Identification of self-incompatibility alleles and pollen incompatibility groups in sweet cherry by PCR based s-allele typing and controlled pollination

A total of 17 pollen incompatibility groups in sweet cherry (Prunusavium L.) were identified among 46 accessions by PCR based S-alleletyping analysis and by controlled test pollinations. Two putativeS-alleles different from S ₁ to S ₆,S _z and S _y were identified. Five S-genotypes, S ₁ S ₅, S ₁ S ₆,S ₂ S ₆, S ₄ S ₆, andS ₅ S ₆, combinations of S ₁ toS ₆ alleles that had not previously been identified from cultivars in NYSAES, were positively confirmed by PCR based S-genotyping analysis. Also, the S-genotypes of cultivars in some pollen incompatibility groups that had previously been incorrectly reported have been clarified. Several popular cultivars, which were previously used as testers for S-allele typing analysis, were found to have been inaccurately genotyped. In addition, the S-genotypes and self-incompatibility groups of some relatively recentlyintroduced cultivars were identified. The molecular typing system ofS-genotypes based on PCR is a useful and rapid method for identifying newS-alleles and incompatibility groups in sweet cherry.     

Correlation of stylar ribonuclease zymograms with incompatibility alleles in sweet cherry

Summary Protein stylar extracts of 16 cultivars of sweet cherry (Prunus avium), from the 10 different incompatibility groups to which incompatibility alleles have been assigned, were separated on acrylamide gels using isoelectric focusing (IEF) and were stained for ribonuclease activity. When two cultivars from the same incompatibility group were analyzed they gave identical zymograms and the cultivars of the 10 different incompatibility groups gave in all eight distinct zymograms. The ribonuclease polymorphism could be correlated with the reported S allele constitutions of the cultivars. Three ribonuclease bands were identified that each consistently corresponded to one of the six known incompatibility alleles (S ₁, S₂ and S ₆), a fourth band apparently corresponded to S ₃ and to the combination of S ₄ and S ₅, and a fifth band to S ₄ and S ₅ in other combinations. Thus, it seems that S alleles of cherry have ribonuclease activity and that IEF is useful for distinguishing S allele constitutions. The ribonuclease pattern of Summit, a cultivar of unknown incompatibility group, indicated its incompatibility genotype to be S ₁S₂, and this was confirmed by controlled pollination. The same band corresponded to S ₄ and S ₄', the mutant allele in self-compatible cultivars. IEF and ribonuclease staining promise to be useful tools for exploring the incompatibility relationships of cherry cultivars and perhaps of other self-incompatible Prunus crops.     

New self-incompatibility alleles in apricot (Prunus armeniaca L.) revealed by stylar ribonuclease assay and S-PCR analysis

Apricot (Prunus armeniaca L.) shows gametophytic self-incompatibility controlled by a single locus with several allelic variants. An allele for self-compatibility (S_C) and seven alleles for self-incompatibility (S₁–S₇) were described previously. Our experiments were carried out to ascertain whether the number of allelic variants of apricot S-locus was indeed so small. Twenty-seven apricot accessions were analysed for stylar ribonucleases by non-equilibrium pH gradient electrofocusing (NEpHGE) to determine their S-genotype. To validate the results of electrofocusing, the applicability of the S-gene-specific consensus PCR primers designed from sweet cherry sequences was tested. NEpHGE revealed 12 bands associated with distinct S-alleles in newly genotyped cultivars. Cherry consensus primers amplified 11 alleles out from 16 ones, which indicated that these primers could also recognize most of the S-RNase sequences in apricot, and provided an efficient tool to confirm or reject NEpHGE results. By combining the protein and DNA-based methods, complete or partial S-genotyping was achieved for 23 apricot accessions and nine putatively new alleles (provisionally labelled S₈–S₁₆) were found. Their identity needs to be confirmed by pollination tests or S-allele sequencing. This study provides evidence that similarly to other Prunus species, the S-locus of apricot is more variable than previously believed.     

Identification of self-incompatibility alleles in pear cultivars (Pyrus communis L.)

