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.     

An S-allele survey of cabbage (Brassica oleracea var. Capitata)

Summary A total of 31 S-alleles was found in a survey of 197 cabbage plants representing 11 cultivars of diverse type. Most of these S-alleles also occurred in either kale or Brussels sprouts, but five of them have not been found previously and apparently occur only in cabbage. A more detailed study of five cultivars of spring cabbage showed only 12 S-alleles in all, with 6–10 S-alleles in four older cultivars and only 3 S-alleles in the newer more highly selected cultivar. S2 was by far the commonest S-allele, as it is in B. oleracea as a whole. The highly recessive alleles S5 and S15 were not particularly common in cabbage and this may partly explain why the sib problem in F₁ hybrids is apparently less in cabbage than in Brussels sprouts. Three cases were found in which an S-allele was completely recessive in both the stigma and the pollen. The problems for the breeder created by this rather unusual situation are discussed.     

Inbred lines with dominant incompatibility alleles for use as parents of F1 hybrid brussels sprouts

Summary To improve the chances of obtaining highly self-incompatible inbred lines for use as parents of F₁ hybrid Brussels sprouts and to extend the range of mutually cross-compatible combinations available, new inbred lines are being produced from plants selected for the presence of dominant S-alleles. The material comprises 42 different inbred families representing 15 cultivars and contains 12 dominant S-alleles of uncommon occurrence in Brussels sprouts. Data on S-allele interactions in the material are presented.Tests showed that whilst many of the parent plants containing dominant S-alleles were highly self-incompatible, a few had only weak self-incompatibility. Although the chances of obtaining strongly self-incompatible inbreds may be increased by using material with dominant S-alleles, it remains necessary to test and select for strong self-incompatibility during the breeding programme.Production of such inbred lines requires the application of two independent selection procedures, one for agronomic type and one for S-allele constitution. Only a small proportion of the plants of a parent cultivar are acceptable on both counts. Thus large populations of the cultivar and large numbers of selections are necessary: this in turn involves much expensive S-allele screening. To alleviate this problem a wide-based panmix containing only dominant S-alleles is being produced. It is hoped that from this it will be possible to extract inbred lines which carry only dominant S-alleles, so avoiding the need to screen each parent plant for its S-allele content.     

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.     

Assessment of serological techniques for S-allele identification in Brassica oleracea

Summary Eleven rabbits were injected with stigma homogenates of Brussels sprout, cabbage and kale. They varied considerably in their reactions; only four produced S-protein-antibodies, and two of these only after booster injections. Seven different S-alleles were involved in the studies and the four which gave S-protein-antibodies were intermediate (S₂ and S₄₅) and high (S₁₆ and S₂₃) in the dominance series. The highest titre of S-protein-antibody was 1/48, produced to the S₁₆ protein. However, results of double diffusion tests were clearest when its titre was 1/16 as the lengthened injection schedule led to reduced specificity. The other S-protein-antibodies were of much lower titre. Without further improvement the method is unsuitable for routine S-allele identification.     

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.     

Dominance relationships between S-alleles in the stigmas of Brussels sprouts (Brassica oleracea var. Gemmifera)

Summary Partial dominance between S-alleles in the stigma was found to be common in 412 plants of Brussels sprouts in which two S-alleles could be detected. Amongst a further 168 plants in which only one S-allele could be detected, 5 plants showed the detected allele to be completely dominant to the other allele present, but further examples are expected. Dominance relationships are given for 34 pairs of S-alleles representing 16 different S-alleles. The most recessive S-alleles in the stigma appear to be S5, S14, S15 and S45, while the most dominant are S23, S29, S35, S36 and S39. The significance of dominance between S-alleles in connection with the breeding system of Brussels sprouts is discussed, and an explanation proposed for the high frequency of S2 based on its unusual dominance behaviour.     

A molecular technique for selection of self-compatible varieties of Japanese pear (Pyrus pyrifolia Nakai)

The pear cultivar ‘Osa-Nijisseiki’ (S₂S^sm ₄; sm = stylar-part mutant) has been used as a parent to breed self-compatible cultivars that produce excellent fruits. However, determination of the self-compatibility of ‘Osa-Nijisseiki’ offspring requires a lot of time, 6 years or more, by conventional cross breeding. We have designed a rapid reliable method for the identification of self-compatible varieties of ‘Osa-Nijisseiki’ offspring based on the polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) with S-allele specific restriction endonucleases. By using this method, 8 self-compatible varieties were selected among 16 selections resulting from a cross between the self-compatible cultivar ‘Osa-Nijisseiki’ (S₂S^sm ₄) and the self-incompatible cultivars ‘Niitaka’ (S₃S₉), ‘Whasan’ (S₃S₅), ‘Chuwhangbae’ (S₄S₆). The S-genotypes of 16 ‘Osa-Nijisseiki’ offsprings were also determined. This revised version was published online in August 2006 with corrections to the Cover Date.     

