Genomic selection for fruit quality traits in apple (Malus×domestica Borkh.)

The genome sequence of apple (Malus×domestica Borkh.) was published more than a year ago, which helped develop an 8K SNP chip to assist in implementing genomic selection (GS). In apple breeding programmes, GS can be used to obtain genomic breeding values (GEBV) for choosing next-generation parents or selections for further testing as potential commercial cultivars at a very early stage. Thus GS has the potential to accelerate breeding efficiency significantly because of decreased generation interval or increased selection intensity. We evaluated the accuracy of GS in a population of 1120 seedlings generated from a factorial mating design of four females and two male parents. All seedlings were genotyped using an Illumina Infinium chip comprising 8,000 single nucleotide polymorphisms (SNPs), and were phenotyped for various fruit quality traits. Random-regression best liner unbiased prediction (RR-BLUP) and the Bayesian LASSO method were used to obtain GEBV, and compared using a cross-validation approach for their accuracy to predict unobserved BLUP-BV. Accuracies were very similar for both methods, varying from 0.70 to 0.90 for various fruit quality traits. The selection response per unit time using GS compared with the traditional BLUP-based selection were very high (>100%) especially for low-heritability traits. Genome-wide average estimated linkage disequilibrium (LD) between adjacent SNPs was 0.32, with a relatively slow decay of LD in the long range (r(2)?=?0.33 and 0.19 at 100 kb and 1,000 kb respectively), contributing to the higher accuracy of GS. Distribution of estimated SNP effects revealed involvement of large effect genes with likely pleiotropic effects. These results demonstrated that genomic selection is a credible alternative to conventional selection for fruit quality traits.     

Mapping quantitative physiological traits in apple (Malus × domestica Borkh.)

Efficient breeding and selection of high-quality apple cultivars requires knowledge and understanding of the underlying genetics. The availability of genetic linkage maps constructed with molecular markers enables the detection and analysis of major genes and quantitative trait loci contributing to the quality traits of a genotype. A segregating population of the cross between the apple varieties `Fiesta' (syn. `Red Pippin') and `Discovery' has been observed over three years at three different sites in Switzerland and data on growth habit, blooming behaviour, juvenile period and fruit quality has been recorded. QTL analyses were performed, based on a genetic linkage map consisting of 804 molecular markers and covering all 17 apple chromosomes. With the maximum likelihood based interval mapping method, the investigated complex traits could be dissected into a number of QTLs affecting the observed characters. Genomic regions participating in the genetic control of stem diameter, plant height increment, leaf size, blooming time, blooming intensity, juvenile phase length, time of fruit maturity, number of fruit, fruit size and weight, fruit flesh firmness, sugar content and fruit acidity were identified and compared with previously mapped QTLs in apple. Although `Discovery' fruit displayed a higher acid content, both acidity QTLs were attributed to the sweeter parent `Fiesta'. This indicated homozygosity at the acidity loci in `Discovery' preventing their detection in the progeny due to the lack of segregation.     

Molecular characterization of newS-RNases (‘S31’ and ‘S32’) in apple (Malus ×domestica Borkh.)’) in apple (Malus ×domestica Borkh.)

Apple exhibits gametophytic self-incompatibility (GSI) that is controlled by the multiallelic S-locus. This S-locus encodes polymorphicS ribonuclease (S-RNase) for the pistil-part 5 determinant. Information aboutS-genotypes is important when selecting pollen donors for fruit production and breeding of new cultivars. We determined the 5-genotypes of ‘Charden’ (S₂S₃S₄), ‘Winesap’ (S₁S₂₈), ‘York Imperial’ (S₂S₃₁), ‘Stark Earliblaze’ (S₁S₂₈), and ‘Burgundy’ (S₂₀S₃₂), byS-RNase sequencing and S-allele-specific PCR analysis. Two newS-RNases, S₃₁ and S₃₂, were also identified from ‘York Imperial’ and ‘Burgundy’, respectively. These newS-alleles contained the conserved eight cysteine residues and two histidine residues essential for RNase activity. Whereas S₃₁ showed high similarity to S₂₀ (94%), S₃₂ exhibited 58% (to S₂₄) to 76% (to S₂₅) similarity in the exon regions. We designed newS-allele-specific primers for amplifying S₃₁- and S₃₂-RNasc-specific fragments; these can serve as specific gene markers. We also rearranged the apple S-allele numbers containing those newS-RNases. They should be useful, along with anS-RNase-based PCR system, in determining S-genotypes and analyzing new alleles from apple cultivars.     

Cytogenetics in fruit breeding - localization of ribosomal RNA genes on chromosomes of apple (Malus×domestica Borkh.)

