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COMMON PART


Project Number16-14-10009

Project titleThe evolution of some groups of vertebrates. Conventional and molecular cytogenetics and ancient DNA.

Project LeadGraphodatsky Alexander

AffiliationInstitute of Molecular and Cellular Biology, Siberian Branch of the Russian Academy of Sciences,

Implementation period2016 - 2018

Research area 04 - BIOLOGY AND LIFE SCIENCES, 04-104 - General genetics

Keywordsanimal genetics, evolution of genomes, chromosomes, selection, ancient DNA, comparative genomics


 

PROJECT CONTENT


Annotation
From the very outset, marked by the one of the world's first chairs of genetics that of Professor YA Filipchenko in 1919 at St. Petersburg University, the study of chromosomes has been decisive in the development of genetics in Russia and has became a trademark of Russian biology. Already in the twenties of the last century, during the civil war, on the initiative of Filipchenko several of his outstanding assistants, including the future evolutionist Theodosius G. Dobzhansky, were conducting expeditions to Central Asia with attempts to describe the chromosomes of yak, sheep and Bactrian camel. The Russian part of the team includes members that are the third and fourth generation of the school of Filipchenko and Dobrzhansky, which focused primarily on chromosomal studies. In 2006, the monograph - ATLAS of Mammalian Chromosomes (John Wiley and Sons) was published. This monograph in a way completed a 30-year period of research on mammalian cytogenetics. It pointed the way to the next step, based on the methods of molecular cytogenetics and genomics. This major book selected only the best karyotypes for 700 species obtained in from all leading laboratories around the world. Among those selected 122 karyotypes, including banded chromosomes of most species of domestic and laboratory animals, were obtained from our laboratory. No other laboratory even came close to matching such an incredible contribution. It was assumed that the submitted chromosome maps would become the basis for the implementation of full-scale genome projects for many species. The next stage of comparative cytogenetics was associated with the use of sorted chromosomes as molecular probes. The main method of work in this period was a comparative chromosome PAINTING or ZooFISH (Fengtang Yang, Alexander S. Graphodatsky Animal Probes and ZOO-FISH in T.Liehr (ed.) Fluorescence In Situ Hybridization (FISH) - Application Guide, Springer, 2009 P 323-347). The results of this long-term cycle studies were summarized in articles: Malcolm A Ferguson-Smith, Vladimir Trifonov Mammalian Karyotype Evolution. Nature Rev. Genetics, Vol 8, 2007 : 950-962 ; Alexander S. Graphodatsky, Vladimir A. Trifonov and Roscoe Stanyon The genome diversity and karyotype evolution of mammals Molecular Cytogenetics 2011 , 4:22 ; and a special issue Cytogenenetics & Genome Research 2012 ; 137:1-4 ), later published in book form (Stanyon, R.; Graphodatsky, A. (Ed's) Evolutionary Dynamics of Mammalian Karyotypes, Karger, Basel, Switzerland, 2012, 208 pp). In a series of papers were identified regions of homologous chromosomes (conserved syntenies) in more than 300 species of all orders of mammals, and the basic laws of evolutionary change of the genomes at the chromosomal level was discussed for all major taxa. Our group is part of a consortium of genome sequencing 10,000 vertebrate species (Genome 10K) since its foundation (Genome 10K Community of Scientists (David Haussler, Stephen J. O'Brien, ... Alexander S. Graphodatsky, ... Ya-Ping Zhang) A Proposal to Obtain Whole Genome Sequence for 10,000 Vertebrate Species The Journal of Heredity (2009); 100 (6): 659-74.) and participates in the development of methodological standards (Wong PBY, ... Perelman P, ... Murphy RW. Genome 10K community of scientists: Tissue sampling and standards for vertebrate genomics. GigaScience 1: 8, 2012) and a number of projects (Perelman P, ... .Pecon-Slattery J. A molecular phylogeny of living primates. PLoS Genet 7 (3): e1001342 , 2011; G. Tamazian, ... A. Makunin, ... SJ O'Brien, "Annotated features of domestic cat - Felis catus genome," GigaScience, vol. 3, no. 1, p. 13, Aug. 2014 .; Jarvis ED, ... Perelman P, ... Zhang G. Whole-genome analyses resolve early branches in the tree of life of modern birds. Science 346: 1320-1331, 2014). Currently close to completing a number of international projects for the sequencing of genomes (the red fox, the Baikal seal, gray whale, alpaca), in which we have participated since their resource provision, ie select animals, isolation of DNA and ensure their completion in terms of binding to chromosomes using mapping panels radiation hybrid cells and mapping BAC-clones. The resources of these projects already completed, ongoing now or planned for implementation in the near future will be used for the study of chromosome and genome evolution in a number of interesting groups of vertebrates, as first of all mammals. The basic methods - the new generation sequencing (NGS) and FISH on chromosomes, including sprawled, a huge number of BAC clones containing the genomic segment of interest, especially marking crucial evolutionary restructuring. Another important approach, the project will receive using microdissected samples of individual chromosomes and their regions and use them as sequencing and data binding NGS genomes of several species to the chromosomes, the study of such an important component of the genomes of both sex chromosomes, heterochromatin, centromeric regions and the extra chromosome, so and analysis intrachromosomal evolutionary rearrangements. However, in the course of these studies, we found out quite clearly that the chromosomal database rarely provided complete information on intrachromosomal rearrangements such as inversions and changing positions of centromeres. Obtaining data on the number and location of these major evolutionary transformations now seems absolutely relevant. It could be assumed that all of these problems will be resolved as we implement genome projects. However, even with the advent of modern methods of the new generation (NGS), when the full genome sequencing has become a routine procedure, genome assembly remains a huge problem and is now almost never comes to anchoring genomic data to chromosomes. Only physical mapping, cytogenetics, was able to document the new phenomena of Centromere Shifts also known as Evolutionary New Centromere. So, the last of more than 100 completed projects genome sequencing mammalian species do not contain any information about the linkage groups and chromosomes Evolution of unusual karyotypic elements - B chromsomes and sex chromosomes - is one of the most interesting and rapidly developing directions of modern cytogenetics. Development of new generation sequencing technologies allows to study sex chromosomes by direct sequencing of chromosome specific libraries. We possess a large collection of cell cultures, metaphase suspensions, and chromosome specific libraries of different vertebrate species, besides we established a collaboration and agreement about material donations with leading international laboratories in the UK, Italy, USA, Czech Republic, Spain, Netherlands and China. In our work we will apply programms of genomic data analysis elaborated in our laboratory. As part of the ideology of the project Genome 10K we began work with ancient DNA of different species of mammals Siberia. Naturally, work with "chromosomal" components of extinct animals is not possible. However, analysis of the DNA fragments species extinct Pleistocene megafauna as the "ancestors" of modern fauna, including the ancestors of domestic species is of great interest. Our data on DNA, "of fauna" of the famous Denisova Cave, a description of the mitochondrial genome of ancient "dogs" of the robber cave Altai and its comparison with the same genomes of ancient and modern dogs and wolves (Druzhkova et al., Ancient DNA analysis affirms the canid from Altai as a primitive dog. PLoS ONE 8(3): e57754, (2013). O.Thalmann,… A.Graphodatsky, A. Druzhkova,… Wayne RK., Complete mitochondrial genomes of ancient canines suggest a European origin of domestic dogs. Science, (2013) Vol. 342 no. 6160 pp. 871-874) have been very perspective, including for solving actual problems of the project proposed here, both in terms of studying the genomes of of extinct species Pleistocene and Holocene and the problems of domestication of species such as the dog and the horse, is largely determined the history of man.

