Evaluation of apple varieties of the Sverdlovsk horticultural breeding station according to the ethylene biosynthesis genes using molecular markers

One of the directions of apple breeding in the Middle Urals is the development of varieties with a long-term storability. The ability of apples to maintain their consumer qualities for a long period is one of the most important indicators of the variety. A significant role in the storage of apples is played by the amount of ethylene produced in them. The paper presents the results of identification of genes involved in the control of ethylene biosynthesis in apple varieties selected by the Sverdlovsk horticultural breeding station. A total of 21 apple varieties were analyzed. The main objective of the study was to detect Md-ACO1-1 and Md-ACS1-2 alleles in the homozygous state. The combination of these alleles in one genotype reduces the production of ethylene in fruits, which contributes to their long-term storability. The analysis showed the availability of polymorphism in the two studied genes. The Md-ACO1 gene is characterized by the availability of two alleles in most varieties. The homozygous Md-ACO1-1 allele was identified in the Isetskoe pozdnee variety. Analysis of the Md-ACS1 gene revealed the predominance of the Md-ACS1-1 allele form. The Md-ACS1-2 allelic form was observed only in heterozygous samples. No combination of Md-ACO1-1 and Md-ACS1-2 alleles was found in the homozygous state. However, heterozygous forms are also of interest for breeding. They can serve as a source of a character of reduced ethylene biosynthesis when creating varieties with a long-term storability. Such varieties are Sokol yasnyy, Aksena, Rozovatoe zimnee, Sverdlovchanin, Isetskoe pozdnee, Blagaya vest'. The comparison of fruit storage periods and the genotype of the variety has been made. Allelesassociated with a reduced level of ethylene biosynthesis are typical both for the varieties with low and high storability.

Polymorphism in coding region of human CYP2D6 gene contributes to failed radical cure of vivax malaria using primaquine

Background: Recent studies found that damaged enzyme activity of cytochrome P450 isoenzyme 2D6 (CYP2D6) can lead to compromised efficacy of primaquine in killing Plasmodium during liver dormancy and consequently relapsed malaria. Therefore, WHO has listed the decrease enzyme activity of CYP2D6 as one of the four scenarios that are not suitable for primaquine therapy. Based on analysis of CYP2D6 gene polymorphisms in relapsed vivax malaria patients, this study preliminarily revealed the genetic association between human CYP2D6 genotype and the declined efficacy of primaquine treatment in vivax malaria. Methods: Blood samples of vivax malaria cases treated with "chloroquine/primaquine eight-day therapy" from 2014 to 2018 in Yunnan Province were collected, and the vivax malaria relapsed cases after clinical curing were determined by identifying the infected Plasmodium isolates through gene traceability analysis. Human genomic DNA was extracted from blood samples, and 9 exons of CYP2D6 gene were amplified by PCR and then sequenced. The CDS of CYP2D6 gene was obtained by alignment and splicing with the wild-type reference sequence. The mutation types of coding DNA sequences and their association with vivax malaria relapse were analyzed, and the activity phenotype of CYP2D6 isozyme was predicted by its allelic form. Results: One hundred and fifty-six blood samples from 75relapsed cases of vivax malaria and the same number of 75 non-relapsed vivax malaria cases were collected for PCR amplification. Two amplification products (2411bp and 2388bp) containing exon1-4 and exon5-9 of CYP2D6 gene were obtained from every sample. The 1491-bp-length CDS chains of CYP2D6 gene were obtained from those 150 samples, and were defined as 24 haplotypes (Hap_1~Hap_24).17 haplotypes were determined from the sequences of vivax malaria relapsed patients and 15 haplotypes were from those of non-relapsed patients.Hap_6 sequencing showed G>C, C>T and G>C base substitutions at three loci of c.408, c.886 and c.1457, resulting in V136V invariance and R296C and S486T variation at 136th, 296th and 486th amino acid, and the odds ratio of Hap_6 to vivax malaria relapse was 5.615 (P˂0.05).The diploids of CYP2D6 gene in relapsed cases were 100% mutation homozygous (C/C, 10/10) at c.408, 80% (8/10) at c.1457 and 20% (2/10) at c.886, respectively. However, the coding DNA sequences from non-relapsed cases only showed mutation heterozygote (C/T, 2/2) at c.886 locus. The allelic form of relapsed patients was CYP2D6 *2, and diploid score was used to indirectly predict the CYP2D6 enzyme activity of Hap_6, and the relapsed patients belong to normal metabolizer (NM).Conclusion: Among the numerous mutations of CYP2D6 gene, the joint mutations at three loci (c.408, c.886, c.1457) are most closely related to the damagedCYP2D6 enzyme activity. Whether c.886 locus mutation plays a critical role contributing to such damage warrants further verification with expanded sample size.