 The localization of rRNA genes was studied by fluorescent in situ hybridization (FISH) on chromosomes of the cultivated apple, M.×domestica ‘Pinova’ (2n=34). The 18S/25S rRNA loci were detected in terminal positions of the short arms of two submetacentric and two metacentric chromosome pairs. One 5S rRNA gene locus was found in the proximal region of the short arm of a small metacentric chromosome pair. Received : 21 June 1996 / Accepted : 28 June 1996     

Heat-shock-mediated elimination of the nptII marker gene in transgenic apple (Malus×domestica Borkh.)

Production of marker-free genetically modified (GM) plants is one of the major challenges of molecular fruit breeding. Employing clean vector technologies, allowing the removal of undesired DNA sequences from GM plants, this goal can be achieved. The present study describes the establishment of a clean vector system in apple Malus×domestica Borkh., which is based on the use of the neomycin phosphotransferase II gene (nptII) as selectable marker gene and kanamycin/paramomycin as selective agent. The nptII gene can be removed after selection of GM shoots via site-specific excision mediated by heat-shock-inducible expression of the budding yeast FLP recombinase driven by the soybean Gmhsp17.5-E promoter. We created a monitoring vector containing the nptII and the flp gene as a box flanked by two direct repeats of the flp recognition target (FRT) sites. The FRT-flanked box separates the gusA reporter gene from the Cauliflower Mosaic Virus 35S (CaMV 35S) promoter. Consequently, GUS expression does only occur after elimination of the FRT-flanked box. Transformation experiments using the monitoring vector resulted in a total of nine transgenic lines. These lines were investigated for transgenicity by PCR, RT-PCR and Southern hybridization. Among different temperature regimes tested, exposure to 42 °C for 3.5 to 4h led to efficient induction of FLP-mediated recombination and removal of the nptII marker gene. A second round of shoot regeneration from leaf explants led to GM apple plants completely free of the nptII gene.     

Linkage maps of the apple ( Malus × domestica Borkh.) cvs ‘Ralls Janet’ and ‘Delicious’ include newly developed EST markers

Two apple genetic linkage maps were constructed using amplified fragment length polymorphisms (AFLPs), simple sequence repeats (SSRs), random amplified polymorphic DNAs (RAPDs), and expressed sequence tag (EST)-derived markers in combination with a pseudo-testcross mapping strategy in which the cultivars ‘Ralls Janet’ and ‘Delicious’ were used as the respective seed parents. Mitsubakaido (Malus sieboldii) was used as the pollen parent for each of the segregating F₁ populations. Expressed sequence tag data were obtained from the random sequencing of cDNA libraries constructed from in vitro cultured shoots and maturing fruits of cv ‘Fuji’, which is the offspring of a cross between ‘Ralls Janet’ and ‘Delicious’. In addition, a number of published gene sequences were used to develop markers for mapping. The ‘Ralls Janet’ map consisted of 346 markers (178 AFLPs, 95 RAPDs, 54 SSRs, 18 ESTs, and the S locus) in 17 linkage groups, with a total length of 1082 cM, while that of ‘Delicious’ comprised 300 markers (120 AFLPs, 81 RAPDs, 64 SSRs, 32 ESTs, and the S, Rf, and MdACS-1 loci) on 17 linkage groups spanning 1031 cM. These maps are amenable to comparisons with previously published maps of ‘Fiesta’ and ‘Discovery’ (Liebhard et al., Mol Breed 10:217–241, 2002; Liebhard et al., Theor Appl Genet 106:1497–1508, 2003a) because several of the SSRs (one to three markers per linkage group) were used in all of the maps. Distorted marker segregation was observed in three and two regions of the ‘Ralls Janet’ and ‘Delicious’ maps, respectively. These regions were localized in different parts of the genome from those in previously reported apple linkage maps. This marker distortion may be dependent on the combinations of cultivars used for map construction.     

Genetic and physical characterisation of the locus controlling columnar habit in apple (Malus × domestica Borkh.)

A better understanding of the genetic control of tree architecture would potentially allow improved tailoring of newly bred apple cultivars in terms of field management aspects, such as planting density, pruning, pest control and disease protection. It would also have an indirect impact on yield and fruit quality. The Columnar (Co) locus strongly suppresses lateral branch elongation and is the most important genetic locus influencing tree architecture in apple. Co has previously been mapped on apple linkage group (LG) 10. In order to obtain fine mapping of Co, both genetically and physically, we have phenotypically analysed and screened three adult segregating experimental populations, with a total of 301 F₁ plants, and one substantial 3-year old population of 1,250 F₁ plants with newly developed simple sequence repeat (SSR) markers, based on the ‘Golden delicious’ apple genome sequence now available. Co was found to co-segregate with SSR marker Co04R12 and was confined in a region of 0.56 cM between SSR markers Co04R11 and Co04R13, corresponding to 393 kb on the ‘Golden delicious’ genome sequence. In this region, 36 genes were predicted, including at least seven sequences potentially belonging to genes that could be considered candidates for involvement in control of shoot development. Our results provide highly reliable, virtually co-segregating markers that will facilitate apple breeding aimed at modifications of the tree habit and lay the foundations for the cloning of Co.     

