Publications by authors named "Mikhail G Divashuk"

19 Publications

  • Page 1 of 1

Case Report: Next Generation Sequencing in Clinical Practice-A Real Tool for Ending the Protracted Diagnostic Odyssey.

Front Cardiovasc Med 2021 13;8:778961. Epub 2022 Jan 13.

Department of Fundamental and Applied Aspects of Obesity, National Medical Research Center for Therapy and Preventive Medicine of the Ministry of Healthcare of the Russian Federation, Moscow, Russia.

We reported a case of sitosterolemia, which is a rare genetic disease, characterized by increased plant sterol absorption and great heterogeneity of clinical manifestations. Our patient was initially referred to the lipid clinic due to high cholesterol levels and premature cardiovascular disease. Diagnosis of familial hypercholesterolemia was established in accordance with the Dutch Lipid Clinic Network criteria. Next-generation sequencing was later performed, which revealed a nonsense mutation in the gene, which led to the diagnosis of sitosterolemia. The aim of our report is to demonstrate, how genetic testing helped to make the correct diagnosis and to explain many of the patient's health problems, which etiology remained unclear for many years.
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http://dx.doi.org/10.3389/fcvm.2021.778961DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8792487PMC
January 2022

Transposons Hidden in Genome Assembly Gaps and Mobilization of Non-Autonomous LTR Retrotransposons Unravelled by Nanotei Pipeline.

Plants (Basel) 2021 Dec 6;10(12). Epub 2021 Dec 6.

All-Russia Research Institute of Agricultural Biotechnology, Timiryazevskaya Str. 42, 127550 Moscow, Russia.

Long-read data is a great tool to discover new active transposable elements (TEs). However, no ready-to-use tools were available to gather this information from low coverage ONT datasets. Here, we developed a novel pipeline, nanotei, that allows detection of TE-contained structural variants, including individual TE transpositions. We exploited this pipeline to identify TE insertion in the genome. Using nanotei, we identified tens of TE copies, including ones for the well-characterized ONSEN retrotransposon family that were hidden in genome assembly gaps. The results demonstrate that some TEs are inaccessible for analysis with the current (TAIR10.1) genome assembly. We further explored the mobilome of the mutant with elevated TE activity. Nanotei captured all TEs previously known to be active in and also identified transposition of non-autonomous TEs. Of them, one non-autonomous TE derived from (AT5TE33540) belongs to TR-GAG retrotransposons with a single open reading frame (ORF) encoding the GAG protein. These results provide the first direct evidence that TR-GAGs and other non-autonomous LTR retrotransposons can transpose in the plant genome, albeit in the absence of most of the encoded proteins. In summary, nanotei is a useful tool to detect active TEs and their insertions in plant genomes using low-coverage data from Nanopore genome sequencing.
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http://dx.doi.org/10.3390/plants10122681DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8704663PMC
December 2021

Alleles of the GRF3-2A Gene in Wheat and Their Agronomic Value.

Int J Mol Sci 2021 Nov 16;22(22). Epub 2021 Nov 16.

Laboratory of Applied Genomics and Crop Breeding, All-Russia Research Institute of Agricultural Biotechnology, 127550 Moscow, Russia.

The Growth-regulating factors (GRF) are a family of plant-specific transcription factors that have roles in plant growth, development and stress response. In this study the diversity of the (TraesCS2A02G435100) gene was investigated in Russian bread wheat germplasm by means of next generation sequencing and molecular markers, and the results compared with those from multiple wheat genome and exome sequencing projects. The results showed that an allele possessing c.495G>T polymorphism found in Bezostaya 1 and designated as , is connected with earlier heading and better grain filling under conditions of the Krasnodar Krai. is more frequent among Russian winter wheat cultivars than in other germplasms found in the world, implying that it is adaptive for the Chernozem region. A new rare mutation of the was found in the spring wheat cultivar Novosibirskaya 67. The molecular markers developed will facilitate utilization of mutations in future agronomic studies and wheat improvement. Albeit may be good at maintaining high milling quality of the grain, it should be used with caution in breeding of winter wheat cultivars in the perspective of climate change.
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http://dx.doi.org/10.3390/ijms222212376DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8622619PMC
November 2021

Targeted Sequencing of 242 Clinically Important Genes in the Russian Population From the Ivanovo Region.

