Comparison of S-alleles and S-glycoproteins between two wild populations of Brassica campestris in Turkey and Japan

Summary The number of identical S-alleles between two wild populations of B. campestris, one in Turkey, the other in Japan, that have been independent of one another for a long time was investigated. Diallel pollination tests between 38 S-allele homozygotes, i.e., 16 S-allele homozygotes from Turkey and 22 from Japan, revealed that these were 29 different S-alleles only 4 common ones. These S-alleles were differentiated by the iso-electric focusing (IEF) analysis of S-locus glycoproteins (SLGs) stained with an antiserum against SLG⁸. All identical S-alleles had the major SLG band at the same pI value without exception, even though they were collected from different populations. However, the number of minor bands of SLGs varied between the two populations; the S-alleles in Balcesme had generally fewer minor bands than those in Oguni. The 29 independent S-alleles were numbered from S ²¹ to S ⁴⁹ according to the pI value of the major SLG band. The major bands whose pI values were 7.5–8.5 were most common. Blot-hybridization patterns of genomic DNA hybridized with SLG ⁸ cDNA were not always the same among the strains of identical S-alleles obtained from different populations. Because about 20% of the S-alleles were shared between the two populations, it can be inferred that more than hundreds of S-alleles have been accumulated by mutation in B. campestris throughout the world.     

Isolation of S-alleles from a wild population of Brassica campestris L. at Balcesme,Turkey and their characterization by S-glycoproteins

Summary S-alleles of self-incompatibility were isolated from a wild population of Brassica campestris growing at Balcesme, Turkey. Out of 88 plants observed, 73 were self-incompatible and 4 were self-compatible. In certain families, selfed progenies from a self-incompatible plant segregated into fewer than three incompatibility classes, which is consistent with a one-locus sporophytic genetic control of self-incompatibility. Out of 25 combinations of S-alleles tested, dominance interactions were observed in 6 of them on the pollen side and on 5 of them on the stigma side. The 35 S-homozygotes thus isolated consisted of 18 independent S-alleles. The number of S-alleles in this population was estimated to be more than 30. The S-locus glycoproteins (SLGs) corresponding to the respective S-alleles were identified by iso-electric focusing (IEF)-gel immunoblotting with a polyclonal antiserum against SLG⁸. SLGs in a stigma were generally composed of several bands, one major and a few minor ones, whose molecular weight was similar to each other, and the major and minor bands were heritable in correlation with each other. SDS-PAGE analysis of SLGs differentiated a few juxtaposed bands between 50 and 60 kDa, and the variations in these bands were considered to be due to differences in the number of polysaccharide residues. General features of the variation of S-genes and their SLGs between the populations in Balcesme, Turkey and Oguni, Japan, were comparatively similar to one another, despite the different surroundings and history of these populations.     

Cloning and sequencing of cDNAs encoding two self-incompatibility associated proteins in Solanum chacoense

Summary We have isolated and sequenced cDNAs for S₂- and S₃-alleles of the self-incompatibility locus (S-locus) in Solanum chacoense Bitt., a wild potato species displaying gametophytic self-incompatibility. The S₂-and S₃-alleles encode pistil-specific proteins of 30 kDa and 31 kDa, respectively, which were previously identified based on cosegregation with their respective alleles in genetic crosses. The amino acid sequence homology between the S₂- and S₃-proteins is 41.5%. This high degree of sequence variability between alleles is a distinctive feature of the S-gene system. Of the 31 amino acid residues which were previously found to be conserved among three Nicotiana alata S-proteins (S₂, S₃, and S₆) and two fungal ribonucleases (R Nase T₂ and R Nase Rh), 27 are also conserved in the S₂- and S₃-proteins of S. chacoense. These residues include two histidines implicated in the active site of the R Nase T₂, six cysteines, four of which form disulfide bonds in R Nase T₂, and hydrophobic residues which might form the core structure of the protein. The finding that these residues are conserved among S-proteins with very divergent sequences suggests a functional role for the ribonuclease activity of the S-protein in gametophytic self-incompatibility.     

