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The apple cultivar McIntosh Wijcik, which is a mutant of ‘McIntosh’, exhibits a columnar growth phenotype (short internodes, few lateral branches, many spurs, etc.) that is controlled by a dominant Co gene. The candidate gene (MdDOX-Co), encoding a 2-oxoglutarate-dependent dioxygenase, is located adjacent to an insertion mutation. Non-columnar apples express MdDOX-Co in the roots, whereas columnar apples express MdDOX-Co in the aerial parts as well as in the roots. However, the function of MdDOX-Co remains unknown. Here, we characterized tobacco plants overexpressing MdDOX-Co. The tobacco plants showed the typical dwarf phenotype, which was restored by application of gibberellin A3 (GA3). Moreover, the dwarf tobacco plants had low concentrations of endogenous bioactive gibberellin A1 (GA1) and gibberellin A4 (GA4). Similarly, ‘McIntosh Wijcik’ contained low endogenous GA4 concentration and its dwarf traits (short main shoot and internodes) were partially reversed by GA3 application. These results indicate that MdDOX-Co is associated with bioactive GA deficiency. Interestingly, GA3 application to apple trees also resulted in an increased number of lateral branches and a decrease in flower bud number, indicating that gibberellin (GA) plays important roles in regulating apple tree architecture by affecting both lateral branch formation (vegetative growth) and flower bud formation (reproductive growth). We propose that a deficiency of bioactive GA by ectopic expression of MdDOX-Co in the aerial parts of columnar apples not only induces dwarf phenotypes but also inhibits lateral branch development and promotes flower bud formation, and assembly of these multiple phenotypes constructs the columnar tree form.

We previously identified the FLOWERING LOCUS C (FLC)-like gene, a MADS-box transcription factor gene that belongs to Arabidopsis thaliana L. FLC clade, in apple (Malus $\times$ domestica Borkh.), and its expression in dormant flower buds is positively correlated with cumulative cold exposure. To elucidate the role of the MdFLC-like in the dormancy process and flower development, we first characterized the phenotypes of MdFLC-like overexpressing lines with the Arabidopsis Columbia-0 background. The overexpression of MdFLC-like significantly delayed the bolting date and reduced the plant size, but it did not significantly affect the number of rosette leaves or flower organ formation. Thus, MdFLC-like may affect vegetative growth and development rather than flowering when expressed in Arabidopsis, which is not like Arabidopsis FLC that affects development of flowering. We compared seasonal expression patterns of MdFLC-like in low-chill ‘Anna’ and high-chill ‘Fuji’ and ‘Tsugaru’ apples collected from trees grown in a cold winter region in temperate zone and found an earlier upregulation in ‘Anna’ compared with ‘Fuji’ and ‘Tsugaru’. Expression patterns were also compared in relation to developmental changes in the flower primordia during the chilling accumulation period. Overall, MdFLC-like was progressively upregulated during flower primordia differentiation and development in autumn to early winter and reached a maximum expression level at around the same time as the genotype-dependent chilling requirements were fulfilled in high-chill cultivars. Thus, we hypothesize MdFLC-like may be upregulated in response to cold exposure and flower primordia development during the progress of endodormancy. Our study also suggests MdFLC-like may have a growth-inhibiting function during the end of endodormancy and ecodormancy when the temperature is low and unfavorable for rapid bud outgrowth.

Background The mechanism underlying the interaction between host plant and host-selective toxin (HST)-producing Alternaria alternata during infection is of particular interest for sustainable crop production. Alternaria blotch of apple (Malus × domestica Borkh.) caused by A. alternata apple pathotype is a major disease particularly in East Asia, which is the largest producer of apples globally. A single dominant gene, Alt, controls the susceptibility of the apple cultivar ‘Delicious’ to Alternaria blotch. In this study, we fine mapped the Alt locus and characterized three potential candidate genes. Results We used 797 F1 individuals derived from 15 crosses between apple accessions susceptible (Alt/alt) and resistant (alt/alt) to Alternaria blotch to construct physical and genetic maps of the Alt locus located on the top of chromosome 11. Susceptible accessions were derived from ‘Delicious.’ To fine map the Alt locus, we constructed a BAC library of ‘Starking Delicious,’ a sport of ‘Delicious,’ and used graphical genotyping to delimit the Alt locus to a region of 43 kb. Three genes predicted within the candidate Alt region were potentially involved in plant defense response, among which the gene encoding a coiled coil-nucleotide binding-leucine rich repeat (CC-NB-LRR) type disease resistance protein was the most promising. Moreover, a 12-bp insertion was uniquely identified in the 5′ untranslated region of the Alt-associated allele of this gene, the presence or absence of which co-segregated with the susceptibility or resistance to A. alternata apple pathotype, respectively, among 43 tested cultivars including old ones and founders of modern apple breeding. Conclusion A disease resistance protein has been suggested as a determinant of susceptibility/resistance to HST-producing A. alternata for the first time. Our finding provides new insight into the mechanism of HST-mediated disease control used by A. alternata against host plants. Electronic supplementary material The online version of this article (10.1186/s12870-019-1737-7) contains supplementary material, which is available to authorized users.