Self-incompatibility in Brassica napus: seed set on crossing 19 self-incompatible lines

Summary Seed set after self-pollination confirmed that 19 lines of Brassica napus were self-incompatible. Eight lines, H, J, Q, W, X, K, P and Z, were fully cross-compatible. Line R was cross-compatible with these lines but often had a low seed set as female parent. These results are consistent with the activity of nine distinct S-alleles. Line S was cross-incompatible with K, as was V with P, and F with Z. With each of the lines A, E, B, O, G, L and M at least one reciprocal difference was found so that the number of additional distinct S-alleles could not be inferred, but there must be a minimum of seven.Pollen tube counts of intra- and inter-line pollinations using M, B and X confirmed the homozygosity of these lines with respect to self-incompatibility status and the presence of non-reciprocal compatibility between M and B.The results are interpreted in terms of the activity of both B. oleracea and B. campestris S-loci and the implications for hybrid breeding are discussed.     

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.     

Identification and breeding behaviour of a major deletion of chromosome 5D ofTriticum aestivum

Summary A study was undertaken to evaluate the breeding behaviour and to identify a spontaneously produced putative chromosomal deletion in the winter wheat (Triticum aestivum L. em. Thell.) cv Norstar. Male and female transmission studies of plants heterozygous for the deletion chromosome indicated 9.5% and 48.8% transmission through the pollen and the egg, respectively. Meiotic analyses of progeny from deletion heterozygotes indicated that the deletion chromosome was eliminated from half of the plants, which agreed with the male and female transmission frequencies. Testcrosses of the deletion chromosome with telocentrics and nullisomic-tetrasomic combinations suggested that the deletion involved the long arm of chromosome 5D. This was confirmed by restriction fragment length polymorphism (RFLP) analysis. Also, monosomic plants obtained in progeny of deletion heterozygotes were shown to be monosomic for 5D. Studies of plants homozygous for the deletion showed relatively normal pairing between the deletion chromosomes, and with the short arm (5DS), but not the long arm (5DL). Deletion homozygotes were self-sterile, and morphologically similar to plants nullisomic for 5D, but plants that also contained 5DL, or a homoeologous chromosome were self-fertile and had normal morphology. Studies of chromosome morphology indicated that the deletion chromosome was metacentric, and the length of the long arm was reduced by approximately 60%. RFLP studies showed that, in terms of genetic distance, 90% of the arm was missing.     

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.     

The effect of alternating temperatures on the self-incompatibility of some clones of Brussels sprouts (Brassica oleracea L. var. Gemmifera (DC.) Schulz)

Summary The influence of temperature on self-incompatibility of six S-homozygous clones of Brussels sprouts was studied. The clones were treated with constant temperatures of 14°, 17° and 20°C and alternating day and night temperatures of 17/14°, 20/14°, 23/14° and 26/14°C. To determine the degree of incompatibility the mean number of pollen tubes per style after selfing was calculated.The clone with the weak S-allele S-5 was less self-incompatible in the 23/14° and 26/14°C treatments than in the other treatments. The other clones with the weak S-alleles S-15 and S-45 remained sufficiently self-incompatible during all treatments. The clone with the rather strong S-39 showed the highest level of self-incompatibility at 26/14°C.In the two weakly self-incompatible clones with S-5 and S-39 a clear difference in self-incompatibility was found between young and old flowers. The strongly SI clones with S-39 showed no difference and in the two clones with S-15 the differences were small and significant in one case only.     

Identification and polymorphism of cultivars and wild ecotypes of safflower based on isozyme patterns

Summary Acid phosphatase and cathodal peroxidase isozyme patterns were used to identify nine introduced and five local cultivars of common safflower (Carthamus tinctorius L.) and seven ecotypes of wild safflower (C. oxyacantha Bieb.). Nineteen and nine bands, respectively, were readily observed for the phosphatase and peroxidase systems in the extracts from seedling shoots. Results indicated that both systems could be used jointly for complete identification of all the 21 seed sources used.Band frequencies and polymorphic indices (PI) were determined from acid phosphatase patterns using at least 92 individual seedlings of five introduced and five local cultivars and three wild ecotypes. Band frequencies ranged from complete absence in some to total presence in other populations. The mean PI for the introduced, local and wild safflowers were found to be 0.069, 0.052 and 0.058, respectively.The banding patterns of either enzyme system were similar in some instances for the cultivated and wild safflowers and there was as much variation between two cultivars as between a cultivar and a wild ecotype, indicating close genetic relationships between C. oxyacantha and C. tinctorius.     

