Microsatellite markers spanning the apple (Malus x domestica Borkh.) genome

A new set of 148 apple microsatellite markers has been developed and mapped on the apple reference linkage map Fiesta x Discovery. One-hundred and seventeen markers were developed from genomic libraries enriched with the repeats GA, GT, AAG, AAC and ATC; 31 were developed from EST sequences. Markers derived from sequences containing dinucleotide repeats were generally more polymorphic than sequences containing trinucleotide repeats. Additional eight SSRs from published apple, pear, and Sorbus torminalis SSRs, whose position on the apple genome was unknown, have also been mapped. The transferability of SSRs across Maloideae species resulted in being efficient with 41% of the markers successfully transferred. For all 156 SSRs, the primer sequences, repeat type, map position, and quality of the amplification products are reported. Also presented are allele sizes, ranges, and number of SSRs found in a set of nine cultivars. All this information and those of the previous CH-SSR series can be searched at the apple SSR database () to which updates and comments can be added. A large number of apple ESTs containing SSR repeats are available and should be used for the development of new apple SSRs. The apple SSR database is also meant to become an international platform for coordinating this effort. The increased coverage of the apple genome with SSRs allowed the selection of a set of 86 reliable, highly polymorphic, and overall the apple genome well-scattered SSRs. These SSRs cover about 85% of the genome with an average distance of one marker per 15 cM.E. Silfverberg-Dilworth and C. L. Matasci contributed equally to this work.     

Effect of rootstock on apple (Malus domestica) tree water relations

The effects of rootstock on mid-season water relations, under orchard conditions of non-limiting soil moisture, were determined for bearing 'Empire' apple trees ( Malus domestica Borkh.) on the clonal rootstocks M9, M26, M7, MM106, and MM104 (most to least dwarfing) in their sixth and seventh growing seasons. Stem water potentials (ψ^stem) of trees on M9 and M26 were more negative at midday, under warm, sunny conditions, than were the trees on the other three rootstocks. However, change in ψ^stem per change in stem distance through the canopy (water potential gradient) did not vary among rootstocks at midday. There was no rootstock effect on diurnal variation in transpiration or stomatal conductance. Differences in water storage capacitance, relative to tree size, were determined in a separate study but did not account for the differences observed in ψ^stem. Calculated hydraulic conductivities of xylem water transport suggest that rootstocks differ in their ability to conduct water to the scion, but hydraulic conductivity of the scion was not affected by rootstock. Root-stock differences in hydraulic conductivity were not accounted for by differences in tree size.     

Metabolic and gene expression analysis of apple (Malus x domestica) carotenogenesis

Carotenoid accumulation confers distinct colouration to plant tissues, with effects on plant response to light and as well as health benefits for consumers of plant products. The carotenoid pathway is controlled by flux of metabolites, rate-limiting enzyme steps, feed-back inhibition, and the strength of sink organelles, the plastids, in the cell. In apple (Malus × domestica Borkh), fruit carotenoid concentrations are low in comparison with those in other fruit species. The apple fruit flesh, in particular, begins development with high amounts of chlorophylls and carotenoids, but in all commercial cultivars a large proportion of this is lost by fruit maturity. To understand the control of carotenoid concentrations in apple fruit, metabolic and gene expression analysis of the carotenoid pathway were measured in genotypes with varying flesh and skin colour. Considerable variation in both carotenoid concentrations and compound profile was observed between tissues and genotypes, with carotenes and xanthophylls being found only in fruit accumulating high carotenoid concentrations. The study identified potential rate-limiting steps in carotenogenesis, which suggested that the expression of ZISO, CRTISO, and LCY-ε, in particular, were significant in predicting final carotenoid accumulation in mature apple fruit.     

Biochemical characterization of cytosolic fructose-1,6-bisphosphatase from apple (Malus domestica) leaves