In apple (Malus spp.), crown gall disease, caused by the bacterial pathogens Rhizobium radiobacter (Ti) and R. rhizogenes (Ti), can be severe. To control the disease, breeding of apple rootstocks that exhibit crown gall resistance is a promising approach. In this study, we used a full-sib F1 population derived from a ‘JM7’ (susceptible) × Sanashi 63 (resistant) cross to identify quantitative trait loci (QTLs) that control resistance to tumour-inducing Rhizobium isolates found in apple production areas in Japan. Three stable QTLs, associated with a wide range of crown gall resistances, were identified in three linkage groups (LGs). QTLs for resistance to isolates Peach CG8331, Nagano 1 and Nagano 2 co-localised at the middle of LG 2 in Sanashi 63, where the crown gall resistance gene Cg (renamed as Rrr1 in this study) was previously identified. Similarly, in ‘JM7’, QTLs were identified on LG 11 for resistance to isolates ARAT-001, ARAT-002 and Kazuno 2, and on LG 15 for resistance to isolate ARAT-001. Fine-mapping of Rrr1 and nucleotide sequencing of a contig obtained from two bacterial artificial chromosome (BAC) clones delimited the Rrr1 gene to a region spanning 217 kb. In silico gene prediction from the region identified four genes encoding the Toll-interleukin-1 receptor/nucleotide-binding site/leucine-rich repeat (TIR-NBS-LRR) class plant resistance genes.

Haplotypes provide useful information for genomics-based approaches, genomic prediction, and genome-wide association study. As a small number of superior founders have contributed largely to the breeding history of fruit trees, the information of founder haplotypes may be relevant for performing the genomics-based approaches in these plants. In this study, we proposed a method to estimate 14 haplotypes from 7 founders and automatically trace the haplotypes forward to apple parental (185 varieties) and breeding (659 F1individuals from 16 full-sib families) populations based on 11,786 single-nucleotide polymorphisms, by combining multiple algorithms. Overall, 92% of the single-nucleotide polymorphisms information in the parental and breeding populations was characterized by the 14 founder haplotypes. The use of founder haplotype information improved the accuracy of genomic prediction in 7 traits and the resolution of genome-wide association study in 13 out of 27 fruit quality traits analyzed in this study. We also visualized the significant propagation of the founder haplotype with the largest genetic effect in genome-wide association study over the pedigree tree of the parental population. These results suggest that the information of founder haplotypes can be useful for not only genetic improvement of fruit quality traits in apples but also for understanding the selection history of founder haplotypes in the breeding program of Japanese apple varieties.

Background: Sweetness is one of the most important traits determining fruit quality. Sweetness is controlled not only by the total sugar content but also by the contents of individual sugars. The major sugars in mature Rosaceae fruits are sucrose, fructose, glucose, and sorbitol, which have different levels of sweetness. Among these, sucrose and fructose have high sweetness, whereas glucose and sorbitol have low sweetness. The objective of this study was to identify the quantitative trait loci (QTLs) associated with fruit traits including individual sugar accumulation and conversion, to infer the candidate genes underlying the QTLs, and to assess the potential of genomic selection for breeding pear fruit traits.Results: We evaluated 10 fruit traits and conducted genome-wide association studies (GWAS) for 106 cultivars and 17 breeding populations (1112 F1 individuals) using 3484 tag single-nucleotide polymorphisms (SNPs) genotyped by double-digest restriction-site associated DNA sequencing (ddRAD-Seq). By implementing a mixed linear model and a Bayesian multiple-QTL model in GWAS, 56 SNPs associated with fruit traits were identified. Four loci were presumed to be associated with sugar conversion because the SNPs were significant for more than one individual sugar and the individual sugar contents associated with each SNP genotype were negatively correlated. In particular, a SNP located close to acid invertase gene PPAIV3 on chromosome 7 and a newly identified SNP on chromosome 11 had quite large effects on sugar conversion. We used ‘Golden Delicious’ doubled haploid (GDDH) 13, an apple reference genome to infer the candidate genes for the identified SNPs. In the region flanking the SNP on chromosome 11, there is a tandem repeat of early responsive to dehydration (ERD6)-like sugar transporter genes which might play a role in the phenotypes observed.Conclusions: SNPs associated with sugar accumulation and conversion were newly identified at several loci, and candidate genes underlying QTLs were inferred using advanced apple genome information. Several QTLs showed clear effects with more than 10% of phenotypic variance explained by those SNPs in the breeding populations. By combining the effects of multiple QTLs, breeders would be able to select seedlings that will later bear fruit with high sucrose and fructose content.

