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Diploid potato populations between a primitive cultivated species, Solanum phureja, and a weedy species, S. chacoense, were used to examine the segregation of microsatellite markers and three traits in backcrosses. Two of the traits, anther culture competence and 2n pollen production, originated from S. phureja whereas the third, leptine production (a specific glycoalkaloid known to convey resistance to the Colorado potato beetle) originated from S. chacoense. Using CP2, a self-incompatible F₁ hybrid originating from a cross between S. chacoense clone 80-1 and S. phureja clone 1-3, three populations were developed: 1-3 x CP2 (PBCp), CP2 x 1-3 (PBCc), and CP2 x 80-1 (CBC). For the microsatellite study, four simple sequence repeat (SSR) primer pairs that amplified fragments within potato sequences found in the GenBank were used to look at segregation ratios in our backcross populations and to eliminate possible spurious genotypes bearing non-parental alleles in these populations. Seventeen spurious genotypes were discarded from PBCp; none was found in PBCc or CBC. Two SSR loci showed skewed segregation in PBCp (favoring transmissnion of the allele originally found in 80-1), PBCc showed normal segregation at all loci, and CBC showed distorted segregation at one locus (revealing a deficiency of homozygotes). In the study of anther culture, three components of ACR were investigated in a preliminary study: 1) embryos produced per anther (EPA), 2) embryo regeneration rate and 3) percentage of monoploids (2n=1x=12) among regenerants. CP2 was intermediate, 80-1 was low, and 1-3 was high for ACR. Only EPA was selected for further characterization in our populations. PBCp (78 genotypes) and CBC (57 genotypes), were characterized for anther culture response ACR/EPA in a series of studies. Nine high and ten low selections were identified in CBC, and ten high and ten low selections were identified in PBCp. EPA selections were used for bulk segregant analysis (BSA) using 214 RAPD primers. Two bands, one amplified by OPQ-10 and another by OPZ-4 were linked in coupling and in repulsion, respectively, to ACR in PBCp. One band amplified by OPW-14 primer was linked in coupling to ACR in CBC. One-way ANOVAs for data from remaining genotypes of the populations verified linkage of the markers to ACR/EPA. For 2n pollen production, a total of 77 PBCp genotypes was characterized; 80-1 produces low % 2n pollen, and 1-3 produces high % 2n pollen. Pollen samples were stained with propidium iodide and examined by flow cytometry. The frequency of 2n pollen varied continuously from 1.7 % to 40.6 % among the 41 genotypes that flowered sufficiently to allow three separate pollen collections. Variation due to the environment was observed where the frequency of 2n pollen appeared greater over a range of genotypes on single collection days. BSA could not be used due to limited population size and a low number of selections at the extremes of the distribution of phenotypes. The continuous variation for 2n pollen production suggests multigenic control of the trait. In the study of leptine content in reciprocal backcross populations, 87 genotypes within PBCp, and 42 genotypes within PBCc were characterized using gas chromatography of leaf samples. CP2 was intermediate, 1-3 had zero, and 80-1 was high for leptine content in the foliage. Leptines were present in low levels in 43 of 87 genotypes in PBCp, indicating simple genetic control. In PBCc, only 7 of 42 genotypes expressed leptines, generally at a higher level than in PBCp, indicating cytoplasmic inheritance. Ten high and ten nil selections within PBCp, and seven high and eight nil selections within PBCc were used for BSA using 214 RAPD primers. Three primers OPQ-2, OPT-16 and OPT-20 amplified bands segregating with high bulks in both populations. These markers were linked in coupling to leptine content in PBCp. Linkage was verified by ANOVAs for leptine content in the entire population.