Expected results
During the implementation of the present project, we plan to obtain the following results: 1. Building a saturated genetic markers of radiation hybrid maps for the two species of camelids (alpaca and one-humped camel ) by creating a radiation hybrid panel on the basis of the merger targeted cell lines with a line of Chinese hamster fibroblasts with thymidine kinase deficiency and mapping panel using high-performance methods of genotyping SNPs chip or sequencing . Camelids Group occupies a basal position in the unit cetartiodactyla, and is extremely important for understanding the evolution of the taxa and the whole group laurasiatheria. Mapping the genome camelid offers opportunities for modern genetic research and its applications for the identification and characterization of genetic abnormalities, phenotypic traits for breeding and selection. 2. Study intrachromosomal rearrangements, representatives of all the families of the order Cetartiodactyla, the detection of "hot spots" of the evolution of chromosomes common ancestor ruminants (Ruminantia). As objects of research to identify the evolutionary breakpoint chromosome selected: Indian muntjac (Muntiacus muntjak), which has the least number of chromosomes in mammals (2n = 6); Giraffe (Giraffa camelopardalis), as a representative of the basal family infraorder higher ruminants; and small mouse-deer (Tragulus javanicus), a representative of the basal family of the suborder of ruminants. Using bioinformatics analysis of selected ancestral ruminant evolutionary point of chromosome breaks - those areas that are at the beginning of the evolution of ruminants were severed and then reconnected in a different combination or orientation. To determine the anticipated fracture points 9 chromosomes to be used assemblies genomes ruminants available database of genomic sequencing (G10K). Presumably, using flanking region BAC-clones of cows, we localize at least 80 points rearrangements that are supposedly specific to ruminants, using the build seven ruminant and five non-ruminant species. To build a high-resolution comparative map of the X chromosome karyotypes cetartiodactyla be selected BAC clones of cows with the most conservative regions of the chromosomes at intervals of 1-2 Megabase and localized on the chromosomes of each species of the family unit. It is hoped that the identification of the structural changes in the genome, will link these changes with the morphological evolution of cloven-hoofed animals, such as the appearance of scars in ruminants (an important organ of great importance in agriculture). 3.With using microdissection libraries and localization of BAC-clones are identified intrachromosomal restructuring, including the establishment of mutual orientation of the segments and evolutionarily new centromere in the most actively evolving group of mammals - voles (Arvicolinae, Cricetidae, Rodentia). With the help of data analysis NGS (next-generation sequencing) chromosome-specific libraries will be identified in the genome of arctic lemming restructuring interchromosomal arctic lemming (Dicrostonyx torquatus, species with an unusual system of sex determination and accessory chromosomes) relative to other species of rodents. 4. Using sequencing data of microdissection and sorting derived chromosome specific libraries we will compare sex and B chromosoems of different vertebrate species and study the genomic segments responsible for sex determination. Especially big interest represent lizards, as this taxon contains different kinds of sex determination. Except reptile we will concentrate on sex chromosome sof fish as most representatives of this large group remain unstudied. We will try to understand, why sex determining systems in fish are evolutionary highly plastic while in some other vertebrate groups they become the most conserved component of the genome. Besides we have a colletion of animals with B chromosome, whose contatin has been studied yet, and using sequencing we will shed light to evolution of these enigmatic elements. 5. Will continue our participation in international projects for the sequencing of genomes of the red fox (Vulpes vulpes), gray whale (Eschrichtius robustus) and the Baikal seal ((Pusa sibirica), in which we provide the resource (tissue samples and DNA) and cytogenetic part. Naturally, not all of these major collaborative work depends on our team, but we can hope that there will be some decent publications in the reporting period of three years. 6. DNA will be extracted (and performed sequencing on a platform MiSeq) from bone samples of horses, wolves, bears, bison, yaks and deer from different regions of Eurasia, but above all from the famous caves of Altai (Denisovskaya, Razboynich'ya, etc.), where they are very well preserved. The samples were dated to 80 thousand years or less. Will be analyzed mitochondrial genomes and the most interesting locus of nuclear genomes. With RAD-seq method is studied changes in the structure of populations of these species in the evolutionary and temporal dynamics. In addition, work has begun to study the DNA of the horse of prehistoric and historic graves in Eurasia. Horse - a species domesticated it in the steppes of the Altai and the Northern Kazakhstan, and we have the opportunity to study this process in the dynamics of the horses to the domestication of horses many of the nomadic peoples, successive over 4 thousand years (during the domestication of the horse), from famous as the burial mounds Ukok, to modern breeds. Actually we offer hiking and expected results are reduced to a description of biological resources, in particular Russia and adjacent territories, at the genomic and chromosomal level. It is not ruled out that other types and groups will be involved in the learning process. The key to success of the project lies in the fact that the team has already proved its ability to work with chromosomes and genomes of species of vertebrate taxa, fish, amphibians, birds and reptiles. Participants of the project are significant publications on genomics and chromosomics of mammals from platypus and marsupials of Australia, sloths and anteaters of South America, to Africa and Asia, elephants and cetaceans of the oceans, naturally including almost all species of mammalian taxa from Russia. It is assumed that all planned our work, just as its predecessors, will be conducted in close cooperation with leading international experts and centers and will meet the highest international standards enough. At the end of the three-year cycle, we had planned to publish the work 9, and hoping that there will be more publications, including journals with a sufficiently high status.