Cytosolic fructose-1,6-bisphosphatase was purified to apparent homogeneity from the leaves of apple, a sorbitol synthesizing species. The enzyme was a homotetramer with a subunit mass of 37 kDa, and was highly specific for fructose 1,6-bisphosphate (F1,6BP) with a Km of 3.1 micro M and a Vmax of 48 units (mg protein)(-1). Either Mg2+ or Mn2+ was required for its activity with a Km of 0.59 mM and 62 micro M, respectively. Li+, Ca2+, Zn2+, Cu2+ and Hg2+ inhibited whereas Mn2+ enhanced the Mg2+ activated enzyme activity. Fructose 6-phosphate (F6P) was found to be a mixed type inhibitor with a Ki of 0.47 mM. Fructose 2,6-bisphosphate (F2,6BP) competitively inhibited the enzyme activity and changed the substrate saturation curve from hyperbolic to sigmoidal. AMP was a non-competitive inhibitor for the enzyme. F6P interacted with F2,6BP and AMP in a synergistic way to inhibit the enzyme activity. Dihydroxyacetone phosphate slightly inhibited the enzyme activity in the presence or absence of F2,6BP. Sorbitol increased the susceptibility of the enzyme to the inhibition by high concentrations of F1,6BP. High concentrations of sorbitol in the reaction mixture led to a reduction in the enzyme activity.     

Microsatellites in Malus X domestica (apple): abundance, polymorphism and cultivar identification

Screening of an apple genomic library with (GA)15 and (GT)15 probes demonstrated that these repeats are abundant, occurring about every 120 and 190 kb, respectively. Microsatellites isolated from a small insert library enriched for (GA) repeats contained numbers of repeats ranging from 7 to 39. Primers to these microsatellite loci were able to direct the amplification of the repeats in 21 different cultivars. The majority of markers were highly polymorphic, diploid, and showed simple Mendelian inheritance, although about 25% of markers generated complex banding patterns consistent with the amplification of more than one locus. As few as three microsatellite markers were sufficient to differentiate between all 21 cultivars. Received: 20 February 1996 / Accepted: 24 May 1996     

Ribonuclease inhibitors in Malus x domestica (common apple): isolation and partial characterization

A ribonuclease inhibitory activity was detected in the fruits of common apple, Malus x domestica, cv. Fuji, and purified by affinity chromatography on ribonuclease A-Sepharose. It inhibited hydrolysis of cyclic-2':3'-CMP by bovine pancreatic ribonuclease A with an apparent inhibition constant of about 5 x 10(-8) M. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of the purified protein gave two peaks corresponding to the mass numbers of 55,658 and 62,839, while three bands of 43-, 34-, and 21-kDa were detected by SDS-PAGE. These results suggested that the inhibitor preparation was a mixture of two proteins comprised of 43- and 21-kDa subunits or of 34- and 21-kDa subunits. Attempts to separate these two proteins were unsuccessful. Amino acid composition and N-terminal amino acid sequence of these subunits were also identified and N-terminal sequences showed some similarity to that of cottonseed storage globulin. The significance of the presence of ribonuclease inhibitors in apple fruits is not clear, but it might allow some speculation about their possible involvement in the control of the self-incompatibility ribonuclease of Rosaceae plants.     

Characterisation of the DELLA subfamily in apple (Malus x domestica Borkh.)

The hormone gibberellic acid (GA) regulates growth and development throughout the plant life cycle. DELLA proteins are key components of the GA signalling pathway and act to repress GA responses. The “DELLA” amino acid motif is highly conserved among diverse species and is essential for GA-induced destruction of DELLA proteins, which relieves repression. Six genes encoding the DELLA motif were identified within an apple expressed sequence tag (EST) database. Full-length cDNA clones were obtained by RACE and these were designated MdRGL1a/b, MdRGL2a/b, and MdRGL3a/b. Sequence alignment of the predicted proteins indicates that the MdDELLAs are 37–93% homologous to one another and 44–65% to the Arabidopsis DELLAs. The MdDELLAs cluster into three pairs, which reflect the presumed allopolyploid origins of the Maloideae. Expression analysis using quantitative real-time PCR indicates that all three pairs of MdDELLA mRNAs are expressed at the highest levels in summer arrested shoot tips and in autumn vegetative buds. Transgenic Arabidopsis expressing MdRGL2a have smaller leaves and shorter stems, take longer to flower in short days, and exhibit a reduced response to exogenous GA₃, indicating significant conservation of gene function between DELLA proteins from apple and Arabidopsis. Electronic supplementary material Supplementary material is available for this article at and is accessible for authorized users.     

