The inherent asymmetry of female meiosis presents an opportunity for genetic material to gain an evolutionary advantage during the formation of the egg. Since centromeres mediate chromosomal segregation by forming the bridge between microtubules and chromosomes during cell division, they are loci that can drive, or selfishly evolve, during female meiosis by manipulating the process of entering the egg. Mimulus guttatus, a species of yellow monkeyflowers, has the best documented case of centromeric drive (Fishman and Saunders, 2008). Since homozygotes for drive have decreased pollen viability, lower seed counts, and poor reproductive success, CENH3 —the gene that encodes the H3 histone specific to centromeres— has evolved to suppress centromeric drive. CENH3 is duplicated in Mimulus, and the sequence variation of CENH3_A suggests that this paralog can suppress centromeric drive during female meiosis (Finseth et al. 2015). Our analysis of gene expression levels in meiotic and mitotic tissues indicates that both CENH3_A and CENH3_B are still expressed at similar levels, suggesting that the paralogs have not specialized for different roles in cell divisions. However, qPCR was only performed with nine tissue samples, so further analysis of gene expression with a larger sample set is needed to confirm whether or not the CENH3 paralogs have specialized roles in meiosis and mitosis.

Submitted by Irene Nascimento (irene.kessia@ufpe.br) on 2016-08-15T17:53:44Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) A_Marques_PhD_thesis_2016_FINAL.pdf: 9324057 bytes, checksum: 92c1de27f6fa947d7e6b3e74ae09a849 (MD5) / Made available in DSpace on 2016-08-15T17:53:46Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) A_Marques_PhD_thesis_2016_FINAL.pdf: 9324057 bytes, checksum: 92c1de27f6fa947d7e6b3e74ae09a849 (MD5) Previous issue date: 2015-02-15 / capes / Cromossomos holocêntricos são caracterizados pela ausência de constrição primária e apresentam normalmente a proteína centromérica CENH3 distribuída ao longo de um eixo em cada cromátide. Embora muitos organismos com cromossomos monocêntricos apresentem sequências de DNA centroméricas específicas e associadas com a CENH3, nenhuma sequência centromérica havia sido identificada em organismos com cromossomos holocêntricos até o momento. Além disso, vários estudos reportam adaptações meióticas em espécies com cromossomos holocêntricos. Sendo observada em alguns casos uma inversão da ordem dos eventos meióticos (meiose invertida ou pós-reducional). Assim, o presente trabalho objetivou estudar a organização centromérica e a meiose de espécies com cromossomos holocêntricos do gênero Rhynchospora (Cyperaceae). Foi realizada uma análise citogenômica da organização e composição dos holocentrômeros de Rhynchospora pubera (2n = 10), sendo reportada a primeira descoberta de sequências centroméricas em espécies com cromossomos holocêntricos. Foi observado que os holocentrômeros de R. pubera são compostos principalmente por arranjos de DNA satélite (Tyba) e retroelementos centroméricos (CRRh) distribuídos pelo genoma. A análise detalhada da sucessão dos eventos meióticos de R. pubera e R. tenuis (2n = 4) reportou uma prófase inicial semelhante a de monocêntricos. No entanto, foi verificado que as cromátides-irmãs separam para polos opostos durante a anáfase I e os homólogos segregam somente durante a meiose II, comprovando uma meiose invertida para ambas as espécies. Curiosamente, durante a meiose de R. pubera foi observado uma organização diferencial dos centrômeros. Ao contrário do observado em mitose, durante meiose não foi observado a formação de holocentrômeros em forma de linha, sendo, na verdade, observado estruturas centroméricas aglomeradas. O restabelecimento de holocentrômeros em forma de linha se deu durante a primeira mitose do pólen. / Holocentric chromosomes are characterized by the absence primary constriction and normally show the centromeric protein CENH3 distributed along the axis of each chromatid. Although many monocentric organisms show centromere-specific DNA sequences associated to CENH3, no centromeric sequences had been identified in any holocentric organism so far. Furthermore, many studies report meiotic adaptations in holocentric species. In some cases is observed an inversion of the order of meiotic events. This type of meiosis has been named of inverted or post-reductional meiosis and would be exclusive of holocentric organisms. Thus, the present work aimed to study the centromere organization and meiosis of holocentric species of the genus Rhynchospora (Cyperaceae). A cytogenomic analysis of the composition and organization of the holocentromeres of Rhynchospora pubera (2n = 10) has been performed, being reported the first centromeric sequences from a holocentric species. It was observed that the holocentromeres of R. pubera are composed mainly by arrays of satellite DNA (Tyba) and centromeric retrotransposons (CRRh) distributed genomewide. The detailed analysis of the succession of meiotic events of R. pubera and R. tenuis (2n = 4) demonstrated an early meiotic prophase similar to that of monocentric. However, it was verified that sister chromatids separate to opposite poles during anaphase I, while homologs only segregate at meiosis II. These results prove the inverted meiosis for both species. Curiously, it was observed during meiosis of R. pubera a differential organization of centromere units. In contrast to the observed in mitosis, during meiosis we did not observed the formation of line-like holocentromeres, being in fact observed the formation of cluster-like holocentromeres. The reestablishment of a line-like holocentromere occurred during the first pollen mitosis.