Colchicine, an antimicrotubule agent, has enhanced the androgenic response of cruciferous and graminaceous species. Our objectives were to determine if colchicine treatments enhanced androgenic embryo production in diploid potato, and if colchicine treatments during anther culture affected the ploidy of anther-derived plants. An additional study, analysis of genetic identity among regenerated anther-derived plants based on RAPD markers, was also conducted. Anther culture of a hybrid between Solanum chacoense and S. phureja (clone CP2) was conducted with five colchicine treatments (0, 25, 50, 100, and 200 mg/L) for 24 h. Anthers containing late uninucleate pollen were cultured on modified liquid LS medium. A mean of 0.25 embryos per anther was obtained; however, there was no significant difference among the colchicine treatments, which ranged from a mean of 0.17 to 0.28 embryos per anther for 200 mg/L colchicine and control, respectively. In total 56 plants were regenerated of which 11% were monoploid. The second experiment using the same colchicine treatments was conducted with 54 hybrids derived by crossing six anther-derived doubled monoploids with three heterozygous pollinators. A mean of 1.78 embryos per anther was obtained; again, there was no significant difference among the colchicine treatments, which ranged from 1.27 to 2.75 for 50 mg/L colchicine and control, respectively. In total 679 plants were regenerated. The 312 plantlets for which ploidy was determined, 75% were monoploid. The third experiment was conducted to study various durations (0, 90 sec vacuum infiltration, 24, 48, and 72 h) of high colchicine treatment (200 mg/L) applied to S. phureja family DM 13-14 202 x ID 5. There was nearly significant difference among treatments (α = 0.05). Mean embryos per anther ranged from 0.96 to 1.90 for 48 h and 90 sec vacuum infiltration, respectively. In an additional study, 26 regenerated anther-derived monoploid plants, from the DM 13-14 202 x ID 5 family, from the second experiment, were genetically characterized based on RAPD markers. Forty-three loci were scored from 13 primers for groups of monoploids derived from the same flasks of anther culture. From one flask, two pairs of monoploids were found to be genetically identical. From a second flask 6 of 7 were genetically identical, and from a third 3 of 7 were genetically identical. The presence of genetically identical individuals within three monoploid populations indicates the prevalence of secondary embryogenesis during anther culture such that a single embryogenic microspore can generate many anther-derived plants.

Monopoloid potato (2n = 1x = 12) can be derived either paternally through anther/microspore culture or maternally through crossing with a haploid-inducing pollinator. Evidence from other genera suggests that androgenic and gynogenic haploid populations derived from the same parent may differ due to gametic selection and/or epigenetic factors. Our objectives were to derive androgenic and gynogenic monoploid populations from each of two diploid (2n = 2x = 24) Solanum phureja clones and compare their phenotypic characteristics in a greenhouse study. A haploid-inducing pollinator, S. phureja IVP101, was crossed to two selections of S. phureja (PP5 and BARD1-3). A total of 185 fruit was obtained from PP5 and 398 from BARD1-3, resulting from 85% and 65% fruit set, respectively. Seed lacking the dominant embryo spot marker carried by IVP101 were selected and germinated in vitro. From 29,300 PP5 x IVP101 seeds, 278 were spotless, resulting in 27 monoploids. Approximately 37 monoploids were obtained from the 99,500 BARD1-3 x IVP101 seeds of which 500 were spotless. In anther culture, PP5 and BARD1-3 yielded 0.16 and 1.67 embryos per anther respectively of which 51% and 44% of the regenerants were monoploid. A total of 32 anther-derived monoploids has been obtained from PP5 and 130 from BARD1-3. Rooted cuttings of 21 androgenic and 21 gynogenic PP5 monoploids were established in a greenhouse in each of three randomized complete blocks. Although the anther-derived monoploids appeared more vigorous, none of the differences between the two populations were significant. Flow cytometry revealed that anther derived progeny of PP5 and BARD1-3 had 3-8% greater nuclear DNA content than gynogenic monoploids derived from the same parental clones.

