Wild and Rare Self-Incompatibility Allele S17 Found in 24 Sweet Cherry (Prunus avium L.) Cultivars

The pollination of self-incompatible diploid sweet cherry is determined by the S-locus alleles. We resolved the S-alleles of 50 sweet cherry cultivars grown in Estonia and determined their incompatibility groups, which were previously unknown for most of the tested cultivars. We used consensus primers SI-19/20, SI-31/32, PaConsI, and PaConsII followed by allele-specific primers and sequencing to identify sweet cherry S-genotypes. Surprisingly, 48% (24/50) of the tested cultivars, including 17 Estonian cultivars, carry the rare S-allele S₁₇, which had initially been described in wild sweet cherries in Belgium and Germany. The S₁₇-allele in Estonian cultivars could originate from ‘Leningradskaya tchernaya’ (S₆|S₁₇), which has been extensively used in Estonian sweet cherry breeding. Four studied cultivars carrying S₁₇ are partly self-compatible, whereas the other 20 cultivars with S₁₇ have not been reported to be self-compatible. The recommended pollinator of seven self-incompatible sweet cherries is of the same S-genotype, including four with S₁₇-allele, suggesting heritable reduced effectiveness of self-infertility. We classified the newly genotyped sweet cherry cultivars into 15 known incompatibility groups, and we proposed four new incompatibility groups, 64–67, for S-locus genotypes S₃|S₁₇, S₄|S₁₇, S₅|S₁₇, and S₆|S₁₇, respectively, which makes them excellent pollinators all across Europe. Alternatively, the frequency of S₁₇ might be underestimated in Eastern European populations and some currently unidentified sweet cherry S-alleles might potentially be S₁₇.

     

Genomic characterization of self-incompatibility ribonucleases in the Central Asian pear germplasm and introgression of new alleles from other species of the genus Pyrus

The Pyrus species exhibit the so-called S-RNase-based gametophytic self-incompatibility system, which is considered to be the most widespread self-incompatibility system among flowering plants. In this study, 57 Iranian pear (Pyrus communis L.) domestic cultivars and wild genotypes, plus 21 European pear cultivars used as references, were genotyped adopting a PCR-based genotyping assay using consensus and allele-specific primers. The results revealed traces of significant genetic contribution in the Iranian traditional varieties and genotypes from other Pyrus species; the genetic contribution of Japanese pear clearly emerged with the detection of some Pyrus pyrifolia S-RNase alleles. Moreover, our results highlighted the presence of three new S-RNase alleles (named S₁₂₆, S₁₂₇, and S₁₂₈) that were not previously identified in P. communis, possibly introduced in the germplasm of cultivated pear through gene transfer from other cultivated or wild species.     

Inheritance of Hetero-Diploid Pollen S-Haplotype in Self-Compatible Tetraploid Chinese Cherry (Prunus pseudocerasus Lindl)

The breakdown of self-incompatibility, which could result from the accumulation of non-functional S-haplotypes or competitive interaction between two different functional S-haplotypes, has been studied extensively at the molecular level in tetraploid Rosaceae species. In this study, two tetraploid Chinese cherry (Prunus pseudocerasus) cultivars and one diploid sweet cherry (Prunus avium) cultivar were used to investigate the ploidy of pollen grains and inheritance of pollen-S alleles. Genetic analysis of the S-genotypes of two intercross-pollinated progenies showed that the pollen grains derived from Chinese cherry cultivars were hetero-diploid, and that the two S-haplotypes were made up of every combination of two of the four possible S-haplotypes. Moreover, the distributions of single S-haplotypes expressed in self- and intercross-pollinated progenies were in disequilibrium. The number of individuals of the two different S-haplotypes was unequal in two self-pollinated and two intercross-pollinated progenies. Notably, the number of individuals containing two different S-haplotypes (S₁- and S₅-, S₅- and S₈-, S₁- and S₄-haplotype) was larger than that of other individuals in the two self-pollinated progenies, indicating that some of these hetero-diploid pollen grains may have the capability to inactivate stylar S-RNase inside the pollen tube and grow better into the ovaries.     

