Modified High-Resolution Melting (HRM) Marker Systems Increasing Discriminability Between Homozygous Alleles.

Targeted single-nucleotide polymorphism (SNP) genotyping, especially for functional nucleotide polymorphism, is widely used for current breeding programs in crops. One of the cost- and time-effective approaches for genotyping is high-resolution melting (HRM) analysis for polymerase chain reaction (PCR) amplicons, including target SNP. The reliability of a genotype obtained from an HRM marker depends on the difference in Tm values between two amplicons. Increasing the reliability of HRM marker genotypes could be archived with the selection of the best nearest neighboring nucleotide substitution (NNNs) in primer sequences surrounding SNPs. This chapter provides an easy-way protocol to design primer sequences for NNNs-HRM markers with table and web service, as well as several tips to develop HRM markers that distinguish between homozygous alleles (e.g., between A/A and C/C).

Molecular characterization of Satsuma mandarin (Citrus unshiu Marc.) VASCULAR PLANT ONE-ZINC FINGER2 (CuVOZ2) interacting with CuFT1 and CuFT3.

Shortening the juvenility is a burning issue in breeding fruit trees such as Satsuma mandarin (Citrus unshiu Marc.). Decreasing the breeding period requires a comprehensive understanding of the flowering process in woody plants. Throughout the Arabidopsis flowering system, FLOWERING LOCUS T (FT) interacts with other transcription factors (TFs) and functions as a transmissible floral inducer. In a previous study, a VASCULAR PLANT ONE-ZINC FINGER1 (VOZ1)-like TF from the Satsuma mandarin, CuVOZ1, showed protein-protein interaction with two citrus FTs in a yeast two-hybrid (Y2H) system and precocious flowering in Arabidopsis. In this study, another VOZ, CuVOZ2, was isolated from the Satsuma mandarin 'Aoshima' and protein-protein interaction was confirmed between CuVOZ2 and CuFTs. No apical meristem (NAM) and zinc coordination motifs were identified within the N-terminal of CuVOZ2. Docking simulation predicted that interactions between CuVOZ2 and CuFTs might occur in domain B of CuVOZ2, which contains a zinc finger motif. According to docking predictions, the distances between the amino acid residues involved ranged from 1.09 to 4.37 Å, indicating weak Van der Waals forces in the interaction. Cys216, Cys221, Cys235, and His239 in CuVOZ2 were suggested to bond with a Zn2+ in the Zn coordination motif. Ectopic expression of 35SΩ:CuVOZ2 in Arabidopsis affected the flowering time, length of inflorescence and internode, and number of siliques, suggesting that CuVOZ2 might regulate both vegetative and reproductive development, act as a trigger for early flowering, and be involved in the elongation of inflorescence possibly in a slightly different way than CuVOZ1.

A new secoiridoid glucoside from Olea europaea.

A new phenolic glucoside (1), olerikaside, and other known secoiridoid glucosides [oleuropein (2), demethyl oleuropein (3), oleoside 11-methyl ester (4), oleoside 7, 11-dimethyl ester (5), 7-ß-D-glucopyranosyl 11-methyl oleoside (6), secoxyloganin (7), ilicifolioside B (8), hydroxytyrosol (9), and hydroxytyrosol glucosides (10-12)] were isolated from unprocessed olive fruits of Olea europaea cv. "Lucca". The chemical structure of olerikaside (1) was clarified based on spectroscopy and chemical analysis data.

RAD-Seq analysis of typical and minor Citrus accessions, including Bhutanese varieties.

We analyzed the reduced-representation genome sequences of Citrus species by double-digest restriction site-associated DNA sequencing (ddRAD-Seq) using 44 accessions, including typical and minor accessions, such as Bhutanese varieties. The results of this analysis using typical accessions were consistent with previous reports that citron, papeda, pummelo, and mandarin are ancestral species, and that most Citrus species are derivatives or hybrids of these four species. Citrus varieties often reproduce asexually and heterozygosity is highly conserved within each variety. Because this approach could readily detect conservation of heterozygosity, it was able to discriminate citrus varieties such as satsuma mandarin from closely related species. Thus, this method provides an inexpensive way to protect citrus varieties from unintended introduction and to prevent the provision of incorrect nursery stocks to customers. One Citrus variety in Bhutan was morphologically similar to Mexican lime and was designated as Himalayan lime. The current analysis confirmed the previous proposition that Mexican lime is a hybrid between papeda and citron, and also suggested that Himalayan lime is a probable hybrid between mandarin and citron. In addition to Himalayan lime, current analysis suggested that several accessions were formed by previously undescribed combinations.

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).

Apple S locus region represents a large cluster of related, polymorphic and pollen-specific F-box genes.

