Use of the multi-allelic self-incompatibility gene in apple to assess homozygocity in shoots obtained through haploid induction

 To obtain homozygous genotypes of apple, we have induced haploid development of either the female or the male gametes by parthenogenesis in situ and anther culture, respectively. Of the shoots obtained, which were mainly of a non-haploid nature, some could be derived from fertilised egg cells or from sporophytic anther tissue. In order to select the shoots having a true haploid origin, and thus homozygotes, we decided to use the single multi-allelic self-incompatibility gene as a molecular marker to discriminate homozygous from heterozygous individuals. The rationale behind this approach was that diploid apple cultivars contain 2 different alleles of the S-gene and therefore the haploid induced shoots obtained from them should have only one of the alleles of the single parent. The parental cultivars used were ‘Idared’ (parthenogenesis in situ) and ‘Braeburn’ (androgenesis), and their S-genotypes were known, except for 1 of the ‘Braeburn’S-alleles. To stimulate parthenogenetic development ‘Idared’ styles were pollinated with irradiated ‘Baskatong’ pollen, the S-alleles of the latter (2n) cultivar were also unknown. The cloning and sequence analysis of these 3 unidentified S-alleles, 1 from ‘Braeburn’ and 2 from ‘Baskatong’ is described, and we show that they correspond to the S ₂₄ -, S ₂₆ - and S ₂₇ -alleles. We have optimised a method for analysis of the S-alleles of ‘Idared/Baskatong’- or ‘Braeburn’-derived in vitro plant tissues and have shown that this approach can be applied for the screening of the in vitro shoots for their haploid origin. Received: 18 August 1997 / Accepted: 10 September 1997     

Theoretical study of crown ethers with incorporated azobenzene moiety

A series of crown ethers containing the azobenzene moiety incorporated into crowns of various sizes [Cr(O₆)_, Cr(O₇) and Cr(O₈)] and their corresponding alkali metal cation (Li⁺, Na⁺, K⁺, Rb⁺) complexes have been studied theoretically. The density functional theory (DFT) method was employed to elucidate the stereochemical structural natures and thermodynamic properties of all of the target molecules at the B3LYP/6-31 G(d) and LANL2DZ level for the cation Rb⁺. The fully optimized geometries had real frequencies, thus indicating their minimum-energy status. In addition, the bond lengths between the metal cation and oxygen atoms, atomic torsion angles and thermodynamic energies for complexes were studied. Natural bond orbital (NBO) analysis was used to explore the origin of the internal forces and the intermolecular interactions for the metal complexes. The calculated results show that the most significant interaction is that between the lone pair electrons of electron-donating oxygens in the cis-forms of azobenzene crown ethers (cis-ACEs) and the LP* (1-center valence antibond lone pair) orbitals of the alkali-metal cations (Li⁺, Na⁺, K⁺ and Rb⁺). The electronic spectra for the cis-ACEs [cis-Cr(O₆), cis-Cr(O₇) and cis-Cr(O₈)] are obtained by the time-dependent density functional theory (TDDFT) at the B3LYP/6-31 G(d) level. The spectra of the cis-isomers show broad π → π* (S₀ → S₂) absorption bands at 310–340 nm but weaker n → π* (S₀ → S₁) bands at 480–490 nm. The calculated results are in good agreement with the experimental results.     

Molecular characterization of newS-RNases (‘S31’ and ‘S32’) in apple (Malus ×domestica Borkh.)’) in apple (Malus ×domestica Borkh.)

