A two-loci system of gametophytic incompatibility in Solanum phureja and S. stenotomum

Summary A two-loci gametophytic incompatibility system is indicated in our experiments involving hybrids between S. phureja accessions and between S. stenotomum and S. phureja. The S locus is epistatic to the R locus. An allele R _fi at the latter locus when present in homozygous condition, leads to female incompatibility, independent of the genotype of the pollen. The two-loci system is assumed to have evolved from doubling the chromosome number of ancestral species with one locus for incompatibility. The irregularities observed in the studies are tentatively ascribed to recombining modifier complexes. These modifiers may be maintained in cultivated species owing to their preponderantly vegetative propagation.     

Production of intraspecific aneuploids in the genus Solanum. III. Intraspecific aneuploids

Summary The triploids recovered from 4x×2x crosses in three Solanum species were very vigorous and although few seeds/fruit were obtained when the triploids were crossed to diploids, the extensive crossing programme produced sufficient seed of four species cph, chc, pnt and tar. The average seed set for the 3x-2x crosses was an extremely low 3.5 seeds/fruit.Approximately 90% of the progeny of the 3x–2x crosses were aneuploids with chromosome numbers of 2n=25–29. The frequency of the aneuploids in the three species that were studied was chc 93%, pnt 94% and tar 92%. The aneuploids of chc and tar were extremely vigorous and fertile and they were used as females in crosses to the marker stocks. The aneuploids of pnt were vigorous, but the crossability barrier of pnt prevented their use in crosses to the marker stocks. A number of the aneuploids produced seed upon being selfed, but the ability to produce self seed may be related to the pseudo-compatibility of the parental clones. In only 7 aneuploids was there an indication that the self fertility was due to overcoming the self-incompatibility barrier as a result of competition-interaction of the aneuploid gamete.Plants in the families from the triploid-diploid crosses had a tremendous amount of variation in morphological characteristics (leaf shape, size and color; berry shape, color and degree or verrucose spotting: and plant habit and vigor). A large portion of variation exhibited in these families was due to the normal genetic segregation of the heterozygous parents. It was impossible to distinguish the aneuploids from their diploid sibs especially those having only one or two additional chromosomes because they were as vigorous and fertile as the diploids.There were some preliminary indications of the existence of distinct morphological characteristics among the aneuploids (separate petals, long berries, and extreme verrucose berries). However, there was no indication that these traits were due to the dosage effect of the extra chromosome. If it should be determined that this was true, it would be extremely useful in associating genes with chromosomes and determining the phenotypic effects due to the presence of an additional chromosome.Cooperative investigations of the College of Agriculture and Life Sciences, University of Winconsin and the Agricultural Research Service, U.S. Department of Agriculture, Madison, Wisconsin. Supported in part by grants from the Research Committee of the Graduate School, University of Wisconsin.First and third author respectively, formally Graduate Research Assistant Department of Horticulture, University of Wisconsin, Madison, and Geneticist Agriculture Research Service, U.S. Department of Agriculture, University of Wisconsin, Madison. Present address: Department of Genetics. University of Wisconsin, Madison, Wisconsin 53706 and Head Breeding and Genetics Department, International Potato Center, Lima, Peru.     

Chlorophyll inheritance patterns and extent of natural self-pollination in timothy

Three hundred sixty-five S₁ and 234 S₂ seedling populations of timothy (Phleum pratense L.) were examined for their segregations for chlorophyll deficiencies. Arrays of both self-and open-pollination families indicated a lack of concentration at definite levels, with ratios expressed forming a continuous series from 4.6:1 to 572:1 for green: chlorophyll-deficient types in the S₁ generation. The S₂ populations examined ranged from 1.8:1 to 244:1. Arrays were very similar in their distributions.Sixty-three plants yielded chlorophyll-deficient types in both S₁ and S₀ populations. From these, it was calculated that 7 to 8 percent of natural self-pollination may occur in timothy.Except at two levels (28:1 and 143:1 tetradisomic) assumption of tetradisomic or simpler inheritance patterns resulted in lower calculated percentages of natural self-pollination than resulted from assumed hexasomic inheritance. No apparent explanation is available to account for these divergences from the regular pattern encountered.Results of cooperative work of the Crops Research Division, Agriculture Research Service, U.S. Department of Agriculture and the Wisconsin Agricultural Experiment Station, Madison.Research Agronomist, Crops Research Division, Agriculture Research Service, U.S. Department of Agriculture and Associate Professor, and Professor of Agronomy, Wisconsin Agricultural Experiment Station, respectively.     

