DNA fingerprinting and genetic relationships among willow (Salix spp.)

Given that morphological identification of willow is difficult, willow lines being investigated for their suitability for use as short rotation crops for biomass production in Saskatchewan were investigated with various molecular techniques as possible tools for DNA fingerprinting. Flow cytometry was used to assess variation in nuclear DNA content and thus ploidy level of the lines of the five species (Salix purpurea, Salix eriocephala, Salix sachalinensis, and Salix dasyclados) and three hybrids (S. purpurea x S. miyabeana, S. sachalinensis x S. miyabeana, S. viminalis x S. miyabeana). The DNA content varied between 1.14 and 3.00pg. Ploidy levels of the species varied from triploid to hexaploid while all hybrids were tetraploid. RAPD and ISSR marker systems were used to assess genetic and taxonomic relationships among all willow lines. Of 90 RAPD primers tested, 60 were selected and 99 polymorphic bands scored. Of 35 ISSR primers tested, 19 were selected and 35 polymorphic bands scored. Both RAPD and ISSR dendrograms clustered together lines belonging to the same species and same hybrid combination. A combination of strong and reproducible RAPD and ISSR bands was used to develop identification keys for lines belonging to the same species. The ribosomal RNA gene region, including the entire 5.8S RNA gene and the internal transcribed spacers (ITS1 and ITS2) was amplified and sequenced to assess sequence homology between the five species. The total length of the amplified region was 601bp, with the ITS1, 5.8 S and ITS2 being 223, 163, and 215bp respectively. Intra- and inter-species SNPs were observed, 6 within ITS1, and 3 within ITS2. No polymorphisms were found in the 5.8S gene. The low rate of variation within the sequenced ITS fragment between species supports the monophyly of the five species involved in this study, and confirms their belonging to the subgenus Caprisalix. SCAR primers were designed from species-specific polymorphic nucleotides and applied to the willow collection to test their use for species identification. A species identification key based on SNPs is proposed

Witches' broom disease of cocoa : genome organization, genetic variability and anlysis of the identity and expression of pathogenicity genes of the fungal pathogen Crinipellis perniciosa

Orientador: Gonçalo Amarante Guimarães PereiraTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: A Vassoura-de-Bruxa do cacaueiro constitui um patosistema complexo e até o momento não foi estabelecido um plano de controle efetivo na região produtora da Bahia. O objetivo principal deste trabalho foi aprimorar os conhecimentos sobre a interação C. perniciosa-Cacaueiro com informações referentes à: 1) tamanho e organização do genoma através da obtenção do cariótipo molecular do fungo; 2) variabilidade genética deste fitopatógeno a nível cromossômico na região produtora da Bahia; e 3) expressão gênica do fungo em presença de extratos do hospedeiro. O cariótipo molecular foi estabelecido por eletroforese em gel de campo pulsado. Esta mesma metodologia foi utilizada em combinação com análise tipo microsatélites para um estudo de variabilidade a nível cromossômico de 38 isolados provenientes de três biótipos e diferentes regiões geográficas. Para análise da expressão gênica foram seqüenciadas quatro bibliotecas de cDNA do fungo sob diferentes condições de cultivo e em diferentes estágios do desenvolvimento, incluindo uma biblioteca subtrativa obtida por SSH. Como resultados, apresentamos o cariótipo molecular de C. perniciosa e a existência de polimorfismos cromossômicos. A análise dos 38 isolados permitiu observar que o cariótipo do biótipo-C é estável ao longo do tempo e sua variabilidade na região da Bahia é baixa, mostrando assim a fragilidade do programa de melhoramento genético do cacaueiro na Bahia, onde os clones resistentes selecionados no campo foram desafiados contra apenas dois genótipos do patógeno. Com relação à análise da expressão do fungo, obtivemos 1427 unigenes e sua análise por similaridade com as bases de dados permitiu a identificação de diversos mecanismos pelos quais o fungo poderia estar manipulando o metabolismo da planta para seu benefício e em detrimento da produção de cacau. Assim, consideramos que a partir deste trabalho foi possível obter uma maior compreensão dos mecanismos utilizados por C. perniciosa no desenvolvimento da Vassoura-de-Bruxa em cacaueiro e estudos mais aprofundados, baseados nos resultados aqui obtidos, deverão auxiliar no desenvolvimento de estratégias de controle para esta importante doençaAbstract: Witches' broom disease of cocoa is a complex pathosystem that has evaded an efficient control program in the cacao-producing region of Bahia, Brazil. The main goal of this work was to acquire a better understanding of the C. perniciosa-Cacao interaction by providing new data regarding: 1) the size and organization of the fungal genome through molecular karyotyping; 2) the genetic variability of this phytopathogen at the chromosomal level in the cacao-producing region of Bahia; and 3) the gene expression of the fungus in the presence of host extracts. The molecular karyotype was obtained through pulsed-field gel electrophoresis. This same technique was applied in combination with microsatellite PCR analysis of 38 isolates from different biotypes and geographic regions in order to study the chromosomal-level genetic variability of this pathogen. For the gene expression analysis, four cDNA libraries of the fungus grown under different culture conditions and developmental stages, including a subtractive library using SSH, were sequenced and searched for similarities in the public databases. As results, we present the molecular karyotype of C. perniciosa and the existence of chromosome-length polymorphism. The analysis of the 38 isolates showed that the karyotype of the C-biotype is very stable in time and that the variability of the pathogen in Bahia is very low, thus indicating the fragility of the current cacao-breeding program in Bahia, where the resistant cacao clones selected in the field were challenged against only two different genotypes of the pathogen. With regards to the gene expression analysis, we obtained 1427 unigenes and the similarity searches allowed the identification of various mechanisms through which the fungus might be manipulating the metabolism of the plant for its own benefit and in detriment of cocoa production. Therefore, we consider that this work allowed us to acquire a better understanding of the molecular mechanisms used by C. perniciosa during witches' broom development in cacao and more detailed studies, based on the results presented here, might aid in the development of novel control strategies for this important diseaseDoutoradoBioquimicaDoutor em Biologia Funcional e Molecula