Self-compatibility in aLycopersicon peruvianum variant (LA2157) is associated with a lack of style S-RNase activity

A series of crosses between a naturally-occurring self-compatible accession ofLycopersicon peruvianum and a closely-related self-incompatible accession were used to demonstrate that the mutation to self-compatibility is located at the S-locus. Progeny of the crosses contain abundant style proteins of about 30 kDa that segregate with the S₆and S₇-alleles from the SI parent and the S_c-allele from the SC parent. The S₆and S₇-associated proteins have ribonuclease activity whereas the S_c-associated protein is not an active ribonuclease. This finding indicates that S-RNases are determinants of self-incompatibility in the style and that the ribonuclease activity is essential for their function.     

Identification of self-incompatibility groups in<Emphasis Type="Italic"> Hatiora</Emphasis> and<Emphasis Type="Italic"> Schlumbergera</Emphasis> (Cactaceae)

The Cactaceae, a family of about 1,800 species of succulent perennials, contains numerous species that exhibit self-incompatibility (SI). The objective of the current study was to determine the number of incompatibility groups present among diploid (2n=2x=22) cultivars of the genera Schlumbergera Lem. (Christmas cacti) and Hatiora Britton & Rose (Easter cacti). Two partial diallel crosses were performed, one with 19 cultivars of Christmas cacti [= S. truncata (Haworth) Moran and S. × buckleyi (Buckley) Tjaden] and the other with 10 cultivars of Easter cacti [= H. gaertneri (Regel) Barthlott, H. rosea (Lagerheim) Barthlott, and H. × graeseri Barthlott ex D. Hunt]. The compatibility/incompatibility status of crosses was determined by percent fruit set and presence of seed in mature fruit. None of the cultivars set fruit when selfed or crossed with a cultivar in the same incompatibility group, but fruit set ranged from 35% to 100% following compatible crosses. For the Christmas cacti, eight intra-incompatible but reciprocally compatible groups were identified, with 13 of the 19 cultivars assigned to three incompatibility groups (68%). The ten cultivars of Easter cacti yielded nine intra-incompatible but reciprocally compatible groups, with two cultivars in one incompatibility group and the other eight cultivars each assigned to a unique group. One cultivar of Christmas cactus ('Abendroth 6') was incompatible when crossed as a male with cultivars in incompatibility group 2 but was compatible in reciprocal crosses, results that suggest that this cultivar is an S-allele homozygote. The crossing relationships are consistent with a one-locus, gametophytic SI system with multiple alleles. Allozyme locus Lap-1, shown previously to be linked with the S locus (recombination frequency 7%) in Schlumbergera, exhibited insufficient allelic diversity for determining the S genotypes of the 19 cultivars of Christmas cacti. Based on the number of incompatibility groups in each diallel, at least five S-alleles occur in the 19 Christmas cacti and the 10 Easter cacti.Publication 3337 of the Massachusetts Agricultural Experiment Station. This material is based on work supported in part by the Cooperative State Research, Extension, Education Service, United States Department of Agriculture, Massachusetts Agricultural Experiment Station, under Project No. 746     

Determination of the transmission frequency of chromosome 4S l of Aegilops sharonensis in a range of wheat genetic backgrounds

Summary The transmission of chromosome 4S ^l from Aegilops sharonensis was observed in a range of wheat genetic backgrounds. Chromosome 4S ^l was transmitted at a very high frequency (at least 97.8%) in all crosses. The genetic background appears to only have a small effect on transmission. The frequency of transmission of chromosome 4S ^l was the same in each genetic background through both the male and female gametes.     

The effect of sheltered load on reproduction in Solanum carolinense, a species with variable self-incompatibility

