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

. Sassa for providing antiserum prepared against S -RNase of almond, G. Lang and K. Ushijima for critical reviews of this manuscript, and Hitoshi Mori, Nagoya University, for helpful discussion. The cost of publishing this paper was defrayed in part by

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Gal Sapir, Raphael A. Stern, Martin Goldway, and Sharoni Shafir

, carries the S -RNase-mediated gametophytic self-incompatibility (GSI) system. This system was first identified in Solanaceae ( Anderson et al., 1986 ) and later in Rosaceae ( Sassa et al., 1992 ) and in Scrophulariaceae ( Xue et al., 1996

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Javier Sanzol and Timothy P. Robbins

-locus encodes a ribonuclease (S-RNase) in the Rosaceae ( Bošković and Tobutt, 1996 ; Broothaerts et al., 1995 ) has allowed further development of biochemical and molecular strategies for S-genotyping. The majority of the molecular methods for S

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H. Yamane, R. Tao, A. Sugiura, N. Hauck, and A. Iezzoni

Most fruit tree species of Prunus exhibit gametophytic self-incompatibility, which is controlled by a single locus with multiple alleles (S-alleles). One interesting aspect of gametophytic self-incompatibility is that it commonly “breaks down” as a result of polyploidy, resulting in self-compatible individuals. This phenomenon is exhibited in the diploid sweet cherry (P. avium) and the tetraploid sour cherry (P. cerasus), in which most cultivars are self-compatible. Recently, S-gene products in pistil of Prunus species were shown to be S-RNases. As sour cherry is one Prunus species, it is likely to possess S-alleles encoding pistil S-RNases. To confirm this, we surveyed stylar extracts of 11 sour cherry cultivars, including six self-compatible and five self-incompatible cultivars, by 2D-PAGE. As expected, all 11 cultivars tested yielded glycoprotein spots similar to S-RNases of other Prunus species in terms of Mr, immunological characteristics, and N-terminal sequences. A cDNA clone encoding one of these glycoproteins was cloned from the cDNA library constructed from styles with stigmas of a self-compatible cultivar, `Erdi Botermo'. Deduced amino acid sequence from the cDNA clone contained two active sites of T2/S type RNases and five conserved regions of rosaceous S-RNases. In order to determine the inheritance of self-incompatibility and S-allele diversity in sour cherry, we conducted genomic DNA blot analysis for sour cherry germplasm collections and mapping populations in MSU using the cDNA as a probe. To date, it appears as if self-compatibility in sour cherry is not simply controlled by a self-fertile allele as demonstrated in other Prunus species.

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Avi Matityahu, Raphael A. Stern, Doron Schneider, and Martin Goldway

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Kentaro Kitahara, Shogo Matsumoto, Toshiya Yamamoto, Junichi Soejima, Tetsuya Kimura, Hiromitsu Komatsu, and Kazuyuki Abe

As the parents of the some of the apple cultivars were unknown and others were uncertain, we investigated the parent-offspring relationships of eight apple cultivars by S-RNase analysis and SSR markers. The paternal parent of `Hida' was identified as `Golden Delicious', not the previously mentioned `Orin'. It was indicated that `Ryoka No Kisetsu' and `Korin' showing identical SSR genotype are likely sports of `Fuji'. `Fuji', rather than `Toko', seemed to be a maternal parent of `Kotoku', but was not a paternal parent of `Orei', `Starking Delicious', `Nero 26', `Empire', or `Aori 3'. Previously mentioned `Mutsu', `Indo', and `Shin Indo' were excluded as paternal parents of `Hokuto'. `Tsugaru' and `Jonathan' and were identified as the respective paternal parents of three cultivars described as having unknown paternal parents, i.e., `Aika No Kaori', `Yoko', and `Tsugaru'.

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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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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 fruit tree species in the genus Prunus (Rosaceae), including almond [ Prunus dulcis (Mill.) D.A. Webb.], exhibit the S-RNase-based gametophytic self-incompatibility (GSI) system ( de Nettancourt, 2001 ; Yamane and Tao, 2009 ). The

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Qin Yang and Yan Fu

germination, pollen tube dynamics, fruit set, and average number of seeds per fruit after different pollination treatments was conducted, and the S - RNase gene AS-PCR amplification was used to identify the true hybrid of the obtained hybrid seedlings. The

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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.