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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
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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 1888 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.
KeywordsErwinia amylovora Recombinants Molecular markers BACs Candidate gene
Apple (Malus domestica Borkh.) is one of the most economically important fruit crops worldwide. However, one of the most serious threats to apple production is fire blight disease, caused by the bacterial pathogen, Erwinia amylovora (Burrill) (Winslow et al. 1920). Fire blight is considered to be the most dreadful bacterial disease affecting pome fruit production. E. amylovora possesses the type III secretion system (T3SS) located on the so-called pathogenicity island (PAI) which is encoded by a cluster of hypersensitive response and pathogenicity (hrp) genes (Khan et al. 2012). The T3SS deposits effector proteins into the host and enables the pathogen to cause disease in susceptible host plants. Primary infection occurs during flowering and depending on the degree of susceptibility of a cultivar, could spread within the entire tree in a single growing season. Fire blight has proven over the years to be very difficult to manage. Some preventive measures employed by pome fruit growers include the use of antibiotics and antagonists. Rigid pruning of infected parts of a tree or the complete eradication of affected trees are curative measures. Nevertheless, these control methods are not sustainable and cannot totally mitigate the disease in a season favorable to E. amylovora.
The use of host plant resistance is desirable and could prove to be a reliable strategy for the sustainable management of fire blight. Since 2005, several fire blight resistance quantitative trait loci (QTLs) have been described in Malus (Calenge et al. 2005; Khan et al. 2006; Peil et al. 2007; Durel et al. 2009; Le Roux et al. 2010; Emeriewen et al. 2014a; Emeriewen et al. 2017a; van de Weg et al. 2018). The strongest QTLs reported in wild apple species are located on linkage group 3 (LG3) of Malus ×robusta 5 (Mr5; Peil et al. 2007) and LG10 of Malus fusca (Emeriewen et al. 2014b), explaining 80 and 66% respectively of the phenotypic variation to the same pathogen strain Ea222_JKI. However, resistance to fire blight has been shown to be strain-specific (Norelli and Aldwinkle 1986; Paulin and Lespinasse 1990; Peil et al. 2011; Vogt et al. 2013; Wöhner et al. 2014).
Bacterial effectors, such as the type III effector protein avrRpt2EA (Vogt et al. 2013) play critical roles via the type III secretion system (T3SS) in regulating plant susceptibility and circulating the pathogen (Khan et al. 2012). A single nucleotide mutation in the avrRpt2EA effector of E. amylovora was responsible for a switch to virulence on Mr5 (Vogt et al. 2013). Furthermore, the loss of the avrRpt2EA effector was proven to cause susceptibility in Mr5 (Vogt et al. 2013). Recently, an eop1 mutant of E. amylovora led to significantly higher disease incidences in M. floribunda 821 (Mf821) and the ornamental cultivar 'Evereste,' suggesting at least a partial breakdown of their fire blight resistances and hence indicating a new gene-for-gene relationship in Malus (Wöhner et al. 2017). Although the fire blight resistance QTL of Mr5 on LG3 is broken-down by avrRpt2EA S-type strains (Peil et al. 2011, Vogt et al. 2013), the underlying resistance gene was described by Fahrentrapp et al. (2013). The gene, FB_MR5, an NBS-LRR gene, belonging to class 2 of plant R genes (Kruijt et al. 2005), was hypothesized to monitor RIN4 from Mr5, a homolog to RIN4 of Arabidopsis thaliana (Fahrentrapp et al. 2013). Furthermore, FB_MR5 is the first, and currently the only functionally characterized fire blight resistance gene of apple (Broggini et al. 2014). Additionally, the proposed function of FB_MR5 in a cisgenic apple was demonstrated by Kost et al. (2015).
Taking the strain specificity of fire blight resistance into account, it is important for breeding to aim at pyramiding different fire blight resistance QTLs for durable resistance (Emeriewen et al. 2015). Therefore, the identification of fire blight resistance donors with different mechanisms and the subsequent isolation and functional verification of the underlying genes is necessary. Till date, the M. fusca accession MAL0045 and the corresponding fire blight resistance QTL on linkage group 10 (Mfu10; Emeriewen et al. 2014b) have not been overcome by any strain of E. amylovora tested (Emeriewen et al. 2015, 2017b; Wöhner et al. 2017). Therefore, the M. fusca fire blight resistance QTL on LG10 (Mfu10) can play a critical role in pyramided resistance for durability especially since it possesses a different resistance mechanism from the Mr5 QTL on LG3 and the QTLs from 'Evereste' and Mf821 on LG12 (Emeriewen et al. 2015; Wöhner et al. 2017). Here, we report the construction of a fine genetic map of the region containing Mfu10 after enrichment with 23 tightly linked markers. Genome walking approach led to the isolation of BAC clones in the Mfu10 putative region and the subsequent identification of a candidate gene from the assembled sequence of two clones in this region.
Materials and methods
Plant materials and DNA extraction
All six populations used in this study with a total number of 1888 progeny individuals
Population name
Number of individuals
Number of recombinants in the interval from
CH03d11 to FR149B
FR20D to FR22A
M. fusca × Idared
Idared × M. fusca
M. fusca × Idared
Idared × M. fusca
M. fusca × Idared
M. fusca × Idared
Marker development and polymorphism tests
Molecular markers developed from contigs of pseudo-chromosome 10 of the 'Golden Delicious' genome
Marker name
Forward primer
Reverse primer
Resistant allele (bp)
Susceptible allele (bp)
222 (G)a
Sequencing of marker fragments to detect SNPs
Markers possessing alleles not polymorphic in the population were used to sequence both parents and selected progenies including recombinants (resistant and susceptible) in an attempt to identify SNP sites segregating among the two classes of progenies. Amplified PCR products were purified using MSB® Spin PCRapace KIT (Invitek GmbH, Berlin, Germany) according to the manufacturer's instructions. Sequencing of amplified products was performed by Eurofins MWG Operon (Ebersberg, Germany). Sequences were analyzed using BioEdit program.
Marker application and identification of recombinant individuals
Firstly, the 12228 and 12229 populations, 1133 individuals in total, were screened with the two SSR markers (CH03d11 and FR149B) bracketing the QTL and then with the two SSRs, FRM4 and FR481A, tightly linked to resistance within this interval in order to identify individuals showing recombination events in the 15.79 cM interval (Emeriewen et al. 2014b). Application of these markers was performed using a PCR approach in a volume of 10.2 μl comprising 1 × GeneAmp PCR buffer II (Applied Biosystems, Monza, Italy), 2.4 mM MgCl2, 0.05 mM dNTPs, 0.75 U AmpliTaq Gold DNA polymerase (GeneAmp, Applied Biosystems, Monza, Italy), 2 mM SSR primer mix (forward and reverse primers) with the following profile: 94 °C for 10 min, followed by 30 cycles of 94 °C for 30 s, 60 °C for 1 min and 72 °C for 1 min, and an extension of 72 °C for 5 min. PCR fragments were analyzed on a 3730XL DNA analyzer (Applied Biosystems, Vienna, Austria). Once recombinants from the 12228 and 12229 populations were identified and their linkage to resistance determined, the interesting ones were then genotyped with newly developed markers (Table 2) to more precisely determine the region of interest. For this purpose, PCR was performed using the Type-It Kit (Qiagen, Hilden, Germany) in a 10 μl volume with the following conditions: 95 °C for 5 min, followed by 30 cycles of 95 °C for 1 min, 60 °C for 1 min 30 s, and 72 °C for 30 s, and a final extension for 30 min at 60 °C. PCR fragments were then analyzed on a CEQ 2000XL DNA sequencer (Beckman Coulter, Germany). Sample preparations for the 3730XL DNA analyzer and the CEQ 2000XL DNA sequencer are as previously reported in Emeriewen et al. (2014b). Afterwards, the other populations were screened as described above with newly developed markers to determine informative recombinants in the shortened region of interest.
