Identification of the gene coding for seed cotyledon albumin SCA in the pea (Pisum L.) genome.

Albumins SCA and SAA are short, highly hydrophilic proteins accumulated in large quantities in the cotyledons and seed axes, respectively, of a dry pea (Pisum sativum L.) seed. SCA was earlier shown to have two allelic variants differing in mobility in polyacrylamide gel electrophoresis in acid medium. Using them, the corresponding gene SCA was mapped on Linkage Group V. This protein was used as a useful genetic and phylogeographical marker, which still required electrophoretic analysis of the protein while the DNA sequence of the corresponding SCA gene remained unknown. Based on the length, the positive charge under acidic conditions and the number of lysine residues of SCA and SAA albumins, estimated earlier electrophoretically, the data available in public databases were searched for candidates for the SCA gene among coding sequences residing in the region of the pea genome which, taking into account the synteny of the pea and Medicago truncatula genomes, corresponds to the map position of SCA. Then we sequenced them in a number of pea accessions. Concordance of the earlier electrophoretic data and sequence variation indicated the sequence Psat0s797g0160 of the reference pea genome to be the SCA gene. The sequence Psat0s797g0240 could encode a minor related albumin SA-a2, while a candidate gene for albumin SAA is still missing (as well as electrophoretic variation of both latter albumins). DNA amplif ication using original primers SCA1_3f and SCA1_3r from genomic DNA and restriction by endonuclease HindII made it possible to distinguish the SCA alleles coding for protein products with different charges without sequencing the gene. Thus, the gene encoding the highly hydrophilic albumin SCA accumulated in pea seeds, the alleles of which are useful for classif ication of pea wild relatives, has now been identif ied in the pea genome and a convenient CAPS marker has been developed on its basis.

Wild pea (Pisum sativum L. subsp. elatius (Bieb.) Aschers. et Graebn. s.l.) at the periphery of its range: Zagros Mountains.

Characteristics of wild peas and their habitats at the periphery of the range are interesting with respect to their potential importance for pre-breeding programs aimed at selection for different environmental conditions. However, wild pea diversity in peripheral regions is insufficiently represented in the existing germplasm collections. In such regions, wild pea populations are rare, small in size and suffer from climatic change and land exploitation, hence their focused search is strongly desirable. A two-week-long expedition to Iran in May 2017 revealed two small populations of the wild pea (Pisum sativum subsp. elatius) in the Zagros Mts, in Aligudarz and Khorramabad Districts of Lorestan Province, Iran, at elevations of 1841 and 1971 m a.s.l., respectively. Their habitats are briefly described. Two pea accessions derived from them, CE9 and CE10, were characterised for some visible and molecular characters. These peas appeared to belong to the evolutionary lineage B, recognised by us earlier in P. sativum as opposed to the so-called lineage AC. They contain a unique non-conservative substitution in subtype 5 of histone H1 and turned to be most related to some wild pea accessions originating from southern and south-eastern Turkey and Golan Heights. Scarce information available on wild pea occurrence in Iran suggests their existence in the south-western principal slope of Zagros Mts and southern principal slopes of Elborz and Kopet Dagh Mts. It was found that wild peas representing the evolutionary lineage B produce poorly open and poorly coloured flowers (as reported by us earlier) only in the greenhouse conditions but normally pigmented and open flowers in the wild and mesh houses at open air in Israel. Some issues of pea taxonomy are discussed.

Identification of genome compositions in allopolyploid species of the genus Elymus (Poaceae: Triticeae) in the Asian part of Russia by CAPS analysis.

