Engineering hydrogel nanoparticles to enhance transdermal local anaesthetic delivery in human eyelid skin

Herein, we focused on developing the feasibility of nano-enabled local anaesthetic (LA) delivery to anaesthetise the full thickness of eyelid skin. For this purpose a temperature-responsive hydrogel poly(N-vinylcaprolactam-co-hyaluronic acid) (p(VCL-co-HA)) was prepared through aqueous emulsion polymerization with a Food and Drug Administration (FDA) approved p(VCL) and hyaluronic acid (HA) showing remarkably high LA drug loading capacity

Malus fusca fire blight resistance: identification of a candidate gene on chromosome 10 and a novel minor locus on chromosome 16

Fire blight resistance of the wild apple species Malus fusca (accession MAL0045) has been previously reported. This accession, crossed with the domesticated apple cultivar ‘Idared’, allowed for studies on the genetics of the resistance of this crabapple with the resultant F1 population. A major fire blight locus (Mfu10), found on chromosome 10, explained up to 66% of the phenotypic variance amongst the M. fusca × ‘Idared’ progenies. Although fire blight resistance is strain specific for some Malus accessions, leading to the breakdown of resistance in few resistance donors by highly aggressive strains of Erwinia amylovora; no strain able to breakdown the resistance of M. fusca itself or Mfu10 has been found. This makes this wild apple an interesting model for resistance studies with different wild-type and mutant strains of E. amylovora. A candidate gene (FB_Mfu10), underlying the major locus, was recently proposed. FB_Mfu10 was predicted on the sequence of a bacterial artificial chromosome (BAC) clone, spanning the fire blight locus on chromosome 10 and encodes B-lectin and serine/threonine kinase domains. Preliminary functional analyses showed, that the open reading frame (ORF), together with its border sequences upstream of the start codon and downstream of the stop codon (~ 6000 bp), is present only in resistant F1 genotypes with 8bp distinguishing between susceptibility and resistance. Furthermore, with a dense genetic map of M. fusca and studies with a mutant of an aggressive strain of E. amylovora, a minor fire blight locus has been identifie

Further insights into Malus fusca fire blight resistance

The apple wild species accession Malus fusca MAL0045 had been found to be resistant to fire blight in artificial inoculation trials with Erwinia amylovora strain Ea222_ JKI. Consequently, using a population derived by crossing MAL0045 with Idared, the corresponding fire blight resistance locus of M. fusca (Mfu10) was mapped on chromosome 10 explaining up to 66% of phenotypic variation at a logarithm of the odd (LOD) ratio of 31.0 with the strain Ea222_JKI. Moreover, the very aggressive strain Ea3049 only minimally affected MAL0045 but significantly affected the population although could not overcome the resistance of Mfu10. To further understand the resistance mechanism of M. fusca, we evaluated resistance of the original mapping population, comprised of 134 individuals, to E. amylovora strain ZYRKD3-1 which causes the breakdown of the resistance of M.×robusta 5 (Mr5) and the Mr5 fire blight resistance QTL on LG3. Our results showed that the major QTL of M. fusca on LG10 could still be detected at the same exact position with a higher effect on fire blight resistance, indicating that ZYRKD3-1 has no effect on Mfu10, although the mean percent lesion length of the population was almost doubled compared with Ea222_JK

The fire blight resistance QTL of Malus fusca (Mfu10) is affected but not broken down by the highly virulent Canadian Erwinia amylovora strain E2002A

Recently, Mfu10 - the major QTL for resistance to fire blight was reported on linkage group 10 of Malus fusca which could explain about 66 % of the phenotypic variation in a population artificially inoculated with Erwinia amylovora strain Ea222_JKI. In the meantime, it had been reported that the resistance of the fire blight QTL of Malus × robusta 5 (Mr5), which could explain 80 % of phenotypic variation is strain specific and is completely overcome by the highly virulent strain Ea3049 originating from Canada (E2002A). In contrast to Mr5, the M. fusca donor of Mfu10 is only very slightly affected by this highly virulent strain. In this short communication, we prove that this particular strain is not able to overcome the resistance of M. fusca but affects Mfu10. The same F1 progenies, derived from a cross between the resistant M. fusca and the susceptible apple cultivar, Idared, used to detect Mfu10, were used for phenotyping with Ea3049. Although the mean shoot necrosis of all progenies was 62.4 %, marker-phenotype association determined by Kruskal-Wallis analysis showed that markers on LG10 correlate significantly to resistance levels with SSR markers FR481A and FRM4 having the highest K values of 37.1 and 36.7, respectively. Interval mapping and multiple QTL mapping (MQM) performed with MAP-QTL®, showed that Mfu10 could still be detected on the same position on LG10, but only explaining 41.2 % of phenotypic variation. The implications of these results are discusse