Fire blight phenotypic evaluation of recombinant individuals
For phenotypic evaluation of recombinant individuals, up to 10 replicates of each recombinant individual were grafted onto M9 rootstock and grown in the greenhouse before being transferred to the quarantine greenhouse where artificial E. amylovora inoculations were carried out with the parents M. fusca and 'Idared' as controls. Inoculation was performed on plants with a minimum length of 25 cm by incising the two youngest leaves with a pair of scissors dipped into E. amylovora inoculum of strain Ea222_JKI at a concentration of 109 cfu/ml (Peil et al. 2007; Emeriewen et al. 2014b). Greenhouse conditions were 25–27 °C (day), 20 °C (night), and 85% air humidity. Shoot length and lesion length of replicates of each genotype were measured 28 days post inoculation (dpi), transformed into percentage lesion length per shoot (PLL) as initially described by Peil et al. (2007), and then averaged.
Fine mapping of the fire blight resistance locus
Genotypic data of individuals were analyzed and mapped using JOINMAP 4.0 (Van Ooijen 2006). Phenotypic quantitative data (PLL) were transformed into binary marker data for genetic mapping (Durel et al. 2009). Recombinants were then arranged in ascending order of PLL. The mean and median PLLs in this order were then determined. While recombinants with PLL values lower than the mean PLL were classed as resistant, those with higher PLL were classed as susceptible. 10% of the recombinants between CH03d11 and FR149B from either side around the median PLL which were designated as being on the border between resistant and susceptible were excluded from further analyses. Phenotypic marker data were then compared with genotypic data. Once it was established that phenotypic data conformed to genotypic data of recombinant individuals, the binary marker data for non-recombinant individuals were then deduced by allocating 0 (i.e. resistant) and 1 (i.e. susceptible) to individuals possessing either resistant or susceptible alleles of tightly linked markers. Kruskal-Wallis analysis was performed to determine marker-phenotype association followed by interval mapping and multiple QTL mapping (MQM) using MAP-QTL 4.0 version (Van Ooijen and Maliepaard 1996).
Malus fusca BAC library construction and isolation of clones
The M. fusca bacterial artificial chromosome (BAC) library was produced using high molecular weight genomic DNA prepared at Amplicon Express (Pullman, Washington, USA) as described by Tao et al. (2002). DNA was partially digested with HindIII, size selected and ligated into pCC1BAC® vector (Epicenter, Madison, USA) and transformed into DH10B E. coli cells. Clones were arrayed on 384-well plates with LB media and frozen. The BAC library screening was performed in two separate rounds of PCR on extracted DNA from independently grown, separately pooled BAC clones. The first round of the PCR was performed on 14 Super Pools containing all BAC clones of the M. fusca library. The second round of PCR was then performed on the respective matrix pools to determine plate, row and column (each position representing a single or multiple BAC clones) reacting positively to the applied marker. Markers used to screen the M. fusca BAC library are listed in Table S1. PCR reactions and fragment analysis for the two rounds of screening were performed as described above.
Identified BAC clones were cultured overnight at 37 °C in LB medium containing 12.5 μg/mL Chloramphenicol. Plasmid DNA was thereafter extracted from clone cultures using NucleoBond® Xtra MIDI Plasmid DNA Purification Kit (MACHEREY-NAGEL, Düren, Germany) according to the manufacturer's protocol. Primers pCC1_RP (CTCGTATGTTGTGTGGAATTGTGAGC) and T7 (TAATACGACTCACTATAGGG) were used to sequence the ends of BACs. Sequencing of BAC-ends was performed by Microsynth AG (Balgach, Switzerland). Positions of BACs compared to the Golden Delicious Doubled Haploid genome (GDDH13) sequence (Daccord et al. 2017) were determined by blasting BAC sequences to the GDDH13 sequence. If the BLAST resulted in multiple hits, the most probable position of the BAC was chosen. De novo sequencing and assembly of BAC clones isolated in the resistance region as well as the susceptible homolog region was performed by various companies. Microsynth AG (Balgach, Switzerland) sequenced clones 46H22, 24N24, 36P10 and 70N1 using MiSeq Illumina sequencing. GATC Biotech (Cologne, Germany) and Amplicon Express (Pullman, USA) sequenced clones 62J21 and 39G5, respectively, using PacBio sequencing technique.
The raw sequencing data of the two overlapping resistant BACs, 24N24 and 46H22 were preprocessed together using Trim Galore. Subsequently, KmerGenie (version: 1.6715: default parameter) was run to determine the optimal k-value for a de Brujin assembly. Finally, CLC assembly cell was used to assemble the data into contigs. For aligning the BAC-ends to the assembled contigs, megablast with default parameters was used.
Gene prediction and expression of transcripts of candidate gene
Obtained sequences were used to predict open reading frames (ORFs) using FGENESH with algorithms for dicot plants, namely Arabidopsis, Solanum lycopersicum, and Vitis vinifera. The predicted proteins were analyzed using the National Centre for Biotechnology (NCBI) Blastp program (Altschul et al. 1997, 2005) and ExPASy PROSITE (Bairoch 1991; Hulo et al. 2008) to predict their domains, families, and functional sites.
The putative fire blight resistance gene of M. fusca was amplified with the primer pair named MVH_FB_Mfu10 (forward: GCTAGCTGCAGAACTTGCTTGCTCA and reverse: GAGAAAGAGAAAACCGCCCCGTCT) developed on the ATG start codon and TGA stop codon, respectively, using genomic DNA and BAC DNA as templates. Additionally, the candidate gene ORF including 1537 bp of the genomic regions upstream of the ATG codon and downstream of the TGA codon, respectively, with a length of 6366 bp, was amplified using genomic DNA and BAC DNA as templates with the primer pair named FB_Mfu10_RSeq1 (forward: TTCATATCAGCCCTTTTAACCACTGCTACA and reverse: GCCATAATTAAAACCTAGCCACTATCCCGT). PCR was performed using the DreamTaq Hot Start PCR Master Mix Kit (Thermo Scientific, Berlin, Germany) in a 12 μl volume with the following conditions: 95 °C for 5 min, followed by 34 cycles of 95 °C for 1 min, 68 °C for 90 s and 72 °C for 6 min, and an extension of 72 °C for 10 min.
To ascertain the candidate gene sequence, amplicons amplified by M. fusca, a resistant progeny, a susceptible progeny, and the susceptible BAC clone 94B13 with the developed gene primers were sequenced. Sequencing was performed by Eurofins MWG Operon (Ebersberg, Germany) with sample preparation as initially described. Comparison of obtained sequences was done using the National Centre for Biotechnology (NCBI) Blastn function (Zhang et al. 2000).
