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Made available in DSpace on 2022-04-29T08:30:50Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-01-01 Gynogenesis is a chromosomal manipulation technique where an offspring is generated exclusively from maternal inheritance. In the yellowtail tetra (Astyanax altiparanae) almost all gynogenetic fish were females, suggesting a genetic sex determination system of XX/XY. However, due to environmental (e.g. temperature) or unknown genetic factors, the rise of males are described. Therefore, the aim of this study was to analyze the reproductive parameters of spontaneously gynogenetic males. The parameters analyzed were spermatozoon viability, motility and ploidy. Additionally, the gonads were processed for histology and the genetic inheritance confirmed by genotyping. Sperm parameters of gynogenetic males did not show differences when compared with semen of normal males. These data supports that gynogenetic males presents fertilization capacity and can be used for future studies of chromosome manipulation or possible production of monosex populations in A. altiparanae. Laboratory of Fish Biotechnology National Center for Research and Conservation of Continental Fish Chico Mendes Institute of Biodiversity Conservation Aquaculture Center Sao Paulo State University Universidade Federal Rural de Pernambuco (UFRPE) Unidade Acadêmica de Serra Talhada Aquaculture Center Sao Paulo State University

Made available in DSpace on 2021-06-25T11:57:48Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-02-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) The aim of this study was to evaluate different post-shock temperatures for tetraploid induction in the yellowtail tetra Astyanax altiparanae. Newly fertilized eggs were divided into four groups, three were submitted to heat shock (40 degrees C for 2 min) at 24 min post-fertilization (mpf) and another group remained without shock (control). Groups submitted to temperature shock were further separated at the following temperatures: 22 degrees C, 26 degrees C and 28 degrees C. Survival among embryonic development was counted and at hatching the ploidy was analyzed by flow cytometry. The results showed that the post-shock temperature affects the parameters analyzed and, therefore, must be considered for optimization of the production of tetraploid in A. altiparanae. Those data are innovative and could be used in future studies of basic biology in this species. Fed Univ Grande Dourados UFGD, BR-79825070 Dourados, MS, Brazil Sao Paulo State Univ, Aquacuiture Ctr, Via Acesso Prof Paulo Donato Castaane S-N, BR-14884900 Jaboticabal, SP, Brazil Chico Mendes Inst Biodivers Conservat, Natl Ctr Res & Conservat Continental Fish, Lab Fish Biotechnol, Rodovia Pref Euberto Nemesio Pereira de Godoy, BR-13630970 Pirassununga, SP, Brazil Sao Paulo State Univ, Aquacuiture Ctr, Via Acesso Prof Paulo Donato Castaane S-N, BR-14884900 Jaboticabal, SP, Brazil FAPESP: 2010/17429-1 FAPESP: 2011/11664-1 FAPESP: 2016/12383-0 CNPq: 471140/2012-0

Tetraploid strains are important sources of diploid gametes available for further expansion of ploidy manipulation. Although artificial induction of tetraploid strains has been attempted by inhibition of a mitotic cell division of zygotes using hydrostatic pressure or temperature treatments at the early developmental stage, successful examples of viable and fertile tetraploid fish were very rare. Because most resultant tetraploid progeny exhibited extremely high mortality. Natural tetraploid variants are considered another source of diploid gametes. In the present study, we tried to induce new strains of tetraploid, triploid and androgenetic diploid using diploid sperm of a tetraploid ginbuna (Japanese silver crucian carp Carassius auratus langsdorfii), which caught at the Jounuma lake, Gunma Prefecture, Japan. New tetraploids, i.e., neo-tetraploids were induced by fertilizing eggs of a diploid goldfish Carassius auratus auratus with diploid sperm of the tetraploid ginbuna, followed by heatshock (40°C for 45, 60 and 75 s) to inhibit the second polar body release at 5 min after fertilization. Although a small number of neo-tetraploid fish survived, we could not obtain any fertile gametes from them in the present study. Triploids were successfully induced by fertilizing eggs of a diploid goldfish with diploid sperm of the tetraploid ginbuna. Some resultant triploid males produced aneuploid sperm at the age of maturation. Androgenetic diploids were induced by fertilizing UV-irradiated eggs of diploid goldfish with diploid sperm of the tetraploid ginbuna. A mature androgenetic diploid produced fertile haploid sperm.

