The reliability of genome analysis and proficiency of genetic manipulation in vivo and in vitro are increased by assignment of linkage groups to specific chromosomes, placement of centromeres, orientation with respect to telomeres, and linear alignment with respect to chromosomal features and dimensions. I undertook five studies aimed at integrating sorghum genomics and cytogenetics at several levels. The results help establish an entirely new "cyto-genomics" resource, impacts of which are likely to be broad. In the first study, I developed a FISH-based karyotyping system for Sorghum bicolor Moench. I used integrated structural genomic resources, including linkage maps and large-insert clonal libraries of sorghum genomic DNA to develop a 17-locus probe cocktail for simultaneous fluorescent in situ hybridization (FISH). This probe enabled facile identification of all chromosome pairs in mitotic chromosome spreads. Perhaps just as important, I established time-efficient means to select sorghum BAC clones for multi-probe FISH. Thus, an integrated cyto-genomics system for sorghum can be constructed without need of chromosome flow sorting or microdissection, both of which are difficult and costly. In the second study, hybridization of DNA clones from 37 different genomic regions enabled the assignment of linkage groups and orientation of linkage maps to chromosomes. Comparisons between genetic and physical distances throughout the genome enabled a new nomenclature for linkage group designation in sorghum. The results provide an integrated nomenclature system of Sorghum bicolor chromosomes and linkage groups. In the third study, I created high-resolution maps by FISH to pachytene bivalents for two linkage groups (B and H), and defined relationships between pericentromeric heterochromatin, centromeres, mapped markers and recombination rates. These relationships will help guide the development and use of sorghum genomics. In the fifth study, I used FISH in two ongoing gene-targeted efforts. For the maturity gene ma5 and fertility restoration gene rfl, I estimated physical lengths between currently available flanking molecular markers. This enables estimation of recombination densities in these regions and assessment of the applicability of map-based and -assisted cloning.

To enhance the scientific utility and practical application of the honey bee genome and assign the linkage groups to specific chromosomes, I identified chromosomes and characterized the karyotype of the sequenced strain DH4 of the honey bee. The primary analysis of the karyotype and ideogram construction was based on banding and Fluorescence In Situ Hybridization (FISH) for rDNA detection. FISH confirmed two locations for the NOR on telomeric regions of chromosomes 6 and 12 plus an additional less frequent signal on chromosome 1, all three of which were confirmed with silver staining (AgNO3). 4’6-diamidino-2phenylindole (DAPI), and CBanding methods were used to construct the primary ideograms that served as a basis to further identify the chromosomes and locate important structures. The primary map was compared with Giemsa banding, AgNO3-banding, Trypsin banding, and R-banding. The karyotype of the honey bee was established as two metacentric chromosomes (1 and 10), two submetacentric with ribosomal organizer (6 and 12), four submetacentric heterochromatic chromosomes (16, 15, 4 and 13), four euchromatic subtelocentric chromosomes (2, 8, 11 and 14) and four acrocentric chromosomes (3, 5, 7 and 9). In situ nick-translation banding methods were used to verify the heterochromatin distribution. The cytogenetic maps of the honey bee karyotype represented in the ideograms were subsequently used to place 35 mapped BACs (Solignac et. al. 2004) of Solignac’s BAC library. As the BACs hybridized to multiple sites, the mapping was based on strength and frequency of the signals. Location and position of the BACs was compared with those published in the different version of Map Viewer of the NCBI and BeeBase web sites. 10 BACs were confirmed with the last version of Map Viewer V4, 12 BACs were mapped based on high frequency and agreement with the earlier version of Map Viewer. 14 BACs were mapped as confirmed based on moderate frequency of the signal and agreement with the last version of MVV, most of these BACs hits as a secondary signal.

The reliability of genome analysis and proficiency of genetic manipulation in vivo and in vitro are increased by assignment of linkage groups to specific chromosomes, placement of centromeres, orientation with respect to telomeres, and linear alignment with respect to chromosomal features and dimensions. I undertook five studies aimed at integrating sorghum genomics and cytogenetics at several levels. The results help establish an entirely new "cyto-genomics" resource, impacts of which are likely to be broad. In the first study, I developed a FISH-based karyotyping system for Sorghum bicolor Moench. I used integrated structural genomic resources, including linkage maps and large-insert clonal libraries of sorghum genomic DNA to develop a 17-locus probe cocktail for simultaneous fluorescent in situ hybridization (FISH). This probe enabled facile identification of all chromosome pairs in mitotic chromosome spreads. Perhaps just as important, I established time-efficient means to select sorghum BAC clones for multi-probe FISH. Thus, an integrated cyto-genomics system for sorghum can be constructed without need of chromosome flow sorting or microdissection, both of which are difficult and costly. In the second study, hybridization of DNA clones from 37 different genomic regions enabled the assignment of linkage groups and orientation of linkage maps to chromosomes. Comparisons between genetic and physical distances throughout the genome enabled a new nomenclature for linkage group designation in sorghum. The results provide an integrated nomenclature system of Sorghum bicolor chromosomes and linkage groups. In the third study, I created high-resolution maps by FISH to pachytene bivalents for two linkage groups (B and H), and defined relationships between pericentromeric heterochromatin, centromeres, mapped markers and recombination rates. These relationships will help guide the development and use of sorghum genomics. In the fifth study, I used FISH in two ongoing gene-targeted efforts. For the maturity gene ma5 and fertility restoration gene rfl, I estimated physical lengths between currently available flanking molecular markers. This enables estimation of recombination densities in these regions and assessment of the applicability of map-based and -assisted cloning.

