Comparative seed longevity under genebank storage and artificial ageing: a case study in heteromorphic wheat wild relatives.

Seed longevity is a complex trait that depends on numerous factors. It varies among species and populations, and within different seed morphs produced by the same plant. Little is known about variation in longevity in different seed morphs or the physiological and molecular basis of these differences. We evaluated the longevity and oxidative stress status in heteromorphic seeds aged in two different storage conditions. We compared controlled ageing tests (seed storage at 45°C and 60% relative humidity; a method of accelerated ageing used to estimate longevity in genebank conditions) with storage in a genebank for up to 40 years (-18°C and 8% seed moisture content). We employed as study species two wild wheats characterized by seed heteromorphism: Aegilops tauschii and Triticum monococcum subsp. aegilopoides. We estimated the ROS content and the expression of genes coding for enzymes related to the H2 O2 scavenging pathway. Results confirmed that seed longevity varies between different seed morphs. Different storage environments resulted in different longevity and survival curves. ROS levels, even if with variable patterns, were higher in several aged seed lots. We observed consistency in the expression of two genes (GSR and CAT) related to ROS scavenging in the late phase of pre-germinative metabolism. Differences in seed longevity between morphs were observed for the first time under genebank conditions. Our results suggest also that controlled ageing tests should be used with caution to infer ranks of longevity under cold storage.

The consensus rye microsatellite map with EST-SSRs transferred from wheat.

Microsatellite (SSR) markers with known precise intrachromosomal locations are widely used for mapping genes in rye and for the investigation of wheat-rye translocation lines and triticale highly demanded for mapping economically important genes and QTL-analysis. One of the sources of novel SSR markers in rye are microsatellites transferable from the wheat genome. Broadening the list of available SSRs in rye mapped to chromosomes is still needed, since some rye chromosome maps still have just a few microsatellite loci mapped. The goal of the current study was to integrate wheat EST-SSRs into the existing rye genetic maps and to construct a consensus rye microsatellite map. Four rye mapping populations (P87/P105, N6/N2, N7/N2 and N7/N6) were tested with CFE (EST-SSRs) primers. A total of 23 Xcfe loci were mapped on rye chromosomes: Xcfe023, -136 and -266 on chromosome 1R, Xcfe006, -067, -175 and -187 on 2R, Xcfe029 and -282 on 3R, Xcfe004, -100, -152, -224 and -260 on 4R, Xcfe037, -208 and -270 on 5R, Xcfe124, -159 and -277 on 6R, Xcfe010, -143 and -228 on 7R. With the exception of Xcfe159 and Xcfe224, all the Xcfe loci mapped were found in orthologous positions considering multiple evolutionary translocations in the rye genome relative to those of common wheat. The consensus map was constructed using mapping data from the four bi-parental populations. It contains a total of 123 microsatellites, 12 SNPs, 118 RFLPs and 2 isozyme loci.

Hybrid dwarfness in crosses between wheat (Triticum aestivum L.) and rye (Secale cereale L.): a new look at an old phenomenon.

The existence of hybrid dwarfs from intraspecific crosses in wheat (Triticum aestivum) was described 100 years ago, and the genetics underlying hybrid dwarfness are well understood. In this study, we report a dwarf phenotype in interspecific hybrids between wheat and rye (Secale cereale). We identified two rye lines that produce hybrid dwarfs with wheat and have none of the hitherto known hybrid dwarfing genes. Genetic analyses revealed that both rye lines carry a single allelic gene responsible for the dwarf phenotype. This gene was designated Hdw-R1 (Hybrid dwarf-R1). Application of gibberellic acid (GA3 ) to both intraspecific (wheat-wheat) and interspecific (wheat-rye) hybrids showed that hybrid dwarfness cannot be overcome by treatment with this phytohormone. Histological analysis of shoot apices showed that wheat-rye hybrids with the dwarf phenotype at 21 and 45 days after germination failed to develop further. Shoot apices of dwarf plants did not elongate, did not form new primordia and had a dome-shaped appearance in the seed. The possible relationship between hybrid dwarfness and the genes responsible for the transition from vegetative to generative growth stage is discussed.

