Implementation of a model for identifying Essentially Derived Varieties in vegetatively propagated Calluna vulgaris varieties

<p>Abstract</p> <p>Background</p> <p>Variety protection is of high relevance for the horticultural community and juridical cases have become more frequent in a globalized economy due to essential derivation of varieties. This applies equally to <it>Calluna vulgaris</it>, a vegetatively propagated species from the <it>Ericaceae </it>family that belongs to the top-selling pot plants in Europe. We therefore analyzed the genetic diversity of 74 selected varieties and genotypes of <it>C. vulgaris </it>and 3 of <it>Erica </it>spp. by means of RAPD and iSSR fingerprinting using 168 mono- and polymorphisms. The same data set was utilized to generate a system to reliably identify Essentially Derived Varieties (EDVs) in <it>C. vulgaris</it>, which was adapted from a method suggested for lettuce and barley. This system was developed, validated and used for selected tests of interest in <it>C. vulgaris</it>.</p> <p>Results</p> <p>As expected following personal communications with breeders, a very small genetic diversity became evident within <it>C. vulgaris </it>when investigated using our molecular methods. Thus, a dendrogram-based assay to detect Essentially Derived Varieties in this species is not suitable, although varieties are propagated vegetatively. In contrast, the system applied in lettuce, which itself applies pairwise comparisons using appropriate reference sets, proved functional with this species.</p> <p>Conclusion</p> <p>The narrow gene pool detected in <it>C. vulgaris </it>may be the genetic basis for juridical conflicts between breeders. We successfully tested a methodology for identification of Essentially Derived Varieties in highly identical <it>C. vulgaris </it>genotypes and recommend this for future proof of essential derivation in <it>C. vulgaris </it>and other vegetatively propagated crops.</p

Nutzung molekularer Marker in der Züchtung von Heide (Calluna vulgaris)

ZusammenfassungZur Unterstützung der Züchtung von knospenblühender Sommerheide (Calluna vulgaris) wurde die Vererbung des Merkmals „Knospenblütigkeit“ in verschiedenen spaltenden Rückkreuzungspopulationen untersucht. Aufgrund der analysierten Spaltungsverhältnisse wird von einem monogen-rezessiven Erbgang ausgegangen. RAPD- und ISSR-Marker wurden zur Untersuchung der Größe des genetischen Pools der Art C. vulgaris verwendet, und ein Verfahren zur Identifikation sogenannter „abgeleiteter Sorten“ wurde für diese Pflanzenart angepasst. Zudem wurde nach molekularen Markern für das ökonomisch wichtige Merkmal „Blütentyp“ gesucht, die eine Selektion auf dieses Merkmal bereits im Jungpflanzenstadium ermöglichen würden. Sowohl für RAPD- als auch für AFLP-Marker wurden ausschließlich Marker für das dominante Allel „Einfachblüher“ gefunden.Die dargestellten Ergebnisse fassen mehrere Arbeiten zur Züchtungsforschung an knospenblühender Sommerheide zusammen.Stichwörter: AFLP, ISSR, Knospenblüher, Marker-gestützte Selektion, RAPD, SortenidentifizierungUse of molecular markers in breeding of heather (Calluna vulgaris)AbstractIn order to assist breeding of bud-flowering heather (Calluna vulgaris) the inheritance of the trait “budflowering” has been analyzed in various segregating backcross populations. From the resulting segregation ratios a monogenic recessive inheritance was deduced. RAPD- and ISSR-markers have been used for evaluation of the genetic pool of C. vulgaris, and a technique for identification of “essentially derived varieties” has been adapted for this species. Moreover, it has been searched for molecular markers of the economically important trait “flower type” that would allow selection already in the seedling stage. However, both RAPD as well as AFLP-markers have only been found for the dominant wild-type allele.In the current review various publications on breeding research of bud-flowering heather are summarized.Keywords: AFLP, bud-flowers, cultivar identification, ISSR, marker-assisted selection, RAP

AFLP-based genetic mapping of the " bud-flowering" trait in heather (Calluna vulgaris)

Background: Calluna vulgaris is one of the most important landscaping plants produced in Germany. Its enormous economic success is due to the prolonged flower attractiveness of mutants in flower morphology, the so-called bud-bloomers. In this study, we present the first genetic linkage map of C. vulgaris in which we mapped a locus of the economically highly desired trait " flower type" .Results: The map was constructed in JoinMap 4.1. using 535 AFLP markers from a single mapping population. A large fraction (40%) of markers showed distorted segregation. To test the effect of segregation distortion on linkage estimation, these markers were sorted regarding their segregation ratio and added in groups to the data set. The plausibility of group formation was evaluated by comparison of the " two-way pseudo-testcross" and the " integrated" mapping approach. Furthermore, regression mapping was compared to the multipoint-likelihood algorithm. The majority of maps constructed by different combinations of these methods consisted of eight linkage groups corresponding to the chromosome number of C. vulgaris.Conclusions: All maps confirmed the independent inheritance of the most important horticultural traits " flower type" , " flower colour" , and " leaf colour". An AFLP marker for the most important breeding target " flower type" was identified. The presented genetic map of C. vulgaris can now serve as a basis for further molecular marker selection and map-based cloning of the candidate gene encoding the unique flower architecture of C. vulgaris bud-bloomers.BLE (Federal Office of Agriculture and Food, Germany)/511–06.01-28-1-43.038-0