Allele-specific PCR detection of sweet cherry self-incompatibility (S) alleles S1 to S16 using consensus and allele-specific primers

PCR-based identification of all 13 known self-incompatibility (S) alleles of sweet cherry is reported. Two pairs of consensus primers were designed from our previously published cDNA sequences of S₁ to S₆ S-RNases, the stylar components of self-incompatibility, to reveal length variation of the first and the second introns. With the exception of the first intron of S₁₃, these also amplified S₇ to S₁₄ and an allele previously referred to as S_x, which we now label S₁₆. The genomic PCR products were cloned and sequenced. The partial sequence of S₁₁ matched that of S₇ and the alleles were shown to have the same functional specificity. Allele-specific primers were designed for S₇ to S₁₆, so that allele-specific primers are now available for all 13 S alleles of cherry (S₈, S₁₁ and S₁₅ are duplicates). These can be used to distinguish between S alleles with introns of similar size and to confirm genotypes determined with consensus primers. The reliability of the PCR with allele-specific primers was improved by the inclusion of an internal control. The use of the consensus and allele-specific primers was demonstrated by resolving conflicting genotypes that have been published recently and by determining genotypes of 18 new cherry cultivars. Two new groups are proposed, Group XXIII (S₃S₁₆), comprising 'Rodmersham Seedling' and 'Strawberry Heart', and Group XXIV (S₆S₁₂), comprising 'Aida' and 'Flamentiner'. Four new self-compatibility genotypes, S₃S₃, S₄S₆, S₄S₉ and S₄S₁₃, were found. The potential use of the consensus primers to reveal incompatibility alleles in other cherry species is also demonstrated.Communicated by H.F. Linskens     

Molecular typing of the self-incompatibility locus of Turkish sweet cherry genotypes reflects phylogenetic relationships among cherries and other Prunus species

Self-incompatibility of sweet cherry (Prunus avium L.) is controlled by the multiallelic S-locus. While many cultivars and wild accessions have been S-genotyped, only limited data are available on accessions native to the center of origin of this species. Therefore, this study was carried out to determine the S-genotype of 11 landrace cultivars and 17 local genotypes selected from populations growing wild at the Black Sea coast. Eleven sweet cherries (S ₁–S ₇, S ₁₀, and S ₁₂–S ₁₄) and some wild cherries (S ₁₇–S ₁₉, S _21/25, and S ₃₁) S-RNase alleles were detected. The results indicate that Turkish cultivars represent a broader gene pool as compared with international cultivars. A new (S ₃₇) and a doubtful allele (provisionally labelled as S _7m) as well as the sour cherry S ₃₄-allele were identified in sweet cherry. These data and others (SSR variants within the S ₁₃-RNase introns) confirmed that allele pools of sweet and sour cherries in the Black Sea region are overlapping. A new cross-incompatibility group, XLV (S ₂ S ₁₈), was also proposed. Allele-specific primers were designed for S ₁₇–S ₁₉, S _21/25, S ₃₄, and S ₃₇. A phylogenetic analysis of the cherry S ₃₁-RNase and its trans-specific sister alleles reliably mirrored the assumed length of the time period after the divergence of species in the subgenera Cerasus and Prunophora. Most variations (insertions/deletions and single-nucleotide polymorphisms) in the S-RNase gene were silent and, hence, have not been exposed to natural selection. The results are discussed from the aspects of S-allele evolution and phylogenetic relationships among cherries and other Prunus species.     

S-allele diversity in Prunus L. Cerasus subgenus from Iran

In this study, S-allele diversity of eight wild and two commercial species of the Cerasus subgenus in Iran was investigated using two primer pairs. A high level of S-allele polymorphism was detected among and within the species evaluated. Furthermore, most of wild species showed 2–4 alleles based on S-allele primers and may be considered as tetraploid. Sweet cherry cultivars, Siah-Mashhad, Siah-Shabestar, Takdaneh-Mashhad, Siah-Daneshkadeh and Protiva showed S₃S₁₂, S₃S₁₂, S₃S₁₂, S₃S₅ and S₃S₄ combinations, respectively, allele S₃ showing the highest frequency. Three Iranian sweet cherry cultivars had the same allelic combination (S₃S₁₂) that the same ancestor in genealogy of these cultivars may explain the loss of diversity observed at the S-locus. Wild cherry (mazzard) accessions showed wide range of alleles such as S₁, S₂, S₇, S₁₄ and S₂₀ and unknown alleles, while sour cherries showed S₆, S₉, S₁₃ and S₂₇ alleles. In conclusion, the conservation of these highly diverse native species of Iranian wild Cerasus germplasm is recommended for future breeding activity.     