Plant regeneration from meristem-derived callus protoplasts of apple (Malus3domestica cv. `Fuji')

A procedure has been established for regeneration from meristem-derived callus protoplasts of scion cultivars of apple that have been difficult to regenerate from leaf protoplasts. Calli were induced from the meristem of apples, Malus×domestica cvs `Fuji' and `Jonagold' and Malus prunifolia var `ringo Asami Mo84-A', cultured on MS medium (2 mg/l 2,4-D, 1 mg/l BA, 0.8% agar) and subcultured in a liquid medium. The ability to regenerate plants from suspension calli was studied under eight different combinations with respect to IAA, ABA, and TDZ concentrations. With the materials studied here, two combinations, one with 0.1 mg/l IAA, 0.1 mg/l ABA, and 2.0 mg/l TDZ and another with 0.1 mg/l IAA, 1.0 mg/l ABA, and 2.0 mg/l TDZ, were effective for plant regeneration. Protoplasts were isolated from the above suspension cultures and then cultured in KM8P medium containing IBA (2 mg/l), BA (1 mg/l), 2,4-D (0.4 mg/l), and MES (5 mM, pH 5.7). Shoot formation of protoplast-derived calli was studied in the above-mentioned regeneration media. The high concentration of Gelrite (0.5% and 0.7%) was also shown to be important for shoot formation of protoplast-derived calli. Shoot primordia were formed in the medium containing IAA (0.1 mg/l), ABA (1.0 mg/l), and TDZ (2.0 mg/l). Ultimately, five regenerants of `Fuji' protoplasts were obtained from 200 protoplast-derived calli. Received: 19 June 1998 / Revision received: 9 October 1998 / Accepted: 27 October 1998     

Apple (Malus pumila var. domestica) phenology is advancing due to rising air temperature in northern Japan

Recent studies show advancing onset of plant growing season in many regions for the last several decades. With the well‐established dependence of plant phenology on temperature, these trends are interpreted as an indication of global warming. For several decades, however, other determinants of plant phenology, e.g. varieties and trends in managed systems, may have changed and confounded the phenological trends. In this study, we tested if long‐term changes in phenology of apple (Malus pumila var. domestica) are attributable to long‐term changes in temperature by comparing the phenological response to long‐term trend in air temperature, which is of our interest, with that to year‐to‐year fluctuation in air temperature, which should represent the real effect of temperature on phenology. We collected records of air temperature and phenological events (budding and flowering) in apple from 1977 to 2004 at six locations in Japan. Linear trends in flowering showed advancing rate in the range from 0.21 to 0.35 day yr^?1, statistically significant at three locations (P<0.05). We also found a warming trend in mean air temperature throughout March and April, with which flowering was closely correlated, in the range from 0.047 to 0.077 °C yr^?1, statistically significant at five locations (P<0.05). We separated the temperature time‐series into two components: a long‐term trend and a year‐to‐year fluctuation, by fitting smoothing spline to the trend and taking the residuals as the anomaly. We then fit a multiple regression model of phenological response to air temperature with separate coefficients for long‐term trend and anomaly. Flowering date responded to the long‐term trend at ?3.8 day °C^?1 and to the anomaly at ?4.6 day °C^?1. The temperature coefficients were not statistically different from each other or among locations, suggesting that the advance of apple phenology has predominantly been caused by the temperature increase across the locations studied. The same result was also observed with budding.     

Identification and characterisation of proteinase inhibitors and their genes from seeds of apple (Malus domestica)

Trypsin and papain proteinase inhibitors have been identified and purified from aqueous extracts of apple seeds (Malus domestica). Superdex G75 gel filtration chromatography identified a higher molecular weight (HMW) papain inhibitory fraction (22-26 kDa) and a lower molecular weight papain and trypsin inhibitory fraction (6-12 kDa). The lower molecular weight fraction was separated into a trypsin inhibitor (designated Trp1) and early (designated Pap1) and late (designated Pap2) eluting papain inhibitors after anion exchange (Hitrap SP) chromatography. For Pap2, two inhibitory peaks (designated Pap2-1 and Pap2-2) were identified after further anion exchange (Resource S) chromatography. Each of these lower molecular weight inhibitors was purified by reverse phase HPLC to homogeneity as determined by SDS-PAGE and by mass spectrometry. The HMW papain inhibitory fraction was purified further by anion-exchange (Hitrap Q followed by Resource Q) column chromatography where a minor inhibitor (HMWPap1) and major inhibitor (HMWPap2) fraction were identified. The relative abundance in seeds of apple and the spectrum of proteinase inhibition has been determined for all of these inhibitors. Reverse-phase HPLC separated HMWPap2 into a minor (HMWPap2-1) and a major (HMWPap2-2) inhibitory fraction, and SDS-PAGE and mass spectrometry confirmed that HMWPap2-2 was purified to homogeneity. Amino acid composition data were obtained from Trp1, Pap1, Pap2-2, and HMWPap2-2, and N-terminal sequence data from Trp1, Pap2-1, Pap2-2, and HMWPap2-2, with two of these sequences (Pap2-2 and HMWPap2-2) perfectly matching predicted protein sequences based on EST sequences from an apple database. The relationship of these inhibitors with those of other species is discussed.     

