Зміна експресії гена Wi у поколіннях амфідиплоїдів Triticinae: дисертація на здобуття наукового ступеня кандидата біологічних наук (доктора філософії)
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Date
2018
Authors
Шпильчин, Віталій
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Дисертація на здобуття наукового ступеня кандидата біологічних наук (доктора філософії) за спеціальністю 03.00.15 "Генетика". Дисертаційне дослідження присвячене виявленню причини перманентної зміни прояву ознаки наявність/відсутність воскової осуги у пшеничних амфідиплоїдів, інтрогресивних ліній та гібридів, що від них походять.
Особливості успадкування ознаки вивчалися на геномно-заміщених амфідиплоїдах Авродес (ААBBSS), Аврозис (ААBBSshSsh), Авролата (ААBBUU) та Авротіка (ААВВТТ) та геномно-доданих амфідиплоїдах Міоза (ААВВММ) та МІТ (ААВВDD). Відомо, що наявність воскової осуги є рецесивною ознакою. Однак в F1 від схрещування контрастних батьківських форм майже всі рослини мали воскову осугу або демонстрували третій фенотип – зелений колос, листя вкриті восковою осугою. Враховуючі схему походження рослинного матеріалу, причиною цього слід вважати певну зміну в геномі, що відбувається до утворення рослиною гамет, а також те, що рослини з домінантним фенотипом можуть бути гетерозиготами за домінантним алелем.
Рослинний матеріал був проаналізований за деякими білками, що контролюються генами відомої хромосомної локалізації. Білкові спектри контрастних за восковою осугою рослин виявилися мономорфними за глютенінами, альфа-амілазою, бета-амілазою та гліадинами. Виключенням стали контрастні зразки Авротіки, які виявилися поліморфними в ω-зоні гліадинів. Для проведення генетичного аналізу Авротіки були запропоновані гаплотипи за генами інтересу. Мікросателітний аналіз був проведений з застосуванням праймерів до мікросателітних локусів, специфічних до 2D та 2В хромосом з метою зрозуміти, у якій з вказаних хромосом змінюється експресія гена серії Iw в штучних гексаплоидах, в результаті якої відбувається зміна доминентного фенотипу рослин (зеленый) на рецесивний (блакитні). Для контрастних форм Авротіки і геномно-доданого амфідиплоїда Міоза було показано поліморфізм за мікрсателітними локусами, локалізованими як на короткому плечі 2D хромосоми, так і на обох плечах хромосоми 2В. Популяція, що розщеплюється за ознакою інтересу [Авротіка 1(зелена) х Авротіка 2(блакитна)]F2, була піддана мікросателітному аналізу з застосуванням праймерів до коротких плечей 2B та 2D хромосом. Для локуса Xgwm702 була показана відсутність незалежного успадкування щодо гена Iw2(T). Був проведений аналіз за допомогою маркерних систем IRAP та REMAP. З IRAP-праймерами пари Nikita/Nikita були отримані поліморфні продукти для зразків Авротіка 1 без воскової осуги та Авротіка 2 з восковою осугою. Пара Sukkula/Sukkula ампліфікувала поліморфні продукти з ДНК контрастних морфотипів Міози. Для контрастних форм Авротіки був встановлений поліморфізм за модифікованим методом REMAP, що вказує на наявність перебудов, пов’язаних з активністю мобільних генетичних елементів поблизу гена Iw2(Т), що може бути причиною втрати домінантного стану даного гена.