Apple exhibits gametophytic self-incompatibility (GSI) that is controlled by the multiallelic S-locus. This S-locus encodes polymorphicS ribonuclease (S-RNase) for the pistil-part 5 determinant. Information aboutS-genotypes is important when selecting pollen donors for fruit production and breeding of new cultivars. We determined the 5-genotypes of ‘Charden’ (S₂S₃S₄), ‘Winesap’ (S₁S₂₈), ‘York Imperial’ (S₂S₃₁), ‘Stark Earliblaze’ (S₁S₂₈), and ‘Burgundy’ (S₂₀S₃₂), byS-RNase sequencing and S-allele-specific PCR analysis. Two newS-RNases, S₃₁ and S₃₂, were also identified from ‘York Imperial’ and ‘Burgundy’, respectively. These newS-alleles contained the conserved eight cysteine residues and two histidine residues essential for RNase activity. Whereas S₃₁ showed high similarity to S₂₀ (94%), S₃₂ exhibited 58% (to S₂₄) to 76% (to S₂₅) similarity in the exon regions. We designed newS-allele-specific primers for amplifying S₃₁- and S₃₂-RNasc-specific fragments; these can serve as specific gene markers. We also rearranged the apple S-allele numbers containing those newS-RNases. They should be useful, along with anS-RNase-based PCR system, in determining S-genotypes and analyzing new alleles from apple cultivars.     

Characterization of three new S-alleles and development of an S-allele-specific PCR system for rapidly identifying the S-genotype in apple cultivars

Apple (Malus domestica Borkh), a member of the Rosaceae, shows gametophytic self-incompatibility (GSI) controlled by polymorphic S-alleles. Identifying the S-genotypes of apple cultivars can be applied on correct assignment of apple cultivars to cross-compatibility groups, which is important for the efficient production of apple fruit. This study characterized three new S-alleles (designated S ₄₄ , S ₄₅ , and S ₄₆ ) in apple and developed an efficient analysis method that can be used to characterize S-genotypes by utilizing allele-specific polymerase chain reaction rapidly. Nineteen allele-specific primers were selectively designed to identify different alleles. Using this method, S-genotypes of 157 apple cultivars were identified.     

QTL analysis for aphid resistance and growth traits in apple

The rosy apple aphid (Dysaphis plantaginea), the leaf-curling aphid (Dysaphis cf. devecta) and the green apple aphid (Aphis pomi) are widespread pest insects that reduce growth of leaves, fruits and shoots in apple (Malus × domestica). Aphid control in apple orchards is generally achieved by insecticides, but alternative management options like growing resistant cultivars are needed for a more sustainable integrated pest management (IPM). A linkage map available for a segregating F₁-cross of the apple cultivars ‘Fiesta’ and ‘Discovery’ was used to investigate the genetic basis of resistance to aphids. Aphid infestation and plant growth characteristics were repeatedly assessed for the same 160 apple genotypes in three different environments and 2 consecutive years. We identified amplified fragment length polymorphism (AFLP) markers linked to quantitative trait loci (QTLs) for resistance to D. plantaginea (‘Fiesta’ linkage group 17, locus 57.7, marker E33M35–0269; heritability: 28.3%), and to D. cf. devecta (‘Fiesta’ linkage group 7, locus 4.5, marker E32M39–0195; heritability: 50.2%). Interactions between aphid species, differences in climatic conditions and the spatial distribution of aphid infestation were identified as possible factors impeding the detection of QTLs. A pedigree analysis of simple sequence repeat (SSR) marker alleles closely associated with the QTL markers revealed the presence of the alleles in other apple cultivars with reported aphid resistance (‘Wagener’, ‘Cox’s Orange Pippin’), highlighting the genetic basis and also the potential for gene pyramiding of aphid resistance in apple. Finally, significant QTLs for shoot length and stem diameter were identified, while there was no relationship between aphid resistance and plant trait QTLs. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Determination of partial genomic sequences and development of a CAPS system of the S-RNase gene for the identification of 22 S haplotypes of apple (Malus × domestica Borkh.)

Information about self-incompatibility (S) genotypes of apple cultivars is important for the selection of pollen donors for fruit production and breeding. Although S genotyping systems using S haplotype-specific PCR of S-RNase, the pistil S gene, are useful, they are sometimes associated with false-positive/negative problems and are unable to identify new S haplotypes. The CAPS (cleaved amplified polymorphic sequences) system is expected to overcome these problems, however, the genomic sequences needed to establish this system are not available for many S-RNases. Here, we determined partial genomic sequences of eight S-RNases, and used the information to design new primer and to select 17 restriction enzymes for the discrimination of 22 S-RNases by CAPS. Using the system, the S genotypes of three cultivars were determined. The genomic sequence-based CAPS system would be useful for S genotyping and analyzing new S haplotypes of apple.     

