Creating a saturated reference map for the apple (Malus × domestica Borkh.) genome

The availability of a high quality linkage map is essential for the detection and the analysis of quantitative traits. Such a map should cover a significant part of the genome, should be densely populated with markers, and in order to gain the maximum advantage should be transferable to populations or cultivars other than the ones on which it has been constructed. An apple genetic linkage map has been constructed on the basis of a segregating population of the cross between the cultivars Fiesta and Discovery. A total of 840 molecular markers, 475 AFLPs, 235 RAPDs, 129 SSRs and 1 SCAR, were used for the two parental maps constructed with JoinMap and spanning 1,140 cM and 1,450 cM, respectively. Large numbers of codominant markers, like SSRs, enable a rapid transfer of the map to other populations or cultivars, allowing the investigation of any chosen trait in another genetic background. This map is currently the most advanced linkage map in apple with regard to genome coverage and marker density. It represents an ideal starting point for future mapping projects in Malus since the stable and transferable SSR frame of the map can be saturated quickly with dominant AFLP markers.     

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.     

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.     

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.     

Validation of a functional molecular marker suitable for marker-assisted breeding for fruit texture in apple (Malus × domestica Borkh.)

Molecular markers are nowadays considered fundamental tools in breeding programs, supporting the selection of the most favourable offspring. This role is invaluable in the case of complex agronomic traits in tree fruit crop species, such as fruit texture in apple (Malus × domestica Borkh.). This work presents the validation of a previously identified functional simple sequence repeat marker, named Md-PG1_SSR10kd, suitable for the advanced selection of high texture performance seedlings. Two independent populations were chosen by marker-assisted parent selection, and a specific set of seedlings was selected by marker-assisted seedling selection, to validate the predictive power of this marker. The two groups of seedlings, further phenotyped for fruit texture, showed a clear difference in texture behaviour. The selection of this marker also showed a higher efficiency than Md-ACS1 and Md-ACO1, two functional markers currently implemented in different breeding programs. Finally, the allelic effect was estimated by computing the breeding values in a collection of 83 different apple cultivars. The results reported here confirmed the association of Md-PG1_SSR10kd with texture sub-traits, proposing this as a novel promising selection strategy suitable for apple fruit texture.     

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.     

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.     

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.     

Cryopreservation of apple (Malus×domestica Borkh.) shoot tips following encapsulation-dehydration or encapsulation-vitrification

Axillary shoot tips of apple cv. Golden Delicious isolated from shoot cultures were successfully cryopreserved using the encapsulation-dehydration technique. After encapsulation in alginate gel, embedded shoot tips were dehydrated by exposure to a sterile air flow before being frozen in liquid nitrogen and subsequent slow thawing. A preculture on modified MS medium containing 0.75 M sucrose followed by 6 h of dehydration (21% residual water) led to the highest shoot regrowth of frozen, coated shoot tips (83.7%). Among the sugars tested, sucrose and sorbitol presented the best cryoprotective effect. Four other scion apple varieties and rootstocks were also successfully cryopreserved. Axillary shoot tips of five apple (Malus×domestica Borkh.) scion and rootstock cultivars were cryopreserved using the encapsulation-vitrification technique. Using a one-step freezing method, we successfully cryopreserved axillary shoot tips without the requirement of a cold hardening pretreatment of the shoot cultures. Cryopreserved shoot tips treated with aqueous cryoprotective mixture IV containing 180% (w/v) sucrose and 120% (v/v) ethylene glycol showed the highest shoot regrowth rates, which varied from 64% to 77%, depending on the cultivar. Received: 29 July 1999 / Revision received: 24 September 1999 / Accepted: 26 November 1999     

Differential gene expression analysis of ‘Granny Smith’ apple (Malus domestica Borkh.) during fruit skin coloration

‘Granny Smith’ apples growing under normal sunlight develop green skin, whereas the peel turns red due to anthocyanin accumulation after the removal of a bagging treatment. Two anthocyanins, Cyanidin 3-O-galactoside (cy3-gal) and Cyanidin 3-O-arabinoside (cy3-ara), were detected in the red ‘Granny Smith’ apple peels, and cy3-gal was determined to be chiefly responsible for the red color. The content of cy3-gal was more than 98% of the total anthocyanin in the red ‘Granny Smith’ peels. To better understand the molecular basis of anthocyanin biosynthesis in ‘Granny Smith’ apples, we performed a quantitative real-time PCR (qRT-PCR) analysis of anthocyanin biosynthetic genes (MdCHS, MdF3H, MdDFR, MdANS, MdUFGT, and MdMYB1). Our results indicate that the expression of these genes (except MdCHS) was associated with increased anthocyanin accumulation in the skin of ‘Granny Smith’ apples. Four selected genes obtained from the ‘Granny Smith’ skin cDNA library, phytoene synthase (PSY), WD40 repeat protein, polygalacturonase (PG), and galactosidase (GAL), were also confirmed by qRT-PCR. We found that these genes were differently expressed during ‘Granny Smith’ apple skin coloration, suggesting that they are directly or indirectly involved in pigment accumulation. In conclusion, anthocyanin biosynthesis in ‘Granny Smith’ apples is the result of interactions between multiple enzymes in the anthocyanin biosynthesis pathway, and the coloring mechanism of ‘Granny Smith’ apples may be similar to that of red-skinned cultivars.     

