges from these studies is discussed.

Preparation of pure starch samples requires gentle extraction from the endosperm matrix, followed by removal of contaminating endosperm proteins. Published techniques for removal of these proteins include protease treatment or centrifugation through CsCI. We present a simple method to remove proteins from starch, exploiting the solubility of the major groups of endosperm proteins in aqueous, saline, and alcoholic solvents. The data show that the purity of the starch and retention of B-granules is similar to conventional techniques. Denaturing polyacry-lamide gel electrophoresis reveals very few proteins retained with the starch. One major protein has an apparent MW of 54000 and probably corresponds to the “waxy” protein, granule-bound starch synthase.

Background The LTR (long terminal repeat) retrotransposons of higher plants are replicated by a mutagenic life cycle containing transcription and reverse transcription steps. The DNA copies are often subject to recombination once integrated into the genome. Complex elements, where two elements share an LTR, are not uncommon. They are thought to result from heterologous recombination between two adjacent elements that occurs following their integration. Results Here, we present evidence for another potential mechanism for the creation of complex elements, involving abnormal template switching during reverse transcription. The template switching creates a large, complex daughter element, formed by the fusion of two parent sequences, which is then inserted into the genome. Conclusion Those complex elements are part of the genome structure of plants in the Poaceae, especially in the Triticeae, but not of Arabidopsis. Hence, retrotransposon dynamics shaping the genome are lineage-specific.

Timothy (Phleum pratense L.), a cool-season hexaploid perennial, is the most important forage grass species in Nordic countries. Earlier analyses of genetic diversity in a collection of 96 genebank accessions of timothy with SSR markers demonstrated high levels of diversity but could not resolve population structure. Therefore, we examined a subset of 51 accessions with REMAP markers, which are based on retrotransposons, and compared the diversity results with those obtained with SSR markers. Using four primer combinations, 533 REMAP markers were analyzed, compared with 464 polymorphic alleles in the 13 SSR loci previously. The average marker index, which describes information obtained per experiment (per primer combination or locus) was over six times higher with REMAPs. Most of the variation found was within accessions, with somewhat less, 89 %, for REMAPs, than for SSR, with 93 %. SSRs revealed differences in the level of diversity slightly better than REMAPs but neither marker type could reveal any clear clustering of accessions based on countries, vegetation zones, or different cultivar types. In our study, reliable evaluation of SSR allele dosages was not possible, so each allele had to be handled as a dominant marker. SSR and REMAP, which report from different mechanisms of generating genetic diversity and from different genomic regions, together indicate a lack of population structure. Taken together, this likely reflects the outcrossing and hexaploid nature of timothy rather than failures of either marker system.