Next-generation sequencing (NGS) has revolutionized the field of genetics by providing a means for fast and relatively affordable sequencing. With the advancement of NGS, whole- genome sequencing (WGS) has become more commonplace. However, sequencing an entire genome is still not cost effective or even beneficial in all cases. In studies that do not require a whole-genome survey, WGS yields lower sequencing depth and sequencing of uninformative loci. Targeted sequencing utilizes the speed and low cost of NGS while providing deeper coverage for desired loci. This thesis applies targeted sequencing to the genomes of two different, non-model plants, Artemisia tridentate (sagebrush) and Lupinus luteus (yellow lupine). We first targeted the transcriptomes of three species of sagebrush (Artemisia) using RNA-seq. By targeting the transcriptome of sagebrush we have built a resource of transcripts previously unmatched in sagebrush and identify transcripts related to terpenes. Terpenes are of growing interest in sagebrush because of their ability to identify certain species of sagebrush and because they play a role in the feeding habits of the threatened sage-grouse. Lastly, using paralogs with synonymous mutations we reconstructed an evolutionary time line of ancient genome duplications. Second, we targeted the flanking loci of recognition sites of two endorestriction enzymes in genome of L. luteus genome through genotyping-by-sequencing (GBS). GBS of yellow lupine provided enough single-nucleotide polymorphic loci for the construction of a genetic map of yellow lupine. Additionally we compare GBS strategies for plant species without a reference genome sequence.

Significant alterations in the environmental conditions can have pronounced effects on plant genome stability. Recent evidence argues for a global involvement of the components of epigenetic modules in the regulation of genome homeostasis both immediately after stress exposure and long after environmental cues were acquired. The last observation is of particular interest as the memory of imposing stress can be maintained at the molecular level throughout plant ontogenesis and may be faithfully propagated into the following generation. Our study provides evidence that epigenetic repercussions exerted by stress exposure of parental plants manifest themselves in untreated progeny at all three levels of the epigenetic module: DNA methylation, histone posttranslational modifications and small RNA metabolism. Additionally, the results of our study shed new light on the engagement of the epigenetic machinery in the maintenance of plant genome integrity by counteracting the activity of invading nucleic acids. / xv, 280 leaves : ill. ; 29 cm

M. Tech. (Department of Biosciences, Faculty of Applied and Computer Sciences) Vaal University of Technology / Most cultivated bananas and plantains (Musa spp. sect. Eumusa), originated from two wild diploid species, Musa acuminata Colla (AA) and Musa balbisiana Colla (BB), which contributed the A and B genomes, respectively. The two genomes confer different traits to a banana plant. Intra- and interspecific hybridization between the wild diploid species, somatic mutations and selection over many thousands of years has given rise to considerable genetic variability in cultivated bananas. Bananas are classified according to its genome composition and a number of morphological traits are used to identify the genomes of a plant. Morphological classification can be misleading since the morphology of plants can be affected by environmental factors. Molecular techniques to identify the genomes of banana have many advantages. The objective of this study was to develop a SCAR (sequence characterized amplified region) marker from a previously reported A genome-specific RAPD fragment that distinguish the A genome of banana from the B genome. This fragment designated OPA17600 was cloned, sequenced and used to design longer 20-mer SCAR primers. Verification of the SCAR primers for its fidelity to the A genome was carried out on a sample of 22 homo-and heterogenomic accessions representing landraces and hybrids of different ploidy and genome combinations. Out of six primers sets that were tested one set (SC3) produced a unique 600 bp in all the A genome containing banana accessions. However, these primers also amplified an 800 bp fragment in all the BB genotypes and some accessions containing the A and B genomes. While previous reports suggested that there was considerable differentiation between the A and B genomes, recent evidence points to the contrary. The presence of the A genome fragment in the B genome genotypes and accessions may be due to recombination between the two genomes, translocations and substitutions. The study concluded that the 600-bp SCAR sequence is conserved across the A genome in Musa and can be used to identify the A genome in banana classification and Musa breeding programmes.