To confirm transcription of the candidate gene, RNA was extracted from M. fusca in vitro seedling using InviTrap® Spin Plant RNA Mini Kit according to the manufacturer's protocol (Stratec, Berlin, Germany). Potential DNA contamination was removed using the Invitrogen DNA-free™ Kit (Thermo Scientific, Berlin, Germany). The RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Berlin, Germany) was used to produce cDNA according to the manufacturer's protocol. Primer pair called FR_FB_Mfu10 (forward: AAAGCGGATATTTATTGGCACTGGTATCAC and reverse: AACGGTGTGCTTTGATTTGTCAACATAGAT) was developed to amplify parts of the ORF on cDNA.
Increase of mapping individuals
To fine map the fire blight resistance locus of M. fusca, five more populations besides the original mapping population (05210) were established at different stages of this study. All six populations employed in this study with a total of 1888 individuals are shown in Table 1.
Marker development
Initially, SSR markers FR481A and FRM4 which correlated with the highest significance to fire blight resistance mapped at 35.56 cM and 37.09 cM respectively on LG10 of M. fusca and were bracketed by two other SSRs, CH03d11 and FR149B (Emeriewen et al. 2014b). Therefore, to saturate the interval between CH03d11 and FR149B, 15 more polymorphic SSR markers which were developed from the corresponding pseudo-chromosome 10 of the 'Golden Delicious' genome have been mapped (Table 2). Additionally, two polymorphic SSRs, YO_FC1 and FRG5342 were developed from the assembled sequences of BAC clones. Similarly, four markers were developed from BAC-ends. Two other markers, FRMf7334158i and FRMf7358424, were developed from M. fusca sequences (http://vannocke.hrt.msu.edu/DBI-0922447/Genomic_Mfusca.html). Whereas both markers were developed from SSR motifs, only FRMf7334158i was polymorphic as an SSR marker. Therefore, to identify possible SNPs, fragments amplified with FRMf7358424 were sequenced. Hence, amplicons of M. fusca, 'Idared', three progenies inheriting the 156 bp allele of FRM4 (in coupling with resistance), and three progenies inheriting the 166 bp allele of FRM4 (in repulsion with resistance) were sequenced. Analysis of the sequences revealed a single nucleotide polymorphism (SNP) at position 57 of the amplicons segregating between both classes of progenies. While nucleotide G was inherited by the resistant progenies, the susceptible progenies inherited A from M. fusca. Thus, FRMf7358424 was designated as a segregating marker.
Identification and phenotyping of recombinant individuals for fine mapping of FB_Mfu10
In total, 152 individuals showing recombination events between CH03d11 and FR149B could be identified from the 05210, 12228, and 12229 populations of 1267 individuals. For phenotyping, individuals of 12228 and 12229 populations showing recombination events between the interval of markers FR20D and FRM4 (deemed as definitely containing FB_Mfu10 from mapping results of Fig. 1b) were analyzed. This reduced the recombinants to be analyzed from 152 to 106 individuals. Inoculation was performed using E. amylovora isolate Ea222_JKI with the parents M. fusca and 'Idared' as controls. Results showed that whereas the resistant parent M. fusca showed no sign of lesion in all replicates, an average percentage lesion length (PLL) of 64% was recorded for the susceptible parent—'Idared'. The distribution of the susceptibility/resistance level of the 12228 and 12229 phenotyped recombinants is shown is Fig. S1. A zero PLL was observed in 15 recombinant individuals whereas no 100% lesion length was recorded in all 106 recombinants. The highest PLL recorded was 83.0%. The mean PLL recorded for all 106 recombinant individuals phenotyped was 19.9% with a median of 11.5%.
Isolation and sequencing of BAC clones
Sequencing and assembly of BAC clones 46H22 and 24N24 as well as marker data showed that 46H22 contains the entire sequence of clone 24N24. An assembly with sequence data of both clones comprised of 45 contigs (216 Kbp in total) with 88 Kbp and 205 bp being the longest and shortest contigs, respectively. The orientation of BAC contigs was in conformity with the orientation of BAC-end sequences blasted against the GDDH13 genome (Fig. 4).
Prediction of candidate gene
Resequencing of putative candidate gene and determination of transcript
The ORF including 1537 bp upstream and downstream, respectively (6366 bp), was amplified only on 46H22 and 24N24 BAC clones, resistant progeny, and M. fusca with primer pair FB_Mfu10_RSeq1. The amplified amplicon was fully resequenced and showed 100% identity with the 46H22/24N24 sequence. The ORF (3555 bp) was amplified with primer pair MVH_FB_Mfu10 in M. fusca, resistant progeny, susceptible progeny, BAC clones 46H22, 24N24 as well as BAC clone 94B13, but not in the susceptible parent 'Idared'. Sequencing of the amplicon from the susceptible BAC clone 94B13 and a susceptible progeny resulted in eight base pairs difference in sequence length of the putative gene and a resultant protein sequence with 28 amino acids difference upstream. Furthermore, part of the ORF (1092 bp) was amplified on cDNA of M. fusca with primer pair FR_FB_Mfu10.
We report the identification of a putative fire blight resistance candidate gene of M. fusca encoding protein kinase domains known to confer resistance to plant diseases. FB_Mfu10 was predicted on the sequence of BAC clone 46H22 which was found to span the M. fusca fire blight resistance region where a resistance QTL was previously reported (Emeriewen et al. 2014b). The identification of the putative candidate gene in this region was achieved following substantial increase of the initial mapping population of 134 individuals (Emeriewen et al. 2014b) with 1754 new individuals (Table 1) and mapping of 23 tightly linked molecular markers which allowed for the identification of informative recombinant individuals whose subsequent phenotypic evaluation allowed for the delimiting of the initially reported fire blight region from a 15.79 cM interval (Fig. 1a; Emeriewen et al. 2014b) to 0.33 cM (Fig. 1b). Only 31 recombinants determined the precise position of FB_Mfu10, however, all recombinant individuals identified in this study offered integral information from phenotyping results (Fig. 2 and S1). Despite applying the method of Durel et al. (2009) to reliably distinguish between resistant and susceptible recombinants for the transformation of binary data, i.e., the elimination of 10% of recombinants with PLL each below and above the median PLL, one individual (12229-893) in the region still showed phenotype/genotype incongruity. Such phenotype/genotype incongruity was also shown by Fahrentrapp et al. (2013) when identifying the candidate gene for fire blight resistance in M. ×robusta 5. The subsequent isolation of FB_MR5, the first functional fire blight resistance gene (Broggini et al. 2014), demonstrates that this approach could still be successful even with an individual showing phenotype/genotype incongruity.
