vavilovВавиловский журнал генетики и селекцииVavilov Journal of Genetics and Breeding2500-3259Institute of Cytology and Genetics of Siberian Branch of the RAS10.18699/VJ20.657vavilov-2779Research ArticleМОЛЕКУЛЯРНАЯ И КЛЕТОЧНАЯ БИОЛОГИЯMOLECULAR AND CELL BIOLOGYРайоны, обогащенные повторенными последовательностями ДНК, в хромосомах Macrostomum mirumnovem – вида, недавно прошедшего полногеномную дупликациюRegions enriched for DNA repeats in chromosomes of Macrostomum mirumnovem, a species with a recent Whole Genome DuplicationЗадесенецК. С.ZadesenetsK. S.

Новосибирск

Novosibirsk

РубцовН. Б.RubtsovN. B.

Новосибирск

Novosibirsk

rubt@bionet.nsc.ruФедеральный исследовательский центр Институт цитологии и генетики Сибирского отделения Российской академии наукРоссияInstitute of Cytology and Genetics of Siberian Branch of the Russian Academy of SciencesRussian Federation202027102020246636642Copyright © Задесенец К.С., Рубцов Н.Б., 20202020Задесенец К.С., Рубцов Н.Б.Zadesenets K.S., Rubtsov N.B.This work is licensed under a Creative Commons Attribution 4.0 License.https://vavilov.elpub.ru/jour/article/view/2779

Свободноживущий плоский червь Macrostomum mirumnovem – неополиплоидный вид, его геном претерпел недавнюю полногеномную дупликацию (Whole Genome Duplication, WGD). В результате слияния гаплоидного хромосомного набора в его кариотипе произошло формирование двух новых крупных хромосом, MMI1 и MMI2. Создание микродиссекционных ДНК-библиотек, обогащенных повторенными последовательностями ДНК, и их последующая гибридизация in situ с метафазными хромосомами M. Mirumnovem выявили в этих хромосомах районы, обогащенные повторенными последовательностями ДНК. Разные ДНКпробы устанавливали в хромосомах M. mirumnovem районы, обогащенные разными повторенными последовательностями. Локализация и размер этих районов варьировали в разных копиях крупных хромосом, это предполагало их дивергенцию и снижение уровня гомологии, что может после полной дупликации генома приводить к его редиплоидизации. Помимо возникших de novo районов хромосом основного набора, обогащенных повторенными последовательностями, в кариотипе у большинства исследованных особей обнаружены В-хромосомы, которые варьировали по размеру и морфологии. Различия в составе ДНК у этих В-хромосом были показаны с помощью флуоресцентной гибридизации in situ (FISH) с полученными микродиссекционными ДНК-пробами на хромосомном материале, взятом от разных животных. Флуоресцентная гибридизация in situ этих ДНК-проб по-разному окрашивала В-хромосомы, содержащиеся в кариотипах у разных особей M. mirumnovem. Часть В-хромосом интенсивно окрашивалась при проведении FISH, тогда как на других В-хромосомах гибридизационных сигналов не было. Специфический FISH-сигнал отсутствовал даже в прицентромерных районах таких В-хромосом. В настоящей статье обсуждаются возможные механизмы возникновения и последующей эволюции В-хромосом у M. mirumnovem. Полученные результаты указывают на важную роль повторенных последовательностей, которую они могут играть в процессе реорганизации генома, приводя к быстрой дифференциации дуплицированных копий хромосом. Высокий уровень внутривидового кариотипического разнообразия по численным и структурным хромосомным перестройкам и по формированию новых хромосомных районов, обогащенных повторенными последовательностями, а также небольшой размер тела (~2 мм) и простота поддержания лабораторных культур M. mirumnovem делают этот вид перспективной моделью в исследованиях геномной и кариотипической эволюции видов, недавно прошедших полногеномную дупликацию.

