Widespread introgression of Solatium phureja DNA in potato (S. tuberosum) dihaploids

Dihaploids were assumed to be of parthenogenic origin but cv. ‘Pentland Crown’ dihaploids produced at the Scottish Crop Research Institute (SCRI), Dundee, Scotland, are known to contain DNA from the S. phureja dihaploid inducer. This study investigates whether inducer DNA occurs in dihaploids of cultivars ‘Aminca’, ‘Brio’, ‘Lizen’ and ‘Sirtema’, produced at INRA, Ploudaniel, France. Two microsatellite markers and five simple sequence repeat 5′-anchored polymerase chain reaction (PCR) primers were used to generate markers. Markers originating from the dihaploid inducer were detected in 13 of the 19 INRA dihaploids and in some dihaploids from each cultivar. Greater introgression occurred in ‘Lizen’ dihaploids than in ‘Brio’ dihaploids, suggesting that the female (S. tuberosum) parent influences introgression. The percentage of INRA dihaploids containing inducer DNA was similar to that for ‘Pentland Crown’ dihaploids (c. 65%). The micro-satellite markers provided the first evidence of the transfer of specific, potentially useful, genes from the inducer to the dihaploid offspring. Interspecific introgression during dihaploid induction is more widespread than previously thought and researchers should be aware that it may influence the results of research using dihaploids.     

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.     

Competition between 2x and x pollen in styles of Solanum tuberosum determined by a quick in vivo method

Summary Pollen competition in vivo was studied by placing two pollen samples on one stigma and comparing the length of the tubes under a U.V. microscope. Comparisons involving pollen of Solanum tuberosum and S. phureja showed that 2x pollen grew faster than x pollen, both in 2x and x styles. Consequences for induction of dihaploids in S. tuberosum are discussed.     

Monohaploids (n=x=12) from autotetraploid Solanum tuberosum (2n=4x=48) through two successive cycles of female parthenogenesis

Summary Viable monohaploids with 12 chromosomes in their somatic cells were obtained from an autotetraploid cultivar of Solanum tuberosum (2n=4x=48) by inducing female parthenogenesis successively in the tetraploid cultivar and in dihaploids from that cultivar. Both dihaploids and monohaploids were induced using the diploid S. phureja clones IvP 35 and IvP 48 as pollinators. The average frequencies of dihaploids and monohaploids in 1973 were 2.0 and 0.14 per berry respectively.Non-homologous chromosome associations (bivalents, trivalents and even quadrivalents) were observed at metaphase I of meiosis in pollen mother cells of the two monohaploids studied.The occurrence of non-homologous associations of chromosomes during meiosis is discussed. In addition the potential significance of monohaploids for basic research andfor breeding of potatoes is considered.     

Parthenogenetic monohaploids (2n=x=12) from Solanum tuberosum L. and S. verrucosum Schlechtd. and the production of homozygous potato diploids

Summary Dihaploid and dihaploid derived clones of Solanum tuberosum and diploid genotypes of S. verrucosum produced 85 viable monohaploids by female parthenogenesis. All were induced using diploid S. phureja clones, homozygous for embryo spot, as pollinator. Frequency of S. tuberosum monohaploids per 100 berries was rather constant in three successive years (14, 17 and 17 respectively). No male and female fertility was found in flowering monohaploids.Colchicine-induced chromosome doubling yielded homozygous s. tuberosum diploids with low pollen quality but good seed fertility.Two diploid self-incompatible species (S. multidissectum and S. berthaultii) produced no monohaploids. The presence of genes for female parthenogenesis in some dihaploids is discussed.     

Intra‐ and inter‐specific crosses of Solanum materials after mitotic polyploidization in vitro

Mitotic polyploidization in vitro was used in selected wild Solanum species and Solanum tuberosum dihaploids. The efficiency of polyploidization by colchicine was compared with that of oryzalin. Oryzalin was more effective than colchicine (P = 0.1). The rate of non‐affected to mixoploid to tetraploid regenerants was 22 : 2.5 : 1 (colchicine) and 14 : 2 : 1 (oryzalin). Optimal concentrations and durations were 3.5 mm /24 h for colchicine and 25 or 30 μm for 24 or 48 h for oryzalin (variations in concentration and duration are necessary owing to possible diversity of responses in selected genotypes). Tetraploids were obtained from S. berthaultii, S. bulbocastanum, S. pinnatisectum, S. verrucosum and eleven S. tuberosum dihaploids. The yield of tetraploids derived from tbr dihaploids was lower than that from the wild species (P = 0.01). Tetraploid regenerants were tested in intra‐ and inter‐specific crosses. Three of 43 intra‐specific combinations (298 pollinated flowers) were successful and yielded 440 seeds. Inter‐specific crosses (138 combinations, 1672 pollinated flowers) yielded 48 seedless berries.     