The genus Elymus L., together with wheat, rye, and barley, belongs to the tribe Triticeae. Apart from its economic value, this tribe is characterized by abundance of polyploid taxa formed in the course of remote hybridization. Single-copy nuclear genes are convenient markers for identification of source genomes incorporated into polyploids. In the present work, a CAPS-marker is developed to distinguish basic St, H, and Y genomes comprising polyploid genomes of Asiatic species of the genus Elymus. The test is based on electrophoretic analysis of restriction patterns of a PCR-amplified fragment of the gene coding for beta-amylase. There are about 50 Elymus species in Russia, and most of them are supposed to possess one of three haplome combinations, StH, StY and StHY. Boreal StH-genomic species endemic for Russia are the least studied. On the basis of nucleotide sequences from public databases, TaqI restrictase was selected, as it produced patterns of restriction fragments specific for St, H, and Y haplomes easily recognizable in agarose gel. A sample of 68 accessions belonging to 32 species was analyzed. In 15 species, the earlier known genomic constitutions were confirmed, but in E. kamoji this assay failed to reveal the presence of H genome. This unusual H genome was suggested to originate from a different Hordeum species. In 16 species, genomic constitutions were identified for the first time. Fifteen accessions from Asian Russia possessed the genomic constitution StStHH, and E. amurensis, phylogenetically close to the StY-genomic species E. ciliaris, had the genomic constitution StStYY. It is inferred that the center of species diversity of the StH-genomic group is shifted to the north as compared to the center of origin of StY-genomic species, confined to China. Key words: Elymus; taxonomy; allopolyploids; genome constitution; CAPS markers.

[Evolution of pandemic influenza virus A(H1N1)pdm09 in 2009-2016: dynamics of receptor specificity of the first hemagglutinin subunit (HA1).]

INTRODUCTION: The new reassortant of the swine flu virus A(H1N1)pdm09, which emerged in 2009, overcame the species barrier and caused the 2009-2010 pandemic. One of the key points required for the influenza virus to overcome the species barrier and adapt it to humans is its specific binding to the receptors on the epithelium of the human respiratory tract. PURPOSE: Studying the dynamics of changes in receptor specificity (RS) of the HA1 subunit of the hemagglutinin of the influenza A(H1N1)pdm09 virus strains isolated during the period 2009-2016 on the territory of the Russian Federation, and an analysis of the possible impact of these changes on the incidence rates of the population of the Russian Federation of pandemic influenza in certain epidemic seasons. MATERIAL AND METHODS: Standard methods of collecting clinical materials, isolation of influenza viruses, their typing and genome sequencing were used. For the study of RS of influenza A virus (H1N1)pdm09, the method of solid phase sialosidenzyme analysis was used. RESULTS: It is shown that the change in the parameter W3/6 , which characterizes the degree of a2-3 receptor specificity (a2-3-RS) of the influenza virus A(H1N1) pdm09 over a2-6-RS, coincides with the change in the incidence rates of the Russian Federation's pandemic flu in separate epidemic seasons. There is a tendency to increase the affinity of the virus A(H1N1)pdm09 to α2-3 analogs of the sialyl-glycan receptors of the human respiratory tract epithelium - α2-3-sialoglycopolymers (α2-3-SGP), and falls to α2-6-SGP, with the virus showing the greatest affinity for sulfated sialoglycopolymers. DISCUSSION: Screening for RS strains of influenza A (H1N1)pdm09 virus isolated on the territory of the Russian Federation in 2009-2016 revealed a decrease in the affinity of viruses for a2-6-sialosides, especially for 6'SL-SGP, which is probably due to the presence of amino acid substitutions in the 222 and 223 positions of RBS HA1 viruses. Previous studies have shown that the presence of such substitutions correlates with an increase in the virulence of the influenza A virus (H1N1)pdm09 [16, 23]. Probably, the pandemic virus has evolved towards the selection of more virulent pneumotropic variants. CONCLUSION: Monitoring of the receptor specificity of a pandemic influenza virus makes it possible to identify strains with altered RS to the epithelium of the human respiratory tract and an increased ability to transfer from person to person. Change in the period 2009-2016 the W3/6 parameter characterizing the degree of α2-3-RS excess of the influenza A(H1N1)pdm09 virus over α2-6-RS, coincides with the change in the incidence rates of the pandemic influenza population of the Russian Federation in certain epidemic seasons.

