A Case of Mediastinal Tuberculous Lymphadenitis in a Chronic Dialysis Patient Diagnosed by Endobronchial Ultrasound-Guided Transbronchial Needle Aspiration (EBUS-TBNA).

A 54-year-old woman on dialysis due to chronic renal failure had a fever lasting 2 weeks and was referred to a hospital. Non-enhanced CT and blood tests showed no remarkable findings. She was hospitalized and received an antibacterial drug. Although she was discharged after the fever subsided, she was hospitalized again due to a fever a few days later. A contrast-enhanced CT revealed mediastinal lymphadenopathy, and she was transferred to our hospital for a bronchoscopy. Endobronchial Ultrasound-Guided Transbronchial Needle Aspiration (EBUS-TBNA) for subcarinal lymph nodes was performed in our hospital. The Polymerase Chain Reaction (PCR) test of the obtained specimen was positive for mycobacterium tuberculosis, and histologically, caseous granulomas were found in the specimen. She was diagnosed with mediastinal tuberculous lymphadenitis, and HREZ (isoniazid, rifampicin, ethambutol, and pyrazinamide) treatment was started. The fever subsided immediately, and she was discharged from our hospital 2 weeks after the initiation of treatment. Thereafter, she received treatment as an outpatient. Since the use of a contrast medium was complicated by dialysis, a non-enhanced CT was performed at first, and it was difficult to make a diagnosis from this. We report this as an informative case that could be diagnosed with EBUS-TBNA, which was easily performed on a patient weakened by prolonged fever and dialysis.

Tracing founder haplotypes of Japanese apple varieties: application in genomic prediction and genome-wide association study.

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 F1 individuals 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.

Columnar growth phenotype in apple results from gibberellin deficiency by ectopic expression of a dioxygenase gene.

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.

Identification of candidate genes responsible for the susceptibility of apple (Malus × domestica Borkh.) to Alternaria blotch.

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.

Role of genetic variations of chitinase 3-like 1 in bronchial asthmatic patients.

BACKGROUND: Single nucleotide polymorphisms (SNPs) in chitinase 3-like 1 (CHI3L1) are associated with bronchial severity and pulmonary function. CHI3L1 proteins are involved in both innate and adaptive immune responses; however, to date, the correlation of these SNPs and their age of onset of bronchial asthma has not been demonstrated. METHODS: To address the role of these genetic variations, 390 patients with well-controlled bronchial asthma and living in Japan were recruited, genotyped, and had a pulmonary function test performed on them in this study. To analyze the concentration levels of CHI3L1 protein, bronchial lavage fluids were examined. RESULTS: Forced expiratory volume in one second, %predicted (%FEV1), was significantly decreased in homozygotes of rs1214194 compared to heterozygotes and wild type. The age of onset of adult bronchial asthma was significantly younger in GG homozygotes of rs4950928 and AA homozygotes of rs1214194 than in the other two genotypes. The concentration of CHI3L1 protein in bronchial lavage fluid increased in both homozygotes of rs4950928 and rs1214194. CONCLUSIONS: Our study demonstrated that the homozygotes of rs4950928 and rs1214194 of CHI3L1 might predict an early onset of bronchial asthma and have the propensity to promote airway remodeling.Trial registration JMA-IIA00045 remodeling-ICS.

Impact of the genetic variants of GLCCI1 on clinical features of asthmatic patients.

BACKGROUND: Several gene variants are associated with a response to an inhaled corticosteroids (ICSs) treatment in patients with bronchial asthma. A variant of the glucocorticoid-induced transcript 1 (GLCCI1) genes has previously been associated with decreased lung function improvement upon treatment with ICSs in patients with bronchial asthma. Another report has also demonstrated that this genetic biomarker did not influence the change in flow volume in 1 second. However, no studies have considered the treatment content and the GLCCI1 variants. We were able to determine the relationship between the pulmonary function and clinical features and the variant of the GLCCI1 in Japanese asthmatic patients receiving long-term ICS treatment. MATERIALS AND METHODS: In this study, 405 patients with bronchial asthma, who were receiving ICS and living in Japan, were recruited, genotyped and underwent pulmonary function tests. To identify the GLCCI1 protein expression cells, endobronchial biopsy specimens were examined. RESULTS: We found that the pulmonary function was not significantly different in the homozygotes compared to the wild types. Also, the homozygotes increased the risk of a sustained step-up of the asthma treatment when compared to the wild type and heterozygotes. GLCCI1-positive cells were localized to the bronchial epithelial cells. The amount of GLCCI1 protein that cultured epithelial cells harboring GLCCI1 variants produced was less than the GLCCI1 wild type in the presence of a corticosteroid. CONCLUSIONS: A worsening of pulmonary function caused by GLCCI1 variants could be prevented due to recently used medications based on new action mechanisms.