 

REPORTS


Annotation of the results obtained in 2018
Whole genome sequencing provides ground for search of genetic basis of biological features in mammalian species or of complex processes such as domestication. In frame of famous Farm fox experiment as part of international team we sequenced the genome of the fox and assembled it with the genetic tools developed over the years, such as chromosome painting maps and FISH gene mapping. The sequences from domesticated and aggressive fox lineages were compared and reveled 113 (including SorCS1 gene) candidate regions. Many genes from these regions are linked to hereditary disorders in humans. The identified regions will allow targeted investigation of the genes, involved in domestication. B chromosomes (Bs) represent a variable addition to the main karyotype in some lineages of animals and plants. Bs accumulate through non-Mendelian inheritance and become widespread in populations. Despite the presence of multiple genes, most Bs lack specific phenotypic effects, although their influence on host genome epigenetic status and gene expression are recorded. Previously, using sequencing of isolated Bs of ruminants and rodents, we demonstrated that Bs originate as segmental duplications of specific genomic regions, and subsequently experience pseudogenization and repeat accumulation. Here, we used a similar approach to characterize Bs of the red fox (Vulpes vulpes L.) and the Chinese raccoon dog (Nyctereutes procyonoides procyonoides Gray). We confirm the previous findings of the KIT gene on Bs of both species, but demostrate an independent origin of Bs in these species, with two reused regions. Comparison of gene ensembles in Bs of canids, ruminants, and rodents once again indicates enrichment with cell-cycle genes, development-related genes, and genes functioning in the neuron synapse. The presence of B-chromosomal copies of genes involved in cell-cycle regulation and tissue differentiation may indicate importance of these genes for B chromosome establishment. Constitutive heterochromatin areas are revealed by differential staining as C-positive chromosomal regions. These C-positive bands may greatly vary by location, size and nucleotide composition. CBG-banding is the most commonly used method to detect structural heterochromatin in animals. The main problem of this method is a difficulty in identification of individual chromosomes as the body of the chromosome is stained uniformly and does not have banding pattern beyond C-bands. Here we present method that we called CDAG for sequential heterochromatin staining after differential GTG-banding. The method uses G-banding followed by heat denaturation in the presence of formamide with consecutive fluorochrome staining. New technique is valid for concurrent revealing heterochromatin of different composition and differential banding of chromosomes in complex karyotypes in any mammalian species from different orders. The description of intrachromosomal rearrangements in some conserved segments of 28 species of voles is made. In the areas homologous to AAK1 and AAK7 (= MAG1=MOEC1p) centromere repositions predominated, but in the AAK3 region, paracentric inversions predominated. An unusual arrangement of the nucleolus organizer regions on the two sex chromosomes of the representative of Ruminant (Ruminantia) Javan deer (Tragulus javanicus) was shown. Using FISH (fluorescence in situ hybridization) analysis with samples containing ribosomal DNA sequences (18S, 5.8S, and 28S), revealed a single localization site on two sex chromosomes, X and Y. We are using radiation hybrid mapping to make physical maps of dromedary and alpaca. We developed 44 primers evenly distributed across the genome and analyzed genotyping results of dromedary radiation hybrid clones (186 for 5000 RAD and 93 for 15000 RAD). We selected 93 and 90 clones for 5000 RAD and 15000 RAD final panels with average retention frequencies 50% and 39% by eliminating clones with high discordance and difference rate between repeated PCR and with extreme retention frequencies. The combination of two panels will make it possible to construct maps of high resolution for efficient mapping. In humans, the most common types of extra chromosomes, sSMCs (small supernumerary marker chromosomes), are diagnosed in approximately 1 of 2000 postnatal cases. Due to low frequency in population, human sSMCs are not considered B chromosomes. Genetic content of both B-chromosomes and sSMCs in most cases remains understudied. Here, we apply microdissection of single chromosomes with subsequent low-pass sequencing on Ion Torrent PGM and Illumina MiSeq to identify unique and repetitive DNA sequences present in a single human sSMC and several B chromosomes in mice Apodemus flavicollis and Apodemus peninsulae. Human sSMC was attributed to the proximal part of chromosome 15 long arm, and breakpoints leading to its formation were located into satellite DNA arrays. Genetic content of Apodemus B chromosomes was species-specific, and minor alterations were observed in both species. Common features of Bs in these Apodemus species were satellite DNA and ERV enrichment, as well as the presence of the vaccinia-related kinase gene Vrk1. Supernumerary elements of the genome are often called B chromosomes. They usually consist of various autosomal sequences and, because of low selective pressure, are mostly pseudogenized and contain many repeats. There are numerous reports on B chromosomes in mammals, fish, invertebrates, plants, and fungi, but only a few of them have been studied using sequencing techniques. However, reptilian supernumerary chromosomes have been detected only cytogenetically and never sequenced or analyzed at the molecular level. One