Production of haploids of potato (Solanum tuberosum subsp. tuberosum) and their identification with electrophoretic analysis

Summary The frequency of haploid production following the interspecific pollination of eight tetraploid potato cultivars (Solanum tuberosum subsp. tuberosum) with Solanum phureja clone 1.22 was investigated. A total of 185 haploids were produced with an overall haploid frequency of 3.9 haploids/100 fruits. The haploid frequency was affected by the genotype of the maternal parent. Atlantic, ND860-2, Superior, Saginaw Gold, Spartan Pearl, Nooksack and Onaway had frequencies of 6.2, 5.1, 4.7, 3.9, 2.3 and 0.7 haploids/100 fruits, respectively. There were 60 and 57 haploids produced from Atlantic and Saginaw Gold, respectively, and no haploids were extracted from fruits of Lemhi Russet. Isozyme analysis and visual examination were performed independently to compare the efficiency of discriminating hybrids from haploids. Approximately 80% of total hybrids could be identified by electrophoretic analysis, while 77% were distinguished through visual examination. Pgm-2 ¹, which is unique in the clone 1.22 and absent from all seed parents, was found to be the most useful locus in hybrid identification and 50% of total hybrids could be distinguished by this allele. With similar rationale, Mdh-1 ¹ allozyme, which was absent in six of the eight parents, identified 37% of total hybrids. A combination of both visual and electrophoretic methods made hybrid identification even more efficient, with an average identification efficiency of 91%. A scheme was proposed to develop a new haploid inducer which would be homozygous for both Pgm-2 ¹ and embryo spot.     

Evidence for possible selective survival and function of gametes on progeny characteristics of Bromus inermis Leyss

Summary Approximately 85% of the S₁, S₂ and S₃ plants in 142 smooth bromegrass progenies were morphologically similar within lines. Progenies developed from diallel crosses possessed greater uniformity than was expected from hybridization of S₁ octaploid smooth bromegrass. Behaviors observed are interpreted on the basis of selective survival and functioning of gametes and can be explained by the fusion of like gametes.Results of cooperation between the Crops Research Division, Agricultural Research Service, US Department of Agriculture, and the Research Division, College of Agricultural and Life Sciences, University of Wisconsin, Madison, Wisconsin 53706.Geneticist (deceased).     

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.     

Development of self-incompatible Brassica napus: (II) Brassica oleracea S-allele expression in B. napus

Brassica napus var. oleifera varieties have traditionally been developed as open‐pollinated varieties. The successful introduction of several high‐yielding hybrids based on cytoplasmic male sterility or transgenic pollination control systems has generated interest in the development of new hybrid systems. Self‐incompatibility could be an additional useful pollination control system for B. napus if a sufficient number of S‐alleles could be developed in this species. The S‐alleles, S2, S5, S13, S24 and S39, were identified in five hybrids of B. oleracea var. italica and subsequently transferred to B. napus. Doubled haploid lines were produced for the self‐incompatible (SI) lines in B. napus and intercrossed to produce SI heterozygotes in order to study allele interaction. There was a greater incidence of interallelic dominance in the stigmas and pollen of B. napus than was reported for the S‐alleles in B. oleracea. Allele S24 exhibited the greatest degree of dominance over the other alleles tested, while allele S2 was generally recessive or codominant with other alleles. Self‐incompatible expression was very similar in the SI homozygotes and heterozygotes, thus no weakening of the SI trait in the heterozygote was observed. The implications of S‐allele interaction for the use of SI in B. napus are discussed.     

Primers amplifying a range of Prunus S-alleles

Although various consensus polymerase chain reaction (PCR) primers have been reported for identifying Prunus S‐alleles, they have been developed from and optimized on a limited set of alleles, which may limit their applicability to a broader allele range. To develop a primer set for use across the genus, degenerate consensus primers were designed from conserved regions of 27 S‐RNase sequences available from five Prunus species. The primers were tested in 15 previously genotyped cultivars of cherry, almond and apricot, representing alleles S₁ to S₆ in each crop and also S_c in apricot. Comparisons were made with previously published primers tested in the same 15 cultivars under reported reaction conditions. The new primers generated an amplification product for each of the 19 S‐alleles whereas those previously available amplified no more than 14. The primers will be useful for genotyping and genetic studies in cultivars and wild populations.     