[Mapping of quantitative trait loci (QTL) associated with activity of disulfide reductase and lipoxygenase in grains of durum wheat Triticum aestivum L. seeds].

Activity of two enzymes of thiol-disulfide cell metabolism, lipoxygenase (LOX, EC 1.13.11.12) and disulfide-reductase (TPDO, EC 1.8.4.2) was studied in recombinant inbred lines of common wheat ITMI. Their activity in the caryopsis may be connected with the gluten quality, one of the most important traits significant for selection. The activity of lipoxygenase under favorable and droughty environmental conditions was shown to be associated with the quantitative trait locus (QTL) located on chromosome 4BS near the structural gene of a subunit of this enzyme. However, no QTL common to this enzyme and any characteristic of gluten quality have been found. Four loci responsible for the activity of disulfide reductase were identified on chromosomes 4A, 5D, 6A, and7D. Previously, indicators of grain and flour properties, such as elasticity, flour vigor, and grain hardiness were mapped at the same loci. This indicates that the given enzyme participates in the formation of the protein complex upon maturation of wheatgrain. The detected QTL can be involved in further genetic studies designed to establish the regularities of gluten formation.

[Mapping of the quantitative trait loci (QTL) associated with grain quality characteristics of the common wheat grown under different environmental conditions].

The quantitative trait loci (QTL) associated with individual characteristics of grain and flour quality in wheat lines grown under contrasting environmental conditions were mapped. Overall, 22 QTL that manifested under contrasting environmental conditions with various significances were detected on 10 chromosomes. Grain hardness and vitreousness were associated with three loci on chromosomes 5D, 6A, and 3A, while the gluten content, with two loci on chromosomes 5B and 7A. Dough extensibility was associated with only one QTL localized in the region of Glu-A1 locus. One of the loci determining flour and dough strengths is located in the region of Gli-B1 and Glu-B3 loci and the rest, in various regions of chromosomes 1B, 5D, and 4B, where no particular genes associated with grain quality have been yet found. The detected QTL can be used in further experiments on genetic control of gluten formation and quality in wheat.

Single nucleotide polymorphisms in rye (Secale cereale L.): discovery, frequency, and applications for genome mapping and diversity studies.

To elucidate the potential of single nucleotide polymorphism (SNP) markers in rye, a set of 48 barley EST (expressed sequence tag) primer pairs was employed to amplify from DNA prepared from five rye inbred lines. A total of 96 SNPs and 26 indels (insertion-deletions) were defined from the sequences of 14 of the resulting amplicons, giving an estimated frequency of 1 SNP per 58 bp and 1 indel per 214 bp in the rye transcriptome. A mean of 3.4 haplotypes per marker with a mean expected heterozygosity of 0.66 were observed. The nucleotide diversity index (pi) was estimated to be in the range 0.0059-0.0530. To improve assay cost-effectiveness, 12 of the 14 SNPs were converted to a cleaved amplified polymorphic sequence (CAPS) format. The resulting 12 SNP loci mapped to chromosomes 1R, 3R, 4R, 5R, 6R, and 7R, at locations consistent with their known map positions in barley. SNP genotypic data were compared with genomic simple sequence repeat (SSR) and EST-derived SSR genotypic data collected from the same templates. This showed a broad equivalence with respect to genetic diversity between these different data types.

[Analysis of the short-stem genes in the genotypes of bread wheat cultivars in Ukraine].