BackgroundUnderstanding mechanisms of sugar accumulation and composition is essential to determining fruit quality and maintaining a desirable balance of sugars in plant storage organs. The major sugars in mature Rosaceae fruits are sucrose, fructose, glucose, and sorbitol. Among these, sucrose and fructose have high sweetness, whereas glucose and sorbitol have low sweetness. Japanese pear has extensive variation in individual sugar contents in mature fruit. Increasing total sugar content and that of individual high-sweetness sugars is a major target of breeding programs. The objective of this study was to identify quantitative trait loci (QTLs) associated with fruit traits including individual sugar accumulation, to infer the candidate genes underlying the QTLs, and to assess the potential of genomic selection for breeding pear fruit traits.ResultsWe evaluated 10 fruit traits and conducted genome-wide association studies (GWAS) for 106 cultivars and 17 breeding populations (1112 F1 individuals) using 3484 tag single-nucleotide polymorphisms (SNPs). By implementing a mixed linear model and a Bayesian multiple-QTL model in GWAS, 56 SNPs associated with fruit traits were identified. In particular, a SNP located close to acid invertase genePPAIV3on chromosome 7 and a newly identified SNP on chromosome 11 had quite large effects on accumulation of sucrose and glucose, respectively. We used ‘Golden Delicious’ doubled haploid 13 (GDDH13), an apple reference genome, to infer the candidate genes for the identified SNPs. In the region flanking the SNP on chromosome 11, there is a tandem repeat ofearly responsive to dehydration(ERD6)-like sugar transporter genes that might play a role in the phenotypes observed.ConclusionsSNPs associated with individual sugar accumulation were newly identified at several loci, and candidate genes underlying QTLs were inferred using advanced apple genome information. The candidate genes for the QTLs are conserved across Pyrinae genomes, which will be useful for further fruit quality studies in Rosaceae. The accuracies of genomic selection for sucrose, fructose, and glucose with genomic best linear unbiased prediction (GBLUP) were relatively high (0.67–0.75), suggesting that it would be possible to select individuals having high-sweetness fruit with high sucrose and fructose contents and low glucose content.

Along with the expansion of dwarfing apple rootstocks in Japan, damage through bark-eating by the Japanese field vole (Microtus montebelli Milne-Edwards, 1872) in apple orchards has increased. The most prevalent cultivar, JM7, in particular, is highly susceptible to vole gnawing. Hence, it is a pressing task to reduce vole damage to apple rootstocks by developing and introducing a new dwarfing rootstock cultivar that is less susceptible to voles. In order to achieve this, it is critical to elucidate which rootstock characteristics underlie vole preference. Preferences of the Japanese field vole among seven apple rootstocks were examined by a cafeteria experiment using ten voles. Voles showed a clear preference for the JM7 cultivar. Nutrient and morphological analyses of these rootstocks revealed that voles preferred to eat rootstocks which were poor in total phenolics, rich in nitrogen, and had low edible ratios of shoots. Among the tested seven rootstocks, JM7 was richest in nitrogen, and poorest in total phenolics. Our results indicate that future breeding projects on vole-resistant dwarfing apple rootstock genotypes should aim to develop a cultivar that is rich in total phenolics and poor in nitrogen, while keeping the advantageous traits of the JM7 rootstock, such as high productivity, and ease of propagation by hardwood cutting.

The columnar apple ‘McIntosh Wijcik’, which is a mutation of ‘McIntosh’ shoots, has short internodes, thick stems, upright growth, poor lateral branches, and increased spur density. These columnar traits are controlled by a single dominant gene known as Co. We previously identified a putative dioxygenase gene (designated as 91071) as a promising Co candidate (expressed in the shoot apices of ‘McIntosh Wijcik’). However, tissue expression and function of the 91071 gene in noncolumnar apples is still not clear. In this study, we used reverse transcription polymerase chain reaction to demonstrate that the 91071 gene is mainly expressed in the roots of noncolumnar apples, whereas it is also expressed in shoot apices and leaves of columnar apples. In situ hybridization revealed that the 91071 gene is expressed at the primordium of lateral roots and root tips of both noncolumnar and columnar apple trees and in the shoot meristem and leaf primordium in the columnar apple ‘McIntosh Wijcik’. Grafting experiments of noncolumnar scion onto columnar rootstocks revealed that the columnar growth phenotype is not transmissible from rootstock to scion. These results indicated that ectopic expression of the 91071 gene in aerial parts causes columnar growth, whereas the expression of the 91071 gene in roots does not produce columnar growth. Furthermore, transgenic apples overexpressing the 91071 gene showed larger median adventitious root length and higher median number of lateral roots than control apples. Our result suggests that the 91071 gene may be related to root development.

Juiciness and freshness play a critical role in consumers’ choice of apples, and objective measurements of the traits are essential for adding a premium to fruits with high consumer demand. Juiciness and freshness were sensory evaluated by four apple-breeding experts. The score of sensory juiciness decreased with decreased instrumental measurements of water content (WC), firmness, and apoplastic solution (AS). The influence of firmness on sensory juiciness was not linear, and perception of juiciness rapidly decreased when firmness decreased below 60 N. The sensory freshness score was related to titratable acidity (TA), firmness, and the AS. A nonlinear regression model for freshness indicated that freshness was less likely to be perceived as the AS in flesh tissue decreased, and the perception of freshness rapidly decreased when firmness decreased below 60 N or TA decreased below 0.25 g 100 ml−1. The firmness of fruit treated with 1-MCP hardly decreased during storage. The titratable acidity of fruit treated with 1-MCP decreased; however, the degree of the decrease was significantly lower than that of non-treated fruit. The AS immediately decreased just after harvest and was not significantly different in most cases between 1-MCP-treated and non-treated fruit during storage. Therefore, measurements of firmness and TA were useful for distinguishing 1-MCP-treated fruit from non-treated fruit and the AS is necessary to distinguish between stored fruit and that just after harvest. The performance of calibration models developed by NIR spectroscopy for TA, firmness, and AS was lower than that for soluble solids content (SSC), which is used to grade apples by NIR spectroscopy in Japanese packing houses. Therefore, it is impractical to grade fruit by freshness using calibration models. However, high correlation coefficients were obtained in the calibration model for WC. The correlation coefficient of WC was about as high as that of SSC. Therefore, apples could be graded by juiciness using the calibration model for WC with the auxiliary use of calibration models for firmness and AS.