Front Genet 2021 7;12:709419. Epub 2021 Oct 7.

National Medical Research Center for Therapy and Preventive Medicine, Moscow, Russia.

We performed a targeted sequencing of 242 clinically important genes mostly associated with cardiovascular diseases in a representative population sample of 1,658 individuals from the Ivanovo region northeast of Moscow. Approximately 11% of 11,876 detected variants were not found in the Single Nucleotide Polymorphism Database (dbSNP) or reported earlier in the Russian population. Most novel variants were singletons and doubletons in our sample, and virtually no novel alleles presumably specific for the Russian population were able to reach the frequencies above 0.1-0.2%. The overwhelming majority (99.3%) of variants detected in this study in three or more copies were shared with other populations. We found two dominant and seven recessive known pathogenic variants with allele frequencies significantly increased compared to those in the gnomAD non-Finnish Europeans. Of the 242 targeted genes, 28 were in the list of 59 genes for which the American College of Medical Genetics and Genomics (ACMG) recommended the reporting of incidental findings. Based on the number of variants detected in the sequenced subset of ACMG59 genes, we approximated the prevalence of known pathogenic and novel or rare protein-truncating variants in the complete set of ACMG59 genes in the Ivanovo population at 1.4 and 2.8%, respectively. We analyzed the available clinical data and observed the incomplete penetrance of known pathogenic variants in the 28 ACMG59 genes: only 1 individual out of 12 with such variants had the phenotype most likely related to the variant. When known pathogenic and novel or rare protein-truncating variants were considered together, the overall rate of confirmed phenotypes was about 19%, with maximum in the subset of novel protein-truncating variants. We report three novel protein truncating variants in and one in observed in individuals with hypobetalipoproteinemia and hypertrophic cardiomyopathy, respectively. Our results provide a valuable reference for the clinical interpretation of gene sequencing in Russian and other populations.
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http://dx.doi.org/10.3389/fgene.2021.709419DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8529250PMC
October 2021

Metabolomics for Crop Breeding: General Considerations.

Genes (Basel) 2021 10 12;12(10). Epub 2021 Oct 12.

All-Russia Research Institute of Agricultural Biotechnology, Timiryazevskaya Street, 42, 127550 Moscow, Russia.

The development of new, more productive varieties of agricultural crops is becoming an increasingly difficult task. Modern approaches for the identification of beneficial alleles and their use in elite cultivars, such as quantitative trait loci (QTL) mapping and marker-assisted selection (MAS), are effective but insufficient for keeping pace with the improvement of wheat or other crops. Metabolomics is a powerful but underutilized approach that can assist crop breeding. In this review, basic methodological information is summarized, and the current strategies of applications of metabolomics related to crop breeding are explored using recent examples. We briefly describe classes of plant metabolites, cellular localization of metabolic pathways, and the strengths and weaknesses of the main metabolomics technique. Among the commercialized genetically modified crops, about 50 with altered metabolic enzyme activities have been identified in the International Service for the Acquisition of Agri-biotech Applications (ISAAA) database. These plants are reviewed as encouraging examples of the application of knowledge of biochemical pathways. Based on the recent examples of metabolomic studies, we discuss the performance of metabolic markers, the integration of metabolic and genomic data in metabolic QTLs (mQTLs) and metabolic genome-wide association studies (mGWAS). The elucidation of metabolic pathways and involved genes will help in crop breeding and the introgression of alleles of wild relatives in a more targeted manner.
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http://dx.doi.org/10.3390/genes12101602DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8535592PMC
October 2021

The Effect of Chromosome Arm 1BS on the Fertility of Alloplasmic Recombinant Lines in Bread Wheat with the Cytoplasm.