Harnessing modern biotechnology for tropical tuber crop improvement: Yam (Dioscorea spp.) molecular breeding

Yams (Dioscorea spp.) constitute a staple food crop for over 100 million people in the humid and subhumid tropics. They are polyploid and vegetatively propagated. The Guinea yams, Dioscorea rotundata and D. cayenensis, are the most important yams in West and Central Africa where they are indigenous, while D. alata (referred to as water yam) is the most widely distributed species globally. The genetics of yams is least understood among the major staple food crops due to several biological constraints and research neglect. Research to unravel the apparent complexity of the yam genome will have far-reaching implications for genetic improvement of this important tuber crop. Some progress has been made in recent years in germplasm characterization and the development of molecular markers for genome analysis. A genetic linkage map based on amplified fragment length polymorphism (AFLP) markers has been constructed for Guinea and water yams. These linkage maps were used to scan the genome for quantitative trait loci (QTL) associated with genes conferring resistance to Yam Mosaic Virus (YMV) in D. rotundata and anthracnose (Colletotrichum gloeosporioides) in D. alata. In addition, candidate random amplified polymorphic DNA (RAPD) markers associated with major genes controlling resistance to YMV and anthracnose have been identified that could be used for selection and pyramiding of YMV and anthracnose resistance genes in yam improvement. Also, molecular markers such as RAPDs, AFLPs, and microsatellites or simple sequence repeats (SSRs) have been developed for yam genome analysis. An initial c-DNA library has been constructed in order to develop expressed sequence tags (ESTs) for gene discovery and as a source of additional molecular markers. This paper will review the advances made, discuss the implications for yam genetic improvement and germplasm conservation, and outline the direction for future research. Key words: Genetic mapping, genome analysis, molecular breeding, PCR-based markers, QTLs, resistance genes, yam. African Journal of Biotechnology Vol. 2 (12), pp. 478-485, December 200

Genes, genomes, and transcriptomes of ciliates and their prokaryotic endosymbionts