[The peculiarities of the influenza epidemics in some areas of Russia during 2012-2013 season. The influenza A (H1N1) pdm09 virus domination in European countries].

The peculiarities of the influenza viruses circulation in 2012-2013 are discussed. The results were obtained in 10 cities of Russia, where basic laboratories of the Influenza Ecology and Epidemics Center of on the basis of Ivanovsky Institute of Virology, Ministry of Health of the Russian Federation, are situated. The increasing rate of the ARD morbidity caused by influenza viruses was observed in January-March 2013. The highest indices of the morbidity were detected during 6-7 weeks with the following decreasing rate till threshold levels to week 14. The influenza A (H1N1) pdm09, A (H3N2), and B viruses were the cause of the epidemic, but their activity differed over areas of Russia. The results of study of the antigenic and genetic properties of the influenza strains demonstrated closed relatives with respect to vaccine strains. In addition, some heterogeneity of the circulating strains and their drift variants were found as well. All tested strains were sensitive to oseltamivir (excluding one A (H1N1) pdm09 strain), zanamivir, arbidol, and remained resistant to rimantadine. The ratio of the ARD viruses was comparable with the last epidemic seasons.

Wild peas vary in their cross-compatibility with cultivated pea (Pisum sativum subsp. sativum L.) depending on alleles of a nuclear-cytoplasmic incompatibility locus.

KEY MESSAGE: Divergent wild and endemic peas differ in hybrid sterility in reciprocal crosses with cultivated pea depending on alleles of a nuclear 'speciation gene' involved in nuclear-cytoplasmic compatibility. BACKGROUND: In hybrids between cultivated and wild peas, nuclear-cytoplasmic conflict frequently occurs. One of the nuclear genes involved, Scs1, was earlier mapped on Linkage Group III. RESULTS: In reciprocal crosses of seven divergent pea accessions with cultivated P. sativum, some alleles of Scs1 manifested incompatibility with an alien cytoplasm as a decrease in pollen fertility to about 50 % in the heterozygotes and lack of some genotypic classes among F2 segregants. Earlier, we defined monophyletic evolutionary lineages A, B, C and D of pea according to allelic state of three markers, from nuclear, plastid and mitochondrial genomes. All tested representatives of wild peas from the lineages A and C exhibited incompatibility due to Scs1 deleterious effects in crosses with testerlines of P. sativum subsp. sativum (the common cultivated pea) at least in one direction. A wild pea from the lineage B and a cultivated pea from the lineage D were compatible with the testerline in both directions. The tested accession of cultivated P. abyssinicum (lineage A) was partially compatible in both directions. The Scs1 alleles of some pea accessions even originating from the same geographic area were remarkably different in their compatibility with cultivated Pisum sativum cytoplasm. CONCLUSION: Variability of a gene involved in reproductive isolation is of important evolutionary role and nominate Scs1 as a speciation gene.

[Correlation between the receptor specificity of pandemic influenza A (H1N1)pdm09 virus strains isolated in 2009-2011 and the structure of the receptor-binding site and the probability of fatal primary viral pneumonia].