model squamate species with available genome sequence is Anolis carolinensis. The scope of the present article was to describe the genetic content of A. carolinensis supernumerary chromosomes. In this article, we confirmed the presence of B chromosomes in this species by reverse painting and synaptonemal complex analysis. We applied low-pass high-throughput sequencing to analyze flow-sorted B chromosomes. Anole B chromosomes exhibit similar traits to other supernumerary chromosomes from different taxons: they contain two genes related to cell division control (INCENP and SPIRE2), are enriched in specific repeats, and show a high degree of pseudogenization. Therefore, the present study confirmed that reptilian B chromosomes resemble supernumerary chromosomes of other taxons. Sex/autosome translocation are rare events. The only known example in catarrhines is in the silvered-leaf monkey. Here the Y chromosome was reciprocally translocated with chromosome 1. The rearrangement produced an X1X2Y1Y2 sex chromosome system. At least three chromosomal variants of the intact chromosome 1 are known to exist. We characterized in high resolution the translocation products (Y1 and Y2) and the polymorphic forms of the intact chromosome 1 with a panel of more than 150 human BAC clones. We showed that the translocation products were extremely rearranged, in contrast to the high level of marker order conservation of the other silvered-leaf monkey chromosomes. Surprisingly, each translocation product appeared to form independent “chromosome lineages”; each having a myriad of distinct rearrangements. We reconstructed the evolutionary history of the translocation products by comparing the homologous chromosomes of two other colobine species: the African mantled guereza and the Indian langur. The results showed a massive reuse of breakpoints: only 12, out of the 40 breaks occurred in domains never reused in other rearrangements, while, strikingly, some domains were used up to four times. Such frequent breakpoint reuse if proved to be a general phenomenon has profound implications for mechanisms of chromosome evolution. The draft published 10 articles, two more articles are being reviewed at the moment. 1. Capozzi O, Stanyon R, Archidiacono N, Ishida T, Romanenko SA, Rocchi M. Rapid emergence of independent “chromosomal lineages” in silvered-leaf monkey triggered by Y/autosome translocation. (doi: 10.1038/s41598-018-21509-4) Sci Reports 8: 3250, 2018 2. Perelman PL, Pichler R, Gaggl A, Larkin DM, Raudsepp T, Alshanbari F, Holl HM, Brooks SA, Burger PA, Periasamy K. Construction of two whole genome radiation hybrid panels for dromedary (Camelus dromedarius): 5000RAD and 15000RAD. (doi: 10.1038/s41598-018-20223-5) Sci Reports 8: 1982, 2018 3. Proskuryakova AA, Kulemzina AI, Perelman PL, Serdukova NA, Ryder OA, Graphodatsky AS. The case of X and Y localization of nucleolus organizer regions (NORs) in Tragulus javanicus (Cetartiodactyla, Mammalia). (doi: 10.3390/genes9060312) Genes 9(6): 312, 2018 4. Pavlova SV, Biltueva LS, Romanenko SA, Lemskaya NA, Shchinov AV, Abramov AV, Rozhnov VV. First cytogenetic analysis of lesser gymnures (Mammalia, Galericidae, Hylomys) from Vietnam. (doi: 10.3897/CompCytogen.v12i3.27207) Comp Cytogen 12(3): 361-372, 2018 5. Kukekova AV, Johnson JL, Xiang X, Feng S, Liu S, Rando HM, Kharlamova AV, Herbeck Y, Serdyukova NA, Xiong Z, Beklemischeva V, Koepfli K-, Gulevich RG, Vladimirova AV, Hekman JP, Perelman PL, Graphodatsky AS, O’Brien SJ, Wang X, Clark AG, Acland GM, Trut LN, Zhang G. Red fox genome assembly identifies genomic regions associated with tame and aggressive behaviours. (doi: 10.1038/s41559-018-0611-6) Nat Ecol Evol. 2018;2(9):1479-91. 6. Makunin AI, Romanenko SA, Beklemisheva VR, Perelman PL, Druzhkova AS, Petrova KO, Prokopov DY, Chernyaeva EN, Johnson JL, Kukekova AV, Yang F, Ferguson-Smith MA, Graphodatsky AS, Trifonov VA. Sequencing of supernumerary chromosomes of red fox and raccoon dog confirms a non-random gene acquisition by B chromosomes. (doi: 10.3390/genes9080405) Genes 9(8): 405, 2018 7. Makunin AI, Rajičić M, Karamysheva TV, Romanenko SA, Druzhkova AS, Blagojević J, Vujošević M, Rubtsov NB, Graphodatsky AS, Trifonov VA. Low-pass single-chromosome sequencing of human small supernumerary marker chromosomes (sSMCs) and Apodemus B chromosomes. (doi: 10.1007/s00412-018-0662-0) Chromosoma 127(3): 301–311, 2018 8. Lemskaya NA, Kulemzina AI, Beklemisheva VR, Biltueva LS, Proskuryakova AA, Hallenbeck JM, Perelman PP, Graphodatsky AS. A combined banding method that allows the reliable identification of chromosomes as well as differentiation of AT- and GC-rich heterochromatin. Chromosome Res, 2018, doi: 10.1007/s10577-018-9589-9 9. Kichigin IG, Lisachov AP, Giovannotti M, Makunin AI, Kabilov MR, O’Brien PCM, Ferguson-Smith MF, Graphodatsky AS, Trifonov VA. First report on B chromosome content in a reptilian species: the case of Anolis carolinensis. Mol Genet Genomics, 2018, doi: 10.1007/s00438-018-1483-9 10. Romanenko S, Serdyukova N, Perelman P, Trifonov V, Golenishchev F, Bulatova N, Stanyon R, Graphodatsky A. Multiple intrasyntenic rearrangements and rapid speciation in voles. (doi: 10.1038/s41598-018-33300-6) Sci Reports 8: 14980, 2018 Farré M, Kim J, Proskurjakova AA, Kulemzina AI, Zhang Y, Huang Z, Yao X, Fang X, Rando HM, Perelman P, Li X, Kukekova AV, Zhao W, Zhang G, Wang J, O’Brien SJ, Graphodatsky AS, Ma J, Lewin HA, Larkin DM Coevolution of chromosome changes and gene regulation in ruminants. Genome Res (in press) Elbers JP, Rogers MF, Perelman PL, Horin P, Corander J, Murphy D, Burger PA Improving Illumina assemblies with Hi-C and long-reads: an example with the North African dromedary. Molecular Ecology Resources (in press)