The free-living flatworm Macrostomum mirumnovem is a neopolyploid species whose genome underwent a recent Whole Genome Duplication (WGD). In the result of chromosome fusions of the ancient haploid chromosome set, large metacentric chromosomes were formed. In addition to three pairs of small metacentrics, the current karyotype of M. mirumnovem contains two pairs of large metacentric chromosomes, MMI1 and MMI2. The generation of microdissected DNA libraries enriched for DNA repeats followed by DNA probe preparation and fluorescent in situ hybridization (FISH) were performed. The DNA probes obtained marked chromosome regions enriched for different DNA repeats in the M. mirumnovem chromosomes. The size and localization of these regions varied in different copies of large chromosomes. They varied even in homologous chromosomes, suggesting their divergence due to genome re-diploidization after a WGD. Besides the newly formed chromosome regions enriched for DNA repeats, B chromosomes were found in the karyotypes of the studied specimens of M. mirumnovem. These B chromosomes varied in size and morphology. FISH with microdissected DNA probes revealed that some Bs had a distinct DNA content. FISH could paint differently B chromosomes in different worms and even in the same sample. B chromosomes could carry a bright specific fluorescent signal or could show no fluorescent signal at all. In latter cases, the specific FISH signal could be absent even in the pericentromeric region of the B chromosome. Possible mechanisms of B chromosome formation and their further evolution are discussed. The results obtained indicate an important role that repetitive DNAs play in genome re-diploidization initiating a rapid differentiation of large chromosome copies. Taking together, karyotype peculiarities (a high level of intraspecific karyotypic diversity associated with chromosome number variation, structural chromosomal rearrangements, and the formation of new regions enriched for DNA repeats) and some phenotypic features of M. mirumnovem (small body size, short lifecycle, easy maintenance in the laboratory) make this species a perspective model in the studies of genomic and karyotypic evolution in species passed through a recent WGD event.