Varieties of winter common wheat widely used in breeding programmes for the period of 1912-2002 in Ukraine were investigated using both the microsatellite Xgwm261 marker linked with the gibberellin-sensitive dwarf gene Rht8 and the biochemical test for the sensitivity of seedlings to gibberellic acid. Allelic characteristics of the locus Xgwm261 have been determined for 97 varieties. Near 98% of the modern varieties of winter common wheat derived from Odessa breeding institute and 50% of the varieties derived from the Mironovka wheat institute possess the allel 192 n.p. and gene Rht8 correspondingly. Estimation of sensitivity of the seedlings to gibberellic acid showed that the majority of the varieteis cultivated in Ukraine until the middle of the seventies are sensitive to gibberellic acid. The insusceptibility to gibberellic acid is predominantly manifested in the varieties derived from the south of Ukraine since the eighties owing to the introduction of the dwarf genes into the genotypes. At the same time the varieties of the Mironovka wheat institute still retain the sensitivity.

Comparative mapping of genes for glume colouration and pubescence in hexaploid wheat (Triticum aestivum L.).

Microsatellite markers were used to map the major genes Bg (determining black glume colour), Rg1 and Rg3 (red glume), and a locus determining smokey-grey coloured glume to the distal ends of the short arms of the homoeologous group 1 chromosomes, proximally (or closely linked) to Xgwm1223 and distal to Xgwm0033. On this basis, we propose that these genes represent a set of homoeoloci, designated Rg-A1, Rg-B1, and Rg-D1. Rg3 and Bg appear to be variant alleles of Rg-A1. Both Rg3 and Bg are closely linked with the major glume pubescence gene Hg. Similarly, the hexaploid wheat smokey-grey glume gene and Rg2 represent alleles at Rg-D1. The microsatellite markers linked to the Rg genes were used to analyse a phenotypically and genotypically characterized set of Siberian spring wheats. A coincidence between the presence of the 264-bp allele of Xgwm0136 and Rg-A1b (Rg3) was observed; so Xgwm0136 can probably be used as a diagnostic marker for this gene.

Rationalising germplasm collections: a case study for wheat.

In total 70 genebank accessions comprising 50 hexaploid, 12 tetraploid and 8 diploid wheats of the Gatersleben collection were selected based on the screening of the passport data for identical cultivar names or accession numbers of the donor genebanks. Twelve potential duplicate groups consisting of three to nine accessions with identical names/numbers were selected and analysed with DNA markers (microsatellites). A bootstrap approach based on re-sampling of both microsatellite markers and alleles within marker loci was used to test for homogeneity. Although several homogeneous groups were identified it became clear that cultivar name identity alone did not allow the determination of duplicates. A combination of SSR-analysis followed by the bootstrap method and database survey considering the botanical classification and other data (origin, growth habit and donor) available is recommended in order to determine duplicates. A procedure for the identification of duplicates and their further handling in ex situ genebanks is discussed.

The gibberellic-acid insensitive dwarfing gene sdw3 of barley is located on chromosome 2HS in a region that shows high colinearity with rice chromosome 7L.

In this study, comparative high resolution genetic mapping of the GA-insensitive dwarfing gene sdw3 of barley revealed highly conserved macrosynteny of the target region on barley chromosome 2HS with rice chromosome 7L. A rice contig covering the sdw3-orthologous region was identified and subsequently exploited for marker saturation of the target interval in barley. This was achieved by (1) mapping of rice markers from the orthologous region of the rice genetic map, (2) mapping of rice ESTs that had been physically localized on the rice contig, or (3) mapping of barley ESTs that show strong sequence similarity to coding sequences present in the rice contig. Finally, the sdw3 gene was mapped to an interval of 0.55 cM in barley, corresponding to a physical distance of about 252 kb in rice, after employing orthologous EST-derived rice markers. Three putative ORFs were identified in this interval in rice, which exhibited significant sequence similarity to known signal regulator genes from different species. These ORFs can serve as starting points for the map-based isolation of the sdw3 gene from barley.

Genetic diversity in cultivated plants-loss or stability?

