Academic literature on the topic 'Diversity Array Technology (DArT)'

Recently, a technology known as DArT (diversity array technology) has been developed to increase throughput in marker assisted selection (MAS). DArT utilizes microarray technology as a method to potentially compare thousands of molecular markers in one test to a single DNA sample. We used DArT on two sets of interspecific tomato [Solanum lycopersicum (Fla 7613) × S. pennellii (LA 716 or LA 2963)] segregating populations (BC, F2, and F1). We compared over 300 segregating plants to 3840 random tomato genomic fragments. After the 3840 markers were prepared, it took about 2 weeks of laboratory time to perform the experiments. With experience, this time can be reduced. We identified a total of 654 polymorphic markers usable for developing a DArT tomato genetic map. Depending on the particular cross, 13 to 17 linkage groups were identified (LOD 3) per population. Most recently, the amplified polymorphic DNA (AFLP) technique has been used for rapid genetic mapping of large numbers of anonymous genomic fragments. Besides the additional effort and reagents using AFLPs compared to DArT, a desired AFLP polymorphic band is often difficult to clone and process into a PCR based marker, whereas in DArT all markers are already cloned and immediately available for such experiments. A drawback to DArT is that it requires specialized software and equipment and is technically demanding. However, once the equipment and software are secured, techniques are optimized, and segregating populations developed, marker throughput is increased by orders of magnitude. Although challenging, the application of DArT can dramatically increase MAS throughput, thus facilitating quantitative trait and saturated mapping research.

Knowledge of the genetic structure and diversity of germplasm collections is crucial for sustainable genetic improvement through hybridization programs and rapid adaptation to changing breeding objectives. The objective of this study was to determine the genetic diversity and population structure of 281 International Institute of Tropical Agriculture (IITA) soybean accessions using diversity array technology (DArT) and single nucleotide polymorphism (SNP) markers for the efficient utilization of these accessions. From the results, the SNP and DArT markers were well distributed across the 20 soybean chromosomes. The cluster and principal component analyses revealed the genetic diversity among the 281 accessions by grouping them into two stratifications, a grouping that was also evident from the population structure analysis, which divided the 281 accessions into two distinct groups. The analysis of molecular variance revealed that 97% and 98% of the genetic variances using SNP and DArT markers, respectively, were within the population. Genetic diversity indices such as Shannon’s diversity index, diversity and unbiased diversity revealed the diversity among the different populations of the soybean accessions. The SNP and DArT markers used provided similar information on the structure, diversity and polymorphism of the accessions, which indicates the applicability of the DArT marker in genetic diversity studies. Our study provides information about the genetic structure and diversity of the IITA soybean accessions that will allow for the efficient utilization of these accessions in soybean improvement programs, especially in Africa.

Several molecular marker systems have been developed for assessing genetic diversity in crop germplasm collections. A trade-off often exists between the number of loci that can feasibly be sampled by a marker system and the amount of information provided by each locus. We compared the usefulness of two marker systems for revealing genetic diversity and population structure in cassava (Manihot esculenta Crantz): simple sequence repeats (SSRs) and diversity array technology (DArT) markers. DArTs survey many more loci per reaction than do SSRs; however, as bi-allelic, dominant markers, DArTs provide less polymorphism information per locus. Genetic differentiation was assessed in a randomly selected set of 436 cassava accessions, consisting of 155 African and 281 Latin American accessions. A genome-wide set of 36 SSR markers and a DArT array of approximately 1000 polymorphic clones were used to assess genetic diversity and differentiation. Cluster analyses were performed using principal coordinate analysis (PCoA). Results were compared with a priori expectations of genetic differentiation based on previous genetic analyses. Analyses of the two datasets generated broadly similar clustering patterns. However, SSRs revealed greater differentiation than DArTs, and more effectively recovered patterns of genetic differentiation observed in previous analyses (differentiation between Latin American and African accessions, and some geographical differentiation within each of these groups). These results suggest that SSR markers, while low throughput in comparison with DArTs, are relatively better at detecting genetic differentiation in cassava germplasm collections. Nonetheless, DArTs will likely prove useful in ‘orphan crop’ species, where alternative molecular markers have not been developed.

A diversity array technology (DArT) marker platform was developed for the cotton genome, to evaluate the use of DArT markers compared with AFLP markers in mapping and transferability across the mapping populations. We used a reference genetic map of tetraploid Gossypium L. that already contained ∼5000 loci, which coalesced into 26 chromosomes, to anchor newly developed DArT and AFLP markers with the aim of further improving utility and map resolution. Our results indicated that the percentage of polymorphic DArT markers that could be genetically mapped (78.15%) was much higher than that of AFLP markers (22.28%). Sequence analysis of DArT markers indicated that a majority matched known expressed sequence tag (EST) sequences from tetraploid and diploid Gossypium species. A total of 794 Arabidopsis genes were homologous with various DArT marker sequences. Chromosomes 5(A), 7(A), 19(D), 23(D), and 24(D) had more Arabidopsis syntenic DArT markers than the other chromosomes. Anchoring DArT markers from the reference map to a recombinant inbred line (RIL) map indicated that DArT markers will speed the building of maps in de novo RIL populations.

With increasing demands on the quality and quantity of food required now and in the future, improvements to current agriculture practices are required. Increased food production requires utilisation of more agricultural land, pushing crops into non- traditional areas. The need for advances in agricultural technologies are not only required for current crop varieties, but for new varieties with increased tolerance to environmental stresses. Technological improvement means better crop yields and reduced land, water, fertilizer and pesticide use. Diversity Arrays Technology (DArT) was used to study wheat diversity, specifically to identify polymorphic markers between various wheat cultivars for use in marker- assisted breeding programs. The hybridisation based technology was used to analyse various bread and durum wheat cultivars for increased understanding of genomic diversity. Analysis shows that DArT is able to discriminate between tissue samples from wheat cultivars grown under various environmental stresses with polymorphic markers identified between samples treated with differing salt, light and temperature conditions. Epigenetic diversity was analysed through methylation detection using DArT to identify a list of candidate polymorphic markers. Markers were identified using the methylation sensitive restriction enzyme McrBC to generate control and treated targets. Diversity through cultivar exploration, looking at breeding experiments between cultivars with phenotypic extremes to examine salt tolerance versus in-tolerance using DArT produced a recombinant inbred line genetic linkage map. Bulk segregant analysis was also used to group phenotypic samples. Candidate markers were identified between cultivars that can be used to genotyping tetraploid and hexaploid wheat cultivars for germplasm identification. In addition, the identification of trait-linked molecular markers, such as salt resistance, plant breeders can genotype individual plants and populations of cultivars to determine the most suitable cultivar to plant that best complements to its local environment. This eliminates the need for multiple planting cycles to optimize crop selections, and gives the plant breeder the highest possible chance for crop success (yield, quality, performance and cost).

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
Sugarcane is an important crop, cultivated in more than 90 countries, occupying an area of approximately 20 million of hectares. Modern varieties (Saccharum spp.) are highly heterozygous interspecific hybrids, polyploids and often aneuploids, with chromosome numbers between 100 and 130. Such characteristics explain the common opinion that the genome of sugarcane is the most complex among cultivated species, posing a challenge to breeding programs. As a contribution to the understanding of this complex genomic architecture, this study aimed to build the first linkage maps using exclusively DArT markers in sugarcane. The maps were built using a progeny derived from the cross between varieties largely used in the Brazilian breeding program of RIDESA (RB97327 x RB72454). The initial mapping population comprised 186 individuals. Total genomic DNA was extracted from axial buds, following the protocol of Al-Janabi et al. (1999). Using the DArT P/L core facility to generate DArT data, a total of 7680 markers were analyzed, of which 850 were polymorphic. The analysis of segregation patterns in the progeny revealed that 47% of the individuals in the progeny were in fact derived from selfing of the female parent RB97327. These individuals were analyzed as a distinct generation. Linkage analyses were then performed on two populations (from selfing and crossing) separately. The software OneMap was used to construct the maps. The established linkage criteria for linkage analysis were LOD-score ≥ 3.5 and recombination fraction ≤ 0.4. In the first map, built using data from individuals originated from selfing, from 850 polymorphic markers, 392 markers (segregating in a 3:1 manner) were used to create 80 linkage groups related to the variety RB97327. For the population derived from the biparental crossing, four linkage maps were built: an integrated map composed of 98 linkage groups including 632 markers (1:1 and 3:1); an integrated framework map, using a more conservative ordering criteria for the linkage groups, which was composed of 94 linkage groups; and two other linkage maps, one for each parent (RB97327 and RB72454), built to estimate the genome size of the varieties involved in this study. The total length of the linkage map built using data from individuals derived from selfing of the variety RB97327 was 828 cM. The total length of the integrated linkage map was 2848 cM. The lengths of the maps built for each parent, using data from individuals derived from crossing, were 1465 cM (RB97327) and 1976 cM (RB72454). Using the methodology of Hulbert et al. (1988), the estimated genome sizes for these varieties were 2811 cM e 3471 cM, respectively. The maps obtained in these cases covered a low percentage of the estimated genome sizes (52% and 57%). In spite of the low polymorphism, DArT markers showed to be an efficient technique to perform genotyping of sugarcane. Hundreds of polymorphic markers were generated in only one assay, using two methods of genome complexity reduction. These markers represent a new tool for genetic studies in sugarcane, especially if the low cost (USD/marker) involved in data production is considered.
A cana-de-açúcar é uma importante cultura, cultivada em mais de 90 países, ocupando uma área total de aproximadamente 20 milhões de hectares. As variedades modernas (Saccharum spp.) são híbridos interespecíficos altamente heterozigóticos, poliploides e frequentemente aneuploides, com número cromossômico variando de 100 a 130. Tais características proporcionaram ao genoma da cana-de-açúcar o título de mais complexo entre as espécies cultivadas, o que representa um desafio para os programas de melhoramento genético da cultura. No intuito de contribuir com dados que auxiliem na compreensão dessa complexa arquitetura genômica, o presente estudo objetivou a construção dos primeiros mapas de ligação para cana-de-açúcar utilizando exclusivamente marcadores DArT, avaliados na progênie derivada do cruzamento de variedades amplamente utilizadas nos programas de melhoramento da RIDESA (RB97327 x RB72454). A população inicial de mapeamento foi composta por 186 indivíduos. O DNA genômico foi extraído de gemas axiais, seguindo o protocolo proposto por Al-Janabi et al. (1999). Após a extração, quantificação e homogeneização da concentração de DNA das amostras, o material foi enviado para a empresa DArT P/L para a geração dos marcadores DArT. Um total de 7680 locos foi analisado, dos quais 850 se apresentaram polimórficos. A análise dos padrões de segregação obtidos na progênie revelou que 47% dos indivíduos da progênie avaliada foram provenientes de autofecundação do genitor feminino RB97327. Os indivíduos identificados como provenientes de autofecundação foram analisados como uma geração distinta. As análises de ligação foram realizadas nas duas populações separadamente. O software OneMap foi utilizado para a construção dos mapas. Os critérios estabelecidos para proceder com as análises de ligação foram LOD-score ≥ 3,5 e fração de recombinação ≤ 0,4. No primeiro mapa, originário da população de autofecundação, dos 850 marcadores polimórficos, 392 marcadores com segregação 3:1 foram utilizados para originar 80 grupos de ligação referentes à variedade RB97327. Para a população derivada do cruzamento biparental foram construídos quatro mapas de ligação: um mapa integrado composto por 98 grupos de ligação a partir da análise de 632 marcadores (com segregações 1:1 e 3:1); um mapa framework integrado, construído a partir de uma ordenação mais refinada dos marcadores dentro de cada um dos grupos de ligação, o qual foi composto por 94 grupos de ligação; e, com o objetivo de se estimar o tamanho do genoma das variedades envolvidas neste estudo, dois mapas de ligação, um para cada genitor (RB97327 e RB72454). O comprimento total do primeiro mapa, referente à variedade RB97327, foi de 828cM. O comprimento total do mapa integrado foi de 2848 cM. Os comprimentos totais dos mapas obtidos para cada um dos genitores, gerados a partir de dados da população de cruzamento biparental, foram de 1465Cm (RB97327) e de 1976 cM (RB72454). Utilizando a metodologia de Hulbert et al. (1988), os tamanhos estimados dos genomas das variedades RB97327 e RB72454 foram 2811 cM e 3471 cM, respectivamente. Assim, pode-se afirmar que os mapas obtidos neste caso apresentaram baixa cobertura (52% e 57%), perante o tamanho estimado dos genomas. Apesar do baixo polimorfismo, os marcadores DArT se mostraram eficientes na genotipagem de progênies de cana-de-açúcar, pois, centenas de marcas polimórficas foram geradas em apenas um ensaio, com dois métodos de redução de complexidade. Estes marcadores representam uma nova ferramenta para o desenvolvimento de estudos genéticos em cana-de-açúcar, principalmente se considerado o baixo custo (R$/marcador) envolvido na obtenção dos genótipos.