Gametophytic self-incompatibility (GSI) of Rosaceae, Solanaceae and Plantaginaceae is controlled by a complex S locus that encodes separate proteins for pistil and pollen specificities, extracellular ribonucleases (S-RNases) and F-box proteins SFB/SLF, respectively. SFB/SLFs of Prunus (subfamily Prunoideae of Rosaceae), Solanaceae and Plantaginaceae are single copy in each S haplotype, while recently identified pollen S candidates SFBBs of subfamily Maloideae of Rosaceae, apple and Japanese pear, are multiple; two and three related SFBBs were isolated from each S haplotype of apple and Japanese pear, respectively. Here, we show that apple (Malus x domestica) SFBBs constitute a gene family that is much larger than initially thought. Twenty additional SFBB-like genes/alleles were isolated by screening of a BAC library derived from S (3) S (9) genotype, and tentatively named MdFBX1-20. All but one MdFBX showed S haplotype-specific polymorphisms. All the polymorphic MdFBXs were completely linked to S-RNase in 239 segregants. In addition, FISH revealed that the monomorphic gene MdFBX11 is also located near S-RNase, and the S locus is located in a subtelomeric region of a chromosome and is not close to the centromere. All MdFBXs were specifically expressed in pollen, except for a pseudogene MdFBX4 that showed no expression in any organs analyzed. Phylogenetic analysis revealed that the closest relatives of most MdFBXs were from a different S haplotype, suggesting that proliferation of MdSFBB/FBXs predates diversification of the S haplotypes.

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.

The FLOWERING LOCUS T/TERMINAL FLOWER 1 family in Lombardy poplar.

Genes in the FLOWERING LOCUS T (FT) and TERMINAL FLOWER 1 (TFL1) family have been shown to be important in the control of the switch between vegetative and reproductive growth in several plant species. We isolated nine members of the FT/TFL1 family from Lombardy poplar (Populus nigra var. italica Koehne). Sequence analysis of the members of the FT/TFL1 family revealed considerable homology within their coding regions both among family members and to the members of the same family in Arabidopsis, tomato and grapevine. Moreover, members of this family in all four species examined display a common exon-intron organization. Phylogenetic analysis revealed that the genes fall into four different clades: two into the TFL1 clade; five into the FT clade; and one each into the MOTHER OF FT AND TFL1 and BROTHER OF FT AND TFL1 clades. One gene in the TFL1 clade, PnTFL1, is expressed in vegetative meristems, and transgenic Arabidopsis that ectopically expressed PnTFL1 had a late-flowering phenotype. The expression patterns of two genes in the FT clade, PnFT1 and PnFT2, suggested a role for them in the promotion of flowering, and transgenic Arabidopsis that ectopically expressed either PnFT1 or PnFT2 had an early-flowering phenotype.

Constitutive expression of two apple (Malus x domestica Borkh.) homolog genes of LIKE HETEROCHROMATIN PROTEIN1 affects flowering time and whole-plant growth in transgenic Arabidopsis.

Fruit trees, such as apple (Malus x domestica Borkh.), are woody perennial plants with a long juvenile phase. The biological analysis for the regulation of flowering time provides insights into the reduction of juvenile phase and the acceleration of breeding in fruit trees. In Arabidopsis, LIKE HETEROCHROMATIN PROTEIN1 (LHP1) is involved in epigenetic silencing of the target genes such as flowering genes. We isolated and characterized twin apple LHP1 homolog genes, MdLHP1a and MdLHP1b. These genes may have been generated as a result of ancient genome duplication. Although the putative MdLHP1 proteins showed lower similarity to any other known plant LHP1 homologs, a chromo domain, a chromo shadow domain, and the nuclear localization signal motifs were highly conserved among them. RT-PCR analysis showed that MdLHP1a and MdLHP1b were expressed constantly in developing shoot apices of apple trees throughout the growing season. Constitutive expression of MdLHP1a or MdLHP1b could compensate for the pleiotropic phenotype of lhp1/tfl2 mutant, suggesting that apple LHP1 homolog genes are involved in the regulation of flowering time and whole-plant growth. Based on these results, LHP1 homolog genes might have rapidly evolved among plant species, but the protein functions were conserved, at least between Arabidopsis and apple.

Apple has two orthologues of FLORICAULA/LEAFY involved in flowering.

Two orthologues of FLORICAULA/LEAFY AFL1 and AFL2 (apple FLO/LFY), were isolated from the floral buds of apple trees. Their expression was detected in various tissues and during differentiation of the floral buds. Furthermore, the flowering effectiveness of each gene was assessed with transgenic Arabidopsis. Both AFL1 and AFL2 showed high homology to each other (90%) and a high degree of similarity to PTLF and PEAFLO (70%), which are homologues of FLO/LFY from poplar and pea, respectively. RNA blot analysis showed that AFL1 was expressed only in the floral bud during the transition from vegetative to reproductive growth, whereas AFL2 was expressed in vegetative shoot apex, floral buds, floral organs and root. Genomic Southern analysis showed that apple had other homologues in addition to AFL1 and AFL2. The transgenic Arabidopsis with over-expressed AFL2 showed accelerated flowering and gave rise to several solitary flowers from rosette axils directly. AFL1 had similar effects, but the phenotypes of the transgenic Arabidopsis with AFL1 were weaker than those with AFL2. These results suggest that both genes are involved in flower differentiation in apple.