Plants (Basel) 2021 May 31;10(6). Epub 2021 May 31.

Institute of Cytology and Genetics, SB RAS, Lavrentiev av., 10, 630090 Novosibirsk, Russia.

The genetic mechanisms of fertility restoration in alloplasmic bread wheat with the barley cytoplasm are poorly explored. The effect of the 1BS chromosome arm on the fertility of bread wheat with the cytoplasm was studied depending on the incompleteness/completeness of the cytonuclear compatibility. (i) Three self-fertile (SF) lines and one partially fertile (PF) line with an incomplete cytonuclear compatibility and (ii) four self-fertile (SF) lines with a complete cytonuclear compatibility were studied. For the lines in group (i), the heteroplasmy (simultaneous presence of barley and wheat copies) of the 18S/5S mitochondrial (mt) repeat was revealed as well as the barley-type homoplasmy of chloroplast simple sequence repeats (cpSSRs). In the lines in group (ii), the 18S/5S mt repeat and cpSSRs were found in the wheat-type homoplasmic state. In all of the lines, the 1BS chromosome arm was substituted for the 1RS arm. The F plants of SF(i)-1BS × 1RS hybrids were fertile. The results of a segregation analysis in the F plants of SF(i)-1BS × 1RS showed that 1BS carries a single dominant fertility restorer gene () of bread wheat with the cytoplasm. All of the F plants of PF(i)-1BS × 1RS hybrids were sterile. A single dose of this restorer gene is not sufficient to restore fertility in this alloplasmic PF(i) line. All of the F and F plants of SF(ii)-1BS × 1RS hybrids were self-fertile.
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http://dx.doi.org/10.3390/plants10061120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8228278PMC
May 2021

Case Report: Hypertriglyceridemia and Premature Atherosclerosis in a Patient With Apolipoprotein E Gene εε Genotype.

Front Cardiovasc Med 2020 18;7:585779. Epub 2021 Jan 18.

National Medical Research Center for Therapy and Preventive Medicine of the Ministry of Healthcare of the Russian Federation, Moscow, Russia.

We present a case of a 40-year-old male with premature atherosclerosis, with evidence of both eruptive and tendinous xanthomas, which could imply an increase in both low-density lipoprotein (LDL) and triglyceride (TG) levels. However, his LDL was 2.08 mmol/l, TG -11.8 mmol/l on rosuvastatin 20 mg. Genetic evaluation was performed using a custom panel consisting of 25 genes and 280 variants responsible for lipid metabolism. A rare εε genotype of apolipoprotein E was detected. The combination of clinical manifestations and genetic factors in this patient leads to the diagnosis of familial dysbetalipoproteinemia. Implementation of genetic testing into routine clinical practice could not only improve disease diagnostics and management, but also help prevent their development.
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http://dx.doi.org/10.3389/fcvm.2020.585779DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7847930PMC
January 2021

The Allelic Diversity of the Gibberellin Signaling Pathway Genes in Coss.

Plants (Basel) 2020 Dec 2;9(12). Epub 2020 Dec 2.

Laboratory of Applied Genomics and Crop Breeding, All-Russia Research Institute of Agricultural Biotechnology, Timiryazevskaya Street 42, 127550 Moscow, Russia.