Self and cross-incompatibility determination by means of fruit and seed set experiments or pollen tube growth observations in the style has been frequently reported to be unclear in pear (Pyrus communis L.). Thus,in order to develop a reliable in vivo method to test pollen-pistil incompatibility in pear, pollen tube performance has been studied along the pistil following self and cross-pollinations. Results show that, while pollen tube growth in the style may be an unclear test, ovule observation at the microscope for the presence of pollen tube in the nucellus is a proper method to test incompatibility in this crop. With this analysis we could identify S-alleles of ‘Williams’ (S₁S₂) and ‘Coscia’(S₃S₄), and three of the four possible S-genotypes resulting from the ‘Williams’ × ‘Coscia’ cross, as represented by ‘Butirra Precoz Morettini’ (S₁S₃), ‘Santa Maria Morettini’ (S₂S₃)and ‘Tosca’ (S₁S₄). This result demonstrates that ‘Williams’ and ‘Coscia’ cultivars do not share any allele in common. We also established two new inter-incompatibility groups in pear. Furthermore, the presence of a common allele between ‘Williams’ and ‘Agua de Aranjuez’,and ‘Coscia’ and ‘Agua de Aranjuez’, three apparently unrelated old cultivars, may indicate a narrower genetic base than expected for European pear. This finding together with the fact that 40% of new released cultivars have direct or indirect parental relationship with the cultivars ‘Coscia’ and/or ‘Williams’, anticipates the possibility of new cases of cross-incompatibility for this crop in the future. Both the method described and the determination of the S-genotypes will facilitate the characterisation of self and cross-incompatibility relationships in this species. This revised version was published online in August 2006 with corrections to the Cover Date.     

Determination of self-incompatible genotypes in sweet cherry (Prunus avium L.) accessions and cultivars of the German Fruit Gene Bank and from private collections

Sweet cherries are self-incompatible because of a gametophytic self-incompatibility system. S alleles in the style and pollen determine the crossing relationships. Knowledge of the S allele constitution of cultivars is very important for cherry growers and breeders, and recently, molecular methods have been developed to distinguish the S alleles in sweet cherries. The S allele genotypes of 149 sweet cherry cultivars and clones, including 126 not previously genotyped, were determined by using PCR analysis. Thirteen different S alleles in 40 combinations were distinguished and nine new incompatibility groups were documented. Two new S alleles were identified in five local sweet cherry processing cultivars from southwestern Germany using the second intron primers. The sequence of these alleles was determined and compared to all known sequences available in the NCBI database. The sequences obtained showed high similarities to the alleles S ₁₉ and S ₂₂, previously described only in wild cherries, Prunus avium L.     

Inheritance of tree and fruit characters in progenies from crosses of sweet cherry (Prunus avium L.) cultivars

Summary Approximately 1000 seedlings from 20 combinations crossed in 1979, 1980 and 1981 (Theiler-Hedtrich 1985a) were tested for several characters: fruit set (yield), fruit size, fruit colour, formation of abscission layer and bleeding after fruit removal from fruit stalks, bacterial canker resistance, flowering and harvesting time. From progeny of crosses with Stella as pollinator, 56% (Vittoria × Stella) and 46% (Schüttler × Stella) of the seedlings were self-compatible, of which 14 were high yielding with good fruit size and quality. From the data recorded it can be concluded:fruit set is a recessive character; only 5 to 20% of very good yielding seedlings were obtained in different progeny, even if the parental plants were both very good croppers.Fruit juice and skin colour was in most progenies ‘black’ even if they were from combinations with ‘white’ varieties, e.g., Merton Glory or Schüttler. Only from the combination Schiittler (‘white’) × Stella (‘black’), 50% of the seedlings were ‘white’; Stella therefore is heterozygous for the character of fruit juice and skin colour.Fruit size is evenly distributed in progeny with respect to the fruit size of their parent plants.Abscission layer formation and non-bleeding is a genetically complex character. In combinations where both parent plants formed fruits with complete abscission layers and which were not bleeding after fruit removal from the stalk, this character was inherited only to 50% (Vittoria × Schüttler) or 85% (Vittoria × Frühe von der Weid) in the progeny. For the genetical control of this character further studies are necessary.Bacterial canker susceptibility was evenly distributed in seedlings from all combinations even if the highly resistant cv. Vittoria was used as one parent plant, thereby not confirming the expected results of a higher proportion of resistant seedlings from combinations with Vittoria.Flowering and harvest time of the seedlings from different combinations was within the range of the parent plants. Only in the combination of Vittoria × Stella (mid-to late-ripening season) one seedling out of 99 was found to form ripe fruits two weeks earlier than the parental plants. From the seedlings tested 40 have been chosen for further evaluation or genetical studies.     