 

Publications

1. Кичигин И.Г., Лисачев А.П., Джиованноти М., Макунин А.И., Кабилов М.Р., ОБрайен П.С.М., Фергюсон-Смит М.Ф., Графодатский А.С., Трифонов В.А. First report on B chromosome content in a reptilian species: the case of Anolis carolinensis Molecular Genetics and Genomics, - (year - 2018).

2. Кукекова АВ, Джонсон ДЛ, Ксианг К, Фенг С, Лю С, Рандо ГМ, Харламова АВ, Гербек Ю, Сердюкова НА, Ксионг З, Беклемишева ВР, Перельман ПЛ, Графодатский АС, ОБрайен СД, Ванг К, Кларк АГ, Окланд ГМ, Трут ЛН, Занг Г. Red fox genome assembly identifies genomic regions associated with tame and aggressive behaviours Nature Ecology and Evolution, Том: 2 Выпуск: 9 Стр.: 1479-1491 (year - 2018).

3. Романенко С., Сердюкова Н., Перельман П., Трифонов В., Голенищев Ф., Булатова Н., Стэнион Р., Графодатский А. Multiple intrasyntenic rearrangements and rapid speciation in voles Scientific Reports, Том: 8, Номер статьи: 14980 (year - 2018).

4. Перельман ПЛ, Пихлер Р, Гаггл А, Ларкин ДМ, Раудсепп Т, Алшанбари Ф, Холл ГМ, Брукс СА, Бургер ПА, Периасами К. Construction of two whole genome radiation hybrid panels for dromedary (Camelus dromedarius): 5000RAD and 15000RAD SCIENTIFIC REPORTS, Том: 8 Номер статьи: 1982 (year - 2018).

5. Капоци О, Стэнион Р, Арчидияконо Н, Ишида Т, Романенко СА, Роччи М. Rapid emergence of independent "chromosomal lineages" in silvered-leaf monkey triggered by Y/autosome translocation Scientific Reports, Том: 8 Номер статьи: 3250 (year - 2018).

6. Павлова СВ, Билтуева ЛС, Романенко СА, Лемская НА, Шинов АВ, Абрамов АВ, Рожнов ВВ. First cytogenetic analysis of lesser gymnures (Mammalia, Galericidae, Hylomys) from Vietnam Comparative Cytogenetics, Том: 12 Выпуск: 3 Стр.: 361-372 (year - 2018).