микродиссекция метафазных хромосомДНК-пробыповторенные последовательности ДНКтранспозиция мобильных элементовFISHамплификация ДНКВ-хромосомыmetaphase chromosome microdissectionDNA probesrepetitive DNAmobile element transpositionFISHDNA amplificationB chromosomesThis study was supported by the Russian Science Foundation under the grant 19-14-00211. We are thankful to Professor Lukas Schärer for providing specimens of M. mirumnovemReferencesBarker M.S., Husband B.C., Pires J.C. Spreading winge and flying high: the evolutionary importance of polyploidy after a century of study. Am. J. Bot. 2016;103(7):1­7. DOI 10.3732/ajb.1600272.Barker M.S., Husband B.C., Pires J.C. Spreading winge and flying high: the evolutionary importance of polyploidy after a century of study. Am. J. Bot. 2016;103(7):1­7. DOI 10.3732/ajb.1600272.Bugrov A.G., Karamysheva T.V., Perepelov E.A., Elisaphenko E.A., Rubtsov D.N., Warchalowska-Sliwa E., Tatsuta H., Rubtsov N.B. DNA content of the B chromosomes in grasshopper Podisma kanoi Storozh. (Orthoptera, Acrididae). Chromosome Res. 2007;15(3): 315­326. DOI 10.1007/s10577­007­1128­z.Bugrov A.G., Karamysheva T.V., Perepelov E.A., Elisaphenko E.A., Rubtsov D.N., Warchalowska-Sliwa E., Tatsuta H., Rubtsov N.B. DNA content of the B chromosomes in grasshopper Podisma kanoi Storozh. (Orthoptera, Acrididae). Chromosome Res. 2007;15(3): 315­326. DOI 10.1007/s10577­007­1128­z.Comparative Genomics. Sankoff D., Nadeau J.H. (Eds.). Kluwer Academic Publ., 2000. DOI 10.1007/978­94­011­4309­7.Comparative Genomics. Sankoff D., Nadeau J.H. (Eds.). Kluwer Academic Publ., 2000. DOI 10.1007/978­94­011­4309­7.Dehal P., Boore J.L. Two rounds of whole genome duplication in the ancestral vertebrate. PLoS Biol. 2005;3:e314. DOI 10.1371/journal.pbio.0030314.Dehal P., Boore J.L. Two rounds of whole genome duplication in the ancestral vertebrate. PLoS Biol. 2005;3:e314. DOI 10.1371/journal.pbio.0030314.Egger B., Ishida S. Chromosome fission or duplication in Macrostomum lignano (Macrostomorpha, Plathelminthes) – remarks on chromosome numbers in ‘archoophoran turbellarians’. J. Zool. Syst. Evol. Res. 2005;43(2):127-132. DOI 10.1111/j.1439­0469.2005.00300.x.Egger B., Ishida S. Chromosome fission or duplication in Macrostomum lignano (Macrostomorpha, Plathelminthes) – remarks on chromosome numbers in ‘archoophoran turbellarians’. J. Zool. Syst. Evol. Res. 2005;43(2):127-132. DOI 10.1111/j.1439­0469.2005.00300.x.Fisher K.J., Buskirk S.W., Vignogna R.C., Marad D.A., Lang G.I. Adaptive genome duplication affects patterns of molecular evolution in Saccharomyces cerevisiae. PLoS Genet. 2018;14(5):e1007396. https//doi.org/10.1371/journal.pgen.1007396.Fisher K.J., Buskirk S.W., Vignogna R.C., Marad D.A., Lang G.I. Adaptive genome duplication affects patterns of molecular evolution in Saccharomyces cerevisiae. PLoS Genet. 2018;14(5):e1007396. https//doi.org/10.1371/journal.pgen.1007396.Glasauer S.M.K., Neuhauss S.C.F. Whole-genome duplication in teleost fishes and its evolutionary consequences. Mol. Genet. Genomics. 2014;289(6):1045­1060. DOI 10.1007/s00438­014­0889­2.Glasauer S.M.K., Neuhauss S.C.F. Whole-genome duplication in teleost fishes and its evolutionary consequences. Mol. Genet. Genomics. 2014;289(6):1045­1060. DOI 10.1007/s00438­014­0889­2.Kenny N.J., Chan K.W., Nong W., Qu Z., Maeso I., Yip H.Y., Chan T.F., Kwan H.S., Holland P.W.H., Chu K.H., Hui J.H.L. Ancestral wholegenome duplication in the marine chelicerate horseshoe crabs. Heredity. 2018;116(2):190­199. DOI 10.1038/hdy.2015.89.Kenny N.J., Chan K.W., Nong W., Qu Z., Maeso I., Yip H.Y., Chan T.F., Kwan H.S., Holland P.W.H., Chu K.H., Hui J.H.L. Ancestral wholegenome duplication in the marine chelicerate horseshoe crabs. Heredity. 