Detection and inheritance of stylar ribonucleases associated with incompatibility alleles in apricot

 Stylar proteins were surveyed by non-equilibrium pH gradient electrofocusing to identify S-RNases associated with gametophytic self-incompatibility in nine apricot cultivars. RNase activities associated with the alleles of incompatibility S ₁ , S ₂ , S ₅ , and S ₆ and with the allele of compatibility Sc were clearly identified. Two other bands that we considered related to the alleles S ₃ and S ₄ were unique to cultivars Sunglo and Harcot, respectively. Two generations of 17 seedlings from the cross Moniquí× Pepito and 38 from Gitano × Pepito were used to determine the inheritance of the S-RNases. Inheritance of these RNase bands followed the expected segregation ratios and the band combinations correlated perfectly with the known self-incompatibility status of the seedlings determined after self-pollination and observation of pollen tube growth. All evidence presented in this study strongly suggests that RNases are associated with gametophytic self-incompatibility of apricot and that RNases may be the S-gene products. This is the first report identifying S-RNases and describing the inheritance of these S-RNases in apricot. Received: 19 February 1998 / Revision accepted: 2 April 1998     

Self-incompatibility genotypes in almond re-evaluated by PCR,stylar ribonucleases,sequencing analysis and controlled pollinations

As part of the almond breeding programme at IRTA, we investigated the S genotypes of several cultivars using a combination of RNase zymograms, testcrosses, pollen-tube growth analysis and molecular identification by PCR analysis. For some of the cultivars examined, discrepancies appeared between their S alleles as reported in the literature and those found in this investigation, leading to a re-evaluation of their S genotypes. Analysis of the stylar ribonucleases (RNases), which are known to correlate with S alleles, of cvs. Achaak, Ardechoise, Desmayo Largueta, Ferrastar, Gabaix, Garbí, Glorieta, Languedoc, Primorskiy and Texas revealed inconsistencies with respect to the S₅ and S₁₀ alleles. However, PCR with the conserved primer pair AS1II/AmyC5R failed to detect any of these inconsistencies. When the S alleles from Desmayo Largueta, Gabaix, Primorskiy and Texas were sequenced, Texas and Primorskiy were found to carry the reported S₅ allele, while Desmayo Largueta and Gabaix carried a new allele, which has been tentatively denoted as S₂₅ This new S allele, previously reported to be S₁₀, was also identified in Achaak, Ardechoise and Ferrastar. The proposed new S genotypes are Achaak (S₂S₂₅), Ardechoise (S₁S₂₅), Desmayo Largueta (S₁S₂₅), Ferrastar (S₂S₂₅) and Gabaix (S₁₀S₂₅). The S alleles of Garbí, Glorieta, Languedoc, Texas and Primorskiy remain as reported in the literature. Testcrosses in the field and laboratory confirmed the new S genotypes. One cultivar (Gabaix) could be assigned to the existing cross-incompatibility group O of unique genotypes, and two new groups were established (XVI and XVII) consisting of two cultivars each. The clarification of these S alleles will be useful in almond breeding programmes and for planning new commercial orchards in the future.     

Cloning and characterization of genomic DNA sequences of four self-incompatibility alleles in sweet cherry (<Emphasis Type="Italic">Prunus avium</Emphasis> L.)