Gibberellin-insensitive reduced height genes are widely spread in modern wheat varieties, making them resistant to lodging under conditions of intensive farming. However, the limited diversity of these genes present in wheat germplasm can limit the adaptability of newly created cultivars to the changing climate. The diversity of the gibberellin signaling pathway genes involved in plant height control- ), ) and )-was studied in the diploid wild goatgrass Coss., one of the ancestral species of the bread wheat ( L.) and the donor of its D subgenome, using high-throughput sequencing. The examination of 24 accessions of different geographical origins revealed a large number of new alleles (haplotypes) not found in bread wheat varieties. Some of the detected polymorphisms lead to changes in the amino acid sequence of proteins. Four isoforms (amino acid sequence variants) were found for the RHT-D1 protein, and two isoforms-for the GID1 and GID2 proteins, each. An analysis of the co-occurrence frequencies of various isoforms of the three proteins showed that their combinations were not random in , which may indicate the functional significance of their differences. New alleles of the , , and genes are promising for introgression into bread wheat and studying their effect on plant height and adaptability.
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http://dx.doi.org/10.3390/plants9121696DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7761575PMC
December 2020

Effects of Rht17 in combination with Vrn-B1 and Ppd-D1 alleles on agronomic traits in wheat in black earth and non-black earth regions.

BMC Plant Biol 2020 Oct 14;20(Suppl 1):304. Epub 2020 Oct 14.

Laboratory of Applied Genomics and Crop Breeding, All-Russia Research Institute of Agricultural Biotechnology, Timiryazevskaya str. 42, Moscow, 127550, Russia.

Background: Plant height is an important wheat trait that is regulated by multiple genes, among which Rht is of the utmost value. In wheat, Rht-B1p (=Rht17) is a mutant allele of the Rht gene that encodes for a DELLA-protein and results in the development of gibberellin-insensitive plants with a dwarfing phenotype. The pleiotropic effects of dwarfing genes on yield are highly dependent on both the genetic background and the environmental conditions. In Russia, the Central Non-Black Earth Region and Krasnodar Krai are two economically important regions that require differing management for sustainable wheat production for food, feed and industry. The purpose of our study was to compare the pleiotropic effects of Rht-B1p on the main valuable agronomic traits in the F families of the spring bread wheat Chris Mutant/Novosibirskaya 67 in the genetic background of Vrn-B1a/vrn-B1 (spring/winter phenotype) and Ppd-D1a/Ppd-D1b (insensitivity/sensitivity to photoperiod) alleles in a field experiment in Moscow and Krasnodar Krai.

Results: Plant height was reduced on average by 21 cm (28%) and 25 cm (30%), respectively; Ppd-D1a slightly strengthened the dwarfing effect in Moscow and mitigated it in Krasnodar Krai. Grain weight of the main spike was reduced by Rht-B1p in Moscow and to lesser extent in Krasnodar; Ppd-D1a and Vrn-B1a tended to partially compensate for this loss in Krasnodar Krai. Thousand grain weight was reduced on average by 5.3 g (16%) and 2.9 g (10%) in Moscow and Krasnodar Krai, respectively, but was partially compensated for by Ppd-D1a in Krasnodar Krai. Harvest index was increased due to Rht-B1p by 6 and 10% in Moscow and Krasnodar Krai, respectively. Rht-B1p resulted in a delay of heading by 1-2 days in Moscow. Ppd-D1a accelerated heading by 1 day and 6 days in Moscow and in Krasnodar Krai, respectively.

Conclusions: Rht-B1p could be introduced into wheat breeding along with dwarfing genes such as Rht-B1b and Rht-D1b. Special attention should be paid to its combination with Ppd-D1a and Vrn-B1a as regulators of developmental rates, compensators of adverse effects of Rht-B1p on productivity and enhancers of positive effect of Rht-B1p on harvest index.
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http://dx.doi.org/10.1186/s12870-020-02514-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7556923PMC
October 2020

A Data-Driven Approach to Carrier Screening for Common Recessive Diseases.

J Pers Med 2020 Sep 22;10(3). Epub 2020 Sep 22.

Federal State Institution «National Medical Research Center for Therapy and Preventive Medicine» of the Ministry of Healthcare of the Russian Federation, Petroverigsky per., 10, bld. 3, 101000 Moscow, Russia.