The receptor specificity (RS) of pandemic influenza A(H1N1) pdm09 virus strains deposited into the State Collection of Viruses of the Russian Federation, D. I. Ivanovsky Research Institute of Virology, Ministry of Health and Social Development of Russia, in the 2009-2010 and 2010-2011 epidemic seasons to a panel of 9 sialoglycopolymers (SGP). The strains were divided into 3 groups according to the W(3/6) index proposed by the authors, which was equal to the amount of reactivities to unbranched alpha2-3-SGP to that of reactivities to unbranched alphal-6-SGP: W(3/6) < or = 1.0; 1.0 < W(3/6) < or = 1.5. The W(3/6) < or = 1.5 group showed a predominance of a2-3-RS, attended by the high incidence of fatal primary viral pneumonias (FPVP) (60.0%) and amino acid replacements in the HA1 receptor-binding site (RBS) (80.0%): D222{G, N} and Q223R. The 1.0 < W(3/6) < or = 1.5 group was characterized by mixed alpha2-3/alpha2-6-RS with the incidence of FPVP (29.7%) and amino acid replacements in the HA1 RBS (40.5%) (D222{G, N, V} and Q223), respectively. In the W(3/6) < or = 1.0 group, alpha2-6-RS was prevalent, FPVPs were absent and amino acid replacements in HA1 RBS (D222{G, E}) were seen only in 6.0% of cases. The number of strains with increased specificity to alpha2-3-sialosides increased in the 2010-2011 epidemic season as compared to the previous season. With their further spread among the population, there may be a rise in cases of severe primary viral pneumonias with possible fatal outcomes, which can be, however, accompanied by a decrease in the capacity of mutants to air-dropwise transmission.

[A possible association of fatal pneumonia with mutations of pandemic influenza A/H1N1 sw1 virus in the receptor-binding site of the HA1 subunit].

The paper gives the results of sequence analysis of 150 positive samples in real-time RT-PCR, including 47 autopsy materials from patients (including 10 pregnant women), who died from fatal pneumonia mainly in November-December 2009, in whom the lifetime etiological diagnosis had not been made and hence no early etiotropic therapy performed. 70% of the primary materials from the deceased patients were found to have pandemic influenza A(H1N1) v mutants in the lung tissue with D222G (15%), D222N (15%), D222E (2%) substitutions, as well as a mixture of mutants (38%). Nasopharyngeal lavages from 3 Chukotka deceased patients exhibited only consensus (nonmutant) D222 virus variants; there was a mixture of consensus and mutant virus variants in the trachea and a mixture of mutant ones in the lung. Preliminary data from the study of the interaction of the hemagglutinin of two strains having D222G and D222N mutations with 9 oligosaccharides imitating the variants of cell receptors for influenza A virus suggest that there is a double receptor specificity for alpha2'-3' and alpha2'-6'-sialosides with a preponderance of alpha2'-3'-specificity. Further spread of the mutants that have acquired a high virulence and preserved their capacity for the respiratory route of human infection may lead to the situation similar to that seen in the 1918-1919 pandemic. Another scenario for evolution of the virus is to preserve its receptor specificity for alpha2'-3'-sialosides and high virulence with losses of alpha2'-6' specificity and capacity for aerosol transmission, by damping the pandemic.

[Meiotic abnormalities as expression of nuclear-cytoplasmic incompatibility in crosses of Pisum sativum subspecies].

Meiosis in anthers and mitosis in somatic cells were studied in reciprocal F1 hybrids of the accession VIR320, which belonged to wild Pisum sativum ssp. elatius (Bieb.) Schmal., and the laboratory line Sprint-1. When VIR320 was used as a maternal form, the hybrids displayed nuclear-cytoplasmic conflict, which caused chlorophyll defects and meiotic abnormalities. One or two chromosomes lagged in the equatorial region during chromosome segregation to the poles, distorting cytokinesis and yielding abnormal microspores. Chlorophyll defects were not observed, and meiotic abnormalities were far less frequent in reciprocal hybrids and in the case of an abnormal paternal inheritance of plastids from Sprint-1. Mitosis lacked overt abnormalities in all of the hybrids.

[Enzyme-linked immunosorbent assay for detection of antibodies to porcine reproductive and respiratory syndrome virus, by using recombinant nucleocapsid protein N].

Recombinant nucleocapsid (rN) protein N of porcine reproductive and respiratory syndrome virus (PRRSV) was prepared, by using the E. coli expressiom system. Insertion of a polyhistidine marker into the structure of the protein allowed the latter to be purified by metal-chelate affinity chromatography. The purity of protein was confirmed by PAAG electrophoresis and its immunospecificity was verified by immunoblotting using rN-specific monoclonal antibodies. The protein was used as an antigen to develop indirect ELISA of PRRSV antibodies. ELISA was shown to be highly sensitive and specific.