Orientador: Eliana Regina Forni Martins / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-15T09:44:06Z (GMT). No. of bitstreams: 1 Ferrari_Fernanda_M.pdf: 4024604 bytes, checksum: fadaeedca82d057b615fd5bfb85b4706 (MD5) Previous issue date: 2010 / Resumo: A cana-de-açúcar assumiu grande importância no cenário econômico mundial, não só pela produção de açúcar, mas também de etanol. Variedades modernas de cana-de-açúcar são essencialmente derivadas de hibridações feitas no início do século XX entre duas spécies de Saccharum, S. officinarum (2n = 80) e S. spontaneum (2n = 40-128), seguidas de retrocruzamentos dos híbridos com S. officinarum. Devido à poliploidia natural do gênero e a aneuploidia das variedades híbridas o estudo citogenético em cana-de-açúcar é complexo. O advento da citogenética molecular, mediante a técnica de hibridização de DNA in situ (FISH e GISH), vem propiciando avanços no entendimento da organização genômica de Saccharum e de gêneros relacionados. O objetivo deste trabalho foi realizar análises cromossômicas, de número e sítios de rDNA, nas duas principais espécies do gênero, S. officinarum e S. spontaneum, e em mais três importantes variedades brasileiras, RB72454, RB835486 e RB867515. Foi possível confirmar as identidades das espécies S. officinarum (2n = 80) e S. spontaneum (2n = 64) mediante a contagem do número cromossômico. Foram caracterizados, pela primeira vez, os números cromossômicos das variedades RB72454 e RB835486 (2n = 112) e na RB867515 (2n = 110). Através de técnicas de hibridização in situ fluorescente foram quantificados os sítios de rDNA 45S e 5S. As espécies S. officinarum e S. spontaneum, conforme já descrito na literatura, apresentaram 8 sítios de cada locus. As variedades RB72454 e RB835486 apresentaram 12 sítios de cada locus e a variedade RB867515 apresentou 11 sítios do rDNA 45S e 9 sítios do rDNA 5S. Os loci de rDNA 45S e 5S encontram-se em grupos homó(eó)logos distintos e por isso, esses dois genes caracterizam-se dois marcadores cromossômicos em Saccharum spp. A localização do locus de rDNA 45S em posição distinta nos cromossomos de S. officinarum (terminais) e S. spontaneum (intersticiais), possibilitou a quantificação da contribuição dessas espécies, no respectivo grupo homeólogo, para as variedades RB72454 e RB867515 / Abstract: Sugarcane has assumed an eminent position in the world economical scenario, not only for sugar, but also for ethanol production. Current sugarcane varieties are hybrids from initial interspecific crosses involving mainly two species of Saccharum, S. officinarum (2n = 80) and S. spontaneum (2n = 40-128), followed by backcrossing with S. officinarum. Due to the polyploidy nature of the genus Saccharum and the aneuploidy occurring in the interspecific hybrids, the cytogenetic study of sugarcane is complex. Moreover, chromosomes are small and morphologically similar. The molecular cytogenetics, with technique of DNA in situ hybridization (GISH and FISH), has provided advances in the understanding of genomic organization of this crop. The goal of this study was to realize chromosomal analysis, including chromosome number and sites of rDNA of two species, S. officinarum and S. spontaneum, and of three Brazilian varieties, RB72454, RB835486 and RB867515. The identities of the species S. officinarum (2n = 80) and S. spontaneum (2n = 64) were confirmed counting their chromosome numbers. We also counted the chromosome numbers of the varieties RB72454 and RB835486 (2n = 112) and RB867515 (2n = 110). Using FISH techniques, we could quantify the rDNA 45S and 5S sites of all the accesses. S. officinarum and S. spontaneum, as described in the literature, had 8 sites at each locus. For the varieties RB72454 and RB835486, 12 sites at each locus were detected and for RB867515, 11 sites of rDNA 45S and 9 sites of rDNA 5S were detected. The loci rDNA 45S and 5S are in different homo(eo)logues groups being thus characterized as two chromosomal markers for Saccharum spp. Since the rDNA 45S is located in different positions in S. officinarum (terminals) and S. spontaneum (interstitial), this marker could be applied in the quantification, in this homeologue group, of the chromosome numbers inherited from S. officinarum and S. spontaneum by the varieties RB72454, RB867515 / Mestrado / Biologia Vegetal / Mestre em Biologia Vegetal