In previous studies, we have investigated the strength of self-incompatibility (SI) in Solanum carolinense, a highly successful weed with a fully functional SI system that inhabits early successional and other disturbed habitats. We have found that the SI response in S. carolinense is a plastic trait—its strength being affected by the age of the flowers, and the presence of developing fruits and that there are genetic differences among families in their self-fertility. However, in species with a fully functional SI response, selfing would not be that common. As a result, deleterious recessives scattered though the genome of horsenettle are only occasionally exposed to selection. It has been suggested that deleterious recessives accumulate near S-alleles in strong SI species because the S-locus is located in a non-recombining region of the genome and because strong S-alleles are never in the homozygous state, thus sheltering some of the genetic load near the S-locus from selection. We performed a series of laboratory and greenhouse experiments to determine the extent to which sheltered load adds to the overall magnitude of inbreeding depression in horsenettle. Specifically, we amplified and sequenced the S-alleles from 16 genets collected from a large population in Pennsylvania and performed a series of controlled self-pollinations. We then grew the selfed progeny in the greenhouse; recorded various measures of growth and reproductive output; and amplified and sequenced their S-allele(s). We found that the heterozygous progeny of self-pollinations produce more flowers and have a greater ability to set both self and cross seed than S-homozygous progeny. We also found evidence of variation in the magnitude of load among S-alleles. These results suggest that sheltered load might slow the fixation of weak (partially compatible) S-alleles in this population, thus adding to the maintenance of a mixed mating system rather than leading to the fixation of the selfing alleles.     

High frequncies of fertilization and embryo formation in hexaploid wheat x Tripsacum dactyloides crosses

The Hexaploid wheat variety Fukuho was crossed with Tripsacum dactyloides (2n=4x=72). The total fertilization frequencies for the egg cell, polar nuclei, and both, were 58.3%, 26.8% and 58.9% of the 168 ovaries examined. However, the fertilization frequency of single polar nuclei was much lower at only 0.6%. The total frequency of fertilization was higher than that in wheat x maize crosses. A total of 49 hexaploid wheat varieties, including Hope carrying the dominant genes Kr1 and Kr2, were crossed with T. dactyloides, and most gave embryos. The embryoformation frequencies ranged from 0.5% to 59.0%. A higher frequency of 32.0% embryo formation was obtained following pollination of the variety Hope. In comparison with embryo formation in wheat x maize crosses the difference of embryo-formation frequencies between the two crosses was significant. The results of high frequencies of fertilization and embryo formation in wheat x T. dactyloides crosses indicated that the Kr genes are as inactive in wheat x T. dactyloides, as they are in wheat x maize crosses, and also that the efficiency of fertilization and embryo formation is higher in wheat x T. dactyloides than in what x maize crosses. The potential of wheat x T. dactyloides crosses for wheat haploid production and wheat improvement is discussed.     

Identification of self-incompatibility genotypes of almond by allele-specific PCR analysis

In almond, gametophytic self-incompatibility is controlled by a single multiallelic locus (S-locus). In styles, the products of S-alleles are ribonucleases, the S-RNases. Cultivated almond in California have four predominant S-alleles (S ^a, S ^b, S ^c, S ^d). We previously reported the cDNA cloning of three of these alleles, namely S ^b, S ^c and S ^d. In this paper we report the cloning and DNA sequence analysis of the S ^a allele. The S^a-RNase displays approximately 55% similarity at the amino-acid level with other almond S-RNases (S^b, S^c, and S^d) and this similarity was lower than that observed among the S^b, S^c and S^d-RNases. Using the cDNA sequence, a PCR-based identification system using genomic DNA was developed for each of the S-RNase alleles. Five almond cultivars with known self-incompatibility (SI) geno-types were analyzed. Common sequences among four S-alleles were used to create four primers, which, when used as sets, amplify DNA bands of unique size that corresponded to each of the four almond S-alleles; S ^a (602 bp), S ^b (1083 bp), S ^c (221 bp) and S ^d (343 bp). All PCR products obtained from genomic DNA isolated from the five almond cultivars were cloned and their DNA sequence obtained. The nucleotide sequence of these genomic DNA fragments matched the corresponding S-allele cDNA sequence in every case. The amplified products obtained for the S ^a- and S ^b-alleles were both longer than that expected for the coding region, revealing the presence of an intron of 84 bp in the S ^a-allele and 556 bp in the S ^b-allele. Both introns are present within the site of the hypervariable region common in S-RNases from the Rosaceae family and which may be important for S specificity. The exon portions of the genomic DNA sequences were completely consistent with the cDNA sequence of the corresponding S-allele. A useful application of these primers would be to identify the S-genotype of progeny in a breeding program, new varieties in an almond nursery, or new grower selections at the seedling stage. Received: 21 June 1999 / Accepted: 15 November 1999     