Identification of genes differentially expressed during interaction of resistant and susceptible apple cultivars (Malus × domestica) with Erwinia amylovora

Background  

The necrogenic enterobacterium, Erwinia amylovora is the causal agent of the fire blight (FB) disease in many Rosaceaespecies, including apple and pear. During the infection process, the bacteria induce an oxidative stress response with kinetics similar to those induced in an incompatible bacteria-plant interaction. No resistance mechanism to E. amylovora in host plants has yet been characterized, recent work has identified some molecular events which occur in resistant and/or susceptible host interaction with E. amylovora: In order to understand the mechanisms that characterize responses to FB, differentially expressed genes were identified by cDNA-AFLP analysis in resistant and susceptible apple genotypes after inoculation with E. amylovora.     

One-step reconstruction of multi-generation pedigree networks in apple (Malus × domestica Borkh.) and the parentage of Golden Delicious

The increasing availability of genomic tools improves our ability to investigate the patterns of genetic diversity and relatedness among individuals. The pedigrees of many apple cultivars are completely unknown, often reducing the efficiency of breeding programs. Using a multilocus simple sequence repeat dataset, we applied a novel multi-generation pedigree-network reconstruction procedure based on the software FRANz in a Malus × domestica collection (101 cultivated and 22 wild apples) with partially known pedigree relationships. The procedure produced 78 parent–offspring relationships organized into three networks and showed high power for detecting real pedigree links (98.5 %) and a low false-positive rate (9.0 %). The largest reconstructed pedigree network spanned four generations and involved 65 cultivars. The availability of detailed pedigree connections confirmed that recent genealogical relationships affect population genetic structure in apple. Finally, our analysis enabled us to confirm or discard several pedigrees known only anecdotically, among which the cultivar Grimes Golden was validated as a parent of the widely grown cultivar Golden Delicious. The pedigree reconstruction protocol here described will be of broad applicability to other collections and crop species.     

The Fast-track breeding approach can be improved by heat-induced expression of the FLOWERING LOCUS T genes from poplar (Populus trichocarpa) in apple (Malus × domestica Borkh.)

The Fast-track breeding approach in apple is based on the utilization of the BpMADS4 gene from Betula pendula. However, this approach has several disadvantages which could be solved using other flowering inducing genes and inducible promoters. The FLOWERING LOCUS T genes (PtFT1 and PtFT2) from poplar (Populus trichocarpa) driven by the heat-inducible Gmhsp 17.5-E (HSP)-promoter from soybean (Glycine max) were transferred into apple (Malus × domestica Borkh.) cv. ‘Pinova’ in order to induce flowering. Seven transgenic apple lines were obtained. All transgenic apple lines micrografted onto ‘Golden Delicious’ seedlings used as rootstocks were transferred to the greenhouse. Six out of seven transgenic lines developed flowers after a heat treatment at 42 °C for 1 h daily over a period of 28 days. The transgenic line T836 failed to flower. Flower morphology and pollen vitality of transgenic lines appeared normal. Transgenic plants were successfully used for hybridizations. Pollen from Malus ×robusta 5 applied to flowers of transgenic plants resulted in fruit formation. Heat induced PtFT1, respectively PtFT2 over-expressing rootstocks did not cause flowering in micrografted non-transgenic ‘Pinova’ scions. The mRNA of the PtFT genes was transported from transgenic rootstocks to non-transgenic scions only in one case. As a balance between plant development and flowering is important for the production of early flowering plants usable for a fast-track breeding program the new approach based on heat-induced flowering could be a refinement of the fast breeding program using the possibility of turning-on-turning-off flowering in physiological well developed plants.     

Isolation and characterization of multiple F-box genes linked to the S 9 - and S 10 -RNase in apple (Malus × domestica Borkh.)