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.     

Identification of broad bean cultivars based on isozyme patterns

Summary With the increasing rate of new cultivar production for different crop plants, there is great need for methods of identifying each cultivar discriminatingly. Starch gel electrophoresis was employed to study the differences between the esterase and cathodal peroxidase isozyme patterns of 40 broad bean (Vicia faba L.) cultivars. A total of 10 and 17 medium to darkly stained bands were obtained for esterase and peroxidase systems, respectively. Bands from each enzyme system could be gropuped into three zones. Bands belonging to zone 1 of esterase (E₁) and zones 2 and 3 of peroxidase (P₂ and P₃) were quite distinct, stained intensely, and were especially useful for identification purposes.The differences in banding patterns among cultivars of the same origin were as great as those of cultivars of unrelated origin. A large proportion of the cultivars could be completely differentiated using both of the isozyme systems. There were no bands present in either enzyme system which were common to all cultivars.     

Identifying the alien chromosomes in wheat – Leymus multicaulis derivatives using GISH and RFLP techniques

Genomic in situ hybridization (GISH) and restriction fragment length polymorphism (RFLP) were used to identify the Leymus multicaulis (XXNN, 2n = 28) chromosomes in wheat-L. muliticaulis derivatives. Fifteen lines containing L. multicaulis alien chromosomes or chromosomal fragments were identified. All alien chromosomes or fragments in these 15 lines were from the X genome and none were from the N genome. Eleven L. multicaulis disomic addition lines and four translocation-addition lines were identified with chromosome rearrangements among homoeologous groups 2, 3, 6 and 7. Only homoeologous group 1 lacked rearrangements in addition or translocation chromosomes. The results revealed that translocation in non-homoeologous chromosomes widely exists in the Triticeae and therefore it is necessary to identify the alien chromosomes (segments) in a wheat background using these combined techniques. During the course of the work, probe PSR112, was found to detect X genome addition lines involving L. multicaulischromosomes. This may prove to be a valuable probe for the identification of alien chromosomes in a wheat background. This revised version was published online in August 2006 with corrections to the Cover Date.     

Isoenzyme and cotyledon protein variation for identification of black beans (Phaseolus vulgaris L.) with similar seed morphology

Summary Methods developed to identify genetically diverse varieties of beans (Phaseolus vulgaris L.) were applied to closely related lines that were difficult to distinguish on the basis of seed morphology. Seedling tissues and seeds of black beans, were examined electrophoretically for isoenzyme and cotyledon proteinn protein patterns. Seven enzymes, extracted from seeds or from seedling stem, root or leaf tissues, were compared for polymorphism. Peptidase, polyphenol oxidase, phosphoglucoisomerase and glutamate oxaloacetate transaminase patterns were the same for all lines. Some differences were observed for acid phosphatase, peroxidase and esterase patterns, but complete discrimination of the six selected lines was not possible on the basis of isoenzyme patterns alone. Using polyacrylamide gel electrophoresis (PAGE) of dissociated 0.1 M acetic acid soluble proteins at pH 3.1 (acid-PAGE), or sodium dodecyl sulphate PAGE (SDS-PAGE) of residual protein extracts, all but one pair of samples in each case could be distinguished from the other samples. Using both techniques all of the lines could be identified unequivocally.     

Association of RFLP markers with trait loci affecting clubroot resistance and morphological characters in Brassica oleracea L.

Summary Resistance to Plasmodiophora brassicae Wor. race 7, the causal agent of the disease clubroot, was examined in an F₂ population of a cross between a clubroot resistant broccoli (Brassica oleracea var. italica) and a susceptible cauliflower (B. oleracea var. botrytis). A genetic linkage map was constructed in the same population based on the segregation of 58 dispersed restriction fragment length polymorphism (RFLP) markers. Associations between the inheritance of RFLP marker genotypes and segregation for disease resistance, morphological and maturity characteristics were examined. For each triat examined, several chromosomal regions marked by RFLP probes appeared to contain trait loci, suggesting that each trait was under polygenic control. RFLP marker linkage to a major factor imparting dominance for clubroot resistance from the broccoli parent was observed in this population. Additionally, RFLP marker linkage to an independently segregating factor contributing clubroot resistance from the cauliflower parent was observed, indicating that it should be possible to use RFLP markers to facilitate selection of transgressive segregants having the combined resistance from both parental sources. In some instances, RFLP markers from the same or closely linked chromosomal regions were associated with both clubroot resistance and morphological traits. Analysis of RFLP marker genotypes at linked loci should facilitate the selection of desired disease resistant morphotypes.