Furthermore, the saturation of the fire blight region with tightly linked molecular markers such as SSRs is also important for marker-assisted selection/breeding (MAS; MAB) of pre-breeding materials prior to the introgression of this trait into related cultivars to minimize genetic drag. The initially reported interval of 15.79 cM contained only 2 SSRs (FRM4 and FR481A) bracketed by two other SSRs, CH03d11, and FR149B (Emeriewen et al. 2014b). Of the 23 newly developed molecular markers, one marker, OFE, mapped downstream of FRM4 (i.e., between FRM4 and FR149B) with six markers mapping between FRM4 and FR481A and 16 markers mapped upstream of FR481A. Importantly, with single gene/trait mapping using transformed binary data of recombinant individuals (Durel et al. 2009); FB_Mfu10 was situated in a 0.33 cM interval between FR39G5T7xT7y/FR24N24RP and FRMf7358424/FR46H22 (Fig. 3). In similar studies, Fahrentrapp et al. (2013) showed that markers FEM14/FEM47 and rp16k15 flanked the fire blight resistance locus of Mr5 within a 0.24 cM interval while Parravicini et al. (2011) reported a distance of 0.18 cM between markers ChfbE01 and ChfbE08 flanking the resistance QTL of 'Evereste' with another marker, ChfbE02-7, co-localizing with the locus. In this study, no marker was found to co-segregate with FB_Mfu10.
Identification of clones and physical characterization of FB_Mfu10 region
The direct screening of the M. fusca BAC library DNA Pool with tightly linked markers is a different approach to other chromosome landing studies in Malus which were hybridization-based (Patocchi et al. 1999; Galli et al. 2010; Parravicini et al. 2011; Fahrentrapp et al. 2013). Although several resistant and susceptible BAC clones were identified using this PCR-based approach (Fig. 4; Table S1), not all could be well defined due to unreliable BAC-end sequences or poor sequence alignments. Nevertheless, the clones presented in Fig. 4 were sufficient for the physical characterization of FB_Mfu10 region and its susceptible homolog, and it was possible to confirm BAC clones 46H22 and 94B13 as spanning the resistant region and susceptible homolog region, respectively. Previous fire blight genome walking approaches in Mr5 (Fahrentrapp et al. 2013) and 'Evereste' (Parravicini et al. 2011) also found single BAC clones spanning the respective regions.
In general, alignment of the sequences of the BAC clones and BAC-ends to the M. domestica doubled haploid genome (GDDH13, Daccord et al. 2017) was essential in confirming the orientation of the clones as well as characterizing the FB_Mfu10 regions. The initial 'Golden Delicious' genome (Velasco et al. 2010), though useful especially in the development of new tightly linked molecular markers, was not accurate in BACs orientation due to large sequence gaps found in this genome as well as the overestimation of the distance and sizes in this region. For example, the M. fusca genetic region from markers FR20D to FR22A (interval of 1.72 cM) which definitely contains the fire blight resistance locus, corresponds to at least 1.2 Mbp on the 'Golden Delicious' genome (Velasco et al. 2010), but only about 700 Kbp on GDDH13 (Daccord et al. 2017). Although the reads of two overlapping resistant BACs 46H22 and 24N24 were used for assembly, a single contig sequence could not be achieved. Fahrentrapp et al. (2013) reported four contigs for BAC 16k15 in the fire blight region of Mr5 with an insert length of 162 Kb after pyro-sequencing. These authors were unable to still close the gaps even after Sanger sequencing and resequencing of clone 16k15 with Illumina HiSeq 2000.
A candidate gene encoding receptor-like kinase protein
With the sequence of 46H22/24N24, a single candidate gene was predicted with protein comprising 880 amino acids that encodes a receptor-like kinase protein including a bulb-type mannose-specific binding lectin (B-lectin) domain and a catalytic domain of the serine/threonine kinases and interleukin-1 receptor-associated kinases (STKc_IRAK). Further analyses of the amino acids also revealed the PAN/APPLE-like domain (Fig. 5), predicted to bind proteins or carbohydrates. The first region, the B-lectin domain, is associated with protein-carbohydrate interactions. Upon attack, plants perceive the structures of the invading microbial organism using cell-surface and intracellular immune receptors such as lectins and proteins with one or more lectin domains (Lannoo and Van Damme 2014). Since it is established that E. amylovora requires the exopolysaccharide amylovoran (ams) among others for pathogenicity (Oh and Beer 2005), it is probable that the B-lectin domain of this candidate gene interacts with ams. STKc_IRAK is the serine/threonine signature characteristic of class 5 genes conferring resistance to bacterial diseases (Kruijt et al. 2005). The domains of the putative fire blight resistance candidate gene of M. fusca are the same as the domains of Pi-d2 gene of rice (B-lectin and an intracellular serine-threonine kinase) which confers gene-for-gene resistance to rice blast caused by the fungal pathogen, Magnaporthe grisea (Chen et al. 2006). Similarly, receptor-like kinase genes have been shown to confer resistance to bacterial blight in rice caused by Xanthomonas oryzae pv. oryzae (Sun et al. 2004; Xiang et al. 2006), and more notably the protein kinase gene (Pto gene) conferring resistance to bacterial speck in tomato caused by Pseudomonas syringae pv. tomato (Martin et al. 1993). In apple, serine/threonine kinase genes were proposed by Parravicini et al. (2011) in the fire blight resistance region of 'Evereste.' The most probable candidate gene of seven identified was MdE-EaK7 with 381 amino acids and possessed an ATP-binding signature. Comparison of the amino acid sequence deduced for the putative fire blight resistance gene of M. fusca and those of MdE-EaK7 and the Pto gene uncovered similarities of 34 and 39%, respectively (results not shown). In contrast to Parravicini et al. (2011), only one candidate gene was found by Fahrentrapp et al. (2013) in Mr5 and in our study in the corresponding BACs. Fahrentrapp et al. (2013) argued that the existence of a single resistance gene not embedded in a cluster of paralogs could be as a result of the lack of a long-term co-evolution between Malus species and E. amylovora.
The gene prediction with the amplicon sequence of the homolog from 94B13 comprised a similar protein with a significant 28 amino acids difference upstream. A single amino acid difference was shown to distinguish resistance from susceptible alleles in the Pi-d2 gene (Chen et al. 2006) and virulence from avirulence in the avrRpt2EA effector of E. amylovora (Vogt et al. 2013). Therefore, the observed differences between the two homolog regions in the current study are enough to distinguish resistance from susceptibility.
Concluding remarks
The results presented here are of significance given that they provide the first step towards the map-based cloning of FB_Mfu10. The putative fire blight resistance gene of M. fusca is of importance in breeding as this crabapple has proven to be resistant to all strains of E. amylovora tested till date, including the resistant-breaking strains of FB_MR5 from M. ×robusta 5 as well as a mutant strain causing disease incidence in 'Evereste' and Mf821. It is therefore imperative to prove the function of the proposed candidate gene in complementing studies. When validated, FB_Mfu10 will allow for the breeding of new cultivars as well as the development of cisgenic cultivars with pyramided fire blight resistance genes in order to establish more durable resistance.
Part of this research was funded by the Genomics and Molecular Physiology of Fruits (GMPF) programme of the Fondazione Edmund Mach, San Michele all 'Adige. The later stage of this project was funded by Deutsche Forschungsgemeinschaft (DFG Project Number: AOBJ: 619970). We would like to thank Dr. Steve van Nocker of the Michigan State University, USA for providing Malus fusca sequences. We would also like to thank Gisela Schulz, Christine Grafe, and the orchard and greenhouse staff in Dresden-Pillnitz and Quedlinburg for technical assistances.
Compliance with ethical standards
Conflict of interest
The authors declare no conflict of interest.
