Root symbioses with arbuscular mycorrhizal fungi and rhizobial bacteria share a common signaling pathway in legumes. Among the common symbiosis genes are CASTOR and POLLUX, the twin homologous genes in Lotus japonicus that encode putative ion channel proteins. Orthologs of CASTOR and POLLUX are ubiquitously present in both legumes and non-legumes, but their function in non-legumes remains to be elucidated. Here, we use reverse genetic approaches to demonstrate that the rice (Oryza sativa) ortholog of POLLUX, namely Os-POLLUX, is indispensible for mycorrhizal symbiosis in rice. Furthermore, we show that Os-POLLUX can restore nodulation, but not rhizobial infection, to a M. truncatula dmi1 mutant.

Borage (Borago officinalis) is an annual herb plant for culinary and medicinal uses. Due to a high level of gamma-linolenic acid (GLA) in its seed oil and the health-related benefits of GLA, borage is commercially cultivated. However, a herbicide-resistant variety has not yet been developed for effective weed management in borage farming. Thus, this thesis aimed to create, identify and characterize ethyl methanesulfonate (EMS) induced borage mutants for herbicide imidazolinone resistance. An EMS-mutagenized borage population was generated by using a series of concentrations of EMS to treat M1 seeds. After screening M2 borage plants with the herbicide, tolerant plants were selected, self-pollinated and grown to their maturity. The offsprings were subjected to herbicide screening again to confirm the phenotype, resulting in identification of two genetically stable imidazolinone-resistant lines. Two acetohydroxyacid synthase (AHAS) genes, AHAS1 and AHAS2, involved in the imidazolinone resistance were isolated and sequenced from both mutant (resistant) and wild type (susceptible) borage plants. Comparison of these AHAS sequences revealed that a single nucleotide substitution occurred in the AHAS1 resulting in an amino acid change from serine (S) in the susceptible plant to asparagine (N) in the first resistant line. The similar substitution was later found in the AHAS2 of the second resistant line. A KASP marker was developed for the AHAS1 mutation to differentiate the homozygous susceptible, homozygous and heterozygous resistant borage plants for the breeding purpose. The in vitro assay showed homozygous resistant borage containing the AHAS1 mutation could retain significantly higher AHAS activity than susceptible borage across different imazamox concentrations. The herbicide dose response test showed that the resistant line with the AHAS1 mutation was tolerant to four times the field applied concentration of the “Solo” herbicide.