Inheritance and genetic mapping of resistance to Asian soybean rust in cultivar TMG 803

This study analyzed the inheritance and identified microsatellite markers linked to the resistance gene to Phakopsora pachyrhizi in soybean cultivar TMG 803. Hybridization between the cultivars TMG 803 and BRS Valiosa RR was performed to obtain F1 progenies and the F2 population. The response of the parents 'TMG 803' and 'BRS Valiosa RR' to P. pachyrhizi was, respectively, resistant and susceptible, and among the 116 F2 plants,93 were resistant and 23 susceptible, under natural infection and field conditions. It was found that the resistance of cultivar TMG 803 is controlled by one gene with complete dominance, mapped as resistance locus Rpp4 of linkage group G. Of the 16 tested, one microsatellite marker, sc21_3420, was completely linked to the resistance gene (distance 0.0cM) and the favorable allelic form was present in cultivar TMG 803, which may therefore be useful in assisted selection in segregating populations.

Differences in Transcriptional Activation by the Two Allelic (L162V Polymorphic) Variants of PPARαafter Omega-3 Fatty Acids Treatment

Omega-3 fatty acids (FAs) have the potential to regulate gene expression via the peroxisome proliferator-activated receptorα(PPARα); therefore, genetic variations in this gene may impact its transcriptional activity on target genes. It is hypothesized that the transcriptional activity by wild-type L162-PPARαis enhanced to a greater extent than the mutated variant (V162-PPARα) in the presence of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) or a mixture of EPA:DHA. To examine the functional difference of the two allelic variants on receptor activity, transient co-transfections were performed in human hepatoma HepG2 cells activated with EPA, DHA and EPA:DHA mixtures. Results indicate that the addition of EPA or DHA demonstrate potential to increase the transcriptional activity by PPARαwith respect to basal level in both variants. Yet, the EPA:DHA mixtures enhanced the transcriptional activity to a greater extent than individual FAs indicating possible additive effects of EPA and DHA. Additionally, the V162 allelic form of PPARαdemonstrated consistently lower transcriptional activation when incubated with EPA, DHA or EPA:DHA mixtures than, the wild-type variant. In conclusion, both allelic variants of the PPARαL162V are activated by omega-3 FAs; however, the V162 allelic form displays a lower transcriptional activity than the wild-type variant.

Allele Specificity of Naturally Acquired Antibody Responses against Plasmodium falciparum Apical Membrane Antigen 1

ABSTRACT Antibody responses against proteins located on the surface or in the apical organelles of merozoites are presumed to be important components of naturally acquired protective immune responses against the malaria parasite Plasmodium falciparum. However, many merozoite antigens are highly polymorphic, and antibodies induced against one particular allelic form might not be effective in controlling growth of parasites expressing alternative forms. The apical membrane antigen 1 (AMA1) is a polymorphic merozoite protein that is a target of naturally acquired invasion-inhibitory antibodies and is a leading asexual-stage vaccine candidate. We characterized the antibody responses against AMA1 in 262 individuals from Papua New Guinea exposed to malaria by using different allelic forms of the full AMA1 ectodomain and some individual subdomains. The majority of individuals had very high levels of antibodies against AMA1. The prevalence and titer of these antibodies increased with age. Although antibodies against conserved regions of the molecule were predominant in the majority of individuals, most plasma samples also contained antibodies directed against polymorphic regions of the antigen. In a few individuals, predominantly from younger age groups, the majority of antibodies against AMA1 were directed against polymorphic epitopes. The D10 allelic form of AMA1 apparently contains most if not all of the epitopes present in the other allelic forms tested, which might argue for its inclusion in future AMA1-based vaccines to be tested. Some important epitopes in AMA1 involved residues located in domain II or III but depended on more than one domain.