Supplementary material
- Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402CrossRefPubMedPubMedCentralGoogle Scholar
- Altschul SF, Wootton JC, Gertz EM, Agarwala R, Morgulis A, Schäffer AA, Yu YK (2005) Protein database searches using compositionally adjusted substitution matrices. FEBS J 272:5101–5109CrossRefPubMedPubMedCentralGoogle Scholar
- Bairoch A (1991) PROSITE: a dictionary of sites and patterns in proteins. Nucleic Acids Res 19:2241–2245CrossRefPubMedPubMedCentralGoogle Scholar
- Broggini GAL, Wöhner T, Fahrentrapp J, Kost TD, Flachowsky H, Peil A, Hanke M-V, Richter K, Patocchi A, Gessler C (2014) Engineering fire blight resistance into the apple cultivar 'Gala' using the FB_MR5 CC-NBS-LRR resistance gene of Malus ×robusta 5. Plant Biotechnol J 12:728–733CrossRefPubMedGoogle Scholar
- Calenge F, Drouet D, Denance C, Van de Weg WE, Brisset MN, Paulin JP, Durel CE (2005) Identification of a major QTL together with several minor additive or espistatic QTLs for resistance to fire blight in apple in two related progenies. Theor Appl Genet 111:128–135CrossRefPubMedGoogle Scholar
- Chen X, Shang J, Chen D, Lei C, Zou Y, Zhai W, Liu G, Xu J, Ling Z, Cao G, Ma B, Wang Y, Zhao X, Li S, Zhu L (2006) A B-lectin receptor kinase gene conferring rice blast resistance. Plant J 46:794–804CrossRefPubMedGoogle Scholar
- Daccord N, Celton J-M, Linsmith G, Becker C, Choisne N, Schijlen E, van de Geest H, Bianco L, Micheletti D, Velasco R, di Pierro EA, Gouzy J, Rees DJG, Guérif P, Muranty H, Durel CE, Laurens F, Lespinasse Y, Gaillard S, Aubourg S, Quesneville H, Weigel D, van de Weg E, Troggio M, Bucher E (2017) High-quality de novo assembly of the apple genome and methylome dynamics of early fruit development. Nat Genet 49:1099–1106CrossRefPubMedGoogle Scholar
- Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for small quantities of fresh leafs. Phytochem Bull 19:11–15Google Scholar
- Durel CE, Denance C, Brisset MN (2009) Two distinct major QTL for resistance to fire blight co-localize on linkage group 12 in apple genotypes 'Evereste' and Malus floribunda clone 821. Genome 52:139–147CrossRefPubMedGoogle Scholar
- Emeriewen O, Malnoy M, Richter K, Kilian A, Hanke M-V, Peil A (2014a) Evidence of a major QTL for fire blight resistance in the apple wild species Malus fusca. Acta Hort 1056:289–293. https://doi.org/10.17660/ActaHortic.2014.1056.49 CrossRefGoogle Scholar
- Emeriewen O, Richter K, Killian A, Zini E, Hanke M-V, Malnoy M, Peil A (2014b) Identification of a major quantitative trait locus for resistance to fire blight in the wild apple species Malus fusca. Mol Breed 34:407–419CrossRefGoogle Scholar
- Emeriewen OF, Richter K, Hanke M-V, Malnoy M, Peil A (2015) The fire blight resistance QTL of Malus fusca (Mfu10) is affected but not broken down by the highly virulent Canadian Erwinia amylovora strain E2002A. Eur J Plant Pathol 141:631–635CrossRefGoogle Scholar
- Emeriewen OF, Peil A, Richter K, Zini E, Hanke M-V, Malnoy M (2017a) Fire blight resistance of Malus ×arnoldiana is controlled by a quantitative trait locus located at the distal end of linkage group 12. Eur J Plant Pathol 148:1011–1018CrossRefGoogle Scholar
- Emeriewen OF, Richter K, Hanke M-V, Malnoy M, Peil A (2017b) Further insights into Malus fusca fire blight resistance. J Plant Pathol. https://doi.org/10.4454/jpp.v99i0.3908
- Fahrentrapp J, Broggini GAL, Kellerhals M, Peil A, Richter K, Zini E, Gessler C (2013) A candidate gene for fire blight resistance in Malus x robusta 5 is coding for a CC-NBS-LRR. Tree Genet Genomes 9:237–251CrossRefGoogle Scholar
- Galli P, Broggini GAL, Kellerhals M, Gessler C, Patocchi A (2010) High resolution genetic map of the Rvi15 (Vr2) apple scab resistance locus. Mol Breed 26:561–572CrossRefGoogle Scholar
- Hulo N, Bairoch A, Bulliard V, Cerutti L, Cuche BA, de Castro E, Lachaize C, Langendgijk-Genevaux PS, Sigrist CJA (2008) The 20 years of PROSITE. Nucleic Acids Res 36:245–249CrossRefGoogle Scholar
- Khan MA, Duffy B, Gessler C, Patocchi A (2006) QTL mapping of fire blight resistance. Mol Breed 17:299–306CrossRefGoogle Scholar
- Khan MA, Zhao YF, Korban SS (2012) Molecular mechanisms of pathogenesis and resistance to the bacterial pathogen Erwinia amylovora, causal agent of fire blight disease in Rosaceae. Plant Mol Biol Rep 30:247–260CrossRefGoogle Scholar
- Kost TD, Gessler C, Jänsch M, Flachowsky H, Patocchi A, Broggini GAL (2015) Development of the first cisgenic apple with increased resistance to fire blight. PLoS One 10(12):e0143980. https://doi.org/10.1371/journal.pone.0143980 CrossRefPubMedPubMedCentralGoogle Scholar
- Kruijt M, De Kock MJD, De Wit PJGM (2005) Receptor-like proteins involved in plant disease resistance. Mol Plant Pathol 6(1):85–97CrossRefPubMedGoogle Scholar
- Lannoo N, Van Damme EJM (2014) Lectin domains at the frontiers of plant defense. Front Plant Sci 5:397. https://doi.org/10.3389/fpls.2014.00397 PubMedPubMedCentralCrossRefGoogle Scholar
- Le Roux PMF, Khan MA, Broggini GAL, Duffy B, Gessler C, Patocchi A (2010) Mapping of quantitative trait loci for fire blight resistance in the apple cultivars 'Florina' and 'Nova Easygro'. Genome 53:710–722CrossRefPubMedGoogle Scholar
- Martin GB, Brommonschenkel SH, Chunwongse J, Frary A, Ganal MV, Spivey R, Wu T, Earle ED, Tanskley SD (1993) Map-based cloning of a protein kinase gene conferring disease resistance in tomato. Science 262:1432–1436CrossRefPubMedGoogle Scholar
- Norelli JN, Aldwinckle HS (1986) Differential susceptibility of Malus spp. cultivars Robusta 5, Novole, and Ottawa 523 to Erwinia amylovora. Plant Dis 70:1017–1019CrossRefGoogle Scholar
- Oh C-S, Beer SV (2005) Molecular genetics of Erwinia amylovora involves in the development of fire blight. FEMS Microbiol Lett 253:185–192CrossRefPubMedGoogle Scholar
