O sistema de acasalamentos das abelhas sociais é intrigante. As rainhas acasalam durante uma curta janela de tempo nas fases inicias de suas vidas, armazenando o esperma pelo resto de suas vidas. Enquanto as rainhas das abelhas sem ferrão (Meliponini) e de mamangavas (Bombini) se acasalam com um ou poucos machos (monândricas), as rainhas de espécies representantes do gênero Apis (Apini) acasalam-se com vários machos (poliândricas). Diversas hipóteses têm sido propostas para compreender os benefícios do comportamento extremamente promíscuo das rainhas do gênero Apis. Porém, pouco foi feito para entender as igualmente intrigantes pressões seletivas que mantém a monandria em um grupo tão diverso quanto o das abelhas sem ferrão. No presente estudo, investigamos como as forças seletivas causadas pelas produção de machos diploides (uma consequência natural do sistema de determinação sexual em Hymenoptera) podem afetar o sistema de acasalamento da abelha sem ferrão brasileira, Scaptotrigona depilis. Em particular, rainhas que realizam um acasalamento pareado para o locus sexual (i.e., acasalam com um macho que possui o mesmo alelo sexual) terão o seu fitness reduzido porque elas serão executadas em colônias com 50% de machos diploides entre sua cria diploide. Acasalar-se com mais de um macho aumentam as chances de um acasalamento pareado, mas reduz a proporção de machos diploides na cria. Por meio da manipulação dos favos de cria de colônias experimentais, nós testamos se a mortalidade de rainhas com proporções menores de machos diploides na cria terão taxas de mortalidade similares à de rainhas em colônias com 0% ou 50% de machos diploides na cria. Para isso, obtivemos rainhas produzindo machos diploides nesta espécie e estudamos alguns detalhes de sua biologia, em particular, a viabilidade e morfologia de seus espermatozoides e comportamento fora de suas colônias (Capítulo 1). Nós verificamos se a rainha morre na presença de machos diploides, como este comportamento é predominante na população, e quais os possíveis mecanismos que desencadeiam este comportamento (Capítulo 2). Finalmente, verificamos se a mortalidade de rainhas em colônias com proporções menores de machos diploides na cria (simulando acasalamentos múltiplos) é mais próxima da mortalidade de rainhas em colônias com 0 ou 50% de machos diploides (Capítulo 3). Verificamos que os machos diploides de S. depilis são viáveis e juntam-se a agregados reprodutivos. Seus espermatozoides possuem a mesma viabilidade que os dos machos haploides, porém possuem a cabeça e a cauda maiores. As rainhas são mortas na presença de cerca de 50% de machos diploides na cria e esse comportamento é muito difundido na população estudada, com 100% de morte das rainhas (n=20). O perfil químico de hidrocarbonetos cuticulares dos machos diploides é quantitativamente diferente dos machos haploides, sugerindo que pode conter informações para que a rainha seja executada. No entanto, por meio da contagem de espermatozoides na espermateca de rainhas recém acasaladas e com um ano de idade foi possível observar que o esgotamento de espermatozoides pode afetar a expectativa de vida das rainhas. Isso sugere que o sinal para execução da rainha também pode estar associado a uma quantidade grande de machos emergindo, como um sinal de esgotamento de espermatozoides da rainha, ao invés da ploidia dos machos. Rainhas em colônias com cerca de 25% de machos diploides emergindo foram mortas em proporções semelhantes à de rainhas em colônias com 50% de machos diploides. Isso indica que esse comportamento causa uma pressão seletiva contra os acasalamentos duplos nessa espécie, pois ao acasalar duas vezes a rainha dobra as chances de realizar um acasalamento pareado e a mortalidade é igual à de rainhas que se acasalam uma única vez. / The mating systems of social bees is intriguing. Queens mate within a narrow time window very early in their lives, storing male sperm for their entire lifespan. In bumblebees (Bombini) and stingless bees (Meliponini), queens usually mate with a single male (monandry), while the queens of Apis species (Apini) mate with several males (polyandry). Several hypothesis have been proposed to understand the benefits of the extreme polyandry of honey bees. However, very little have been done in order to understand the equally intriguing selective forces that make such a diverse group as stingless bees as monandrous. In the present study we investigate how the selective force imposed by the chances of producing diploid males (a natural consequence of sex determination system of Hymenoptera) can affect the mating system of the Brazilian stingless bee Scaptotrigona depilis. In particular, queens that make a matched mating for the sex locus (i.e., mating with a male with the same sex allele) will have reduced fitness because queens are executed when producing 50% of diploid males amongst her diploid offspring. Mating with more males increase the chances of a matched mating, but reduces the proportion of diploid males on their brood. By manipulating the brood combs of experimental