The elucidation of the composition and organization of genomes of higher plants is a fundamental problem of modern molecular biology. The genus Beta containing 14 species assigned to the sections Beta, Corollinae, Nanae and Procumbentes provides a suitable system for the comparative study of the nuclear genomes. Sugar beet Beta vulgaris has a genome size of 758 Mbp DNA with estimated 63 % repetitive sequences and the number of chromosomes n=9. The wild beet Beta procumbens is an important natural pool of resistance against pests and tolerance to unfavorable growth conditions. The subject of this research was the isolation and description of new repetitive DNA families from genomes of this Beta species. This work presents the molecular investigation and cytogenetic characterization by high-resolution multicolor fluorescent in situ hybridization (FISH) of the satellite and dispersed repetitive sequences in wild and cultivated beet species and in their hybrids. New repetitive sequences were isolated from the B. procumbens genome. The AluI restriction satellite repeats pAp11 are 229-246 bp long and form subfamilies. The satellite is amplified in the section Procumbentes, but also found in distantly related section Beta. Thus, pAp11 is probably an ancient component of Beta genomes. It could be the ancestor of the satellite subfamily pEV4 in B. vulgaris based on sequence analysis, Southern hybridization and comparative FISH. pAp11 was found at centromeric and a few intercalary sites in B. procumbens and formed intercalary blocks on B. vulgaris chromosomes where it co-localized with pEV4. These remarkable differences in the chromosomal position of pAp11 between Procumbentes and Beta species indicate that both satellites were likely involved in the expansion or rearrangement of the intercalary heterochromatin of B. vulgaris. Other two sequence families characterized on molecular, genomic and chromosomal levels are the non-homologous repeats pAp4 and pAp22, 1354 and 582 bp long. They have a dispersed organization in the genome and are widely scattered along B. procumbens chromosomes. pAp4 and pAp22 are specific for the section Procumbentes and can be used as DNA probes to discriminate parental genomes in interspecific hybrids. High-resolution FISH on meiotic chromosomes showed that the both sequences mostly co-localize. The PCR analysis of their flanking regions revealed that pAp22 is a part of a Long Terminal Repeat (LTR) of an Athila-like env-class retrotransposon. This is the first indication that the retrovirus-like DNA elements exist in Beta. An ancient family of subtelomeric satellite DNA pAv34 was isolated from all four sections of the genus Beta and from spinach, a related Chenopodiaceae. Five clones were analyzed from each of the five species. The genomic organization and species distribution of the satellites were studied by sequencing and Southern hybridization. The repeating units in all families are 344-362 bp long and share 46.2-98.8 % similarity. Each monomer consists of two subunits SU1 and SU2 of 165-184 bp. The maximum likelihood and neighbor joining analyses of the 25 subtelomeric satellite monomers and their subunits indicated, that the duplication leading to the emergence of the 360 bp satellite should have occurred early in the phylogeny. The two directions of diversification are the clustering of satellites in two groups of subunits SU1 and SU2 and the arrangement of satellite repeats in section-specific groups. The comparative chromosomal localization of the telomeric repeat, pAv34 and rDNA was investigated by multicolor FISH. B. vulgaris chromosome termini showed unique physical organization of telomeric repeat and the subtelomeric satellite, as studied by high-resolution FISH on extended DNA fibers. The estimated length of the telomeric array was 0.55 - 62.65 kb, the length of pAv34 was 5.0-125.25 kb, the spacer between these sequences spanned 1.0-16.60 kb. Eight various classes of repeats were used to characterize the minichromosomes of the sugar beet fragment addition lines PRO1 and PAT2 by comparative multi-color FISH. The study allowed to propose a schematic pattern of repetitive DNA organization on the PRO1 and PAT2 minichromosomes. PRO1 has an acrocentric minichromosome, while PAT2 possesses a metacentric or submetacentric chromosome fragment. The functional integrity of the fragment addition line centromeres was confirmed by an immunostaining localization of the proteins specific to the active kinetochore. The serine 10-phosphorylated histone H3 was detected in pericentromeric regions of the PRO1 chromosomes. The microtubuli attachment sites were visualized as parts of kinetochore complexes.