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Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq
The first efforts in sugarcane breeding involved crosses between polyploid species, Saccharum spontaneum L. and Saccharum officinarum L.. These crosses produced interspecific hybrids that were successively backcrossed to S. officinarum. This strategy resulted in a considerable increase in the sugarcane genome complexity. Current varieties exhibit high levels of ploidy and heterozygosity, besides varying levels of aneuploidy. These properties make the understanding of sugarcane genome more difficult; and therefore present a challenge to the development of genetic studies with this culture. Among the different approaches to perform the genetic characterization of a species, the development of genetic maps is useful in providing information about its genomic structure. In this work, we report the first linkage maps for sugarcane using both DArT (Diversity Arrays Technology) and SSR (Single Sequence Repeat) markers. We identified markers significantly associated to characters involved in sugar production. Maps were obtained using two populations: one, consisting of 81 genotypes, was derived from the selfing of a single RB97327 plant; the other, consisting of 91 genotypes, was derived from the crossing RB97327 x RB72454. Genomic DNA was extracted from axillary buds. Genotypes for twenty pairs of SSR primers and 7680 DArT markers were identified. Using mendelian segregation analysis a total of 392 DArT and 57 SSR polymorphic markers, in the population of selfing, and 632 DArT and 79 SSR polymorphic markers, in the outcrossing population, were detected to be segregating as single-dose markers. Both maps were obtained using the OneMap software. Critical values for LOD-score of 3.5 and recombination fraction of 0.3 were chosen. In the map obtained with the selfing population, 449 polymorphic markers with 3:1 segregation were used to originate 95 linkage groups for the variety RB97327. This map had a total length of 1217.2 cM. The estimated size of the genome of RB97327 was 10540.9 cM, which suggests that the obtained coverage (11.5%) is still low. For the population derived from crossing, the 711 polymorphic markers with 3:1 and 1:1 segregation originated 136 linkage groups. The map showed a total length of 2722.2 cM. The SSR markers allowed the identification of six possible homeology groups for the female parent RB97327, and nine homeology groups for the integrated map. For each population, framework maps were produced which were then used to investigate putative associations between markers and characters involved in sugar production. QTL were found both using single marker analysis and composite interval mapping. In the population derived from selfing, using single marker analysis, 63 markers were significantly associated to six variables: number of internodes, number of stems per plant, stem length, stem diameter, stem weight and percentage of soluble solids (°Brix). Using composite interval mapping, three QTL related to stem diameter, length of stem and °Brix were identified. In the population derived from the cross RB97327 x RB724554, using single marker analysis, 60 markers were significantly associated with the same six variables. Using composite interval mapping, two QTL related to diameter and length of stem were detected.
Os primeiros trabalhos de melhoramento genético em cana-de-açúcar envolveram cruzamentos entre espécies poliplóides, Saccharum spontaneum L. e Saccharum officinarum L., os quais originaram híbridos interespecíficos que foram sucessivamente retrocruzados com S. officinarum. Essa estratégia resultou em considerável aumento da complexidade do genoma, de modo que as variedades atuais apresentem elevados níveis de ploidia e heterozigose, além de aneuploidias. Tais características dificultam a compreensão do genoma da cana-de-açúcar e, consequentemente, representam um desafio para o desenvolvimento de estudos genéticos com esta cultura. Dentre os diferentes estudos de caracterização genética, o desenvolvimento de mapas genéticos é importante por fornecer informações acerca da estrutura do genoma de uma espécie. Neste trabalho foram obtidos os primeiros mapas de ligação para cana-de-açúcar utilizando marcadores DArT (Diversity Arrays Technology) e SSR (Single Sequence Repeat). Além disso, foram identificados marcadores significativamente associados aos caracteres envolvidos na produção de açúcar. Para a obtenção dos mapas foram utilizadas duas populações, sendo uma constituída por 81 genótipos derivados da autofecundação de uma planta da cultivar RB97327, e outra constituída por 91 genótipos oriundos do cruzamento RB97327 x RB72454. O DNA genômico foi extraído de gemas axilares. A genotipagem foi realizada a partir de vinte pares de primers SSR e 7.680 marcadores DArT. A análise de segregação mendeliana permitiu a distinção de 392 marcas DArT e 57 marcas SSR polimórficas na população de autofecundação, e 632 DArT e 79 marcas SSR polimórficas na população de fecundação cruzada, com segregação single-dose. Ambos os mapas foram obtidos através do software OneMap utilizando-se um valor crítico de LOD-score igual a 3,5 e de fração de recombinação igual a 0,3. No mapa associado à população de autofecundação, os 449 marcadores DArT e SSR polimórficos com segregação 3:1 foram utilizados para originar 95 grupos de ligação referentes à variedade RB97327. Esse mapa apresentou um comprimento total de 1.217,2 cM. O tamanho estimado do genoma de RB97327 foi de 10.540,9 cM, o que permite afirmar que o mapa obtido apresentou baixa cobertura (11,5%). Para a população derivada de cruzamento, os 711 marcadores DArT e SSR polimórficos com segregação 3:1 e 1:1 originaram 136 grupos de ligação e o mapa apresentou um comprimento total de 2.722,2 cM. Os marcadores SSR também possibilitaram a identificação de seis possíveis grupos de homeologia no mapa referente ao genitor feminino RB97327 e nove no mapa integrado. Para cada população, foram obtidos os mapas framework nos quais foram identificados marcadores DArT e SSR associados aos caracteres envolvidos na produção de açúcar. Em ambas as populações procedeu-se à identificação de QTL a partir de análises de marcas simples e de mapeamento por intervalo composto. Na população derivada de autofecundação foram identificados, pela análise de marcas simples, 63 marcadores significativamente associados às seis variáveis avaliadas: número de entrenós, número de colmos por planta, comprimento de colmos, diâmetro de colmo, peso médio de colmo e teor de sólidos solúveis (brix). Pelo mapeamento por intervalo composto, três QTL relacionados a diâmetro de colmo, comprimento de colmo e brix foram identificados. Na população proveniente do cruzamento RB97327 x RB724554, foram identificados pela análise de marcas simples, 60 marcadores significativamente associados às seis variáveis. Pelo mapeamento por intervalo composto identificou-se dois QTL relacionados ao diâmetro e ao comprimento de colmo.