Made available in DSpace on 2020-12-10T17:27:21Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-02-01 AES Tiete/ANEEL Triploidization is a technique to produce sterile fish on a large scale. In the spotted catfish Pimelodus maculatus, sterile fish serve as a host of germ cell transplantation for subsequent reconstitution of endangered catfish species. This study aimed to establish an efficient protocol for triploidization in P. maculatus using temperature shock. In this study, we tested the efficacy of heat shock treatments (37 degrees C, 38 degrees C, and 39 degrees C) applied at 2 min post-fertilization (mpf) for the duration of 2 min. Intact embryos served as diploid control. Ploidy status was confirmed by flow cytometry, characterization of erythrocytes nuclei, and karyotyping. Developmental stages were verified at cleavage, blastula, gastrula, somite, and hatching stages, as well as the percentages of normal and abnormal larvae. No significant difference was observed between the temperature treatments for percentage of unfertilized oocytes (P = 0.1834), 2-cell stage (P = 0.2348), blastula (P = 0.2972), hatching (P = 0.0668), and abnormal larvae (P = 0.6177). Heat shock decreased the survival at the blastula stage (P = 0.0178), somite (P = 0.0469), and the percentage of abnormal larvae (P = 0.0261). All treatments presented high percentages of triploid individuals, and the highest values were observed for heat shock at 38 degrees C (96.7 %). Based on the results described above, an efficient protocol for triploidization in P. maculatus was established using heat shock (38 degrees C) at 2 mpf for 2 min. Chico Mendes Inst Biodivers Conservat, Natl Ctr Res & Conservat Continental Fish, Lab Fish Biotechnol, Rodovia Pref Euberto Nemesio Pereira de Godoy, BR-13630970 Pirassununga, SP, Brazil Sao Paulo State Univ, Inst Biosci, Rua Prof Doutor Antonio Celso Wagner Zanin S-No, BR-18618689 Botucatu, SP, Brazil Sao Paulo State Univ, Inst Biosci, Rua Prof Doutor Antonio Celso Wagner Zanin S-No, BR-18618689 Botucatu, SP, Brazil AES Tiete/ANEEL: 4690000174

Submitted by Gabriel Marra Schade (gabrielmarras@gmail.com) on 2020-09-13T23:21:18Z No. of bitstreams: 1 Dissertação_de_Mestrado_Gabriel_Marra.pdf: 1091990 bytes, checksum: edbb7361832bdc2015659846f946dfbc (MD5) Rejected by ROSANGELA APARECIDA LOBO null (rosangelalobo@btu.unesp.br), reason: Solicitamos que realize uma nova submissão seguindo as orientações abaixo: problema 1: folha de rosto (natureza do trabalho) Na folha de rosto, natureza do trabalho você coloca: “Defesa de Mestrado”. O correto é: Dissertação (Dissertação apresentada ao curso... ). Na capa não é necessário colocar essa informação: “Defesa de Mestrado” Problema 2: financiamento Capes No formulário de submissão consta a Capes como agência financiadora do seu projeto. No arquivo submetido não localizei uma agradecimento a esta. Isso é uma exigência das agências de fomento. E a Capes sugere que o agradecimento seja feito da seguinte maneira: "O presente trabalho foi realizado com apoio da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Código de Financiamento 001 Assim que tiver efetuado as correções submeta o arquivo, em formato PDF, novamente. Agradecemos a compreensão. on 2020-09-14T13:13:49Z (GMT) Submitted by Gabriel Marra Schade (gabrielmarras@gmail.com) on 2020-09-30T10:33:12Z No. of bitstreams: 1 FINAL_Dissertação_Gabriel_Marra.pdf: 1672112 bytes, checksum: 31b22cd342193a774b5374d28d29f188 (MD5) Approved for entry into archive by ROSANGELA APARECIDA LOBO null (rosangelalobo@btu.unesp.br) on 2020-09-30T14:29:11Z (GMT) No. of bitstreams: 1 schade_gm_me_bot_par.pdf: 487366 bytes, checksum: 9abe49ca71e4ef62c6f1d41e06b383c3 (MD5) Made available in DSpace on 2020-09-30T14:29:11Z (GMT). No. of bitstreams: 1 schade_gm_me_bot_par.pdf: 487366 bytes, checksum: 9abe49ca71e4ef62c6f1d41e06b383c3 (MD5) Previous issue date: 2020-08-13 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Poliploidia é um fenômeno onde um organismo apresenta um conjunto extra de cromossomos, com histórico evolutivo de ocorrência natural em muitos grupos de peixes. Dentre as teorias relacionadas à sua ocorrência, a hibridização interespecífica é plausível por suas possibilidades de recombinações únicas de cromossomos. A hibridização pode causar anormalidades na gametogênese principalmente no tocante à etapa reducional da meiose, levando a ocorrência de gametas não reduzidos como espermatozoides e oócitos 2n ou 4n. Desta maneira, a fertilização com tais gametas poderia produzir progênie naturalmente poliploide. Objetivou-se nesse estudo verificar se a hibridização interespecífica entre Astyanax altiparanae e A. schubarti, e interpopulacional entre A. altiparanae favorece a ocorrência de gametas não reduzidos. Os espécimes apresentaram número diploide de 2n = 50 (A. altiparanae), 2n = 36 (A. schubarti) e 2n = 43 (hibrido interespecífico), com desenvolvimento embrionário similar ao controle (A. altiparanae). Os híbridos interpopulacionais não evidenciaram espermatozoides não reduzidos, com parâmetros espermáticos similares ao controle. Os híbridos interespecíficos indicaram maior quantidade de DNA (P = 0,0024) e espermatozoides não reduzidos (1n, 2n e 4n) viáveis, porém não sem nenhuma motilidade. Embora os híbridos interespecíficos sejam inférteis, a evidência de espermatozoides não reduzidos entre híbridos do gênero Astyanax suporta a hipótese da ocorrência de peixes naturalmente poliploides na região Neotropical. Polyploidy is a phenomenon in which an organism has an extra set of chromosomes, with the evolutionary history of occurring naturally in many fish groups. Among many theories related to its occurrence, interspecific hybridization is plausible due to its unique chromosome recombination possibilities. Hybridization can cause abnormalities in gametogenesis, especially concerning the reductive stage of meiosis, leading to the occurrence of unreduced gametes like 2n or 4n spermatozoa. Thus, fertilization with such gametes could produce naturally polyploid progeny. The objective of this study was to verify if the interspecific hybridization between Astyanax altiparanae and A. schubarti, and interpopulation hybridization between A. altiparanae favors the occurrence of unreduced gametes. The specimens had a diploid number of 2n = 50 (A. altiparanae), 2n = 36 (A. schubarti) and 2n = 43 (interspecific hybrid), and showed embryonic development similar to the control group (A. altiparanae). The interpopulation hybrids did not present unreduced sperm and showed sperm parameters similar to the control group. The interspecific hybrids showed a higher amount of DNA (P = 0.0024) and unreduced sperm (1n, 2n and 4n), but it did not show any motility. Although interspecific hybrids are infertile, the evidence of unreduced sperm among hybrids of the Astyanax genus supports the hypothesis that hybridization events are associated with the occurrence of naturally polyploid fish in the Neotropical region. 88887.341931/2019-00

