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Hisayo Yamane, Yukinobu Kashiwa, Tomomi Ooka, Ryutaro Tao, and Keizo Yonemori

To understand the molecular basis of the endodormancy of buds of perennial plants, we searched for the genes that are expressed preferentially in endodormant lateral buds of the deciduous fruit tree japanese apricot (Prunus mume Sieb. et Zucc.) using suppression subtractive hybridization with mirror orientation selection (SSH/MOS). We generated two SSH/MOS libraries containing gene pools that are expressed preferentially in endodormant buds in comparison with paradormant or ecodormant buds to search for the genes that are upregulated by endodormancy induction or down-regulated by endodormancy release, respectively. Differential screening and sequencing indicated that genes involved in gibberellin metabolism, stress resistance, cell wall modification, and signal transduction, such as transcription factors, are upregulated in endodormant buds. After a further expression survey and full-length cDNA cloning, we found that a gene similar to the SVP/AGL24-type MADS-box transcription factor showed endodormancy-associated expression. Seasonal expression analysis suggested that the SVP/AGL24 homolog in japanese apricot might be involved in endodormancy regulation of its lateral buds.

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Ryutaro Tao, Tsuyoshi Habu, Hisayo Yamane, Akira Sugiura, and Kazuya Iwamoto

Self-compatible cultivars of Japanese apricot (Prunus mume Sieb. et Zucc.) have a horticultural advantage over self-incompatible ones because no pollinizer is required. Self-incompatibility is gametophytic, as in other Prunus species. We searched for molecular markers to identify self-compatible cultivars based on the information about S-ribonucleases (S-RNases) of other Prunus species. Total DNA isolated from five self-incompatible and six self-compatible cultivars were PCR-amplified by oligonucleotide primers designed from conserved regions of Prunus S-RNases. Self-compatible cultivars exhibited a common band of ≈1.5 kbp. Self-compatible cultivars also showed a common band of ≈12.1 kbp when genomic DNA digested with HindIII was probed with the cDNA encoding S 2-RNase of sweet cherry (Prunus avium L.). These results suggest that self-compatible cultivars of Japanese apricot have a common S-RNase allele that can be used as a molecular marker for self-compatibility.

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Nathanael R. Hauck, Amy F. Iezzoni, Hisayo Yamane, and Ryutaro Tao

Correct assignment of self-incompatibility alleles (S-alleles) in sweet cherry (Prunus avium L.) is important to assure fruit set in field plantings and breeding crosses. Until recently, only six S-alleles had been assigned. With the determination that the stylar product of the S-locus is a ribonuclease (RNase) and subsequent cloning of the S-RNases, it has been possible to use isoenzyme and DNA analysis to genotype S-alleles. As a result, numerous additional S-alleles have been identified; however, since different groups used different strategies for genotype analysis and different cultivars, the nomenclature contained inconsistencies and redundancies. In this study restriction fragment-length polymorphism (RFLP) profiles are presented using HindIII, EcoRI, DraI, or XbaI restriction digests of the S-alleles present in 22 sweet cherry cultivars which were chosen based upon their unique S-allele designations and/or their importance to the United States sweet cherry breeding community. Twelve previously published alleles (S1, S2, S3, S4, S5, S6, S7, S9, S10, S11, S12, and S13) could be differentiated by their RFLP profiles for each of the four restriction enzymes. Two new putative S-alleles, both found in `NY1625', are reported, bringing the total to 14 differentiable alleles. We propose the adoption of a standard nomenclature in which the sweet cherry cultivars `Hedelfingen' and `Burlat' are S3S5 and S3S9, respectively. Fragment sizes for each S-allele/restriction enzyme combination are presented for reference in future S-allele discovery projects.

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Yuto Kitamura, Hisayo Yamane, Akira Yukimori, Hiroyoshi Shimo, Koji Numaguchi, and Ryutaro Tao

Flower bud development and the timing of blooming are mainly affected by genotype-dependent chilling requirements (CRs) during endodormancy and subsequent heat requirements (HRs) during ecodormancy. However, little information is available regarding the responses of flower buds to temperatures during endodormancy and ecodormancy in japanese apricot. We exposed japanese apricot ‘Nanko’ trees to various temperatures to estimate the CRs and HRs using development index (DVI) models specific for the endodormant (DVIendo) and ecodormant (DVIeco) stages. These models were based on the experimentally determined development rate (DVR). The DVRendo value was calculated as the reciprocal of the chilling time required to break endodormancy. The relationship between the DVRendo value and temperature was estimated using a three-dimensional curve. Our results indicated that 5–6 °C was the most effective temperature for breaking endodormancy in ‘Nanko’ flower buds. Additionally, exposure to −3 °C negatively affected endodormancy release, whereas 15 °C had no effect. We also determined that the DVReco values for temperatures between 5 and 20 °C were the reciprocal values of the time required for blooming after endodormancy release. The values outside this range were estimated using linear functions. The DVI was defined as the sum of the DVR values ranging from 0 to 1. Models for predicting the blooming date were constructed using the functions of sequentially combined DVIendo and DVIeco models. The accuracy of each model was assessed by comparing the predicted and actual blooming dates. The prediction of the model in which DVIeco = 1 corresponded to a 40% blooming level and DVIeco = 0 was set to DVIendo = 0.5 had the lowest root mean square error (RMSE) value (i.e., 3.11) for trees in commercial orchards exposed to different climates. Our results suggest that the developed model may have practical applications.