[Enzyme immunoassay for detection of porcine circovirus type 2, by using the recombinant capsid protein ORF-2].

Recombinant antigen ORF2 from porcine circovirus type 2 (PCV-2) was produced, by using the baculovirus expression system, with histidine tags to allow purification by metal-chelate affinity chromatography. The purity of the protein was verified by polyacrylamide gel electrophoresis; and its immunospecificity was confirmed by the immunoblotting test using reference PCV-2-positive and PCV-2-negative porcine sera and monoclonal antibodies. The protein was used as an antigen to develop an indirect enzyme immunoassay (EIA) of PCV-2 antibodies. EIA was shown to have a high sensitivity and specificity as compared with indirect immunofluorescence test. Porcine serum samples from 15 pig-breeding farms of the Russian Federation were studied. Seropositive samples were found in all age pig groups in all the farms, The number of seropositive animals was shown to be directly related to its age.

[The role of laparoscopy in the diagnosis and treatment of early postoperative complications].

Results of 276 laparoscopic operations performed for diagnostic and therapeutic purposes in the early postoperative period in 198 patients are analyzed. All the patients had been previously operated on the organs of the abdominal cavity and retroperitoneal space: in 131 patients the investigations were performed with a diagnostic purpose; in 67 patients a laparoscopic method was used for treatment of complications of the early postoperative period. This problem can be solved by using the methods allowing diagnosing the complication as early as possible, before the development of irreversible alterations, and allowing to choose the most rational methods of treatment.

Structure of allelic variants of subtype 5 of histone H1 in pea Pisum sativum L.

The pea genome contains seven histone H1 genes encoding different subtypes. Previously, the DNA sequence of only one gene, His1, coding for the subtype H1-1, had been identified. We isolated a histone H1 allele from a pea genomic DNA library. Data from the electrophoretic mobility of the pea H1 subtypes and their N-bromosuccinimide cleavage products indicated that the newly isolated gene corresponded to the H1-5 subtype encoded by His5. We confirmed this result by sequencing the gene from three pea lines with H1-5 allelic variants of altered electrophoretic mobility. The allele of the slow H1-5 variant differed from the standard allele by a nucleotide substitution that caused the replacement of the positively charged lysine with asparagine in the DNA-interacting domain of the histone molecule. A temperature-related occurrence had previously been demonstrated for this H1-5 variant in a study on a worldwide collection of pea germplasm. The variant tended to occur at higher frequencies in geographic regions with a cold climate. The fast allelic variant of H1-5 displayed a deletion resulting in the loss of a duplicated pentapeptide in the C-terminal domain.

[Genetic variability of the nucleocapsid protein of the virus of the porcine reproductive and respiratory syndrome]. / Variabel'nost' gena belka nukleokapsida virusa reproduktivnogo i respiratornogo sindroma svinei (RRSS).

The porcine reproductive and respiratory syndrome (PRRS) is a contagious viral pathology caused by PRRS virus. There are 2 types of the above virus--the European and American ones. Distribution patterns of the PRRS virus were studied for Russia and Byelorussia. Above 700 porcine sera obtained from 32 households of 21 Russia's administrative regions and from 19 households of 6 Byelorussia's administrative regions were tested for presence of antibodies to the PRRS virus. Simultaneously, the samples were tested for virus presence by polymerase chain reaction (PCR). It was proven serologically that the PRRS virus is widespread in the territories of Russia and Byelorussia. Noteworthily, all field isolates found in Russia and Byelorussia belong to the European type. Not a single viral isolate of the American PRRS type was found. The nucleocapsid (N) recombinant protein was obtained on the basis of the Russian field isolate of the PRRS virus by using the E. coli. expression system. Finally, it was shown as possible to use the recombinant protein in indirect immune enzyme assay for the sake of detecting the antibodies to the PRRS virus.

