Thesis describes main attributes and principles of Evolutionary and Genetic algorithms. Crossover, mutation and selection are described as well as termination options. There are examples of practical use of evolutionary and genetic algorithms. Optimization of distribution routes using PHP&MySQL and Google Maps API technologies.

Wheat (genome AABBDD) is one of the essential crops, offering approximate 20% of human calorie consumption worldwide. Allopolyploidization of three diploid ancestors led to hexaploid wheat with narrowed genetic variation. Chromosome engineering is an applicable approach to restore the evolutionarily-omitted genetic diversity by homoeologous chromosomes recombination between wheat and its relatives. Two diploid relatives of wheat, Thinopyrum elongatum (genome EE) and Aegilops speltoides (genome SS), containing favorable genes, are used as gene resources for alien introgression and genome diversification in wheat. An advanced and effective experiment procedure was developed and applied for the production, recovery, detection, and characterization of homoeologous recombinants. Meanwhile, a novel recombinant chromosome recovery strategy was exploited with improved efficiency and accuracy. In this study, recombinants of wheat chromosomes 3B and 7B with their homoeologous chromosomes in Th. elongatum and Ae. speltoides (i.e. 3B-3E, 7B-7E, and 7B-7S) were produced and detected. Totolly, 81 3B-3E recombinants and four aberrations involving in distinct chromosomal regions were developed in three recombination cycles by fluorescent genomic in situ hybridization (FGISH). The secondary and tertiary recombination breakpoints occurred toward the proximal regions comparing to the primary recombination under this advanced recombination procedure. A novel recovery strategy was used to recover 7B-7E and 7B-7S homoeologous recombinants by chromosome-specific markers and FGISH verification. Marker-based pre-screening and subsequent FGISH verification identified 29 7B-7E and 61 7B- 7S recombinants, seven 7B-7E and four 7B-7S Robertsonian translocations, one 7E and five 7S telocentric chromosomes, and three 7S deletions. All the recombinants and aberrations were genotyped by high-throughput wheat 90K single nucleotide polymorphism (SNP) assay and the recombination breakpoints were physically mapped to wheat chromosome 3B or 7B according to their FGISH patterns, SNP results, and wheat reference genome sequence. Chromosome 3B was physically partitioned into 38 bins with 429 SNPs. Meanwhile, 44 distinct bins were resolved for chromosome 7B with 523 SNPs. A composite bin map was constructed for chromosomes 3B and 7B, respectively, with a comprehensive analysis of FGISH and SNPs results. In summary, this project provides a unique physical framework for further wheat genome studies and diversifies the wheat genome for germplasm development in wheat breeding.

Y-chromosome testing has become more prevalent in recent years as a means of identifying forensic samples using STRs or identifying biomarkers for disease or determining geographic origins of populations. Additionally, Y-chromosome analysis is especially useful in paternity testing as the Y chromosome is inherited paternally and the male-specific region of the Y chromosome does not undergo any recombination events, allowing the genotypic data of both the father and son to be identical. Though in most cases a father-son pair will have the same Y-allelic data, random mutations like allele insertions and deletions can occur, which can interfere and result in incorrect conclusions in regards to paternity testing, forensic analysis, or genealogy. Though the exact mechanism of Y loci mutability is unknown, postulations of factors that can cause mutations have been studied, as well as attempts to determine mutation rate specific to each locus. A multi-generational pedigree consisting of 9 males was analyzed using two different methodologies: capillary electrophoresis and next-generation sequencing. The samples were amplified using either a ForenSeq™ Signature DNA Prep Kit (Verogen, San Diego, CA) or a YFiler™ Plus PCR Amplification Kit (Thermo Fisher Scientific, Waltham, MA). Between the two methods, five Y-STR loci were identified as being discordant between a father-son pair. Next-generation sequencing identified an allele insertion at DYS385a/b, resulting in a potential tri-allelic locus, but was disproved after comparison with the capillary electrophoresis data of the sample. The capillary electrophoresis data identified four discordances between father-son pairs, one of which was an allele mutation with a gain of a repeat at DYS458. At DYS 389II, an allele insertion was identified, but was contradicted after comparison with the next-generation sequencing data. There was a potential null allele at DYS518 and either an OL variant allele or a 2 base pair deletion at DYS481. Following peak height ratio, stutter, and comparative analysis between the genotypic data of the two analysis methods, two of these discordances were proven to be errors, one was a definitive mutational event, and the other two could neither be confirmed nor denied due to differences in loci tested in each kit.