Improved discrimination of self-incompatibility S-RNase alleles in cherry and high throughput genotyping by automated sizing of first intron polymerase chain reaction products

A novel polymerase chain reaction (PCR) approach to determine and confirm the self‐incompatibility (S) genotype of cherries is reported. The method involves PCR amplification with a new pair of consensus primers that immediately flank the first intron of cherry S‐RNases, one of which is fluorescently labelled. Fluorescent amplification products range from 234 to c. 460 bp and can be sized accurately on an automated sequencer. Thirteen S alleles reported in sweet cherry can be distinguished, except for S₂ and S₇, which have an amplification product of exactly the same size. S₁₃, which is also amplified, gives a microsatellite‐like trace which shows minor intra‐allelic length variation. This method gives fast and accurate results and should be especially useful for medium/high‐throughput genotyping of wild and cultivated cherries.     

Sequence of the S 9-RNase cDNA and PCR-RFLP system for discriminating S 1- to S 9-allele in Japanese pear

A new S ₉-allele was discovered in 6 Japanese pear cultivars, ‘Shinkou’, ‘Shinsei’, ‘Niitaka’, ‘Amanogawa’, ‘Nangetsu’ and ‘Nansui’. cDNA encoding S ₉-RNase, a stylar product of S ₉-allele, was cloned from pistils of ‘Shinkou’ and ‘Shinsei’ by 3' and 5' RACE. The S ₉-RNase gene had an open reading frame of 684 nucleotides encoding 228 amino acid residues. S ₉-RNase had a hypervariable (HV) region different from S ₁- to S ₈-RNase and shared higher similarity (95.2%) with apple S ₃-RNase than with 8 Japanese pear S-RNases (from 61.0% to 70.7%). Genomic PCR with primers ‘FTQQYQ’ and ‘anti-(I/T) IWPNV’ provided S ₁- to S ₉-amplicon (product), but could not discriminate the S ₂ from the S ₉ of ca. 1.3 kb. The S ₂ and S ₉ were distinguished by digestion with AflII and BstBI, respectively. The digestion with nine S-allele-specific restriction endonucleases, SfcI, AflII, PpuMI, NdeI,AlwNI, HincII, AccII, NruI and BstBI, distinguished S ₁ to S ₉, establishing that this PCR-RFLP system is useful for S-genotype assignments in Japanese pear harboring S ₁- to S ₉-allele. ‘Shinkou’, ‘Shinsei’, ‘Nangetsu’ and ‘Nansui’ assigned as S ₄ S ₉ were determined to be cross incompatible. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification of self-incompatibility alleles (S) by PCR-RFLP in radish (Raphanus sativus L.)

From 16 inbred lines of cultivated radishes (Raphanus sativus L.), 6 S-alleles tentatively named S²⁰¹ to S²⁰⁶ were identified, and their dominance relationships were examined. Among the S-alleles, S²⁰¹, S²⁰², S²⁰³ and S²⁰⁴ were found to be co-dominant. These 4 S-alleles showed dominance with S²⁰⁵ in pollen and with S²⁰⁶ in both pollen and stigma, while S²⁰⁵ and S²⁰⁶ were co-dominant. Polymerase chain reaction (PCR) was performed using the radish inbred lines randomly selected from the 6 S-allele groups. The primers were based on the highly conserved sequences of the S-locus specific glycoprotein (SLG) genes in Brassica oleracea. As a result of the PCR, a single DNA fragment of about 1.16kb was amplified as expected from the original sequence of B.oleracea. The S-allele specific pattern in the restriction fragments of the PCR products (PCR-RFLP) was confirmed for the first group of S-alleles (S²⁰¹, S²⁰², S²⁰³ and S²⁰⁴). However, for the second group of the S-alleles (S²⁰⁵ and S²⁰⁶), no PCR products were obtained. The usefulness of the PCR-RFLP in a radish breeding program is described. This revised version was published online in July 2006 with corrections to the Cover Date.     