7. Проскурякова АА, Кулемзина АИ, Перельман ПЛ, Сердюкова НА, Райдер ОА, Графодатский АС The case of X and Y localization of nucleolus organizer regions (NORs) in Tragulus javanicus (Cetartiodactyla, Mammalia) GENES, Том: 9 Выпуск: 6 (year - 2018).

8. Макунин АИ, Райчич М, Карамышева ТВ, Романенко СА, Дружкова АС, Благоевич Д, Вуешевич М, Рубцов НБ, Графодатский АС, Трифонов ВА Low-pass single-chromosome sequencing of human small supernumerary marker chromosomes (sSMCs) and Apodemus B chromosomes Chromosoma, Том: 127 Выпуск: 3 Стр.: 301-311 (year - 2018).

9. Макунин АИ, Романенко СА, Беклемишева ВР, Перельман ПЛ, Дружкова АС, Петрова КО, Прокопов ДЮ, Черняква ЕН, Джонсон ДЛ, Кукекова АВ, Янг Ф, Фергюсон-Смит МА, Графодатский АС, Трифонов ВА Sequencing of supernumerary chromosomes of red fox and raccoon dog confirms a non-random gene acquisition by B chromosomes GENES, Том: 9 Выпуск: 8 (year - 2018).

10. Лемская НА, Кулемзина АИ, Беклемишева ВР, Билтуева ЛС, Проскурякова АА, Халленбек ДМ, Перельман ПЛ, Графодатский АС. A combined banding method that allows the reliable identification of chromosomes as well as differentiation of AT- and GC-rich heterochromatin Chromosome Research, - (year - 2018).

11. Трифонов ВА, Лисачев АП, Кичигин ИГ, Макунин АИ, Перейра ДК, Дружкова АС, Фергюсон-Смит МА, Джиованнотти М. Evolutionary sex chromosome translocations in amniotes COMPARATIVE CYTOGENETICS, Том: 12 Выпуск: 3 Стр.: 304-305 Аннотация к встрече: L7 (year - 2018).

12. Беклемишева ВР, Перельман ПЛ, Лемская НА, Кулемзина АИ, Проскурякова АА, Бурканов ВН, ОБрайен СД, Графодатский АС Pinniped karyotype evolution substantiated by comparative chromo-some painting of 10 pinniped species (Pinnipedia, Carnivora) COMPARATIVE CYTOGENETICS, Том: 12 Выпуск: 3 Стр.: 306-307 Аннотация к встрече: O2 (year - 2018).

13. Проскурякова АА, Кулемзина АИ, Перельман ПЛ, Макунин АИ, Лемская НА, Беклемишева ВР, Ларкин ДМ, Фарре М, Кукекова АВ, Райдер ОА, ОБрайен С, Графодатский АС. X chromosome evolution in Cetartiodactyla. COMPARATIVE CYTOGENETICS, Том: 12 Выпуск: 3 Стр.: 307-308 Аннотация к встрече: O3 (year - 2018).

14. - Sequenced fox genome hints at genetic basis of behavior сайт РНФ, http://rscf.ru/ru/node/3313 (year - ).

15. Фарре М., Ким Дж., Проскурякова АА., Занг И., Кулемзина АИ., Ли К., Жу И., Ксонг И., Джонсон Дж., Перельман П., Джонсон ВЕ., Варрен ВК., Кукекова АВ., Занг Г., ОБрайен СДж., Райдер ОА., Графодатский АС., Ма Дж., Люин ХА., Ларкин ДМ Evolution of gene regulation in ruminants differs between evolutionary breakpoint regions and homologous synteny blocks Genome Research, 29: 576-589 (year - 2019).