Genomic dissection of a 'Fuji' apple cultivar: re-sequencing, SNP marker development, definition of haplotypes, and QTL detection.

'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.

Expression of a putative dioxygenase gene adjacent to an insertion mutation is involved in the short internodes of columnar apples (Malus × domestica).

Determining the molecular mechanism of fruit tree architecture is important for tree management and fruit production. An apple mutant 'McIntosh Wijcik', which was discovered as a bud mutation from 'McIntosh', exhibits a columnar growth phenotype that is controlled by a single dominant gene, Co. In this study, the mutation and the Co gene were analyzed. Fine mapping narrowed the Co region to a 101 kb region. Sequence analysis of the Co region and the original wild-type co region identified an insertion mutation of an 8202 bp long terminal repeat (LTR) retroposon in the Co region. Segregation analysis using a DNA marker based on the insertion polymorphism showed that the LTR retroposon was closely associated with the columnar growth phenotype. RNA-seq and RT-PCR analysis identified a promising Co candidate gene (91071-gene) within the Co region that is specifically expressed in 'McIntosh Wijcik' but not in 'McIntosh'. The 91071-gene was located approximately 16 kb downstream of the insertion mutation and is predicted to encode a 2-oxoglutarate-dependent dioxygenase involved in an unknown reaction. Overexpression of the 91071-gene in transgenic tobaccos and apples resulted in phenotypes with short internodes, like columnar apples. These data suggested that the 8202 bp retroposon insertion in 'McIntosh Wijcik' is associated with the short internodes of the columnar growth phenotype via upregulated expression of the adjacent 91071-gene. Furthermore, the DNA marker based on the insertion polymorphism could be useful for the marker-assisted selection of columnar apples.

Fine mapping of the gene for susceptibility to black spot disease in Japanese pear (Pyrus pyrifolia Nakai).

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.

Mitigation of tight junction protein dysfunction in lung microvascular endothelial cells with pitavastatin.

BACKGROUND: Statin use in individuals with chronic obstructive pulmonary disease (COPD) with coexisting cardiovascular disease is associated with a reduced risk of exacerbations. The mechanisms by which statin plays a role in the pathophysiology of COPD have not been defined. To explore the mechanisms involved, we investigated the effect of statin on endothelial cell function, especially endothelial cell tight junctions. METHOD: We primarily assessed whether pitavastatin could help mitigate the development of emphysema induced by continuous cigarette smoking (CS) exposure. We also investigated the activation of liver kinase B1 (LKB1)/AMP-activated protein kinase (AMPK) signaling, which plays a role in maintaining endothelial functions, important tight junction proteins, zonula occludens (ZO)-1 and claudin-5 expression, and lung microvascular endothelial cell permeability. RESULTS: We found that pitavastatin prevented the CS-induced decrease in angiomotin-like protein 1 (AmotL1)-positive vessels via the activation of LKB1/AMPK signaling and IFN-γ-induced hyperpermeability of cultured human lung microvascular endothelial cells by maintaining the levels of AmotL1, ZO-1, and claudin-5 expression at the tight junctions. CONCLUSION: Our results indicate that the maintenance of lung microvascular endothelial cells by pitavastatin prevents tight junction protein dysfunctions induced by CS. These findings may ultimately lead to new and novel therapeutic targets for patients with COPD.

Optimization of Inversion Time for Postmortem Fluid-attenuated Inversion Recovery (FLAIR) MR Imaging at 1.5T: Temperature-based Suppression of Cerebrospinal Fluid.

PURPOSE: Signal intensity (SI) and image contrast on postmortem magnetic resonance (MR) imaging are different from those of imaging of living bodies. We sought to suppress the SI of cerebrospinal fluid (CSF) sufficiently for fluid-attenuated inversion recovery (FLAIR) sequence in postmortem MR (PMMR) imaging by optimizing inversion time (TI). MATERIALS AND METHODS: We subject 28 deceased patients to PMMR imaging 3 to 113 hours after confirmation of death (mean, 27.4 hrs.). PMMR imaging was performed at 1.5 tesla, and T1 values of CSF were measured with maps of relaxation time. Rectal temperatures (RT) measured immediately after PMMR imaging ranged from 6 to 32°C (mean, 15.4°C). We analyzed the relationship between T1 and RT statistically using Pearson's correlation coefficient. We obtained FLAIR images from one cadaver using both a TI routinely used for living bodies and an optimized TI calculated from the RT. RESULTS: T1 values of CSF ranged from 2159 to 4063 ms (mean 2962.4), and there was a significantly positive correlation between T1 and RT (r = 0.96, P < 0.0001). The regression expression for the relationship was T1 = 74.4 * RT + 1813 for a magnetic field strength of 1.5T. The SI of CSF was effectively suppressed with the optimized TI (0.693 * T1), namely, TI = 0.693 * (77.4 * RT + 1813). CONCLUSION: Use of the TI calculated from the linear regression of the T1 and RT optimizes the FLAIR sequence of PMMR imaging.