Human activities like urbanisation, the replacement of traditional agriculture systems by modern industrial methods or the introduction of modern high-yielding varieties may pose a danger to the biological diversity. Using microsatellite markers, we analysed samples of cultivated wheat ( Triticum aestivum L.) collected over an interval of 40-50 years in four comparable geographical regions of Europe and Asia. No significant differences in both the total number of alleles per locus and in the PIC values were detected when the material collected in the repeated collection missions in all four regions were compared. About two-thirds of the alleles were common to both collection periods, while one-third represented collection mission-specific alleles. These findings demonstrate that an allele flow took place during the adaptation of traditional agriculture to modern systems, whereas the level of genetic diversity was not significantly influenced.

Molecular studies on genetic integrity of open-pollinating species rye (Secale cereale L.) after long-term genebank maintenance.

The genetic integrity of six accessions represented by 14 sub-populations of the open-pollinating species rye ( Secale cereale L.) was investigated. Seeds available from a herbarium collection (first regeneration) and from the cold store (most recent regeneration) were multiplied two to fourteen times and fingerprinted using microsatellite markers. Four accessions had significantly different allele frequencies. These were multiplied seven to thirteen times. Nearly 50% of the alleles discovered in the original samples were not found in the material present in the cold store. However alleles were detected in the most recently propagated sub-populations, that were not observed in the investigated plants of the original one. The change in allele frequencies is a continuous process. Reasons for the occurrence of genetic changes and consequences for managing open pollinating species maintained in ex situ genebanks are discussed.

Influence of rhTSH on [(18)F]fluorodeoxyglucose uptake by differentiated thyroid carcinoma.

The influence of serum TSH levels on fluorine-18 fluorodeoxyglucose (FDG) uptake by recurrences or metastases of differentiated thyroid carcinomas has not yet been clarified. The aim of this study was to ascertain whether the administration of recombinant human thyrotropin (rhTSH) stimulates FDG uptake by such lesions. In this prospective study, 30 patients with positive or equivocal thyroglobulin (Tg) levels and negative or equivocal iodine-131 and/or morphological imaging results (ultrasound, MRI, CT) underwent FDG positron emission tomography (PET) under exogenous TSH suppression and under exogenous TSH stimulation of serum levels by injection of rhTSH. The mean interval between the FDG-PET studies under these two conditions was 9.3+/-8.8 weeks. Serum TSH levels and free thyroid hormones were determined on each occasion. FDG uptake was quantitated using tumour to background ratios (TBRs) and standardised uptake values (SUVs). Under TSH suppression there was focal FDG accumulation in nine subjects (22 tumour-like lesions). The total number of foci was 45. After exogenous TSH stimulation, the number of patients in whom FDG foci were detected was 19, and the number of foci identified was 82 (78 tumour-like lesions). TBR of regions showing positive FDG contrast with either of the modalities averaged 2.54+/-1.89, and under stimulated TSH levels, 5.51+/-2.99 ( P<0.0001). Corresponding SUVs were 2.05+/-1.45 versus 2.77+/-1.58 ( P<0.001). In a small number ( n=4) of foci related to inflammatory lymph nodes, TBR and SUV were only marginally increased under TSH stimulation (2.01+/-0.38 and 1.07+/-0.38, respectively), and the values did not differ significantly from those obtained under suppression. These results provide the first direct evidence that TSH stimulates FDG uptake by differentiated thyroid carcinoma and that, therefore, FDG-PET is more accurate under rhTSH than under suppression.

Assessing genetic diversity of wheat ( Triticum aestivum L.) germplasm using microsatellite markers.

