S-allele identification by PCR analysis in sweet cherry cultivars

Gametophytic self‐incompatibility, governed by the S‐locus, operates in sweet cherry. The knowledge of the S‐genotype of sweet cherry cultivars is therefore essential to establish productive orchards by defining compatible combinations. The isolation of sweet cherry S‐R Nases has allowed the use of different molecular techniques to characterize the S‐genotypes of sweet cherry cultivars. Previously, incompatibility group assignment could only be carried out on mature trees through pollination tests. In this work, PCR analysis with primers designed on the conserved sequences of sweet cherry S‐R Nases has been used to characterize the S‐genotype of 71 sweet cherry cultivars, including 26 cultivars whose S‐allele constitution had not been previously described. This approach has allowed the detection of alleles that had not been amplified by PCR before, to identify six putative new S‐alleles, to define three new self‐incompatibility groups and to compile the standards for a PCR‐based S‐allele typing method in sweet cherry.     

Selection of vigorous and fertile S-homo- and heterozygous tester clones from self-incompatible diploid potato, Solanum tuberosum L.

For the selection of diploid (2n = 2x = 24) potato (Solanum tuberosum) genotypes that are useful for the molecular and genetic analysis of the phenomenon of gametophytic self-incompatibility, three different types of basic populations were investigated. These populations were derived from three primary dihaploid clones, G609, G254 and B16, which possessed the S-allele combinations S1S2, S1S3 and S3S4 respectively. In order to select highly vigorous, profusely flowering, fertile and tuberising progenies, three types of populations, derived from the above mentioned diploid genotypes, were screened for performance and classified for the expression of self-incompatibility. Although the selection for well defined S-genotypes was sometimes complicated due to the occurrence of pseudo-compatibility and of a self-compatibilising factor, the use of a combination of criteria, viz., Iso Electric Focusing (IEF), pollen tube growth in the styles and the extent of berry and seed set made the selection of sufficient representatives of all six types of S-heterozygotes (S1S2, S1S3, S1S4, S2S3, S2S4 and S3S4) possible. After evaluating the strength of the self-incompatibility reaction in these heterozygotes, those with high expression were selfed, and intercrossed within their S-allele incompatibility group through the method of counterfeit pollination. In these progenies, well-performing S-homozygotes (S1S1; S2S2; S3S3; S4S4) for all four S-alleles with high expression of self-incompatibility were selected. As a result, all possible S-homo- and heterozygous genotypes with a predictable type of self-incompatibility are available and maintained both vegetatively and as botanical seed. The development of this material has paved the way for more critical analysis of molecular factors involved in self-incompatibility in diploid potato. This revised version was published online in July 2006 with corrections to the Cover Date.     

苹果自交不亲和基因型(S基因)研究进展

 苹果是蔷薇科一个典型的配子体自交不亲和果树树种。本文详细介绍了苹果S等位基因研究现状,系统介绍了基于PCR技术的苹果S 基因型鉴定的理论基础和主要方法,列出了迄今国内外应用这些方法鉴定出的500多个苹果品种的S 基因型。本文对于苹果品种授粉树的合理配置和杂交育种亲和性亲本选择具有重要的指导意义。     

The production of inbred lines of brussels sprouts with dominant S-alleles

Summary The best sources of Brussels sprout inbred lines with both good agronomic characters and high self-incompatibility are likely to be cultivars of reasonably good agronomic type which have not been too intensively selected. Comparison of three cultivars of different agronomic quality showed that the cultivars of poor and moderate quality had about 55% of plants with a dominant S-allele, but the most highly selected cultivar had only 25% of such plants. A programme of S-allele screening is suggested which incorporates the minimum number of tests required to determine whether or not a particular plant has a dominant S-allele. A survey of S-alleles present in commercial F₁ hybrids showed that the frequency of dominant S-alleles was only 19% in hybrids released prior to the end of 1971, but was 50% in hybrids released since 1971.     

Classification of S-alleles by their activity in S-heterozygotes of brussels sprouts (Brassica oleracea var. Gemmifera (DC.) Schultz)

Summary Over a period of many years, data on dominance relationships of S-alleles in Brussels sprouts were collected at URL and IVT. The level of activity of S-alleles in heterozygotes was assessed on the basis of the number of pollen tubes that penetrated into the stigma. 209 out of 210 possible combinations between 21 S-alleles were used for this investigation. The S-alleles were grouped separately for activity in pollen and style on the basis of their sensitivity to lose activity in S-heterozygotes. Besides S-allele interaction per se, activity was found to be influenced by environment and genetic background.Results suggest that in stigma, co-dominance is the normal pattern and that deviations are caused by factors other than S-allele interaction as such.In pollen, only three truly recessive alleles were found. Besides several combinations with mutual weakening in pollen, examples of independent weakening were found.     