colonies, we tested whether queens with smaller proportions of diploid males (25%) will have mortality rates similar to queens in colonies with 0% or 50% diploid males. For that, we obtained queens producing diploid males and studied details of diploid males biology, in particular their sperm viability and morphology, and their behaviour out of their mother colonies (Chapter 1). We verified how prevalent queen execution behaviour under diploid male production is on this species, and the possible proximate mechanisms that trigger this behaviour (Chapter 2). Finally, we verified whether queen mortality in colonies with lower proportion of diploid males (simulating multiple mating) is closer to colonies with 50% or 0% of diploid males (Chapter 3). We verified that diploid males of S. depilis are viable and can join reproductive aggregations. Their sperm cells have the same viability of haploid males, but have longer sperm head and tail cells. Queens are executed in the presence of 50% of diploid males, and this behaviour is very prevalent on the studied population, with 100% of queen death (n=20). The cuticular hydrocarbon chemical profile of diploid males is quantitatively different from haploid males, suggesting that workers could use it as a signal for queen execution. However, sperm count of newly mated queens and one-year-old queens showed that sperm depletion might affect queen lifespan. This suggests that the signal for queen execution could also be related to a high number of males emergence, as a signal of queen sperm depletion, instead of male ploidy. Queens mortality in colonies with 25% of diploid males was similar to queens in colonies with 50%. This indicates that double mating is selected against regarding this selective pressure, since mating twice will increase the chances of a matched mating and queen mortality will be the same as single mated queens.

Background: Over the relatively short period of its evolutionary history, Boechera (Brassicaceae) has undergone rapid radiation that has produced 70+ morphologically distinct, sexual diploids. However, reproductive isolation has moved more slowly than morphological divergence in this group and the diploids appear to hybridize frequently where they coexist. Boechera duchesnensis appears to be the result of hybridization between its putative parents Boechera thompsonii and Boechera formosa. Objectives: The objectives of this study are to (i) analyze and document genetic diversity patterns in the population structure, - including allelic and heterozygosity frequencies - of B. thompsonii and B. formosa in concert with their geographic distribution to determine clustering relationships within these populations, (ii) confirm and expand the morphological characteristics of B. thompsonii and B. formosa, as initially proposed in the literature, including pollen and trichome structure using Scanning Electron Microscopy (SEM) to confirm ploidy level and to determine whether both putative parent species share morphological characteristics with their apomictic diploid offspring, and (iii) use genetic and morphologic evidence to show that B. thompsoii and B. formosa are, in fact, the parents of B. duchesnensis by comparing the genetic diversity patterns, population structure, and morphological characteristics of B. duchesnensis, to those of its proposed putative parents (B. thompsonii and B. formosa) and to confirm that B. duchesnensis shares characteristics of Boechera. Methods: Microsatellite data from 14 loci previously identified in Boechera were used to reexamine the current classifications and taxonomic foundations of three Boechera spp. GenAlEx 6.501 (Peakall and Smouse, 2006, 2012) was used to analyze genetic population structures of two divergent sexual diploids in the genus Boechera: B. thompsonii and B. formosa and to later compare those with the population structure of B. duchesnensis. Geographicaldata were plotted using ArcGIS 10.1 (Esri, 2012) to map heterozygosity distribution. Cluster analysis was run with STRUCTURE 2.3.3 (Pritchard et al., 2000; Falush et al., 2003, 2007) and distribution of allelic diversity and heterozygosity was subsequently compared within each taxon and correlated with geographic distribution characteristics. Resultant data were then compared with B. duchesnensis data to document genetic diversity patterns, population structure, and morphological characteristics. Key Results: Analysis of genetic diversity patterns, allelic distribution of the populations, and heterozygosity of B. thompsonii and B. formosa across their geographic range identified four genetically distinct clusters within B. thompsonii, and one genetically distinct cluster in B. formosa. Allelic frequencies in all four discrete population clusters of B. thompsonii and in one discrete population cluster of B. formosa were close to values found in species on the decline. Reproductive isolation, genetic variability, and allelic frequencies were determined, specimen elevations reported, and morphological characteristics reported in the literature were confirmed and