Identification of self-incompatibility-related glycoproteins in styles of apple (Malus x domestica)

In this study, stylar proteins of apple (Malus x domestica) which correlate with known intervarietal incompatibility relationships and have similar characteristics to the S-glycoproteins of Japanese pear (Pyrus serotina) were surveyed by two-dimensional gel electrophoresis (2D-PAGE). Varietal differences were detected in a group of glycoproteins having Mrs and pIs similar to those of the S-glycoproteins of Japanese pear. 2D-PAGE profiles of these glycoproteins were correlated with intervarietal incompatibility relationships. These glycoproteins reacted with antiserum raised against the S ⁴-glycoprotein of Japanese pear, a result suggesting that they may be the products of S-alleles in styles of apple. On the basis of the profiles of the putative S-glycoproteins, S-genotypes were proposed for each of the apple cultivars examined.     

Procedures for identifying S-allele genotypes of Brassica

Summary Procedures are described for efficient selection of: (1) homozygous and heterozygous S-allele genotypes; (2) homozygous inbreds with the strong self- and sib-incompatibility required for effective seed production of single-cross F₁ hybrids; (3) heterozygous genotypes with the high self- and sib-incompatibility required for effective seed production of 3- and 4-way hybrids.From reciprocal crosses between two first generation inbred (I₁) plants there are three potential results: both crosses are incompatible; one is incompatible and the other compatible; and both are compatible. Incompatibility of both crosses is useful information only when combined with data from other reciprocal crosses. Each compatible cross, depending on whether its reciprocal is incompatible or compatible, dictates alternative reasoning and additional reciprocal crosses for efficiently and simultaneously identifying: (A) the S-allele genotype of all individual I₁ plants, and (B) the expressions of dominance or codominance in pollen and stigma (sexual organs) of an S-allele heterozygous genotype. Reciprocal crosses provide the only efficient means of identifying S-allele genotypes and also the sexual-organ x S-allele-interaction types.Fluorescent microscope assay of pollen tube penetration into the style facilitates quantitation within 24–48 hours of incompatibility and compatibility of the reciprocal crosses. A procedure for quantitating the reciprocal difference is described that maximizes informational content of the data about interactions between S alleles in pollen and stigma of the S-allele-heterozygous genotype.Use of the non-inbred I_o generation parent as a known heterozygous S-allele genotype in crosses with its first generation selfed (I₁) progeny usually reduces at least 7 fold the effort required for achieving objectives 1, 2, and 3, compared to the method of making reciprocal crosses only among I₁ plants.Identifying the heterozygous and both homozygous S-allele genotypes during the I₁ generation facilitates, during subsequent inbred generations, strong selection for or against modifier genes that influence the intensity of self- and sib-incompatibility. Selection for strong self and sib incompatibility can be effective for both homozygous inbreds and also for the S-allele heterozygote, thus facilitating production of single-cross F₁ hybrids and also of 3-and 4-way hybrids.Department of Plant Breeding and Biometry paper No. 690     

Abortion and determination of stages for embryo rescue in crosses between sweet-potato,Ipomoea batatas Lam. (2n=6x=90) and its wild relative,I. trifida (H. B. K.) G. Don. (2n=2x=30)