The Malus fusca fire blight resistance locus: validation of Mfu10 and genetic resolution of the region containing the locus

The apple wild species accession Malus fusca MAL0045 was found to be resistant in artificial inoculation trials with fire blight. Erwinia amylovora strain Ea222_JKI, usually used for mapping purposes in Dresden, as well as the highly virulent strain Ea3049 did not affect MAL0045. The corresponding fire blight resistance locus of M. fusca (Mfu10) was previously mapped on chromosome 10. This quantitative trait locus explaining up to 66 % of phenotypic variation at a logarithm of the odd (LOD) ratio of 31.0 was based on 134 individuals derived from a cross of M. fusca × Idared and phenotyped with Erwinia amylovora strain Ea222_JKI. To gain more insight into the resistance mechanism of M. fusca, we phenotyped the original mapping population of 134 individuals with the highly virulent E. amylovora resistance-breaking strain of M. ×robusta 5 - Ea3049, and fine mapped the region containing Mfu10. Ea3049 could not break down the resistance of Mfu10 but significantly affected it. Furthermore, the closest tightly linked SSR markers to Mfu10 were used to genotype an increased population of 1,202 individuals, leading to the identification of interesting recombinants in the region of interest. The increase in population, addition of more tightly linked SSR markers to linkage group 10 as well as the phenotypic evaluation of recombinants ensured the genetic resolution of the resistance locus

DArT and SSR markers are linked with a strong QTL for resistance to fire blight in the apple wild species Malus fusca.

The most important bacterial disease affecting pome fruit is fire blight caused by Erwinia amylovora. Antibiotics treatment is the only reliable control measure for this devastating disease, yet their use is strictly regulated if not completely banned in many European countries due to environmental and sustainable issues. Since resistance can be genetically determined, planting of fire blight-resistant cultivars seems to be a highly probable approach. In this study, we explored a segregating population derived from a cross between the apple wild species Malus fusca and the Malus x domestica cultivar ‘Idared’. The mapping population was artificially inoculated with Erwinia amylovora strain Ea222_JKI at a concentration of 109 cfu/ml in 3 different years. A new library of DArT clones was constructed using the complexity reduction method that identified 1080 DArT markers after images were processed using DArTsoft. SSR markers were sourced from literature and used to genotype the population. The averages of percentage lesion length (PLL) of all replicates of each genotype were used as numerical traits for statistical analysis. A Kruskal-Wallis analysis, used to determine marker-phenotype association revealed one linkage group containing DArT markers significantly linked to fire blight resistance. To determine the corresponding linkage group, the DArT markers were sequenced and sequences aligned to the ‘Golden Delicious’ reference genome. SSR markers were developed from the ‘Golden Delicious’ genome to replace highly significant DArT markers and to determine the QTL region. Interval mapping revealed a strong QTL on LG10 explaining about 65% of the phenotypic variation. Through genome walking approach aimed at determining the resistance region, additional SSR markers have been developed and mappe

Identification of a major quantitative trait locus for resistance to fire blight in the wild apple species Malus fusca

Fire blight, caused by the Gram-negative bacterium Erwinia amylovora, is the most important bacterial disease affecting apple (Malus × domestica) and pear (Pyrus communis) production. The use of antibiotic treatment, though effective to some degree, is forbidden or strictly regulated in many European countries, and hence an alternative means of control is essential. The planting of fire blight-resistant cultivars seems to be a highly feasible strategy. In this study, we explored a segregating population derived from a cross between the wild apple species Malus fusca and the M. × domestica cultivar Idared. F1 progenies used for mapping were artificially inoculated with Erwinia amylovora strain Ea222_JKI at a concentration of 109 cfu/ml in three different years. The averages of percentage lesion length of all replicates of each genotype were used as numerical traits for statistical analysis. A Kruskal–Wallis analysis was used to determine marker–phenotype association and revealed a linkage group with Diversity Arrays Technology (DArT) markers significantly linked with fire blight. After locating the positions of the DArT markers on the Golden Delicious genome, simple sequence repeat (SSR) markers were developed from chromosome 10 to replace the DArT markers and to determine the quantitative trait locus (QTL) region. Multiple QTL mapping (MQM) revealed a strong QTL (Mfu10) on linkage group 10 of M. fusca explaining about 65.6 % of the phenotypic variation. This is the first report on a fire blight resistance QTL of M. fusc

Vergleichende genetische Kartierung der Feuerbrandresistenz bei Malus sp. = Comparative genetic mapping of resistance to fire blight in Malus sp.