Genetic screening is an advanced tool for reducing recessive disease burden. Nowadays, it is still unclear as to the number of genes or their variants that are necessary for effective screening. This paper describes the development of a carrier screening custom panel for cystic fibrosis, phenylketonuria, alpha-1 antitrypsin deficiency, and sensorineural hearing loss consisting of 116 variants in the , , and genes. The approach is based on the cheapest and fastest method, on using a small number of genes, and on the estimation of the effectiveness of carriers' detection. The custom panel was tested on a population-based cohort that included 1244 participants. Genotypes were determined by the TaqMan OpenArray Genotyping platform on the QuantStudio 12K Flex Real-Time PCR System. The frequency of heterozygotes in the Russian population was 16.87% or 1:6 (CI95%: 14.76-19.00% by Clopper-Pearson exact method): in -2.81% (1:36), -2.33% (1:43), -4.90% (1:20), and -6.83% (1:15). The data on allele frequencies were obtained for the first time on a Russian population. The panel allows us to identify the vast majority of carriers of recessive diseases in the population. It is an effective approach to carrier screening for common recessive diseases.
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http://dx.doi.org/10.3390/jpm10030140DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7563953PMC
September 2020

Development of Specific Cytogenetic Markers for Wheat-Wheatgrass Hybrids Using Sequencing and qPCR Data.

Int J Mol Sci 2020 Jun 24;21(12). Epub 2020 Jun 24.

Laboratory of Applied Genomics and Crop Breeding, All-Russia Research Institute of Agricultural Biotechnology, Timiryazevskaya str. 42, Moscow 127550, Russia.

The cytogenetic study of wide hybrids of wheat has both practical and fundamental values. Partial wheat-wheatgrass hybrids (WWGHs) are interesting as a breeding bridge to confer valuable genes to wheat genome, as well as a model object that contains related genomes of . The development of cytogenetic markers is a process that requires long and laborious fluorescence in situ hybridization (FISH) testing of various probes before a suitable probe is found. In this study, we aimed to find an approach that allows to facilitate this process. Based on the data sequencing of we selected six tandem repeat (TR) clusters using RepeatExplorer2 pipeline and designed primers for each of them. We estimated the found TRs' abundance in the genomes of , , and four different WWGH accessions using real-time qPCR, and localized them on the chromosomes of the studied WWGHs using fluorescence in situ hybridization. As a result, we obtained three tandem repeat cytogenetic markers that specifically labeled wheatgrass chromosomes in the presence of bread wheat chromosomes. Moreover, we designed and tested primers for these repeats, and demonstrated that they can be used as qPCR markers for quick and cheap monitoring of the presence of certain chromosomes of wheatgrass in breeding programs.
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http://dx.doi.org/10.3390/ijms21124495DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7349979PMC
June 2020

Allele mining of TaGRF-2D gene 5'-UTR in Triticum aestivum and Aegilops tauschii genotypes.

PLoS One 2020 16;15(4):e0231704. Epub 2020 Apr 16.

Laboratory of Applied Genomics and Crop Breeding, All-Russia Research Institute of Agricultural Biotechnology, Moscow, Russia.

The low diversity of the D-subgenome of bread wheat requires the involvement of new alleles for breeding. In grasses, the allelic state of Growth Regulating Factor (GRF) gene is correlated with nitrogen uptake. In this study, we characterized the sequence of TaGRF-2D and assessed its diversity in bread wheat and goatgrass Aegilops tauschii (genome DD). In silico analysis was performed for reference sequence searching, primer pairs design and sequence assembly. The gene sequence was obtained using Illumina and Sanger sequencing. The complete sequences of TaGRF-2D were obtained for 18 varieties of wheat. The polymorphism in the presence/absence of two GCAGCC repeats in 5' UTR was revealed and the GRF-2D-SSR marker was developed. Our results showed that the alleles 5' UTR-250 and 5' UTR-238 were present in wheat varieties, 5' UTR-250 was presented in the majority of wheat varieties. In Ae. tauschii ssp. strangulata (likely donor of the D-subgenome of polyploid wheat), most accessions carried the 5' UTR-250 allele, whilst most Ae. tauschii ssp. tauschii have 5' UTR-244. The developed GRF-2D-SSR marker can be used to study the genetic diversity of wheat and Ae. tauschii.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0231704PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7162470PMC
July 2020

Copy Number Variation of Transposable Elements in and Its Diploid Relative Species.