- Parravicini G, Gessler C, Denance C, Lasserre-Zuber P, Vergne E, Brisset MN, Patocchi A, Durel CE, Broggini GAL (2011) Identification of serine/threonine kinase and nucleotide-binding-site-leucine-rich repeat (NBS-LRR) genes in the fire blight resistance quantitative trait locus of apple cultivar 'Evereste'. Mol Plant Pathol 12:493–505CrossRefPubMedGoogle Scholar
- Patocchi A, Vinatzer BA, Gianfranceschi L, Tartarini S, Zhang HB, Sansavini S, Gessler C (1999) Construction of a 550 kb BAC contig spanning the genomic region containing the apple scab resistance gene Vf. Mol Gen Genet 262:884–891CrossRefPubMedGoogle Scholar
- Paulin JP, Lespinasse Y (1990) Pathogenicity of strains of Erwinia amylovora to some apple cultivars in the greenhouse. Acta Hort 273:319–326CrossRefGoogle Scholar
- Peil A, Garcia-Libreros T, Richter K, Trognitz FC, Trognitz B, Hanke MV, Flachowsky H (2007) Strong evidence for a fire blight resistance gene of Malus robusta located on linkage group 3. Plant Breed 126:270–475CrossRefGoogle Scholar
- Peil A, Flachowsky H, Hanke M-V, Richter K, Rode J (2011) Inoculation of Malus × robusta 5 progeny with a strain breaking resistance to fire blight reveals a minor QTL on LG5. Acta Hort 986:357–362CrossRefGoogle Scholar
- Rozen S, Skaletsky HJ (2000) Primer3 on the WWW for general users and for biologist programmers. In: Krawetz S, Misener S (eds) Bioinformatics methods and protocols: methods in molecular biology. Humana, Totowa, NJ, pp 365–386Google Scholar
- Schuelke M (2000) An economic method for the fluorescent labelling of PCR fragments. Nat Biotechnol 18:233–234CrossRefPubMedGoogle Scholar
- Sun X, Cao Y, Yang Z, Xu C, Li X, Wang S, Zhang Q (2004) Xa26, a gene conferring resistance to Xanthomonas oryzae pv. Oryzae in rice, encodes an LRR receptor kinase-like protein. Plant J 37:517–527CrossRefPubMedGoogle Scholar
- Tao Q, Wang A, Zhang HB (2002) One large-insert plant-transformation-competent BIBAC library and three BAC libraries of Japonica rice for genome research in rice and other grasses. Theor Appl Genet 105:1058–1066CrossRefPubMedGoogle Scholar
- van de Weg E, Di Guardo M, Jänsch M et al (2018) Epistatic fire blight resistance QTL alleles in apple cultivar 'Enterprise' and selection X-6398 discovered and characterized through pedigree-informed analysis. Mol Breed 38(2018):5. https://doi.org/10.1007/s11032-017-0755-0 CrossRefGoogle Scholar
- Van Ooijen JW, Maliepaard C (1996) MapQTL 4.0. Software for the calculation of QTL positions on genetic maps. CPRO-DLO, WageningenGoogle Scholar
- Van Ooijen JW (2006) JoinMap ® 4, software for the calculation of genetic linkage maps in experimental populations. Kyazma B.V, WageningenGoogle Scholar
- Velasco R, Zharkikh A, Affourtit J, Dhingra A, Cestaro A, Kalyanaraman A, Fontana P, Bhatnagar SK, Troggio M, Pruss D, Salvi S, Pindo M, Baldi P, Castelletti S, Cavaiuolo M, Coppola G, Costa F, Cova V, Dal Ri A, Goremykin V, Komjanc M, Longhi S, Magnago P, Malacarne G, Malnoy M, Micheletti D, Moretto M, Perazzolli M, Si-Ammour A, Vezzulli S, Zini E, Eldredge G, Fitzgerald LM, Gutin N, Lanchbury J, Macalma T, Mitchell JT, Reid J, Wardell B, Kodira C, Chen Z, Desany B, Niazi F, Palmer M, Koepke T, Jiwan D, Schaeffer S, Krishnan V, Wu C, Chu VT, King ST, Vick J, Tao Q, Mraz A, Stormo A, Stormo K, Bogden R, Ederle D, Stella A, Vecchietti A, Kater MM, Masiero S, Lasserre P, Lespinasse Y, Allan AC, Bus V, Chagné D, Crowhurst RN, Gleave AP, Lavezzo E, Fawcett JA, Proost S, Rouzé P, Sterck L, Toppo S, Lazzari B, Hellens RP, Durel CE, Gutin A, Bumgarner RE, Gardiner SE, Skolnick M, Egholm M, van de Peer Y, Salamini F, Viola R (2010) The genome of the domesticated apple (Malus x domestica Borkh.). Nat Genet 42:833–839CrossRefPubMedGoogle Scholar
- Vogt I, Wöhner T, Richter K, Flachowsky H, Sundin GW, Wensing A, Savory EA, Geider K, Day B, Hanke MV, Peil A (2013) Gene-for-gene relationship in the host-pathogen system Malus xrobusta 5-Erwinia amylovora. New Phytol 197:1262–1275CrossRefPubMedGoogle Scholar
- Winslow CEA, Broadhurst J, Buchanan RE, Krumwiede C, Rogers LA, Smith GH (1920) The families and genera of the bacteria. Final report of the Committee of the Society of American Bacteriologists on the characterization and classification of bacterial types. J Bacteriol 5:191–229PubMedPubMedCentralGoogle Scholar
- Wöhner TW, Flachowsky H, Richter K, Garcia-Libreros T, Trognitz F, Hanke M-V, Peil A (2014) QTL mapping of fire blight resistance in Malus ×robusta 5 after inoculation with different strains of Erwinia amylovora. Mol Breed 34:217–230CrossRefGoogle Scholar
- Wöhner T, Richter K, Sundin GW, Zhao Y, Stockwell VO, Sellmann J, Flachowsky H, Hanke M-V, Peil A (2017) Inoculation of Malus genotypes with a set of Erwinia amylovora strains indicates a gene-for-gene relationship between the effector gene eop1 and both Malus floribunda 821 and Malus 'Evereste'. Plant Pathol 67:938–947. https://doi.org/10.1111/ppa.12784 CrossRefGoogle Scholar
- Xiang Y, Cao Y, Xu C, Li X, Wang S (2006) Xa3, conferring resistance for rice bacterial blight and encoding a receptor kinase-like protein, is the same as Xa26. Theor Appl Genet 113:1347–1355CrossRefPubMedGoogle Scholar
- Zhang Z, Schwartz S, Wagner L (2000) Miller W (2000) a greedy algorithm for aligning DNA sequences. J Comput Biol 7(1–2):203–214CrossRefPubMedGoogle Scholar
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Governor H R Bhardwaj has written to Chief Minister Siddaramaiah to take "immediate" and "effective" steps to implement the recommendations of the Criminal Investigation Department (CID) that probed the irregularities in selection of candidates for the posts of Gazetted Probationers Group 'A' and 'B,' by the Karnataka Public Service Commission (KPSC).
Bhardwaj's move comes in the wake of a delegation of members of the Loksatta party submitting a memorandum to him, along with the CID report, on the alleged large-scale irregularities in the KPSC.
The Governor's office despatched the letter to the chief minister on January 18, copies of which the Loksatta party released to the media on Wednesday.