We isolated and confirmed two S-RNases, denoted as mpS1 and mpS2, from apple rootstock ``Marubakaido`` (Malus prunifolia Borkh. Var. ringo Asami). These S-RNases contained and conserved five cysteine residues and two histidine residues, which are essential for RNase activity. The mpS1 showed high similarity to S5 (99.1%) of Malus spectabilis, whereas the mpS2 showed 99.5% nucleotide sequence similarity to S26 of (Malus × domestica) and 99.6% to S35 of (Malus sieversii) when compared with reported S-RNases. In amino acid sequences, the mpS1-RNase was almost similar to the S5-RNase of Malus spectabilis, and the mpS2-RNase was similar to the S35 of Malus sieversii, with only one bp being different from the S26-RNase of Malus × domestica. The 57 S-RNases of Malus species were renamed and rearranged containing the new S-RNases, as mprpS35 (mpS2) and mprpS57 (mpS1), for determining S-genotypes and identifying new alleles from apple species (Malus spp.).

'Fuji' is one of the most popular and highly-produced apple cultivars worldwide, and has been frequently used in breeding programs. The development of genotypic markers for the preferable phenotypes of 'Fuji' is required. Here, we aimed to define the haplotypes of 'Fuji' and find associations between haplotypes and phenotypes of five traits (harvest day, fruit weight, acidity, degree of watercore, and flesh mealiness) by using 115 accessions related to 'Fuji'. Through the re-sequencing of 'Fuji' genome, total of 2,820,759 variants, including single nucleotide polymorphisms (SNPs) and insertions or deletions (indels) were detected between 'Fuji' and 'Golden Delicious' reference genome. We selected mapping-validated 1,014 SNPs, most of which were heterozygous in 'Fuji' and capable of distinguishing alleles inherited from the parents of 'Fuji' (i.e., 'Ralls Janet' and 'Delicious'). We used these SNPs to define the haplotypes of 'Fuji' and trace their inheritance in relatives, which were shown to have an average of 27% of 'Fuji' genome. Analysis of variance (ANOVA) based on 'Fuji' haplotypes identified one quantitative trait loci (QTL) each for harvest time, acidity, degree of watercore, and mealiness. A haplotype from 'Delicious' chr14 was considered to dominantly cause watercore, and one from 'Ralls Janet' chr1 was related to low-mealiness.

Black spot disease, which is caused by the Japanese pear pathotype of the filamentous fungus Alternaria alternata (Fries) Keissler, is one of the most harmful diseases in Japanese pear cultivation. We mapped a gene for susceptibility to black spot disease in the Japanese pear (Pyrus pyrifolia Nakai) cultivar 'Kinchaku' (Aki gene) at the top of linkage group 11, similar to the positions of the susceptibility genes Ani in 'Osa Nijisseiki' and Ana in 'Nansui'. Using synteny-based marker enrichment, we developed novel apple SSR markers in the target region. We constructed a fine map of linkage group 11 of 'Kinchaku' and localized the Aki locus within a 1.5-cM genome region between SSR markers Mdo.chr11.28 and Mdo.chr11.34. Marker Mdo.chr11.30 co-segregated with Aki in all 621 F1 plantlets of a 'Housui' × 'Kinchaku' cross. The physical size of the Aki region, which includes three markers (Mdo.chr11.28, Mdo.chr11.30, and Mdo.chr11.34), was estimated to be 250 Kb in the 'Golden Delicious' apple genome and 107 Kb in the 'Dangshansuli' Chinese pear genome. Our results will help to identify the candidate gene for susceptibility to black spot disease in Japanese pear.

The objective of this study was to investigate the effects of temperature treatments on anthocyanin accumulation and ethylene production in the fruit of early- and medium-maturing cultivars that were harvested early during fruit ripening. We first investigated the effects of various temperature treatments on anthocyanin accumulation in detached apples of ‘Tsugaru’, ‘Tsugaru Hime’, ‘Akane’ and ‘Akibae’ using an incubator. Three years of experiments demonstrated that at harvest, the lower-temperature treatments induced anthocyanin accumulation in ‘Tsugaru’, ‘Tsugaru Hime’, and ‘Akibae’ fruits, whereas the increases in anthocyanin accumulation under the 25 °C treatment were similar to those under the 15 and 20 °C treatments in ‘Akane’ fruit. The rate of ethylene production did not increase substantially during the temperature treatments in any of the four cultivars, except after the treatments of ‘Tsugaru’ fruit at harvest. The inhibition of ethylene action by the application of 1-methylcyclopropene (1-MCP) to detached fruits at harvest suppressed anthocyanin development under 15 and 20 °C temperature treatments in ‘Tsugaru’, ‘Tsugaru Hime’, and ‘Akibae’, but not in ‘Akane’. In the second experiment, we investigated changes in the anthocyanin concentration in attached fruit of ‘Misuzu Tsugaru’ under different temperature conditions in a greenhouse. At harvest, the anthocyanin concentration in fruit under the hotter climatic condition (29 °C 12 hours/19 °C 12 hours) was lower than that under the control condition (25 °C 12 hours/15 °C 12 hours). During the last week before harvest, anthocyanin development proceeded rapidly in fruit skin not only under the control condition, but also under the hotter climatic condition. The rapid accumulation of anthocyanin in the fruit skin of ‘Misuzu Tsugaru’ at harvest under a relatively high temperature (25 °C) condition was confirmed by the experiment using an incubator. At harvest, the maximum level of ethylene production in fruits sampled from trees grown under the hotter climatic condition was 9-fold higher than that in fruits from trees grown under the control condition. These results indicate that the comparison of pigmentation potential after the 15 or 25 °C treatments using detached fruit was effective for estimating anthocyanin accumulation in fruit skins under hotter climatic conditions in early- and medium-maturing cultivars that were harvested early and that a hotter climatic condition during ripening increased ethylene production in apple fruit after harvest.