The objective of this study was to develop and implement an advanced generation breeding programme at the Institute for Commercial Forestry Research (ICFR) to manage and integrate the many and disjunct breeding and production populations of Eucalyptus nitens established by various entities over the past 30 years at multiple sites in South Africa. To develop such a breeding strategy, a good understanding of the population genetics, and the underlying assumptions made by tree breeders about the species, was needed. Eucalyptus nitens is an important forestry species grown for pulp and paper production in the temperate, summer rainfall regions of South Africa. A tree improvement programme has been ongoing at the ICFR for three decades. The measurement and statistical analysis of data from eight F1 trials established during the 1980s and 1990s have enabled characterisation of the ICFR’s breeding population. Provenance testing showed that the more northerly New South Wales (Australia) Eucalyptus nitens provenances of Barren Mountain and Barrington Tops are distinctly better suited to growth in South Africa than the southern New South Wales provenances and the Victorian provenances, Penny Saddle and Bendoc. Generally, the species was not badly affected by Coniothyrium canker. High Type B genetic correlations for all sites pairs, except one comparison, ranged from 0.75 to 0.99 for diameter at breast height at 76 to 113 months, indicating very little, or no, genotype by environment interaction for diameter at breast height for the genotypes tested in the F1 generation. Narrow sense heritability estimates ranged from 0.01 to 0.34, indicating that the species provides a breeding opportunity for improvement of diameter growth. High genetic correlations of greater than 0.90 between diameter measurements at 52 to 62 months after establishment and diameter measurements at 94 or 113 months were found, indicating that selections can be made reliably at five or six years. Diameter measurements at both 60 months and full rotation (94 to 113 months) were highly correlated with the final height measurements in these trial series (rg > 0.71 and > 0.83, respectively). Predicted genetic gains for the F2 over the F1 generation were highest in the trials at Goedehoop and Arthur’s Seat, with predicted increases in diameter at breast height of 3.07 cm (17.1%) and 3.17 cm (20.7%), respectively, at full rotation. Genetic improvement in the species has been slower than anticipated due to delayed and infrequent flowering and seed production. Three genetic gain trials were established, firstly, to quantify the gains that have been made in the first generation of improvement in the breeding programme; and secondly, to establish whether a number of seed source and orchard variables influence the performance of the progeny. These variables were: the number of flowering trees in the seed orchard, year of seed collection, seed orchard origin and composition of seed orchard seed bulks. Diameter at breast height and tree height were measured in the trials at between 87 and 97 months after establishment, and timber volumes and survival were calculated. Improved seed orchard bulks performed significantly better (p < 0.01) than unimproved controls in the field trials, and genetic gains ranging from 23.2 to 164.8 m3ha-1 were observed over the unimproved commercial seed. There were significant differences (p < 0.01) in progeny growth between the levels of flowering, with higher levels of flowering (R 40 %) producing substantially greater progeny growth than lower flowering levels (S 20 %). The seed orchard origin had no effect on progeny growth in this trial series. This suggests that seed collected from any of the four seed orchards tested will produce trees with significant improvement in growth. Various scenarios investigating a range of assumptions were developed and used to predict genetic gain in the F2 populations. These were compared with realised gains achieved in the genetic gain trials. The family nested within provenance scenarios proved to be closer to realised gain than the family across provenance predictions. Two scenarios were used for family nested within provenance: Firstly, actual flowering for family nested within provenance; and secondly, estimated flowering after a 30% roguing of poor families. For both scenarios, a coefficient of relationship of 0.33 predicted gains closest to the realised gains. Indications were that the effects were additive, and that very little or no heterosis had occurred. The statistical information suggested that outcrossing in the seed orchards was > 80%. This study provides an objective and quantitative assessment of the underlying assumptions used for estimating genetic parameters, and predicting gain in this population of Eucalyptus nitens. At the same time that genetic gain trials were established, F2 trials were planted, using seedlots collected from F1 seed orchards. Analysis of the two F2 trials showed that realised gains for diameter at breast height at 87 months were close to the predicted values and ranged from 1.02 cm to 1.90 cm. Two exceptions were the sites at Helvetia and Babanango, where gains were under- and over-predicted, respectively. Realised heritability estimates, which are related directly to the realised gain and the actual selection intensities used in the seed orchards, reflected this trend. Estimation of breeding values allowed for selection of elite individuals in top families. Both grand-maternal provenance origin and F1 maternal effects were significant in the F2 trials. A Type B genetic correlation of 0.61 for diameter at 87 months indicated the possible presence of genotype by environment interactions for the two F2 sites. A low narrow sense heritability estimate of 0.06 for diameter at breast height at 87 months at one F2 site indicated that more emphasis should be placed on family information rather than individual information at this site. A heritability estimate of 0.17 for diameter at breast height at 87 months at the second site, however, indicated that further improvement is possible in this population of Eucalyptus nitens. Modelling of predicted genetic gain using various breeding strategy scenarios can be a useful tool in assisting with the decision on which strategy or management plan will deliver the most genetic gains per unit time. Such modelling, using the parameters established in the first part of the study, played an important role in developing the advanced generation breeding strategy for Eucalyptus nitens. In addition, the modelling exercise highlighted various management options which could be used to increase gains in the existing production populations or orchards. Indications are that additional roguing of 1) existing Clonal Seed Orchards based on results of F2 trials (i.e., backward selection); and 2) F1 Breeding Seed Orchards based on stricter provenance selection, will markedly increase the quality of the seed produced from these orchards within one season. This study also highlighted the importance of shortening the breeding cycle in Eucalyptus nitens, particularly in view of the delays caused by reticent flowering and seed production in the species. The information and understanding gathered from this study led to the development of a proposal for an advanced generation breeding strategy in Eucalyptus nitens. This proposal uses parental reconstruction of open-pollinated progeny to secure pedigree information of forward selections, thus combining the benefits of increased genetic gain with a shortened breeding cycle. Recommendations on the management and adaption of current production populations to increase gains have been made, because establishment and management of improved material in seed orchards to ensure a sustainable supply of improved seed to the South African forestry industry, is a key objective of the ICFR Eucalyptus nitens breeding programme. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.