[Synthesis of recombinant proteins from the American and European viruses of the porcine reproductive and respiratory syndrome in baculovirus expression system and their antigenic properties]. / Sintez v bakulovirusnoi sisteme rekombinantnykh belkov amerikanskogo i evropeiskogo tipov virusa reproduktivnogo i respiratornogo sindroma svinei i ikh antigennye svoistva.

The baculovirus expression system was made use of to derive the recombinant nucleocapsid (N) proteins of the American and European virus types and of the porcine reproductive and respiratory syndrome (PRRS). The obtained products were purified by metal-affine chromatography and their specificity was confirmed in immunechemical reactions with reference monoclonal antibodies. The antigenic activity of recombinant proteins was studied by indirect immune enzyme assay (IEA) with porcine serum, which had been in advance characterized by the "HerdCheck" kit (IDEXX Co.). It was shown as possible to apply the derived recombinant antigens in determining, by indirect IEA, antibodies to the PRRS virus.

Tertiary trisomics in the garden pea as a model of B chromosome evolution in plants.

It is hypothesized that, in plants, genetically empty B chromosomes may originate from the extra chromosome (E) of tertiary trisomics if (i) the region of basic chromosomes homologous to the E (H-region) harbors a sporophytic lethal covered by the wild-type allele in E, and (ii) crossing-over between E and the H-region is suppressed. Under these conditions, most loss-of-function mutations occurring in the H-region are deleterious for haploid gametophytes, whereas those occurring in E are neutral or advantageous for hyperploid (n+1) gametophytes. As a result, natural selection at the gametophyte level can lead to the degeneration of E, leaving the H-region intact. Using Hammarlund translocation T(3-6)a, we synthesized two trisomic lines of the garden pea (Pisum sativum L.), where E was composed of the short arms of chromosomes 3 and 6 and the H-region carried recessive markers. In the trisomic line TRIS, we found few crossovers between E and the H-region. In the trisomic line TRUST, obtained after a change of basic chromosome constitution, recombination in this region was completely suppressed. After induction in the H-region of TRUST of a recessive sporophytic mutation rmv, two 15-chromosome lines of stable trisomics were established. One of them passed 11 generations, having produced more than 6000 individuals, all of them trisomic, and E remained present as a single element with no pairing partners. No tetrasomics were detected in these lines. If such trisomics occurred in nature, their extra chromosomes are likely to become a B chromosome.

Mortality of pollen grains may result from errors of meiosis: study of pollen tetrads in Typha latifolia L.

In the cattail Typha latifolia the four haploid products of meiosis remain attached and form the flat tetrad of pollen grains. Gametophytic lethals arisen de novo in diploid cells of sporophyte must manifest themselves as pollen tetrads with two dead grains. This could allow to estimate the rate of recessive lethals arresting pollen grain development. We studied pollen samples collected from 44 sprouts in two populations in the vicinity of Novosibirsk. The anomalous tetrads T1, T2, T3, and T4 carrying one, two, three, and four dead grains, respectively, were detected in each sampled individual. The mean frequency of all anomalous tetrads in the two populations was 3.4% and 8.7%. The frequencies of tetrad classes varied widely among the individuals with correlation coefficient up to 0.94, but their ratios remained nearly constant. The majority of anomalous tetrads were presented by T1 and T2 classes (their sum comprising 72.7 and 74.0% in two populations), T1 being a little more abundant. The observed pattern of frequencies of tetrads with dead grains can be explained by errors of male meiosis such as chromosome non-disjunction in both meiotic divisions. The tetrads with two dead pollen grains may result mostly from non-disjunction in anaphase I, and those with one pollen grain from non-disjunction in anaphase II, thus making tetrad analysis ineffective for estimating the rate of gametophytic lethals.