Chez Arabidopsis, l’enveloppe nucléaire constitue un site de nucléation des microtubules à partir des complexes à gamma-tubuline. Conservées des plantes à l'Homme, les protéines GIPs/MZT1 ont été initialement découvertes comme partenaires d’AtGCP3. J’ai consacré ma thèse à la caractérisation moléculaire et fonctionnelle des AtGIPs et de leurs partenaires à l’interface nucléocytoplasmique. Mes résultats confirment l’appartenance des GIPs aux complexes à gamma-tubuline, et démontrent leur association entre elles et avec TSA1 (TonSoKu [TSK]-Associating protein 1) et l'histone centromérique CenH3. Les interactions génétiques entre les gènes GIPs, TSA1 et TSK révèlent des anomalies sévères à l'échelle de l'organisme, des cellules et des noyaux. Les mutants gip1gip2 démontrent une diminution de la cohésion des régions centromériques. L’ensemble de nos résultats suggère un rôle des AtGIPs dans un continuum nucléocytoplasmique inédit, la régulation de l'architecture nucléaire et du centromère. / In Arabidopsis, the nuclear envelope is a nucleation center where gamma-tubulin complexes initiate the polymerization of microtubules. Conserved from plants to humans, GIPs/MZT1 proteins were initially discovered as AtGCP3 interacting partners. Our investigations were devoted to the molecular and functional characterization of AtGIPs and their associated proteins at the nucleocytoplasmic interface. We confirmed that AtGIPs are integral components of gamma-tubulin complexes, and showed that they interact with each other, TSA1 (TonSoKu [TSK]-Associating protein 1) and centromeric histone H3 (CenH3). Genetic interactions between GIPs, TSA1 and TSK reveal severe defects at the organism, cellular and nuclear scales. gip1gip2 mutants exhibit a decrease of centromeric and pericentromeric cohesion. Altogether, this is the first evidence for the role of a gamma–tubulin complex component in the structural maintenance of centromeric regions, and in defining nuclear morphology and architecture.

We evaluated the genetic diversity of Penstemon's Centromeric Histone 3 (CENH3), which localizes to chromosome centromeres in eukaryotes. From RNA extractions of 56 species, we successfully amplified two CENH3s (termed CENH3a and CENH3b), which share approximately 69% sequence homology across the length of the gene and about 85% across the histone fold domain (HFD). CENH3a is generally 72 bp longer than CENH3b and contains seven exons while CENH3b is made of five. Unlike studies of CENH3 in other plants, Penstemon's CENH3 N-tail was found to be highly conserved, indicative that the genus has undergone a short evolutionary history. Surprisingly, of the 99 CENH3 sequences obtained during this study, 32 appeared to be mis-spliced and contained premature stop codons. Of those aberrant transcripts, 84.4% originated from CENH3b genomic DNA. Most mis-spliced transcripts resulted from the retention of all or part of an intron. In some cases, all or portions of an exon were missing, including one that was missing the L1 motif. Second, we systematically cataloged interspecific breeding data in Penstemon, on which we then conducted a network analysis. The resulting network provides breeders with a better visualization of successful parental combinations and also identifies gaps in interspecific breeding. This method allowed for the identification of species with a high degree of interspecific compatibility, which we compared to the CENH3 sequencing data. Finally, we studied the performance of soil streaming in high tunnel production of tomatoes (Solanum lycopersicum L.). Our experiments revealed that soil steaming and mulch reduced weed coverage of Palmer amaranth (Amaranthus palmeri S. Watson), large crabgrass (Digitaria sanguinalis L.), and yellow nutsedge (Cyperus esculentus L.). Steam and mulch increased the tomato plant size, fruit size, fruit number, and fruit yield. Additionally, soil steaming reduced tomato southern blight, caused by Sclerotium rolfsii, by 5.8-fold. These findings provide promising results for high tunnel tomato producers, particularly those involved in organic production where pesticide and fumigation use is limited.