No description available.

Thesis describes main attributes and principles of Evolutionary and Genetic algorithms. Crossover, mutation and selection are described as well as termination options. There are examples of practical use of evolutionary and genetic algorithms. Optimization of distribution routes using PHP&MySQL and Google Maps API technologies.

I developed and tested a protocol for determining the sex of individual pinnipeds using the sex-chromosome specific genes ZFX and ZFY. I screened a total of 368 seals (168 crabeater, Lobodon carcinophagus; 159 Weddell, Leptonychotes weddellii; and 41 Ross, Ommatophoca rossii) of known or unknown sex and compared the molecular sex to the sex assigned at the time of collection in the Ross and Amundsen seas, Antarctica. Discrepancies ranged from 0.0% - 6.7% among species. It is unclear, however, if mis-assignment of sex occurred in situ or in the laboratory. It also is possible, however, that the assigned morphological and molecular sex both are correct, owing perhaps to developmental effects of environmental pollution. I sequenced a portion (ca 475 bp) of the mitochondrial control region of Weddell seals (N = 181); crabeater seals (N = 143); and Ross seals (N = 41). I resolved 251 haplotypes with a haplotype diversity of 0.98 to 0.99. Bayesian estimates of Θ from the program LAMARC ranged from 0.075 for Weddell seals to 0.576 for crabeater seals. I used the values of theta to estimate female effective population sizes (NEF), which were 40,700 to 63,000 for Weddell seals, 44,400 to 97,800 for Ross seals, and 358,500 to 531,900 for crabeater seals. Weddell seals and crabeater seals had significant, unimodal mean pairwise difference mismatch distributions (p = 0.56 and 0.36, respectively), suggesting that their populations expanded suddenly around 731,000 years ago (Weddell seals) and around 1.6 million years ago (crabeater seals). Both of these expansions occurred during times of intensified glaciations and may have been fostered by expanding pack ice habitat. Autosomal microsatellite based NEs were 147,850 for L. Weddellii, 344,950 for O. rossii, and 939,600 for L. carcinophagus. I screened one X-linked microsatellite (Lw18), which yielded a larger NE estimate for O. rossii than the other two species. Microsatellite NE estimates are compared with previously published mitochondrial NE estimates and this comparison indicates that the Ross seal may have a serially monogamous system of mating. I find no sign of a recent, sustained genetic bottleneck in any of the three species.

In this thesis, we study the physics of the pulled polymer loops motivated by a biological problem of chromosome alignment during meiosis in fission yeast. During prophase I of meiotic fission yeast, the chromosomes form a loop structure by binding their telomeres to the Spindle Pole Body (SPB). SPB nucleates the growth of microtubules in the cytoplasm. Molecular motors attached to the cell membrane can exert the force on the microtubules and thus pull the whole nucleus. The nucleus performs oscillatory motion from one to the other end of the elongated zygote cell. Experimental evidence suggests that these oscillations facilitate homologous chromosome alignment which is required for the gene recombination. Our goal is to understand the physical mechanism of this alignment. We thus propose a model of pulled polymer loops to represent the chromosomal motion during oscillations. Using a freely-jointed bead-rod model for the pulled polymer loop, we solve the equilibrium statistics of the polymer configurations both in 1D and 3D. In 1D, we find a peculiar mapping of the bead-rod system to a system of particles on a lattice. Utilizing the wealth of tools of the particle system, we solve exactly the 1D stationary measure and map it back to the polymer system. To address the looping geometry, the Brownian Bridge technique is employed. The mean and variance of beads position along the loop are discussed in detail both in 1D and 3D. We then can calculate the three-dimensional statistics of the distance between corresponding beads from a pair of loops in order to discuss the pairing problem of homologous chromosomes. The steady-state shape of a three-dimensional pulled polymer loop is quantified using the descriptors based on the gyration tensor. Beyond the steady state statistics, the relaxation dynamics of the pinned polymer loop in a constant external force field is discussed. In 1D we show the mapping of polymer dynamics to the well-known Asymmetric Simple Exclusion Process (ASEP) model. Our pinned polymer loop is mapped to a half-filled ASEP with reflecting boundaries. We solve the ASEP model exactly by using the generalized Bethe ansatz method. Thus with the help of the ASEP theory, the relaxation time of the polymer problem can be calculated analytically. To test our theoretical predictions, extensive simulations are performed. We find that our theory of relaxation time fit very well to the relaxation time of a 3D polymer in the direction of the external force field. Finally, we discuss the relevance of our findings to the problem of chromosome alignment in fission yeast.