Double-bridge hybrids of Solanum bulbocastanum and cultivars of Solanum tuberosum

Summary Solanum bulbocastanum (2n=2x=24) has valuable characters for potato breeding, but cannot be hybridized directly with S. tuberosum cultivars. Both S. acaule (2n=4x) and S. phureja (2n=2x) were used as bridging species. Triploid S. acaule × S. bulbocastanum were doubled with colchicine and the resulting fertile hexaploid F₁'s crossed with S. phureja. The triple hybrids obtained were tetraploid or nearly so. The two genomes of S. acaule in these triple hybrids probably pair preferentially, which may provoke pairing and possibly crossing over between the chromosomes of S. bulbocastanum and S. phureja.More than 20000 pollinations of the triple hybrids with four potato cultivars had to be made to produce 40 quadruple hybrids. These highly vigorous hybrids varied greatly in many morphological characters, resistance to Phytophthora infestans, fertility and crossability. The chromosome numbers are 48 (24 hybrids), 49 and 46, but some higher ploidy levels (65, 66, 72 chromosomes) were found as well. Their origin is to be sought in the fusion of an unreduced egg cell from triple hybrids (either euploid or hypoploid) and a reduced male gamete from the cultivars. This view is corroborated by their extreme resistance to Phytophthora. Also some 48-chromosome hybrids are highly resistant, which may indicate introgression from S. bulbocastanum.Most quadruple hybrids are readily inter-crossable and crossable as females with cultivars; several also as males. Two could be hybridized with S. bulbocastanum, but the few seeds dit not germinate.Studies of pachytene stage of meiosis revealed the presence of a S. bulbocastanum chromosome in at least one tetraploid hybrid, which is highly resistant to Phytophthora. At metaphase I of meiosis chromosome associations higher than quadrivalents were not found. Except in one hybrid, the frequency of quadrivalents did not exceed one per cell and the average proportion of chromosomes associated as bivalents amounted to 90%.The quadruple hybrids (double-bridge hybrids) appear good starting material for breeding programmes aimed at introducing genes from S. bulbocastanum into S. tuberosum cultivars.     

Anther culture of Solanum tuberosum L.

Summary Embryoid formation was induced in pollen of Solanum tuberosum, cv. Pentland Crown, Maris Piper and Record, by culturing anthers at or just prior to the first pollen mitosis. The composition of the medium was not critical in respect of either nutrients or hormones. In the presence of relatively high hormone levels, callus formation also occurred from somatic tissues of the anthers.     

Somatic chromosome elimination and meiotic chromosome pairing in the triple hybrid 6x-(Solanum acaule × S. bulbocastanum) × 2×-S. phureja

Summary Two selected hexaploid F₁ clones from the cross Solanum acaule x S. bulbocastanum were intercrossed and the resulting hybrid plants pollinated with the diploid species S. phureja, in order to obtain tetraploid triple hybrids with the same ploidy level as S. tuberosum cultivars (2n=4x=48).Apart from three trihaploids a large population of triple hybrids was obtained, showing chromosome mosaicism in root tip cells (euploid + hypoploid chromosome numbers) and a uniform, mostly hypoploid chromosome number in the pollen mother cells. It is demonstrated that somatic chromosome elimination in the early stages of development is the most probable cause.From detailed pachytene observations as well as from the chromosome associations observed at metaphase I it is evident, that there is normal pairing between the four genomes in the triple hybrids. Although S. bulbocastanum is a quite distinct species with a rigid crossability barrier with S. phureja, the chromosomes of these two species appear to have a high degree of homology. Especially the formation of quadrivalents involving all twelve groups of four homeologous chromosomes, indicated that the four parental genomes (two from S. acaule, one from S. bulbocastanum and one from S. phureja) are not differentiated to the extent of affecting normal pairing and chiasma formation. These results support the view, that the transfer of valuable characters from S. bulbocastanum to S. tuberosum cultivars is feasible even when these characters are polygenically controlled.     