Spatial and Temporal Genetic Variation of Green Mussel, Perna viridis in the Gulf of Thailand and Implication for Aquaculture

The culture of green mussel (Perna viridis) in the Gulf of Thailand depends on natural spat which are believed to come from spawning grounds adjacent to major river mouths. In the present paper, genetic diversity of spatial and temporal populations of green mussel in the Gulf of Thailand was investigated using five microsatellite loci. The results showed moderate genetic variation of all 11 populations (averaged number of alleles per locus, A = 10.4–12.2; effective number of alleles per locus, A _e = 5.36–6.59; mean allelic richness, A _r = 10.23–12.06; observed heterozygosity, H _o = 0.52–0.63, and expected heterozygosity, H _e = 0.66–0.73) without significant differences among populations. No sign of bottleneck or genetic disequilibrium was observed. Genetic differentiation among spatial populations was low (F _ST = 0.0046, CI_0.95 = 0.0020–0.0083 for the samples collected in January, 2007, and F _ST = 0.0088, CI_0.95 = 0.0010–0.0162 for the samples collected in July, 2007) while temporal variation was significant as revealed by the analysis of molecular variance. Multidimensional scaling separated temporal population groups with minor exception. The assignment test revealed that most of the recruits were from other populations.     

The effects of Quaternary glaciations in Patagonia as evidenced by chloroplast DNA phylogeography of Southern beech Nothofagus obliqua

Climatic oscillations during the Quaternary strongly affected the distribution of warm-temperate tree species, which experienced local restrictions into ice-free areas and posterior expansions. To evaluate the impact of these range movements on the genetic structure of populations, we performed a phylogeographical analysis of the species Nothofagus obliqua with chloroplast DNA markers. A total of 27 populations covering the whole natural distribution range were analyzed using polymerase chain reaction-restriction fragment length polymorphism. Diversity (h _T, h _S), allelic richness (r _g), and differentiation among populations for unordered (G _ST) and ordered alleles (N _ST) were calculated. The relationships among haplotypes were evaluated by the construction of a minimum spanning network. The spatial distribution of the genetic variation was analyzed through a Mantel test and with a nested analysis of molecular variance to differentiate between geographic regions. The screening of 11 non-coding regions allowed the identification of 14 haplotypes. A high genetic differentiation was detected (N _ST = 0.875 and G _ST = 0.824) with the existence of phylogeographic structure (p < 0.05). The distribution of the genetic variation was partially explained by the topography of the region when the populations were divided longitudinally into Coastal Mountains, Central Valley, and Andes Mountains (ϕ _RT = 0.093, p = 0.001). In agreement with pollen records, our results support the hypothesis of Coastal refuges since the region harbors high diversity together with older and private haplotypes. Long-lasting persistence of some Coastal populations without contribution to re-colonization is proposed. Additional refuges are also postulated along the Andes and Longitudinal Valley. Survival in multiple glacial refuges is discussed together with the possible migratory routes.     

Polymorphism of <Emphasis Type="Italic">SFBB</Emphasis><Superscript>−γ</Superscript> and its use for <Emphasis Type="Italic">S</Emphasis> genotyping in Japanese pear (<Emphasis Type="Italic">Pyrus pyrifolia</Emphasis>)

Japanese pear (Pyrus pyrifolia) exhibits the S-RNase-based gametophytic self-incompatibility where the pollen-part determinant, pollen S, had long remained elusive. Recent identification of S locus F-box brothers (SFBB) in Japanese pear and apple suggested that the multiple F-box genes are the pollen S candidates as they exhibited pollen specific expression, S haplotype-specific polymorphisms and linkage to the S locus. In Japanese pear, three SFBBs were identified from a single S haplotype, and they were more homologous to other haplotype genes of the same group (i.e., α-, β- and γ-groups). In this study, we isolated new seven PpSFBB ^−γ genes from different S genotypes of Japanese pear. These genes showed S haplotype-specific polymorphisms, however, sequence similarities among them were very high. Based on the sequence polymorphisms of the PpSFBB ^−γ genes, we developed a CAPS/dCAPS system for S genotyping of the Japanese pear cultivars. This new S genotyping system was found to not only be able to discriminate the S ¹–S ⁹, but also be suitable for identification of the mutant S ^4sm haplotype for the breeding of self-compatible cultivars, and detection of new S haplotypes such as S ^k.     