A set of 24 wheat microsatellite markers, representing at least one marker from each chromosome, was used for the assessment of genetic diversity in 998 accessions of hexaploid bread wheat ( Triticum aestivum L.) which originated from 68 countries of five continents. A total of 470 alleles were detected with an average allele number of 18.1 per locus. The highest number of alleles per locus was detected in the B genome with 19.9, compared to 17.4 and 16.5 for genomes A and D, respectively. The lowest allele number per locus among the seven homoeologous groups was observed in group 4. Greater genetic variation exists in the non-centromeric regions than in the centromeric regions of chromosomes. Allele numbers increased with the repeat number of the microsatellites used and their relative distance from the centromere, and was not dependent on the motif of microsatellites. Gene diversity was correlated with the number of alleles. Gene diversity according to Nei for the 26 microsatellite loci varied from 0.43 to 0.94 with an average of 0.77, and was 0.78, 0.81 and 0.73 for three genomes A, B and D, respectively. Alleles for each locus were present in regular two or three base-pair steps, indicating that the genetic variation during the wheat evolution occurred step by step in a continuous manner. In most cases, allele frequencies showed a normal distribution. Comparative analysis of microsatellite diversity among the eight geographical regions revealed that the accessions from the Near East and the Middle East exhibited more genetic diversity than those from the other regions. Greater diversity was found in Southeast Europe than in North and Southwest Europe. The present study also indicates that microsatellite markers permit the fast and high throughput fingerprinting of large numbers of accessions from a germplasm collection in order to assess genetic diversity.

Molecular mapping, phenotypic expression and geographical distribution of genes determining anthocyanin pigmentation of coleoptiles in wheat ( Triticum aestivumL.).

Three major gene loci determining the anthocyanin pigmentation of coleoptiles were mapped on the short arms of chromosomes 7A, 7B and 7D, respectively. All three genes map about 15 to 20 cM distal from the centromere and, therefore, it may be concluded that they are members of a homoeologous series and should be designated Rc-A1, Rc-B1 and Rc-D1, respectively. Further homoeologous loci exist in Triticum durum, Triticum tauschii, and most probably in Secale cereale and Hordeum vulgare. By analyzing a syntheticxcultivated wheat cross (ITMI mapping population) under different environmental conditions it was shown that the expression of the genes determining anthocyanin pigmentation of the coleoptiles varies. One additional locus was detected on chromosome 4BL. Beside the mapping data, results of a screening for red coleoptile color genes in 468 mainly European wheat varieties are presented.

Mapping of quantitative trait loci determining agronomic important characters in hexaploid wheat ( Triticum aestivum L.).

A set of 114 recombinant inbred lines of the 'International Triticeae Mapping Initiative' mapping population was grown during the seasons 1997, 1998, 1999 and 2000 under several environments. Twenty morphological (glume colour, awn colour, waxiness, leaf erectness, peduncle length), agronomical (ear emergence time, flowering time, grain filling time, ear length, plant height, lodging, grain number, thousand-grain-weight, grain weight per ear, grain protein content, winter hardiness) and disease resistance (powdery mildew, yellow rust, leaf rust, fusarium) traits were studied. Not all traits were scored in each experiment. In total 210 QTLs with a LOD threshold of >2.0 (minor QTLs) were detected of which 64 reached a LOD score of >3.0 (major QTLs). Often QTLs were detected in comparable positions in different experiments. Homologous and homoeologous relationships of the detected QTLs, and already described major genes or QTLs determining the same traits in wheat or other Triticeae members, are discussed.

Biomimetic asymmetric hydrogenation.

Enzymes and synthetic organometallic catalysts utilize different approaches for the creation of chiral centers in prochiral substrates. While chiral organometallic catalysts realize the transfer of chirality mainly by repulsive interactions, several enzymes use preferentially stereodiscriminating hydrogen bonding. To investigate if hydrogen bonding within the catalyst-substrate assembly can also have a benefit on the rhodium diphosphine-catalyzed asymmetric hydrogenation, some model metal complexes and substrates were investigated. As 'biomimetically acting' functionalities, hydroxy groups were incorporated in the chiral ligand. Three secondary interactions could be identified by different analytical methods which influence rate and enantioselectivity of the catalytic reaction: 1) HO/Rh-interactions, 2) HO/HO-interactions within the backbone of the ligand, and 3) hydrogen bonding between HO-groups of the ligand and functional groups of an appropriate substrate. Due to the effect of the additional hydroxy groups, enantioselectivities by up to 99% ee could be induced in the hydrogenation product even with water as solvent.