Popcorn is increasingly becoming popular as a snack and is consumed widely all over the world. It is a high value crop, with possible multiplier effects like income generation for the under-resourced communities in the second economy. Despite its popularity, developing countries are battling to meet the demand and rely on importing popcorn grain due to challenges which include poor agronomic traits and slow breeding progress. Most of the imported varieties are not adapted to stress-prone local environments, which are prevalent in tropical sub-Saharan Africa. The objective of the study was to evaluate newly developed hybrids and inbred lines for agronomic and popping quality traits with the possibility for commercialization in future. The study aimed at determining variability for popping ability in inbred lines and hybrids, grain yield and its secondary traits, the nature of gene action, relationships among agronomic and popping quality traits, effect of genotype x environment interaction on agronomic traits and popping method x genotype interaction effects. To determine popping ability, 128 inbred lines were evaluated at the University of KwaZulu-Natal, South Africa, in June 2011 using two popping methods, the microwave method and the hot-air method. The popping quality attributes measured were flake volume, popping fold, number of unpopped kernels, kernel size and quality score. Variability among inbred lines was significant (P<0.05) for all traits. Flake volume ranged from 63 cm3 to 850 cm3, popping fold ranged from 2.5 to 34 times the original volume. Kernel size had a significant positive correlation (r= 0.49) with the number of unpopped kernels. There was a significant strong and negative correlation between flake volume and the number of unpopped kernels (r= -0.62), indicating that either of the two traits would be effective for measuring popping ability. Experimental hybrids were then developed from 87 out of the possible 128 inbred lines. Only the inbred lines with sufficient seed were crossed to develop hybrids. Random crosses were generated at Makhathini Research Station during the winter season of 2011. Crosses were made at random among parents that managed to synchronize their flowering dates, resulting in 119 hybrids with sufficient seed for planting in trials. To determine agronomic superiority, the 119 experimental hybrids and the standard check P618 were evaluated at the Cedara Research Station and Ukulinga Research Farm in the Midlands of KwaZulu-Natal during the summer of 2011/2012. The experiments were laid out as 10 x 12 alpha lattice design, with two replications at each site. Standard cultural practices for maize were followed. The data were subjected to analysis of variance and line x tester analysis in Genstat and SAS statistical programmes. Results indicated that hybrids were significantly different for all agronomic traits. Means for grain yield ranged from 1.0 t/ha to 5.2 t/ ha. General combining ability effects were significant for all agronomic traits, suggesting that additive gene effects were governing these traits. Specific combining ability effects were significant for ear length, number of ears per plant and yield indicating, that non-additive gene effects were influential for these traits. Generally, agronomic traits were highly heritable. Grain yield showed significant and positive correlation with ear length, plant height, ear position, shelling percentage and number of ears per plant, indicating that these were the major yield-determining secondary traits which should be enhanced in popcorn. Although site main effects were highly significant for secondary traits, the hybrid x site interaction was not significant. The results therefore indicate that the hybrids were ranked similarly at both sites. The 119 experimental hybrids and the standard check P618 were evaluated for popping quality, using the microwave and the hot-air popping method. There was a significant variability observed among hybrids for popping quality traits. Flake volume across sites and across popping methods ranged from 734 cm3 to 1288 cm3. Popping fold ranged from 14.69 to 25.75 times the original volume. Additive gene action was more prominent than non-additive action for all popping quality traits. The SCA effects were significant for flake volume, popping fold and number of kernels per 10 g. All popping quality traits had high heritability, indicating that selection would be effective to improve popping. Flake volume was negatively correlated to quality score, indicating that popping expansion is reflected on the quality score and a significant negative correlation between flake volume and number of unpopped kernels. There was significant and strong positive correlation between kernel size and number of unpopped kernels. Hybrid x site interaction was only significant for quality score and kernel size. Hybrid x method interaction was not significant, indicating that popping ability was not dependent on the method. Inbred lines showed significant variation for popping quality and therefore have utility for hybrid development. Significant genotypic variation was also observed among hybrids for agronomic and popping quality traits. Additive gene action was predominantly responsible for both agronomic and popping quality traits. Both agronomic and popping quality traits were highly heritable and positive relationships were identified among traits. Overall, the study indicates opportunities for further breeding progress through selection. / Thesis (M.Sc.)-Unversity of KwaZulu-Natal, Pietermaritzburg, 2012