Physical mapping of three fruit ripening genes：Endopolygalacturonase,ACC oxidase and ACC synthase from apple（Malus x domestica） in an apple rootstock A106（Malus sieboldii

The apple rootstock,A106(Malus sieboldii),had 17 bivalents in pollen mother cells at meiotic metaphase 1,and 17 chromosomes in a haploid pollen cell.Karyotypes were prepared from root-tip cells with 2n=34 chromosomes,Seven out of 82 karyotypes(8.5%) showed one pari of satellites at the end of the short arm of chromosome 3.C-bands were shown on 6 pairs of chromosomes 2,4,6,8,14,and 16 near the telomeric regions of short arms.Probes for three ripening-related genes from Malus x domestica:endopolygalacturonase(EPG,0.6kb),ACC oxidase(1.2kb),and ACC synthase(2kb)were hybridized in situ to metaphase chromosomes of A106.Hybridization sites for the EPG gene were observed on the long arm of chromosome 14 in 15 out of 16 replicate spreads and proximal to the centromere of chromosomes 6 and 11.For the ACC oxidase gene,hylridization sites were observed in the telomeric region of the short arm of chromosomes 5 and 11 in 87% and 81% of 16 spreads respectively,proxiaml to the centromere of chromosome 1 in 81% of the spreads,and on the long arm of chromosome 13 in 50% of the spreads. Physical mapping of three fruit ripening genes in an apple rootstock A106.Twenty five spreads were studied for the ACC synthase gene and hybridization sites were observed in the telomeric region of the short arm of chromosome 12 in 96% of the spreads.chromosomes 9 and 10 in 76% of the spreads,and chromosome 17 in 56% of the spreads.     

The consequences of clone size for paternal and maternal success in domestic apple (Malus x domestica)

Clonal growth in plants can increase pollen and ovule production per genet. However, paternal and maternal reproductive success may not increase because within-clone pollination (geitonogamy) can reduce pollen export to adjacent clones (pollen discounting) and pollen import to the central ramets (pollen limitation). The relationship between clone size and mating success was investigated using clones of Malus × domestica at four orchards (blocks of 1-5 rows of trees). For each block, maternal function was measured as fruit and seed set in all rows and paternal function as siring rate estimated from isozyme profiles in the first row of the adjacent block. Expected relations between reproductive success and clone size were generated from simulations and data on pollen dispersal in this species. Siring rate per clone averaged 70% and did not increase significantly with block size, consistent with simulations of pollen dispersal under pollen discounting. Simulations also indicated that the ratio of compatible to incompatible pollen received by a tree should decline with increased block size and from the periphery to the center of blocks. However, female function was not significantly reduced among block sizes or within blocks. The results suggest that paternal function may be more sensitive to the effects of clonality than female function.     

Development and characterisation of 140 new microsatellites in apple (Malus x domestica Borkh.)

The availability of suitable genetic markers is essential to efficiently select and breed apple varieties of high quality and with multiple disease resistances. Microsatellites (simple sequence repeats, SSR) are very useful in this respect since they are codominant, highly polymorphic, abundant and reliably reproducible. Over 140 new SSR markers have been developed in apple and tested on a panel of 7 cultivars and 1 breeding selection. Their high level of polymorphism is expressed with an average of 6.1 alleles per locus and an average heterozygosity (H) of 0.74. Of all SSR markers, 115 have been positioned on a genetic linkage map of the cross Fiesta × Discovery. As a result, all 17 linkage groups, corresponding to the 17 chromosomes of apple, were identified. Each chromosome carries at least two SSR markers, allowing the alignment of any apple molecular marker map both with regard to identification as well as to orientation of the linkage groups. To test the degree of conservation of the SSR flanking regions and the transferability of the SSR markers to other Rosaceae species, 15 primer pairs were tested on a series of Maloideae and Amygdaloideae species. The usefulness of the newly developed microsatellites in genetic mapping is demonstrated by means of the genetic linkage map. The possibility of constructing a global apple linkage map and the impact of such a number of microsatellite markers on gene and QTL mapping is discussed.     