The CID in its recommendations has sought prosecution of former KPSC chairperson Gonal Bhimappa, member Mangala Sridhar and others for their alleged involvement in the scam. It has also recommended fresh interviews for the 326 gazetted posts. "It is a matter of concern that the affairs of the Public Service Commission, a constitutional body, led to large public outcry in the State", the Governor stated.
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Glades Electric Cooperative News & Hot Topics
Report Outages and Make Payments with the SmartHub app
Members can now report an outage and access accounts with the SmartHub App! Check usage, pay your bill, manage account settings and more, through your mobile device or computer. Visit www.smarthubapp.com, the Apple App Store or the Google Play Store and search for the SmartHub app.
2019 Washington, DC Youth Tour Applications are available!
All high school juniors in Glades Electric Cooperative's service territory are eligible to apply for an all-expenses paid trip to Washington, DC. Students chosen will join their peers for a tour of our nation's capital, meetings with congressional representatives, and educational opportunities June 15-20, 2019. To apply, download and complete the application by February 7, 2019.
Reduce Clutter - Go Paperless!
Want a chance to receive a $250 credit to your account? Sign up for paperless billing today! One member enrolled in paperless billing will be chosen randomly each month to receive a $250 credit. Login to SmartHub to "turn off paper bills" or call GEC at 863-946-6200 to go paperless!
Considering Solar?
Don't be surprised by unexpected issues with a solar project installation. There are many factors to consider so please contact our Engineering Department at (863) 946-6200 BEFORE you sign an agreement with a solar contractor.
We Support Our Community's Students
We are proud to offer programs providing educational opportunities to students served by Glades Electric Cooperative, Inc.. Visit our Student Programs and Information Page to learn more about scholarship and Washington, DC Youth Tour opportunities for high school students. Applications are available now!


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Essendon VFL has named an extended squad to face Geelong on Sunday.
The Bombers will be looking to finish off the home-and-away season strong, with a win over the Cats at Simonds Stadium.
The Dons are sitting in sixth place on the ladder, with only one win separating them from a top four spot.
Nash Holmes is set to return, after missing the majority of the VFL Bombers season due to an ankle injury.
Essendon v Geelong
Simonds Stadium
Sunday 27th August
B 49. M Dea 28. M Brown 10. A Francis
HB 5. B Stanton 50. J Ferry 39. H Hocking
C 57. N Hind 30. K Langford 19. K Mutch
HF 27. M Redman 44. McKernan 54. N O'Brien
F 56. J Peters 63. M Darby 37. D Clarke
R 38. S Draper 24. C Bird 40. B Howlett
Int 64. C Lill, 25. A Morgan, 59. A Heppell, 55. D Younan,
62. M Luxford, 70. A Carr, 68. E Ogden, 71. J Cornell,
65. B Graham, 69. A Marklew, 14. J Ridley, 77. N Holmes
23P 72. J Ottavi
You can keep up to date with the game by following @essendonvfl on Twitter


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More Bad News for Canon's 6D Mark II
Does Canon's new camera actually perform worse than the cheaper EOS 80D?
Canon released its 6D Mark II last month to a pretty unanimously disappointed group of DIY filmmakers. Many had hoped that Canon's popular, cost effective little brother to the 5D would have the ability to record in 4K. It does not.
4K is quickly becoming a necessary format for cinema cameras, so this is a significant spec for Canon to leave out if it is courting the filmmaker market at all. Others are quick to point out that the photography company has been distancing their line of DSLRs from film users, perhaps in an attempt to draw more buyers towards its true cinema line of C-series cameras. The 6D was clearly updated with the photographer and not the filmmaker in mind.
The big upgrades are a swivel screen, image stabilization, and dual pixel auto focus. As Charles Haine noted in an earlier piece, "Dual pixel, a technology that many love, hasn't come in such an affordable package before, which will excite photographers. However, filmmakers, who still tend to manually focus more often, aren't necessarily dying for the feature. The swivel screen is a nice addition, but it's also something that pretty much all the major competitors have already, and many, many filmmakers are attaching external monitors for a larger image and extreme articulation, so the feature isn't likely to be a strong draw."
More bad news came this week as Camera tester William J. Claff of Photons to Photos released his Dynamic Range vs ISO chart for the 6D Mark II. As you can see, in some cases, the camera actually seems to perform worse than the last model.
The dynamic range is almost identical to the original 6D, with the main difference seemingly lying in an improvement at higher ISOs, but with the consequence of a worse performance at lower ISOs. Perhaps, the most indicative reading from the graph is that the 6D Mark II somehow performs worse than the significantly cheaper 80D DSLR at lower ISOs. The full frame 6D Mark II has a retail price of $1,999 while the APS-C 80D goes for $1,099.
It appears that the camera's highly touted newly designed sensor is actually worse. DPReview wrote that "it seems the benefits that appeared in the sensors used in the EOS 80D and EOS 5D IV have not been applied to the latest EOS 6D II, and the new camera has less dynamic range than we've become used to," adding that test shots from both cameras revealed the 80D footage, "shot with the same exposures look cleaner, when brightened to the same degree."
This is a major step backward for Canon's DSLRs, regardless of whether or not you're using it for video. As you can see in the second chart above, the current number one digital camera manufacturer on the market now faces some stiff competition from the likes of Sony and FujiFilm—especially for filmmakers—and it looks like the 6D won't do much in the ways of keeping them at the top.
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6 Comments
Have started my transition to sony for DSLR filmmaking... Canon slacking off and I'm tired of it.
July 22, 2017 at 2:00AM
Damn, looks like the 5D IV is a better camera than the 6D. Who would've thunk it?
Although, if I was shooting at ISO 100 all of the time and did not care about full frame vs crop frame, I would probably prefer the 80D. Truthfully, I just have a few EOS M's that I pull out whenever I need to take photos or timelapse since I am really more of a videographer than a photographer. If I really wanted to make money off of photography I would probably go for the 1DX II. Or maybe a 5D IV if the weight was an issue.
What does any of this have to do with the article? Nothing. If you want to shoot video with photo camera, get a Sony.
July 22, 2017 at 10:53AM
@NFS, wouldn't a more appropriate title for this article be, "Why the hell are filmmakers still paying any attention to Canon?"
Haha, in all honesty... this is a company who got lucky off the 5D and just DOES NOT GET US! Nor do they care about "us" (filmmakers). If that's not clear to you yet & your still expecting something worthwhile from the Canon-Camp, maybe you should write for a photography blog.
Canon is dead in my eyes and I have over $10k in EF glass, so it's not like I wasn't pulling for them, but I knew the score back when the 7DII first dropped.
These guys have no interest in catering to filmmakers via DSLR. They would rather you spend 5x more cash to buy one of their plastic Documentary-Cameras.
When they release a 6DII in 2017 with no 4K, it's lights out.
July 22, 2017 at 9:32PM
If you look at the bottom of any page on the site, you'll see 'The DSLR Cinematography Guide'. It's one of the things this site was started and built on; the DSLR revolution. Canon was the first to kick start it, and things haven't been the same since. It's like your first love moving on; you keep clinging to hope, even though not much is there, but you can't pretend nothing happened. Who knows? Maybe Canon might just hear the outcry, and mix everything up (They're mixed plenty up in the past!)