The inheritance of partial self-compatibility in Brassica oleracea L.: Results from a half diallel homozygous for a moderately recessive S-allele

Summary In a study of partial self-compatibility in Brassica oleracea, flower number, seeded siliqua and seed production were recorded on self-and cross-pollinated inflorescences of the progenies of a half diallel between six in bred Brussels sprout plants homozygous for the same moderately recessive incompatibility allele S₄₅.On both self-and cross-pollinated inflorescences significant amounts of additively controlled genetic variation were found for seed set per flower. For cross-pollinated inflorescences this was also the case for the two components of seed set, seeded siliquae per flower and seeds per seeded siliquae, but for self-pollinated ones only seeded siliquae production showed significant additive variation. Considerable heterosis and gene interaction were always present and a simple additive dominance model did not explain the variation.Two of the parents transmitted lower levels of partial self-compatibility to their progenies and, in one of these, dominant genes appeared to be responsible. The most important feature determining the production of self seeds was found to be the number of flowering sites at which the incompatibility mechanism failed rather than the number of seeds produced at each site.     

Analysis of pollen tube growth in situ to investigate self-incompatibility in the wild potato Solanum commersonii

Summary Pollen tube growth was investigated in a diallelic crossing design with seven genotypes of the diploid wild potato species Solanum commersonii, accession O/S UR-9, CIP 762459. Pollen tube growth in the style was recorded using a combined quantitative and qualitative evaluation scale. Clear-cut differences in pollen tube growth behavior in compatible and in partially or completely incompatible crosses were detected. Diallelic crossing of the seven randomly chosen genotypes, intercrossing within two progeny families, and backcrossing of two progeny populations to the parents revealed the existence of a one-locus gametophytic system of stylar incompatibility. The S-allele status of all genotypes investigated was determined.     

Identification of cherry incompatibility alleles by microarray

We have developed a microarray for identification of sweet cherry incompatibility alleles. Using intron sequence information of the S-RNase gene, we have created a microarray chip that allows the specific recognition of the incompatibility alleles present in a cultivar. Most of the probes designed showed high specificity towards their alleles. In the original set of probes, cross-hybridization was observed between a few alleles with high sequence similarity. As our identification system is based on the combined hybridization information from both introns, we were able to identify false positive and unspecific probes which could be eliminated from our microarray. The optimized microarray was tested on cultivars with known alleles. The chip correctly identified all alleles tested. Furthermore, it was also possible to identify alleles in other cultivars where, so far, only one allele has been determined and also to determine in sour cherry the alleles originating from the sweet cherry parent. Our results demonstrate the great promise of microarray technology for this novel application.     

Development of self-incompatible Brassica napus: (III) B. napus genotype effects on S-allele expression

Use of self‐incompatibility (SI) as a pollination control method for Brassica napus hybrid production requires the development of a sufficient number of S‐alleles that are expressed consistently in a range of B. napus lines. Self‐incompatibility (SI) alleles have been transferred from Brassica oleracea and Brassica rapa into B. napus var. oleifera. An understanding of expression of these alleles in B. napus is essential for their commercial use. Four SI B. napus doubled haploids containing the B. oleracea S‐alleles S2, S5, S13 and S24 were crossed to three B. napus cultivars to measure the B. napus genetic background effect on S‐allele expression. A line x tester analysis indicated that the largest source of variation in the expression rate of SI was the S‐allele itself. The B. napus genotypes tested contained modifier gene(s), some that enhanced SI expression and others that inhibited SI expression. The B. napus Canadian cultivar ‘Westar’ generally had a negative effect on SI expression while the European cultivar ‘Topas’ had a positive effect on the B. oleracea S‐allele expression. The B. oleracea S‐allele S24 was very similar in expression to the B. rapa allele W1. The application of these results for the use of B. oleracea S‐alleles for hybrid production in B. napus is discussed.     

Identification of S-alleles in Brassica oleracea

Summary S-alleles of Brassica oleracea were identified using a method which is based on the amplification of S-sequences from genomic DNA, followed by digestion of the PCR products with selected restriction enzymes (PCR-RFLP). A study was made in which the same S-allele was present in the homozygous state in a range of different crop types. This showed that, with minor exceptions, characteristic restriction patterns were obtained, and therefore that it was possible to identify the S-allele. To test whether the method was also suitable for the identification of both the S-alleles present in heterozygotes, a number of S-heterozygotes together with an F₂ population were screened. The results showed that the standard method was not very reliable for the identification of both of the S-alleles. This is because firstly, one of the S-alleles may be amplified preferentially, and secondly, the restriction patterns are not unique to a particular combination of S-alleles. Finally, although it is not possible to identify unequivocally both S-alleles of heterozygotes using a standard technique, the procedure can be modified for particular combinations of alleles to enable the identification to be made.