>Magister Scientiae - MSc
The Rosaceae family contains major temperate crops such as the domesticated apple(Malus x domestica Borkh.), peach (Prunus persica L. Batsch) and European pear (Pyrus communis L.). However, despite its evident economic importance, it is generally poorly studied in genomic terms, relative to the other major crop groups. Microsatellite and Diversity Array Technology (DArT) genetic markers have been exploited in this work and are essential tools in genetic map construction and marker-assisted selection (MAS) of high quality apples and other rosaceous crops. Microsatellites are advantageous in that they are co-dominant, highly polymorphic, abundant, transferable and reliably reproducible; hence their use in this study. In order for budbreak to take place in a timely and homogenous fashion, apple trees need a period of exposure to low temperatures.Within orchards the application of chemicals that induce budbreak in unsuitable environments is required to produce apples from cultivars that require high chilling levels. However, this and other practices using chemicals in orchards tend to pollute the environment. One of the solutions to this problem is to breed low chill apples such as ‘Anna’ cultivar, which was used as one of the parents in this study.This work was aimed at understanding the underlying genetic factors that determine chilling requirements for the time of initial vegetative budbreak trait in the apple cross ‘Anna’ x ‘Austin’. This was achieved through linkage map construction using SSR and DArT molecular markers followed by QTL analysis. This thesis has therefore exploited the large number of Expressed Sequence Tags (ESTs) and genome sequence data for the apple, using Tandem Repeats Finder, to design a total of 98 new SSR primers pairs. The other 369 SSR markers used in this work were from published work. JoinMap! 4.1 software was used to create an integrated genetic map with 17 linkage groups, for the domesticated apple cultivar, ‘Austin’ x ‘Anna’ mapping population with 80 individuals.The result of this process was a genetic map 1 212cM in length, and a total of 429 markers (314 DArT and 115 SSR), at an average density of a marker every 4 cM. This map was used identify the Quantitative Trait Loci (QTLs) determining chilling requirements for time of vegetative budbreak (IVB). In this process, putative IVB QTLs were identified in the ‘Anna’ x ‘Austin’ mapping population using the rMQM analysis function of MapQTL! 6.0, for both adult and seedling data collected over 3 growing seasons from 1996 to 1998. These QTLs were detected on linkage groups 2, 9 and 14,and explained 0.3 to 12.8 % of the observed phenotypic variation for the adult population,and 5.3 - 21 % for the seedling population. Seedling (LG 14) and adult (LGs 5, 7, 10) specific QTLs were also detected for the ‘Anna’ x ‘Austin’ cross. These QTLs will provide the basis for marker validation on related mapping populations in the apple breeding programme, and for the future identification of candidate genes controlling the process of budbreak.

Tese (doutorado)—Universidade de Brasília, Departamento de Biologia Celular, 2012.
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Espécies de Eucalyptus tem sido utilizadas com sucesso para plantios florestais devido ao seu rápido crescimento, sua capacidade de adaptação às diversas condições edafo-climáticas e pelo seu potencial econômico na produção de energia, fibra e madeira sólida, reduzindo assim a pressão sobre as florestas tropicais e a biodiversidade associada. Marcadores moleculares tais como RAPD, AFLP, microssatélites, e mais recentemente SFP e SNPs têm contribuído para a caracterização e conservação dos recursos genéticos do gênero Eucalyptus, auxiliando também na compreensão da evolução do gênero, além de permitir a construção de mapas de ligação e identificação de QTLs (Quantitative trait loci). Entretanto, estas técnicas são lentas, laboriosas, apresentam limitações de cobertura genômica e envolvem custos elevados para a análise de muitos indivíduos. Diversity Arrays Technology (DArT) é um método baseado em hibridização que permite genotipar centenas a milhares de marcadores num simples ensaio. Esta tecnologia gera um perfil genômico com um alto rendimento e um grande poder de transferibilidade entre espécies. Neste trabalho é apresentado o desenvolvimento da primeira plataforma de genotipagem de alto desempenho de marcadores DArTs em microarranjo para o gênero Eucalyptus, e demonstrada sua eficiência em estudos de diversidade genética, filogenia e mapeamento genético. Foram desenvolvidas 18 bibliotecas genômicas de complexidade reduzida a partir de 64 espécies diferentes do gênero. Um total de 23.808 fragmentos de DNA foram avaliados para revelação de polimorfismos DArT, e 13.300 (56%) destes declarados polimórficos entre um painel de triagem composto por 284 indivíduos. Destes, 7.680 marcadores foram selecionados para a construção de um microarranjo de genotipagem para uso em rotina. Em um estudo de diversidade intra-específica, 4.752 marcadores foram polimórficos e 5.013 mostraram segregação mendeliana em seis populações segregantes não relacionadas, com uma média de 2.211 marcadores polimórficos por população. Na etapa seguinte do trabalho, foi otimizada a tecnologia de genotipagem por sequenciamento DArT-seq para a construção de um mapa genético de alta densidade para uma população segregante do cruzamento entre árvores elite de E. grandis (BRASUZ1 x M4D31). A população foi genotipada com o microarranjo DArT e com a técnica DArTseq. Enquanto o microarranjo DArT forneceu 1.088 marcadores, a genotipagem DArT-seq forneceu 2.449. No total, um mapa de ligação integrado por 564 marcadores DArT, 1.930 marcadores DArTseq e 29 microssatélites foi construído. Além destes marcadores, mais de 1.500 SNPs derivados da metodologia DArT-seq foram obtidos proporcionando uma vantagem adicional pela inclusão de marcadores co-dominantes no mapa. O desenvolvimento de metodologias de genotipagem por sequenciamento (GbS) via enzimas de restrição como DArT-seq ou captura com sondas, representa uma aplicação adicional das tecnologias de "next generation sequencing" além do sequenciamento, 2 potencializando a análise genética com marcadores moleculares. A combinação do elevado número de marcadores, baixo custo, metodologia relativamente accessível e uso de reagentes universais, aponta para um uso crescente de GbS nos próximos anos nas mais diversas aplicações em estudos de genética de populações, investigações evolutivas e em apoio ao melhoramento acelerando e aumentando a precisão da seleção direcional de características multifatoriais complexas. _______________________________________________________________________________________ ABSTRACT
Species of Eucalyptus have been successfully used for forest plantations due to its rapid growth, its ability to adapt to various soil and climatic conditions and its economic use in energy, fiber and solid wood, reducing pressure on tropical forests and associated biodiversity. Molecular markers such as RAPD, AFLP, microsatellites, and more recently SFP and SNPs have contributed to the characterization and conservation of genetic resources of the genus, to the understanding of the evolution of the genus, and allowing the construction of linkage and QTLs maps. However, these techniques are slow, laborious, provide limited genome coverage and are costly for the analysis of large sample sizes. Diversity Arrays Technology (DArT) is a hybridization-based method that allows genotyping hundreds to thousands of markers in a single assay. This technology generates a genomic profile with high throughput and transferability between species. This study presents the development of the first high throughput genotyping platform for species of Eucalyptus based on a DArT microarray and demonstrates its use for diversity, phylogeny and mapping studies. A total of 18 reduced representation genomic libraries from 64 different species of the genus were developed. A total of 23,808 DNA fragments were screened for polymorphism and 13,300 (56%) of them declared polymorphic in a panel of 284 individuals. Out of these, 7,680 markers were selected to populate a routine DArT genotyping microarray. In an inter-specific diversity study, 4,752 were deemed polymorphic while 5,013 showed Mendelian segregation when assessed in six inter-specific mapping pedigrees, with an average of 2,211 polymorphic markers per pedigree. The subsequent step of the study, involved the optimization of the genotyping-by-sequencing technology called DArT-seq to construct a high density genetic map for a segregating population derived from the E. grandis elite trees (BRASUZ1 x M4D31). The population was genotyped both with the DArT microarray and with DArT-seq. While the DArT microarray yielded 1,088 markers, the DArT-seq method supplied 2,449 markers. In total, an integrated linkage map with 564 DArT markers, 1,930 DArT-NGS markers and 29 microsatellites was built. Besides these mapped markers, an additional set of over 1,500 SNPs derived from DArT-seq were scored providing an additional advantage by the inclusion of co-dominant markers on the map. The development of genotyping by sequencing (GBS) 3 methods via restriction enzymes as DART-seq or capture probes, represents a further application of the technologies of "next generation sequencing" beyond sequencing, empowering the genetic analysis with molecular markers. The combination of the large number of markers, low cost, relatively accessible methodology and use of universal reagents, points to an increased use of GBS in the coming years in several applications in studies of population genetics, evolutionary investigations and in support to plant breeding accelerating and increasing accuracy of directional selection for complex multi-factorial traits.

Tese (doutorado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, Pós-Graduação em Biologia Molecular, 2013.
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Métodos de genotipagem de polimorfismos no DNA em larga escala a baixos custos são fundamentais para ampliar a resolução de estudos filogenéticos e populacionais, viabilizar a integração das tecnologias genômicas na prática do melhoramento genético e auxiliar a montagem de genomas. Neste trabalho, tendo Pinus (Pinus teada L. - Pinaceae) como espécie de interesse, foram desenvolvidos: (1) um estudo filogenético de espécies americanas de Pinus baseado no desenvolvimento de um microarranjo de marcadores moleculares DArT; (2) um mapa genético de alta densidade para P. taeda com base em microssatélites e marcadores genotipados via redução de complexidade genômica e sequenciamento pela metodologia DArT-seq. Para o desenvolvimento da tecnologia DArT, amostras de 16 espécies de Pinus, oito procedências de P. taeda e cinco árvores-elite de P. taeda foram utilizadas para a construção de um microarranjo com 7680 sondas de DNA derivadas de uma fração do genoma com complexidade reduzida via enzimas de restrição. Um total de 4.171 marcadores polimórficos foi identificado entre as espécies de Pinus e 1.211 marcadores entre procedências de P. taeda. Uma análise filogenética de 12 espécies de Pinus da América com 3.273 marcadores DArT, revelou importantes diferenças em relação à proposta mais recente de classificação taxonômica baseada na análise de sequências de DNA cloroplastidial. Nossos resultados sugerem a possibilidade de que P. taeda e demais espécies do norte da América venham a ser classificadas em outra subseção. Um mapa genético com base na análise de 288 megagametófitos haploides da árvore 7-56 de P. taeda foi construído com microssatélites e marcadores DArT-seq (sequências de 69 bases) desenvolvidos a partir da redução da complexidade genômica via enzimas de restrição (PstI-HhaI), e sequenciamento das representações genômicas reduzidas. De um total de 4.367 marcadores DArT-seq de alta qualidade, 2.469 e 32 microssatélites foram mapeados e ordenados em 12 grupos de ligação (n=12 cromossomos em P. taeda) cobrindo 1.226,47 cM, com média de 203 marcadores por grupo de ligação e densidade de 0,62 cM por marcador. O mapa genético foi em seguida utilizado para ancorar 75 clones de BACs (cromossomos artificiais de bactéria) de Pinus taeda disponíveis no NCBI, demonstrando sua potencial utilidade para fins de montagem do genoma, porém corroborando também a natureza repetitiva do genoma ao ancorar múltiplos BACs em diferentes grupos de ligação. O mapa genético aumentou em cerca de 20 vezes a densidade de marcadores dos mapas existentes para a espécie, fornecendo uma ferramenta adicional para estudos de mapeamento genético e montagem do genoma de P. taeda. __________________________________________________________________________________ ABSTRACT
Genome-wide, high-throughput and cost-efficient DNA genotyping methods are key to increase the resolution and speed of phylogenetic and populational studies, allow the integration of genomics into breeding and support the assembly of genomes. In this work having loblolly pine (Pinus taeda L. - Pinaceae) as model species we developed: (1) a phylogenetic study of American Pinus species based on the development of a microarray of DArT markers for species of the genus and (2) a high density genetic map for Pinus taeda using microsatellite markers and sequence based genotyping carried out by the DArT (DArT-seq) method using next-generation sequencing. For DArT microarray probe development, DNA samples of 16 species of Pinus, eight provenances of P. taeda and five elite trees of P. taeda were used to build an array of 7,680 probes derived from a restriction enzyme complexity reduced fraction of the pine genome. A total of 4,171 markers were found polymorphic across samples of Pinus species and 1,211 markers across provenances of P. taeda. A phylogenetic study involving 12 American Pinus species using 3,273 DArT markers revealed important differences from the most recent classification proposed based on chloroplast DNA amplified sequences, additionally suggesting the possibility that P. taeda and its close species from North America could be classified into a new subsection. A genetic map, based on a set of 288 haploid megagametophytes of P. taeda tree 7-56 was built with microsatallites and DArT-seq (69 bases tags) markers developed from complexity reduction with double digest (PstI-HhaI) and sequencing. From a total of 4,367 high quality DArT-seq markers, 2,469 and 32 microsatallites were mapped and ordered into 12 linkage groups (n=12 chromosomes in Pinus taeda), covering 1,226.47 cM with an average of 203 markers per linkage group and marker density of 0.62 cM. This genetic map was subsequently used to anchor 75 BAC clones (bacterial artificial chromosomes) of Pinus taeda available in NCBI, demonstrating its potential utility to aid genome assembly, although also corroborating the repetitive nature of the genome as several BAC clones were anchored in multiple positions in different linkage groups. The genetic map presented in this study increased marker density by approximately 20 times the current density of the existing microsatellite maps providing an additional tool for genetic mapping studies and assembly of the P. taeda genome.