Made available in DSpace on 2020-12-10T19:50:25Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-04-15 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Tetraploid fish are an important source of diploid gametes for polyploid production, and they can be induced by inhibition of the first mitotic cell division. Dispite its potential, very few protocols regarding tetraploidization and subsequent application to mass production of triploid fish for aquaculture exist. In the yellowtail tetra Astyanax altiparanae, triploids present sterility and increased carcass yield, suggesting the generation of tetraploid for large-scale production of such triploids. In this study, the efficacy of heat shock treatments (40 degrees for 2 min) at 16, 18, 20, 22, 24, and 26 min post-fertilization (mpf) at an incubation temperature of 22 degrees C were compared on tetraploid induction of A. altiparanae. Ploidy status was confirmed by flow cytometry, karyotyping, and nuclear diameter of erythrocytes. Afterwards, sperm parameters and fertility capacity of tetraploid males were evaluated. The results showed that heat shock decreased the hatching rate, especially at 22 (19.1 +/- 9.6%), 24 (16.0 +/- 9.1%), and 26 mpf (10.3 +/- 6.2%) in comparison with the control group (62.2 +/- 7.7%). The flow cytometric analysis showed that the control group and the treatments with heat shock at 16 and 18 mpf presented only diploid individuals. However, from 20 mpf onwards, tetraploid individuals were observed, and the highest percentage arose at 26 mpf (94.55%). When mature, tetraploid males are capable of producing diploid spermatozoa with fertilization capacity, generating a 100% triploid offspring. The results indicate that the heat shock treatment at 26 mpf (40 degrees C for 2 min) is optimum for tetraploid induction in the A. altiparanae. Additionally, tetraploid mature males generated diploid spermatozoa and can be used to mass production of triploid fish. The current data are the first report of tetraploids in family Characidae, presenting an application in basic and applied sciences. Sao Paulo State Univ, Aquaculture Ctr, Via Acesso Prof Paulo Donato Castellane S-N, BR-14884900 Jaboticabal, SP, Brazil Chico Mendes Inst Biodivers Conservat, Natl Ctr Res & Conservat Continental Fish, Lab Fish Biotechnol, Rodovia Pref Euberto Nemesio Pereira de Godoy, BR-13630970 Pirassununga, SP, Brazil Univ Fed Rural Rio de Janeiro, BR-465 Km 7, BR-23897000 Seropedica, RJ, Brazil Hokkaido Univ, Fac Fisheries Sci, 3-1-1 Minato Cho, Hakodate, Hokkaido 0418611, Japan Sao Paulo State Univ, Aquaculture Ctr, Via Acesso Prof Paulo Donato Castellane S-N, BR-14884900 Jaboticabal, SP, Brazil FAPESP: 2010/17429-1 FAPESP: 2011/11664-1 FAPESP: 2016/12383-0 CNPq: 471140/2012-0