Gametophytic self-incompatibility (GSI) in sweet cherry is determined by a locus S with multiple alleles. In the style, the S-locus codifies for an allele-specific ribonuclease (S-RNase) that is involved in the rejection of pollen that carries the same S allele. In this work we report the cloning and genomic DNA sequence analysis including the 5 flanking regions of four S-RNases of sweet cherry (Prunus avium L., Rosaceae). DNA from the cultivars Ferrovia, Pico Colorado, Taleguera Brillante and Vittoria was amplified through PCR using primers designed in the conserved sequences of sweet cherry S-RNases. Two alleles were amplified for each cultivar and three of them correspond to three new S-alleles named S ₂₃ , S ₂₄ and S ₂₅ present in 'Pico Colorado', 'Vittoria' and 'Taleguera Brillante' respectively. To confirm the identity of the amplified fragments, the genomic DNA of these three putative S-RNases and the allele S ₁₂ amplified in the cultivar Ferrovia were cloned and sequenced. The nucleotide and deduced amino-acid sequences obtained contained the structural features of rosaceous S-RNases. The isolation of the 5-flanking sequences of these four S-RNases revealed a conserved putative TATA box and high similarity among them downstream from that sequence. However, similarity was low compared with the 5-flanking regions of S-RNases from the Maloideae. S ₆ - and S ₂₄ -RNase sequences are highly similar, and most amino-acid substitutions among these two RNases occur outside the rosaceous hypervariable region (RHV), but within another highly variable region. The confirmation of the different specificity of these two S-RNases would help elucidate which regions of the S-RNase sequences play a role in S-pollen specific recognition.Communicated by H.F. Linskens     

Characterisation of novel S-alleles from cherry (Prunus avium L.)

In plant populations exhibiting gametophytic self-incompatibility, individuals harbouring rare S alleles are likely to have a reproductive advantage over individuals having more common alleles. Consequently, determination of the self-incompatibility haplotype of individuals is essential for genetic studies and the development of informed management strategies. This study characterises six new S alleles identified in wild cherry (Prunus avium L.). Investigations to determine the S genotype of individuals in recently planted woodland through length polymorphisms of introns associated with the stylar S-RNase gene and the pollen SFB gene revealed six S intron profiles which did not correspond to those of known S alleles. These are now attributed to S ₂₇ to S ₃₂ . Consensus primers, annealing in the S-RNase sequence coding for the signal peptide and C5 regions, were used to isolate the S-RNase alleles associated with the novel S intron profiles. The proteins corresponding to the new alleles were separated by isoelectric focusing from stylar extracts and their pI values determined. Similarities between the deduced amino acid sequence for the new alleles isolated and other cherry S-RNase sequences available on the databases ranged from 40% to 86%. Amplification products for SFB introns ranged from 172 to 208bp. New sequence regions exposed to positive selection were identified and the significance of the PS3 region reinforced. A phylogenetic relationship between P. avium S-RNases for S ₁₀ and S ₁₃ and between corresponding SFB alleles may indicate co-evolution of allele specificities of these two genes. The nucleotide sequences reported in this paper have been submitted to the EMBL/GenBank database under the following accession numbers: S ₂₇ (DQ266439), S ₂₈ (DQ266440), S ₂₉ (DQ266441), S ₃₀ (DQ266442), S ₃₁ (DQ266443), S ₃₂ (DQ266444).     

An isoenzyme marker linked to the incompatibility locus in cherry

Analysis of two cherry progenies from semi-compatible crosses for the esterase enzyme system showed extremely distorted segregation ratios for Est-5. Analysis of two progenies from compatible crosses for esterase and for stylar ribonuclease proved that Est-5 is linked with the incompatibility locus S. The recombination fraction is 4%. About a fifth of some 50 cultivars or selections genotyped for Est-5 were heterozygous. The various heterozygotes could provide ’testers’ for the presence in cultivars of unknown genotype of 8 of the 11 known S alleles. A seedling suitable for testing S ₉ has been identified and crosses have been made to raise testers for S ₁₀ and S ₁₁ . Isoenzyme analysis of the four progenies for glutamate oxaloacetate transaminase, and of one of them for isocitrate dehydrogenase, showed no evidence for the linkage of Got-1 or Idh-2 with S, contrary to a previous report. Estimation of linkage with S in semi-compatible crosses is discussed. Received: 16 April 1999 / Accepted: 22 June 1999     

Revised dominance hierarchy for S-alleles in Corylus avellana L.