Analysis of genetic relationships among 22 European barley varieties based on two PCR markers

Two long primers of 19 (F17) and 20 (F13) nucleotides, respectively,were used in polymerase chain reactions to amplify DNA from differentcultivated barley accessions. These primers can distinguish closely relatedvarieties and, with a unique primer, all the barley accessions analysedshowed a characteristic fingerprint. Sixty per cent and 76% of thefragments generated using F13 and F17, respectively, were polymorphic.The genetic similarity values between accessions were estimated from F13and F17 data. The dendrogram and principal coordinate analysis performedwith F13 data revealed a clear separation of these varieties in accord withtheir pedigree relationships.     

Osmoregulation in birdseed millet under conditions of water stress II. Variation in F3 lines of Setaria italica and its relationship to plant morphology and yield

We report, for the first time, there generation of four homozygous lines in sweet cherry (Prunus avium L.) byin situ parthenogenesis followed by embryo and cotyledon culture. The sweet cherry cultivar ‘Altenburger’ was pollinated with marked pollen irradiated by γ-rays at doses ranging from 250 to1200 Gy. Pollination with such irradiated pollen affected fruit set and the quality of the embryos, and induced the formation of parthenogenic embryos. The immature embryos extracted from the stones, 35 or 75days after pollination, were cultivatedin vitro in an embryo or cotyledon culture. Although flow cytometrical analysis demonstrated the diploid level for all regenerants, four lines could be characterized as homozygous using isoenzyme analysis. This revised version was published online in July 2006 with corrections to the Cover Date.     

The myrobalan (<Emphasis Type="Italic">Prunus cerasifera</Emphasis> L.): a useful diploid model for studying the molecular genetics of self-incompatibility in plums

A series of PCR methods were used to detect S-RNase alleles and SFB alleles and to determine S-genotypes in 25 accessions of myrobalan (Prunus cerasifera L.). Firstly, primers flanking the polymorphic second intron were used to identify S-RNases in agarose gels. These primers amplified one or two bands per accession in 25 accessions. Then consensus primers were designed for amplifying the polymorphic first intron, unique to Prunus S-RNases, for automated fluorescent detection. Each accession produced one or two peaks. New primers were then developed to amplify the intron in the SFB gene, for detection by fluorescence. Cross-referencing PCR bands and peaks indicated 15 S-alleles were present in the 25 accessions. Cloning, sequencing and comparison with published data indicated that the amplified products were S-RNase alleles. Sequence information was used to design primers specific for each S-RNase. Full and consistent S-genotypes were obtained by cross-comparing PCR data for 23 of the 25 accessions, and two accessions appeared to have a single allele. Pollen-tube microscopy indicated function of some but not all of the S-alleles sequenced.     

The exploitation of genetic resources of Brassica oleracea in breeding for resistance to clubroot (Plasmodiophora brassicae)

Summary About 1000 Brassica oleracea accessions were evaluated in glasshouse tests for response to Plasmodiophora brassicae (clubroot). Resistance was confirmed in some north and west European kales and cabbage. A new source of resistance in cabbage, from Eire, is reported. Most other accessions were highly susceptible but lower levels of susceptibility were observed in open pollinated Brussels sprouts and forms of south European cabbage, cauliflower and broccoli. Modern breeding (as in the production of hybrid cultivars) appears to have resulted in increased susceptibility in several crop types. The implications of these results for the exploitation of germplasm are discussed.     

Occurrence of self-compatibility, self-incompatibility and unilateral incompatibility after crossing diploid S. tuberosum (SI) with S. verrucosum (SC): I. Expression and inheritance of self-compatibility