The topic of my PhD research was the study of symbiotic relationships between ciliated protozoa (phylum Ciliophora) and their bacterial symbionts, employing mainly molecular techniques with the goal of characterizing both partners in the symbiosis. My projects can be grouped into four categories, each corresponding to one of the parts of this thesis. In the first part I present the description of 6 bacterial species, providing information on their classification, relationship with the host, and phylogeny. The peculiar features of some of these prokaryotes, like the presence of flagella in representatives of lineages previously considered non-flagellated, allowed to propose evolutionary hypotheses of broader interest, including inferences on the characteristics of the mitochondria ancestor. The second part of the thesis contains the first genomic analysis of a bacterial symbiont, Polynucleobacter necessarius, harbored by a ciliate, Euplotes. The genomic structure and the gene content were comparatively studied with closely related free-living organisms in order to understand the physiological bases of the ciliate/symbiont interaction and study the process of genome reduction, that is seldom investigated outside of model arthropodan systems. In the third part of the thesis, I present systematic and multi-disciplinary surveys of symbiont-harboring ciliate genera, combining old and new data on many aspects of their diversity, including phylogeny and biogeography. The fourth part briefly reports the results of my most recent line of research, ciliate transcriptomics, and in particular the successful optimization of a single-cell RNA-seq protocol suitable for protists. In summary, this thesis provides characterization works on previously understudied organisms, hypotheses on the evolution of varied features, and the first results in two areas in their infancy: genomics of ciliate symbionts, and single-cell transcriptomics of protists. [ITA] L’argomento della mia ricerca di dottorato è stato lo studio delle relazioni simbiotiche tra protozoi ciliati (Phylum Ciliophora) e i loro endosimbionti batterici, utilizzando principalmente tecniche molecolari al fine di caratterizzare entrambi i partner della simbiosi. I miei progetti possono essere suddivisi in quattro categorie, corrispondenti ciascuna a una delle quattro parti di questa tesi. Nella prima parte presento la descrizione di 6 specie batteriche, fornisco informazioni sulla loro classificazione, rapporto con l’ospite, e filogenesi. Le caratteristiche più peculiari di alcuni di questi procarioti, come la presenza di flagelli in rappresentanti di gruppi precedentemente considerati non flagellati, ha consentito di proporre ipotesi evolutive di più ampio interesse, incluse ricostruzioni delle possibili caratteristiche dell’antenato dei mitocondri. La seconda parte di questa tesi contiene la prima analisi genomica su un simbionte batterico, Polynucleobacter necessarius, ospite di un ciliato, Euplotes. La struttura genomica e il contenuto genico sono stati studiati comparativamente rispetto a organismi a vita libera strettamente imparentati, per comprendere le basi fisiologiche dell’interazione ciliato/simbionte e studiare il processo di riduzione genomica, raramente investigato al di fuori dei sistemi modello negli artropodi. Nella terza parte della tesi presento indagini sistematiche e multidisciplinari su generi di ciliati che ospitano simbionti, integrando dati vecchi e recenti su molti aspetti della loro diversità, incluse filogenesi e biogeografia. La quarta parte riporta brevemente i risultati della mia più recente linea di ricerca, la trascrittomica dei ciliati, e in particolare il successo nell’ottimizzazione di un protocollo di RNA-seq su singola cellula efficace sui protisti. In sintesi, questa tesi fornisce caratterizzazioni di organismi precedentemente poco noti, ipotesi sull’evoluzione di diversi caratteri, e i primi risultati in due campi appena agli inizi: la genomica dei simbionti di ciliati, e la trascrittomica su singola cellula di protisti

Mutation induction by gamma irradiation in chrysanthemum [Chrysanthemum x grandiflorum (Ramat.) Kitam.]

Doctor of Philosophy (Biology), 201

A comparative investigation of nuclear DNA content and its phenotypic impacts in Silene marizii and S. latifolia

Considerable variation exists both within and between species in nuclear DNA content. Despite there being no obvious functional role for much of this DNA, many studies have reported phenotypic correlations with genome size at various taxonomic levels. This suggests that DNA plays a functional role beyond the traditionally understood mechanisms. One such example of a phenotypic correlation with DNA content is present in the genus Silene, where a negative correlation between DNA content and flower size exists within and between species. This relationship is consistent with the direction of sexual dimorphism in DNA content (caused by heteromorphic sex-chromosomes) and flower size in the most studied species in the genus: S. latifolia. This thesis takes a comparative approach between two closely related species in the genus (S. latifolia and S. marizii), which differ markedly in their nuclear DNA content, in order to investigate the nature and phenotypic impacts of variation in DNA content. A phenotypic survey from a number of S. marizii populations reveals that the pattern of DNA content variation in this species is very different to that in S. latifolia. In particular, phenotypic correlations with DNA content appear be much weaker, whilst sexual dimorphism in DNA content, when present, appears to occur in either direction. A survey of interspecific hybrids suggests that this may be due to an enlarged S. marizii X-chromosome and that DNA content in hybrids may be biased with regard to their parents. Repetitive elements may be significant constituents of plant genomes. A study of Ty1-copia class retrotransposons in the two species reveals that they are present as a large and highly heterogeneous population. Phylogenetic analysis of these elements suggests a substantial degree of genetic isolation between the two species. Finally, an assessment of the flow-cytometric method, used to estimate DNA content, reveals substantial error associated with the method, but only limited evidence for stoichiometric effects

Genotypic Variation in Sweetpotato Ipomoea Batatas (L.) Lam. Clones.