Detection of bovine spongiform encephalopathy, ovine scrapie prion-related protein (PrPSc) and normal PrPc by monoclonal antibodies raised to copper-refolded prion protein

Prion-related protein (PrP) is a glycosylphosphatidylinositol-linked cell-surface protein expressed by a wide variety of cells, including those of the nervous system and the immune system. Several functions of normal cellular PrP (PrPc) have been proposed that may be associated with the capacity of this protein to bind copper. In the present study, we describe the generation of a panel of monoclonal antibodies raised to copper-refolded PrP, which may be used to analyse the normal and disease-associated forms of this protein. The anti-PrP monoclonal antibodies were reactive by Western blot and ELISA with recombinant murine PrPc refolded in the presence or absence of either copper or manganese, and with the disease-susceptible allelic form V136R154Q171 ('VRQ'; where single-letter amino-acid notation has been used) and disease-resistant allelic form A136R154R171 ('ARR') of recombinant ovine PrPc. FACS analysis of lymphoid cells using these monoclonal antibodies showed that wild-type non-activated mouse lymphocytes expressed little, if any, PrPc. These monoclonal antibodies were shown to react with the unglycosylated and monoglycosylated forms of PrPSc (abnormal disease-specific conformation of PrP) in prion-infected tissue samples from all of the different species tested by Western blot. In addition, this analysis allowed one to make a distinction between bovine spongiform encephalopathy ('BSE') and scrapie PrPSc isolates from experimentally infected sheep on the basis of their different electrophoretic mobilities.

pfcrt Polymorphism and Chloroquine Resistance in Plasmodium falciparum Strains Isolated in Cambodia

ABSTRACT Plasmodium falciparum chloroquine resistance was first detected in Cambodia in the early sixties. Treatment with chloroquine was abandoned 20 years ago. In vitro chloroquine sensitivity monitoring indicates that all eastern Cambodian isolates were sensitive to chloroquine, whereas most isolates collected from western provinces displayed reduced susceptibility to chloroquine. This indicates that the rate of chloroquine resistance remains high and stable in this region in the absence of chloroquine pressure. Characterization of codons 72 to 78 and 218 to 220 of pfcrt revealed six distinct haplotypes, four of which had never been described. The frequency of each haplotype depended on the geographical origin of the samples. The CVIETIF//ISS haplotype was detected in 92% of western Cambodian isolates and in 11% of isolates collected from the eastern province, where CVMNKIF//ISA and CVIDTIF//ISS predominate. The detection of an intermediate haplotype from a susceptible area with 76T/220A, suggests that acquisition of chloroquine resistance might be a stepwise process, during which accumulation of point mutations modulates the response to chloroquine. The association of the K76T mutation with chloroquine resistance was not clear. The mutation was detected in resistant and susceptible samples, suggesting that additional factors are involved in chloroquine resistance. By contrast, the pfcrt D/N75E mutation was strongly associated with the in vitro chloroquine resistance in Cambodian isolates. The N86 allelic form of pfmdr1 was detected in all isolates, consistent with a poor association with resistance to chloroquine. This indicates that in vitro resistance to chloroquine was associated with accumulation of point mutations in pfcrt.

Poliovirus-Induced Apoptosis Is Reduced in Cells Expressing a Mutant CD155 Selected during Persistent Poliovirus Infection in Neuroblastoma Cells

ABSTRACT Poliovirus (PV) can establish persistent infections in human neuroblastoma IMR-32 cells. We previously showed that during persistent infection, specific mutations were selected in the first extracellular domain of the PV receptor (CD155) of these cells (N. Pavio, T. Couderc, S. Girard, J. Y. Sgro, B. Blondel, and F. Colbère-Garapin, Virology 274:331-342, 2000). These mutations included the Ala 67 → Thr substitution, corresponding to a previously described allelic form of the PV receptor. The mutated CD155Thr67 and the nonmutated IMR-32 CD155 (CD155IMR) were expressed independently in murine LM cells lacking the CD155 gene. Following infection of the cells with PV, we analyzed the death of cells expressing these two forms of CD155. Levels of DNA fragmentation, caspase activity, and cytochrome c release were lower in LM-CD155Thr67 cells than in LM-CD155IMR cells. Thus, the level of apoptosis was lower in cells expressing mutated CD155 selected during persistent PV infection in IMR-32 than in cells expressing the wild-type receptor.