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Rayane Barcelos Bisi, Rafael Pio, Daniela da Hora Farias, Guilherme Locatelli, Caio Morais de Alcântara Barbosa, and Welison Andrade Pereira

increased. In this sense, breeding programs for cultivars adapted to regions with high temperatures must be intensified. This study aimed to characterize S-alleles in hybrid pear cultivars ( P. communis × P. pyrifolia ) adapted to subtropical climates and

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Shogo Matsumoto, Kentaro Kitahara, Sadao Komori, and Junichi Soejima

S-allele genotypes of nine apple (Malus ×domestica Borkh.) cultivars were identified using S-allele–specific polymerase chain reaction (PCR)–restriction fragmentlength polymorphism (RFLP) analysis. A new S-allele, Sg, was proposed to be present in `American Summer Pearmain', `Indo', `Kitanosachi', and `Meku 10'. This allele is very similar to Sf at the nucleotide sequence (92%) and deduced amino acid sequence (94%) levels.

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Javier Sanzol and Timothy P. Robbins

-genotyping are based on polymorphisms of the S-RNase gene. Initial attempts to identify S-RNases associated with S-alleles were based on the existing knowledge about the S-genotype constitutions of cultivars deduced from the assessment of their S-phenotypes after

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Sadao Komori, J. Soejima, Y. Ito, and H. Bessho

Cross-incompatible combinations among the main cultivars in apple are rarely reported in Japan. Recently, however, most new Japanese cultivars are progenies of `Ralls Janet', `Delicious', `Golden Delicious', `Jonathan', and `Indo'. Cross incompatibility in apple, therefore, will become a serious problem in the near future. Since the analysis of the S-allele genotypes were not performed, especially in Japanese apple cultivars, the fruit set percentage were examined in several combinations of `Hatsuaki' (`Jonathan' × `Golden Delicious') and `Iwakami' (`Fuji' × `Jonathan') progenies using back crossings. As a result, we found that `Golden Delicious' and `Jonathan' had no common S-allele, while `Fuji' and `Jonathan' had one common S-allele. These facts were used as basics for the S-allele genotype analysis, and fruit set percentage and seed number per fruit were investigated on a large scale. The cross seedlings between `Delicious' and `Jonathan', `Ralls Janet' and `Jonathan', `Iwakami' and `Golden Delicious', `Golden Delicious' and `Delicious', `Hatsuaki' and `Fuji', `Hatsuaki' and `Delicious', `Hatsuaki' and `Jonathan', and `Hatsuaki' and `Golden Delicious' were analyzed. In addition, incompatibility between `Redgold' and `Kinesei' (`Golden Delicious' × `Ralls Janet'), `Senshu' (`Toukou' × `Fuji') and `Iwakami', and progenies of `Northern Spy' also were analyzed. As a result, we have found the existence of six alleles and 15 genotypes, and we have established S-allele standard cultivars and strains as follows: (Sa, Sb) = `Golden Delicious'; (Sa, Sc) (4)-354, (4)-425; (Sa, Sd) = `Toukou'; (Sa, Se) = `Redgold', `Kinsei'; (Sa, Sf) = `Narihokou', (4)-4195; (Sb, Sc) = `Hatsuaki', `Kuifua', `Sekaiichi'; (Sb, Sd) = `Tsugaru', (4)-300; (Sb, Se) = (4)-150, (4)-743; (Sb, Sf) = `Northern Spy', M.9, `Umezawa'; (Sc, Sd) = `Jonathan', `Himekami'; (Sc, Sf) = `Fuji', `Shinkou'; (Sd, Se) =; (Sd, Sf) = `Senshu', `Iwakami'; (Se, Sf) = `Ralls Janet'.

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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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Kristi K. Barckley, Sandra L. Uratsu, Thomas M. Gradziel, and Abhaya M. Dandekar

The California almond industry is the largest supplier of almonds [Prunus dulcis (Miller) D.A. Webb] in the United States and throughout the world. Self-incompatibility is a major issue in almond production as it greatly affects nut set. In this study, we determined full-length sequences for alleles Sa - Si, determined the genotypes of 44 California cultivars, and assigned the cultivars to cross-incompatibility groups (CIGs). Newly identified S-alleles led to an increase in the number of CIGs. A pairwise distance tree was constructed using the aligned amino acid sequences showing their similarity. Four pairs of alleles (Sc and Se, Sg and Sh, Sd and Sj, and Sb and Sf) showed high sequence similarity. Because of its simplicity, reproducibility, and ease of analysis, PCR is the preferred method for genotyping S-alleles.

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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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Wim Broothaerts, Ilse Van Nerum, and Johan Keulemans

Apple cultivars display a self-incompatibility system that restricts self-fertilization and fertilization between cultivars bearing identical S-alleles. There has been considerable progress in identification of S-alleles in apple in recent years and methods are now available for the accurate S-genotyping of cultivars. Following a recently revised numerical identification system for apple S-alleles, we present the first extensive compilation of apple cultivars with their S-genotypes. This list contains data from our own investigations using S-allele-specific PCR methodology, including a number of new data, as well as published data from various other sources. Eighteen different S-alleles are discriminated, which allowed the determination of the S-genotypes for 150 diploid or triploid European, American, and Japanese cultivars. Many of these cultivars are cultivated worldwide for their fruit. Also included are a number of old, obsolete cultivars and a few nondomestic genotypes. We observed a wide variation in the frequency of S-alleles in the apple germplasm. Three S-alleles (S2, S3, and S9) are very common in the cultivars evaluated, presumably as a result of the widespread use of the same breeding parents, and seven alleles are very rare (S4, S6, S8, S16, S22, S23, S26).

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Kentaro Kitahara, Junichi Soejima, Hiromitsu Komatsu, Hirokazu Fukui, and Shogo Matsumoto

The S-locus genes in the pistil (S-RNases) were cloned from the apple (Malus ×domestica Borkh.) cultivar Akane (S-genotype SdSh from pollination analysis). The Sd- and Sh-RNase corresponded to S7- and S24-RNase, which have been cloned from `Idared' and `Braeburn', respectively. Sh-RNase was very similar to Sf- and Sg-RNases at the deduced amino acid-sequence levels (93%). We developed an S-allele specific polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis method for distinguishing the Sh from Sf and Sg, and the Sh-alleles of `Akane', `Touhoku 2', `Vista Bella', and `Worcester Pearmain' were identified. We also identified the S-allele genotypes of 16 apple cultivars.

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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.