Plants (Basel) 2019 Dec 21;9(1). Epub 2019 Dec 21.

Laboratory of Applied Genomics and Crop Breeding, All-Russia Research Institute of Agricultural Biotechnology, Moscow 127550, Russia.

Diploid and polyploid wild species of have complex relationships, and the understanding of their evolution and speciation could help to increase the usability of them in wheat breeding as a source of genetic diversity. The diploid species (St), (J), (V) derived from a hypothetical common ancestor are considered to be possible subgenome donors in hexaploid species (JJSt, where indices r, v, and s stand for the partial relation to the genomes of , , and , respectively). We quantified 10 families of transposable elements (TEs) in , , (per one genome), and (per one average subgenome) using the quantitative real time PCR assay and compared their abundance within the studied genomes as well as between them. was the most abundant among all studied elements in , , and and among elements in all studied species. Among elements, and showed the highest and close abundance with the exception of , and comprised the majority of all studied elements in . , and showed similar abundance among diploids and in hexaploid; and demonstrated sharp differences between diploid genomes. The relationships between genomes of species based on the studied TE abundance and the role of TEs in speciation and polyploidization in the light of the current phylogenetic models is discussed.
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http://dx.doi.org/10.3390/plants9010015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7020174PMC
December 2019

Development of new cytogenetic markers for (Podp.) Z.-W. Liu & R.-C. Wang.

Comp Cytogenet 2019 13;13(3):231-243. Epub 2019 Aug 13.

Laboratory of Applied Genomics and Crop Breeding, All-Russia Research Institute of Agricultural Biotechnology, Timiryazevskaya str. 42, Moscow 127550, Russia All-Russia Research Institute of Agricultural Biotechnology Moscow Russia.

(Podpěra, 1902) Z.-W. Liu & R.-C.Wang, 1993 is an important polyploid wild perennial Triticeae species that is widely used as a source of valuable genes for wheat but its genomic constitution has long been debated. For its chromosome identification, only a limited set of FISH probes has been used. The development of new cytogenetic markers for chromosomes is of great importance both for cytogenetic characterization of wheat-wheatgrass hybrids and for fundamental comparative studies of phylogenetic relationships between species. Here, we report on the development of five cytogenetic markers for based on repetitive satellite DNA of which sequences were selected from the whole genome sequence of Cosson, 1849. Using real-time quantitative PCR we estimated the abundance of the found repeats: P720 and P427 had the highest abundance and P132, P332 and P170 had lower quantity in genome. Using fluorescence hybridization (FISH) we localized five repeats to different regions of the chromosomes of . Using reprobing multicolor FISH we colocalized the probes between each other. The distribution of these found repeats in the Triticeae genomes and its usability as cytogenetic markers for chromosomes of are discussed.
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http://dx.doi.org/10.3897/CompCytogen.v13i3.36112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6702164PMC
August 2019

Variation in Copy Number of Ty3/Gypsy Centromeric Retrotransposons in the Genomes of Thinopyrum intermedium and Its Diploid Progenitors.

PLoS One 2016 27;11(4):e0154241. Epub 2016 Apr 27.

Centre for Molecular Biotechnology, Russian State Agrarian University-Moscow Timiryazev Agricultural Academy, Timiryazevskaya St. 49, 127550, Moscow, Russia.