Plant breeding and the development of new or better varieties of plants are essential parts of modern agronomy, horticulture and forestry. The basis for such improvement is a knowledge of the factors and principles of genetics. A number of genetic studies have been made with barley in recent years. This is partly because of the many distinct heritable characters of barley plants. The cultivated species of barley offers the plant breeder and geneticist a wealth of material for genetic studies. Varieties differ in a great many readily distinguishable characters, species hybridize readily, and their small number of chromosomes make it good material for inheritance studies. The barley genetic work has been divided among the principal workers in the U.S., each being responsible for one linkage group. This station has been assigned group IV of which this study is a part. A study of the inheritance of other genes not located in linkage group IV, but appearing in the crosses used, has also been made. This investigation is a by-product of the cereal breeding and improvement program being carried on the Utah Agricultural Experiment Station.

In plants, P450 enzymes encoded by P450 genes play a central role in numerous biosynthetic pathways, such as the production of secondary metabolites, stress responses and disease resistance. This thesis reports upon the utilization of molecular biology techniques to study P450 gene(s) in barley (Hordeum vulgare L.).Using several combinations of degenerate primers, a large number of barley P450 gene fragments were cloned and sequenced from two commercial varieties, Chebec and Harrington. Among 247 isolated sequences, twenty six percent were homologous to genes of known function. The abundance of these sequences differed between the two cultivars. Variations in the motif sequences of the cloned genes were also found between these two cultivars. In addition to the cloned fragments, twenty-two putative barley P450 encoding genes were identified from 24,000 cereal sequences in the International Triticeae EST Cooperative (ITEC) database by homology search. Among these Expressed Sequence Tag (EST) sequences, a full-length P450 sequence was selected for further investigation in this study.This novel P450 gene, CYP72A39, was expressed at a very early vegetative stage, but no expression was detected at the reproductive stage. Comparison of expression profiles of this gene and “digital expression” databases confirmed that this gene was homologous to several cereal EST clones with tissue-specific transcripts responding to various environmental stimuli, such as stresses and disease. Among these, many transcripts in barley were obtained from stressed tissues at the vegetative stage, and two transcripts in wheat (Triticum aestivum L.) were expressed after being challenged by barley powdery mildew pathogen (Blumeria graminis f. sp. hordei L.). This suggested that CYP72A39 may play a defence role in the barley seedling.The secondary structure of CYP72A39 was investigated in a comparative analysis using a computational approach. However, attempts to express CYP72A39 in a heterologous system and functional assays of the native protein in barley did not give decisive results, due to the disordered structure of the native protein and limitations of the current method. Screening the 3’ UnTranslated Region (3’UTR) of this gene in 158 genotypes of domesticated, landrace and wild barley revealed two haplotypes, which differed by a 12 base indel positioned between two transversions. The presence of both haplotypes in wild and cultivated barley suggests this polymorphism predates the domestication of barley. This indel was mapped to the long arm of chromosome 6H, less than 10 centi-Morgans (cM) from the gene encoding resistance to powdery mildew in barley (B. graminis). A comparison between haplotype diversity and powdery mildew resistance data for over 102 genotypes showed a weak link between the ‘long’ haplotype and resistance, while the ‘short’ haplotype was associated with susceptibility. There was no evidence for a strong correlation between haplotype and quality type (malt or feed); however, more malting varieties had the long haplotype, suggesting a possible association with some attributes in modern malting barley. There was no evidence for association with other characteristics such as geographic origin, growth habit or row number. Phylogenetic relationship of the CYP72A39 to other CYP72 members was also investigated.