Since apple fruit skin reddens poorly under warmer climates, new apple cultivars are desired that are adapted to global warming in terms of bearing well-reddened fruit. We developed a simple sequence repeat marker, Mdo.chr9.4, which is suitable for red skin color selection. It amplified four alleles (Mdo.chr9.4-R-0, Mdo.chr9.4-Y-3, Mdo.chr9.4-Y-9, and Mdo.chr9.4-Y-15) distinguished by length. Mdo. chr9.4-R-0 associated with MdMYB1-1 which confers red fruit skin. The presence of Mdo. chr9.4-R-0 was consistent with empirical skin color in all 160 tested accessions. Mdo. chr9.4 was identified as the only significant marker that contributed to red skin color intensity by a genome wide association study (GWAS), and it accounted for 52.0% of phenotypic variation, confirming that MdMYB1 was the major and principal determinant of fruit skin color in apples. Individuals with a homozygous state of Mdo.chr9.4-R-0 (dose 2) were significantly redder than those showing a heterozygote state (dose 1) in both the accession set and full-sib families, indicating a partially dominant effect of MdMYB1-1. Therefore, the selection of dose 2 individuals would target individuals with intensive red skin. We applied Mdo.chr9.4 to several application populations using a time and cost-efficient genotyping system developed in the present study. This system, along with Mdo.chr9.4, provide advanced marker-assisted breeding for intensive red skin color apples adapted to a global warming climate.

Many important apple (Malus × domestica Borkh.) fruit quality traits are regulated by multiple genes, and more information about quantitative trait loci (QTLs) for these traits is required for marker-assisted selection. In this study, we constructed genetic linkage maps of the Japanese apple cultivars 'Orin' and 'Akane' using F1 seedlings derived from a cross between these cultivars. The 'Orin' map consisted of 251 loci covering 17 linkage groups (LGs; total length 1095.3 cM), and the 'Akane' map consisted of 291 loci covering 18 LGs (total length 1098.2 cM). We performed QTL analysis for 16 important traits, and found that four QTLs related to harvest time explained about 70% of genetic variation, and these will be useful for marker-assisted selection. The QTL for early harvest time in LG15 was located very close to the QTL for preharvest fruit drop. The QTL for skin color depth was located around the position of MYB1 in LG9, which suggested that alleles harbored by 'Akane' are regulating red color depth with different degrees of effect. We also analyzed soluble solids and sugar component contents, and found that a QTL for soluble solids content in LG16 could be explained by the amount of sorbitol and fructose.

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.

Rosaceae is a large family, however, our understanding of its phylogeny is based largely on morphological observations. To understand the relationship between subfamilies Rosoideae, Amygdaloideae, Maloideae and Spiraeoideae at a molecular level, we isolated and compared the plant phosphatidyl ethanolamine-binding protein-like genes TERMINAL FLOWER1 (TFL1)-like and CENTRORADIALIS (CEN)-like, which are involved in the control of shoot meristem identity and flowering time. A comparison of gene structures and phylogenetic tree analyses by the Neighbor-Joining method showed that each of the two TFL1-like (MdTFL1-1 and MdTFL1-2) and CEN-like genes (MdCENa and MdCENb) in Maloideae were classified into two distinct clades. The TFL1-like and CEN-like genes of Gillenia in Spiraeoideae belonged to monophyletic Maloideae groups, suggesting that Gillenia and Maloideae have a common near ancestor. However, the Gillenia TFL1-like gene does not contain the insertion sequence of the third intron that is found in MdTFL1-2-like genes of the members of Maloideae such as apple, Korean whitebeam, quince, and Siberian mountain ash. Therefore, after the Maloideae ancestor genome became polyploid through hybridization between Gillenia-like species or genome doubling, an insertion sequence of the third intron of MdTFL1-2-like genes was generated.