expanded. A codominant genetic analysis performed for 14 different loci for B. duchesnensis against those of its parents showed that B. duchesnensis inherits alleles from both putative parents and confirms B. thompsonii and B. formosa as the parents of B. duchesnensis. Observed levels of heterozygosity of B. thompsonii and B. formosa were lower than expected levels and lower than those of other outcrossing diploids. The mean overall observed heterozygosities for each cluster were determined and documented by geographic location. A substantially higher level of observed heterozygosity in B. duchesnensis (Ho = 0.908) consistent with genetic fixation of a heterozygote and apomixis, supports hybridization as a speciation mechanism and apomixis as a mode of reproduction accounting for genotypic and phenotypic diversity. Morphological characteristics, especially those of pollen and trichomes were confirmed, expanded, and documented with SEM imagery. Discussion: This study provides an analysis of the genetic diversity patterns inherent in the population structure, allelic frequencies, allelic variation among individuals of the rare sexual diploids B. thompsonii, B. formosa, and the apomictic diploid B. duchesnensis in correlation with their geographic distribution. There is an implication of a reproductive barrier, within populations of the same species, that contributes to genetic isolation between clusters. I analyze the tendency of reduced heterozygosity to lead to genetic fixation, reproductive isolation, and how the heightened heterozygosity supports the classification of B. duchesnensis as an apomict. Assessing potential populations that might exist based on similar characteristics could possibly provide inferences about where future research might find similar examples of this hybridization. Reproductive isolation is hypothesized to limit gene flow between identified clusters of B. thompsonii and B. formosa exacerbating low observed heterozygosity levels and low allelic frequency levels. Population studies and cluster analysis have implications for offering future conservation strategies for both taxa.

As abelhas sem ferrão exercem importante papel ecológico como polinizadores de muitas espécies vegetais das regiões tropicais e tem significativo potencial para uso na polinização de culturas agrícolas. Contudo, com a contínua degradação de habitats, as populações de inúmeras espécies tem se tornado cada vez menores e separadas umas das outras por grandes distâncias. A criação de espécies de abelhas é um componente essencial para a conservação da biodiversidade, além de uma alternativa de fonte de renda. Para tanto, esforços de conservação e programas de criação em escala comercial requerem uma combinação de fatores, como o conhecimento biológico mais amplo, principalmente os relacionados à produção de sexuados e à diversidade genética necessária para manter a viabilidade de pequenas populações destas abelhas. Nesse contexto, os principais objetivos desta tese foram avaliar a variabilidade genética em populações manejadas, sob condições de isolamento genético ou não, e a produção de machos e rainhas nessas populações e o papel na reprodução. Para isso estudamos duas populações de Melipona scutellaris mantidas em regiões geográficas distintas, uma no município de Igarassu (PE; 7°50S 34°54W), onde a espécie ocorre naturalmente e outra no município de São Simão (SP; 21°26 47°34W), onde a população foi iniciada com duas colônias e criada por mais de 10 anos, quando chegou, a partir de sucessivas multiplicações, a 55 ninhos. Assim, embora a população de S. Simão tivesse maior redução na diversidade alélica e maior frequência de machos diplóides, quando comparada à mantida em Igarassu, ela foi criada com sucesso por um extenso período (ca. 10 anos). Provavelmente o baixo número de alelos sexuais em S. Simão, e a conseqüente produção de machos diplóides, foi a principal explicação para a freqüência significativamente maior de sexuados criados nessa população. Como contraponto à alta produção de machos diplóides, as substituições das rainhas-mãe foram mais frequentes e as colônias produziram mais rainhas. Além disso, a alta produção de machos e rainhas também pode ser entendida em termos de benefícios reprodutivos individuais. Tanto as rainhas fisogástricas como as operárias poedeiras foram responsáveis pela maternidade de machos haplóides. Contudo, 80% dos machos filhos de operárias foram produzidos por operárias filhas da rainha-mãe substituída, indicando que essas operárias especiais tem sobrevida maior que as demais e parasitam reprodutivamente a força de trabalho da geração seguinte, que são menos relacionadas a elas. Quanto à super-produção de rainhas, detectou-se que 25% das colônias órfãs, após a perda da rainha-mãe, eram invadidas por rainhas que foram produzidas e vieram de colônias próximas. Nessas colônias não-natais, elas iniciaram suas atividades de postura. Este importante resultado muda as bases para melhoramento genético destas abelhas estabelecidas até o momento. Outro estudo relacionado à alta produção