Diploid Solanum tuberosum (tbr), 2n=2x=24,can be crossed with S. verrucosum (ver) only when the latter is used as a pistillate parent but not reciprocally. This conforms to the phenomenon of unilateral incompatibility (UI) where a self-compatible species, like ver (SC) cannot be used as a male parent to cross with a self-incompatible (SI) parent like tbr. Even if ver × tbr hybrids are made, the F1 hybrids possess cytoplasmic male sterility and thus hinder genetic analysis of crossing barriers. Exceptionally, however, some diploid genotypes of tbr (SI) can be used as pistillate parents to cross with ver, and such exceptional tbr clones are called `acceptors'. Repeated backcrossing of acceptors to ver have resulted in male fertile genotypes that possess tbr cytoplasm and ver nucleus. These genotypes were used for the genetic analysis of `acceptance' and UI in thse experiments. It was found that acceptance of ver-pollen by tbr-pistils is based on a dominant gene A that expresses only in the absence of an inhibitor I. In the F₁ hybrids, only the S-allele of tbr was expressedbut not that of ver. Concomitant with this observation, it was shown that ver does not produce style-specific S-glycoproteins that are responsible for self-incompatible reaction in diploid potato. Although the the F₁ populations were SC, they segregated into SC and SI genotypes giving skewed segregation ratios for this trait. Because of this as well as the disappearance and re-appearance of SC trait in the offspring generations, it was necessary to postulate a more complex interaction between A and I. Models are presented in order to explain acceptance, non-acceptance and the expression of UI. It is concluded that at least four different loci are involved in the expression of UI. This revised version was published online in July 2006 with corrections to the Cover Date.     

Variation in digestibility in nine populations of perennial ryegrass (Lolium perenne) and its genetic basis

Summary Thirty five individuals from each of nine perennial ryegrass populations were sampled from set-stocked and zero-grazed swards and from an old permanent pasture. The swards were 6, 18, 30 months and 11 years old when sampled. The permanent pasture was at least 40 years old.Dry matter digestibility (DMD) and organic matter digestibility (OMD) were determined for green leaf material harvested 4 days after ear emergence. Significant differences were found between the populations, but genotypic differences were found only for OMD. The largest proportion of the total variation was due to differences between blocks. In the zero-grazed populations, DMD and OMD declined significantly with increasing sward age. This was not found for the set-stocked swards. Two sets of pair crosses, from which mid-parent, progeny regressions could be determined, were carried out.The regressions of offspring on mid-parent for set-stocked and zero-grazed swards were used to assess the presence of additive genetic variation and to estimate narrow sense heritability of DMD. The coefficients of correlation, 0.122 and 0.226 respectively, were non-significant, indicating an absence of additive genetic variation.     

Identification of reciprocal translocations observed in several Melilotus species (subgenus Eumelilotus) by interspecific triple crossings

Summary Melilotus alba differs by a reciprocal translocation from 7 other species which are categorized into M. officinalis and M. dentata groups. The two species groups, however, remained to be studied in relation to their cytological relations because of early degeneration of hybrid embryo. Interspecific triple crosses were successfully made in order to examine whether or not the reciprocal translocations observed were of the same origin. When F₁ hybrids between M. alba and M. officinalis group were crossed with M. dentata group, about a half of hybrids plants were heterozygous for a reciprocal translocation while the remaining plants were normal chromosome pairing, showing a segregation ratio of 1:1. This result indicates that reciprocal translocations observed among the three groups are of the same origin. Accordingly, it is expected that M. officinalis group and M. dentata group have the same chromosomal constitutions.     

Cultivar identification and genetic relationship among selected breeding lines and cultivars in chickpea (Cicer arietinum L.)

Twenty two RAPD and 22 ISSR markers were evaluated for their potential use in determination of genetic relationships in chickpea (Cicer arietinum L.) cultivars and breeding lines. We were able to identify six chickpea cultivars/breeding lines by cultivar-specific markers. All of the cultivars tested displayed a different phenotype generated either by the RAPD or ISSR primers. Though ISSR primers generated less markers than RAPD primers, the ISSR primers produced higher levels of polymorphism (% of polymorphic markers per primer) than RAPD primers. A high level of within cultivar homogeneity was observed in chickpea. Cultivars/breeding lines originating from a common genetic background showed closer genetic relationship. Chickpea lines with similar seed type(kabuli or desi) had a tendency to cluster together. This revised version was published online in August 2006 with corrections to the Cover Date.     

