Karyotype analysis and visualization of 45S rRNA genes using fluorescence in situ hybridization in aroids (Araceae).

Karyotype analysis and FISH mapping using 45S rDNA sequences on 6 economically important plant species Anthuriumandraeanum Linden ex André, 1877, Monsteradeliciosa Liebmann, 1849, Philodendronscandens Koch & Sello, 1853, Spathiphyllumwallisii Regel, 1877, Syngoniumauritum (Linnaeus, 1759) Schott, 1829 and Zantedeschiaelliottiana (Knight, 1890) Engler, 1915 within the monocotyledonous family Araceae (aroids) were performed. Chromosome numbers varied between 2n=2x=24 and 2n=2x=60 and the chromosome length varied between 15.77 µm and 1.87 µm. No correlation between chromosome numbers and genome sizes was observed for the studied genera. The chromosome formulas contained only metacentric and submetacentric chromosomes, except for Philodendronscandens in which also telocentric and subtelocentric chromosomes were observed. The highest degree of compaction was calculated for Spathiphyllumwallisii (66.49Mbp/µm). B-chromosome-like structures were observed in Anthuriumandraeanum. Their measured size was 1.87 times smaller than the length of the shortest chromosome. After FISH experiments, two 45S rDNA sites were observed in 5 genera. Only in Zantedeschiaelliottiana, 4 sites were seen. Our results showed clear cytogenetic differences among genera within Araceae, and are the first molecular cytogenetics report for these genera. These chromosome data and molecular cytogenetic information are useful in aroid breeding programmes, systematics and evolutionary studies.

Antioxidant changes during domestic food processing of the white shaft and green leaves of leek (Allium ampeloprasum var. porrum).

BACKGROUND: Evaluating the effect of domestic cooking on the health benefits of vegetables has great practical importance. However, only a limited number of reports provide information on the effect of these treatments on the antioxidant capacity, polyphenol and S-alk(en)yl-L-cysteine sulfoxide (ACSO, e.g. isoalliin and methiin) content of the white shaft and green leaves of leek (Allium ampeloprasum var. porrum). RESULTS: In the present study, the antioxidant capacity of leek was highly influenced by cooking (blanching, boiling and steaming). Boiling had a negative effect on total phenolic content in the white shaft and green leaves. An obvious increase could be observed in the antioxidant capacity of the steamed green leaves, while steaming did not influence the polyphenolic content. Remarkably, blanching resulted in a slight increase in the ACSO content. Subjecting leek samples to a longer thermal treatment appeared to have a negative influence on the ACSO content in leek. Steaming was also responsible for a decrease in ACSOs. Methiin was less susceptible to heat treatment than isoalliin. CONCLUSION: In general, steaming appeared to be responsible for better retention of the bioactive compounds present in leek compared with boiling.

Progress in plant protoplast research.

In this review we focus on recent progress in protoplast regeneration, symmetric and asymmetric hybridization and novel technology developments. Regeneration of new species and improved culture techniques opened new horizons for practical breeding in a number of crops. The importance of protoplast sources and embedding systems is discussed. The study of reactive oxygen species effects and DNA (de)condensation, along with thorough phytohormone monitoring, are in our opinion the most promising research topics in the further strive for rationalization of protoplast regeneration. Following, fusion and fragmentation progress is summarized. Genomic, transcriptomic and proteomic studies have led to better insights in fundamental processes such as cell wall formation, cell development and chromosome rearrangements in fusion products, whether or not obtained after irradiation. Advanced molecular screening methods of both genome and cytoplasmome facilitate efficient screening of both symmetric and asymmetric fusion products. We expect that emerging technologies as GISH, high resolution melting and next generation sequencing will pay major contributions to our insights of genome creation and stabilization, mainly after asymmetric hybridization. Finally, we demonstrate agricultural valorization of somatic hybridization through enumerating recent introgression of diverse traits in a number of commercial crops.

How to perform RT-qPCR accurately in plant species? A case study on flower colour gene expression in an azalea (Rhododendron simsii hybrids) mapping population.