Für die vergleichende Kartierung von Feuerbrandresistenz bei Malus wurden vier Populationen bearbeitet, die als einen Elter einen Donor für Feuerbrandresistenz und als zweiten Elter die anfällige Sorte ‘Idared’ hatten. Als Donoren für Widerstandsfähigkeit wurden definierte Abstammungen der Wildarten M. baccata, M. fusca, M. ×robusta und die widerstandsfähige Pillnitzer Apfelsorte ‘Rewena’ genutzt. Die Phänotypisierung der Populationen erfolgte in unterschiedlichen Jahren durch Inokulation von vegetativen Trieben mit dem Erwinia amylovora-Isolat Ea222 und anderen definierten Isolaten unter Gewächshausbedingungen. Der Befall der Triebe wurde als Befallsrate, dem Verhältnis der befallenen Trieblänge zur Gesamttrieblänge, ermittelt. Je nach Isolat und Population variierte die durchschnittliche Befallsrate zwischen 10 bis 82%. Die Genotypisierung der Nachkommen zur Erstellung genetischer Karten erfolgte mit Mikrosatelliten-, SNP- und DArT-Markern. Die genetischen Kopplungskarten wurden mittels Joinmap 4.0 und die Resistenz gegenüber Feuerbrand mit MapQTL5.0 kartiert. Major-QTLs konnten für M. ×robusta auf Kopplungsgruppe 3, für M. baccata auf Kopplungsgruppe 12 und für M. fusca auf Kopplungsgruppe 10 detektiert werden. Während die Feuerbrand-QTLs von M. ×robusta 5 und M. fusca je nach Jahr bis zu ca. 87% der phänotypischen Varianz erklären können, erklärt der QTL von M. baccata nur ca. 45%, was die Existenz eines zweiten QTL nahe legt. Für die resistente Sorte ‘Rewena’ konnte kein QTL detektiert werden. Mit den Markern, die im Bereich der detektierten QTLs liegen, kann auf Resistenz gegenüber Feuerbrand selektiert werden. Damit ist eine Kombinierung unterschiedlicher Feuerbrandresistenz-QTLs möglich. Auf dieser Basis können zukünftig Sorten gezüchtet werden, die dauerhaft widerstandsfähig gegenüber Feuerbrand sind, um diese dann dem deutschen Obstbau zur Verfügung stellen zu könne

Towards map-based cloning of FB_Mfu10: identification of a receptor-like kinase candidate gene underlying the Malus fusca fire blight resistance locus on linkage group 10

Breeding for resistance against the destructive fire blight disease of apples is the most sustainable strategy to control the menace of this disease, and has become increasingly important in European apple breeding programs. Since most cultivars are susceptible, wild accessions have been explored for resistance with quantitative trait loci detected in a few wild species. Fire blight resistance of Malus fusca was described following phenotypic evaluations with a C-type strain of Erwinia amylovora, Ea222_JKI, and the detection of a major QTL on chromosome 10 (Mfu10) of this crabapple. The stability of the resistance of M. fusca and Mfu10 has been evaluated using two other strains, the highly aggressive Canadian S-type strain – Ea3049, and the avrRpt2EA mutant – ZYRKD3-1, both of which overcome the resistance of Malus ×robusta 5, a wild species accession with an already described fire blight resistance gene. To pave the way for positional cloning of the underlying fire blight resistance gene of M. fusca, we have fine mapped the QTL region on linkage group 10 using 1,888 individuals and 23 newly developed molecular markers, thus delimiting the interval of interest to 0.33 cM between markers FR39G5T7xT7y/FR24N24RP and FRMf7358424/FR46H22. Tightly linked SSR markers are suitable for marker-assisted selection in breeding programs. Furthermore, a bacterial artificial chromosome (BAC) clone spanning FB_Mfu10 region was isolated and sequenced. One putative fire blight resistance candidate gene of M. fusca was predicted on the sequence of BAC 46H22 within the resistance region that encodes B-lectin and serine/threonine kinase domains