Single-seed PCR-RFLP analysis for the identification of S haplotypes in commercial F1 hybrid cultivars of broccoli and cabbage

 Several simple methods of DNA preparation from plant tissues were evaluated for PCR-RFLP analyses of SLG and SRK alleles, which can be used for the identification of S haplotypes of breeding lines in broccoli and cabbage (Brassica oleracea L.) and in purity tests of F₁ hybrid seeds. On the five methods tested, the NaI method was found to be the most suitable for the amplification of the SLG and SRK alleles. This method enables the use of a single seed as testing material. Using this method, we identified S haplotypes of 31 broccoli and 31 cabbage cultivars. Ninety-four percent of the cultivars of broccoli and 97% of those of cabbage were-single cross F₁ hybrids. Nine and 15 S haplotypes were found in broccoli and cabbage, respectively. The small number of S haplotypes in broccoli suggests the importance of incorporating new S haplotypes in the breeding program. Received: 18 February 1999 / Revision received: 4 May 1999 / Accepted: 14 May 1999     

An Assessment of the Phylogenetic Relationship Among Sugarcane and Related Taxa Based on the Nucleotide Sequence of 5S rRNA Intergenic Spacers

5S rRNA intergenic spacers were amplified from two elite sugarcane (Saccharumhybrids) cultivars and their related taxa by polymerase chain reaction (PCR) with 5S rDNA consensus primers. Resulting PCR products were uniform in length from each accession but exhibited some degree of length variation among the sugarcane accessions and related taxa. These PCR products did not always cross hybridize in Southern blot hybridization experiments. These PCR products were cloned into a commercial plasmid vector PCR™ 2.1 and sequenced. Direct sequencing of cloned PCR products revealed spacer length of 231–237 bp for S. officinarum, 233–237 for sugarcane cultivars, 228–238 bp for S. spontaneum, 239–252 bp for S. giganteum, 385–410 bp for Erianthusspp., 226–230 bp for Miscanthus sinensisZebra, 206–207 bp for M. sinensisIMP 3057, 207–209 bp for Sorghum bicolor, and 247–249 bp for Zea mays. Nucleotide sequence polymorphism were found at both the segment and single nucleotide level. A consensus sequence for each taxon was obtained by Align X. Multiple sequences were aligned and phylogenetic trees constructed using Align X, CLUSTAL and DNAMAN programs. In general, accessions of the following taxa tended to group together to form distinct clusters: S. giganteum, Erianthusspp., M. sinensis, S. bicolor, and Z. mays. However, the two S. officinarumclones and two sugarcane cultivars did not form distinct clusters but interrelated within the S. spontaneumcluster. The disclosure of these 5S rRNA intergenic spacer sequences will facilitate marker-assisted breeding in sugarcane. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification of random amplified polymorphic DNA (RAPD) markers for self-incompatibility alleles in Corylus avellana L.