Pollen-stigma compatibility was studied in cultivars and more than 1800 seedlings of the European hazelnut (Corylus avellana L). Four new S-alleles were identified, bringing the total to 25 unique alleles within C. avellana. The new alleles are the recessive alleles in ‘Tonda di Giffoni’ and ‘Segorbe’ (S₂₃), in ‘Neue Riesennuss’ (S₂₅), in ‘Gasaway’ (S₂₆), and a dominant allele in a seedling of Turkish origin (S₂₄). Dominance relationships in 233 of the possible 300 pairs of alleles were determined in both pistil and pollen. All alleles exhibited independent action in the pistil, whereas in the pollen either dominance or codominance was exhibited. The dominance hierarchy of alleles in the pollen was revised in light of the new information obtained. All 25 alleles have been assigned to a level in the hierarchy that is linear and now has eight levels. S₆ and S₉ were reassigned to lower levels in the hierarchy. Thirteen of the alleles are on the level of S₁, while S₄, S₆, S₁₁, and S₂₃ occupy unique positions in the hierarchy. Improved pollen tester clones were identified for several S-alleles. The alleles in 55 cultivars were determined. The alleles identified in ‘DuChilly’ (S₁₀ S₁₄) did not agree with previous reports. Four cultivars have the same alleles as ‘Römische Nuss’ (S₁₀ S₁₈) and are morphologically indistinguishable from it: ‘Frutto-grosso’, ‘Istarski Okrogloplodna’, ‘Payrone’, and ‘Romai’. ‘Belle di Giubilino’ and ‘Tonda di Biglini’ are both S₁ S₁₀ and appear to be synonyms for the same cultivar.     

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.     

cDNA cloning of nine S alleles and establishment of a PCR-RFLP system for genotyping European pear cultivars

Nine full-length cDNAs of S ribonucleases (S-RNases) were cloned from stylar RNA of European pear cultivars by RT-PCR and 3′ and 5′ RACE. Comparison of the nucleotide sequences between the nine S-RNases cloned and 13 putative S alleles previously amplified by genomic PCRs revealed that seven corresponded to Sa, Sb, Sd, Se, Sh, Sk and Sl alleles, and the other two were new S alleles (designated as Sq and Sr alleles). Genomic PCR with a set of ȁ8FTQQYQȁ9 and ȁ8EP-anti-IIWPNVȁ9 primers was used to amplify nine S alleles; 1,414 bp (Sl), ca. 1.3 kb (Sk and Sq), 998 bp (Se), 440 bp (Sb) and ca. 350 bp (Sa, Sd, Sh and Sr). Among these, S alleles of similar size were discriminated by digestion with BaeI, BglII, BssHII, HindIII, EcoO109I and SphI. The PCR amplification of S allele following digestion with the restriction enzymes provided a PCR-RFLP system for rapid S-genotyping European pear cultivars harboring nine S alleles. The PCR-RFLP system assigned a total of 63 European pear cultivars to 25 genotypes. Among these, 14 genotypes were shared by two or more cultivars, which were cross-incompatible. These results suggested that the genes cloned represented the S-RNases from European pear, and that there were many cross-incompatible combinations among European pear varieties.     

Identification of new S-RNase self-incompatibility alleles and characterization of natural mutations in Iranian almond cultivars

The two main objectives of this research were to identify new S-RNase alleles in Iranian almond cultivars and to characterize naturally occurring mutations in these alleles that may cause self-compatibility. We investigated S genotypes of 22 Iranian almond cultivars using stylar RNase electrophoresis, PCR and DNA sequencing. We report six previously unidentified P. dulcis S-RNase alleles (S ₄₅ , S ₄₆ , S ₄₇ , S ₄₈ , S ₄₉ and S ₅₀ ). Four of 12 tested S-RNases were found to be non-functional in vitro: S ₄₉ , S ₅₀ , S ₂₄ /S _na and S ₂₅ /S ₄₇ . Detected point mutations in the C3 coding region of S ₄₉ - and S ₅₀ -RNase, leading to the replacement of a highly conserved cysteine and histidine residues, are with the highest probability the reason of these S-RNases inactivity. Results also suggested that ten Iranian almond cultivars display unique S genotype. All presented data confirm Iranian cultivars as valuable almond sources which are of interest to almond breeding and conservation programs.     

