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

and reverse transcriptase–polymerase chain reaction analysis of four SFBs. Genomic DNA blot analysis showed that SFB a , SFB b , SFB c , and SFB e are cosegregated with the S a -, S b -, S c -, and S e -haplotypes of japanese