Arbitrarily-primed PCR-based assays established the presence of sweetpotato intra-clonal genetic variability. These DNA polymorphism assays provided benchmark information regarding cultivar genetic uniformity in sweetpotato foundation seed programs. Arbitrarily-primed markers were also used to compare the genetic uniformity among sweetpotato clones derived conventionally, i.e., through adventitious sprouts, and nodally-based propagation systems. Initially, 38 primers generated 110 scorable DNA fragments using two virus-indexed plants from each clone source. Twenty-one bands (18.6%) were scored as putative polymorphic markers based on the presence or absence of amplified products. A subset of 14 marker loci generated by four selected primers was used to further assay 10 sample plants per clone group. Polymorphism ranged from 7.1% to 35.7% in five of eight clone groups. Field studies show variation in nearly all yield grades measured. In three tests during the 1991 and 1992 seasons, yield differences ranged from 27% to 46% within the economically important U.S. No. 1 root grade. The results suggest the usefulness of arbitrarily-primed markers in detecting intra-clonal genomic variability in the crop. To determine the role of propagation method in sweetpotato genotypic uniformity, a single sprout each of \u27Jewel,\u27 \u27Sumor,\u27 and L87-95 served as source of clonal plants simultaneously propagated through conventional adventitious procedures and an in vitro-based nodal technique. Fifteen arbitrary primers generated 64 scorable amplified fragments, 29 of which were putatively polymorphic across n = 60 samples (10 each of nodal and adventitiously derived plants/genotype). Within adventitiously derived materials, putative polymorphisms ranged from 4.7% to 31.3% depending upon genotypic class. In contrast, putative polymorphisms ranged from 0.0% to 3.1% among nodally-derived samples. The marker loci differentiated the genotypes and putative marker phenotype variants as revealed through multidimensional scaling analysis. An \u27analysis of molecular variance\u27 shows that genotypic effects accounted for 88.7% of the total marker variability, while propagation effects (within genotypic groups) accounted for 11.3%. The results suggest variability associated with propagation, wherein clonal plants derived from pre-existing meristematic regions are more genetically uniform than plants propagated from adventitious origins

Genomic tools and sex determination in the extremophile brine shrimp Artemia franciscana

The aim of this study was the construction of a genomic Artemia toolkit. Sex-specific AFLP-based genetic maps were constructed based on 433 AFLP markers segregating in a 112 full-sib family, revealing 21 male and 22 female linkage groups (2n = 42). Fifteen putatively homologous linkage groups, including the sex linkage groups, were identified between the female and male linkage maps. Eight sex-linked markers, heterozygous in female animals, mapped to a single locus on a female linkage group, supporting the hypothesis of a WZ/ZZ genetic sex-determining system and showing primary sex determination is likely directed by a single gene. To fine-map the sex locus, bulked segregant analysis was performed. Candidate primary sex-determining genes were identified, including Cytochrome P450 which, through transcriptomic studies, is already known as a candidate sex-determining gene for Macrobrachium nipponense. The 1,310-Mbp Artemia draft genome sequence (N50 = 14,784 bp; GC-content = 35%; 176,667 scaffolds) was annotated, predicting 188,101 genes with an average length of 692 bp. Ninety-two percent of the transcriptome reads of Artemia in different conditions were present in the Artemia genome, indicating that the functional part of the genome under the RNAseq sampling conditions is virtually fully represented in the assembly. Several steps were taken in this study to introduce Artemia as a new genomic model for crustaceans. Although the functional part of the Artemia genome under the RNAseq sampling conditions is virtually fully represented in the assembly, thus making it useful for qualitative research, genome finishing strategies will still be necessary to complete the genome project. The further development of genomic resources for Artemia will add a completely new dimension to Artemia research and its use as live food in aquaculture

Characterisation of Miscanthus genetic resources: a combined analysis of plastid and nuclear microsatellites, nrDNA sequences, flow cytometry and morphology.