Bei der Untersuchung von Dequalinium Chloride wurden alle relevanten genetischen Endpunkte berücksichtigt. Einer absolut negativen Datenlage steht lediglich ein spezifischer mutagener In vitro-Effekt im Ames-Test entgegen: In einem Bakterienstamm wurden eine bestimmte Art von Genmutationen, Frameshift-Mutationen, ausgelöst. Die Relevanz dieses Ergebnisses wurde in vivo im Spot Test überprüft. Der Spot Test war deshalb ausgewählt worden, da er die Erfassung von Mutagenen erlaubt, welche spezifisch Frameshift-Mutationen auslösen (Fahrig, 1995). Dies war eine notwendige Voraussetzung für den Einsatz des Tests, da im Ames-Test gerade diese Art von Mutationen ausgelöst worden war und deshalb dieser Endpunkt auch verfolgt werden musste. Die Verfolgung dieses genetischen Endpunkts in vivo im Spot Test mit Mäusen brachte trotz Verabreichung toxischer Dosen (5 und 7 mg/kg) ein negatives Ergebnis. Da zuvor schon der HPRT-Test ein negatives Ergebnis gezeigt hatte, stehen der spezifischen Frameshift-induzierenden Wirkung von Dequalinium Chloride in Bakterien eindeutig negative Ergebnisse im Säuger in vitro und in vivo gegenüber. Für die Relevanz der Ergebnisse im Spot Test und damit für die Wertung ist entscheidend, dass Dequalinium Chloride die Zielzellen in genügenden Konzentrationen erreicht, um eine Wirkung ausüben zu können. Bedenkt man, dass Dequalinium Chloride toxische Wirkung auf Muttertiere und Neugeborene ausübte und daß die Häufigkeit von gelben Missdifferenzierungssflecken und weißen, auf der Abtötung von Pigmentzellen beruhenden Bauchflecken gegenüber der Kontrolle erhöht war, so kann davon ausgegangen werden, dass Dequalinium Chloride die Zielzellen erreicht, aber weder mutagene noch rekombinogene Wirkung ausgeübt hat. Isoliert dastehenden positiven In-vitro-Befunden sollte von vornherein keine große Bedeutung beigemessen werden. Im vorliegenden Falle konnte gezeigt werden, dass eine mutagene Wirkung nur bei Bakterien und weder in vivo noch in vitro in Säugerzellen auftrat. Aus diesem Grunde kann dem Befund mit Bakterien keine Bedeutung für den Menschen beigemessen werden.:I. Datenlage 1. Genmutationen in vitro 1.1 Salmonella-Mikrosomen (Ames)-Test 1.2. HPRT-Test mit V79 Zellen des Chinesischen Hamsters 2. Chromosomenaberrationen in vitro 2.1 Chromosomenaberrationen in menschlichen Lymphozyten 3. Genmutationen in vivo 3.1 Spot Test mit Mäusen II. Wertung