Annotation of the results obtained in 2016
Rapid karyotype evolution in Lasiopodomys voles. The generic status of Lasiopodomys and its division into subgenera Lasiopodomys (L. mandarinus, L. brandtii) and Stenocranius (L. gregalis, L. raddei) are not generally accepted because of contradictions between the morphological and molecular data. To obtain cytogenetic evidence for the Lasiopodomys genus and its subgenera and to test the autosome to sex chromosome translocation hypothesis of sex chromosome complex origin in L. mandarinus proposed previously we hybridized chromosome painting probes from the field vole (Microtus agrestis, MAG) and the Arctic lemming (Dicrostonyx torquatus, DTO) onto the metaphases of a female Mandarin vole (L. mandarinus, 2n = 47) and a male Brandt's vole (L. brandtii, 2n = 34). In addition, we hybridized Arctic lemming painting probes onto chromosomes of a female narrow-headed vole (L. gregalis, 2n = 36). Cross-species painting revealed three cytogenetic signatures (MAG12/18, 17a/19, and 22/24) that could validate the genus Lasiopodomys and indicate the evolutionary affinity of L. gregalis to the genus. Moreover, all three species retained the associations MAG1bc/17b and 2/8a detected previously in karyotypes of all arvicolins studied. The associations MAG2a/8a/19b, 8b/21, 9b/23, 11/13b, 12b/18, 17a/19a, and 5 fissions of ancestral segments appear to be characteristic for the subgenus Lasiopodomys. We also validated the autosome to sex chromosome translocation hypothesis on the origin of complex sex chromosomes in L. mandarinus. Two translocations of autosomes onto the ancestral X chromosome in L. mandarinus led to a complex of neo-X1, neo-X2, and neo-X3 elements. Our results demonstrate that genus Lasiopodomys represents a striking example of rapid chromosome evolution involving both autosomes and sex chromosomes. Multiple reshuffling events including Robertsonian fusions, chromosomal fissions, inversions and heterochromatin expansion have led to the formation of modern species karyotypes in a very short time, about 2.4 MY. Gladkikh O.L., Romanenko S.A., Lemskaya N.A., Serdyukova N.A., O’Brien P.C.M., Kovalskaya J.M., Smorkatcheva A.V., Golenishchev F.N., Perelman P.L., Trifonov V.A., Ferguson-Smith M.A., Yang F., Graphodatsky A.S.) Rapid karyotype evolution in Lasiopodomys involved at least two autosome – sex chromosome translocations PLOS ONE (2016 г.) DOI: 10.1371/journal.pone.0167653 Chromosomes and genomes of Anolis lizards. Squamate reptiles show a striking diversity in modes of sex determination, including both genetic (XY or ZW) and temperature-dependent sex determination systems. The genomes of only a handful of species have been sequenced, analyzed and assembled including the genome of Anolis carolinensis. Despite a high genome coverage, only macrochromosomes of A. carolinensis were assembled whereas the content of most microchromosomes remained unclear. Most of the Anolis species have homomorphic XY sex chromosome system. However, some species have large heteromorphic XY chromosomes (e.g., A. sagrei) and even multiple sex chromosomes systems (e.g. A. pogus), that were shown to be derived from fusions of the ancestral XY with microautosomes. We applied next generation sequencing of flow sorting-derived chromosome-specific DNA pools to characterize the content and composition of microchromosomes in A. carolinensis and A. sagrei. Comparative analysis of sequenced chromosome-specific DNA pools revealed that the A. sagrei XY sex chromosomes contain regions homologous to several microautosomes of A. carolinensis. We suggest that the sex chromosomes of A. sagrei are derived by fusions of the ancestral sex chromosome with three microautosomes and subsequent loss of some genetic content on the Y chromosome. http://link.springer.com/article/10.1007%2Fs00438-016-1230-z In the first year we localized X-chromosome and autosomes specific BACs onto chromosomes of the remaining Cetartiodactyla families, begin to construct radiation hybrid maps for two camelid species, establish region-specific microdissected probes for chromosomes of Microtus, used DOP-PCR and WGA to construct B- and sex chromosome specific libraries for some vertabrates and continue to study ancient DNA of horses, moose and bears.

 

Publications

1. Кичигин И.Г., Джиованнотти М., Макунин А.И., Нг Б.Л., Кабилов М.Р., Тупикин А.Е., Баручи В.К., Сплендиани А., Руджери П., Ренс В., ОБрайен П.С.М., Фергюсон-Смит М.А., Графодатский А.С., Трифонов В.А. Evolutionary dynamics of Anolis sex chromosomes revealed by sequencing of flow sorting-derived microchromosome-specific DNA Molecular Genetics and Genomics, Volume 291, Issue 5, pp 1955–1966 (year - 2016).

2. Гладких О.Л., Романенко С.А., Лемская Н.А., Сердюкова Н.А., ОБрайен П.С.М., Ковальская Ю.М., Сморкачева А.В., Голенищев Ф.М., Перельман П.Л., Трифонов В.А., Фергюсон-Смит М.А., Графодатский А.С. Rapid karyotype evolution in Lasiopodomys involved at least two autosome – sex chromosome translocations PLOS ONE, - (year - 2016).