 Random amplified polymorphic DNA (RAPD) markers were identified for self-incompatibility (SI) alleles that will allow marker-assisted selection of desired S-alleles in hazelnut (Corylus avellana L.). DNA was extracted from young leaves collected from field-planted parents and 26 progeny of the cross OSU 23.017 (S₁S₁₂)×VR6-28 (S₂S₂₆) (OSU23×VR6). Screening of 10-base oligonucleotide RAPD primers was performed using bulked segregant analysis. DNA samples from 6 trees each were pooled into four ‘bulks’, one for each of the following: S₁ S₂, S₁ S₂₆ ^, S₂ S₁₂, and S₁₂ S₂₆. ‘Super bulks’ of 12 trees each for S₁, S₂, S₁₂, and S₂₆ were then created for each allele by combining the appropriate bulks. The DNA from these four super bulks and from the parents was used as a template in the PCR assays. A total of 250 primers were screened, and one RAPD marker each was identified for alleles S₂ (OPI07₇₅₀) and S₁ (OPJ14₁₇₀₀). OPJ14₁₇₀₀ was identified in 13 of 14 S₁ individuals of the cross OSU23×VR6 used in bulking and yielded a false positive in 1 non-S₁ individual. This same marker was not effective outside the original cross, identifying 4 of 5 S₁ progeny in another cross, ‘Willamette’×VR6-28 (‘Will’×VR6), but yielded false positives in 4 of 9 non-S₁ individuals from the cross ‘Casina’×VR6-28 (‘Cas’×VR6). OPI07₇₅₀ served as an excellent marker for the S₂ allele and was linked closely to this allele, identifying 12 of 13 S₂ individuals in the OSU23×VR6 population with no false positives. OPI07₇₅₀ was found in 4 of 4 S₂ individuals from ‘Will’×VR and 7 of 7 S₂ individuals of ‘Cas’×VR6 with no false positives, as well as 10 of 10 S₂ individuals of the cross OSU 296.082 (S₁S₈)×VR8-32 (S₂S₂₆), with only 1 false positive individual out of 21 progeny. OPI07₇₅₀ was also present in 5 of 5 cultivars carrying the S₂ allele, with no false-positive bands in non-S₂ cultivars, and correctly identified all but 2 S₂ individuals in 57 additional selections in the breeding program. In the OSU23×VR6 population, the recombination rate between the marker OPJ14₁₇₀₀ and the S₁ allele was 7.6% and between the OPI07₇₅₀ marker and the S₂ allele was 3.8%. RAPD marker bands were excised from gels, cloned, and sequenced to enable the production of longer primers (18 or 24 bp) that were used to obtain sequence characterized amplified regions (SCARs). Both the S₁ and S₂ markers were successfully cloned and 18 bp primers yielded the sole OPJ14₁₇₀₀ product, while 24-bp primers yielded OPI07₇₅₀ as well as an additional smaller product (700 bp) that was not polymorphic but was present in all of the S-genotypes examined. Received: 10 January 1998 / Accepted: 26 January 1998     

Genomic characterization of self-incompatibility ribonucleases (S-RNases) in European pear cultivars and development of PCR detection for 20 alleles

Sexual self-incompatibility in European pear (Pyrus communis L.) is controlled by a single locus (S-locus) encoding a polymorphic stylar ribonuclease (S-RNase) that is responsible for the female function in pollen–pistil recognition. In this study, genomic DNA sequences corresponding to five new S-RNase alleles (named S ₂₀ , S ₂₁ , S ₂₂ , S ₂₃ , and S ₂₄ ) and to S _m were characterized in European pear cultivars. Re-sequencing S _q from ‘General Le Clerc’ showed this S-RNase to encode the same protein as S ₁₂ . Based on these findings, a polymerase chain reaction (PCR)-based method was developed for the molecular typing of cultivars bearing 20 S-RNases (S ₁ –S ₁₄ , S _m , and S ₂₀ –S ₂₄ ) using consensus and allele-specific primers. Genomic PCR with consensus primers amplified product sizes characteristic of the S-RNases S ₁ , S ₂ , S ₄ , S ₁₀ , S ₁₃ , and S ₂₀ . However, the allele groups S ₃ /S ₁₂ , S ₆ /S ₈ /S ₁₁ /S ₂₂ and S ₅ /S ₇ /S ₉ /S ₁₄ /S _m /S ₂₁ /S ₂₃ /S ₂₄ amplified PCR products of similar size. To discriminate between alleles within these groups, primers to specifically amplify each S-RNase were developed. Application of this approach in 19 cultivars with published S-alleles allowed re-evaluation of one of the alleles of ‘Passe Crassane,’ ‘Conference,’ and ‘Condo.’ Finally, this method was used to assign S-genotypes to 37 cultivars. Test crosses confirmed molecular results. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

An AFLP-based survey of genetic diversity and relationships of major farmed cultivars and geographically isolated wild populations of <Emphasis Type="Italic">Saccharina japonica</Emphasis> (Phaeophyta) along the northwest coasts of the Pacific