Selection of reference genes for normalization of quantitative real-time PCR in cell cultures of Cyclamen persicum

As a prerequisite for gene expression analyses in cell cultures of the ornamental crop Cyclamen persicum basic parameters for quantitative real-time polymerase chain reaction (qRT-PCR) have been established including the selection of reference genes using the software tools \u2018geNorm\u2019 and \u2018NormFinder\u2019. Five potential reference genes have been tested (elongation factor tu (Ef-Tu), putative ABC transporter ATPase, putative conserved oligomeric Golgi (COG) complex component, V-ATPase G subunit 1 and Histone H3-K9 methyltransferase 4 (H3-K9-HMTase 4)). \u2018NormFinder\u2019 as well as \u2018geNorm\u2019 identified Ef-Tu to be the least stable reference gene while the ranking of the most stable genes differed depending on the algorithm. According to \u2018NormFinder\u2019 COG complex component displayed the most stable expression whereas \u2018geNorm\u2019 indicated V-ATPase G subunit 1 and a putative ABC transporter ATPase to be the most reliable reference genes. Hence, we concluded to use a normalization factor calculated from the four reference genes V-ATPase G subunit 1, ABC transporter ATPase, Histone H3-K9 methyltransferase 4 (H3-K9-HMTase 4) and COG complex component for normalization of qRT-PCR in cell cultures of Cyclamen persicum

Entwicklung von „near isogenic lines“ als Basis zur nachhaltigen Züchtung von Basilikum-Sorten mit Resistenz gegen den Falschen Mehltau

Die Produktion von Basilikum-Frischware erfolgt in hoher Intensität in spezialisierten Betrieben. Der Anbau ist seit einigen Jahren regelmäßig durch das Auftreten des Falschen Mehltaus, verursacht durch den Erreger Peronospora belbahrii, bedroht, für deren Kontrolle derzeit keine geeigneten Verfahren zur Verfügung stehen. Eine umwelt-, verbraucher- und produzentenfreundliche Strategie, dem Auftreten des Falschen Mehltaus entgegen zu wirken, ist der Anbau von resistenten Sorten. In einem voraus-gegangenen Projekt konnte eine Resistenzquelle bzw. resistenter Basilikumgenotyp aufgefunden wer-den, die genutzt wurde, um die Resistenz in den vom Verbraucher bevorzugten Genoveser-Typ einzukreuzen. Ziel dieses Projektes war die Erstellung von Near Isogenic Lines (NILs) durch wiederholte Rückkreuzung von bestimmten Elterlinien von Basilikum mit Resistenz gegenüber dem Erreger des Falschen Mehltaus als Basis für eine Marker-basierte Züchtung von Basilikumsorten. Eine vorliegende F2-Population aus Kreuzungen von Genoveser-Typ und dem Wildtyp ‚Apfelbasilikum‘ wurde eine F3-Generation generiert, in der sich einige Pflanzen als resistent erwiesen. Aus weiteren Rückkreuzungen mit dem rekurrenten Elter und einer abschließenden Selbstung wurde die F3BC3-S1 Generation generiert mit 140 resistenten bzw. wenig anfälligen Pflanzen gegenüber dem Falschen Mehltau. Ein weiteres Ziel des Projektes war, zu prüfen, ob die epidemiologische Entwicklung des Falschen Mehltaus im Basilikumbestand durch Reduktion der relativen Luftfeuchtigkeit unter Gewächshausbedingungen reduziert werden kann. Der Erreger P. belbahrii benötigt für die Keimung der Sporen und die Infektion von Basilikum eine bestimmte Blattnässe und –dauer, die durch die Luftfeuchtigkeit im Bestand beeinflusst wird. Um die relative Luftfeuchtigkeit im Bestand von Basilikum zu reduzieren, wurden Gewächshauskabinen mit entsprechenden Heizrohren ausgestattet. In mehreren Sätzen von Basilikum wurde die relative Luftfeuchtigkeit im Bestand in Abhängigkeit von der Vorlauftemperatur in den Heizrohren (40°C) und dem Abstand der Heizrohre zum Tischboden (ca. 9, 15 und 22 cm) im Vergleich zu einem Bestand ohne Wärmezufuhr untersucht. Die Ergebnisse von fünf Versuchen zeigten, die niedrigste rel. Luftfeuchtigkeit im Bestand wurde durch eine Vorlauftemperatur von 40°C mit einem Abstand der Heizrohre von 9 und 15 cm erzielt. Die epidemiologische Entwicklung des Falschen Mehltaus im Bestand wurde daher in Abhängigkeit von den genannten Bedingungen geprüft. Die Ausbringung des Erreger-Inokulums erfolgte mittels infizierter Pflanzen, die in den Bestand gesetzt wurden. Nachfolgend wurde die epidemische Entwicklung des Falschen Mehltaus im Bestand bonitiert. Durch Reduzierung der relativen Luftfeuchtigkeit konnte die epidemische Ausbreitung des Erregers deutlich reduziert werden. Die Beeinflussung der relativen Luftfeuchtigkeit kann in der Praxis genutzt werden, um dem Auftreten des Falschen Mehltaus entgegen zu wirken