Identification of S-genotypes in Chinese cherry cultivars (Prunus pseudocerasus Lindl.)

Self-incompatibility has been studied extensively at the molecular level in Solanaceae, Rosaceae, and Scrophulariaceae, all of which exhibit gametophytic self-incompatibility. In the present study, we successfully isolated nine S-RNase alleles from cultivars of Chinese cherry by PCR amplification from genomic DNA and stylar cDNA combining with cleaved amplified polymorphic sequence marker. Analysis of amino acid sequences revealed five novel S-alleles, S ₂ , S ₄ , S ₆ , S ₈ , and S ₉ , with respective accession numbers in the NCBI database of EF541168, EF541173, EF541172, FJ628598, and FJ628599. Results showed that “Dongtang” and “Yinzhu” contained six S-alleles (S ₁ , S ₃ , S ₅ , S ₇ , S ₈ , and S ₉ ); “Taishanganying” contained four S-alleles (S ₁ , S ₂ , S ₄ , and S ₆ ); “Daiba”, “Dayingzui”, and “Xiaomizi” contained four S-alleles (S ₁ , S ₂ , S ₅ , and S ₈ ); “Laiyangduanzhi”, “Shuangquanchangba”, and “Daqingye” contained three S-alleles (S ₁ , S ₂ , and S ₈ ). It is interesting that different cultivars collected from the same place hold the same S-genotypes. Moreover, pollination tests and pollen tube growth assays showed that nine cultivars were self-compatible. Chinese cherry presented in this article are naturally polyploidy, which is a very useful material for the study of self-compatibility, and much of this information will be valuable for further work on self-(in)compatibility of fruit tree in Rosaceae.     

Self-(in)compatibility of the almonds P. dulcis and P. webbii: detection and cloning of 'wild-type Sf ' and new self-compatibility alleles encoding inactive S-RNases

Prunus dulcis, the almond, is a predominantly self-incompatible (SI) species with a gametophytic self-incompatibility system mediated by S-RNases. The economically important allele S _f , which results in self-compatibility in P. dulcis, is said to have arisen by introgression from Prunus webbii in the Italian region of Apulia. We investigated the range of self-(in)compatibility alleles in Apulian material of the two species. About 23 cultivars of P. dulcis (14 self-compatible (SC) and nine SI) and 33 accessions of P. webbii (16 SC, two SI and 15 initially of unknown status), all from Apulia, were analysed using PCR of genomic DNA to amplify S-RNase alleles and, in most cases, IEF and staining of stylar protein extracts to detect S-RNase activity. Some amplification products were cloned and sequenced. The allele S _f was present in nearly all the SC cultivars of P. dulcis but, surprisingly, was absent from nearly all SC accessions of P. webbii. And of particular interest was the presence in many SI cultivars of P. dulcis of a new active allele, labelled S ₃₀ , the sequence of which showed it to be the wild-type of S _f so that S _f can be regarded as a stylar part mutant S ₃₀ °. These findings indicate S _f may have arisen within P. dulcis, by mutation. One SC cultivar of P. dulcis, ‘Patalina’, had a new self-compatibility allele lacking RNase activity, S _n5 , which could be useful in breeding programmes. In the accessions of P. webbii, some of which were known to be SC, three new alleles were found which lacked RNase activity but had normal DNA sequences.     