Role of VA-mycorrhiza in growth and mineral nutrition of apple (Malus pumila var.domestica) rootstock cuttings

One-year-old apple cuttings (Malus pumila var.domestica cv. M26) were grown for 6 months in pot culture with and without inoculum of the VA-mycorrhizal fungus (VAMF)Glomus macrocarpum in soil from a long-term fertilizer field experiment with different P availability (20, 210, and 280 mg CAL-extractable P kg⁻¹). The indigenous VAMF propagule density was reduced by 0.5 Mrad X-irradiation. At harvest, non-inoculated and inoculated plants had similar proportions of root length bearing vesicles. Net dry weight of tree cuttings was significantly increased by inoculation only at 20 mg P kg⁻¹ (+62%). Increasing P availability from 210 to 280 mg P kg⁻¹ led to a 4-week depression of shoot elongation rate only in the inoculated plants. Uptake of P was significantly enhanced by inoculation at 20 and 210 mg P kg⁻¹ (+64 and +12%, respectively). On average, inoculated plants had significantly higher concentrations of Zn in leaves and in roots (+16 and +14%, respectively) and of copper in stems and in roots (+13 and +126%, respectively). Proportion of vesicle bearing root length was significantly correlated with root caloric content. A lipid content of 0.9–4.5% in the root dry matter was attributed to the presence of vesicles corresponding to 1.6–8.2% of total root caloric content. As the control plants were also infected, the beneficial effect of VA-mycorrhiza on nutrient uptake and growth of apple cuttings was underestimated at all P levels. Furthermore, VAM-potential at the lowest P level was not fully exploited as onset of infection was most certainly delayed because of a decreased photosynthetic rate due to P deficiency. Energy drain by VAMF-infection was most probably underestimated considerably, due to, among others, loss of infected root cortex during root growth, sampling and staining. It is concluded that apple cuttings rely on VA-mycorrhizal P-uptake at least in low P soils. In high P soils, apple cuttings may profit predominantly from the uptake of Zn and Cu by the fungal symbionts.     

Inheritance of pollen enzymes and polyploid origin of apple (Malus x domestica Borkh.)

Summary Electrophoresis of 7 pollen enzymes was applied to 5 progenies from controlled crosses and one self-progeny of apple. Segregation data were examined according to three kinds of hypotheses: monogenic disomic, bigenic disomic and tetrasomic inheritance Twenty codominant alleles and a recessive null were identified. Results provided evidence of bigenic disomic inheritance in most cases: 6 pairs of homoeologous loci carrying identical homoeoalleles were revealed; only 2 enzymes exhibited a simple monogenic control. Preferential pairing between pairs of homologous chromosomes in meiosis can be postulated. These results indicated an allopolyploid origin of apple genome. Fixed heterozygosity occurred for several enzymes, which is a typical feature of allopolyploidy. Loss of duplicate gene expression can account for the monogenic control of 2 of the enzymes.The results reported in this paper are part of a thesis by the first author for the degree of Docteur Ingénieur     

Genomic cloning and linkage mapping of the Mal d 1 (PR-10) gene family in apple (Malus domestica)

Fresh apples can cause birch pollen-related food allergy in northern and central European populations, primarily because of the presence of Mal d 1, the major apple allergen that is cross-reactive to the homologous and sensitizing allergen Bet v 1 from birch. Apple cultivars differ significantly in their allergenicity. Knowledge of the genetic basis of these differences would direct breeding for hypoallergenic cultivars. The PCR genomic cloning and sequencing were performed on two cultivars, Prima and Fiesta, which resulted in 37 different Mal d 1 gDNA sequences. Based on the mapping of sequence-specific molecular markers, these sequences appeared to represent 18 Mal d 1 genes. Sixteen genes were located in two clusters, one cluster with seven genes on linkage group (LG) 13, and the other cluster with nine genes on the homoeologous LG 16. One gene was mapped on LG 6, and one remained unmapped. According to sequence identity, these 18 genes could be subdivided into four subfamilies. Subfamilies I–III had an intron of different size that was subfamily and gene-specific. Subfamily IV consisted of 11 intronless genes. The deduced amino acid sequence identity varied from 65% to 81% among subfamilies, from 82% to 100% among genes within a subfamily, and from 97.5% to 100% among alleles of one gene. This study provides a better understanding of the genetics of Mal d 1 and the basis for further research on the occurrence of allelic diversity among cultivars in relation to allergenicity and their biological functions.