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Abstract 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 markers derived from putative fire blight resistance genes associated with the QTL 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 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 ‘Malling 9’ X ‘Robusta 5’ population inoculated with E. amylovora ICMP11176, the proximal QTL co-located with SNP markers derived from a leucine-rich repeat, receptor-like protein ( MxdRLP1) and a closely linked class 3 peroxidase gene. While the QTL detected in the German ‘Idared’ X ‘Robusta 5’ population inoculated with E. amylovora strains Ea222_JKI or ICMP11176 was approximately 6 cM distal to this, directly below a SNP marker derived from a heat shock 90 family protein gene ( HSP90). In the US ‘Otawa3’ X ‘Robusta5’ population inoculated with E. amylovora strains Ea273 or E2002a, 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’ 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. Mapping markers derived from putative fire blight resistance genes has proved a useful aid in defining these QTLs and developing markers for marker-assisted breeding of fire blight resistance.

Background: Most published genome sequences are drafts, and most are dominated by computational gene prediction. Draft genomes typically incorporate considerable sequence data that are not assigned to chromosomes, and predicted genes without quality confidence measures. The current Actinidia chinensis (kiwifruit) 'Hongyang' draft genome has 164 Mb of sequences unassigned to pseudo-chromosomes, and omissions have been identified in the gene models. Results: A second genome of an A. chinensis (genotype Red5) was fully sequenced. This new sequence resulted in a 554.0 Mb assembly with all but 6 Mb assigned to pseudo-chromosomes. Pseudo-chromosomal comparisons showed a considerable number of translocation events have occurred following a whole genome duplication (WGD) event some consistent with centromeric Robertsonian-like translocations. RNA sequencing data from 12 tissues and ab initio analysis informed a genome-wide manual annotation, using the WebApollo tool. In total, 33,044 gene loci represented by 33,123 isoforms were identified, named and tagged for quality of evidential support. Of these 3114 (9.4%) were identical to a protein within 'Hongyang' The Kiwifruit Information Resource (KIR v2). Some proportion of the differences will be varietal polymorphisms. However, as most computationally predicted Red5 models required manual re-annotation this proportion is expected to be small. The quality of the new gene models was tested by fully sequencing 550 cloned 'Hort16A' cDNAs and comparing with the predicted protein models for Red5 and both the original 'Hongyang' assembly and the revised annotation from KIR v2. Only 48.9% and 63.5% of the cDNAs had a match with 90% identity or better to the original and revised 'Hongyang' annotation, respectively, compared with 90.9% to the Red5 models. Conclusions: Our study highlights the need to take a cautious approach to draft genomes and computationally predicted genes. Our use of the manual annotation tool WebApollo facilitated manual checking and correction of gene models enabling improvement of computational prediction. This utility was especially relevant for certain types of gene families such as the EXPANSIN like genes. Finally, this high quality gene set will supply the kiwifruit and general plant community with a new tool for genomics and other comparative analysis. [ABSTRACT FROM AUTHOR]

Genotyping by sequencing (GBS) is a restriction enzyme based targeted approach developed to reduce the genome complexity and discover genetic markers when a priori sequence information is unavailable. Sufficient coverage at each locus is essential to distinguish heterozygous from homozygous sites accurately. The number of GBS samples able to be pooled in one sequencing lane is limited by the number of restriction sites present in the genome and the read depth required at each site per sample for accurate calling of single-nucleotide polymorphisms. Loci bias was observed using a slight modification of the Elshire et al. method: some restriction enzyme sites were represented in higher proportions while others were poorly represented or absent. This bias could be due to the quality of genomic DNA, the endonuclease and ligase reaction efficiency, the distance between restriction sites, the preferential amplification of small library restriction fragments, or bias towards cluster formation of small amplicons during the sequencing process. To overcome these issues, we have developed a GBS method based on randomly tagging genomic DNA (rtGBS). By randomly landing on the genome, we can, with less bias, find restriction sites that are far apart, and undetected by the standard GBS (stdGBS) method. The study comprises two types of biological replicates: six different kiwifruit plants and two independent DNA extractions per plant; and three types of technical replicates: four samples of each DNA extraction, stdGBS vs. rtGBS methods, and two independent library amplifications, each sequenced in separate lanes. A statistically significant unbiased distribution of restriction fragment size by rtGBS showed that this method targeted 49% (39,145) of BamH I sites shared with the reference genome, compared to only 14% (11,513) by stdGBS. [ABSTRACT FROM AUTHOR]

The molecular genetic mechanisms underlying fruit size remain poorly understood in perennial crops, despite size being an important agronomic trait. Here we show that the expression level of a micro RNA gene ( mi RNA172) influences fruit size in apple. A transposon insertional allele of mi RNA172 showing reduced expression associates with large fruit in an apple breeding population, whereas over-expression of mi RNA172 in transgenic apple significantly reduces fruit size. The transposon insertional allele was found to be co-located with a major fruit size quantitative trait locus, fixed in cultivated apples and their wild progenitor species with relatively large fruit. This finding supports the view that the selection for large size in apple fruit was initiated prior to apple domestication, likely by large mammals, before being subsequently strengthened by humans, and also helps to explain why signatures of genetic bottlenecks and selective sweeps are normally weaker in perennial crops than in annual crops. [ABSTRACT FROM AUTHOR]

Buds were sampled from non-flowering spurs that had developed on 1 year-old wood of four apple (Malus domestica Borkh.) cultivars during the 1998-99 growing season in order to determine the effect of cultivar on appendage formation, doming and flower morphogenesis. Cultivars differed in their pattern of appendage formation over time. The rate of appendage formation was highest in 'Fuji' and 'Pacific Rose™' and lowest in 'Braeburn' during the first 60 d after bloom (DAB). A high proportion of buds were floral in all cultivars at the end of the growing season (75-100% depending on cultivar). However, the probability of observing doming was never greater than 0.13, indicating that flower morphogenesis proceeded rapidly once buds were committed to floral development. The four cultivars each exhibited a unique pattern of floral development, as determined by fitting response probabilities to each of five ordinal stages of development with time. Doming occurred significantly earlier in 'Fuji' buds (peaking 86 DAB) than in buds of the other cultivars (peaking 104-112 DAB). Doming was initiated at the same time in buds of 'Braeburn', 'Royal Gala' and 'Pacific Rose™, but was completed earlier in 'Braeburn' than in 'Royal Gala'. The timing of floral commitment was not related either to the time of flowering, or to the time of fruit maturity of the cultivar. These observations indicate that the timing of specific events during flower morphogenesis differed between cultivars. [ABSTRACT FROM AUTHOR]

Highlights: [•] A model has been developed to describe relationships between storage temperature and duration, fruit dry matter at harvest, and disorder incidence after storage. [•] The model developed is a logistic non-linear mixed effects model and its simplified version with normal errors on the logit linear predictor. [•] The model gives a good general description of the relationship between disorders and dry matter at harvest, storage temperature and storage duration. [•] Predictions of disorder at the orchard level are likely to have large prediction errors because of the high variability among orchards and seasons. [Copyright &y& Elsevier]