Polyphenol oxidase (PPO) catalyses undesirable darkening in wheat products such as Asian noodles. Genetic variation for PPO activity is characterized in bread wheat. Australian wheat breeding programmes recognize that reduced PPO activity is an important quality target. Despite this interest from breeders, no varieties possessing extremely low and null PPO activity exist. The development of null PPO wheat varieties is dependant on an understanding of the genetic control of the null phenotype. Knowledge of these factors will accelerate efforts to develop them. The inheritance of PPO activity was investigated in two populations that were derived from hybrids between a null PPO genotype and Australian wheat varieties Lang and QAlBis. Observed genetic ratios were consistent with two and three gene control, respectively in these populations. QTL mapping was performed in the QALBis x VAW08-A17 population. The Diversity Array Technology (DArT) approach was employed to genotype the QALBis x VAW08-A17 population. Three highly significant QTLs that control PPO activity were identified on chromosomes 2AL, 2BS and 2DL. Close associations between PPO activity and DArT marker loci wPt-7024, wPt-0094 and wPt-2544 were observed, respectively. Collectively, these loci explained 74% of the observed variation in PPO activity across seasons. Significant QTLs on chromosomes 1B and 3B were also identified that together explained an additional 17% of variation in PPO activity. The relationship between PPO activity and yellow alkaline noodles (YAN) colour stability parameters was investigated in a DM5637*B8 x H45 doubled haploid population. PPO activity and changes in YAN brightness (ΔL* 0-24h) and yellowness (Δb* 0-24h) in both seasons were analysed. Quantitative trait analyses of PPO activity, flour yellowness (b*) and YAN colour stability was also conducted in this population. QTL mapping of variation in PPO activity in the DM5637*B8 x H45 DH population identified a highly significant QTL on chromosome 2AL, which explained 52% of the observed variation across seasons. Regression analysis identified that wPt-7024 was highly significantly associated with PPO activity in this population. A highly significant association between this marker and PPO was also identified in the QALBis x VAW08-A17 population. Collectively, the three identified QTLs (on chromosomes 2AL, 7A and 7B) explained 71% of variation in PPO activity across seasons. A highly significant (P<0.001) QTL on chromosome 2B along with significant (P<0.01) QTLs on the chromosomes 1A, 3B, 4B and 5B were found to control flour yellowness. The QTLs on 2B, 4B and 5B were detected in both seasons analysed and accounted for 90% of variation in flour b* across seasons. The study on YAN colour stability located two highly significant (P<0.001) QTLs and two significant (P<0.01) QTLs that controlled the change in brightness of yellow alkaline noodle. The 2A QTL accounted for 64% of observed variation across seasons. It was in the same location as the PPO QTL and shared a common closest marker wPt-7024. Only one significant QTL for YAN a* (0-24h) was identified. It accounted for 12% of variation across seasons and was only detected in one season. One highly significant (P<0.001) QTL and two significant (P<0.01) QTLs were identified that controlled the change in yellowness of yellow alkaline noodle. The 2A QTL accounted for 68% of observed variation across seasons. The location of this QTL corresponded with that of 2A QTLs for PPO activity and L* of YAN in this study. Furthermore, wPt-7024 was also identified as the marker with the most significant association with L*. The identification of a correlation between the characters and a common location of a highly significant QTL for each of these characters indicates that it is likely that PPO activity is directly responsible for a large proportion of the changes in brightness and yellowness of YAN. QTLs for L* and b* of YAN were detected in a common location on chromosome 1A. However, no corresponding QTL was identified that controls PPO activity, highlighting the complexity of the relationship between these traits. Resistance to three rust pathogens (Puccinia graminis, Puccinia striiformis, and Puccinia triticina) was also investigated in the DM5637*B8 x H45 DH population because they are major yield limiting diseases in wheat. Disease response data at the seedling stage were converted to genotypic scores for rust genes Sr24/Lr24, Sr36, Lr13 and Yr7 to construct a genetic linkage map. No recombination was observed between rust resistance genes Sr36, Lr13 and Yr7 in this DH population. Therefore, these genes mapped in the same position on chromosome 2B. The Lr24/Sr24 locus was incorporated into the chromosome 3D map. Interval mapping analysis identified QTLs on chromosomes 2B, 3B, 4B and 5B that control adult plant resistance (APR) to stripe rust. Two QTLs on chromosomes 2B and 3D were identified that controlled APR to leaf rust in this DH population.

Doctor of Philosophy
Polyphenol oxidase (PPO) catalyses undesirable darkening in wheat products such as Asian noodles. Genetic variation for PPO activity is characterized in bread wheat. Australian wheat breeding programmes recognize that reduced PPO activity is an important quality target. Despite this interest from breeders, no varieties possessing extremely low and null PPO activity exist. The development of null PPO wheat varieties is dependant on an understanding of the genetic control of the null phenotype. Knowledge of these factors will accelerate efforts to develop them. The inheritance of PPO activity was investigated in two populations that were derived from hybrids between a null PPO genotype and Australian wheat varieties Lang and QAlBis. Observed genetic ratios were consistent with two and three gene control, respectively in these populations. QTL mapping was performed in the QALBis x VAW08-A17 population. The Diversity Array Technology (DArT) approach was employed to genotype the QALBis x VAW08-A17 population. Three highly significant QTLs that control PPO activity were identified on chromosomes 2AL, 2BS and 2DL. Close associations between PPO activity and DArT marker loci wPt-7024, wPt-0094 and wPt-2544 were observed, respectively. Collectively, these loci explained 74% of the observed variation in PPO activity across seasons. Significant QTLs on chromosomes 1B and 3B were also identified that together explained an additional 17% of variation in PPO activity. The relationship between PPO activity and yellow alkaline noodles (YAN) colour stability parameters was investigated in a DM5637*B8 x H45 doubled haploid population. PPO activity and changes in YAN brightness (ΔL* 0-24h) and yellowness (Δb* 0-24h) in both seasons were analysed. Quantitative trait analyses of PPO activity, flour yellowness (b*) and YAN colour stability was also conducted in this population. QTL mapping of variation in PPO activity in the DM5637*B8 x H45 DH population identified a highly significant QTL on chromosome 2AL, which explained 52% of the observed variation across seasons. Regression analysis identified that wPt-7024 was highly significantly associated with PPO activity in this population. A highly significant association between this marker and PPO was also identified in the QALBis x VAW08-A17 population. Collectively, the three identified QTLs (on chromosomes 2AL, 7A and 7B) explained 71% of variation in PPO activity across seasons. A highly significant (P<0.001) QTL on chromosome 2B along with significant (P<0.01) QTLs on the chromosomes 1A, 3B, 4B and 5B were found to control flour yellowness. The QTLs on 2B, 4B and 5B were detected in both seasons analysed and accounted for 90% of variation in flour b* across seasons. The study on YAN colour stability located two highly significant (P<0.001) QTLs and two significant (P<0.01) QTLs that controlled the change in brightness of yellow alkaline noodle. The 2A QTL accounted for 64% of observed variation across seasons. It was in the same location as the PPO QTL and shared a common closest marker wPt-7024. Only one significant QTL for YAN a* (0-24h) was identified. It accounted for 12% of variation across seasons and was only detected in one season. One highly significant (P<0.001) QTL and two significant (P<0.01) QTLs were identified that controlled the change in yellowness of yellow alkaline noodle. The 2A QTL accounted for 68% of observed variation across seasons. The location of this QTL corresponded with that of 2A QTLs for PPO activity and L* of YAN in this study. Furthermore, wPt-7024 was also identified as the marker with the most significant association with L*. The identification of a correlation between the characters and a common location of a highly significant QTL for each of these characters indicates that it is likely that PPO activity is directly responsible for a large proportion of the changes in brightness and yellowness of YAN. QTLs for L* and b* of YAN were detected in a common location on chromosome 1A. However, no corresponding QTL was identified that controls PPO activity, highlighting the complexity of the relationship between these traits. Resistance to three rust pathogens (Puccinia graminis, Puccinia striiformis, and Puccinia triticina) was also investigated in the DM5637*B8 x H45 DH population because they are major yield limiting diseases in wheat. Disease response data at the seedling stage were converted to genotypic scores for rust genes Sr24/Lr24, Sr36, Lr13 and Yr7 to construct a genetic linkage map. No recombination was observed between rust resistance genes Sr36, Lr13 and Yr7 in this DH population. Therefore, these genes mapped in the same position on chromosome 2B. The Lr24/Sr24 locus was incorporated into the chromosome 3D map. Interval mapping analysis identified QTLs on chromosomes 2B, 3B, 4B and 5B that control adult plant resistance (APR) to stripe rust. Two QTLs on chromosomes 2B and 3D were identified that controlled APR to leaf rust in this DH population.