An efficient method for the generation of adventitious shoots from the cotyledons of Japanese pear and related species was developed. Cotyledons from seeds of the Japanese pear “Okusankichi” and the Asian pea pear “Hokushimamenashi” were used to determine the optimum concentrations of phytohormones in the medium. The rates of generation of adventitious shoots and the numbers of adventitious shoots per explant were highest when the media contained 5 μM 1-naphthaleneacetic acid combined with 10 or 25 μM 6-benzylaminopurine. These growth regulators were used to generate adventitious shoots from the cotyledons of 33 cultivars of Japanese, Chinese, Asian pea, and European pears. A high number of adventitious shoots per explant (1.3–2.3) and high rates of regeneration of adventitious shoots (60–76 %) were obtained from the cotyledons of Japanese pears “Imamuraaki” and “Agenosho Shinanashi.” Although pollination was not controlled, cotyledons from mother trees of old Japanese cultivars and Chinese pears tended to be more regenerable than those from other pear species. Since the rooting ability of the adventitious shoots was very low, micro-grafting was applied to obtain regenerated potted plants from adventitious shoots. Grafted regenerated plants were recovered at a rate of more than 60 %, regardless of cultivar. To our knowledge, this is the first report to evaluate shoot regeneration from cotyledons of major Pyrus species.

With 5 figures and 2 tables Abstract Fruit acidity is an important characteristic to determine the marketability of apple (Malus × domestica Borkh.). To reveal the mode of inheritance in fruit acidity and to estimate the genetic parameters, we performed segregation analysis using a population from an apple breeding programme. Four models (mixed, Mendelian, polygenic and environmental) were compared to find the most likely mode of the inheritance of acidity. The phenotypic variance of acidity in the population was properly explained using a mixed model of a major gene and polygenes. The genotypic values of the major gene (AA, Aa and aa) were estimated to be 0.45, 0.52 and 0.92 g/100 ml in titratable acidity, respectively. The values of the homozygote (AA) and the heterozygote (Aa) were very close and lower than that of the other homozygote (aa), indicating that an allele of the major gene appeared to have complete dominance with a function of lowering acidity. The estimate of the heritability after accounting for the major gene was moderately high, 0.43, in the mixed model. This means that even with removal of the effect of the major gene, acidity could fluctuate considerably by the effect of polygenes. The proportions of progeny with adequate acidity differed greatly, depending on which genotype was used as parents for the crossing. Therefore, it is very important to know the genotypes of parental cultivars before crossing.

Understanding the flowering process in apple (Malus × domestica Borkh.) is essential for developing methods to shorten the breeding period and regulate fruit yield. It is known that FLOWERING LOCUS T (FT) acts as a transmissible floral inducer in the Arabidopsis flowering network system. To clarify the molecular network of two apple FT orthologues, MdFT1 and MdFT2, we performed a yeast two-hybrid screen to identify proteins that interact with MdFT1. We identified several transcription factors, including two members of the TCP (TEOSINTE BRANCHED1, CYCLOIDEA and PROLIFERATING CELL FACTORs) family, designated MdTCP2 and MdTCP4, and an Arabidopsis thaliana VOZ1 (Vascular plant One Zinc finger protein1)-like protein, designated MdVOZ1. MdTCP2 and MdVOZ1 also interacted with MdFT2 in yeast. The expression domain of MdTCP2 and MdVOZ1 partially overlapped with that of MdFT1 and MdFT2, most strikingly in apple fruit tissue, further suggesting a potential interaction in vivo. Constitutive expression of MdTCP2, MdTCP4 and MdVOZ1 in Arabidopsis affected plant size, leaf morphology and the formation of leaf primordia on the adaxial side of cotyledons. On the other hand, chimeric MdTCP2, MdTCP4 and MdVOZ1 repressors that included the ethylene-responsive transcription factors (ERF)-associated amphiphilic repression (EAR) domain motif influenced reproduction and inflorescence architecture in transgenic Arabidopsis. These results suggest that MdFT1 and/or MdFT2 might be involved in the regulation of cellular proliferation and the formation of new tissues and that they might affect leaf and fruit development by interacting with TCP- and VOZ-family proteins. DDBJ accession nos. AB531019 (MdTCP2a mRNA), AB531020 (MdTCP2b mRNA), AB531021 (MdTCP4a mRNA), AB531022 (MdTCP4b mRNA) and AB531023 (MdVOZ1a mRNA).

SummaryTo elucidate the influence of temperature on water flow from soil to stem in a dwarfing rootstock, sap flow was measured under different temperature conditions in 1-year-old trees of apple rootstocks of different vigour. Sap flow was largely determined by solar radiation, and increased linearly as solar radiation increased when the temperatures above and below ground were held constant. The adjusted means of sap flow in the trees tested were not significantly influenced by the total leaf area, but were significantly influenced by the root mass. The adjusted means of sap flow at 30oC were two- to three-times greater than those at 20oC in all rootstocks tested, except one super-dwarfing rootstock, during the period of shoot extension. As the degree of rootstock vigour increased, the adjusted means of sap flow increased. The difference in sap flow between an invigorating rootstock and a dwarfing one increased under high temperature conditions. Because the degree of dwarfing was clearly expressed as th...

Differentiation of cultivars with simple sequence repeat (SSR) markers is a very useful technique for the true-to-type characterization of cultivars and clarification of parent-offspring relationships. We developed an SSR marker set for cultivar identification comprising 15 markers that were screened from 46 previously published SSRs. This marker set could be used for apple varieties including Malus × domestica and/or other Malus species. These SSRs successfully characterized 95 apples, including the leading and major founding cultivars used worldwide for modern apple breeding. Therefore, this marker set could be applied to almost all apple cultivars. We also analyzed the parent-offspring relationships of 69 cultivars by considering allele transmissions. This analysis revealed the true parentage of the following seven cultivars: ‘Kizashi’, ‘Chinatsu’, ‘Honey Queen’, ‘Haruka’, ‘Seirin’, ‘Ozenokurenai’, and Morioka #48. This analysis also revealed a parentage discrepancy for ‘Hacnine’. From the parent-offspring analysis, two microsatellite mutation events at alleles inherited from pollen parents were observed.