Inheritance and interactions of incompatibility alleles in the tetraploid sour cherry

Three progenies of sour cherry (Prunus cerasus) were analysed to correlate self-(in)compatibility status with S-RNase phenotype in this allotetraploid hybrid of sweet and ground cherry. Self-(in)compatibility was assessed in the field and by monitoring pollen tube growth after selfing. The S-RNase phenotypes were determined by isoelectric focusing of stylar proteins and staining for RNase activity and, for the parents, confirmed by PCR. Seedling phenotypes were generally consistent with disomic segregation of S-RNase alleles. The genetic arrangements of the parents were deduced to be ‘Köröser’ (self-incompatible) S ₁ S ₄ .S _B S _D , ‘Schattenmorelle’ (self-compatible) S ₆ S ₁₃ .S _B S _B , and clone 43.87 (self-compatible) S ₄ S ₁₃ .S _B S _B , where “.” separates the two homoeologous genomes. The presence of S ₄ and S ₆ alleles at the same locus led to self-incompatibility, whereas S ₁₃ and S _B at homoeologous loci led to self-compatibility. The failure of certain heteroallelic genotypes in the three crosses or in the self-incompatible seedlings indicates that S ₄ and S ₆ are dominant to S _B . However, the success of S ₁₃ S _B pollen on styles expressing corresponding S-RNases indicates competitive interaction or lack of pollen-S components. In general, the universal compatibility of S ₁₃ S _B pollen may explain the frequent occurrence of S ₁₃ and S _B together in sour cherry cultivars. Alleles S _B and S _D , that are presumed to derive from ground cherry, and S ₁₃ , presumably from sweet cherry, were sequenced. Our findings contribute to an understanding of inheritance of self-(in)compatibility, facilitate screening of progenies for self-compatibility and provide a basis for studying molecular interactions in heteroallelic pollen.     

Allele-specific hybridization using streptavidin-coated magnetic beads for species identification, S genotyping, and SNP analysis in plants

Dot-blot hybridization has been successfully used for the construction of single nucleotide polymorphism (SNP)-based linkage maps, quantitative trait locus analysis, marker-assisted selection, and the identification of species and cultivars. This method is, however, time-consuming, even for a small number of plant samples. We propose a method in which streptavidin-coated magnetic beads replace the nylon membrane for immobilization of the PCR products and are hybridized with allele-specific oligonucleotide probes and a digoxigenin-labeled oligonucleotide hybridized with the allele-specific oligonucleotide probe. After amplification of plant DNA by PCR with the biotinylated primers, those oligonucleotide probes having species-specific or allele-specific sequences were mixed together with the digoxigenin-labeled oligonucleotide and the streptavidin-coated magnetic beads at a temperature suitable for each probe. Species-specific internal transcribed spacer 1 (ITS1) sequences and allele-specific sequences of the hypervariable region I of S-locus receptor kinase (SRK) specifically detected ITS1 sequences and SRK alleles in Brassica species, respectively. SNPs were also successfully analyzed by using allele-specific oligonucleotide probes and competitive oligonucleotides. In the SNP analysis, PCR products were indirectly captured by magnetic beads. SNP alleles of eight cultivars each of Brassica rapa and Raphanus sativus were analyzed using streptavidin-coated magnetic beads. The genotyping results corresponded well with those of dot-blot-SNP analysis. Although allele-specific hybridization using streptavidin-coated magnetic beads is somewhat costly, it is easier and more rapid than dot-blot hybridization. This method is suitable for the analysis of a small number of plant samples with a large number of DNA markers.     

An allele responsible for seedling death in Pinus radiata D. Don

 When inbred, most outcrossing species show high mortality, manifested at several life stages. The occurrence of homozygotes for deleterious or lethal alleles is believed to be responsible. Here, we report the identification of an allele responsible for the death of selfed Pinus radiata D. Don seedlings in their first month after germination. Among 291 S₁ seedlings of plus-tree 850.55, 76 died within 1 month of emergence. Their death appears to be caused by a single recessive lethal allele, SDPr (seedling death in Pinus radiata). SDPr is located in a linkage group with 28 RAPD markers, the closest of which is ai05800a. Of the 76 seedlings that died, megagametophytes of 73 could be genotyped. Of these, 71 had the null (no band) allele of ai05800a; only two had the band allele. Of the 190 surviving S₁ diploids that were genotyped, only two individuals were homozygous for the null allele of ai05800a. By two different methods, the map distance between SDPr and ai05800a was estimated to be between 1.0 and 2.7 cM respectively. The frequency of band and null alleles in the combined population of dead and surviving seedlings and in un-sown seeds shows no evidence of selection at this locus prior to germination. Received: 30 September 1997 / Accepted: 29 October 1997