de rainhas foi realizado em colônia poligínica de M. quadrifasciata, em que oito rainhas fisogástricas co-existiam. Ao contrário da hipótese de que alguma das rainhas poderia ter vindo de outro ninho, todas eram irmãs completas. Isto sugere novas estratégias reprodutivas ainda desconhecidas para as abelhas do gênero Melipona. / Stingless bees play an important ecological role as pollinators of many wild plant species in the tropics and have significant potential for the pollination of agricultural crops. However, as a consequence of habit degradation, populations of a number of bee species have became increasingly small and separated from one another by large distances. Thus, stingless beekeeping is an essential component of biodiversity conservation, as well as a profitable business. Therefore, conservation efforts and breeding programme on a large scale require a combination of factors, including a broader biological knowledge, especially those related to production of sexuals and to the genetic diversity needed to maintain the viability of small population. In this context, the main goals were to evaluate the genetic variability in managed populations under or not genetic isolation and the production of males and queens. Two Melipona scutellaris populations were studied and they were kept in different geographic regions, one in Igarassu (PE; 7°50\'S 34°54\'W), in the species natural area of occurrence, and the other in São Simão (SP; 21°26 47°34W), where the population was started from only two foundress colonies and which after a breeding programme of ten years increased to about 55. Despite a great reduction in the number of alleles and an increased frequency of diploid males in the S. Simão population, it could be successfully bred and maintained for a prolonged period (ca. 10 years). Probably the low number of sex alleles in S. Simão population, leading to production of diploid males, was the main reason for the highest level of sexual production. To counter-balance the high production of diploid males, the replacement of mother queen was more frequent leading to higher levels of queen production by the colonies. Furthermore, the high production of males and queens can also be explained by the individual fitness benefits. Queen and reproductive workers were haploid males mothers. However, 80% of the workers sons had genotypes that were compatible with them being the sons of workers that were the offspring of a superseded queen, indicating that these workers greatly outlive all other workers and reproductively parasitize the next-generation workforce, that are less related individuals. Related to queen overproduction, 25% of all queenless colonies were invaded by unrelated queens that fly in from unrelated hives nearby. In these non-natal colonies, the alien queens started their egg laying activities. Another study related to the high queen production was conducted in a polygyne colony of M. quadrifasciata, where eight physogastric queens coexisted. Contrary to the hypothesis that some of these queens could be an alien queen, it was confirmed that they were full-sisters. This suggests new reproductive strategies that are unknown for Melipona bees.

O sistema de acasalamentos das abelhas sociais é intrigante. As rainhas acasalam durante uma curta janela de tempo nas fases inicias de suas vidas, armazenando o esperma pelo resto de suas vidas. Enquanto as rainhas das abelhas sem ferrão (Meliponini) e de mamangavas (Bombini) se acasalam com um ou poucos machos (monândricas), as rainhas de espécies representantes do gênero Apis (Apini) acasalam-se com vários machos (poliândricas). Diversas hipóteses têm sido propostas para compreender os benefícios do comportamento extremamente promíscuo das rainhas do gênero Apis. Porém, pouco foi feito para entender as igualmente intrigantes pressões seletivas que mantém a monandria em um grupo tão diverso quanto o das abelhas sem ferrão. No presente estudo, investigamos como as forças seletivas causadas pelas produção de machos diploides (uma consequência natural do sistema de determinação sexual em Hymenoptera) podem afetar o sistema de acasalamento da abelha sem ferrão brasileira, Scaptotrigona depilis. Em particular, rainhas que realizam um acasalamento pareado para o locus sexual (i.e., acasalam com um macho que possui o mesmo alelo sexual) terão o seu fitness reduzido porque elas serão executadas em colônias com 50% de machos diploides entre sua cria diploide. Acasalar-se com mais de um macho aumentam as chances de um acasalamento pareado, mas reduz a proporção de machos diploides na cria. Por meio da manipulação dos favos de cria de colônias experimentais, nós testamos se a mortalidade de rainhas com proporções menores de machos diploides na cria terão taxas de mortalidade similares à de rainhas em colônias com 0% ou 50% de machos diploides na cria. Para isso, obtivemos rainhas produzindo machos diploides nesta espécie e estudamos alguns detalhes de sua biologia, em particular, a viabilidade e morfologia de seus espermatozoides e comportamento fora de suas colônias (Capítulo 1). Nós verificamos se a rainha morre na presença de machos diploides, como este comportamento é predominante na população, e quais os possíveis mecanismos que desencadeiam este comportamento (Capítulo 2). Finalmente, verificamos se a mortalidade de rainhas em colônias com proporções menores de machos diploides na cria (simulando acasalamentos múltiplos) é mais próxima da mortalidade de rainhas em colônias com 0 ou 50% de machos diploides (Capítulo 3). Verificamos que os machos diploides de S. depilis são viáveis e juntam-se a agregados reprodutivos. Seus espermatozoides possuem a mesma viabilidade que os dos machos haploides, porém possuem a cabeça e a cauda maiores. As rainhas são mortas na presença de cerca de 50% de machos diploides na cria e esse comportamento é muito difundido na população estudada, com 100% de morte das rainhas (n=20). O perfil químico de hidrocarbonetos cuticulares dos machos diploides é quantitativamente diferente dos machos haploides, sugerindo que pode conter informações para que a rainha seja executada. No entanto, por meio da contagem de espermatozoides na espermateca de rainhas recém acasaladas e com um ano de idade foi possível observar que o esgotamento de espermatozoides pode afetar a expectativa de vida das rainhas. Isso sugere que o sinal para execução da rainha também pode estar associado a uma quantidade grande de machos emergindo, como um sinal de esgotamento de espermatozoides da rainha, ao invés da ploidia dos machos. Rainhas em colônias com cerca de 25% de machos diploides emergindo foram mortas em proporções semelhantes à de rainhas em colônias com 50% de machos diploides. Isso indica que esse comportamento causa uma pressão seletiva contra os acasalamentos duplos nessa espécie, pois ao acasalar duas vezes a rainha dobra as chances de realizar um acasalamento pareado e a mortalidade é igual à de rainhas que se acasalam uma única vez. / The mating systems of social bees is intriguing. Queens mate within a narrow time window very early in their lives, storing male sperm for their entire lifespan. In bumblebees (Bombini) and stingless bees (Meliponini), queens usually mate with a single male (monandry), while the queens of Apis species (Apini) mate with several males (polyandry). Several hypothesis have been proposed to understand the benefits of the extreme polyandry of honey bees. However, very little have been done in order to understand the equally intriguing selective forces that make such a diverse group as stingless bees as monandrous. In the present study we investigate how the selective force imposed by the chances of producing diploid males (a natural consequence of sex determination system of Hymenoptera) can affect the mating system of the Brazilian stingless bee Scaptotrigona depilis. In particular, queens that make a matched mating for the sex locus (i.e., mating with a male with the same sex allele) will have reduced fitness because queens are executed when producing 50% of diploid males amongst her diploid offspring. Mating with more males increase the chances of a matched mating, but reduces the proportion of diploid males on their brood. By manipulating the brood combs of experimental colonies, we tested whether queens with smaller proportions of diploid males (25%) will have mortality rates similar to queens in colonies with 0% or 50% diploid males. For that, we obtained queens producing diploid males and studied details of diploid males biology, in particular their sperm viability and morphology, and their behaviour out of their mother colonies (Chapter 1). We verified how prevalent queen execution behaviour under diploid male production is on this species, and the possible proximate mechanisms that trigger this behaviour (Chapter 2). Finally, we verified whether queen mortality in colonies with lower proportion of diploid males (simulating multiple mating) is closer to colonies with 50% or 0% of diploid males (Chapter 3). We verified that diploid males of S. depilis are viable and can join reproductive aggregations. Their sperm cells have the same viability of haploid males, but have longer sperm head and tail cells. Queens are executed in the presence of 50% of diploid males, and this behaviour is very prevalent on the studied population, with 100% of queen death (n=20). The cuticular hydrocarbon chemical profile of diploid males is quantitatively different from haploid males, suggesting that workers could use it as a signal for queen execution. However, sperm count of newly mated queens and one-year-old queens showed that sperm depletion might affect queen lifespan. This suggests that the signal for queen execution could also be related to a high number of males emergence, as a signal of queen sperm depletion, instead of male ploidy. Queens mortality in colonies with 25% of diploid males was similar to queens in colonies with 50%. This indicates that double mating is selected against regarding this selective pressure, since mating twice will increase the chances of a matched mating and queen mortality will be the same as single mated queens.