Speciation and allopolyploidization in cereals may be accompanied by dramatic changes in abundance of centromeric repeated transposable elements. Here we demonstrate that the reverse transcriptase part of Ty3/gypsy centromeric retrotransposon (RT-CR) is highly conservative in the segmental hexaploid Thinopyrum intermedium (JrJvsSt) and its possible diploid progenitors Th. bessarabicum (Jb), Pseudoroegneria spicata (St) and Dasypyrum villosum (V) but the abundance of the repeats varied to a large extent. Fluorescence in situ hybridization (FISH) showed hybridization signals in centromeric region of all chromosomes in the studied species, although the intensity of the signals drastically differed. In Th. intermedium, the strongest signal of RT-CR probe was detected on the chromosomes of Jv, intermediate on Jr and faint on Js and St subgenome suggesting different abundance of RT-CR on the individual chromosomes rather than the sequence specificity of RT-CRs of the subgenomes. RT-CR quantification using real-time PCR revealed that its content per genome in Th. bessarabicum is ~ 2 times and P. spicata is ~ 1,5 times higher than in genome of D. villosum. The possible burst of Ty3/gypsy centromeric retrotransposon in Th. intermedium during allopolyploidization and its role in proper mitotic and meiotic chromosome behavior in a nascent allopolyploid is discussed.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0154241PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4847875PMC
March 2017

Molecular cytogenetic analysis of monoecious hemp (Cannabis sativa L.) cultivars reveals its karyotype variations and sex chromosomes constitution.

Protoplasma 2016 May 7;253(3):895-901. Epub 2015 Jul 7.

Center for Molecular Biotechnology, Russian State Agrarian University-MTAA, Timiryazevskaya St. 49, Moscow, 127550, Russia.

Hemp (Cannabis sativa L., 2n = 20) is a dioecious plant. Sex expression is controlled by an X-to-autosome balance system consisting of the heteromorphic sex chromosomes XY for males and XX for females. Genetically monoecious hemp offers several agronomic advantages compared to the dioecious cultivars that are widely used in hemp cultivation. The male or female origin of monoecious maternal plants is unknown. Additionally, the sex chromosome composition of monoecious hemp forms remains unknown. In this study, we examine the sex chromosome makeup in monoecious hemp using a cytogenetic approach. Eight monoecious and two dioecious cultivars were used. The DNA of 210 monoecious plants was used for PCR analysis with the male-associated markers MADC2 and SCAR323. All monoecious plants showed female amplification patterns. Fluorescence in situ hybridization (FISH) with the subtelomeric CS-1 probe to chromosomes plates and karyotyping revealed a lack of Y chromosome and presence of XX sex chromosomes in monoecious cultivars with the chromosome number 2n = 20. There was a high level of intra- and intercultivar karyotype variation detected. The results of this study can be used for further analysis of the genetic basis of sex expression in plants.
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http://dx.doi.org/10.1007/s00709-015-0851-0DOI Listing
May 2016

Use of laser microdissection for the construction of Humulusjaponicus Siebold et Zuccarini, 1846 (Cannabaceae) sex chromosome-specific DNA library and cytogenetics analysis.

Comp Cytogenet 2014 10;8(4):323-36. Epub 2014 Dec 10.

Centre for Molecular Biotechnology, Russian State Agrarian University - Moscow Timiryazev Agricultural Academy, Moscow 127550, Timiryazevskaya street, 49, Russia.

