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The efficacy of ploidy breeding using unreduced pollen in japanese persimmon (Diospyros kaki Thunb.) is not high because of the low frequency of unreduced pollen in most cultivars. This study was conducted in 2002 and 2003 to determine if the exposure to a low temperature before flowering could enhance the unreduced pollen formation in five cultivars of japanese persimmon including two cultivars that barely produce unreduced pollen under the field condition. The results showed that low-temperature treatment (4 °C for 48 hours) increased the occurrence of unreduced pollen at 15 to 17 and 17 to 18 days after the end of the low-temperature treatment in 2002 and 2003, respectively, in all five cultivars tested. Naturally occurring temperatures below 5 °C in the field also appeared to enhance the unreduced pollen formation in the cultivars that naturally produce unreduced pollen in the field.

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Distribution of pollen diameter of Japanese persimmon cv. Zenjimaru (2n = 6x, × = 15) was determined using pollen grains hydrated with CPW solution supplemented with 0.9 M mannitol. Mean diameter of giant pollen grains (65 μm) was 1.3 times longer than that of normal pollen grains (50 μm). The occurrence of giant pollen was estimated to be about 5% of the pollen population. The hydrated giant pollen grains could be sorted out from normal pollen grains by filtering through a layer of nylon mesh (62 μm). Flow cytometric analysis of nuclear DNA content confirmed that giant pollen was unreduced 2n pollen. 2n giant pollen grains were pollinated to cn. Jiro (2n = 6x) callie and plantlets could be obtained from immature embyros excised from seeds 70 days after pollination.

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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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Multi-color genomic in situ hybridization (MCGISH) was performed for mitotic cells of the somatic hybrids of Diospyros kaki (2n = 6x = 90) and D. glandulosa (2n = 2x = 30). Total DNA of D. kaki and D. glandulosa were isolated and labeled with biotin-16-UTP and digoxigenin (DIG)-11-UTP, respectively. The labeled DNAs were used as probes to differentiate parental chromosomes. The biotin-labeled probe was detected with avidin-rhodamine, and the DIG-labeled probe was detected with anti-DIG-FITC (fluorescein isothiocyanate). Ninety chromosomes from D. kaki that showed reddish-orange and 30 chromosomes from D. glandulosa that showed greenish-yellow were observed under a fluorescence microscope. Some chromosomes showed cross-hybridization with both probes at their terminal or other chromosome regions. These results indicated that MCGISH could be used to analyze genomes of Diospyros species whose chromosomes are small and numerous.

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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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Research is described on the development of an automated inspection system which uses digital images and artificial intelligence techniques. Procedures have been developed for evaluating size, shape, and color of apples, potatoes, and mushrooms. Current emphasis is being placed on developing algorithms for detection of surface defects. A major effort will also be expended toward the development of an overall “quality” score for automated inspection of fruit and vegetables. The automated results are compared with those obtained using conventional manual inspection methods. Apples, potatoes, and mushrooms are the primary crops being inspected although the algorithms and techniques are applicable to many different fruits and vegetables. Color and monochromatic image processing components in “MS-DOS” and “Macintosh” computers are being used in this study.

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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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S4′ is a pollen-part mutant in sweet cherry (Prunus avium L.) that is extensively used to develop self-compatible cultivars. The S4′ -haplotype is known to have a functional stylar component and a nonfunctional pollen component. The pollen component in sweet cherry necessary for the specificity of the pollen reaction is believed to be an S-haplotype specific F-box protein gene, called SFB. This study describes two molecular markers that distinguish between SFB4 and SFB4′ by taking advantage of a four base pair deletion in the mutant allele. The resulting polymerase chain reaction (PCR) products can either be separated directly on a polyacrylamide gel or they can be subjected to restriction enzyme digestion and the different sized products can be visualized on an agarose gel. The latter technique utilizes restriction sites created in the PCR products from the SFB4′ allele, but not the SFB4 allele. Because the primer sets created differential restriction sites, these primer sets were termed dCAPS (derived cleaved amplified polymorphism sequence) markers. These molecular assays can be used to verify self-compatibility conferred by the S4′ -haplotype.

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Stylar proteins of four Prunus species, P. avium, P. dulcis, P. mume, and P. salicina, were surveyed by 2D-PAGE combined with immunoblot and N-terminal amino acid sequence analyses to identify S-proteins associated with gametophytic SI in the Prunus. All four S-allelic products tested for P. dulcis could be identified in the highly basic zone of the gel. These S-proteins had Mr of about 28–30 kDa and reacted with the anti-S4 -serum prepared from Japanese pear (Pyrus serotina). Two of six S-allelic products tested for P. avium could be also identified in the 2D-PAGE profiles, with roughly the same pI and Mr as those of S-proteins of P. dulcis. Putative S-proteins for P. mume and P. salicina were found in the same area of 2D-PAGE as the area where S-proteins of P. avium and P. dulcis were located. N-terminal amino acid sequence analysis of these proteins revealed that they were similar to S-RNases reported previously.

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