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.     

Expression patterns of several floral genes during flower initiation in the apical buds of apple (Malus × domestica Borkh.) revealed by in situ hybridization

The apple (Malus?×?domestica Borkh.) is one of the commercially important fruit crops in the worldwide. The apple has a relatively long juvenile period (up to 4?years) and a long reproductive period between the flower initiation and the mature fruit (14?C16?months), which prevent the fruit breeding. Therefore, the understanding of the flowering system is important to improve breeding efficiency in the apple. In this study, to examine the temporal and spatial expression patterns of the floral genes, MdTFL1, MdAP1 (MdMASD5), AFL2, and MdFT, we conducted in situ hybridization analysis in the apple shoot apex. In vegetative phase, MdTFL1 was expressed on the rib meristem zone. When vegetative meristem began converting into inflorescence meristem, the expression level of MdTFL1 was drastically decreased. At the early stage of inflorescence meristem, the expression levels of AFL2, MdFT, and MdAP1 were up-regulated in the leaf primordia and the upper region of cell layers on the shoot apex. In late stage, the expression levels of AFL2 and MdAP1 were up-regulated in the young floral primordia. At a more advanced stage, high expression of MdAP1 was observed in the inflorescence primordium through the inner layer of sepal primordia and the outer layer of receptacle primordia and floral axis. Our results suggest that AFL2, MdFT, and MdAP1 affect to convert from the vegetative meristem into the inflorescence meristem after the decline of MdTFL1 expression. After that, AFL2 and MdAP1 promote the formation of the floral primordia and floral organs.     

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     

Characterization of the flanking regions of the S-RNase genes of Japanese pear (Pyrus serotina) and apple (Malus×domestica)

Genomic sequences of the self-incompatibility genes, the S-RNase genes, from two rosaceous species, Japanese pear and apple, were characterized. Genomic Southern blot and sequencing of a 4.5-kb genomic clone showed that the S⁴-RNase gene of Japanese pear is surrounded by repetitive sequences as in the case of the S-RNase genes of solanaceous species. The flanking regions of the S²- and S^f-RNase genes of apple were also cloned and sequenced. The 5′ flanking regions of the three alleles bore no similarity with those of the solanaceous S-RNase genes, although the position and sequence of the putative TATA box were conserved. The putative promoter regions of the Japanese pear S⁴- and apple S^f-RNase genes shared a stretch of about 200 bp with 80% sequence identity. However, this sequence was not present in the S²-RNase gene of apple, and thus it may reflect a close relationship between the S⁴- and S^f-RNase genes rather than a cis-element important in regulating gene expression. Despite the uniform pattern of expression of the rosaceous S-RNase genes, sequence motifs conserved in the 5′ flanking regions of the three alleles were not found, implying that the cis-element controlling pistil specific gene expression also locates at the intragenic region or upstream of the analyzed promoter region.     

Primigenic and positional dominance among reproductive buds in shoots of two apple (Malus × domestica (Borkh.)) cultivars in a warmer and cooler site

Key message

In two apple cultivars, fruit set was due to primigenic dominance within the annual shoot in areas with insufficient winter chilling while positional dominance took precedence when chilling was sufficient.

Abstract

The purpose of our study was to use fruit set and inflorescence size to characterize the positional (position along the shoot) and/or temporal (relative time of budburst and flowering) influences on competition between reproductive laterals within an annual shoot. The relative time of budburst and flowering, and the relative position within the shoot of reproductive buds were recorded on 2-year-old shoots of ‘Granny Smith’ and ‘Golden Delicious’ apple (Malus × domestica (Borkh.)) trees. The trees were grown at two locations in South Africa, a cool area, Koue Bokkeveld, and a warm area, Warm Bokkeveld, with sufficient and insufficient winter chilling, respectively. Inflorescence size (leaf number, leaf area, and flower number) did not differ temporally or with position. For both cultivars, fruit set in the cool area was acrotonic and independent of relative flowering time, while it was more influenced by temporal (primigenic) dominance in the warm area. Therefore, there is a clear positional advantage within the shoot to fruit set in cool areas (i.e., better local climate conditions for the growing fruit), while there is a clear temporal advantage (first bud to burst sets a fruit), or a “first come, first serve” approach to fruit set, in warm areas, which have limited and delayed budbreak. Inflorescence size and fruit set indicate a separation of environmental (degree of winter chilling) and innate factors in competition among reproductive buds along the 2-year-old annual shoot.