Using 11 consensus primer pairs designed from S-linked F-box genes of apple and Japanese pear, 10 new F-box genes (MdFBX21 to 30) were isolated from the apple cultivar ‘Spartan’ (S ₉ S ₁₀ ). MdFBX21 to 23 and MdFBX24 to 30 were completely linked to the S ₉ -RNase and S ₁₀ -RNase, respectively, and showed pollen-specific expression and S-haplotype-specific polymorphisms. Therefore, these 10 F-box genes are good candidates for the pollen determinant of self-incompatibility in apple. Phylogenetic analysis and comparison of deduced amino acid sequences of MdFBX21 to 30 with those of 25 S-linked F-box genes previously isolated from apple showed that a deduced amino acid identity of greater than 88.0 % can be used as the tentative criterion to classify F-box genes into one type. Using this criterion, 31 of 35 F-box genes of apple were classified into 11 types (SFBB1–11). All types included F-box genes derived from S ₃ - and S ₉ -haplotypes, and seven types included F-box genes derived from S ₃ -, S ₉ -, and S ₁₀ -haplotypes. Moreover, comparison of nucleotide sequences of S-RNases and multiple F-box genes among S ₃ -, S ₉ -, and S ₁₀ -haplotypes suggested that F-box genes within each type showed high nucleotide identity regardless of the identity of the S-RNase. The large number of F-box genes as candidates for the pollen determinant and the high degree of conservation within each type are consistent with the collaborative non-self-recognition model reported for Petunia. These findings support that the collaborative non-self-recognition system also exists in apple.     

Effects of apple (Malus?×?domestica Borkh.) phenolic compounds on proteins and cell wall-degrading enzymes of Venturia inaequalis

Cell wall-degrading enzymes of Venturia inaequalis are supposed to be fungal virulence factors whereas phenolic compounds of the host plant may be involved in defence. Since phenolic structures are predestined for an interaction with proteins we studied the effects on enzymes and proteins in course of in vitro culture and with preparations from culture filtrates and mycelia, respectively. The native compounds epicatechin, catechin, phloridzin, chlorogenic acid, caffeic acid, p-coumaric acid and phloridzin tested under non-oxidizing conditions had no or weak effects on enzyme activities. A significant inhibition of pectinase was only detected with the highest concentrations of procyanidins and phloretin. Aerobe conditions resulted in a fast oxidation of most phenolics which was enhanced by fungal phenoloxidases. Generally, no inhibition of fungal growth occurred in vitro but distinct irreversible effects on proteins and enzymes were detected with oxidized phenolics in course of in vitro-cultures as well as with the corresponding preparations. Efficacy of inhibitory activity in in vitro-cultures depended on media, culture technique and time course. Direct treatment of enzyme preparations with the oxidized phenolics resulted in a distinct inhibition of cellulolytic and especially pectinolytic activity. Apart from cellulase pattern altered by phenolics, in vitro-culture zymograms revealed a non specific reduction of enzymatic activities, whereas action on total culture filtrate proteins resulted in specific effects due to phenolic compounds and incubation time. An attempt was made to characterize the oxidation products of epicatechin. Chromatographic fractionation revealed a non-resolvable complex of inhibitory compounds which were not consistent with the typical yellow oxidation products.     

Endophytic bacterial community living in roots of healthy and ‘Candidatus Phytoplasma mali’-infected apple (Malus domestica, Borkh.) trees

‘Candidatus Phytoplasma mali’, the causal agent of apple proliferation (AP) disease, is a quarantine pathogen controlled by chemical treatments against insect vectors and eradication of diseased plants. In accordance with the European Community guidelines, novel strategies should be developed for sustainable management of plant diseases by using resistance inducers (e.g. endophytes). A basic point for the success of this approach is the study of endophytic bacteria associated with plants. In the present work, endophytic bacteria living in healthy and ‘Ca. Phytoplasma mali’-infected apple trees were described by cultivation-dependent and independent methods. 16S rDNA sequence analysis showed the presence of the groups Proteobacteria, Acidobacteria, Bacteroidetes, Actinobacteria, Chlamydiae, and Firmicutes. In detail, library analyses underscored 24 and 17 operational taxonomic units (OTUs) in healthy and infected roots, respectively, with a dominance of Betaproteobacteria. Moreover, differences in OTUs number and in CFU/g suggested that phytoplasmas could modify the composition of endophytic bacterial communities associated with infected plants. Intriguingly, the combination of culturing methods and cloning analysis allowed the identification of endophytic bacteria (e.g. Bacillus, Pseudomonas, and Burkholderia) that have been reported as biocontrol agents. Future research will investigate the capability of these bacteria to control ‘Ca. Phytoplasma mali’ in order to develop sustainable approaches for managing AP.     