BACKGROUND: Flower colour variation is one of the most crucial selection criteria in the breeding of a flowering pot plant, as is also the case for azalea (Rhododendron simsii hybrids). Flavonoid biosynthesis was studied intensively in several species. In azalea, flower colour can be described by means of a 3-gene model. However, this model does not clarify pink-coloration. The last decade gene expression studies have been implemented widely for studying flower colour. However, the methods used were often only semi-quantitative or quantification was not done according to the MIQE-guidelines. We aimed to develop an accurate protocol for RT-qPCR and to validate the protocol to study flower colour in an azalea mapping population. RESULTS: An accurate RT-qPCR protocol had to be established. RNA quality was evaluated in a combined approach by means of different techniques e.g. SPUD-assay and Experion-analysis. We demonstrated the importance of testing noRT-samples for all genes under study to detect contaminating DNA. In spite of the limited sequence information available, we prepared a set of 11 reference genes which was validated in flower petals; a combination of three reference genes was most optimal. Finally we also used plasmids for the construction of standard curves. This allowed us to calculate gene-specific PCR efficiencies for every gene to assure an accurate quantification. The validity of the protocol was demonstrated by means of the study of six genes of the flavonoid biosynthesis pathway. No correlations were found between flower colour and the individual expression profiles. However, the combination of early pathway genes (CHS, F3H, F3'H and FLS) is clearly related to co-pigmentation with flavonols. The late pathway genes DFR and ANS are to a minor extent involved in differentiating between coloured and white flowers. Concerning pink coloration, we could demonstrate that the lower intensity in this type of flowers is correlated to the expression of F3'H. CONCLUSIONS: Currently in plant research, validated and qualitative RT-qPCR protocols are still rare. The protocol in this study can be implemented on all plant species to assure accurate quantification of gene expression. We have been able to correlate flower colour to the combined regulation of structural genes, both in the early and late branch of the pathway. This allowed us to differentiate between flower colours in a broader genetic background as was done so far in flower colour studies. These data will now be used for eQTL mapping to comprehend even more the regulation of this pathway.

Antioxidant changes of leek (Allium ampeloprasum var. porrum) during spontaneous fermentation of the white shaft and green leaves.

BACKGROUND: Leek is grown for its thickened cylindrical white shaft made up of long leaf bases. Despite the potentially valuable nutritional profile of the green leaves, a large portion remains unused owing its restricted culinary applications. This large quantity of this plant biomass could be valorized given an adequate stabilization method. In this study, we examined leek fermentation with regard to antioxidant changes. RESULTS: The oxygen radical absorbance capacity (ORAC) increased by 62% when the green leaves were fermented for 21 days, while 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity did not increase significantly. Fermentation resulted in an increase of endogenous polyphenolic compounds such as ferulic acid, astragalin, luteolin and naringenin. Moreover, fermentation stimulated the production of a series of polyphenolic compounds that were not present in the fresh leek. The flavour precursors in leek, i.e. methiin and isoalliin, were reduced by 91-93% and 100%, respectively, when spontaneous fermentation was allowed to occur on the white shaft and green leaves. CONCLUSION: Our results demonstrated that application of fermentation resulted in a higher ORAC value and polyphenol content of the leek plant, especially in the green leaves. These results indicate the nutritional relevance of fermentation, which hold promise as a stabilization technique.

Influence of cultivar and harvest time on the amounts of isoalliin and methiin in leek (Allium ampeloprasum var. porrum).

Extracts of 31 leek cultivars were analyzed using liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) to determine the distribution of the two most abundant S-alk(en)yl-l-cysteine sulfoxides (ACSOs) in leek, that is, isoalliin and methiin. The isoalliin concentration of the white shaft and green leaves of the 31 leek cultivars varied from 15 to 53 mg/g dry weight (dw) and from 9 to 45 mg/g dw, respectively, whereas the methiin concentration varied from 3 to 16 mg/g dw and from 1 to 10 mg/g dw, respectively. Leek cultivar and tissue had an effect on the ACSO amounts. Cultivars Artico and Apollo F1 rated highest for the mean isoalliin and methiin concentration, respectively. In general, the whole leek plant of the winter leek cultivars contained a significantly higher ACSO amount than the summer and autumn cultivars. To determine whether this difference was attributed to the cultivar background or time of harvest, ACSOs were also quantitated in nine leek hybrids at four different stages during the next growth season. The amounts of ACSO changed significantly during the growth season, indicating the importance of harvest at specific time moments, although there was still an effect of cultivar on the ACSO amounts.

Antioxidant capacity, total phenolic and ascorbate content as a function of the genetic diversity of leek (Allium ampeloprasum var. porrum).