Submitted by Rafaela Manchin Bertolini (rafaelambertolini@hotmail.com) on 2019-01-22T12:48:45Z No. of bitstreams: 1 Dissertaçao de mestrado anexar repositorio.pdf: 3078434 bytes, checksum: 2da481903f76ada25e5f710ff2bc3d35 (MD5) Approved for entry into archive by Luciana Pizzani null (luciana@btu.unesp.br) on 2019-01-22T14:01:05Z (GMT) No. of bitstreams: 1 bertolini_rm_me_bot_par.pdf: 1360655 bytes, checksum: 07260ea94f9bb82b35dae2abffe595b6 (MD5) Made available in DSpace on 2019-01-22T14:01:05Z (GMT). No. of bitstreams: 1 bertolini_rm_me_bot_par.pdf: 1360655 bytes, checksum: 07260ea94f9bb82b35dae2abffe595b6 (MD5) Previous issue date: 2018-02-26 Outra A triploidização é uma ferramenta interessante para produzir peixes estéreis. No Mandi amarelo, Pimelodus maculatus isso pode ser aplicada como uma ferramenta de reconstituição de espécies ameaçadas através de transplante de células germinativastronco. No capítulo I, objetivou-se estabelecer um protocolo eficiente para a triploidização da espécie P. maculatus empregando choques de térmicos. As temperaturas testadas foram de 37°C, 38°C e 39°C, 2 minutos pós-fertilização durante 2 minutos. Os embriões intactos serviram como grupo controle diplóide. A ploidia foi confirmada por citometria de fluxo, diâmetro nuclear dos eritrócitos e citogenética. Taxas de fertilização e sobrevivência foram verificadas nos principais estágios de desenvolvimento embrionário (clivagem, blástula, gástrula, segmentação e eclosão), assim como a porcentagem de larvas normais e anormais, e eficiência da triploidização. O choque térmico reduziu significativamente a sobrevivência no estágio de gástrula (P = 0,0178), somito (P = 0,0469) e incrementou o porcentual de larvas anormais (P = 0,0261). A menor sobrevivência foi observada para o tratamento a 39°C. Todos os tratamentos apresentaram altas porcentagens de indivíduos triploides, sendo o maior valor observado para o choque a 38°C (96,7%). Com base nos resultados acima, foi obtido um eficiente protocolo de triploidização em P. maculatus utilizado choque quente (38°C, 2 mpf e duração de 2 minutos). No capitulo II, foram avaliados aspectos relacionados ao desempenho reprodutivo e crescimento de indivíduos diploides (2n) e triploides (3n) da espécie P. maculatus. O objetivo foi avaliar crescimento entre as duas diferentes ploidias através do peso, comprimento, rendimento de carcaça, índice gonodassomatico e a possível esterilidade em peixes triploides por meio de análise histológica. Fêmeas 2n apresentaram valores de IGS significativamente maiores (P= 0,0157). A análise histológica mostrou fêmeas 2n em fase de maturação. Por outro lado, gônadas de fêmeas 3n apresentaram somente oogônias, células apoptóticas e poucos oócitos primários isolados. Machos 2n apresentaram gametogênese normal, ao passo que em machos 3n não foram observados esprmatozoides formados. Portanto, não foram observados gametas em fêmeas e machos 3n de P. maculatus, sendo considerados estéreis. Essas características são aplicáveis tanto nas ciências básicas, como modelo experimental, bem como nas ciências aplicadas, para a produção em larga escala. Triploidization is an interesting tool to produce sterile fish. In the spotted catfish Pimelodus maculatus, this may be used as a tool for reconstitution of endangered species based on stem germ cell transplantation. In Chapter I, the study aimed to establish an efficient protocol for triploidization in the spotted catfish P. maculatus using temperature shock. The temperatures tested were 37°C, 38°C e 39°C, 2 min postfertilization during 2 minutes. Intact embryos served as diploid control. Ploidy status was confirmed by flow cytometry, nuclear diameter of erythrocytes and karyotyping. Fertilization and hatching rates were verified at the main embryo stages (cleavage, blastula, gastrula, somite stage and hatching), the percentage of normal and abnormal larvae and also the efficacy of triploidization. Heat shock decrease the survival at blastula stage (P= 0,0178), somite (P = 0,0469) and increased the percentage of abnormal larvae (P = 0,0261). The lowest survival was observed at 39 °C. All treatments presented high percentages of triploid individuals, and the highest values were observed for heat shock at 38°C (96,7%). Based on results above, it was obtained an efficient protocol for triploidization in P. maculatus using heat shock (38°C, 2 min postfertilization during 2 minutes). In Chapter II, aspects related to the growth and reproductive performance of diploid (2n) and triploid (3n) of P. maculatus were evaluated. The objective was to evaluate growth according with the ploidy status, through length, weight, carcass yield, gonadassometic index and possible sterility in triploid fish by means of histological analysis. Diploid females presented significantly higher IGS values (P = 0.0157). Histological analysis showed 2n females in maturation stage. On the other hand, gonads of 3n-females presented only oogonia, apoptotic cells and few isolated primary oocytes. Diploid males presented normal gametogenesis, although in 3n-males spermatozoa were not observed. However, gametes were not observed in females and males 3n of P. maculatus, considered then as sterile sterile. AES Tietê Projeto #4690000174