Solanum phureja genes are expressed in the leaves and tubers of aneusomatic potato dihaploids

Summary The expression of leaf isozymes and tuber patatin in dihaploids derived from the Solanum tuberosum cv. Pentland Crown was investigated. Seven of the dihaploids were aneusomatic containing additional chromosomes from the S. phureja dihaploid inducer. Of these, four genotypes expressed leaf isozymes characteristic of the S. phureja dihaploid inducer, and the tubers of three aneusomatic dihaploids contained a S. phureja form of patatin. Aneusomatic dihaploids in which the proportion of cells containing additional S. phureja chromosomes was relatively small (i.e. 1–15%) did not express leaf isozyme markers or patatin bands characteristic of the dihaploid inducer or showed only faint expression of one or two markers. However, those with a high proportion of cells containing additional chromosomes (50–55%) had a range of strongly expressed leaf isozymes that were characteristic of the dihaploid inducer and also expressed the S. phureja tuber patatin.One dihaploid genotype was exclusively euploid (2n<24), yet is expressed a S. phureja leaf isozyme marker and S. phureja tuber patatin, suggesting recombination or chromosome substitution between the genome of the S. phureja dihaploid inducer and the cultivar Pentland Crown.     

Determination of incompatibility genotypes in almond using first and second intron consensus primers: detection of new S alleles and correction of reported S genotypes

The work aimed to develop a reliable and convenient PCR approach for determining incompatibility S genotypes in almond. Initially, genomic DNAs of 24 accessions of known S genotype were amplified with novel consensus primers flanking the first and second introns of the S‐RNase gene. The PCR products separated on agarose showed length polymorphisms and correlated well with the reference alleles S1‐S₂3 and Sf. In addition, to improve discrimination between alleles of similar sizes, the same sets of primers but fluorescently labelled were used, and the products sized on an automated sequencer. These fluorescent primers were particularly informative in the case of the first intron, variation in the length of which has not been used previously for S genotyping in almond. Some reference alleles showed the same patterns with first and second intron primers, and others showed a microsatellite‐like trace. Subsequently, the S genotypes of 26 cultivars not genotyped previously and of four of uncertain genotype were determined. An allele described in Australian work as putative S₁₀ was shown to be a ‘new’ allele and ascribed to S₂₄ and evidence of five more ‘new’S alleles was found, for which the labels S₂₅‐S₂₉ are proposed. This PCR approach should be useful for genotyping in other Prunus crops.     

Analysis of S‐allele genetic diversity in Sicilian almond germplasm comparing different molecular methods

Italian almond germplasm is characterized by a wide diversity in several growing areas among which Sicily is one of the most important. Analysis with consensus and specific primers and DNA sequencing was performed to investigate S‐RNase genetic diversity and to elucidate the homology rate within a genetic pool of 27 Italian accessions. Interestingly, some of the self‐compatible cultivars did not show the presence of S_f allele. Amplicons from consensus and allele‐specific PCR primers revealed a high level of variability. Sequencing of all the S‐RNase amplicons derived from consensus primers allowed the identification of two new S‐RNase alleles (S₅₁ and S₅₂). Surprisingly, despite the AA replacement mutation, S₅₁ did not exhibit any change of its S‐RNase function. Additionally, several mutations, with no effect on amino acid composition, were detected in the intron and/or in the ORF of four known alleles (S_g, S₁₀, S₃₁ and S₃₅). Genetic variation, regarding point mutations and only detected by sequencing, was revealed among 11 of 27 tested cultivars. The new sources of variability might have an interest for product traceability.     

Identification of S-alleles in almond using multiplex PCR

The S-genotypes of eight almond (Prunus dulcis Miller (D.A. Webb)) cultivars from different geographical origins and of nine new selections from the CEBAS-CSIC (Murcia, Spain) breeding program were determined using single and multiplex PCR with different sets of specific oligonucleotide primers. The results of PCR using the AS1II- and AmyC5R-specific primers showed amplification in a single reaction of 10 different self-incompatibility alleles and of the self-compatibility allele S _f. However, the amplified fragments of the S _f allele were of similar sizes to those amplified from the S ₃ self-incompatibility allele. For this reason, a specific PCR primer CEBASf was designed from the intron sequence of S _f. A multiplex-PCR reaction using the AS1II, CEBASf and AmyC5R primers permitted unequivocal identification of the 10 self-incompatibility alleles and of the self-compatibility allele. Multiplex PCR opens the possibility to identify new S-alleles using different sets of primers. The applications of these PCR markers in the almond-breeding programs are discussed.     