Extracts of the white shaft and green leaves of 30 leek cultivars were investigated for their antioxidant properties, total phenolic (TP) and l-ascorbic acid (AA) content. The measured antioxidant properties included free radical scavenging activities against peroxyl (ORAC) and 2,2-diphenyl-1-picrylhydrazyl radicals (DPPH) and their Fe(3+) reducing capacity (FRAP). The results from this study suggest that the green leek leaves generally have significantly stronger antioxidant properties than the white shaft. Correlation analysis between the TP and the AA content and the antioxidant activity showed that phenolics and ascorbic acid contribute significantly to the antioxidant activity of leek. The three antioxidant activity assays were all correlated for the extracts of the white shaft of the 30 leek cultivars. Principal component analysis (PCA) elucidated the influence of part and type of cultivar on the antioxidant capacity, TP, and l-ascorbic acid content, whilst the breeding strategy and seed company had no influence.

Low melting point agarose beads as a standard method for plantlet regeneration from protoplasts within the Cichorium genus.

KEY MESSAGE: A standard method has been developed with which we are able to fully regenerate protoplasts of different Cichorium species. For the first time, endive protoplasts have been regenerated into plantlets. Protoplast regeneration is essential for somatic hybridizations. In this study, a standard method for plantlet regeneration from Cichorium protoplasts was developed. We evaluated the effect of the low melting point agarose (LMPA) bead technique on the regeneration capacity of protoplasts of seven C. intybus and four C. endivia genotypes. The LMPA bead technique was more efficient than culture in liquid or solid medium and allowed us to obtain plating efficiencies up to 4.9 % in C. intybus genotypes and efficiencies of up to 0.7 % in C. endivia genotypes. Moreover, the LMPA bead technique offers great advantages over liquid and solid culture systems: the media can be readily refreshed, protoplasts can be monitored separately, and microcalli can easily be removed from the beads. This increased efficiency was observed for all of the 11 Cichorium genotypes tested. Shoot formation was induced more efficiently when using 0.5 mg l(-1) indole-3-acetic acid-enriched medium (up to 87.5 % of the protoplast-derived calli started shoot development) compared to 1-naphthaleneacetic acid-enriched medium. The LMPA bead technique optimized in this study enabled for the first time the full plantlet regeneration from protoplasts of C. endivia genotypes and increased the protoplast regenerating ability in other Cichorium species. This fine-tuned LMPA bead technique can therefore be applied for protoplast regeneration after protoplast fusions of the genus Cichorium.

A critique of widely used normalization software tools and an alternative method to identify reliable reference genes in red clover (Trifolium pratense L.).

Determination of appropriate reference genes is crucial to normalization of gene expression data and prevention of biased results in qRT-PCR studies. This study is the first attempt to systematically compare potential reference genes to detect the most constitutively expressed reference genes for accurate normalization in red clover tissues including leaves, stems and roots. To identify the best-suited reference gene(s) for normalization, several statistical algorithms such as geNorm, BestKeeper and NormFinder have been developed. All these algorithms are based on the key assumption that none of the investigated candidate reference genes show systematic variation in their expression profile across the samples being considered. However, this assumption is likely to be violated in practice. The authors therefore suggest a simple and novel stability index based on the analysis of variance model which is free from the assumption made by the algorithms. We assessed the expression stability of eight candidate reference genes including actin (ACT), glyceraldehyde-3-phosphate-dehydrogenase (GADPH), elongation factor-1alpha (EF-1α), translation initiation factor (EIF-4a), ubiquitin-conjugating enzyme E2 (UBC2), polyubiquitin (UBQ10), sand family protein (SAND) and yellow-leaf-specific protein 8 (YLS8). Our results indicated that UBC2 and UBQ10 ranked as the two most stably expressed genes in leaf tissue. UBC2 and YLS8 were defined as optimal control genes for stem tissue. EIF-4a and UBC2 were found to be the most stable reference gene for root tissue. GAPDH and SAND showed relatively low stability in expression study of red clover. When all tested tissues were considered, we observed that YLS8 and UBC2 showed remarkable stability in their expression level across tissues.

Towards an optimal sampling strategy for assessing genetic variation within and among white clover (Trifolium repens L.) cultivars using AFLP.

Cost reduction in plant breeding and conservation programs depends largely on correctly defining the minimal sample size required for the trustworthy assessment of intra- and inter-cultivar genetic variation. White clover, an important pasture legume, was chosen for studying this aspect. In clonal plants, such as the aforementioned, an appropriate sampling scheme eliminates the redundant analysis of identical genotypes. The aim was to define an optimal sampling strategy, i.e., the minimum sample size and appropriate sampling scheme for white clover cultivars, by using AFLP data (283 loci) from three popular types. A grid-based sampling scheme, with an interplant distance of at least 40 cm, was sufficient to avoid any excess in replicates. Simulations revealed that the number of samples substantially influenced genetic diversity parameters. When using less than 15 per cultivar, the expected heterozygosity (He) and Shannon diversity index (I) were greatly underestimated, whereas with 20, more than 95% of total intra-cultivar genetic variation was covered. Based on AMOVA, a 20-cultivar sample was apparently sufficient to accurately quantify individual genetic structuring. The recommended sampling strategy facilitates the efficient characterization of diversity in white clover, for both conservation and exploitation.