Application of a high-speed breeding technology to apple (Malus × domestica) based on transgenic early flowering plants and marker-assisted selection

Breeding of apple (Malus × domestica) remains a slow process because of protracted generation cycles. Shortening the juvenile phase to achieve the introgression of traits from wild species into prebreeding material within a reasonable time frame is a great challenge. In this study, we evaluated early flowering transgenic apple lines overexpressing the BpMADS4 gene of silver birch with regard to tree morphology in glasshouse conditions. Based on the results obtained, line T1190 was selected for further analysis and application to fast breeding. The DNA sequences flanking the T-DNA were isolated and the T-DNA integration site was mapped on linkage group 4. The inheritance and correctness of the T-DNA integration were confirmed after meiosis. A crossbred breeding programme was initiated by crossing T1190 with the fire blight-resistant wild species Malus fusca. Transgenic early flowering F(1) seedlings were selected and backcrossed with 'Regia' and 98/6-10 in order to introgress the apple scab Rvi2, Rvi4 and powdery mildew Pl-1, Pl-2 resistance genes and the fire blight resistance quantitative trait locus FB-F7 present in 'Regia'. Three transgenic BC'1 seedlings pyramiding Rvi2, Rvi4 and FB-F7, as well as three other BC'1 seedlings combining Pl-1 and Pl-2, were identified. Thus, the first transgenic early flowering-based apple breeding programme combined with marker-assisted selection was established.     

Map position and functional allelic diversity of Md-Exp7, a new putative expansin gene associated with fruit softening in apple (Malus × domestica Borkh.) and pear (Pyrus communis)

Fruit ripening can be considered as a complex set of biochemical and physiological changes occurring at the end of the developmental stage. Ripe fruit texture notably affects overall quality and consumer appreciation. Excessive softening limits shelf-life and storability, thereby increasing disease susceptibility and economic loss. Fruit softening is a process due to the depolymerisation of different polysaccharide classes, an event controlled by a synergic and coordinated action of several enzymes among which expansins play a fundamental role. To date, six expansin genes are known to be expressed during apple fruit ontogeny, from full bloom up to fruit ripening. We identified a novel expansin apple homolog (Md-Exp7) sharing high sequence similarity with specific-ripening expansin genes of other crops. A functional marker (Md-Exp7_SSR) based on an SSR motif located within the untranslated region of the gene was developed and mapped on Linkage Group 1 of the apple and pear genomes in a region where one major apple QTL for fruit firmness had been previously identified. The allelic composition of 31 apple varieties for the SSR marker was associated with differences in fruit softening.     

Determination of partial genomic sequences and development of a CAPS system of the S-RNase gene for the identification of 22 S haplotypes of apple (Malus × domestica Borkh.)

Information about self-incompatibility (S) genotypes of apple cultivars is important for the selection of pollen donors for fruit production and breeding. Although S genotyping systems using S haplotype-specific PCR of S-RNase, the pistil S gene, are useful, they are sometimes associated with false-positive/negative problems and are unable to identify new S haplotypes. The CAPS (cleaved amplified polymorphic sequences) system is expected to overcome these problems, however, the genomic sequences needed to establish this system are not available for many S-RNases. Here, we determined partial genomic sequences of eight S-RNases, and used the information to design new primer and to select 17 restriction enzymes for the discrimination of 22 S-RNases by CAPS. Using the system, the S genotypes of three cultivars were determined. The genomic sequence-based CAPS system would be useful for S genotyping and analyzing new S haplotypes of apple.     

Branch development in custard apple (cherimoya Annona cherimola Miller × sugar apple A. squamosa L.) in relation to tip-pruning and flowering, including effects on production

Custard apple has cryptic axillary buds, hidden from view by the base of the petiole. This has led to confusion about custard apple’s flowering habit. Flowering only occurs during early branch development, and can be forced at any time of the growing season simply by removing leaves. Here, we show that flowering is terminal, not extra-axillary, and that the apparent continuation of the main stem beyond the flower is, instead, a sympodial branch. Secondary (including sympodial) branching only occurs during early branch development. Thereafter, axillary bud release is inhibited by the subtending leaf. Here, we show that summer tip-pruning of all branches arrests canopy development until the following spring owing to this inhibition. Although summer tip-pruning prevented new vegetative growth in the canopy, fruit size decreased relative to the control trees by ca. 23%. The reason for this decrease was probably related to increased carbon limitation to growth given that dawn water soluble and total non-structural carbohydrate concentrations were lower in the tip-pruned trees. Thus, it appears that the reduced competition between fruit development and new vegetative growth in the tip-pruned trees was more than matched by lower photosynthetic capacity in the arrested canopy. Trees grown inside a shade-house were more vigorous than those grown outside. The difference in vigour had little effect on fruit size.