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

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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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Ali Lansari and Amy Iezzoni

Self-incompatibility was investigated in sour cherry (Prunus cerasus L.) by examining pollen growth in the pistil by use of ultraviolet fluorescence microscopy following self- and cross-pollination. The sour cherry cultivars Tschernokorka and Crisana exhibit pollen tube inhibition in the style characteristic of gametophytic self-incompatibility. `Meteor' and `Montmorency' appear to be partially self-incompatible, with few self-pollen tubes reaching the ovary. Several hybrid seedlings from crosses between self-compatible cultivars were self-incompatible, suggesting that these self-compatible parental cultivars carry self-incompatibility alleles.

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Tsuyoshi Habu, Fumio Kishida, Miki Morikita, Akira Kitajima, Toshiaki Yamada and Ryutaro Tao

Japanese apricot (Prunus mume Sieb. et Zucc.) exhibits S-RNase-based gametophytic self-incompatibility as do other Prunus species. Both self-incompatible and self-compatible Japanese apricot cultivars are grown commercially in Japan. These self-compatible cultivars are shown to have a common S-haplotype called S f that contains S f-RNase and SFB f (S-haplotype-specific F-box protein). This study describes a simple and rapid detection of SFB f, in Japanese apricot, based on loop-mediated isothermal amplification (LAMP) method. A set of 4 primers, F3, B3, FIP, and BIP primer, were designed from the exon and the putative inserted sequence of SFB f. Optimal reaction time at 63 C was determined to be 90 minutes. It appeared that the LAMP method combined with the ultrasimple DNA extraction efficiently detected SFB f. The advantage of the marker-assisted selection of self-compatibility based on the LAMP method was discussed.

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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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Bindu Chawla, Robert Bernatzki and Michael Marcotrigiano

Lycopersicon peruvianum is a wild species of tomato that exhibits gametophytic self-incompatibility (S), wherein the SI response is controlled by the genotype of the pollen. Cultivated tomato (L. esculentum) is a self-compatible species. Assisted by phenotypic markers, periclinal graft chimeras between these two species have been obtained. Fruit set analysis following breeding demonstrated that the available five chimeras (PPE, PEE, PEP, EPP, and EEP) are able to accept pollen from L. peruvianum, suggesting that there is a failure of the SI response. SI response is known to be dependent on S-locus associated proteins. These proteins are present in the style, which is mainly derived from the L1 and L2 layers of meristem. RNA analysis of the style tissue using a cloned S-locus cDNA as a probe showed that, except for EEP, all chimeras expressed the S-allele. This was also confirmed by SDS-PAGE analysis of stylar proteins that were present in variable amounts depending on the periclinal combination. Thus, the breakdown of SI is not associated with the lack of expression of the S-locus. Further work is being conducted to understand the nature of this breakdown.

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Sandra M. Reed

The objectives of this study were to evaluate self-incompatibility in Hydrangea paniculata Sieb. and H. quercifolia Bartr. and to determine optimum time for pollination of these two species. Flowers from three genotypes of each species were collected 1, 2, 4, 8, 24, 48, and 72 hours after cross- and self-pollination, stained with aniline blue and observed using a fluorescence microscope. In both species, pollen germination was observed on stigmas of over half of the flowers collected 4 to 72 hours after cross- or self-pollination. Differences in pollen tube length between cross- and self-pollinated flowers were noted from 8 to 72 hours after pollination in H. paniculata and from 24 to 72 hours after pollination in H. quercifolia. By 72 hours after pollination, most self-pollen tubes had only penetrated the top third of the style but cross-pollen tubes had grown to the base of the style and entered 40% to 60% of the ovules. Stigmas of H. paniculata were receptive to pollen from anthesis to 5 days after anthesis, while stigmas of H. quercifolia were receptive from 1 to 5 days after anthesis. This study provides evidence of a gametophytic self-incompatibility system in H. paniculata and H. quercifolia. Occasional self-seed set previously observed in these species was theorized to have been due to pseudo-self compatibility.

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Sandra M. Reed

Little information is available on the reproductive behavior of Hydrangea macrophylla (Thunb. Ex J.A. Murr.) Ser. The objectives of this study were to investigate time of stigma receptivity, viability of pollen from sterile flowers, and self-incompatibility in this popular ornamental shrub. Pollen germination and pollen tube growth in styles were examined using fluorescence microscopy. Stigma receptivity was examined in cross-pollinations made from 1 day before anthesis to 8 days after anthesis. Maximum stigma receptivity for the two cultivars examined occurred from anthesis to 4 days after anthesis. Viability of pollen from sterile flowers was evaluated through pollen staining and observations of pollen tube growth. No significant difference in percent stainable pollen between fertile and sterile flowers was observed in any of the six taxa examined. Pollen germination and pollen tube growth were studied in cross-pollinations made using pollen from fertile and sterile flowers of two cultivars. For both cultivars, pollen tubes from fertile and sterile flowers grew to the same length and had entered ovules by 72 hours after pollination. Self-incompatibility was evaluated by comparing pollen germination and pollen tube growth in cross- and self-pollinations. In the five taxa examined, self pollen tubes were significantly shorter than cross pollen tubes in flowers that were examined 72 hours after pollination. This finding indicates the presence of a gametophytic self-incompatibility system in H. macrophylla.

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Hai-nan Liu, Jian-rong Feng, Xiao-fang Liu, Wen-hui Li, Wen-juan Lv and Ming Luo

°25′E). The fruit of ‘Xiaobiaxing’ is oval, light yellow, and glabrous. ‘Xiaobaixing’ exhibits typical S - RNase -based gametophytic self-incompatibility (GSI) controlled by the S-allele. The S-allele contains at least one pollen determinant and one