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Takanori Takeuchi, Miwako Cecile Matsushita, Soichiro Nishiyama, Hisayo Yamane, Kiyoshi Banno, and Ryutaro Tao

Endodormancy release and the fulfillment of the chilling requirement (CR) are critical physiological processes that enable uniform blooming in fruit tree species, including apple (Malus ×domestica). However, the molecular mechanisms underlying these traits have not been fully characterized. The objective of this study was to identify potential master regulators of endodormancy release and the CR in apple. We conducted RNA-Sequencing (RNA-seq) analyses and narrowed down the number of candidates among the differentially expressed genes (DEGs) based on the following two strict screening criteria: 1) the gene must be differentially expressed between endodormant and ecodormant buds under different environmental conditions and 2) the gene must exhibit chill unit (CU)–correlated expression. The results of our cluster analysis suggested that global expression patterns varied between field-grown buds and continuously chilled buds, even though they were exposed to similar amounts of chilling and were expected to have a similar dormancy status. Consequently, our strict selection strategy resulted in narrowing down the number of possible candidates and identified the DEGs strongly associated with the transition between dormancy stages. The genes included four transcription factor genes, PHYTOCHROME-INTERACTING FACTOR 4 (PIF4), FLOWERING LOCUS C (FLC)-LIKE, APETALLA2 (AP2)/ETHYLENE-RESPONSIVE 113 (ERF113), and MYC2. Their expressions were upregulated during endodormancy release, and were correlated with the CU, suggesting that these transcription factors are closely associated with chilling-mediated endodormancy release in apple.

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Hisayo Yamane, Megumi Ichiki, Ryutaro Tao, Tomoya Esumi, Keizo Yonemori, Takeshi Niikawa, and Hino Motosugi

Fruit size is one of the most important traits that affect the economic value of fruit. In persimmon (Diospyros kaki Thunb.), somatic and bud-sport mutations that affect the fruit traits are frequently observed. Recently, a small-fruit mutant, ‘Totsutanenashi’ (TTN), was discovered in Japan as a bud-sport mutant of the leading cultivar Hiratanenashi (HTN). In this study, we investigated the morphological and physiological characteristics of TTN and HTN focusing on the tree architecture, fruit size, and the fruit flesh chemical composition. The objectives of the study were to evaluate the potential horticultural use of TTN and to characterize the differences between HTN and TTN. Both TTN and HTN are nonaploid plants, indicating that a difference in ploidy is not the cause of the small-fruit mutation. The vegetative growth of trees and tissue-cultured shoots of TTN was more compact than that of HTN. The floral organs of TTN appeared similar to those of HTN before flowering, but the TTN flowers opened earlier, resulting in smaller ovaries than in HTN flowers. The fruit size of TTN was consistently lower than that of HTN at all fruit developmental stages. TTN fruit had a higher sugar content and a higher proportion of sucrose to total sugars than HTN fruit. TTN fruits contained lower levels of secondary metabolites such as soluble tannins and ascorbate than HTN fruits. These results suggest that the fruit size mutation also affects the fruit biochemistry, leading to alterations in the fruit flesh composition. TTN may be a valuable genetic resource because compact trees require less labor and maintenance, and small, sweeter fruits may meet the various needs of consumers. The use of TTN in studies of the genetic control of fruit size is also discussed.

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Hisayo Yamane, Ryutaro Tao, Akira Sugiura, Nathanael R. Hauck, and Amy F. Iezzoni

This report demonstrates the presence of S-ribonucleases (S-RNases), which are associated with gametophytic self-incompatibility (SI) in Prunus L., in styles of self-incompatible and self-compatible (SC) selections of tetraploid sour cherry (Prunus cerasus L.). Based on self-pollen tube growth in the styles of 13 sour cherry selections, seven selections were SC, while six selections were SI. In the SI selections, the swelling of pollen tube tips, which is typical of SI pollen tube growth in gametophytic SI, was observed. Stylar extracts of these selections were evaluated by two-dimensional polyacrylamide gel electrophoresis. Glycoproteins which had molecular weights and isoelectric points similar to those of S-RNases in other Prunus sp. were detected in all selections tested. These proteins had immunological characteristics and N-terminal amino acid sequences consistent with the S-RNases in other Prunus sp. Two cDNAs encoding glycoproteins from `Erdi Botermo' were cloned. One of them had the same nucleotide sequence as that of S4-RNase of sweet cherry (Prunus avium L.), while the amino acid sequence from the other cDNA encoded a novel S-RNase (named Sa-RNase in this study). This novel RNase contained two active sites of T2/S type RNases and five regions conserved among other Prunus S-RNases. Genomic DNA blot analysis using cDNAs encoding S-RNases of sweet cherry as probes indicated that three or four S-RNase alleles are present in the genome of each selection regardless of SI. All of the selections tested seemed to have at least one S-allele that is also found in sweet cherry. Genetic control of SI/SC in tetraploid sour cherry is discussed based on the results obtained from restriction fragment length polymorphism analysis.