July 23, 2017 at 12:42AM
The C-Series is what Canon is offering videographers. They regard shooters still using one of their photo cameras primarily for video as a novelty, and the spec's on the 6DII & 5D MKIV were a not so subtle way of telling video shooters that their DSLR line is "just not that into them."
July 24, 2017 at 11:35AM


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SOURCE: World Street Fundamentals
October 04, 2011 09:00 ET
HONG KONG--(Marketwire - Oct 4, 2011) - Today, www.WorldStreetFundamentals.com released its industry report highlighting Sanderson Farms, Inc. (NASDAQ: SAFM) and Pilgrim's Pride Corporation (NYSE: PPC). Full fundamental and technical analysis is available at www.WorldStreetFundamentals.com/Reports.php?id=SAFM+PPC.
Short-term outlooks within US and global economies continue to adjust downward, as US Treasury yields touched their lowest levels in over 65 years. International Monetary Fund downgraded estimates for US growth by 1.0% this year, down from 2.5%. 'Significant downside risks' to the US economy were confirmed by the Federal Reserve pressured by 'strains in global financial markets.'
As the US dollar trades above an eight-month high against the Euro, it highlights a deepening of the Eurozone sovereign debt crisis -- furthered by continued policy inaction from European governments. We expect continued volatility until economic data stabilizes and we see decisive policy action to deal with the Eurozone debt crisis.
Our members are preparing for a turn in tides. Currently, the Credit Suisse Risk Appetite Indicator is at a 30-year low. For the past three occurrences, this level has acted as a leading indicator of large stock market rallies 18 months following.
World Street Fundamentals has highlighted Sanderson Farms, Inc. for its current position within the consumer goods industry. Sanderson Farms, Inc. is a poultry processing company engaged in the production, processing, marketing and distribution of fresh and frozen chicken products. In addition, the Company is engaged in the processing, marketing and distribution of prepared chicken through its wholly owned subsidiary, Sanderson Farms, Inc. (Foods Division). The entire report on Sanderson Farms, Inc. (NASDAQ: SAFM) is available here: www.WorldStreetFundamentals.com/Report.php?id=SAFM.
World Street has featured Pilgrim's Pride Corporation for its development within the consumer goods industry. Pilgrim's Pride Corporation (Pilgrim's) is a chicken producer with operations in the United States, Mexico and Puerto Rico. The Company is engaged in the production, processing, marketing and distribution of fresh, frozen and value-added chicken products to retailers, distributors and foodservice operators. The entire report on Pilgrim's Pride Corporation (NYSE: PPC) is available here: www.WorldStreetFundamentals.com/Report.php?id=PPC.
About World Street
World Street Fundamentals is an online portal for professionals, investors and new-comers to the markets to find in depth comprehensive research and research tools to help guide you through the ever changing financial markets.
Adam RedfordEmail Contactwww.WorldStreetFundamentals.com
Adam RedfordEmail Contactwww.WorldStreetFundamentals.com
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Maio Restaurants, Food and Beaches
a corn meal served hot, and Maio's renowned goat cheese, chicken and the favorite: Cachupa (a-hearty-everything-in-the-pot stew, mainly corn) surprisingly tasty, depending on the cook and the ingredients.
Local Dishes
Some of the other favorites indigenous dishes are: Pastel con Diablo Dentro (pastry with the devil inside)...fresh tuna fish, onions and tomatoes wrapped in a pastry made from boiled potatoes and corn flour, then deep fried.
Coldo de Peixe (fish stew)...fish, various spices, vegetables thickened with cassava root flour.
Food Markets
Vila has several mini-markets. There is soon to be a discount supermarket in Morro which will sell retail as well as wholesale at substantial savings.
Vila has good, modern restaurants and more coming, at a medium price range of about US$7.
DRINK BOTTLED WATER which is available at all hotels, restaurants and small shops. At restaurants, ask the waiter for "boia de agua" (1.5 liter) and take it with you.
Maio has splendid beaches which makes it a fabulous tourist attraction for beach goers, sail and surf boarding and scuba diving. The best beaches to be found are those visible from the road on the west coast: beginning with Vila's excellent beach, then stretching north where the turn in the road makes it possible to visually encounter the continuing beaches past Funchago until Morro.
For those sun lovers who dream of lonely virginal beaches, Maio is their fantasy come true; however, isolation also means that one must take swimming safety more seriously. At times large waves might create undercurrents on some of the beaches which makes the sea more conductive to surf board sailing and board surfing than casual swimming. Only the strongest of swimmers should challenge the sea when these conditions exist. Floatation devices are recommended if you are not a strong swimmer or are swimming alone. These warnings are not to frighten but only to remind one that the sea is an ever-changing, whimsical giant which cannot be taken lightly.
The people of Maio are not prudish or formal in their daily attire as many European and North African countries. Wear whatever makes you comfortable in a warm climate: walking shorts, blue jeans, bikinis (at the beach)…all readily accepted. Rubber-soled or jogging shoes are a must when walking on cobbled-stoned streets or highway. A hat and dark glasses are highly recommended, of course, a good sun screen.
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Katy Perry Joins YouTube Red! [Video]
Will you be my witness? That's exactly what Katy Perry is asking in this new behind-the-scenes feature film that will be dropping on YouTube Red.
Remember when Katy Perry live-streamed for four days straight? She played games, she had celebrity visitors, and of course… she slept. You really got a glimpse into who Katy Perry is and what Katy Perry does.
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text: Posted December 19, 2013 Shalom and greetings all my pals.Here is nice Greek holiday flight by Israir Airbus A320 from LLBG Ben Gurion airport located near the suburb of Lod in the southeast outskirts of Tel Aviv 12 miles from Tel Aviv's city center to LGIR Heraklion International Airport located about 5 km east of the main city center of HeraklionJetway disconnected from full boarded planeWaiting for pushback tug to start pushing back from gateNice wing view of busy airport!!Being pushed back from gate while airport's follow me car is waiting for the plane ready to taxi short of runway 30Time to start engines and make final adjustments to the cockpit panel (oppss I forgot to set altitude)Taxiing to short of runway 30 while the follow me airport car returns back to gate area to assist other planesoh darn some light rainNah I do not need wipers to deal with few raindrops on my cockpit window (oppss in state of excitement I still forgot to set altitude)Entering into runway 30 for take off via PURLA departureOn climb to FL340 executing PURLA departureBye bye Tel Aviv!!! I am still totally embarressed by lousy FSX default Tel Aviv city scenery and cannot find city scenery for thatClimbing through clouds is FUNMaking little left turn to avoid strong tuberlanceStill climbing climbing to FL340 through scattered thunderstormIntercepting PURLA and making right turn to fly straight path to island of Crete via UH1B SUVAS UL53 KAROL UM1 SITNow cruising at FL340 across Mediterranean sea Because of regulations of the forums not allowing more than 20 screenshots per post, please be kind enough to go to this hyperlink below to view rest of the flight. Here is the link showing approach to island of Crete: http://vatil.org.il/forum/index.php/topic/41190-approaching-island-of-crete-can-be-fun-for-israeli-pilots/Thank you for viewing!! Stay tuned for next exciting flight!!Regards,Aharon 1 Share this post Link to post Share on other sites