The genetic diversity and relationships of six representative cultivars and six geographically isolated wild populations of Saccharina japonica along the northwest coasts of the Pacific Ocean were investigated using AFLP markers. A total of 547 bands were generated across all samples by ten primer combinations. At the cultivar or population level, the percentage of polymorphic loci (P), gene diversity (H), and Shannon’s information index (I) was highest in Dalian population (P 59.05%; H 0.2057; I 0.3062) and lowest in Lianjiang cultivar (P 9.87%; H 0.0331; I 0.0497). At the species level, P, H, and I were 85.01%, 0.1948, and 0.3096, respectively. Unique bands were detected in all the six wild populations, with Dalian being the most. In comparison, only Yanza cultivar possessed one unique band. The G _ST value was 0.6226 and the gene flow (N _m ) was 0.1515, indicating strong genetic differentiation among cultivars and populations. Two UMPGA dendrograms were constructed based on the Dice similarity coefficients among individuals and on genetic distances among cultivars and populations, which generally revealed three major clades corresponding to three countries. Analysis of molecular variance revealed that a larger proportion (60.21%) of the total genetic variation was attributable to differences among cultivars and populations. The Mantel test suggested that genetic differentiation was positively correlated with geographic distance (r = 0.7962, P = 0.011) in the six wild populations, agreeing with the isolation by distance model. On the whole, low to moderate genetic diversity within cultivars and populations (except Dalian population) and high genetic differentiation among cultivars and populations were detected.     

The reaction mechanism of nitrosothiols with copper(I)

Copper and other transition metal ions and their complexes are catalysts for the decomposition of nitrosothiols. In this way they catalyze the biological functions of nitrosothiols. The kinetics and mechanism of the reaction of two nitrosothiols, S-nitrosothiolactic acid and S-nitrosoglutathione (GSNO), with copper(I) are reported. The kinetics of the reaction of Cu(MeCN) _n ⁺ (n=0–3) with the nitrosothiols were studied. The results indicate that Cu⁺ _aq is the active species in the GSNO system, with k(Cu⁺ _aq+GSNO)=(9.4 ±2.0)×10⁷ dm³ mol⁻¹ s⁻¹. The results also indicate that the Cu(MeCN) _n ⁺ (n=0–3) complexes react with S-nitrosothiolactic acid. Transient species are formed in these processes. The results suggest that these species contain copper(I) and thiol. The results shed light on the catalytic role of copper complexes in the decomposition of S-nitrosothiols. Received 10 April 1999 / Accepted 17 December 1999     

Genetic diversity assessment of sub-samples of cacao, Theobroma cacao L. collections in West Africa using simple sequence repeats marker

Knowledge of genebank and on-farm genetic diversity, particularly in an introduced crop species, is crucial to the management and utilization of the genetic resources available. Microsatellite markers were used to determine genetic diversity in 574 accessions of cacao, Theobroma cacao L., representing eight groups covering parental populations in West Africa, genebank, and farmers’ populations in Nigeria. From the 12 microsatellite markers used, a total of 144 alleles were detected with a mean allelic richness of 4.39 alleles/locus. The largest genetic diversity was found in the Upper Amazon parent population (H _nb  = 0.730), followed by the 1944 Posnette’s Introduction (H _nb  = 0.704), and was lowest in the Local parent population (H _nb  = 0.471). Gene diversity was appreciably high in the farmers’ populations (H _nb  = 0.563–0.624); however, the effective number of alleles was lower than that found in the genebank’s Posnette’s population. Fixation index estimates indicated deficiency of heterozygotes in the Upper Amazon and the Local parent populations (F _is  = 0.209 and 0.160, respectively), and excess of heterozygotes in the Trinitario parent population (F _is  = −0.341). The presence of inbreeding in the Local parent populations and substructure (Wahlund effect) in the Upper Amazon were suggested for the deficiency of heterozygotes observed. Non-significant genetic differentiation observed between the genebank’s and farmers’ populations indicated significant impact of national breeding programs on varieties grown in farmers’ plantations. From this study, we showed that appreciable genetic diversity was present in on-farm and field genebank collections of cacao that can be exploited for crop improvement in West Africa. Suggestions for future conservation of on-farm genetic diversity and local landraces are further discussed.     