2018;116(2):190­199. DOI 10.1038/hdy.2015.89.Makunin A.I., Rajičić M., Karamysheva T.V., Romanenko S.A., Druzhkova A.S., Blagojević J., Vujošević M., Rubtsov N.B., Graphodatsky A.S., Trifonov V.A. Low-pass single-chromosome sequencing of human small supernumerary marker chromosomes (sSMCs) and Apodemus B chromosomes. Chromosoma. 2018;127(3):301-311. DOI 10.1007/s00412­018­0662­0.Makunin A.I., Rajičić M., Karamysheva T.V., Romanenko S.A., Druzhkova A.S., Blagojević J., Vujošević M., Rubtsov N.B., Graphodatsky A.S., Trifonov V.A. Low-pass single-chromosome sequencing of human small supernumerary marker chromosomes (sSMCs) and Apodemus B chromosomes. Chromosoma. 2018;127(3):301-311. DOI 10.1007/s00412­018­0662­0.Mayrose I., Zhan S.H., Rothfels C.J., Magnuson-Ford K., Barker M.S., Rieseberg L.H., Otto S.P. Recently formed polyploid plants diversify at lower rates. Science. 2011;60(333):1257. DOI 10.1126/science.1207205.Mayrose I., Zhan S.H., Rothfels C.J., Magnuson-Ford K., Barker M.S., Rieseberg L.H., Otto S.P. Recently formed polyploid plants diversify at lower rates. Science. 2011;60(333):1257. DOI 10.1126/science.1207205.Moghe G.D., Hufnagel D.E., Tang H., Xiao Y., Dworkin I., Town C.T., Conner J.K., Shiu S.-H. Consequences of whole-genome triplication as revealed by comparative genomic analyses of the wild radish Raphanus raphanistrum and three other Brassicaceae species. Plant Cell. 2014;26(5):1925­1937. DOI 10.1105/tpc.114.124297.Moghe G.D., Hufnagel D.E., Tang H., Xiao Y., Dworkin I., Town C.T., Conner J.K., Shiu S.-H. Consequences of whole-genome triplication as revealed by comparative genomic analyses of the wild radish Raphanus raphanistrum and three other Brassicaceae species. Plant Cell. 2014;26(5):1925­1937. DOI 10.1105/tpc.114.124297.Panopoulou G., Hennig S., Groth D., Krause A., Poustka A.J., Herwig R., Vingron M., Lehrach H. New evidence for genome-wide duplications at the origin of vertebrates using an amphioxus gene set and completed animal genomes. Genome Res. 2003;13:1056-1066. DOI 10.1101/gr.874803.Panopoulou G., Hennig S., Groth D., Krause A., Poustka A.J., Herwig R., Vingron M., Lehrach H. New evidence for genome-wide duplications at the origin of vertebrates using an amphioxus gene set and completed animal genomes. Genome Res. 2003;13:1056-1066. DOI 10.1101/gr.874803.Schärer L., Brand J.N., Singh P., Zadesenets K.S., Stelzer C.­P., Viktorin G. A phylogenetically informed search for an alternative Macrostomum model species with notes on taxonomy, mating behavior, karyology, and genome size. J. Zool. Syst. Evol. Res. 2020;58:41-65. DOI 10.1111/jzs.12344.Schärer L., Brand J.N., Singh P., Zadesenets K.S., Stelzer C.­P., Viktorin G. A phylogenetically informed search for an alternative Macrostomum model species with notes on taxonomy, mating behavior, karyology, and genome size. J. Zool. Syst. Evol. Res. 2020;58:41-65. DOI 10.1111/jzs.12344.Soltis D.E., Segovia-Salcedo M.C., Jordon-Thaden I., Majure L.C., Miles N.M., Mavrodiev E.V., Mei W., Cortez M.B., Soltis P.S., Gitzendanner M.A. Are polyploids really evolutionary dead-ends (again)? A critical reappraisal of Mayrose et al. New Phytol. 2014; 202(4):1105­1117. DOI 10.1111/nph.12756.Soltis D.E., Segovia-Salcedo M.C., Jordon-Thaden I., Majure L.C., Miles N.M., Mavrodiev E.V., Mei W., Cortez M.B., Soltis P.S., Gitzendanner M.A. Are polyploids really evolutionary dead-ends (again)? A critical reappraisal of Mayrose et al. New Phytol. 