Anther culture experiments were conducted to construct a monoploid family. The donor plants used were hybrids between high leptine producing selections of Solanum chacoense Bitt. and anther culture responsive selections of Solanum phureja Juz. et Buk. Several steps of the anther culture process were studied. The results indicated that genotype remains the main factor affecting anther culture response. Growing anther-donor plants in higher greenhouse temperatures (30 degrees C day/20 degrees C night) increased the number of embryos per anther by 40 percent. A heat shock given to anthers in culture for 12h at 35 degrees C was also found to be beneficial resulting in an increase of the anther culture response by 40 percent. However the effect of the high temperature shock resulted in lower regeneration rates. In all experiments a highly significant "date" effect was observed with one or two days differing from the others by showing higher response rates in all hybrids tested. The majority of the regenerated plants was diploid, probably resulting from unreduced gametes. Simple sequence repeat analysis with eight polymorphic primer pairs was used successfully to identify the homozygous diploid plants that were added to the monoploids. In total 34 monoploid plants and 14 homozugous diploids were obtained. The degree of heterozygosity revealed by SSR analysis indicated that the diploid plants originated from unreduced gametes formed by first division restitution (FDR) mechanism. The SSR marker data were used to map the genes with respect to the centromeres by half tetrad analysis. SSR-containing sequences from the public databases, as well as sequences obtained from a genomic library enriched for SSRs, were used to generate 48 primer pairs. Only 12 of them were found to be polymorphic in the monoploid family. Ten primer pairs did not amplify any specific fragment. The monoploid population showed distorted segregation at four of the polymorphic loci, showing overrepresentation of the chacoense alleles in three of them. One of the loci showing distorted segregation (STSTP, amplified by primer pair RV 11+12) is most probably linked to lethal alleles, whereas another one (ST13ST, amplified by primer pair RV 21+22) could be linked to genes affecting anther-culture response. The location of the SSR loci on the potato chromosomes is not known except for one (waxy, primer pair 3+4), but statistical analysis on the segregation data obtained from 70 heterozygous anther-derived diploids showed no linkage between them. The SSR primer pairs developed in this study might be useful in studying genetic relationships among cultivars and accessions in breeding programs. Randomly amplified polymorphic DNA (RAPD) analysis was used in association with bulked segregant analysis to detect linkage with genes controlling leptine biosynthesis. With all the limitations imposed by the population size and contamination from foreign pollen, a band amplified by primer OPA-16 could differentiate the bulks contrasting for leptine content. It is possible that this band is linked to genes suppressing leptine biosynthesis, since it appears only in the plants that do not synthesize leptines. Further investigation with larger populations is needed to confirm this possibility.

Hordeum bulbosum L. is an obligatory outcrossing perennial grass and is considered to be the closest relative of cultivated barley, H. vulgare. H. bulbosum has many useful agronomic traits such as winterhardiness and drought tolerance. The resistance it has to many pathogens, including powdery mildew, yellow rust, net blotch, and scald would be worthwhile transferring to H. vulgare. Although hybrids between H. vulgare and H. bulbosum are easily obtained and homoeologous chromosome pairing occurs in H. vulgare x H. bulbosum hybrids, successful gene transfer is rare. Possible reasons for this include: 1) the diploid constitution of H. vulgare may not tolerate introgressions from an alien species; 2) the existence of certation, where the recombinant male gametes may not be able to compete with non-recombinant gametes to effect fertilisation; 3) the pairing between H. vulgare and H. bulbosum chromosomes may not result in crossing over; 4) the recombinant pollen grains may be sterile and 5) the recombinant chromosomes may be unstable depending on the size of the introgression. In an attempt to overcome the problem of certation, plants were regenerated directly from pollen grains, of H. vulgare-H. bulbosum hybrids, by anther culture. Embryo-like structure (ELS) formation was initially optimised and generally it was found hybrids responded best to anther culture when grown in either a growth chamber at 15°C or a