The use of dihaploids in increasing the homozygosity of tetraploid potatoes

Summary Electrophoresis banding patterns of the tuber proteins of 12 dihaploids of the cultivar Pentland Crown showed that four had types of patatin unlike that of the parent. The patatin types of somatically chromosome-doubled clones derived from three of the dihaploids were identical to those of the dihaploid progenitors. Fourteen dihaploids produced from the chromosome-doubled derivative (PDH40X2) of one dihaploid, which had a variant patatin, had the patatin type of the parent dihaploid.The experiment showed that dihaploids and somatic chromosome doubling could be used to fix variation found in heterozygous tetraploid potatoes. Used together, haploidisation and chromosome doubling can generate highly homozygous tetraploids for use by plant breeders.     

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.     

Recommended isolation distances for the field multiplication of diploid tuber-bearing Solanum species

The sexual propagation of genebank accessions by hand pollination in the greenhouse is a very expensive and time-consuming routine task. The self-incompatibility of most diploid potato species allows their multiplication in isolated field plots, which is coupled with the advantage of the presence of natural pollinators and maintaining a high level of heterozygosity. The aim of this study was to determine the isolation distance required for minimizing pollen flow between neighbouring multiplication plots. The Solanum phureja clone IVP48 was used as the pollen donor since it possesses a dominant seed marker (‘embryo spot’). The centrally located pollen donor was surrounded by S. phureja recipient plots at distances of 10,20,40, and 80 m, respectively. The average pollen contamination ranged from 0.2% in the plots 80 m from the pollen donor to 5.1% in those 10 m away. In this study, procedures are suggested which will help to reduce cross-pollination between accessions.     

Analysis of dihaploid induction and production ability and seed parent x pollinator interaction in potato

Summary The production of dihaploids is the first step in a potato breeding program at the diploid level. Dihaploid induction ability, dihaploid production ability and seed parent x pollinator interaction were analyzed for 28 × 3, seed parent x pollinator combinations. This is the first report on significant interaction between seed parents and pollinators. Despite this interaction, IVP 101 had a significantly higher dihaploid induction ability than the widely used pollinators IVP 35 and IVP 48. Previous findings on significant differences in dihaploid production ability between seed parents were clearly confirmed.     

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.     

Tolerance to low temperatures and tuber soft rot in hybrids between Solanum commersonii and Solanum tuberosum obtained through manipulation of ploidy and endosperm balance number (EBN)

The objectives of this study were to evaluate the tolerance to low temperatures and tuber soft rot in hybrids between Solanum commersonii and Solanum tuberosum. The experimental materials consisted of F₁ triploid, BC₁ pentaploid‐near pentaploid and BC₂ tetraploid–near tetraploid hybrids. The F₁ triploids had a freezing tolerance and acclimatization capacity closest to S. commersonii. This indicated that the endosperm barriers which prevent the introgression of 1EBN S. commersonii into 4EBN S. tuberosum had been overcome. Indeed, the triploids produced 2n eggs, thus giving a compatible maternal to paternal EBN ratio in the hybrid endosperm generated by the 3x(2EBN) × 4x(3EBN) crosses. The tolerance to low temperatures of BC₁ and BC₂ hybrids was lower than that of the F₁. However, a number of genotypes were identified which were able to withstand temperatures down to ‐5°C. Some BC₂ hybrids were also tested for their tolerance to tuber soft rot, and some resistant hybrids were detected. A number of them combined the capacity for cold acclimatization with tolerance to tuber soft rot. These hybrids have an EBN of 4; they are fertile and have been used in backcrosses with 4EBN S. tuberosum.     

Intra- and interspecific transmission of androgenetic competence in diploid potato species

Summary In an attempt to determine the transmission of androgenetic competence, 10 families resulting from intra- and interspecific hybrids including three reciprocal hybrids were examined in anther culture. Hybrid families were generated between competent clones of Solanum phureja and incompetent clones of S. phureja, S. microdontum and S. berthaultii. S. phureja clones PP5 and A95 (derived by androgenesis of a 2n microspore of PP5) were found to be consistently competent for androgenesis. Androgenetic competence was observed to segregate in all hybrid families with some highly responsive and some unresponsive genotypes in all families. A total of 9,465 cultured anthers have yielded 936 embryoids and 91 plants, including 29 monoploids. The cytoplasm of species lacking competence appeared to have greater influence on the expression of androgenesis in intraspecific than in interspecific hybrids. Expression of androgenesis varied among half-sib hybrid families indicating that competence for androgenesis was influenced by the parents lacking competence. The anther culture data on a backcross between a highly responsive hybrid and its unresponsive parent indicated that competence may be under control of a single dominant gene.     