Summary The frequency of aborted fruits and the changes and abnormalities that occur during the embryo development in intraspecific crosses of sweet-potato Ipomoea batatas (2n=6x=90) and interspecific crosses between I. batatas and I. trifida (2n=2x=30) were investigated in order to study the causes of the low seed production. Three genotypes of I. batatas and 18 genotypes of I. trifida were intermated. The frequency of aborted fruits was below 25% in the intraspecific crosses and over 90% in the interspecific crosses. Paraffin sections were used to examine the developmental stages of fruits and seeds. Embryos in different developmental stages were observed to determine the stage of abortion. These observations permitted the identification of developmental stages of embryo rescue in interspecific crosses. There were no significant differences in the frequency of embryo abortion before the early globular stage among female sweet-potato progenitors for the intraspecific and interspecific crosses. The frequency of the late occurrence of embryo abortion (when embryo abortion occurs after the pre-globular stage) was higher in interspecific crosses (19.1%) than in intraspecific crosses (5.5%). The frequency of the late occurrence of embryo abortion in interspecific crosses was higher at the globular stage (9.6%) than at the heart stage (4.3%). Providing that embryo rescue is conducted in interspecific crosses, the estimated number of potentially viable embryos could be increased: 30 times with embryos at the globular stage; 20 times with embryos at the heart stage; and 11 times if embryos at the torpedo stage were used for the rescue with respect to the seed set. The results suggested that the appropriate time for embryo rescue in interspecific crosses is at the globular stage. If embryos could be rescued at the globular stage, it would be possible to increase the number of surviving embryos up to 30 times in interspecific crosses and 0.02 times in intraspecific crosses with respect to natural conditions without embryo rescue.This research was initiated during sabbatical of M.I. at the Asian Vegetable Research and Development Center (AVRDC) in Taiwan     

Breakdown of self-incompatibility in a natural population of Petunia axillaris (Solanaceae) in Uruguay containing both self-incompatible and self-compatible plants

 Many members of the Solanaceae display a type of gametophytic self-incompatibility which is controlled by a single multiallelic locus, called the S-locus. From our previous survey of more than 100 natural populations of Petunia axillaris (a solanaceous species) in Uruguay, we had found that the majority of the populations of subspecies axillaris were comprised of virtually all self-incompatible individuals. The rest were ”mixed populations” which contained mostly self-incompatible and some self-compatible individuals. In this study, we examined the self-incompatibility behavior and determined the S-genotypes of 33 plants raised from seeds obtained from one such mixed population, designated U1. We found that 30 of the 33 plants (designated U1–1 through U1–33) were self-incompatible and a total of 18 different S-alleles were represented. To determine the S-genotypes of the three self-compatible plants (U1–2, U1–16, and U1–22) and the possible causes for the breakdown of their self-incompatibility, we carried out reciprocal crosses between each of them and each of the 18 S-homozygotes (S ₁ S ₁ through S ₁₈ S ₁₈ ) obtained from bud-selfed progeny of 14 of the 30 self-incompatible plants. For U1–2 and U1–16, we also carried out additional crosses with U1–25 (with S ₁ S ₁₃ genotype) and an S ₁₃ S ₁₅ plant (obtained from a cross between an S ₁₃ -homozygote and an S ₁₅ -homozygote), respectively. Based on all the pollination results and analysis of the production of S-RNases, products of S-alleles in the pistil, we determined the S-genotypes of U1–2, U1–16, and U1–22, and propose that the breakdown of self-incompatibility in these three plants is caused by suppression of the production of S₁₃-RNase from the S ₁₃ -allele they all carry. We have termed this phenomenon ”stylar-part suppression of an S-allele” or SPS. Received: 25 September 1998 / Revision accepted: 22 December 1998     

Conservation of S-locus for self-incompatibility in Brassica napus (L.) and Brassica oleracea (L.)

 Self-incompatibility (SI) in Brassica is a sporophytic system, genetically determined by alleles at the S-locus, which prevents self-fertilization and encourages outbreeding. This system occurs naturally in diploid Brassica species but is introduced into amphidiploid Brassica species by interspecific breeding, so that in both cases there is a potential for yield increase due to heterosis and the combination of desirable characteristics from both parental lines. Using a polymerase chain reaction (PCR) based analysis specific for the alleles of the SLG (S-locus glycoprotein gene) located on the S-locus, we genetically mapped the S-locus of B. oleracea for SI using a F₂ population from a cross between a rapid-cycling B. oleracea line (CrGC-85) and a cabbage line (86-16-5). The linkage map contained both RFLP (restriction fragment length polymorphism) and RAPD (random amplified polymorphic DNA) markers. Similarly, the S-loci were mapped in B. napus using two different crosses (91-SN-5263×87-DHS-002; 90-DHW-1855-4×87-DHS-002) where the common male parent was self-compatible, while the S-alleles introgressed in the two different SI female parents had not been characterized. The linkage group with the S-locus in B. oleracea showed remarkable homology to the corresponding linkage group in B. napus except that in the latter there was an additional locus present, which might have been introgressed from B. rapa. The S-allele in the rapid-cycling Brassica was identified as the S₂₉ allele, the S-allele of the cabbage was the S ₅ allele. These same alleles were present in our two B. napus SI lines, but there was evidence that it might not be the active or major SI allele that caused self-incompatibility in these two B. napus crosses. Received: 7 June 1996/Accepted: 6 September 1996     