Androgenetic doubled haploids (DHs) were induced in the loach Misgurnus anguillicaudatus (Cobitidae) without irradiation of the eggs. The eggs of wild-type females were activated with the intact sperm of an orange-phenotype male, and treated (within 10 s of activation) at 3 ± 0.5 °C for 30 min, to eliminate the female nucleus. The eggs were then incubated in a water bath at 20 ± 0.5 °C for 35 min. Finally, diploidy was restored (65 min after activation) by heat-shock treatment at 42 ± 0.5 °C for 2 min. Under these conditions, the yield rate (mean ± SD) of putative DHs relative to the total number of eggs used was 10.43 ± 1.69%, which was significantly higher (P < 0.05) than the yield rates obtained under the remaining heat-shock initiation conditions (55 min, 60 min, and 70 min after activation). We analyzed the ploidy status of the putative DH by using flow cytometry. All-male inheritance was confirmed by the expression of the recessive orange body color trait and microsatellite genotypes. We detected no maternally derived alleles or heterozygous genotypes at any of the 28 loci (covering 27 linkage groups) of loach, indicating the exclusively paternal inheritance and homozygosity of the obtained androgenetic DHs.

Tetraploid loaches have been discovered amongst specimens recovered from fish markets in Japan. These tetraploids can be used as a source of diploid gametes to assist the further expansion of ploidy manipulation. Here, we produced a first generation neo-tetraploid strain by fertilizing eggs from a normal diploid female with diploid spermatozoa from a natural tetraploid male, followed five minutes later by heat shock (42°C, 2 min duration) to inhibit release of the second polar body. Diploid spermatozoa from the neo-tetraploid males produced were then used to create androgenetic diploid progeny by fertilizing UV-irradiated eggs from a normal diploid female. Triploid progeny were produced by crossing a normal diploid female with a neo-tetraploid male. Tetraploid progeny were produced by cold shock (1°C, 40 min duration), beginning 5 min after fertilizing normal eggs with diploid spermatozoa from first generation neo-tetraploid males. Reproductive performance of second generation progeny was also examined. Androgenetic diploid males generated fertile haploid spermatozoa. Triploid males were sterile, but a triploid female laid fertile haploid eggs. Second generation neo-tetraploid males were considered sterile.

In the loach, Misgurnus anguillicaudatus (Teleoste: Cobitidae), diploid sperm can be obtained from natural tetraploid individuals with four sets of homologous chromosomes. Using diploid sperm, various kinds of polyploids and androgenetic diploids have been produced. Cryptic clonal lineages are also recognized in wild populations of the loach. They produce unreduced diploid eggs genetically identical to somatic cells of the mother fish and most diploid eggs develop gynogenetically as a member of the clone. However, some eggs develop to triploid and/or diploid-triploid mosaic individuals by incorporation of sperm nucleus. Diploid-triploid mosaic males exclusively generate fertile diploid sperm with clonal genotypes. Such diploid sperm can also be obtained from artificially sex-reversed clonal individuals. Recent population studies suggested that Japanese M. anguillicaudatus might not be a single species, but a complex involving cryptic species, because wild populations exhibited genetic differentiation at interspecific level. This implies possible relationship between atypical reproduction and natural hybridization in the loach.