The two FLOWERING LOCUS T (FT)-like genes of apple (Malus x domestica Borkh.), MdFT1 and MdFT2, have been isolated and characterized. MdFT1 and MdFT2 were mapped, respectively, on distinct linkage groups (LGs) with partial homoeology, LG 12 and LG 4. The expression pattern of MdFT1 and MdFT2 differed in that MdFT1 was expressed mainly in apical buds of fruit-bearing shoots in the adult phase, with little expression in the juvenile tissues, whereas MdFT2 was expressed mainly in reproductive organs, including flower buds and young fruit. On the other hand, both genes had the potential to induce early flowering since transgenic Arabidopsis, which ectopically expressed MdFT1 or MdFT2, flowered earlier than wild-type plants. Furthermore, overexpression of MdFT1 conferred precocious flowering in apple, with altered expression of other endogenous genes, such as MdMADS12. These results suggest that MdFT1 could function to promote flowering by altering the expression of those genes and that, at least, other genes may play an important role as well in the regulation of flowering in apple. The long juvenile period of fruit trees prevents early cropping and efficient breeding. Our findings will be useful information to unveil the molecular mechanism of flowering and to develop methods to shorten the juvenile period in various fruit trees, including apple.

Although division site positioning in rod-shaped bacteria is generally believed to occur through the combined effect of nucleoid occlusion and the Min system, several lines of evidence suggest the existence of additional mechanisms. Studies using outgrown spores of Bacillus subtilis have shown that inhibiting the early stages of DNA replication, leading up to assembly of the replisome at oriC, influences Z ring positioning. Here we examine whether Z ring formation at midcell under various conditions of DNA replication inhibition is solely the result of relief of nucleoid occlusion. We show that midcell Z rings form preferentially over unreplicated nucleoids that have a bilobed morphology (lowering DNA concentration at midcell), whereas acentral Z rings form beside a single-lobed nucleoid. Remarkably however, when the DnaB replication initiation protein is inactivated midcell Z rings never form over bilobed nucleoids. Relieving nucleoid occlusion by deleting noc increased midcell Z ring frequency for all situations of DNA replication inhibition, however not to the same extent, with the DnaB-inactivated strain having the lowest frequency of midcell Z rings. We propose an additional mechanism for Z ring positioning in which the division site becomes increasingly potentiated for Z ring formation as initiation of replication is progressively completed.

Crown gall, caused by Agrobacterium tumefaciens, causes severe damage to apple saplings resulting in weak growth and loss of commercial value. Developing molecular markers linked to crown gall resistance genes, and establishing a marker-assisted selection (MAS) for such a trait would be an effective way to improve rootstock breeding for crown gall resistance. The wild apple Malus sieboldii Sanashi 63 carries the crown gall resistance gene Cg effective against the A. tumefaciens strain Peach CG8331 (biovar 2). Applying the genome scanning approach on the mapping population JM7 (cgcg) × Malus sieboldii Sanashi 63 (Cgcg), Cg was mapped on the linkage group (LG) 2. The constructed linkage map of LG 2 of Sanashi 63 spans 59.8 cM and has an average marker density of 3.5 cM per marker. The 191 bp allele of the simple sequence repeat (SSR) NZmsEB119405 co-segregated perfectly with Cg in a segregating population of 119 individuals. Quantitative trait loci, accounting for 75.3% to 84.3% of phenotypic variation were detected in the same position. Testing eight additional rootstocks with the NZmsEB119405 SSR marker revealed that the 191 bp allele is also present in crown gall-susceptible rootstock accessions. Only the markers CH03b01 and NZmsPal92 mapping at 0.9 and 4.3 cM from Cg, respectively, showed “private” alleles associated to Cg.

To make progress in columnar apple breeding, we constructed genetic linkage maps of the Co genomic region and identified useful DNA markers that enable correct and efficient selection of columnar-type seedlings in a practical columnar apple breeding program. Linkage maps of three mapping populations were constructed on the apple linkage group 10, and the Co gene was mapped on two populations, ‘Fuji’ × 8H-9-45 and ‘Fuji’ × 5-12786. Closely linked marker genotypes of SCAR682, SCAR216, CH03d11, and Hi01a03 were determined on 33 columnar-type cultivars and/or selections, and non-columnar-type cultivars contained 7 ancestral cultivars of apple breeding in Japan. A 174 bp fragment for primer Hi01a03 was amplified in all columnar-type cultivars, selections, and ‘McIntosh’, but was not detected in other ancestral apples. Amplified products of 682 bp for SCAR682 and 177 bp for CH03d11 were detected in almost all columnar genotypes except for one selection, and ‘McIntosh’, but the products were not found in other ancestral apples. A 177 bp allele of CH03d11, characteristic of a columnar phenotype, was detected in all 170 columnar-type plants obtained from 18 crosses, suggesting that it is one of the most tightly linked DNA markers to the Co. These results indicated that CH03d11 was the most reliable marker to distinguish columnar and non-columnar phenotypes for the MAS system. In our two most advanced breeding selections, the Co gene-associated alleles of the original columnar mutant (‘Wijcik’) on CH03d11 (177 bp) and Hi01a03 (174 bp) remained, indicating that using these two breeding selections as Co donors in the breeding program, CH03d11 and Hi01a03 enable the selection of seedlings with columnar phenotype.