Dioecy is relatively rare among plant species, and distinguishable sex chromosomes have been reported in few dioecious species. The multiple sex chromosome system (XX/XY1Y2) of Humulusjaponicus Siebold et Zuccarini, 1846 differs from that of other members of the family Cannabaceae, in which the XX/XY chromosome system is present. Sex chromosomes of Humulusjaponicus were isolated from meiotic chromosome spreads of males by laser microdissection with the P.A.L.M. MicroLaser system. The chromosomal DNA was directly amplified by degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR). Fast fluorescence in situ hybridization (FAST-FISH) using a labeled, chromosome-specific DOP-PCR product as a probe showed preferential hybridization to sex chromosomes. In addition, the DOP-PCR product was used to construct a short-insert, Humulusjaponicus sex chromosomes-specific DNA library. The randomly sequenced clones showed that about 12% of them have significant homology to Humuluslupulus and 88% to Cannabissativa Linnaeus, 1753 sequences from GenBank database. Forty-four percent of the sequences show homology to plant retroelements. It was concluded that laser microdissection is a useful tool for isolating the DNA of sex chromosomes of Humulusjaponicus and for the construction of chromosome-specific DNA libraries for the study of the structure and evolution of sex chromosomes. The results provide the potential for identifying unique or sex chromosome-specific sequence elements in Humulusjaponicus and could aid in the identification of sex chromosome-specific repeat and coding regions through chromosome isolation and genome complexity reduction.
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http://dx.doi.org/10.3897/CompCytogen.v8i4.8473DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4296719PMC
January 2015

Molecular cytogenetic characterization of the dioecious Cannabis sativa with an XY chromosome sex determination system.

PLoS One 2014 21;9(1):e85118. Epub 2014 Jan 21.

Centre for Molecular Biotechnology, Russian State Agrarian University - Moscow Timiryazev Agricultural Academy, Moscow, Russia.

Hemp (Cannabis sativa L.) was karyotyped using by DAPI/C-banding staining to provide chromosome measurements, and by fluorescence in situ hybridization with probes for 45 rDNA (pTa71), 5S rDNA (pCT4.2), a subtelomeric repeat (CS-1) and the Arabidopsis telomere probes. The karyotype has 18 autosomes plus a sex chromosome pair (XX in female and XY in male plants). The autosomes are difficult to distinguish morphologically, but three pairs could be distinguished using the probes. The Y chromosome is larger than the autosomes, and carries a fully heterochromatic DAPI positive arm and CS-1 repeats only on the less intensely DAPI-stained, euchromatic arm. The X is the largest chromosome of all, and carries CS-1 subtelomeric repeats on both arms. The meiotic configuration of the sex bivalent locates a pseudoautosomal region of the Y chromosome at the end of the euchromatic CS-1-carrying arm. Our molecular cytogenetic study of the C. sativa sex chromosomes is a starting point for helping to make C. sativa a promising model to study sex chromosome evolution.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0085118PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3897423PMC
October 2014

Sex chromosome differentiation in Humulus japonicus Siebold & Zuccarini, 1846 (Cannabaceae) revealed by fluorescence in situ hybridization of subtelomeric repeat.

Comp Cytogenet 2012 10;6(3):239-47. Epub 2012 Jul 10.

Centre for Molecular Biotechnology, Russian State Agrarian University - Moscow Timiryazev Agricultural Academy, Moscow 127550, Timiryazevskaya Street, 49, Russia.

Humulus japonicus Siebold et Zucc (Japanese hop) is a dioecious species of the family Cannabaceae. The chromosome number is 2n = 16 = 14 + XX for females and 2n = 17 = 14 + XY1Y2 for male. To date, no fluorescence in situ hybridization (FISH) markers have been established for the identification of Humulus japonicus sex chromosomes. In this paper, we report a method for the mitotic and meiotic sex chromosome differentiation in Humulus japonicus by FISH for HJSR, a high copy subtelomeric repeat. The signal is present in the subtelomeric region of one arm of the X chromosome. We demonstrate that males have two Y chromosomes that differ in FISH signal with the HJSR probe. Indeed, the HJSR probe hybridizes to a subtelomeric region on both arms of chromosome Y1 but not of chromosome Y2. The orientation and position of pseudoautosomal regions (PAR1 and PAR2) were also determined.
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http://dx.doi.org/10.3897/CompCytogen.v6i3.3261DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3833800PMC
November 2013
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