The application of Y-chromosome analysis is expanding in fields such as forensic science and genealogy. By researching the potential polymorphisms this chromosome can present, we can further our ability to assess DNA profiles for these disciplines to avoid erroneous exclusions of paternal linkage, wrongful convictions based on forensic evidence, and other misinformed genetic conclusions. The conservation of Y-haplotypes during transmission occurs due to a relative lack of genetic recombination events in the inheritance of the Y-chromosome [1]. However, random mutation events can occur in a paternal line resulting in haplotype changes. These changes can include allele duplications and deletions that occur at the STR and SNP loci used in forensic DNA analysis. This can become important in cases of sexual assault where male-female mixture samples have low amounts of male DNA such that the male signal is not amplified in currently used STR multiplexes [7]. In this study, we analyzed a father and his eleven sons using two different methodologies for genetic analysis; next generation sequencing and capillary electrophoresis. The samples were obtained from the Coriell Institute for Medical Research located in Hamden, NJ, in the form of frozen DNA extracts isolated from a blood-sourced lymphocyte cell culture [22]. DNA from these samples was tested with the ForenSeqTM DNA Signature Prep Kit [14] (Verogen, San Diego, CA) primer set A and the YFilerTM Plus PCR Amplification Kit [24] (Thermo Fisher Scientific, Waltham, MA). Using these two platforms, three Y-STR loci were identified as discordant between the father and all of his eleven sons. In all three instances, the father possessed the same allele as the sons as well as one additional allele. At two of these loci (DYS449 and DYS635), the additional allele was one repeat (4bp) longer than that of the shared allele. At the other locus (DYS458), the additional allele was three repeats (12bp) longer than that of the shared allele. Following read count and peak height analysis, it was concluded that these double allele loci are not the product of stutter and are potentially the product of a non-inheritable mutation. With the knowledge that the DNA was extracted from a blood lymphocyte cell culture, it is believed that a somatic mutation may be present in the cell line. We are not able to determine whether the mutations exist in the blood of the father (true somatic mutations) or occurred as a result of the cell culture process. Throughout the study, details concerning the position of these loci on the Y-chromosome, the repeat motifs of the alleles, and the potential for duplication and/or stutter as the originating event are discussed in an effort to further understand this phenomenon. Potential locus duplications were compared to those reported on the National Institute of Standards and Technology STRBase [21] list of allele variations and also to information found in literature. The observed DYS635 locus had an allele designation of 21,22 which is reported on STRBase. The DYS449 and DYS458 loci showed potential allele-specific locus duplications that were not found on STRBase. The implications of potentially undocumented non-inheritable allele patterns in the Y-chromosome, such as this, are significant when considering comparisons between DNA obtained from germline cells (sperm) versus a known casework sample which is usually obtained from blood or saliva [7].

This thesis presents the work done during my master degree projects under the supervision of Alexander Suh and Francisco J. Ruiz-Ruano. My work focused on the development of in-silico methods to improve the assembly of the Germline Restricted Chromosome (GRC) of songbirds, more specifically that of zebra finch.GRCs are a good example of the popular saying "The exception that proves the rule". For a very long time, it was assumed that every cell in a healthy multicellular organism carries the same genetic information. Cytogenetic evidence dating back as far as early XX century suggests that this is not always the case, as it has been documented that certain organisms carry supernumerary B chromosomes, which are dispensable chromosomes that are not part of the normal karyotype of a species. GRCs are often regarded as a special case of B chromosomes, where every individual from a species carries an additional chromosome whose presence is restricted to germline cells only. GRCs presence has been documented in insects, hagfishes and songbirds. A peculiar case of GRCs is that of zebra finch, whose GRC has an estimated size of over 150 Mb, accounting for over 10% of zebra finch total genome size. Despite the first cytogenetic evidence of zebra finch GRC dating back to 1998, it was only last year that the first comprehensive genomic study about this relatively large chromosome was published. This study shed some light on the gene content of the GRC in zebra finch, revealing that the GRC of zebra finch mostly consists of paralogs of A chromosomal genes. The GRC assembly and annotation that were published as part of this study included 115 GRC-linked genes that were identified through germline/soma read mapping, as well as 36 manually curated scaffolds with a median length of 3.6 kb. Considering the conspicuous size of the GRC of zebra finch, it is clear that this is a very fragmented and likely incomplete GRC assembly. There are many factors that can have a negative impact on assembly completeness and contiguity. In the GRC case, these factors collectively affect coverage in ways that are not properly handled by available genome assemblers. In the course of my master degree project I developed kFish, a bioinformatic software to perform alignment-free enrichment of GRC-linked barcodes from a 10x Genomics linked-read DNA Chromium library. kFish uses an iterative approach where the k-mer content of a set of GRC-linked sequences is compared with that of reads corresponding to each individual 10x Genomics barcode. This comparison allows kFish to identify likely GRC-linked barcodes, and then only use reads corresponding to these barcodes when trying to assemble the GRC. First benchmarking results generated using five GRC-linked genes from zebra finch as reference sequences, show that kFish is not only capable of assembling already known GRC-linked sequences, but also new ones with high confidence. kFish can do all of this in a matter of hours, using only few gigabytes of system memory, while previous efforts took over two days to assemble zebra finch genome and identify GRC-linked scaffolds using an approach based on read mapping. High quality genome assemblies and annotations are the foundations of modern genomics research, the lack of which greatly limits the breadth of the questions that can be answered. There is still a lot that we do not understand about GRCs, and part of this is due to the lack of high quality GRC assemblies and annotations. Producing such an assembly will likely require an integrated approach, where multiple sequencing technologies as well as bleeding edge bioinformatic tools such as kFish, are combined together to produce an high quality assembly, which will be crucial to unravel the mystery of GRCs function and evolutionary history.