Genetic analysis of resistance to barley scald (Rhynchosporium secalis) in the Ethiopian line `Abyssinian' (CI668)

A doubled haploid barley (Hordeum vulgare L.) population from a cross between the cultivar `Ingrid' and the Ethiopian landrace `Abyssinian' was mapped by AFLP, RFLP, SSR and STS markers and tested for resistance to isolates`4004', `2', `16-6', `17', `22' and `WRS 1872' of Rhynchosporium secalis (Oudem.) J.J. Davis, the causal agent of leaf scald. Resistance tests were conducted on parents, DH-lines, a near-isogenic line of `Abyssinian' (NIL) into `Ingrid', and an F₂ population descended from the same F₁ plants as the DHs. The DH population segregated for at least two major R. secalis resistance QTL. All isolates tested identified a major QTL on chromosome 3 (3H) associated with R. secalis resistance, in a 4 cM support interval between the co-segregating markers Bmac0209/Falc666 and MWG680. The QTL was linked with the markers Falc666 (2.3 cM), YLM/ylp (0.3 cM), MWG680 (1.7 cM), cttaca2 (2.5 cM) and agtc17 (9.8 cM). The second QTL was located on chromosome 1 (7H).However, this QTL was only detected by one isolate and was located in an interval of 16 cM in the distal part of the chromosome. At this QTL the allele for improved scald resistance originated from the parent `Ingrid'. There were a number of minor QTL on chromosomes 2 (2H), 4 (4H) and 6 (6H) that were not repeatable either across replications or analysis methods. The importance of checking QTL-models by cross-validation is stressed. This revised version was published online in August 2006 with corrections to the Cover Date.     

Traditional management of cassava morphological and genetic diversity by the Makushi Amerindians (Guyana, South America): Perspectives for on-farm conservation of crop genetic resources

In this paper we present original data on morphological and genetic diversity of cassava managed by the Makushi Amerindians from Guyana. Although they propagate cassava exclusively vegetatively by means of stem cuttings, many Amerindian farmers also use and multiply volunteer plants grown from seeds produced by sexual reproduction. Morphological characters were recorded for 29 varieties cultivated by the Makushi and two populations of plants originating from volunteer cassava seedlings. Genetic characterisation with AFLP markers was available for 21 of the examined varieties. The morphological and agronomic characters were highly variable among varieties. Every variety could be differentiated from any other one, except for one pair of varieties. However, high intra-varietal variability existed, which might lead to confusions between phenotypically similar varieties by the Makushi. Seedlings were on average different from the pool of the varieties studied, but 67% were found to resemble closely enough one of the varieties to be liable to be assigned to it. Confusion between very similar varieties, as well as assignment of seedlings to a variety, should generate genetic variability within varieties, which was detected with AFLP markers. As in other sites in Amazonia, there was only a weak correlation between inter-varietal distances assessed with molecular and with morphological markers, suggesting that diversification of morphological characters has taken place repeatedly and independently across the Amazonian range of the crop. Diversifying selection, exchanges of varieties between farmers, and incorporation of sexually produced volunteer plants are key mechanisms responsible for the high diversity observed. Strategies of conservation of genetic resources should take these dynamic processes into account. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic diversity evaluation of a loquat (<Emphasis Type="Italic">Eriobotrya japonica</Emphasis> (Thunb) Lindl) germplasm collection by SSRs and <Emphasis Type="Italic">S</Emphasis>-allele fragments

Loquat (Eriobotrya japonica (Thunb.) Lindl.) is a minor Rosaceae fruit of growing interest as an alternative to the main fruit crops. In this context, the selection of new cultivars to satisfy the market demand will request the suitable characterization of the available germplasm. In this work, genetic relationships among 83 loquat accessions from different countries belonging to the European loquat germplasm collection, held at the Instituto Valenciano de Investigaciones Agrarias (IVIA) in Moncada (Spain) were evaluated using microsatellites and S-allele fragments. A total of nine single sequence repeats (SSRs) from Malus and Eriobotrya genera revealed 53 informative alleles and the S-RNases consensus primers detected 11 self-incompatibility putative alleles. The combined data allow to distinguish unambiguously 80 out of the 83 accessions studied. Unweighted pair-group method (UPGMA) cluster and principal coordinates analysis (PCoA), based on Dice’s genetic distance, generally grouped genotypes according to their geographic origins and pedigrees. Discrepancies and similarities of the results obtained with other variability analysis, based on pomological traits or molecular markers, on the same loquat collection are discussed.