Doctoral ThesisMiscanthus is a highly important forage and horticultural genus of perennial grasses (Poaceae) primarily native to South East Asia. Miscanthus is under intense global investigation as a biomass source for renewable energy production and several breeding initiatives are underway to develop new genotypes optimized for improved biomass and tolerance to a range of environmental stress conditions. A collection of 128 accessions belonging to the genus Miscanthus was established in Oak Park, Teagasc, Carlow, in 2008 and was investigated for morphological and molecular variation. Morphological traits were measured at the end of the second growing season and were compared with herbarium specimens of Miscanthus. Vegetative and inflorescence traits were scored and analysed using basic summary statistics, tests of normality and Principal Components Analysis (PCA). A large degree of morphological variation was recorded in the collections. The PCA of herbarium specimens was able to separate some species from others but there was also considerable overlap among species in the ordination, especially M. sacchariflorus, M. sinensis, M. condensatus and M. floridulus. These are known to be closely related and can interbreed. The PCA of the specimens from the Oak Park collection was less informative because of missing data due to lack of inflorescences (accessions did not flower). It was clear that morphology alone is often insufficient to distinguish taxa especially when inflorescence characters and ploidy information is lacking. The ploidy level of the accessions in the collection was evaluated through flow cytometry. The ploidy included di-, tri- and tetraploids. All individuals labelled as M. ×giganteus showed a triploid status, together with the newly bred M. sacchariflorus×M. sinensis hybrids. Most M. sinensis were diploids. Miscanthus sinensis Tea-62 was triploid and comparable to the value of the M. ×giganteus. A different situation was found for other non-diploid M. sinensis, in particular four M. sinensis ‘Goliath’ and the M. sinensis ‘Zebrinus’ Tea-33. In these the ratio measured by the flowcytometer was in between the values of the triploid M. giganteus and tetraploid M. sacchariflorus standards. The ‘Goliath-like’ hybrid is likely an autotriploid with three M. sinensis haploid sets, whereas M. ×giganteus is an allotriploid that is supposed to have two genomes from M. sinensis and one from M. sacchariflorus, which has a lower amount of DNA per haploid genome. DNA sequences of the internal transcribed spacer of the nrDNA were obtained for 76 genotypes in the collection and compared for polymorphism. The SNPs were particularly VI useful for differentiating M. sinensis, M. sacchariflorus and M. ×giganteus accessions and in combination with ploidy and morphology offer high potential for taxon identification. To gather more markers for population level diversity and differentiation studies, new microsatellite markers for both plastid and nuclear genomes were developed. For the development of plastid markers the chloroplast genome information of Saccharum officinarum was used. The nuclear SSRs (nSSRs) were developed from the sequences of 192 clones obtained from microsatellite enriched library. New primer pairs for the amplification of nineteen nuclear loci and six chloroplast loci were developed. Both chloroplast (cpSSR) and nSSR primers were used to characterise DNA variation, to help establish gene pools and to better understand hybridization and introgression. Huge genotypic variation was found within the genus, mostly in the species M. sinensis. The markers showed wide utility across a large number of Miscanthus species and also some closely related genera. The analysis of the cpSSRs showed a high number of different haplotypes but with a clear bias in allele composition between M. sinensis and the two species M. sacchariflorus and M. ×giganteus, thus confirming M. sacchariflorus as the maternal lineage of the hybrid M. xgiganteus. The nSSRs were found to be highly polymorphic across the collection and transferable to closely related genera such as Saccharum. The new markers were also used in UPGMA clustering and Bayesian structuring analysis to group individuals according to their similarity. Three major clusters of individuals were defined using the Bayesian STRUCTURE analysis with nuclear markers (nSSRs) and two with plastid markers (cpSSRs). In conclusion, the morphological, ploidy, sequence and microsatellite results highlighted the high level of diversity still unexplored in the genus and have clarified taxon identity of many accessions in the collection. A large set of new markers have been developed for the plant breeding and systematics community. The newly developed markers will be useful to further explore this diversity and to select useful traits for breeding of new and improved genotypes for biomass production.Teagasc Walsh Fellowship; National Development Plan Irelan