Analysis of genetic diversity among persimmon cultivars using microsatellite markers

In the Mediterranean area, the production of persimmon (Diospyros kaki Thumb) [2n = 6x = 90] has increased recently as an alternative to the major fruit crops. In Spain, production relies almost exclusively on the cultivar “Rojo Brillante” which accounts for 83% of the crop. A crop based on a monovarietal culture implies several commercial risks that can compromise the future of the crop. Although the species was introduced in Europe very recently, it is well adapted to the climate of southern Europe. However, the recent introduction from Japan, the mistakes on the identity of varieties in the collections due to a bad translation of variety names from Japanese, and the lack of genetic characterization of many varieties have caused difficulties for effective management of the available genetic resources. The present paper was aimed at exploring the genetic diversity among different persimmon cultivars, including those collected in the European survey as well as Japanese cultivars. Seventy-one persimmon cultivars coming from two European collections that included accessions from Japan, Italy, and Spain were analyzed using 19 polymorphic microsatellite markers. A total of 206 alleles were obtained, with a mean value of 10.8 alleles per locus. A neighbor joining dendrogram and a principal coordinate analysis arranged the cultivars according to their genetic relationships. Analysis of molecular variance revealed significant genetic variability between and within groups, 73.3% and 85.2% for astringent-type and country origin, respectively. The simple sequence repeat markers classified the persimmon cultivars according to their genetic relationship.     

Electrochemistry of the CuA domain of Thermus thermophilus cytochrome ba 3

 The electrochemistry of a water-soluble fragment from the Cu_A domain of Thermus thermophilus cytochrome ba ₃ has been investigated. At 25  °C, Cu_A exhibits a reversible reduction at a pyridine-4-aldehydesemicarbazone-modified gold electrode (0.1 M Tris, pH 8) with E° = 0.24 V vs NHE. Thermodynamic parameters for the [Cu(Cys)₂Cu]^+/0 electrode reaction were determined by variable-temperature electrochemistry (ΔS°_rc = –5.4(12) eu, ΔS° = –21.0(12) eu, ΔH° = –11.9(4) kcal/mol;ΔG° = –5.6 (11) kcal/mol). The relatively small reaction entropy is consistent with a low reorganization energy for [Cu(Cys)₂Cu]^+/0 electron transfer. An irreversible oxidation of [Cu(Cys)₂Cu]⁺ at 1 V vs NHE confirms that the Cu^II:Cu^II state of Cu_A is significantly destabilized relative to the Cu^II state of analogous blue-copper proteins. Received: 3 June 1996 / Accepted: 26 August 1996     

Determination of self-locompatibility genotypes of Korean apple cultivars based on S-RNase PCR

To prevent self-fertilization, apple has a gametophytic self-incompatibility mechanism, part of a widespread intraspecific system, that is controlled by a multi-allelic locus. This attribute has been exploited in breeding programs for new cultivars. Likewise, many apple orchards depend on artificial pollination. Therefore, molecular analysis and early identification of the self-incompatibility (S) genotype could greatly improve breeding schemes and pollen donors selection. Here, we PCR-amplified the S-RNase PCR fragments from a total of 14 cultivars and parents, using new primers (ASPF3+ASPR3) common to 23 S-alleles in apple. The S-genotypes were determined for the following: ‘Hongro’ (S₁S₃), ‘Gamhong’ (S₁S₉), ‘Saenara’ (S₁S₃), ‘Chukwang’ (S₃S₉), ‘Hwahong’ (S₃S₉), ‘Seokwang’ (S₃S₃), ‘Hwarang’ (S₁S₉), ‘Sunhong’ (S₃S₉), ‘S.E.B.’ (S₁S₁₉), ‘S.G.D.’ (S₂S₃), and ‘Mollie’s Delicious’ (S₃S₇). We also confirmed the characteristics of the S-genotypes for eight Korean apple cultivars by PCR-Southern blot analysis, using seven S-RNases as probes.