2014; 202(4):1105­1117. DOI 10.1111/nph.12756.Wendel J.F. Genome evolution in polyploids. Plant Mol. Biol. 2000;42: 225­249. DOI 10.1023/A:1006392424384.Wendel J.F. Genome evolution in polyploids. Plant Mol. Biol. 2000;42: 225­249. DOI 10.1023/A:1006392424384.Zadesenets K.S., Ershov N.I., Berezikov E., Rubtsov N.B. Chromosome evolution in the free­living flatworms: first evidence of intrachromosomal rearrangements in karyotype evolution of Macrostomum lignano (Platyhelminthes, Macrostomida). Genes. 2017a;8:298. DOI 10.3390/genes8110298.Zadesenets K.S., Ershov N.I., Berezikov E., Rubtsov N.B. Chromosome evolution in the free­living flatworms: first evidence of intrachromosomal rearrangements in karyotype evolution of Macrostomum lignano (Platyhelminthes, Macrostomida). Genes. 2017a;8:298. DOI 10.3390/genes8110298.Zadesenets K.S., Ershov N.I., Rubtsov N.B. Whole-genome sequencing of eukaryotes: from sequencing of DNA fragments to a genome assembly. Russ. J. Genet. 2017b;53(6):631-639. DOI 10.1134/S102279541705012X.Zadesenets K.S., Ershov N.I., Rubtsov N.B. Whole-genome sequencing of eukaryotes: from sequencing of DNA fragments to a genome assembly. Russ. J. Genet. 2017b;53(6):631-639. DOI 10.1134/S102279541705012X.Zadesenets K.S., Jetybayev I.Y., Schärer L., Rubtsov N.B. Genome and karyotype reorganization after whole genome duplication in freeliving flatworms of the genus Macrostomum. Int. J. Mol. Sci. 2020; 21:680. DOI 10.3390/ijms21020680.Zadesenets K.S., Jetybayev I.Y., Schärer L., Rubtsov N.B. Genome and karyotype reorganization after whole genome duplication in freeliving flatworms of the genus Macrostomum. Int. J. Mol. Sci. 2020; 21:680. DOI 10.3390/ijms21020680.Zadesenets K.S., Rubtsov N.B. Genome duplication in animal evolution. Russ. J. Genet. 2018;54(10):1125-1136. DOI 10.1134/S1022795418090168.Zadesenets K.S., Rubtsov N.B. Genome duplication in animal evolution. Russ. J. Genet. 2018;54(10):1125-1136. DOI 10.1134/S1022795418090168.Zadesenets K.S., Rubtsov N.B. Generation of microdissected DNA probes from metaphase chromosomes in case of an impossibility of chromosomes identification by routine staining. Vavilovskii Zhurnal Genetiki i Selektsii = Vavilov Journal of Genetics and Breeding. 2020;24(5)519­524. DOI 10.18699/VJ20.46­o.Zadesenets K.S., Rubtsov N.B. Generation of microdissected DNA probes from metaphase chromosomes in case of an impossibility of chromosomes identification by routine staining. Vavilovskii Zhurnal Genetiki i Selektsii = Vavilov Journal of Genetics and Breeding. 2020;24(5)519­524. DOI 10.18699/VJ20.46­o.Zadesenets K.S., Schӓrer L., Rubtsov N.B. New insights into the karyotype evolution of the free­living flatworm Macrostomum lignano (Platyhelminthes, Turbellaria). Sci. Rep. 2017c;7:6066. DOI 10.1038/s41598­017­06498­0.Zadesenets K.S., Schӓrer L., Rubtsov N.B. New insights into the karyotype evolution of the free­living flatworm Macrostomum lignano (Platyhelminthes, Turbellaria). Sci. Rep. 2017c;7:6066. DOI 10.1038/s41598­017­06498­0.Zadesenets K.S., Vizoso D.B., Schlatter A., Konopatskaia I.D., Berezikov E., Schärer L., Rubtsov N.B. Evidence for karyotype polymorphism in the free­living flatworm, Macrostomum lignano, a model organism for evolutionary and developmental biology. PLoS One. 2016;11:e0164915. DOI 10.1371/journal.pone.0164915.Zadesenets K.S., Vizoso D.B., Schlatter A., Konopatskaia I.D., Berezikov E., Schärer L., Rubtsov N.B. Evidence for karyotype polymorphism in the free­living flatworm, Macrostomum lignano, a model organism for evolutionary and developmental biology. PLoS One. 2016;11:e0164915. DOI 10.1371/journal.pone.0164915.

The authors declare that there are no conflicts of interest present.