The development of mouse (Mus musculus) haploid parthenogenetic embryos does not reach the same level as normal embryos. The aim of this diploma thesis was to find out whether haploid parthenogenetic embryos of mice differ in the nucleocytoplasmic ratio. The volume of the nucleus increases with ploidity. The nucleocytoplasmic ratios of haploid embryos do not significantly change between the two-cell and four-cell stage (p = 0.052), there is a significant difference (p < 0.001) for diploid and tetraploid embryos. Non-standard nucleocytoplasmic ratio could be related to the problematic development. Understanding the regulation of preimplantational development of parthenogenetic embryos will increase the efficiency of haploid embryonic stem cell derivation.

Chenopodium quinoa Willd., an ancient Andean pseudocereal almost exclusively consumed in South America, jumped onto the global stage when Western cultures noted quinoa's advantageous nutritional profile. Quinoa seed's high protein content, nutritionally balanced amino acid profile, low glycemic index, and high fiber, vitamin, and mineral content, make it a highly sought-after 'superfood'. Pitseed goosefoot (C. berlandieri Moq.), a closely related North American species sharing quinoa's genome composition (AABB), grows across the North American continent, inhabiting diverse environments including the saline coastal soils of the Gulf of Texas and the drought-prone regions of the Southwest. Quinoa and pitseed goosefoot, along with South American avian goosefoot (C. hircinum Schrad.), make up the Allotetraploid Goosefoot Complex (ATGC). We hypothesize that an ancient hybridization event between A- and B-genome diploids, with a subsequent whole-genome duplication, gave rise to the common ancestor of the ATGC. Prior data indicate that allopolyploidization most likely occurred within North America, with long-range dispersal of the ATGC to South America. We have sequenced the genome of the North American AA-genome diploid C. watsonii and identified via DNA marker analyses the closest extant species to the AA-genome diploid ancestor of the ATGC from among a panel of 41 AA-genome diploid resequenced accessions, encompassing 30 putative AA-genome diploid species, from North and South America. We also present evidence for reciprocal long-range dispersal of Chenopodium diploids between North and South America.

Perennial warm-season grasses constitute the backbone of many forage production systems, whether for grazing or harvested feed. North American native plants, specifically grasses, forbs and legumes offer unique ecosystem benefits along with forage quality and digestibility that are unmatched by introduced species. The disparity in breeding and research focused on improvement of introduced species as opposed to native genera has led to inflated use of introduced species as forage types in lieu of native options, due to their unimproved nature. Eastern gamagrass [Tripsacum dactyloides (L.) L.] is proven to be a widely adapted, highly productive forage species in the southeast, Great Plains and northeast United States. A major limitation to more widespread use of eastern gamagrass is high seed dormancy, which leads to increased seed cost. Here, research used recurrent phenotypic selection breeding methods to reduce seed dormancy, with the ultimate goal of developing a population of individuals that produce non-dormant eastern gamagrass seed.

The genus Chenopodium contains many economically important species in the New World, but is relatively understudied and poorly understood, especially in terms of evolutionary relationships. A better understanding of the structure of this genus could significantly help in breeding efforts on its cultivated members, notably the tetraploid C. quinoa and also certain varieties of C. berlandieri, also tetraploid. Of special concern is determining which diploid weed species are the most likely ancestors for C. quinoa, C. berlandieri, and the other tetraploid members of subsection Cellulata. The phylogeny can be understood in part by examining the ribosomal RNA loci and observing how many copies of the 5S and 45S loci each New World species contains. In this work, the 5S and 45S ribosomal RNA loci are characterized by means of fluorescence in situ hybridization in 23 Chenopodium species collected in the New World, with the 5S locus labeled red and the 45S locus labeled green. Based on these results, the pool of most likely candidate ancestor species for C. quinoa and C. berlandieri includes C. fremontii, C. incanum, C. neomexicanum, and C. watsonii.