A dissertation submitted to acquire the degree of Doctor of Philosophy (PhD) in Biology, speciality 03.00.15 – genetics. – Institute of Cell Biology and Genetic Engineering of National Academy of Sciences of Ukraine, Kyiv, 2018. The work on dissertation was conducted in the National University of "Kyiv-Mohyla Academy" Ministry of Education and Science of Ukraine. The dissertation is devoted to the discovery of the phenomena of permanent change in a manifestation of a glaucousness trait in wheat amphidiploids, introgressive lines and hybrids derived from them. The variability of the trait is observed among artificial genome substituted, genome added hexaploids, and introgressive lines. Variability in its essence is illegitimate, as it was found among the populations of theoretically homozygous plants, permanent, because glaucous plants unexpectedly appeared constantly in each subsequent generation and irreversibly, since plants with a changed phenotype in subsequent generations never returned to the original phenotype. Unexpected variability of this trait is the result of processes occurring in natural and artificial amphidiploids during the formation of their genomes. The inheritance of the trait was studied on the genome substituted lines Aurodes (AABBSS), Aurosis (AABBSshSsh), Aurolata (AABBUU) and Aurotica (AABBTT) and genome added amphidiploids Miosa (AAВBMM) and MIT (AAВВDD). Although the glaucousness is a recessive trait, in the F1 from contrast parent phenotypes crossing, almost all plants were glaucous or had non-glaucous spike and glaucous leaves. Taking in account the origin of the plant material, we consider that the reason for this may lie in certain changes that occur in the plant genome before gametogenesis. Moreover, it should be noted that plants with a dominant phenotype can be heterozygous for dominant allele. Plant material was analyzed with protein-based molecular markers that have known chromosome location. Protein electrophoretic spectra of glaucous and non-glaucous plants were monomorphic by glutenins, alpha-amylase, beta-amylase and gliadins. Only glaucous and non-glaucous Aurotica samples were polymorphic in the ω-zone of gliadins. A microsatellite analysis with primers to SSR loci specific to the 2D chromosome was performed to determine changes in the expression of the gene located the 2nd homeologous group chromosomes from the 3rd subgenome of the corresponding artificial hexaploids. The amplification products in denaturing conditions were identified for introgressive lines for three SSR loci: Xcfd51, Xcfd56, and Xbarc159. The microsatellite analysis of glaucous and non-glaucous Aurotica plants and genome added amphidiploid Miosa showed polymorphism for loci located on the short arm of chromosome 2D and for the loci on the short and the long arms of chromosome 2B respectively. For the genetic analysis of amphidiploid Aurotica 5 contrast on glaucousness trait samples were used: Aurotica 1 (non-glaucous), Aurotica 1 (intermediate phenotype), Aurotica 2 (non-glaucous), Aurotica 2 (intermediate phenotype), and Aurotica 2 (glaucous). Based on our knowledge about the origin of amphidiploids and the genetic control of this trait, haplotypes were suggested for the genes of interest. The initial amphidipiloid (non-glaucous) had the following haplotype – iw3 W1 iw1 Iw2 (T) Iw3 (T), an intermediate form that differs from the initial one for one gene – iw3 W1 iw1 iw2 (T) Iw3 (T), a haplotype of the glaucous form – iw3 W1 iw1 iw2 (T) iw3 (T) differs from the initial haplotype by two genes. Segregating population [Aurotica 1 (non-glaucous) x Aurotica 2 (glaucous)] F2 was subjected to microsatellite analysis using SSR primers specific to the short arms of 2B and 2D chromosomes. For the Xgwm702 locus no independent inheritance with the Iw2(T) gene was shown, hence, they may be linked on the 2T chromosome. Contrasting on glaucousness trait hybrid plants have been analyzed using IRAP and REMAP markers. From the IRAP primers of the Nikita/Nikita pair, polymorphic products were obtained for DNA samples of Aurotica 1 (non-glaucous) and Aurotica 2 (glaucous). With the Sukkula/Sukkula pair polymorphic DNA products were produced of contrasting Miosa morphotypes. In almost all cases, the electrophoretic spectra of amphidiploids differed from the Aurora spectra. For a more specific analysis of contrast forms (Aurotica 2 (glaucous) and some F4 lines) + (Aurotica 1 non-glaucous and some F4 lines), the modified variant of REMAP method was used when one of the primers was a specific microsatellite locus. Almost all combinations produced monomeric products, except for the combination Xgwm702F/REMAPCAn. Amplification with this pair of primers produced an additional component in an electrophoretic spectrum of DNA of glaucous hybrid lines. According to our data, the Xgwm702 locus is linked to the Iw2(T) gene and amplification of polymorphic products with the primer to LTR of retrotransposon indicates alterations caused by the activity of mobile genetic elements near the Iw2(T) gene. Such activity can affect expression of Iw2(T) gene resulting in a permanent changes in the manifestation of glaucousness trait in wheat amphidiploids.