Applications of flow cytometry in plant pathology for genome size determination, detection and physiological status.

Flow cytometers are probably the most multipurpose laboratory devices available. They can analyse a vast and very diverse range of cell parameters. This technique has left its mark on cancer, human immunodeficiency virus and immunology research, and is indispensable in routine clinical diagnostics. Flow cytometry (FCM) is also a well-known tool for the detection and physiological status assessment of microorganisms in drinking water, marine environments, food and fermentation processes. However, flow cytometers are seldom used in plant pathology, despite FCM's major advantages as both a detection method and a research tool. Potential uses of FCM include the characterization of genome sizes of fungal and oomycete populations, multiplexed pathogen detection and the monitoring of the viability, culturability and gene expression of plant pathogens, and many others. This review provides an overview of the history, advantages and disadvantages of FCM, and focuses on the current applications and future possibilities of FCM in plant pathology.

Aberrant genome size and instability of Phytophthora ramorum oospore progenies.

The functionality of the sexual cycle in the heterothallic pathogen Phytophthora ramorum, causal agent of Sudden Oak Death, has recently been demonstrated. Sexual reproduction could create genotypic variation and increase the pathogen's ability to adapt to other host plants or changing environments. Genetic characterization using co-dominant microsatellite markers and flow cytometry of single-oospore progeny of crosses between a European A1 isolate and North American or European A2 isolates revealed a considerable number of non-Mendelian inheritance events. This includes inheritance of more than two alleles at a locus and non-inheritance of alleles from one parent at another locus. The progenies were mitotically unstable: zoospore and hyphal tip derivatives of the progenies showed genotypic rearrangements and phenotypic variation. Flow cytometry confirmed variation and instability in DNA content of the single-oospore progenies. This indicates that single-oospore progenies not only display aberrant genomic and phenotypic variation due to meiotic irregularities, but also extra variation as a result of post-meiotic genomic rearrangements.

Validation of reference genes for gene expression analysis in chicory (Cichorium intybus) using quantitative real-time PCR.

BACKGROUND: Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) is a sensitive technique for quantifying gene expression levels. One or more appropriate reference genes must be selected to accurately compare mRNA transcripts across different samples and tissues. Thus far, only actin-2 has been used as a reference gene for qRT-PCR in chicory, and a full comparison of several candidate reference genes in chicory has not yet been reported. RESULTS: Seven candidate reference genes, including nicotinamide adenine dinucleotide dehydrogenase (NADHD), actin (ACT), beta-tubulin (TUB), glyceraldehyde-3-phosphate-dehydrogenase (GADPH), histone H3 (H3), elongation factor 1-alpha (EF) and 18S rRNA (rRNA) were selected to study the expression stability for normalisation of gene expression in chicory. Primer specificity and amplification efficiency were verified for each gene. The expression stability of these genes was analysed across chicory root and leaf tissues using geNorm, NormFinder and BestKeeper software. ACT, EF, and rRNA were the most stable genes as identified by the three different analysis methods. In addition, the use of ACT, EF and GAPDH as reference genes was illustrated by analysing 1-FEHII (FEHII) expression in chicory root and leaf tissues. These analyses revealed the biological variation in FEHII transcript expression among the tissues studied, and between individual plants. CONCLUSIONS: geNorm, NormFinder, and BestKeeper analyses indicated that ACT, EF and rRNA had the highest expression stability across leaf and root tissues, while GAPDH and NADHD showed relatively low expression stability. The results of this study emphasise the importance of validating reference genes for qRT-PCR analysis in chicory. The use of the most stable reference genes such as ACT and EF allows accurate normalisation of gene expression in chicory leaf and root tissues.

Multipoint-likelihood maximization mapping on 4 segregating populations to achieve an integrated framework map for QTL analysis in pot azalea (Rhododendron simsii hybrids).