Annotation of the results obtained in 2017
X chromosome maps of 18 cetartiodactyl species had been done in current year. The analysis of rearrangements, the search for phylogenetic markers and the ancestral X chromosomes were conducted. Based on this work the article "X chromosome evolution in Cetartiodactyla" had been published. Also, in the course of X chromosomes study a unique location of the nucleolus organizer on Javan mouse deer sex chromosomes was revealed. By comparative G-banding analysis variety of Bovidae X chromosome morphology was revealed. Within the framework of this project, dromedary camel and human chromosome libraries were localized on metaphase chromosomes of musk ox - the surviving megafauna representative. Now there is an active preparation for the publication of these data. The work of identification ruminants evolutionary breakpoints regions (EBR) have begun by the localization of BAC clones on the metaphase chromosomes of the Javan mouse deer and the giraffe. At the moment, majority probes have been localized on chromosomes of this species. The probes homologous to q-arm of the chromosomes MOEC1 (=MAG2/8=AAK1) and MOEC7 (=MAG3=AAK3) were localized on chromosomes of 17 arvicoline species. The full description of intrachromosomal rearrangements in the conserved segments of 28 species is made. In the areas homologous to AAK1 and AAK7 (= MAG1=MOEC1p) centromere repositions predominated, but in the AAK3 region, paracentric inversions predominated. The complete description of C. sokolovi karyotype with C- and G-banding and chromosome painting was made. The results confirm the species status of C. sokolovi and show that its karyotype was formed due to at least 4 Robertsinian events and one centromere reposition. Chromosomal fan of Ellobius alaicus is being described. Chromosome level genome assemblies are required for the next step in development of highly efficient genomic tools genetic analysis of already sequenced mammalian genomes. There is no chromosome level assembly for camelids and we are working toward it. We are using radiation hybrid mapping to make physical maps of dromedary and alpaca. We developed 44 primers evenly distributed across the genome and analyzed genotyping results of dromedary radiation hybrid clones (186 for 5000 RAD and 93 for 15000 RAD). We selected 93 and 90 clones for 5000 RAD and 15000 RAD final panels with average retention frequencies 50% and 39% by eliminating clones with high discordance and difference rate between repeated PCR and with extreme retention frequencies. The combination of two panels will make it possible to construct maps of high resolution for efficient mapping. We continued RH mapping of alpaca by constructing maps of X and chromosome 17. The primary fibroblast cell line of dromedary was used for third generation technology of genome assembly. We obtained chromosome spreads for rare camel species Camelus ferus to conduct molecular-cytogenetic analyses of chromosomes. Development of such resources is necessary step toward full genome characterization of camelids essential for further investigation of genetics and evolution of this important mammalian group. Whole genome sequencing provides ground for search of genetic basis of biological features in mammalian species or of complex processes such as domestication. In frame of famous Farm fox experiment as part of international team we sequenced the genome of the fox and assembled it with the genetic tools developed over the years, such as chromosome painting maps and FISH gene mapping. The sequences from domesticated and aggressive fox lineages were compared and reveled 113 candidate regions. Many genes from these regions are linked to hereditary disorders in humans. The identified regions will allow targeted investigation of the genes, involved in domestication. Our study of sex chromosomes and B chromosomes using high throughput sequencing revealed unusual details of those elements evolution: we suggested that B chromosomes of two rodent species - yellow-necked wood mice and collared lemming are derived from sex chromosomes; revealed uneven rates of common origin Bs evolution in the red fox and raccoon dog, and found unusually high rates of rearrangements in sex chromosomes of iguanid lizards. Besides, a technique was elaborated for rapid characterization of chromosome specific repeated elements in species with a high number of similar chromosomes in karyotypes. DNA from Ovodov's horse (32 thousand years old from Denisova cave, Altai) was extracted, high-throughput sequencing of libraries enriched with the target fragments was done. Bioinformatic analysis showed that E. ovodovi is closer to zebras than to donkeys (E. asinus, E. hemionus), or E. kiang, while the previous study (Vilstrup et al., 2013) points on the basal position of E.ovodovi among non-caballine equds. Our data expanded the geographic and temporal ranges of E. ovodovi species. Analysis of fragments of the mitochondrial DNA D-loop from 17 sheep bone samples (about 4000-1000 years old), found in archeological complexes in the South Altai (Western Siberia), showed that 15 samples belong to the previously described haplogroups A, B, C, D and E and two samples have unique haplotypes and have a basal position relative to the rest of the samples analyzed. A wide diversity of haplotypes may indicate that Altai region was a transport route more than 1000 years ago.

 

Publications

1. Дымова МА, Задорожный АВ, Мишукова ОВ, Храпов ЕА, Дружкова АС, Трифонов ВА, Кичигин ИГ, Тишкин АА, Грушин СП, Филипенко МЛ Mitochondrial DNA analysis of ancient sheep from Altai Anim Genet, 48(5): 615-618 (year - 2017).

2. Райчич М, Романенко СА, Карамышева ТВ, Благоевич Дж, Аднадевич Т, Будински И, Богданов АС, Трифонов ВА, Рубцов НБ, Вуёшевич М. The origin of B chromosomes in yellow-necked mice (Apodemus flavicollis) - Break rules but keep playing the game PLoS ONE, 12(3): e0172704 (year - 2017).

3. Проскурякова АА, Кулемзина АИ, Перельман ПЛ, Макунин АИ, Ларкин ДМ, Фарре М, Кукекова АВ, Джонсон Дж.Л, Лемская НА, Беклемишева ВР, Рёлке-Паркер МЕ, Белицци Дж., Райдер ОА, ОБрайен СДж., Графодатский АС X chromosome Evolution in Cetartiodactyla GENES, 8(9): 216 (year - 2017).

4. Дружкова АС, Макунин АИ, Воробьева НВ, Васильев СК, Оводов НД, Шуньков МВ, Трифонов ВА, Графодатский АС Complete mitochondrial genome of an extinct Equus (Sussemionus) ovodovi specimen from Denisova cave (Altai, Russia) MITOCHONDRIAL DNA PART B: RESOURCES, Volume 2, Issue 1, Pages 79-81 (year - 2017).

5. Романенко СА, Сердюкова НА, Перельман ПЛ, Павлова СВ, Булатова НШ, Голенищев ФН, Стэнион Р, Графодатский АС Intrachromosomal rearrangements in rodents from the perspective of comparative region-specific painting Genes, Genes 8(9): 215 (year - 2017).

6. Билтуева ЛС, Прокопов ДЮ, Макунин АИ, Комиссаров АС, Кудрявцева АВ, Лемская НА, Воробьева НВ, Сердюкова НА, Романенко СА, Гладких ОЛ, Графодатский АС, Трифонов ВА Genomic Organization and Physical Mapping of Tandemly Arranged Repetitive DNAs in Sterlet ( Acipenser ruthenus ) Cytogenet Genome Res, - (year - 2017).

7. Поплавская НС, Романенко СА, Сердюкова НА, Трифонов ВА, Янг Ф, Ни В, Ванг Дж, Банникова АА, Суров АВ, Лебедев ВС Karyotype Evolution and Phylogenetic Relationships of Cricetulus sokolovi Orlov et Malygin 1988 (Cricetidae, Rodentia) Inferred from Chromosomal Painting and Molecular Data Cytogenet Genome Res, 152: 65-72 (year - 2017).