Inheritance of male sterility in Trifolium hirtum All

The inheritance of male sterility was studied using a set of intra and interpopulation crosses from Californian populations of rose clover (Trifolium hirtum All.). The F2 gave higher frequencies of male steriles in interpopulation crosses than in intrapopulation crosses. Several F2 families gave ratios that were compatible with models of two and three complementary genes. However, the frequency distribution of male sterility among F3 families rejected such models. Given that single and two gene models were rejected, we interpret the results using a cytoplasmic-nuclear model with more than two restorer loci which is at least consistent with the observed results.     

Pollen tube expression of pseudo-self-compatibility (PSC) inPetunia hybrida

Summary AnS _1.1 self-incompatible (SI) petunia plant which showed atypical seed set was found in an I₇ population. This plant showed a strong SI reaction when selfed but produced varying amounts of seed when used as the seed parent in crosses with unrelated individuals homozygous for the sameS allele. Reciprocal crosses yielded no seed indicating that the reaction was a stylar response. Self seed obtained by high temperature treatments produced 18 plants, all of which exhibited the parental characteristics, the ability to reject self pollen but accept, to varying degrees, pollen bearing the sameS allele from unrelated plants. Several petunias homozygous forS ₁, and exhibiting various levels of PSC as determined by self seed set, progeny tests and temperature treatments, were used as pollen parents. The mean seed set of these crosses produced a ranking of the pollen parents which reflected the PSC levels obtained by other methods. The behavior of the F₁ and F₂ populations suggests that the pollen discriminating ability may be a simply inherited, dominant character in these plants. The styles of these unusual petunias illustrate the participation of the pollen tube in determining PSC.Scientific Journal Series Paper Number 10.479 of the Minnesota Agricultural Experiment Station     

Interactions of S alleles in sporophytically controlled self incompatibility of Brassica

Summary The expressed activity in pollen and stigma was determined for both S alleles of sixteen S-alíele heterozygous genotypes and for one of the two S alleles of two additional heterozygotes. Activities were measured using pollen tube penetration and seed set data from reciprocal crosses between each S-allele heterozygote and its two corresponding S-allele homozygotes.In pollen the S-allele activities ranged from zero to 100% inhibition of pollen tube penetration and seed set, and in the stigma they ranged from 8 to 100% inhibition. Of the sixty-eight S-allele activities measured, thirty-three (48%) were 90 to 100% inhibition, nine (13%) were 80 to 89% inhibition and one to five were within each ten-unit range below 80% inhibition.In an S-allele heterozygote, each subset of two S alleles had an activity for each allele in both pollen and stigma which was highly repeatable among duplicate pollinations within and among successive years. Each subset of two S alleles had a specific S-allele interaction in the pollen, and the same or another specific interaction in the stigma. In pairings with six other S alleles, allele S ₂ had four calculated levels of activity in pollen that ranged from 88 to 94%, and five levels in the stigmas between 15 and 94%. When paired in a heterozygote, alleles S ₃ and S ₅ had activities ranging between 42 and 59%, representing mutual weakening of S-allele activity. Also, heterozygote S ₁₅ S ₃ had pollen activities, respectively, of 25 and 6%, i.e. mutual weakening in the pollen.These results indicate that in heterozygous combination with a series of other S alleles, each S-allele may have activity in pollen and also in stigma that potentially is between zero and 100% inhibition. They further indicate that the defined sexual-organ X S-allele-interaction Types I, II, III and IV are extremes; all intermediate variations including complete weakening of both alleles are possible. Recessiveness is weakening of the activity of but one of the two S alleles. The pollen tube penetrations into the style and seed set were highly correlated.Department of Plant Breeding and Biometry Paper No. 683     

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     

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.