The elucidation of the composition and organization of genomes of higher plants is a fundamental problem of modern molecular biology. The genus Beta containing 14 species assigned to the sections Beta, Corollinae, Nanae and Procumbentes provides a suitable system for the comparative study of the nuclear genomes. Sugar beet Beta vulgaris has a genome size of 758 Mbp DNA with estimated 63 % repetitive sequences and the number of chromosomes n=9. The wild beet Beta procumbens is an important natural pool of resistance against pests and tolerance to unfavorable growth conditions. The subject of this research was the isolation and description of new repetitive DNA families from genomes of this Beta species. This work presents the molecular investigation and cytogenetic characterization by high-resolution multicolor fluorescent in situ hybridization (FISH) of the satellite and dispersed repetitive sequences in wild and cultivated beet species and in their hybrids. New repetitive sequences were isolated from the B. procumbens genome. The AluI restriction satellite repeats pAp11 are 229-246 bp long and form subfamilies. The satellite is amplified in the section Procumbentes, but also found in distantly related section Beta. Thus, pAp11 is probably an ancient component of Beta genomes. It could be the ancestor of the satellite subfamily pEV4 in B. vulgaris based on sequence analysis, Southern hybridization and comparative FISH. pAp11 was found at centromeric and a few intercalary sites in B. procumbens and formed intercalary blocks on B. vulgaris chromosomes where it co-localized with pEV4. These remarkable differences in the chromosomal position of pAp11 between Procumbentes and Beta species indicate that both satellites were likely involved in the expansion or rearrangement of the intercalary heterochromatin of B. vulgaris. Other two sequence families characterized on molecular, genomic and chromosomal levels are the non-homologous repeats pAp4 and pAp22, 1354 and 582 bp long. They have a dispersed organization in the genome and are widely scattered along B. procumbens chromosomes. pAp4 and pAp22 are specific for the section Procumbentes and can be used as DNA probes to discriminate parental genomes in interspecific hybrids. High-resolution FISH on meiotic chromosomes showed that the both sequences mostly co-localize. The PCR analysis of their flanking regions revealed that pAp22 is a part of a Long Terminal Repeat (LTR) of an Athila-like env-class retrotransposon. This is the first indication that the retrovirus-like DNA elements exist in Beta. An ancient family of subtelomeric satellite DNA pAv34 was isolated from all four sections of the genus Beta and from spinach, a related Chenopodiaceae. Five clones were analyzed from each of the five species. The genomic organization and species distribution of the satellites were studied by sequencing and Southern hybridization. The repeating units in all families are 344-362 bp long and share 46.2-98.8 % similarity. Each monomer consists of two subunits SU1 and SU2 of 165-184 bp. The maximum likelihood and neighbor joining analyses of the 25 subtelomeric satellite monomers and their subunits indicated, that the duplication leading to the emergence of the 360 bp satellite should have occurred early in the phylogeny. The two directions of diversification are the clustering of satellites in two groups of subunits SU1 and SU2 and the arrangement of satellite repeats in section-specific groups. The comparative chromosomal localization of the telomeric repeat, pAv34 and rDNA was investigated by multicolor FISH. B. vulgaris chromosome termini showed unique physical organization of telomeric repeat and the subtelomeric satellite, as studied by high-resolution FISH on extended DNA fibers. The estimated length of the telomeric array was 0.55 - 62.65 kb, the length of pAv34 was 5.0-125.25 kb, the spacer between these sequences spanned 1.0-16.60 kb. Eight various classes of repeats were used to characterize the minichromosomes of the sugar beet fragment addition lines PRO1 and PAT2 by comparative multi-color FISH. The study allowed to propose a schematic pattern of repetitive DNA organization on the PRO1 and PAT2 minichromosomes. PRO1 has an acrocentric minichromosome, while PAT2 possesses a metacentric or submetacentric chromosome fragment. The functional integrity of the fragment addition line centromeres was confirmed by an immunostaining localization of the proteins specific to the active kinetochore. The serine 10-phosphorylated histone H3 was detected in pericentromeric regions of the PRO1 chromosomes. The microtubuli attachment sites were visualized as parts of kinetochore complexes.