AFLP analysis of Solanum phureja DNA introgressed into potato dihaploids

Introgression of Solanum phureja DNA into S. tuberosum dihaploids was analysed by the use of amplified fragment length polymorphism (AFLP) markers. Five dihaploids, derived from crosses between S. tuberosum cv. Pentland Crown and two different S. phureja pollinators (IVP48 and EC90) were investigated by use of 17 AFLP primer pairs. Also 30 dihaploids, derived from pollination of five different S. tuberosum seed parents with S. phureja IVP101, were investigated for the presence of S. phureja‐specific markers. In total approximately 680 and 850 AFLP products were detected in the diploid S. phureja clones and in the tetraploid S. tuberosum genotypes, respectively. A total of 68 S. phureja IVP48‐specific markers were detected, while the total number of S. phureja IVP101‐specific markers was in the range of 72‐96, depending on the S. tuberosum seed parent. Introgression of DNA in the S. tuberosum cv. Pentland Crown dihaploids, after pollination with S. phureja IVP48 and S. phureja EC90, was demonstrated by the detection of 14 of 68 IPV48‐specific markers in the dihaploids. However, no DNA introgression was found in any of the 30 S. tuberosum dihaploids derived from S. phureja IVP101. Hence, S. phureja IVP101 is regarded as an excellent pollinator in the production of S. tuberosum dihaploids in potato breeding programmes because of the high yield of dihaploids per 100 berries, and because no introgression of DNA into the S. tuberosum dihaploids was evidenced.     

The genetic system for reaction of field beans (Phaseolus vulgaris L.) to infection by three physiologic races of Colletotrichum lindemuthianum

Genetic resistance to race alpha of C. lindemuthianum in the nine varieties of beans studied can be accounted for by dominant alleles at either of two loci, which are unlinked and which behave as duplicate factor loci in the classical sense. A similar system of loci governs reaction to races beta and gamma, with evidence favoring multiple allelism and dominance of alleles conferring susceptibility in certain instances in the case of beta. In addition, for reaction to races beta and gamma, there are distinct systems of complementary factors, again with multiple allelism at the beta loci. Finally, there is genetic linkage between genes of the duplicate factor set governing reaction to gamma and genes of the duplicate factor set, and complementary factor set conditioning reaction to beta. The genes that are members of the set(s) pertaining to any one race alone are independent of each other in a linkage sense.Paper based on work conducted by the senior author in partial fulfilment of requirements for the Doctor of Philosophy degree at Michigan State University and supported by the Rockefeller Foundation.Formerly Rockefeller Foundation Student in Crop Science, now Director, Instituto Nacional de Investigaciones Agricolas, Veracruz, Mexico; Professor of Crop Science, Michigan State University; and Research Pathologist, Crops Research Division, Agricultural Research Service, USDA.     

Identification and characterization of genomic DNA sequences of the S-ribonuclease gene associated with self-incompatibility alleles S1 to S5 in European pear

The stylar products of the S‐locus for the gametophytic self‐incompatibility (GSI) system in the Rosaceae are ribonucleases (S‐RNases). Recently, sequences for 13 pear S‐RNase alleles have been published and named following a letter–symbol nomenclature (S_a to S_d and S_h to S_p). To establish the correspondence between these sequences and the self‐incompatibility alleles we have described previously (S₁ to S₅), we have amplified genomic DNA with consensus primers from the cultivars, ‘Williams’ (S₁S₂), ‘Coscia’ (S₃S₄), ‘Butirra Precoce Morettini’ (S₁S₃), ‘Santa Maria Morettini’ (S₂S₃) and ‘Doyenne du Comice’ (S₄S₅) and identified PCR products specifically associated with each S allele. Cloning and sequencing of the amplification products has revealed that they correspond to European pear sequences already deposited in the database. This allowed us to link S‐RNase sequences with S allele phenotypes and to determine a correspondence between the symbol–letter nomenclature used to name S‐RNase sequences and the number‐based nomenclature used to name S alleles. Based on this result the prediction of new cross‐incompatibilities among pear cultivars is discussed. Finally, we propose a unified number‐based nomenclature to avoid future confusion denominating S alleles in pear.     