BACKGROUND: Azalea (Rhododendron simsii hybrids) is the most important flowering pot plant produced in Belgium, being exported world-wide. In the breeding program, flower color is the main feature for selection, only in later stages cultivation related plant quality traits are evaluated. As a result, plants with attractive flowering are kept too long in the breeding cycle. The inheritance of flower color has been well studied; information on the heritability of cultivation related quality traits is lacking. For this purpose, QTL mapping in diverse genetic backgrounds appeared to be a must and therefore 4 mapping populations were made and analyzed. RESULTS: An integrated framework map on four individual linkage maps in Rhododendron simsii hybrids was constructed. For genotyping, mainly dominant scored AFLP (on average 364 per population) and MYB-based markers (15) were combined with co-dominant SSR (23) and EST markers (12). Linkage groups were estimated in JoinMap. A consensus grouping for the 4 mapping populations was made and applied in each individual mapping population. Finally, 16 stable linkage groups were set for the 4 populations; the azalea chromosome number being 13. A combination of regression mapping (JoinMap) and multipoint-likelihood maximization (Carthagène) enabled the construction of 4 maps and their alignment. A large portion of loci (43%) was common to at least two populations and could therefore serve as bridging markers. The different steps taken for map optimization and integration into a reference framework map for QTL mapping are discussed. CONCLUSIONS: This is the first map of azalea up to our knowledge. AFLP and SSR markers are used as a reference backbone and functional markers (EST and MYB) were added as candidate genes for QTL analysis. The alignment of the 4 maps on the basis of framework markers will facilitate in turn the alignment of QTL regions detected in each of the populations. The approach we took is thoroughly different than the recently published integrated maps and well-suited for mapping in a non-model crop.

Genome size variation in Begonia.

The genome sizes of a Begonia collection comprising 37 species and 23 hybrids of African, Asiatic, Middle American, and South American origin were screened using flow cytometry. Within the collection, 1C values varied between 0.23 and 1.46 pg DNA. Genome sizes were, in most cases, not positively correlated with chromosome number, but with pollen size. A 12-fold difference in mean chromosome size was found between the genotypes with the largest and smallest chromosomes. In general, chromosomes from South American genotypes were smaller than chromosomes of African, Asian, or Middle American genotypes, except for B. boliviensis and B. pearcei. Cytological chromosome studies in different genotypes showed variable chromosome numbers, length, width, and total chromosome volume, which confirmed the diversity in genome size. Large secondary constrictions were present in several investigated genotypes. These data show that chromosome number and structure exhibit a great deal of variation within the genus Begonia, and likely help to explain the large number of taxa found within the genus.

Morphological and AFLP-based differentiation within the taxonomical complex section Caninae (subgenus Rosa).

BACKGROUND AND AIMS: The taxonomical structure of the polymorphic subgenus Rosa section Caninae is highly complex due to the combination of some unusual features: the unique polyploid chromosomal constitution, the heterogamic canina meiosis, the ability to hybridize interspecifically, and the predominantly matroclinal inheritance. Although most taxonomists agree on the subdivision of the section into three morphologically well-defined groups (Rubigineae, Vestitae, and Caninae), they disagree on the existence of smaller groups such as Tomentellae. The aim was to gain insight in the taxonomical structure and investigate the interpopulation differentiation of the polymorphic section Caninae by analysing morphological and AFLP-based characters of the seven most common Belgian dog-rose taxa. METHODS: The intersubsectional and -specific relationships within the dog-roses were examined using morphological and molecular-genetic markers. AFLP data were analysed with basic descriptive genetic statistics because of the lack of Hardy-Weinberg equilibrium due to the polyploid genetic structure and heterogamic meiosis. KEY RESULTS: Both the morphological and AFLP-based analyses supported the subdivision of the dog-roses in three well-defined though partly overlapping groups, Rubigineae, Vestitae and Caninae. However, it was not possible to distinguish between the morphologically well-defined taxa within the same subsection using AFLP-based data. In addition, the results suggested a high similarity of Rosa balsamica with subsection Caninae taxa. Small-scale geographical AFLP-based differentiation was observed within several dog-rose taxa. Surprisingly, individuals sampled at one locality and belonging to morphologically distinct dog-rose taxa displayed higher genetic similarities in comparison to their congeners sampled at different localities. CONCLUSIONS: The hybridogenic character of the dog-roses was reflected in the vague boundaries between the subsections and on the species level within the subsections. Indications were found for current or historical hybridization on the genetic structure of the population. No morphological or AFLP-based evidence was obtained to support the existence of the separate subsection Tomentellae.

Mapping of markers related to self-incompatibility, disease resistance, and quality traits in Lolium perenne L.