This thesis reports the outcomes of research evaluating the application of Diversity Array Technology (DArT) to two important tropical legume crops, mungbean (Vigna radiata (L.) Wilczek) and soybean (Glycine max (L). Merrill.). Mungbean is comparatively under-studied, and is therefore likely to benefit from research to improve adaptation and productivity. Soybean has received more breeding attention than mungbean, but is still under-researched when compared with modern cereals like rice, wheat and maize. Drought remains an important constraint to soybean productivity in rainfed areas. DArT is a novel molecular marker technology that has been successfully applied in genetic studies in several plant species, but had not been previously applied to either of the crops of interest. A DArT marker library/ array for mungbean was created using two mungbean cultivars, Berken and Kiloga, and two wild accessions V. radiata. ssp. sublobata, ACC 1 and ACC 87, and one or more accessions of five other Vigna species, viz. V. lanceolata, V. mungo, V. mungo var. sylvestris, V. trilobata and V. vexillata. A DArT library/ array for soybean was developed using two oilseed type cultivars, CPI 26671 and Valder, a landrace G2120, a wild soybean (G. soja) accession and one or more accessions of two wild Glycine species, G. falcata and G. tomentella. For each crop, two genomic complexity reduction methods, utilizing PstI/TaqI and PstI/BstNI restriction digests, were selected for DNA clonal library development and for the isolation in each case of 7,680 DArT clones from genomic representations of pooled DNA samples. While the PstI/BstNI method produced more polymorphic clones than PstI/TaqI for the soybean library, there was no significant difference between the two methods for the mungbean library. In the initial library evaluation, there were nearly 1,500 polymorphic clones identified for soybean. Polymorphism frequencies in mungbean were around twice those in soybean, reflecting greater diversity in the mungbean germplasm samples. The DArT marker transferability from soybean to mungbean (13.6%) was nearly five times higher than that from mungbean to soybean (3.1%). The percentage of DArT marker transferability between mungbean and several other Vigna species ranged from 3.4 to 20.2%. The genetic similarities among 11 diverse Vigna spp. samples, evaluated using the DArT mungbean library, were consistent with published information on these taxa. These mungbean and soybean arrays were then used to evaluate the application of DArT markers for construction of genetic linkage maps and identifying putative qualitative and quantitative trait loci (QTLs). In mungbean, four F₅ recombinant inbred line (RIL) populations were derived from crosses between cultivars Berken and Kiloga and wild accessions ACC 1 and ACC 87. The F₅ RIL populations were evaluated for 54 qualitative and quantitative traits using plants grown in large pots on outdoor benches. There were large differences between the cultivated and wild parents and individual lines for all traits. Broad sense heritability estimates were moderate to high in most cases, with significant phenotypic correlations between many traits. A large number of polymorphic DArT markers were selected for the four RIL populations (1062 – 2013). The four mungbean linkage maps contained 672 to 981 DArT markers with segregation distortion levels higher in the ACC 87 (44.1 – 47.8%) than in the ACC 1 crosses (33.7 – 42.4%). Maps consisted of 15 – 19 linkage groups (LGs) and spanned lengths of 629.7 to 883.5 cM with average inter-marker distances of 0.9 – 1.2 cM. Various putative QTLs (77 – 122 QTLs) were identified for the vast majority of the 54 evaluated traits. In addition, the level of congruence across populations was reasonably strong. In soybean, one F₇ and two F₆ RIL populations derived from crosses between CPI 26671, Valder and G2120 were used in phenotypic evaluation and QTL mapping with DArT markers for physiological drought stress response traits. The RILs were grown in deep cylindrical pots in the glasshouse, and exposed to severe water deficit followed by re-watering. Traits recorded included relative water content (RWC), epidermal conductance (gₑ), and recovery in growth following re-watering. The drought stress responses in the parental plants and RIL populations were broadly consistent with prior studies: As plant available water (PAW) in the soil declined, both RWC and gₑ declined, with the relation between RWC and gₑ exponential rather than linear as in previous studies. Analysis of variance showed significant differences at both population and genotypic levels for all key traits. However, there were large environmental effects on most traits, which resulted in high coefficients of variation and low estimates of broad sense heritability. The three individual linkage maps contained 196 – 409 DArT markers and 15 – 22 LGs with the aggregate length ranging from 409.4 to 516.7 cM. An integrated soybean map was constructed consisting of 759 DArT markers, 27 LGs and an expanded length of 762.2 cM. Total numbers of putative QTLs identified in the CPI 26671 x G2120 (CG) and VG (Valder x G2120) populations were 106 and 34 respectively. In each of the population, 10 LGs harboured QTLs associated with RWC, gₑ and recovery ability, of which five similar LGs contributed to drought tolerance. A BLAST (Basic Local Alignment Search Tool) search for sequences of 19 selected DArT markers linked to QTLs conditioning drought response traits indicated that 18 DArT markers were unique and aligned to 12 soybean chromosomes. Comparison of these DArT markers with other markers associated with drought-related QTLs in previous studies confirmed that five of them overlapped whereas the remaining 13 had not been previously identified. However, except for chromosome 15, the chromosomes with which the DArT QTLs in the CG and VG populations were associated were ones that had been shown to harbour drought-related QTLs in previous studies. This study is the first showing DArT development in mungbean and soybean and its application for manipulating QTLs associated with wild physiological traits in mungbean and drought tolerance in soybean. DArT was successfully developed for both species, with more polymorphisms evident in the mungbean than in the soybean arrays. The study demonstrated that DArT provides high quality markers which can be used for diversity analyses, the construction of high-density genetic linkage maps and for QTL analysis. Meanwhile the marker transferability between arrays enabled plausible discrimination of genetic relationships between related taxa. In both mungbean and soybean, the large numbers of DArT markers that were generated contributed to relatively tight resolution in the genetic maps, enhancing the power for QTL detection. Potentially useful QTLs/ markers were identified for many traits in mungbean, including some potentially useful ones such as resistance to powdery mildew and thrips, late flowering, hardseededness and perenniality, and in soybean, for RWC, gₑ and recovery after drought stress. In mungbean, further research is needed to identify an appropriate approach for the construction of an integrated map from the four RIL populations used in this study.In both species, follow-up research is required to verify the QTLs detected in this study before they can be used for marker-assisted selection in mungbean and soybean breeding programs. Nonetheless, the QTL analyses based on DArT markers in this study have been shown to be useful in the genetic dissection of both qualitative and quantitative traits in both species, and it is apparent that DArT markers will offer advantages for a range of molecular breeding and genomics applications.

Prior to 1994, the media operated in an environment that can best be described as ‘suppressed’. Diversity of thoughts, views and opinions on media platforms were non-existent as the regime, at the time, ruled with an iron-fist. A variety of print media outlets sought to reflect reality, but it was a steady struggle especially for those with meagre resources, and exacerbated by the constant clampdowns. The state-run broadcaster, if anything, entrenched discriminatory principles and practices. Given our precarious past, the birth of democracy proved to be the perfect panacea for a promising pathway for the media fraternity. Transformation, in more ways than one, permeated the sector. Reflections of the South African Media: 1994-2019 is a compilation by authors who have peculiar insight of and excelled in the different areas of the fast-developing industry in the first 25 years of South Africa’s democracy. And they are no ordinary authors. Every chapter contributed came from women and men who had, through the years, a direct link with ML Sultan Technikon, Technikon Natal, Durban Institute of Technology (DIT) or Durban University of Technology (DUT) * either as a student, lecturer, visiting professor, speaker or associate. Compiling and editing this book has been an incredibly invigorating experience. It was never in doubt whose image will adorn the cover of the book, so it was beautifully uplifting that many authors, not knowing my choice, gave Nelson Mandela due recognition. My brief to the authors was simple: let me have your personal lookback in your own style on the topic that you are most comfortable with. All of them stepped up to the plate, and the vast array of content in the book bares strong testimony. A section titled Journeys in Journalism encapsulates input from alumni of DUT Journalism – they were afforded free reign to trace the territory they traversed. I’m indebted to each and every contributor for generously volunteering their precious time and talent to the book. They were simply magnificent. It has to be said that this publication far exceeded my expectations as it, initially, was a humble idea to celebrate 25 years of the media industry with a handful of contributions. Little did I realise that my desk will be flooded with 40 pieces of excellence and a Foreword penned by the brilliant Jeremy Thompson. My eternal gratitude must also be extended to the small team of assistants for understanding my vision upfront and rallying remarkably throughout. Once you’ve enjoyed the read, I invite you to share Reflections of the South African Media: 1994-2019 with whoever you believe can benefit from its rich and diverse content!