Large impact of the apoplast on somatic embryogenesis in Cyclamen persicum offers possibilities for improved developmental control in vitro

<p>Abstract</p> <p>Background</p> <p>Clonal propagation is highly desired especially for valuable horticultural crops. The method with the potentially highest multiplication rate is regeneration via somatic embryogenesis. However, this mode of propagation is often hampered by the occurrence of developmental aberrations and non-embryogenic callus. Therefore, the developmental process of somatic embryogenesis was analysed in the ornamental crop <it>Cyclamen persicum </it>by expression profiling, comparing different developmental stages of embryogenic cell cultures, zygotic vs. somatic embryos and embryogenic vs. non-embryogenic cell cultures.</p> <p>Results</p> <p>The analysis was based on a cDNA microarray representing 1,216 transcripts and was exemplarily validated by realtime PCR. For this purpose relative transcript abundances of homologues of a putative receptor kinase, two different glutathione S-transferases (GST), a xyloglucan endotransglycosylase (XET) and a peroxidase (POX) were quantitatively measured by realtime PCR for three different comparisons. In total, 417 genes were found to be differentially expressed. Gene Ontology annotation revealed that transcripts coding for enzymes that are active in the extracellular compartment (apoplast) were significantly overrepresented in several comparisons. The expression profiling results are underpinned by thorough histological analyses of somatic and zygotic embryos.</p> <p>Conclusions</p> <p>The putative underlying physiological processes are discussed and hypotheses on improvement of the protocol for <it>in vitro </it>somatic embryogenesis in <it>Cyclamen persicum </it>are deduced. A set of physiological markers is proposed for efficient molecular control of the process of somatic embryogenesis in <it>C. persicum</it>. The general suitability of expression profiling for the development and improvement of micropropagation methods is discussed.</p

Molecular Reconstruction of an Old Pedigree of Diploid and Triploid Hydrangea macrophylla Genotypes

The ornamental crop species Hydrangea macrophylla exhibits diploid and triploid levels of ploidy and develops lacecap (wild type) or mophead inflorescences. In order to characterize a H. macrophylla germplasm collection, we determined the inflorescence type and the 2C DNA content of 120 plants representing 43 cultivars. We identified 78 putative diploid and 39 putative triploid plants by flow cytometry. In our collection 69 out of 98 flowering plants produced lacecap inflorescences, whereas 29 plants developed mophead inflorescences. Surprisingly, 12 cultivars included diploid as well as triploid plants, while 5 cultivars contained plants with different inflorescence types. We genotyped this germplasm collection using 12 SSR markers that detected 2–7 alleles per marker, and identified 51 different alleles in this collection. We detected 62 distinct fingerprints, revealing a higher genetic variation than the number of cultivars suggested. Only one genotype per cultivar is expected due to the vegetative propagation of Hydrangea cultivars; however we identified 25 cultivars containing 2–4 different genotypes. These different genotypes explained the variation in DNA content and inflorescence type. Diploid and triploid plants with the same cultivar name were exclusively mix-ups. We therefor assume, that 36% of the tested plants were mislabeled. Based on the “Wädenswil” pedigree, which includes 31 of the tested cultivars, we predicted cultivar-specific fingerprints and identified at least 21 out of 31 cultivars by SSR marker-based reconstruction of the “Wädenswil” pedigree. Furthermore, we detected 4 putative interploid crosses between diploid and triploid plants in this pedigree. These interploid crosses resulted in diploid or/and triploid offspring, suggesting that crosses with triploids were successfully applied in breeding of H. macrophylla

Selection of reference genes for normalization of quantitative real-time PCR in cell cultures of Cyclamen persicum

As a prerequisite for gene expression analyses in cell cultures of the ornamental crop Cyclamen persicum basic parameters for quantitative real-time polymerase chain reaction (qRT-PCR) have been established including the selection of reference genes using the software tools ‘geNorm’ and ‘NormFinder’. Five potential reference genes have been tested (elongation factor tu (Ef-Tu), putative ABC transporter ATPase, putative conserved oligomeric Golgi (COG) complex component, V-ATPase G subunit 1 and Histone H3-K9 methyltransferase 4 (H3-K9-HMTase 4)). ‘NormFinder’ as well as ‘geNorm’ identified Ef-Tu to be the least stable reference gene while the ranking of the most stable genes differed depending on the algorithm. According to ‘NormFinder’ COG complex component displayed the most stable expression whereas ‘geNorm’ indicated V-ATPase G subunit 1 and a putative ABC transporter ATPase to be the most reliable reference genes. Hence, we concluded to use a normalization factor calculated from the four reference genes V-ATPase G subunit 1, ABC transporter ATPase, Histone H3-K9 methyltransferase 4 (H3-K9-HMTase 4) and COG complex component for normalization of qRT-PCR in cell cultures of Cyclamen persicum