glasshouse at 22°C, when spikes were cold pretreated for 28 days and when ELS were induced on either of the two medium tested (modified medium 1 and BAC3 medium). The two hybrid parents most responsive to anther culture, genotypes 3028 and VG2 558/9, were triploid hybrids and contained 14 H. vulgare and 7 H. bulbosum chromosomes. Both ELS induction and green plant regeneration was genotype dependent. A total of 43 green plants were obtained, three of these were triploid, three were tetraploid, one was aneusomatic and the remainder were haploid or diploid. The most responsive genotype to anther culture, 3028, was subjected to spike inflorescence culture, backcrossing to a diploid H. bulbosum genotype Cb2920/4, and selfing. Regenerated plants produced by anther culture, backcrossing and selfing were analysed at the morphological, cytological and molecular level (by RFLP analysis). It was found that each plant regeneration system produced genetically unique plants. Great variation was observed in the locations of H. bulbosum introgressions, which were detected using single or low copy telomeric probes. The reactions of regenerants to three fungal diseases, powdery mildew, scald and net blotch, were scored. Some regenerants were more resistant than the hybrid parent from which they were derived, reflecting DNA rearrangements had occurred resulting in increased resistance. RFLP analysis of green and albino anther culture-derived plants using the methylsensitive endonucleases MspI and HpaII, showed albino plants had much higher levels of methylation than the corresponding green regenerants. These findings supported the hypothesis that changes in DNA methylation could affect the frequency of green plant regeneration.

Crosses between Hordeum vulgare L. (cultivated barley) and H. bulbosum L. (bulbous barley grass) have been carried out for several decades to introgress desirable genes from the wild species into cultivated barley, but these projects have met with little success. Since H. bulbosum is considered to be the most closely related species to H. vulgare it was hypothesized in this thesis that interspecific genetic recombination is prevented by pre- or post-fertilization barriers. A series of investigations using H. vulgare x H. bulbosum hybrids was initiated to assess: 1 environmental effects on chromosome pairing by growing hybrids at two different temperatures and analyzing pollen mother cells at metaphase I; 2 crossing-over between homoeologous chromosomes at meiosis. This was carried out by producing diploid hybrids from crosses between a H. vulgare paracentric inversion stock and H. bulbosum. Pollen mother cells were analyzed at first and second anaphases, and anomalies (bridges and fragments) were related to frequencies of crossovers; 3 chromosome instability and elimination associated with particular parental combinations. 'VVB' hybrids had never been directly produced from crosses between H. vulgare (4x) and H. bulbosum (2x), so hybridizations were carried out at a temperature less than 17°C using genotypes known to promote chromosome retention; 4 production of progeny from crosses involving H. vulgare and partially fertile triploid hybrids ('VBB') of H. vulgare (2x) x H. bulbosum (4x). Any viable plants obtained were to be screened cytologically, pathologically and morphologically; 5 the use of anther culture to avoid certation effects after hybridizing H. vulgare with pollen from hybrids. Anthers from a range of diploid, triploid and tetraploid hybrids were cold pretreated and cultured according to protocols developed specifically for barley. It was concluded from the investigations that optimum environmental conditions may be determined for individual hybrids to promote chromosome pairing but that crossing-over between paired homoeologues is greatly reduced compared with intraspecific H. vulgare hybrids. Novel triploid 'VVB' hybrids were obtained and certain genotypic combinations proved to be relatively stable. However, because of a lack of allosyndesis their use for gene introgression will be limited. Progeny from crosses between H. vulgare and partially male fertile 'VBB' hybrids included the first reported monosomic substitutions of H. bulbosum chromosomes into H. vulgare. These will be valuable for transferring genes from H. bulbosum to H. vulgare and locating them on particular chromosomes. Finally, although only few green regenerants were obtained from culture of anthers from hybrids one of these was characterized as a chromosomally modified diploid 'VB' hybrid with partial anther dehiscence. Progeny from crosses between this plant and H. vulgare demonstrated that chromosome additions and substitutions can be developed.