Nationalism: Past as Prologue began as a single volume being compiled by Ad Akande, a scholar from South Africa, who proposed it to me as co-author about two years ago. The original idea was to examine how the damaging roots of nationalism have been corroding political systems around the world, and creating dangerous obstacles for necessary international cooperation. Since I (Bruce E. Johansen) has written profusely about climate change (global warming, a.k.a. infrared forcing), I suggested a concerted effort in that direction. This is a worldwide existential threat that affects every living thing on Earth. It often compounds upon itself, so delays in reducing emissions of fossil fuels are shortening the amount of time remaining to eliminate the use of fossil fuels to preserve a livable planet. Nationalism often impedes solutions to this problem (among many others), as nations place their singular needs above the common good. Our initial proposal got around, and abstracts on many subjects arrived. Within a few weeks, we had enough good material for a 100,000-word book. The book then fattened to two moderate volumes and then to four two very hefty tomes. We tried several different titles as good submissions swelled. We also discovered that our best contributors were experts in their fields, which ranged the world. We settled on three stand-alone books:” 1/ nationalism and racial justice. Our first volume grew as the growth of Black Lives Matter following the brutal killing of George Floyd ignited protests over police brutality and other issues during 2020, following the police assassination of Floyd in Minneapolis. It is estimated that more people took part in protests of police brutality during the summer of 2020 than any other series of marches in United States history. This includes upheavals during the 1960s over racial issues and against the war in Southeast Asia (notably Vietnam). We choose a volume on racism because it is one of nationalism’s main motive forces. This volume provides a worldwide array of work on nationalism’s growth in various countries, usually by authors residing in them, or in the United States with ethnic ties to the nation being examined, often recent immigrants to the United States from them. Our roster of contributors comprises a small United Nations of insightful, well-written research and commentary from Indonesia, New Zealand, Australia, China, India, South Africa, France, Portugal, Estonia, Hungary, Russia, Poland, Kazakhstan, Georgia, and the United States. Volume 2 (this one) describes and analyzes nationalism, by country, around the world, except for the United States; and 3/material directly related to President Donald Trump, and the United States. The first volume is under consideration at the Texas A & M University Press. The other two are under contract to Nova Science Publishers (which includes social sciences). These three volumes may be used individually or as a set. Environmental material is taken up in appropriate places in each of the three books. * * * * * What became the United States of America has been strongly nationalist since the English of present-day Massachusetts and Jamestown first hit North America’s eastern shores. The country propelled itself across North America with the self-serving ideology of “manifest destiny” for four centuries before Donald Trump came along. Anyone who believes that a Trumpian affection for deportation of “illegals” is a new thing ought to take a look at immigration and deportation statistics in Adam Goodman’s The Deportation Machine: America’s Long History of Deporting Immigrants (Princeton University Press, 2020). Between 1920 and 2018, the United States deported 56.3 million people, compared with 51.7 million who were granted legal immigration status during the same dates. Nearly nine of ten deportees were Mexican (Nolan, 2020, 83). This kind of nationalism, has become an assassin of democracy as well as an impediment to solving global problems. Paul Krugman wrote in the New York Times (2019:A-25): that “In their 2018 book, How Democracies Die, the political scientists Steven Levitsky and Daniel Ziblatt documented how this process has played out in many countries, from Vladimir Putin’s Russia, to Recep Erdogan’s Turkey, to Viktor Orban’s Hungary. Add to these India’s Narendra Modi, China’s Xi Jinping, and the United States’ Donald Trump, among others. Bit by bit, the guardrails of democracy have been torn down, as institutions meant to serve the public became tools of ruling parties and self-serving ideologies, weaponized to punish and intimidate opposition parties’ opponents. On paper, these countries are still democracies; in practice, they have become one-party regimes….And it’s happening here [the United States] as we speak. If you are not worried about the future of American democracy, you aren’t paying attention” (Krugmam, 2019, A-25). We are reminded continuously that the late Carl Sagan, one of our most insightful scientific public intellectuals, had an interesting theory about highly developed civilizations. Given the number of stars and planets that must exist in the vast reaches of the universe, he said, there must be other highly developed and organized forms of life. Distance may keep us from making physical contact, but Sagan said that another reason we may never be on speaking terms with another intelligent race is (judging from our own example) could be their penchant for destroying themselves in relatively short order after reaching technological complexity. This book’s chapters, introduction, and conclusion examine the worldwide rise of partisan nationalism and the damage it has wrought on the worldwide pursuit of solutions for issues requiring worldwide scope, such scientific co-operation public health and others, mixing analysis of both. We use both historical description and analysis. This analysis concludes with a description of why we must avoid the isolating nature of nationalism that isolates people and encourages separation if we are to deal with issues of world-wide concern, and to maintain a sustainable, survivable Earth, placing the dominant political movement of our time against the Earth’s existential crises. Our contributors, all experts in their fields, each have assumed responsibility for a country, or two if they are related. This work entwines themes of worldwide concern with the political growth of nationalism because leaders with such a worldview are disinclined to co-operate internationally at a time when nations must find ways to solve common problems, such as the climate crisis. Inability to cooperate at this stage may doom everyone, eventually, to an overheated, stormy future plagued by droughts and deluges portending shortages of food and other essential commodities, meanwhile destroying large coastal urban areas because of rising sea levels. Future historians may look back at our time and wonder why as well as how our world succumbed to isolating nationalism at a time when time was so short for cooperative intervention which is crucial for survival of a sustainable earth. Pride in language and culture is salubrious to individuals’ sense of history and identity. Excess nationalism that prevents international co-operation on harmful worldwide maladies is quite another. As Pope Francis has pointed out: For all of our connectivity due to expansion of social media, ability to communicate can breed contempt as well as mutual trust. “For all our hyper-connectivity,” said Francis, “We witnessed a fragmentation that made it more difficult to resolve problems that affect us all” (Horowitz, 2020, A-12). The pope’s encyclical, titled “Brothers All,” also said: “The forces of myopic, extremist, resentful, and aggressive nationalism are on the rise.” The pope’s document also advocates support for migrants, as well as resistance to nationalist and tribal populism. Francis broadened his critique to the role of market capitalism, as well as nationalism has failed the peoples of the world when they need co-operation and solidarity in the face of the world-wide corona virus pandemic. Humankind needs to unite into “a new sense of the human family [Fratelli Tutti, “Brothers All”], that rejects war at all costs” (Pope, 2020, 6-A). Our journey takes us first to Russia, with the able eye and honed expertise of Richard D. Anderson, Jr. who teaches as UCLA and publishes on the subject of his chapter: “Putin, Russian identity, and Russia’s conduct at home and abroad.” Readers should find Dr. Anderson’s analysis fascinating because Vladimir Putin, the singular leader of Russian foreign and domestic policy these days (and perhaps for the rest of his life, given how malleable Russia’s Constitution has become) may be a short man physically, but has high ambitions. One of these involves restoring the old Russian (and Soviet) empire, which would involve re-subjugating a number of nations that broke off as the old order dissolved about 30 years ago. President (shall we say czar?) Putin also has international ambitions, notably by destabilizing the United States, where election meddling has become a specialty. The sight of Putin and U.S. president Donald Trump, two very rich men (Putin $70-$200 billion; Trump $2.5 billion), nuzzling in friendship would probably set Thomas Jefferson and Vladimir Lenin spinning in their graves. The road of history can take some unanticipated twists and turns. Consider Poland, from which we have an expert native analysis in chapter 2, Bartosz Hlebowicz, who is a Polish anthropologist and journalist. His piece is titled “Lawless and Unjust: How to Quickly Make Your Own Country a Puppet State Run by a Group of Hoodlums – the Hopeless Case of Poland (2015–2020).” When I visited Poland to teach and lecture twice between 2006 and 2008, most people seemed to be walking on air induced by freedom to conduct their own affairs to an unusual degree for a state usually squeezed between nationalists in Germany and Russia. What did the Poles then do in a couple of decades? Read Hlebowicz’ chapter and decide. It certainly isn’t soft-bellied liberalism. In Chapter 3, with Bruce E. Johansen, we visit China’s western provinces, the lands of Tibet as well as the Uighurs and other Muslims in the Xinjiang region, who would most assuredly resent being characterized as being possessed by the Chinese of the Han to the east. As a student of Native American history, I had never before thought of the Tibetans and Uighurs as Native peoples struggling against the Independence-minded peoples of a land that is called an adjunct of China on most of our maps. The random act of sitting next to a young woman on an Air India flight out of Hyderabad, bound for New Delhi taught me that the Tibetans had something to share with the Lakota, the Iroquois, and hundreds of other Native American states and nations in North America. Active resistance to Chinese rule lasted into the mid-nineteenth century, and continues today in a subversive manner, even in song, as I learned in 2018 when I acted as a foreign adjudicator on a Ph.D. dissertation by a Tibetan student at the University of Madras (in what is now in a city called Chennai), in southwestern India on resistance in song during Tibet’s recent history. Tibet is one of very few places on Earth where a young dissident can get shot to death for singing a song that troubles China’s Quest for Lebensraum. The situation in Xinjiang region, where close to a million Muslims have been interned in “reeducation” camps surrounded with brick walls and barbed wire. They sing, too. Come with us and hear the music. Back to Europe now, in Chapter 4, to Portugal and Spain, we find a break in the general pattern of nationalism. Portugal has been more progressive governmentally than most. Spain varies from a liberal majority to military coups, a pattern which has been exported to Latin America. A situation such as this can make use of the term “populism” problematic, because general usage in our time usually ties the word into a right-wing connotative straightjacket. “Populism” can be used to describe progressive (left-wing) insurgencies as well. José Pinto, who is native to Portugal and also researches and writes in Spanish as well as English, in “Populism in Portugal and Spain: a Real Neighbourhood?” provides insight into these historical paradoxes. Hungary shares some historical inclinations with Poland (above). Both emerged from Soviet dominance in an air of developing freedom and multicultural diversity after the Berlin Wall fell and the Soviet Union collapsed. Then, gradually at first, right wing-forces began to tighten up, stripping structures supporting popular freedom, from the courts, mass media, and other institutions. In Chapter 5, Bernard Tamas, in “From Youth Movement to Right-Liberal Wing Authoritarianism: The Rise of Fidesz and the Decline of Hungarian Democracy” puts the renewed growth of political and social repression into a context of worldwide nationalism. Tamas, an associate professor of political science at Valdosta State University, has been a postdoctoral fellow at Harvard University and a Fulbright scholar at the Central European University in Budapest, Hungary. His books include From Dissident to Party Politics: The Struggle for Democracy in Post-Communist Hungary (2007). Bear in mind that not everyone shares Orbán’s vision of what will make this nation great, again. On graffiti-covered walls in Budapest, Runes (traditional Hungarian script) has been found that read “Orbán is a motherfucker” (Mikanowski, 2019, 58). Also in Europe, in Chapter 6, Professor Ronan Le Coadic, of the University of Rennes, Rennes, France, in “Is There a Revival of French Nationalism?” Stating this title in the form of a question is quite appropriate because France’s nationalistic shift has built and ebbed several times during the last few decades. For a time after 2000, it came close to assuming the role of a substantial minority, only to ebb after that. In 2017, the candidate of the National Front reached the second round of the French presidential election. This was the second time this nationalist party reached the second round of the presidential election in the history of the Fifth Republic. In 2002, however, Jean-Marie Le Pen had only obtained 17.79% of the votes, while fifteen years later his daughter, Marine Le Pen, almost doubled her father's record, reaching 33.90% of the votes cast. Moreover, in the 2019 European elections, re-named Rassemblement National obtained the largest number of votes of all French political formations and can therefore boast of being "the leading party in France.” The brutality of oppressive nationalism may be expressed in personal relationships, such as child abuse. While Indonesia and Aotearoa [the Maoris’ name for New Zealand] hold very different ranks in the United Nations Human Development Programme assessments, where Indonesia is classified as a medium development country and Aotearoa New Zealand as a very high development country. In Chapter 7, “Domestic Violence Against Women in Indonesia and Aotearoa New Zealand: Making Sense of Differences and Similarities” co-authors, in Chapter 8, Mandy Morgan and Dr. Elli N. Hayati, from New Zealand and Indonesia respectively, found that despite their socio-economic differences, one in three women in each country experience physical or sexual intimate partner violence over their lifetime. In this chapter ther authors aim to deepen understandings of domestic violence through discussion of the socio-economic and demographic characteristics of theit countries to address domestic violence alongside studies of women’s attitudes to gender norms and experiences of intimate partner violence. One of the most surprising and upsetting scholarly journeys that a North American student may take involves Adolf Hitler’s comments on oppression of American Indians and Blacks as he imagined the construction of the Nazi state, a genesis of nationalism that is all but unknown in the United States of America, traced in this volume (Chapter 8) by co-editor Johansen. Beginning in Mein Kampf, during the 1920s, Hitler explicitly used the westward expansion of the United States across North America as a model and justification for Nazi conquest and anticipated colonization by Germans of what the Nazis called the “wild East” – the Slavic nations of Poland, the Baltic states, Ukraine, and Russia, most of which were under control of the Soviet Union. The Volga River (in Russia) was styled by Hitler as the Germans’ Mississippi, and covered wagons were readied for the German “manifest destiny” of imprisoning, eradicating, and replacing peoples the Nazis deemed inferior, all with direct references to events in North America during the previous century. At the same time, with no sense of contradiction, the Nazis partook of a long-standing German romanticism of Native Americans. One of Goebbels’ less propitious schemes was to confer honorary Aryan status on Native American tribes, in the hope that they would rise up against their oppressors. U.S. racial attitudes were “evidence [to the Nazis] that America was evolving in the right direction, despite its specious rhetoric about equality.” Ming Xie, originally from Beijing, in the People’s Republic of China, in Chapter 9, “News Coverage and Public Perceptions of the Social Credit System in China,” writes that The State Council of China in 2014 announced “that a nationwide social credit system would be established” in China. “Under this system, individuals, private companies, social organizations, and governmental agencies are assigned a score which will be calculated based on their trustworthiness and daily actions such as transaction history, professional conduct, obedience to law, corruption, tax evasion, and academic plagiarism.” The “nationalism” in this case is that of the state over the individual. China has 1.4 billion people; this system takes their measure for the purpose of state control. Once fully operational, control will be more subtle. People who are subject to it, through modern technology (most often smart phones) will prompt many people to self-censor. Orwell, modernized, might write: “Your smart phone is watching you.” Ming Xie holds two Ph.Ds, one in Public Administration from University of Nebraska at Omaha and another in Cultural Anthropology from the Chinese Academy of Social Sciences, Beijing, where she also worked for more than 10 years at a national think tank in the same institution. While there she summarized news from non-Chinese sources for senior members of the Chinese Communist Party. Ming is presently an assistant professor at the Department of Political Science and Criminal Justice, West Texas A&M University. In Chapter 10, analyzing native peoples and nationhood, Barbara Alice Mann, Professor of Honours at the University of Toledo, in “Divide, et Impera: The Self-Genocide Game” details ways in which European-American invaders deprive the conquered of their sense of nationhood as part of a subjugation system that amounts to genocide, rubbing out their languages and cultures -- and ultimately forcing the native peoples to assimilate on their own, for survival in a culture that is foreign to them. Mann is one of Native American Studies’ most acute critics of conquests’ contradictions, and an author who retrieves Native history with a powerful sense of voice and purpose, having authored roughly a dozen books and numerous book chapters, among many other works, who has traveled around the world lecturing and publishing on many subjects. Nalanda Roy and S. Mae Pedron in Chapter 11, “Understanding the Face of Humanity: The Rohingya Genocide.” describe one of the largest forced migrations in the history of the human race, the removal of 700,000 to 800,000 Muslims from Buddhist Myanmar to Bangladesh, which itself is already one of the most crowded and impoverished nations on Earth. With about 150 million people packed into an area the size of Nebraska and Iowa (population less than a tenth that of Bangladesh, a country that is losing land steadily to rising sea levels and erosion of the Ganges river delta. The Rohingyas’ refugee camp has been squeezed onto a gigantic, eroding, muddy slope that contains nearly no vegetation. However, Bangladesh is majority Muslim, so while the Rohingya may starve, they won’t be shot to death by marauding armies. Both authors of this exquisite (and excruciating) account teach at Georgia Southern University in Savannah, Georgia, Roy as an associate professor of International Studies and Asian politics, and Pedron as a graduate student; Roy originally hails from very eastern India, close to both Myanmar and Bangladesh, so he has special insight into the context of one of the most brutal genocides of our time, or any other. This is our case describing the problems that nationalism has and will pose for the sustainability of the Earth as our little blue-and-green orb becomes more crowded over time. The old ways, in which national arguments often end in devastating wars, are obsolete, given that the Earth and all the people, plants, and other animals that it sustains are faced with the existential threat of a climate crisis that within two centuries, more or less, will flood large parts of coastal cities, and endanger many species of plants and animals. To survive, we must listen to the Earth, and observe her travails, because they are increasingly our own.