Identification of QTLs for fruit quality traits in Japanese apples: QTLs for early ripening are tightly related to preharvest fruit drop.

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.

Isolation and characterization of multiple F-box genes linked to the S9- and S10-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.

Identification of a Skp1-like protein interacting with SFB, the pollen S determinant of the gametophytic self-incompatibility in Prunus.

Many species in Rosaceae, Solanaceae, and Plantaginaceae exhibit S-RNase-based self-incompatibility (SI). In this system, the pistil and pollen specificities are determined by S-RNase and the S locus F-box protein, respectively. The pollen S determinant F-box protein in Prunus (Rosaceae) is referred to by two different terms, SFB (for S-haplotype-specific F-box protein) and SLF (for S locus F box), whereas it is called SLF in Solanaceae and Plantaginaceae. Prunus SFB is thought to be a molecule indispensable for its cognate S-RNase to exert cytotoxicity and to arrest pollen tube growth in incompatible reactions. Although recent studies have demonstrated the molecular function of SCF(SLF) in the SI reaction of Solanaceae and Plantaginaceae, how SFB participates in the Prunus SI mechanism remains to be elucidated. Here we report the identification of sweet cherry (Prunus avium) SFB (PavSFB)-interacting Skp1-like1 (PavSSK1) using a yeast (Saccharomyces cerevisiae) two-hybrid screening against the pollen cDNA library. Phylogenetic analysis showed that PavSSK1 belongs to the same clade as Antirrhinum hispanicum SLF-interacting Skp1-like1 and Petunia hybrida SLF-interacting Skp1-like1 (PhSSK1). In yeast, PavSSK1 interacted not only with PavSFBs from different S haplotypes and Cullin1-likes (PavCul1s), but also with S-locus F-box-likes. A pull-down assay confirmed the interactions between PavSSK1 and PavSFB and between PavSSK1 and PavCul1s. These results collectively indicate that PavSSK1 could be a functional component of the SCF complex and that PavSFB may function as a component of the SCF complex. We discuss the molecular function of PavSFB in self-/nonself-recognition in the gametophytic SI of Prunus.

Apple FLOWERING LOCUS T proteins interact with transcription factors implicated in cell growth and organ development.

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).

Molecular characterization of FLOWERING LOCUS T-like genes of apple (Malus x domestica Borkh.).

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.

Four TFL1/CEN-like genes on distinct linkage groups show different expression patterns to regulate vegetative and reproductive development in apple (Malus x domestica Borkh.).

Recent molecular analyses in several plant species revealed that TERMINAL FLOWER1 (TFL1) and CENTRORADIALIS (CEN) homologs are involved in regulating the flowering time and/or maintaining the inflorescence meristem. In apple (Malusxdomestica Borkh.), four TFL1/CEN-like genes, MdTFL1, MdTFL1a, MdCENa and MdCENb, were found and mapped by a similar position on putatively homoeologous linkage groups. Apple TFL1/CEN-like genes functioned equivalently to TFL1 when expressed constitutively in transgenic Arabidopsis plants, suggesting that they have a potential to complement the TFL1 function. Because MdTFL1 and MdTFL1a were expressed in the vegetative tissues in both the adult and juvenile phases, they could function redundantly as a flowering repressor and a regulator of vegetative meristem identity. On the other hand, MdCENa was mainly expressed in fruit receptacles, cultured tissues and roots, suggesting that it is involved in the development of proliferating tissues but not in the control of the transition from the juvenile to the adult phase. In contrast, MdCENb was silenced in most organs probably due to gene duplication by the polyploid origin of apple. The expression patterns of MdTFL1 and MdCENa in apple were also supported by the heterologous expression of beta-glucuronidase fused with their promoter regions in transgenic Arabidopsis. Our results suggest that functional divergence of the roles in the regulation of vegetative meristem identity may have occurred among four TFL1/CEN-like genes during evolution in apple.