A dissertation submitted to acquire the degree of Doctor of Philosophy (PhD) in Biology, speciality 03.00.15 – genetics. – Institute of Cell Biology and Genetic Engineering of National Academy of Sciences of Ukraine, Kyiv, 2018. The work on dissertation was conducted in the National University of "Kyiv-Mohyla Academy" Ministry of Education and Science of Ukraine. The dissertation is devoted to the discovery of the phenomena of permanent change in a manifestation of a glaucousness trait in wheat amphidiploids, introgressive lines and hybrids derived from them. The variability of the trait is observed among artificial genome substituted, genome added hexaploids, and introgressive lines. Variability in its essence is illegitimate, as it was found among the populations of theoretically homozygous plants, permanent, because glaucous plants unexpectedly appeared constantly in each subsequent generation and irreversibly, since plants with a changed phenotype in subsequent generations never returned to the original phenotype. Unexpected variability of this trait is the result of processes occurring in natural and artificial amphidiploids during the formation of their genomes. The inheritance of the trait was studied on the genome substituted lines Aurodes (AABBSS), Aurosis (AABBSshSsh), Aurolata (AABBUU) and Aurotica (AABBTT) and genome added amphidiploids Miosa (AAВBMM) and MIT (AAВВDD). Although the glaucousness is a recessive trait, in the F1 from contrast parent phenotypes crossing, almost all plants were glaucous or had non-glaucous spike and glaucous leaves. Taking in account the origin of the plant material, we consider that the reason for this may lie in certain changes that occur in the plant genome before gametogenesis. Moreover, it should be noted that plants with a dominant phenotype can be heterozygous for dominant allele. Plant material was analyzed with protein-based molecular markers that have known chromosome location. Protein electrophoretic spectra of glaucous and non-glaucous plants were monomorphic by glutenins, alpha-amylase, beta-amylase and gliadins. Only glaucous and non-glaucous Aurotica samples were polymorphic in the ω-zone of gliadins. A microsatellite analysis with primers to SSR loci specific to the 2D chromosome was performed to determine changes in the expression of the gene located the 2nd homeologous group chromosomes from the 3rd subgenome of the corresponding artificial hexaploids. The amplification products in denaturing conditions were identified for introgressive lines for three SSR loci: Xcfd51, Xcfd56, and Xbarc159. The microsatellite analysis of glaucous and non-glaucous Aurotica plants and genome added amphidiploid Miosa showed polymorphism for loci located on the short arm of chromosome 2D and for the loci on the short and the long arms of chromosome 2B respectively. For the genetic analysis of amphidiploid Aurotica 5 contrast on glaucousness trait samples were used: Aurotica 1 (non-glaucous), Aurotica 1 (intermediate phenotype), Aurotica 2 (non-glaucous), Aurotica 2 (intermediate phenotype), and Aurotica 2 (glaucous). Based on our knowledge about the origin of amphidiploids and the genetic control of this trait, haplotypes were suggested for the genes of interest. The initial amphidipiloid (non-glaucous) had the following haplotype – iw3 W1 iw1 Iw2 (T) Iw3 (T), an intermediate form that differs from the initial one for one gene – iw3 W1 iw1 iw2 (T) Iw3 (T), a haplotype of the glaucous form – iw3 W1 iw1 iw2 (T) iw3 (T) differs from the initial haplotype by two genes. Segregating population [Aurotica 1 (non-glaucous) x Aurotica 2 (glaucous)] F2 was subjected to microsatellite analysis using SSR primers specific to the short arms of 2B and 2D chromosomes. For the Xgwm702 locus no independent inheritance with the Iw2(T) gene was shown, hence, they may be linked on the 2T chromosome. Contrasting on glaucousness trait hybrid plants have been analyzed using IRAP and REMAP markers. From the IRAP primers of the Nikita/Nikita pair, polymorphic products were obtained for DNA samples of Aurotica 1 (non-glaucous) and Aurotica 2 (glaucous). With the Sukkula/Sukkula pair polymorphic DNA products were produced of contrasting Miosa morphotypes. In almost all cases, the electrophoretic spectra of amphidiploids differed from the Aurora spectra. For a more specific analysis of contrast forms (Aurotica 2 (glaucous) and some F4 lines) + (Aurotica 1 non-glaucous and some F4 lines), the modified variant of REMAP method was used when one of the primers was a specific microsatellite locus. Almost all combinations produced monomeric products, except for the combination Xgwm702F/REMAPCAn. Amplification with this pair of primers produced an additional component in an electrophoretic spectrum of DNA of glaucous hybrid lines. According to our data, the Xgwm702 locus is linked to the Iw2(T) gene and amplification of polymorphic products with the primer to LTR of retrotransposon indicates alterations caused by the activity of mobile genetic elements near the Iw2(T) gene. Such activity can affect expression of Iw2(T) gene resulting in a permanent changes in the manifestation of glaucousness trait in wheat amphidiploids.
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Keywords
воскова осуга, геномно-заміщені амфідиплоїди, геномнододані амфідиплоїди, мікростателітні локуси, IRAP, REMAP, мінливість, дисертація, glaucousness, genome substitution amphidiploid, genome addition amphidiploids, microsatellites, variability, thesis