AbstractThe concept of change includes a variety of topics, situations, disciplines, dimensions, and aspects. Its diversity and impact on individuals and organizations has led to an array of definitions, models, and theories. Thus, changes constitute a response to values, transformations that are interpreted as opportunities to improve an organization’s resilience and increase its achievements. This chapter will further discuss the concept of change, leading to a deep analysis of teachers’ moral and ethical role in one of the most impactful changes in schools: the technological revolution. If teachers believe that change is necessary, they will make great efforts to implement it effectively both in class and at school. International examples will be shown (e.g., Israel, USA, UK, and Turkey), and conclusions will be drawn regarding the need to specifically train teachers to raise their ICT awareness and understand the drawbacks and risks of technology in the twenty-first century. Schools’ transformation in information and communication is not just a technological revolution but also a social and ethical change that involves teachers in a complex weave of technologies, its creators and users, their interactions, and the social context.

The activity described in this chapter is centered on learning dance and uses YouTube as a primary resource. YouTube can help supplement a teacher’s instruction to more effectively communicate a wide array of styles. The activity builds on a relatively traditional procedure of modeling, except that it gives learners more flexibility in terms of time usage, scheduling, as well as an opportunity to experiment with various ideas. It also gives learners a level of authority over what, when, and how they learn after the teacher’s initial introduction. The lessons outlined were born from a desire to better represent the diversity of styles that exist in various regions of Kenya. By using YouTube, lessons are not limited to the knowledge of the teacher and as a result, students can have more freedom in what they learn.

This chapter uses Korean indie groups to reveal the diversity of music under the K-pop umbrella and the ramifications of such diversity for music aesthetics, authenticity, and globalization. Placing K-pop within a global R&B tradition highlights K-pop as a diverse style of music largely informed by African American popular music. It reveals the extent to which Black popular music has informed K-pop. Technology enables fans to access a wide array of K-pop, foster fan communities, and act as critics and arbiters of taste. K-pop’s place in a global R&B tradition also reveals the diversity of global influences, challenging the focus on a generalized West and United States that obscures the impact of African American culture.

DNA micro-array is a fastest-growing technology in molecular biology and bioinformatics. Based on series of microscopic spots of DNA sequences, they allow the measurement of gene expression in specific conditions at a whole genome scale. Micro-array experiments result in wide sets of expression data that are useful to the biologist to investigate various biological questions. Experimental micro-arrays data and sources of biological knowledge are now available on public repositories. As a consequence, comparative analyses involving several experiments become conceivable and hold potentially relevant knowledge. Nevertheless, the task of manually navigating and searching for similar tendencies in such huge spaces is mainly impracticable for the investigator and leads to limited results. In this context, the authors propose a semantic data warehousing solution based on semantic web technologies that allows to monitoring both the diversity and the volume of all related data.

Public health stands for the study and practice of those activities and initiatives that result in the prevention and reduction of incidences of illnesses and diseases in the population. The application of Information and Communication Technologies (ICT) can considerably facilitate Public Health project initiatives. In spite of the huge benefits of using ICT in Public Health, it can also pose considerable challenges in certain populations, pertaining to the access and comprehension of information shared through modern technology stemming from a range of issues such as illiteracy, demographic and linguistic diversity, differing economic strata of people, and differing priorities. In this chapter, after presenting a discussion on the issues faced by the public and relevant systems thinking approaches that may enable addressing the same, we propose a visionary architectural framework for ICT in Public Health through the eye of systems-thinking. We have called this framework e-System for Public Health (ePH). The understanding draws heavily from the Indian context as the country presents an interesting array of the challenges that we have mentioned above.