Targeted BSA mapping of Scmv1 and Scmv2 conferring resistance to SCMV using PstI/MseI compared with EcoRI/MseI AFLP markers

In a previous study, two chromosome regions (Scmv1 and Scmv2), conferring sugarcane mosaic virus (SCMV) resistance in maize, were enriched with EcoRI/MseI AFLP (Eco‐AFLP) markers (methylation insensitive) by targeted bulked segregant analysis (tBSA). The objective of the present study was to further saturate these two regions with PstI/MseI AFLP (Pst‐AFLP) markers (methylation sensitive) using the same tBSA approach, and to compare the genomic distribution of both Pst‐AFLP and Eco‐AFLP markers. Out of 470 PstI/MseI primer combinations screened, four Pst‐AFLP markers were identified in the Scmv1 region (chromosome 6), and none in the Scmv2 region (chromosome 3). These Pst‐AFLP markers were more closely linked to the Scmv1a gene than any of the Eco‐AFLP markers, and could be useful for marker‐assisted selection and even map‐based cloning. In addition, Pst‐AFLP and Eco‐AFLP markers were dissimilarly distributed in both target regions. Pst‐AFLP markers were equally distributed across both regions, while Eco‐AFLPs were clustered in the Scmv2 region.     

Identification of molecular markers linked with crown rust (Puccinia coronata f. sp. lolii) resistance in perennial ryegrass (Lolium perenne) using AFLP markers and a bulked segregant approach

Crown rust resistance is an important selection criterion in ryegrass breeding. The fungal disease caused by P. coronata causes yield loss and a reduced quality of the fodder crop. Molecular markers were used to unravel the genomic organization of crown rust resistance in a segregating L. perenne population. Two genomic regions involved in crown rust resistance were identified that together explained 35% of the phenotypic variance present. Bulked segregant analysis in combination with AFLP markers was a suitable method to identify DNA markers associated with genomic regions of major effect. One cluster of AFLP markers explained 6.1% of the variance and mapped to linkage group 2, a genomic region known to contain crown rust resistance genes. A second cluster of AFLP markers detected a novel genomic region of major effect that explained 27.7% of the phenotypic variance in crown rust resistance. This cluster was unlinked to the cluster on linkage group 2. Divergent selections performed within the segregating F₁ population on the basis of genotype and phenotype revealed that the markers associated with crown rust resistance identified in this study have potential for marker assisted selection. Selection of plants on the basis of markers was more straightforward than the selection on the basis of phenotype.     

Breaking the crossability barriers between disomic tetraploid Solanum acaule and tetrasomic tetraploid S. tuberosum

Summary A combination of compatible second pollinations and embryo rescue was applied for systematic production of true tetraploid hybrids from crosses between disomic tetraploid Solanum acaule and tetrasomic tetraploid potato, S. tuberosum. Several genotypes of tetraploid potatoes were pollinated with S. acaule, and the compatible second pollinations were made on the following day, with a genotype of S. phureja, IvP 35 to promote fruit development. Embryo rescue was carried out in 21 families, 14 to 27 days after the first pollination. A total of eight plants were obtained from the embryo rescue and their chromosome numbers were counted in the root tips. Three of the eight plants were identified as tetraploid, and five others as diploid. Morphology, isozyme banding patterns, and pollen stainability, as well as potato spindle tuber viroid (PSTVd) resistance, indicated the hybrid nature of the three plants. This is the first report of successful tetraploid hybrid production between disomic tetraploid S. acaule (4x) and tetrasomic tetraploid potatoes. Seed set from the crosses between one of hybrids and diploid potatoes indicated workable levels of both male and female fertility for introgression of valuable genes from S. acaule into the cultivated potato gene pool. The methodology used may be applied to other disomic tetraploid tuber-bearing Solanum species and with some modifications also to distantly related solanaceous species and genera.