In plants, P450 enzymes encoded by P450 genes play a central role in numerous biosynthetic pathways, such as the production of secondary metabolites, stress responses and disease resistance. This thesis reports upon the utilization of molecular biology techniques to study P450 gene(s) in barley (Hordeum vulgare L.).Using several combinations of degenerate primers, a large number of barley P450 gene fragments were cloned and sequenced from two commercial varieties, Chebec and Harrington. Among 247 isolated sequences, twenty six percent were homologous to genes of known function. The abundance of these sequences differed between the two cultivars. Variations in the motif sequences of the cloned genes were also found between these two cultivars. In addition to the cloned fragments, twenty-two putative barley P450 encoding genes were identified from 24,000 cereal sequences in the International Triticeae EST Cooperative (ITEC) database by homology search. Among these Expressed Sequence Tag (EST) sequences, a full-length P450 sequence was selected for further investigation in this study.This novel P450 gene, CYP72A39, was expressed at a very early vegetative stage, but no expression was detected at the reproductive stage. Comparison of expression profiles of this gene and “digital expression” databases confirmed that this gene was homologous to several cereal EST clones with tissue-specific transcripts responding to various environmental stimuli, such as stresses and disease. Among these, many transcripts in barley were obtained from stressed tissues at the vegetative stage, and two transcripts in wheat (Triticum aestivum L.) were expressed after being challenged by barley powdery mildew pathogen (Blumeria graminis f. sp. hordei L.). This suggested that CYP72A39 may play a defence role in the barley seedling.The secondary structure of CYP72A39 was investigated in a comparative analysis using a computational approach. However, attempts to express CYP72A39 in a heterologous system and functional assays of the native protein in barley did not give decisive results, due to the disordered structure of the native protein and limitations of the current method. Screening the 3’ UnTranslated Region (3’UTR) of this gene in 158 genotypes of domesticated, landrace and wild barley revealed two haplotypes, which differed by a 12 base indel positioned between two transversions. The presence of both haplotypes in wild and cultivated barley suggests this polymorphism predates the domestication of barley. This indel was mapped to the long arm of chromosome 6H, less than 10 centi-Morgans (cM) from the gene encoding resistance to powdery mildew in barley (B. graminis). A comparison between haplotype diversity and powdery mildew resistance data for over 102 genotypes showed a weak link between the ‘long’ haplotype and resistance, while the ‘short’ haplotype was associated with susceptibility. There was no evidence for a strong correlation between haplotype and quality type (malt or feed); however, more malting varieties had the long haplotype, suggesting a possible association with some attributes in modern malting barley. There was no evidence for association with other characteristics such as geographic origin, growth habit or row number. Phylogenetic relationship of the CYP72A39 to other CYP72 members was also investigated.

Diss. (sammanfattning) Alnarp : Sveriges lantbruksuniversitet, 2005. / Härtill 4 uppsatser.

Diss. (sammanfattning) Alnarp : Sveriges lantbruksuniv., 2002. / Härtill 4 uppsatser.

Diss. (sammanfattning) Umeå : Sveriges lantbruksuniversitet, 2006. / Härtill 3 uppsatser.