Molecular characterization of intergeneric hybrids between Malus and Pyrus

Apple (Malus) and pear (Pyrus) are economically important fruit crops well known for their unique textures, flavours, and nutritional qualities. Both genera are characterised by a distinct pattern of secondary metabolites, which directly affect not only resistance to certain diseases, but also have significant impacts on the flavour and nutritional value of the fruit. The identical chromosome numbers, similar genome size, and their recent divergence date, together with DNA markers have shown that apple and pear genomes are highly co-linear. This study utilized comparative genomic approaches, including simple sequence repeats, high resolution single nucleotide polymorphism melting analysis, and single nucleotide polymorphism chip analysis to identify genetic differences among hybrids of Malus and Pyrus, and F2 offspring. This research has demonstrated and validated that these three marker types, along with metabolomics analysis are very powerful tools to detect and confirm hybridity of progeny derived from crosses between apple and pear in both cross directions. Furthermore, this work analysed the genus-specific metabolite patterns and the resistance to fire blight (Erwinia amylovora) in progeny. The findings of this work will enhance and accelerate the breeding of novel tree fruit crops that benefit producers and consumers, by enabling marker assisted selection of desired traits introgressed between pear and apple

Identification of Pyrus Single Nucleotide Polymorphisms (SNPs) and Evaluation for Genetic Mapping in European Pear and Interspecific Pyrus Hybrids

We have used new generation sequencing (NGS) technologies to identify single nucleotide polymorphism (SNP) markers from three European pear (Pyrus communis L.) cultivars and subsequently developed a subset of 1096 pear SNPs into high throughput markers by combining them with the set of 7692 apple SNPs on the IRSC apple Infinium® II 8K array. We then evaluated this apple and pear Infinium® II 9K SNP array for large-scale genotyping in pear across several species, using both pear and apple SNPs. The segregating populations employed for array validation included a segregating population of European pear (‘Old Home’בLouise Bon Jersey’) and four interspecific breeding families derived from Asian (P. pyrifolia Nakai and P. bretschneideri Rehd.) and European pear pedigrees. In total, we mapped 857 polymorphic pear markers to construct the first SNP-based genetic maps for pear, comprising 78% of the total pear SNPs included in the array. In addition, 1031 SNP markers derived from apple (13% of the total apple SNPs included in the array) were polymorphic and were mapped in one or more of the pear populations. These results are the first to demonstrate SNP transferability across the genera Malus and Pyrus. Our construction of high density SNP-based and gene-based genetic maps in pear represents an important step towards the identification of chromosomal regions associated with a range of horticultural characters, such as pest and disease resistance, orchard yield and fruit quality

Phototriggered release of tetrapeptide AAPV from coumarinyl and pyrenyl cages

Ala-Ala-Pro-Val (AAPV) is a bioactive tetrapeptide that inhibits human neutrophil elastase (HNE), an enzyme involved in skin chronic inflammatory diseases like psoriasis. Caged derivatives of this peptide were prepared by proper N- and C-terminal derivatisation through a carbamate or ester linkage, respectively, with two photoactive moieties, namely 7-methoxycoumarin-2-ylmethyl and pyren-2-ylmethyl groups. These groups were chosen to assess the influence of the photosensitive group and the type of linkage in the controlled photorelease of the active molecule. The caged peptides were irradiated at selected wavelengths of irradiation (254, 300, and 350 nm), and the photolytic process was monitored by HPLC-UV. The results established the applicability of the tested photoactive groups for the release of AAPV, especially for the derivative bearing the carbamate-linked pyrenylmethyl group, which displayed the shortest irradiation times for the release at the various wavelengths of irradiation (ca. 4 min at 254 nm, 8 min at 300 nm and 46 min at 350 nm).Thanks are due to the Fundação para a Ciência e Tecnologia (FCT, Portugal) for financial support to the portuguese NMR network (PTNMR, Bruker Avance III 400- Univ. Minho), FCT and FEDER (European Fund for Regional Development)- COMPETE-QREN-EU for financial support through the Chemistry Research Centre of the University of Minho (Ref. UID/QUI/00686/2013 and UID/QUI/0686/2016). A PhD grant to A.M.S. (SFRH/BD/80813/2011) is also acknowledged.info:eu-repo/semantics/publishedVersio

Putative resistance gene markers associated with quantitative trait loci for fire blight resistance in Malus 'Robusta 5' accessions

Background Breeding of fire blight resistant scions and rootstocks is a goal of several international apple breeding programs, as options are limited for management of this destructive disease caused by the bacterial pathogen Erwinia amylovora. A broad, large-effect quantitative trait locus (QTL) for fire blight resistance has been reported on linkage group 3 of Malus ‘Robusta 5’. In this study we identified candidate gene markers for fire blight resistance by integrating further genetic mapping studies with bioinformatics analysis of transcript profiling data and genome sequence databases. Results When several defined E.amylovora strains were used to inoculate three progenies from 62 international breeding programs, all with ‘Robusta 5’ as a common parent, two distinct QTLs were detected on linkage group 3, where only one had previously been mapped. In the New Zealand population, the proximal QTL co-located with SNP markers for a leucine-rich repeat, receptor-like protein (MxdRLP1) candidate resistance gene and a closely linked class 3 peroxidase gene. While the QTL detected in the German population was approximately 6 cM distal to this, directly below a SNP marker for a heat shock 90 family protein (HSP90). In the US population, the position of the LOD score peak on linkage group 3 was dependent upon the pathogen strains used for inoculation. One of the five MxdRLP1 alleles identified in fire blight resistant and susceptible cultivars was genetically associated with resistance and used to develop a high resolution melting PCR marker. A resistance QTL detected on linkage group 7 of the US population co-located with another HSP90 gene-family member and a WRKY transcription factor previously associated with fire blight resistance. However, this QTL was not observed in the New Zealand or German populations. Conclusions The results suggest that the upper region of ‘Robusta 5’ 76 linkage group 3 contains multiple genes contributing to fire blight resistance and that their contributions to resistance can vary depending upon pathogen virulence and other factors. Candidate gene mapping has proved a useful aid in defining these QTLs and developing markers for marker-assisted breeding of fire blight resistance