DNA replication is essential to maintain genome integrity in S phase of the cell division cycle. Accumulation of stalled replication forks is a major source of genetic instability, and likely constitutes a key driver of tumorigenesis. The mechanisms of regulation of replication fork progression have therefore been extensively investigated, in particular with DNA combing, an optical mapping technique that allows the stretching of single molecules and the mapping of active region for DNA synthesis by fluorescence microscopy. DNA linearization in nanochannels has been successfully used to probe genomic information patterns along single chromosomes, and has been proposed to be a competitive alternative to DNA combing. Yet this conjecture remains to be confirmed experimentally. Here, two complementary techniques are established to detect the genomic distribution of tracks of newly synthesized DNA in human cells by optical mapping in nanochannels. Their respective advantages and limitations are compared, and applied them to detect deregulations of the replication program induced by the antitumor drug hydroxyurea. The developments here thus broaden the field of applications accessible to nanofluidic technologies, and can be used in the future as part for molecular diagnostics in the context of high throughput cancer drug screening.

Interspecific hybridization is used to introduce desirable characters from related species into allopolyploids such as wheat. The development of substitution and addition lines of related species in bread wheat can be useful as a genetic tool to transfer agronomic traits in the background of bread wheat. However, there is a low level of pairing and recombination between wheat chromosomes and those from the relative species. Different approaches have been carried out to promote pairing and recombination between related chromosomes such as the ph1b mutant, ionizing radiations and gametocidal genes. However, the most important discovery has been the Ph1 locus which regulates pairing and recombination between homologous chromosomes during meiosis in wheat. Thus, in its absence, related chromosomes could associate and recombine. Chapter 2 of this work examines the dynamic of DNA replication during premeiosis and early meiosis in wheat using flow cytometry, which has allowed the quantification of the amount of DNA in wheat anther in each stage meiotic stage. Chromosome replication was detected in wheat during premeiosis and early meiosis until the stage of pachytene, when chromosomes are associated in pairs to further recombine and correctly segregate in the gametes. Also, an important role of the Ph1 locus on the length of meiotic DNA replication in wheat was shown using flow cytometry. Chapter 3 deals with chromosome manipulation to induce meiotic recombination between barley and wheat in the absence of the Ph1 locus. Genetic crosses between the ph1b mutant and both wild and cultivated barley substitution and addition lines in wheat were carried out. More than...

The objective of the present study was to determine the possible production of Persian sturgeon (Acipenser persicus) and Beluga (Huso huso) gynogen / triploids and also to determine the most appropriate type of thermal shock and the duration of induced shock after fertilization. Persian sturgeon and Beluga spawners were collected from Guilan's sturgeon catch stations and transported to the Shahid Beheshti sturgeon hatchery for artificial breeding and restocking programs. Ovulated eggs and sperms were collected based on common procedures in hatcheries. In order to separate the seminal fluids and dilute the milts, sperms were centrifuged at 6000rpm for 20 min. and seminal fluids stored in refrigerator for further use. Sperm motility was investigated. In order to determine the best duration for radiation, the milt was diluted (1:9) with immobilizing solution. Samples of diluted milt were placed for UV irradiation (UV lamp model UVG-54, 254 nm, made by UVP America) for 0.5, 1, 1.5, 1.45, 2, . to 5 min. The motility of radiated sperms and controls were examined under the light microscope and the motility curve was drawn. For application of thermal shock two types of heat shock (32, 34 and 37°C) and cold shock (0±1°C) were used for duration of 2.5 and 60 min respectively. Both thermal shock were applied at 12, 15, 18 min after fertilization. Four experimental groups were designed including; normal eggs as control group and sperms without UV thermal shock), gynogenesis (Sperm irradiated with UV and thermal shock were applied), triploid (thermal shock without radiation by UV on sperm) and haploid group (without thermal shock but using irradiated sperm for fertilization). Verification of the success of treatments was assessed using genetic analysis on sturgeon larvae and fingerlings. In triploids the total surface area, volume of cells and nucleus as well as chromosome number were determined. To identify a gynogenetic larva, microsatellite markers were used to analysis specific loci by using primers designed for lake sturgeon. The results were analyzed using SPSS, Excell software. To determine the significant levels between various parameters and comparison between controls and various treatments, one way of Analysis of Variance (ANOVA) was used. Whenever the significant level was observed to determine its level a Duncan test were examined. Results of present study showed that the best duration for UV radiation on sperms of Beluga was 105-110 seconds. Average fertilization rate for control Beluga was 51%, while in heat shock group it was 2-5 % and in cold shock it was 44.6%. There was a significant difference in fertilization rate in cold shock group compared to heat shock group ( P 0.05), however no difference was observed between 32 and 34°C treatments. The average survival rate of larvae in control group was 51%, while in heat shock treatment (32 and 34° C) it was very low close to zero. However in cold shock treatment the results was better and hatching percentage of larvae was between 30 -35%. Triploid treatment showed better results than gynogenesis group. A minimum triploid larvae obtained from heat shock was zero but using cold shock, the maximum number of 170 specimen was harvested. There was no significant difference in the number of larvae obtained between 32 and 34° C treatments (P 0.05). Although some difference was observed on large and small axes, surface areas and volume of red blood cells but no significant differences were observed between control and triploid groups (P 0.05). In the meantime, the chromosome number in triploid beluga was (3N= 177 ±3) as compared to diploid 2N= 118 ±3, which indicated an extra set of chromosome (n=60) in triploid fish. Totally 26.6% of investigated fish was triploids. Microsatellite molecular markers clearly differentiate gynogenetic fish on the bases of allele inheritance of male and female parents, and were proven that this technique can clearly identify allelic inheritance of parents to offspring. In Persian sturgeon in compare to beluga a different results were observed. Heat shock (37°C) not present any positive results therefore has no application in induce gynogenesis on this species, also no significant difference was observed between 32 and 34 °C treatment. Cold shock showed better results, especially when duration of UV radiation was adjusted to 105 seconds. Molecular analysis using microsatellite marker positively proved the gynogenetic offspring by counting the allelic inheritance. However Persian sturgeon as a tetraploid species (2N=240) has its difficulty on scoring the banding patterns. We highly recommend disomic primers application for allelic inheritance on gynogene Persian sturgeon. Iranian Fisheries Science Research Institute Published