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Ryutaro Tao, Hisayo Yamane, Akira Sugiura, Hideki Murayama, Hidenori Sassa, and Hitoshi Mori

This report identifies S-RNases of sweet cherry (Prunus avium L.) and presents information about cDNA sequences encoding the S-RNases, which leads to the development of a molecular typing system for S-alleles in this fruit tree species. Stylar proteins of sweet cherry were surveyed by two dimensional polyaclylamide gel electrophoresis (2D-PAGE) to identify S-proteins associated with gametophytic self-incompatibility. Glycoprotein spots linked to S-alleles were found in a group of proteins which had Mr and pI similar to those of other rosaceous S-RNases. These glycoproteins were present at highest concentration in the upper segment of the mature style and shared immunological characteristics and N-terminal sequences with those of S-RNases of other plant species. cDNAs encoding these glycoproteins were cloned based on the N-terminal sequences. Genomic DNA and RNA blot analyses and deduced amino acid sequences indicated that the cDNAs encode S-RNases; thus the S-proteins identified by 2D-PAGE are S-RNases. Although S1 to S6-alleles of sweet cherry cultivars could be distinguished from each other with the genomic DNA blot analysis, a much simpler method of PCR-based typing system was developed for the six S-alleles based on the DNA sequence data obtained from the cDNAs encoding S-RNases.

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Akiko Watari, Toshio Hanada, Hisayo Yamane, Tomoya Esumi, Ryutaro Tao, Hideaki Yaegaki, Masami Yamaguchi, Kenji Beppu, and Ikuo Kataoka

Most commercial cultivars of japanese plum (Prunus salicina Lindl.) exhibit S-RNase-based gametophytic self-incompatibility (GSI), although some self-compatible (SC) cultivars exist. In this study, we characterized S-RNase and SFB, the pistil and pollen S determinants of the specificity of the GSI reaction, respectively, from four S-haplotypes, including a SC (Se) and three SI (Sa, Sb, and Sc) S-haplotypes of japanese plum. The genomic organization and structure of the SC Se-haplotype appear intact, because the relative transcriptional orientation of its S-RNase and SFB and their intergenetic distance are similar to those of the other three SI S-haplotypes of japanese plum and other Prunus L. species. Furthermore, there is no apparent defect in the DNA sequences of Se-RNase and SFBe. However, a series of transcriptional analyses, including real-time reverse transcriptase–polymerase chain reaction, showed that the Se-RNase transcript levels in the pistil are significantly lower than those of the Sa-, Sb-, and Sc-RNases, although transcripts of SFBa, SFBb, SFBc, and SFBe are present at similar levels in pollen. Furthermore, no Se-RNase spot was detected in two-dimensional polyacrylamide gel electrophoresis profiles of stylar extracts of the cultivars with the Se-haplotype. We discuss the possible molecular basis of SC observed with the Se-haplotype with special reference to the insufficient Se-RNase accumulation incited by the very low transcriptional level of Se-RNase in pistils.

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Toshio Hanada, Kyoko Fukuta, Hisayo Yamane, Tomoya Esumi, Ryutaro Tao, Thomas M. Gradziel, Abhaya M. Dandekar, Ángel Fernández i Martí, José M. Alonso, and Rafel Socias i Company

Most of the self-compatible (SC) cultivars of almond [Prunus dulcis (Mill.) D.A. Webb. syn. P. amygdalus Batsch] have the Sf haplotype. In this study, we cloned and characterized the S locus region of the Sf haplotype of SC ‘Lauranne’. The relative transcriptional orientation of SFBf and Sf-RNase and the physical distance between them are similar to those of other functional self-incompatible (SI) S haplotypes of Prunus, indicating that the genomic structure of the SC Sf haplotype appears to be intact. Although there is no apparent mutation in the coding sequence of SFBf, the Sf-RNase sequence in this study and previously reported Sf-RNase sequences show discrepancies. First, as opposed to previous indications, the ‘Lauranne’ Sf-RNase sequence encodes a histidine residue in place of a previously reported arginine residue in the conserved C2 region of Prunus S-RNase. Direct sequencing of the polymerase chain reaction products from the Sf-RNase of ‘Tuono’ confirmed that ‘Tuono’ Sf-RNase also encodes the histidine residue. We found another difference in the ‘Lauranne’ Sf-RNase sequence and other reported Sf-RNase sequences. Namely, ‘Lauranne’ Sf-RNase encodes a phenylalanine residue in place of a previously reported leucine residue in the conserved C5 region of Prunus S-RNase. This is also the case for ‘Tuono’ Sf-RNase. Expression analysis of Sf-RNase and SFBf by reverse transcriptase–polymerase chain reaction showed that Sf-RNase transcripts were barely detectable in pistil, whereas SFBf transcripts were accumulated at a similar level to the level that was observed with SFB of other functional SI S haplotypes of almond. We discuss the possible molecular mechanisms of SC observed with the Sf haplotype with special references to the expression of Sf-RNase.