Molecular Identification and Expression Analysis of Filaggrin-2, a Member of the S100 Fused-Type Protein Family

Genes of the S100 fused-type protein (SFTP) family are clustered within the epidermal differentiation complex and encode essential components that maintain epithelial homeostasis and barrier functions. Recent genetic studies have shown that mutations within the gene encoding the SFTP filaggrin cause ichthyosis vulgaris and are major predisposing factors for atopic dermatitis. As a vital component of healthy skin, filaggrin is also a precursor of natural moisturizing factors. Here we present the discovery of a member of this family, designated as filaggrin-2 (FLG2) that is expressed in human skin. The FLG2 gene encodes a histidine- and glutamine-rich protein of approximately 248 kDa, which shares common structural features with other SFTP members, in particular filaggrin. We found that FLG2 transcripts are present in skin, thymus, tonsils, stomach, testis and placenta. In cultured primary keratinocytes, FLG2 mRNA expression displayed almost the same kinetics as that of filaggrin following Ca2+ stimulation, suggesting an important role in molecular regulation of epidermal terminal differentiation. We provide evidences that like filaggrin, FLG2 is initially expressed by upper granular cells, proteolytically processed and deposited in the stratum granulosum and stratum corneum (SC) layers of normal epidermis. Thus, FLG2 and filaggrin may have overlapping and perhaps synergistic roles in the formation of the epidermal barrier, protecting the skin from environmental insults and the escape of moisture by offering precursors of natural moisturizing factors

Fire Blight resistance in apple/pear hybrids may be related to their genomic structure

Although Malus and Pyrus are closely related, with highly co-linear genomes, the two genera are characterised by many specific differences, including disease resistances, secondary metabolites, fruit texture, flavour and shape. Hence, intergeneric hybrids between apple and pear provide a unique germplasm resource for genetic analysis, as well as new cultivar development, using advanced genomic breeding strategies. Fire blight, caused by the Gram-negative bacterium Erwinia amylovora (Enterobacterales; Erwiniaceae), affects apple and pear production worldwide. A number of resistance loci against this disease have been located on genetic maps of both apple and pear.We investigated fire blight resistance in apple-pear hybrids, by studying 31 putative hybrids raised from a ‘Cox’s Orange Pippin’ x ‘Old Home’ cross at The New Zealand Institute for Plant and Food Research Limited. We inoculated up to eight replicates of each hybrid grafted on ‘M9’ rootstock and compared these with ‘Cox’s Orange Pippin’ and ‘Imperial Gala’ grafted on ‘M9’ rootstock and ‘Old Home’ and ‘Williams’ grafted with Pyrus calleryana, using the cut-leaf method (Maas Geesteranus and Heyting, 1981) for inoculation with E. amylovora (Ea 236 at 1 x 106 cfu/ml), as it is widely applied in both apple and pear. Disease progress was observed in the period from 2 to 6 weeks after inoculation. Level of disease was quantified by expressed necrosis length as a percentage of the total shoot length, both measured downwards from the point of inoculation. The result clearly showed that all of the 31 putative hybrids were resistant to fire blight, while the parents and controls exhibited the expected range of resistance and susceptibility according to previous work. Preliminary results using high-resolution melting marker analysis of the seedling genomes indicate there is a hybrid apple/pear genomic region on LG2, while LG7 is represented by apple DNA. Interestingly, fire blight resistance has been reported on LG2 of pear ‘Old Home’ (Montanari et al., 2016), while Khan et al. (2007) have located fire blight resistance on LG7 of the apple ‘Fiesta’, which is related by descent from ‘Cox’s Orange Pippin’.Our next step is to analyse recombination events during the crossing of apple and pear, using the IRSC 9K apple/pear SNP array: this will enable us to further investigate the relationship of these reported QTL resistances to fire blight infection within the genomic structure of our 31 apple/pear hybrid

Molecular characterization of intergeneric hybrids between Malus and Pyrus

Apple (Malus) and pear (Pyrus) are economically important fruit crops well known for their unique textures, flavours and nutritional qualities. Both genera are characterised by a distinct pattern of secondary metabolites, which directly affect not only resistance to certain diseases, but also have significant impacts on the flavour and nutritional value of the fruit. The identical chromosome numbers, similar genome size, and their recent divergence date, together with DNA markers have shown that apple and pear genomes are highly co-linear. This study utilized comparative genomic approaches, including simple sequence repeats, high resolution SNP melting analysis, and SNP-chip analysis to identify genetic differences among putative hybrids of Malus and Pyrus, and F2 offspring. This research has demonstrated and validated that these three marker types, along with metabolomics analysis are very powerful tools to detect and confirm hybridity of progeny derived from crosses between apple and pear in both cross directions. Furthermore, this work analysed the genus-specific metabolite patterns and the resistance to fire blight (Erwinia amylovora) in progeny. The findings of this work will enhance and accelerate the breeding of novel tree fruit crops that benefit producers and consumers, by enabling the specific detection of introgression of desired traits between pear and apple