Haploid induction in potato: Cytological basis for the pollinator effect

Summary A hexaploid endosperm is regularly associated with a haploid embryo following 4x–2x crosses in potato. One explanation for this is that a single 24-chromosome restitution sperm nucleus fuses with the central cell of the megagametophyte. This suggests that a superior pollinator may be one that produces a high frequency of restitution sperm nuclei. To test this, a technique was developed for observing pollen tube mitosis in vitro following germination of potato pollen in a 20% lactose-50 ppm boric acid solution. A single 24-chromosome restitution sperm nucleus was found in up to 38% of the pollen tubes from a superior pollinator. Moreover, pollen from an inferior pollinator soaked in a sucrose-boric acid-colchicine solution produced 100% restitution sperm nuclei in vitro and a haploid frequency from a tetraploid cultivar comparable to that normally induced by an untreated superior pollinator. The data indicate that the effectiveness of a pollinator in inducing haploids in potato is determined by the frequency of restitution sperm nuclei it produces.Cooperative investigations of the Crops Research Division, Agricultural Research Service, U.S. Department of Agriculture, and the Wisconsin Agricultural Experiment Station. Part of a thesis submitted by the senior author in partial fulfilment of the requirements for the Ph. D. degree. Published with the approval of the Director of the Wisconsin Agricultural Experiment Station.Research Assistant, Department of Horticulture, University of Wisconsin, and Research Geneticist, Crops Research Division, Agricultural Research Service, U.S. Department of Agriculture, Madison, Wisconsin 53706.     

Resistance to viruses in F1 hybrids produced by direct crossing between diploid Solanum series Tuberosa and diploid S. brevidens (series Etuberosa) using S. phureja for rescue pollination

Resistance to potato viruses was examined in the F₁ hybrids (TET) obtained from a cross between a diploid (2n = 24), tuber-bearing interspecific hybrid 87HW13.7 (Solanum tuberosum W231 ×S. multi-dissectum PI 473354) and a diploid (2n = 24), nontuber-bearing wild potato species (S. brevidens CPC 2451) using S. phureja IvP35 (2n = 24) for rescue pollination. The parental plants were susceptible to PVX, whereas two hybrids (TET38.2 and TET38.9) and S. phureja IvP35 reacted with hypersensitivity to PVX. Two hybrids (TET 38.9 and TET 38.12) were extremely resistant to PVY°, which was similar to S. brevidens and S. phureja IvP35, whereas the remaining two hybrids were moderately resistant to PVY°. No resistance to PVA and PLRV was observed in the progenies, in contrast to S. brevidens which was extremely resistant to PVA and PLRV. Hypersensitivity to PVX in two progenies suggested (1) integration by somatic translocation or heterofertilization and expression of genes from the rescue pollinator S. phureja IvP35, or (2) transgressive or complementary gene action.     

Fertility,late blight resistance and genome relationship in an interspecific hybrid,Solanum polytrichon Rydb × S.Phureja Juz. et Buk.

Summary Solanum polytrichon (2n = 48) is easily crossable withS. phureja (2n = 24). In the F₁-progeny only triploids are found. The triploid hybrids are highly variable and pollensterile. Sterility and reduced vigour of some F₁ plants are believed to be due to disharmony of the parental genomes and/or due to gene-cytoplasm interaction. The two genomes A₄ and B ofS. polytrichon and the genome A₁ ofS. phureja do not seem to be very much differentiated from each other. More than 70% of the hybrid plants showed resistance toPhytophthora race 1.3.4.7.8. Its easy cross ability,Phytophthora resistance and genomic constitution makeS. polytrichon a promising species for study in practical breeding.Guest worker of the Agronomy Department, Faculty of Agriculture, Cairo University, Cairo, U.A.R. (Egypt).     

Breeding behavior for tuber protein in Solanum tuberosum and Tuberosum-phureja hybrids

Summary True protein content among tubers within a plant of Solanum tuberosum cv. Oneida was found to be negatively correlated with tuber size. A similar study of S. phureja genotype 148-17 revealed no correlation. Tuber protein was determined for 250 genotypes in each of four hybrid potato populations in a factorial mating design with four potato cultivars as stylar parents mated to four groups of pollen parents (4x cultivars, 4x high protein selections, 2x S. phureja, 2x high protein S. phureja). The hybrid population derived from the 4x high protein selections was significantly higher in protein content than the other populations. High estimates of general combining ability for tuber protein content were found for both stylar and pollen parents. Poor photoperiodic adaptation to growing conditions in northern latitudes was a possible explanation for the variable protein phenotypes typical of S. phureja and the lack of transmission of the high protein character in phureja to 4x-2x hybrids. Although protein content was negatively correlated with total yield, high protein segregates with good yield potential were identified in all four populations.Scientific Journal Series Article 11, 616 of the Minnesota Agricultural Experiment Station.     

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.