Changes in turgor and flesh firmness during storage at 20 °C were investigated using 27 apple (Malus ×domestica Borkh.) cultivars for 2 years. Flesh firmness was measured using a penetrometer, and turgor was determined using a thermocouple psychrometer. Firmness and turgor of fruit decreased during storage. The cultivars with little softening during storage had low rates of reduction in turgor. The softening rates in mealy cultivars were high, but there were cultivars with low rates of turgor reduction. When the rates of reduction in turgor after harvest were low, the mealy cultivars of the fruit tended to develop severe mealiness during storage. Therefore, a low rate of reduction in turgor could contribute to cultivars with both good shelf life and severe mealiness. The reduction rates of turgor in progeny cultivars were nearly identical to the mean reduction rates of turgor of their parents. This suggests that a cultivar with a low reduction rate of turgor, although it can be mealy, has the potential to produce a progeny with a low reduction rate of turgor.

GTP-binding proteins are involved in cell proliferation, development, signal transduction, protein elongation, etc. and construct the GTPase superfamily, whose structures and sequence motifs (G-1 to G-5) are highly conserved from prokaryote to eukaryote. Obg of Bacillus subtilis and Obg homologues of other bacteria belong to the GTPase superfamily and have been suggested as being essential for cell growth, development and monitoring of intracellular levels of GTP. We identified the Obg homologue in Escherichia coli, a protein previously known as YhbZ, which we have renamed ObgE. Double cross-over experiments showed that the obgE gene is essential for growth in E. coli. From characterization of the obgE temperature-sensitive mutant, we found that DNA replication was not inhibited, that the nucleoids did not partition and instead remained in the middle of cell, and that the cells elongated. Overproduction of ObgE also resulted in aberrant chromosome segregation. These data suggested that ObgE is involved directly or indirectly in E. coli chromosome partitioning. Characterization studies showed that ObgE is abundant in normal cells, partially associated with the membrane and does not associate with ribosomes such as in Obg of B. subtilis. We purified ObgE protein from a cell extract of E. coli, and the purified ObgE had GTPase activity and DNA-binding ability.

To compare changes in fruit quality during cold storage with those during shelf life conditions, flesh firmness and titratable acidity (TA) were measured during storage in 20 apple (Malus ×domestica Borkh.) cultivars. Fruit of each cultivar were divided into two groups and stored in chambers controlled at 20 ± 2 °C and 85 ± 5% relative humidity (RH) (shelf life conditions) or 0.5 ± 0.3 °C and 95 ± 5% RH (cold storage). Five of the stored fruit were removed for measurements at 5- or 10-d intervals for 40 d and at 1-month intervals until 10 months after harvest at 20 °C and 0.5 °C, respectively. Data for firmness and TA were subjected to a linear regression and a nonlinear regression, respectively. Moreover, to determine the advantages of 0.5 °C storage over 20 °C storage for retaining firmness and TA, the effect of storage type on extending the storage period was introduced as a parameter. The estimate of the effect of storage type showed that firmness and TA could be retained 8.9 and 3.7 times, respectively, longer at 0.5 °C than 20 °C, independently of the cultivar. Therefore, firmness and TA after cold storage could be predicted by the change in firmness and TA during shelf life conditions. Moreover, cultivar differences regarding quality change under cold storage could be determined in a short period after harvest because the cultivar differences under shelf life conditions were detected within 1 month after harvest.

Progenies from 38 unbalanced crosses using 20 apple (Malus ×domestica Borkh.) cultivars/selections as parents were evaluated for changes in flesh firmness after harvest in two seasons to determine the mechanism of inheritance of fruit softening. The change in firmness was fitted by linear regression, and the softening rate (N·d−1) expressed as the regression coefficient was used as the phenotypic value of softening after harvest. Fruit were stored under 20 °C and 85% relative humidity after harvest for up to 40 days. The softening rates in the progeny populations were distributed continuously around the softening rates of parents, despite a distinct segregation in the degree of mealiness at 30 days of storage. The narrow-sense heritability of the softening rate was estimated by parent-offspring regression, and the estimate was high (h2 = 0.93). Because the softening rate can be influenced by mealiness, an undesirable trait in the apple industry, the progenies were divided into individuals with and without mealiness, and the breeding values of the parents were estimated based on the softening rate of the nonmealy progeny. The softening rate of the nonmealy progeny was analyzed using a mixed linear model and the restricted maximum likelihood method, with general combining ability (GCA) as parental effects and specific combining ability (SCA) as parental interaction effects. The variance of GCA was significant, but the variance of SCA was small and nonsignificant. The narrow-sense heritability of the softening rate in the nonmealy progeny was estimated by sib analysis, and the estimate was moderately high (h2 = 0.55). A significant correlation was observed between the phenotypic value and the breeding value (twice the GCA effects) in nonmealy parents, but the phenotypic value did not significantly correlate with the breeding value in mealy parents. Therefore, contribution of a mealy parent to the softening rate of nonmealy progenies cannot be predicted by its phenotypic value.