Several linkage maps, mainly based on anonymous markers, are now available for Lolium perenne. The saturation of these maps with markers derived from expressed sequences would provide information useful for QTL mapping and map alignment. Therefore, we initiated a study to develop and map DNA markers in genes related to self-incompatibility, disease resistance, and quality traits such as digestibility and sugar content in two L. perenne families. In total, 483 and 504 primer pairs were designed and used to screen the ILGI and CLO-DvP mapping populations, respectively, for length polymorphisms. Finally, we were able to map 67 EST markers in at least one mapping population. Several of these markers coincide with previously reported QTL regions for the traits considered or are located in the neighbourhood of the self-incompatibility loci, S and Z. The markers developed expand the set of gene-derived markers available for genetic mapping in ryegrasses.

Evaluation of genetic diversity of Fusarium head blight resistance in European winter wheat.

Genetic diversity in relation to Fusarium head blight (FHB) resistance was investigated among 295 European winter wheat cultivars and advanced breeding lines using 47 wheat SSR markers. Twelve additional wheat lines with known FHB resistance were included as reference material. At least one SSR marker per chromosome arm, including SSR markers reported in the literature with putative associations with QTLs for FHB resistance, were assayed to give an even distribution of SSR markers across the wheat genome. A total of 404 SSR alleles were detected. The number of alleles per locus ranged from 2 to 21, with an average of 8.6 alleles. The polymorphism information content of the SSR markers ranged from 0.13 (Xwmc483) to 0.87 (Xwmc607), with an average of 0.54. Cluster analysis was performed by both genetic distance-based and model-based methods. In general, the dendrogram based on unweighted pair-group method with arithmetic averages showed similar groupings to the model-based analysis. Seven clusters were identified by the model-based method, which did not strictly correspond to geographical origin. The FHB resistance level of the wheat lines was evaluated in field trials conducted over multiple years or locations by assessing the following traits: % FHB severity, % FHB incidence, % diseased kernels, in spray inoculation trials, and % FHB spread and % wilted tips, in point inoculation trials. Association analysis between SSR markers and the FHB disease traits detected markers significantly associated with FHB resistance, including some that have not been previously reported. The percentage of variance explained by each individual marker was, however, rather low. Haplotype analysis revealed that the FHB-resistant European wheat lines do not contain the 3BS locus derived from Sumai 3. The information generated in this study will assist in the selection of parental lines in order to increase the efficiency of breeding efforts for FHB resistance.

Stability of the T-DNA flanking regions in transgenic Arabidopsis thaliana plants under influence of abiotic stress and cultivation practices.

Genetic transformation is often associated with different rearrangements of the plant genome at the site of insertion. Therefore the question remains weather these T-DNA insertion sites are more prone to genotoxic stresses. Here, we studied the impact of propagation through generations, the influence of gene stacking and of photo oxidative stress caused by high light intensity on the stability of the transgene flanking regions in the model plant Arabidopsis thaliana. Conformational Sensitive Capillary Electrophoresis (CSCE), RFLP and sequencing were deployed in this analysis in order to study the proximal 100 bp and the long-range T-DNA flanking sequences. By screening seven transgenic lines no evidence for occurrence of mutation events were found, implying that the nucleotide sequence of the T-DNA flanking regions of the studied events is unlikely to be unstable.

Efficient extraction of high-quality total RNA from various hop tissues (Humulus lupulus L.).

The isolation of high-quality RNA is a prerequisite for gene expression studies. RNA quality is of special relevance if PCR-based strategies such as cDNA-AFLP or RT-PCR are followed. Our molecular investigations of hop (Humulus lupulus L.) focus on genes that determine the biosynthesis of prenylflavonoids with interesting biological activities which accumulate in the lupulin glands. However, optimized protocols for RNA extraction from hop cones are not available. In this study, the RNeasy midi kit protocol was modified to isolate high amounts of total RNA from fresh and freeze-dried hop tissues, specifically leaves, female inflorescences, and lupulin-enriched hop cone fractions. The main difficulties in obtaining high RNA yields were related to specific features of hop, including the abundance of secondary metabolites and their accumulation in the sticky lupulin glands. The introduction of a number of modifications into the RNeasy midi kit protocol resulted in RNA of high quality, as assessed by spectrophotometry and electrophoresis in agarose gels. The protocol developed is currently being used to produce cDNA-AFLP fingerprints of hop cones and leaves for genetic screening. Furthermore, the method could provide RNA for further molecular studies, including Northern blot hybridizations and reverse transcription-polymerase chain reactions.