Master of Science / Genetics Interdepartmental Program / Bikram S. Gill / TILLING (Targeting Induced Local Lesions IN Genomes) is a high-throughput, reverse genetics strategy for scanning mutagenized populations for point mutations in loci of interest. Originally, TILLING was used to investigate gene function in Arabidopsis and has since been similarly applied for gene functional analysis in other organisms. TILLING also allows the generation of novel genetic variation in specific genotypes and, thus, has been implemented as a tool for crop improvement. Ethyl methanesulfonate (EMS) is a widely used mutagen to induce point mutations in most TILLING protocols. M1 plants are then self-pollinated and M2 seed harvested. A single seed is grown from each M2 progeny and tissue taken for DNA isolation. M3 seed is cataloged. DNA is pooled to increase the efficiency and aid in mutation detection. Polymerase chain reaction (PCR) is used to amplify a locus of interest using the M2 DNA pools as a template. The PCR products are digested with an endonuclease that cleaves mismatched, mutant DNA, and the digested products are visualized. The pools for which PCR products are positive for a mutation are deconvoluted to determine which individual plant of the pool was responsible for the mutation. DNA from the positive individual is sequenced to determine the type of mutation (missense, nonsense, synonymous). Individuals with mutations that are more likely to disrupt gene function (nonsense and certain missense) are studied further by growing the corresponding M3 generation. In bread wheat, Triticum aestivum, TILLING is complicated by polyploidy: genes that have homoeologs require that the functionality of each be studied. If functional homoeologs are present for all three genomes, mutants must be identified for each homoeolog, followed by successive intercrossing to produce a triple mutant plant. As a model for wheat genetics, we propose TILLING in diploid wheat. EMS mutant populations were created in diploid wheat (Triticum monococcum ssp. monococcum) and the hexaploid bread wheat cultivar ‘Jagger’. The diploid and hexaploid wheat populations were screened for mutations at the waxy locus, GBSS1, as a validation of our population and for comparative analysis of mutation rates in 2x and 6x wheat. For diploid wheat, GBSSI was screened in 716 M2 plants, and one mutant was found for 1.9 Mb screened. 3 For hexaploid wheat, GBSSI was screened in 518 M2 plants, and 30 mutants were identified within a total of 657 Kb screened, giving a mutation frequency of one mutation per 22 Kb. The reasons for this vast difference in mutation frequency between diploid and hexaploid wheat are discussed. The diploid wheat population was further examined by screening for mutations within four lignin biosynthesis candidate genes, for a total of 2 Mb screened. A single mutant was discovered for both of the lignin genes PAL6 and HCT, giving a mutation frequency of one mutation per 1 Mb screened.

Bean pod mottle virus (BPMV), a member of the genus Comovirus in the family Comoviridae, is widespread in the major soybean-growing areas in the United States. The complete nucleotide sequences of the genomic RNAs of the naturally occurring partial diploid strain IL-Cb1 were determined. Intermolecular RNA1 recombinants were isolated from strain IL-Cb1 and characterized at the molecular level. Structurally similar recombinant RNA1 was also generated after four passages in soybean derived from plants previously inoculated with a mixture of infectious RNA1 transcripts from two distinct strains. BPMV was developed as a plant viral vector that is appropriate for gene expression and virus-induced gene silencing (VIGS) in soybean. The foreign gene was inserted between the movement protein (MP) and the large coat protein (L-CP) coding regions. The recombinant BPMV constructs were stable following several serial passages in soybean and relatively high levels of protein expression were attained. Successful expression of several proteins with different biological activities was demonstrated from the BPMV vector. Double infection of soybean by BPMV and SMV triggers a synergistic interaction leading to a serious disease. To investigate the underlying mechanism, helper componentprotease (HC-Pro) genes from several SMV strains and TEV were expressed from BPMV vectors. The recombinant BPMV vectors carrying the HC-Pro genes from SMV strain G7 or TEV induced very severe symptoms on soybean whereas constructs containing the HC-Pro gene from SMV isolate P10, a mild strain with an apparent defect in synergism, induced only very mild symptoms. Transient agroinfiltration assays using GFP-transgenic Nicotiana benthamiana showed that HC-Pro from SMV isolate P10 was not a RNA silencing suppressor, whereas those of SMV strain G7 and TEV exhibited strong suppressor activities. Analysis of chimeric HC-Pro genes and point mutations indicated that a positively charged amino acid at position 144 is critical for the suppressor function of not only SMV HC-Pro but also other potyvirus HC-Pro proteins. Although amino acid substitution at position 144 resulted in changes in small RNA profile, it did not affect HC-Pro stability.