Genotype manipulation through the introduction of novel genes for improved yield, agronomic qualities and a larger gene pool informed an experimental cross between diploid Solanum melongena 'Golden' (2n = 2x = 24) and tetraploid S. scabrum 'Scabrum' (2n = 4x = 48). The F1 fruit contained eight seeds which had a 100% germination and a chromosome number 2n = 6x = 72. Surviving hybrids were closer to the diploid parent in many characters. Leaves were fairly lobed, sparsely hairy and were 13.5 x 8.6 cm in the hybrid compared to the hairy, deeply lobed, 14.8 x 10.6 cm leaves in the diploid and glabrous, entire and 11.4 x 10.6 cm leaves in the tetraploid parent. The inflorescence in the hybrid was a raceme as in the diploid parent but was umbellate in the tetraploid. Pollen viability was 38.2% in the hybrid but was 71% and 97.4% in the diploid and tetraploid parents, respectively. Fruit was seedless in the F2; it was round and red, containing 384 seeds, globose-shaped, yellow seeds in the diploid, and 67, round, purple seeds in the tetraploid parent. Meiosis was regular in the hybrid with few univalents and impaired bivalents due to dissimilar parental genomes. Mitotic chromosomes were asymmetrical with various sizes. Epistasis and negative gene interaction mechanisms were implicated in the hybrids' low quality and breakdown. Backcross to the tetraploid parent may bring about gene recombination and allelic realignment for desirable phenotypes in the F2 and subsequent generations. Endoduplication of the triploid zygote might have produced an autoallohexaploid hybrid., La manipulación genotípica permite la introducción de genes nuevos para la mejora de la producción y otras cualidades agronómicas. Se llevó a cabo un cruce experimental entre Solanum melongena 'Golden' (diploide, 2n = 2x = 24) y S. scabrum 'Scabrum' (tetraploide, 2n = 4x = 48). Los frutos de la F1 contenían ocho semillas con un 100% de germinación y un número de cromosomas 2n = 6x = 72. Los híbridos que sobrevivieron estaban más próximos, en muchos caracteres, al parental diploide. Las hojas del híbrido eran de 13,5 x 8,6 cm, algo lobuladas y tenían escasos pelos, mientras que las del diploide eran de 14,8 x 10,6 cm, con pelos y profundamente lobuladas, y las del parental tetraploide de 11,4 x 10,6 cm, sin pelos y sin lobular. Las inflorescencias del híbrido y del diploide eran en racimo, mientras que en el tetraploide eran umbeladas. La viabilidad del polen fue del 38,2%, 71% y 97,4% en el híbrido, diploide y tetraploide, respectivamente. El fruto de la F2 no tenía semilla, era redondeado y rojizo; mientras que en el parental diploide contenía 384 semillas, era alargado y amarillo, y en el tetraploide tenía 67 semillas, y era redondeado y púrpura. La meiosis en el híbrido fue regular, con algunos cromosomas sin aparear. Los cromosomas mitóticos fueron asimétricos y de varios tamaños. La baja calidad y fallo de los híbridos se explican por la epistasis y los mecanismos de interacción génica negativos. El retrocruzamiento con el parental tetraploide puede dar una recombinación génica y realineamiento de los alelos para los fenotipos deseables en la F2 y generaciones siguientes. La endoduplicación del cigoto del tetraploide podría haber producido un híbrido autoalohexaploide.