The fundamental premise of chemistry is that all matter consists of molecules. The physical and chemical properties of matter are those of the constituent molecules, and the transformation of matter into different materials (compounds) is the result of their reactions to form new molecules. A molecule consists of two or more atoms held in a relatively fixed array via valence-electron orbital overlap (covalent bonds; chemical bonds). In the nineteenth century chemists focused on the remarkable diversity of molecules produced by living organisms, which have in common the presence of tetravalent carbon atoms. As a result the unique versatility of carbon for the design and synthesis of new molecules was discovered, and the subdiscipline of organic chemistry (the science of carbon-containing molecules) has become the dominant part of the discipline. Clearly, the results from a focus on carbon-based chemistry have been immensely useful to science and to society. Although most molecules in biological systems [and produced by living organisms (particularly aerobic systems)] contain oxygen atoms as well as carbon and hydrogen (e.g., proteins, nucleic acids, carbohydrates, lipids, hormones, and vitamins), there has been a long tradition in all of chemistry to treat oxygen atoms as “neutral counterweights” for the “important,” character-determining elements (C, H, Al, Si, Fe, I) of the molecule. Thus, chemists have tended to take the most important element (oxygen) for granted. The chemistry curriculum devotes one or two year-courses to the chemistry of carbon (“Organic Chemistry”), but only a brief chapter on oxygen is included in the first-year and the inorganic courses. However, if the multitude of hydrocarbon molecules is from the incorporation of oxygen atoms in single-carbon molecules argues against the assignment of a “neutral character” for oxygen atoms [e.g., Cn(graphite), CH4(g), CH3OH(1), CH2(O)(1), HC(O)OH(1), (HO)2C(O)(aq), CO(g), CO2(g)]. Just as the focus of nineteenth century chemists on carbon-containing molecules has produced revolutionary advances in chemical understanding, and yielded the technology to synthesize and produce useful chemicals, polymers, and medicinals; I believe that a similar focus on oxygen chemistry is appropriate and will have analogous rewards for chemistry, biochemistry, and the chemical process technologies.

The fruit of pepper (Capsicum annuum) commonly wilts (or shrivels) during postharvest storage due to rapid water loss, a condition that greatly reduces its shelf life and market value. The fact that pepper fruit are hollow, and thus have limited water content, only exacerbates this problem in pepper. The collaborators on this project completed research whose findings provided new insight into the genetic, physiological, and biochemical basis for water loss from the fruits of pepper (Capsicum annuum and related Capsicum species). Well-defined genetic populations of pepper were used in this study, the first being a series of backcross F₁ and segregating F₂, F₃, and F₄ populations derived from two original parents selected for having dramatic differences in fruit water loss rate (very high and very low water loss). The secondly population utilized in these studies was a collection of 50 accessions representing world diversity in both species and cultivar types. We found that an unexpectedly large amount of variation was present in both fruit wax and cutin composition in these collections. In addition, our studies revealed significant correlations between the chemical composition of both the fruit cuticular waxes and cutin monomers with fruit water loss rate. Among the most significant were that high alkane content in fruit waxes conferred low fruit water loss rates and low permeability in fruit cuticles. In contrast, high amounts of terpenoids (plus steroidal compounds) were associated with very high fruit water loss and cuticle permeability. These results are consistent with our models that the simple straight chain alkanes pack closely together in the cuticle membrane and obstruct water diffusion, whereas lipids with more complex 3-dimensional structure (such as terpenoids) do not pack so closely, and thus increase the diffusion pathways. The backcross segregating populations were used to map quantitative trait loci (QTLs) associated with water loss (using DART markers, Diversity Arrays Technology LTD). These studies resulted in identification of two linked QTLs on pepper’s chromosome 10. Although the exact genetic or physiological basis for these QTLs function in water loss is unknown, the genotypic contribution in studies of near-isogenic lines selected from these backcross populations reveals a strong association between certain wax compounds, the free fatty acids and iso-alkanes. There was also a lesser association between the water loss QTLs with both fruit firmness and total soluble sugars. Results of these analyses have revealed especially strong genetic linkages between fruit water loss, cuticle composition, and two QTLs on chromosome 10. These findings lead us to further speculate that genes located at or near these QTLs have a strong influence on cuticle lipids that impact water loss rate (and possibly, whether directly or indirectly, other traits like fruit firmness and sugar content). The QTL markers identified in these studies will be valuable in the breeding programs of scientists seeking to select for low water loss, long lasting fruits, of pepper, and likely the fruits of related commodities. Further work with these newly developed genetic resources should ultimately lead to the discovery of the genes controlling these fruit characteristics, allowing for the use of transgenic breeding approaches toward the improvement of fruit postharvest shelf life.

Oroginal Objectives: (i) identify DNA markers linked to the avirulence (Avr) locus and locate the Avr locus through genetic mapping with an inter-race Orobanche cumana population; (ii) develop high-throughput fingerprint DNA markers for genotypingO. cumana races; (iii) identify nucleotide binding domain leucine rich repeat (NB-LRR) genes encoding R proteins conferring resistance to O. cumana in sunflower; (iv) increase the resolution of the chromosomal segment harboring Or₅ and related R genes through genetic and physical mapping in previously and newly developed mapping populations of sunflower; and (v) develop high-throughput DNA markers for rapidly and efficiently identifying and transferring sunflower R genes through marker-assisted selection. Revisions made during the course of project: Following changes in O. cumana race distribution in Israel, the newly arrived virulent race H was chosen for further analysis. HA412-HO, which was primarily chosen as a susceptible sunflower cultivar, was more resistant to the new parasite populations than var. Shemesh, thus we shifted sunflower research into analyzing the resistance of HA412-HO. We exceeded the deliverables for Objectives #3-5 by securing funding for complete physical and high-density genetic mapping of the sunflower genome, in addition to producing a complete draft sequence of the sunflower genome. We discovered limited diversity between the parents of the O. cumana population developed for the mapping study. Hence, the developed DNA marker resources were insufficient to support genetic map construction. This objective was beyond the scale and scope of the funding. This objective is challenging enough to be the entire focus of follow up studies. Background to the topic: O. cumana, an obligate parasitic weed, is one of the most economically important and damaging diseases of sunflower, causes significant yield losses in susceptible genotypes, and threatens production in Israel and many other countries. Breeding for resistance has been crucial for protecting sunflower from O. cumana, and problematic because new races of the pathogen continually emerge, necessitating discovery and deployment of new R genes. The process is challenging because of the uncertainty in identifying races in a genetically diverse parasite. Major conclusions, solutions, achievements: We developed a small collection of SSR markers for genetic mapping in O. cumana and completed a diversity study to lay the ground for objective #1. Because DNA sequencing and SNPgenotyping technology dramatically advanced during the course of the study, we recommend shifting future work to SNP discovery and mapping using array-based approaches, instead of SSR markers. We completed a pilot study using a 96-SNP array, but it was not large enough to support genetic mapping in O.cumana. The development of further SNPs was beyond the scope of the grant. However, the collection of SSR markers was ideal for genetic diversity analysis, which indicated that O. cumanapopulations in Israel considerably differ frompopulations in other Mediterranean countries. We supplied physical and genetic mapping resources for identifying R-genes in sunflower responsible for resistance to O. cumana. Several thousand mapped SNP markers and a complete draft of the sunflower genome sequence are powerful tools for identifying additional candidate genes and understanding the genomic architecture of O. cumana-resistanceanddisease-resistance genes. Implications: The OrobancheSSR markers have utility in sunflower breeding and genetics programs, as well as a tool for understanding the heterogeneity of races in the field and for geographically mapping of pathotypes.The segregating populations of both Orobanche and sunflower hybrids are now available for QTL analyses.

The Cucurbitaceae family includes a broad array of economically and nutritionally important crop species that are consumed as vegetables, staple starches and desserts. Fruit of these species, and types within species, exhibit extensive diversity as evidenced by variation in size, shape, color, flavor, and others. Fruit size and shape are critical quality determinants that delineate uses and market classes and are key traits under selection in breeding programs. However, the underlying genetic bases for variation in fruit size remain to be determined. A few species the Cucurbitaceae family were sequenced during the time of this project (cucumber was already sequenced when the project started watermelon and melon sequence became available during the project) but functional genomic tools are still missing. This research program had three major goals: 1. Develop whole genome cucumber and melon SNP arrays. 2. Develop and characterize cucumber populations segregating for fruit size. 3. Combine genomic tools, segregating populations, and phenotypic characterization to identify loci associated with fruit size. As suggested by the reviewers the work concentrated mostly in cucumber and not both in cucumber and melon. In order to develop a SNP (single nucleotide polymorphism) array for cucumber, available and newly generated sequence from two cucumber cultivars with extreme differences in shape and size, pickling GY14 and Chinese long 9930, were analyzed for variation (SNPs). A large set of high quality SNPs was discovered between the two parents of the RILs population (GY14 and 9930) and used to design a custom SNP array with 35000 SNPs using Agilent technology. The array was validated using 9930, Gy14 and F1 progeny of the two parents. Several mapping populations were developed for linkage mapping of quantitative trait loci (QTL) for fruit size These includes 145 F3 families and 150 recombinant inbred line (RILs F7 or F8 (Gy14 X 9930) and third population contained 450 F2 plants from a cross between Gy14 and a wild plant from India. The main population that was used in this study is the RILs population of Gy14 X 9930. Phenotypic and morphological analyses of 9930, Gy14, and their segregating F2 and RIL progeny indicated that several, likely independent, factors influence cucumber fruit size and shape, including factors that act both pre-anthesis and post-pollination. These include: amount, rate, duration, and plane of cell division pre- and post-anthesis and orientation of cell expansion. Analysis of F2 and RIL progeny indicated that factors influencing fruit length were largely determined pre-anthesis, while fruit diameter was more strongly influenced by environment and growth factors post-anthesis. These results suggest involvement of multiple genetically segregating factors expected to map independently onto the cucumber genome. Using the SNP array and the phenotypic data two major QTLs for fruit size of cucumber were mapped in very high accuracy (around 300 Kb) with large set of markers that should facilitate identification and cloning of major genes that contribute to fruit size in cucumber. In addition, a highly accurate haplotype map of all RILS was created to allow fine mapping of other traits segregating in this population. A detailed cucumber genetic map with 6000 markers was also established (currently the most detailed genetic map of cucumber). The integration of genetics physiology and genomic approaches in this project yielded new major infrastructure tools that can be used for understanding fruit size and many other traits of importance in cucumber. The SNP array and genetic population with an ultra-fine map can be used for future breeding efforts, high resolution mapping and cloning of traits of interest that segregate in this population. The genetic map that was developed can be used for other breeding efforts in other populations. The study of fruit development that was done during this project will be important in dissecting function of genes that that contribute to the fruit size QTLs. The SNP array can be used as tool for mapping different traits in cucumber. The development of the tools and knowledge will thus promote genetic improvement of cucumber and related cucurbits.