Genotype manipulation through the introduction of novel genes for improved yield, agronomic qualities and a larger gene pool informed an experimental cross between diploid Solanum melongena 'Golden' (2n = 2x = 24) and tetraploid S. scabrum 'Scabrum' (2n = 4x = 48). The F1 fruit contained eight seeds which had a 100% germination and a chromosome number 2n = 6x = 72. Surviving hybrids were closer to the diploid parent in many characters. Leaves were fairly lobed, sparsely hairy and were 13.5 x 8.6 cm in the hybrid compared to the hairy, deeply lobed, 14.8 x 10.6 cm leaves in the diploid and glabrous, entire and 11.4 x 10.6 cm leaves in the tetraploid parent. The inflorescence in the hybrid was a raceme as in the diploid parent but was umbellate in the tetraploid. Pollen viability was 38.2% in the hybrid but was 71% and 97.4% in the diploid and tetraploid parents, respectively. Fruit was seedless in the F2; it was round and red, containing 384 seeds, globose-shaped, yellow seeds in the diploid, and 67, round, purple seeds in the tetraploid parent. Meiosis was regular in the hybrid with few univalents and impaired bivalents due to dissimilar parental genomes. Mitotic chromosomes were asymmetrical with various sizes. Epistasis and negative gene interaction mechanisms were implicated in the hybrids' low quality and breakdown. Backcross to the tetraploid parent may bring about gene recombination and allelic realignment for desirable phenotypes in the F2 and subsequent generations. Endoduplication of the triploid zygote might have produced an autoallohexaploid hybrid. La manipulación genotípica permite la introducción de genes nuevos para la mejora de la producción y otras cualidades agronómicas. Se llevó a cabo un cruce experimental entre Solanum melongena 'Golden' (diploide, 2n = 2x = 24) y S. scabrum 'Scabrum' (tetraploide, 2n = 4x = 48). Los frutos de la F1 contenían ocho semillas con un 100% de germinación y un número de cromosomas 2n = 6x = 72. Los híbridos que sobrevivieron estaban más próximos, en muchos caracteres, al parental diploide. Las hojas del híbrido eran de 13,5 x 8,6 cm, algo lobuladas y tenían escasos pelos, mientras que las del diploide eran de 14,8 x 10,6 cm, con pelos y profundamente lobuladas, y las del parental tetraploide de 11,4 x 10,6 cm, sin pelos y sin lobular. Las inflorescencias del híbrido y del diploide eran en racimo, mientras que en el tetraploide eran umbeladas. La viabilidad del polen fue del 38,2%, 71% y 97,4% en el híbrido, diploide y tetraploide, respectivamente. El fruto de la F2 no tenía semilla, era redondeado y rojizo; mientras que en el parental diploide contenía 384 semillas, era alargado y amarillo, y en el tetraploide tenía 67 semillas, y era redondeado y púrpura. La meiosis en el híbrido fue regular, con algunos cromosomas sin aparear. Los cromosomas mitóticos fueron asimétricos y de varios tamaños. La baja calidad y fallo de los híbridos se explican por la epistasis y los mecanismos de interacción génica negativos. El retrocruzamiento con el parental tetraploide puede dar una recombinación génica y realineamiento de los alelos para los fenotipos deseables en la F2 y generaciones siguientes. La endoduplicación del cigoto del tetraploide podría haber producido un híbrido autoalohexaploide.

An experiment was conducted to induce triploidy in African catfish, Clarias gariepinus, using heat shock and cold shock techniques. Cold shock at a temperature of 0± 1°C and 5±1°C for a duration of 15, 30, 45 and 60 min and heat shock at a temperature of 40±0.5°C and 41 ±OS C for a duration of 1, 2 and 3 min was given to induce triploidy 5 min after fertilization. Maximum percentage of triploids (91.4%) were obtained in the heat shock at a temperature of 40±0SC for a duration of 1 min whereas cold shock at 0± 1 C for a duration of 60 min yielded 90% of triploids. Chromosome analysis revealed that diploids have 54 chromosomes and triploids have 81 chromosomes. The erythrocyte measurements of the minor axis and major axis were 1.17 times larger in treated fish than in controls. The growth studies showed that the growth rate was not significantly affected in triploids.