Wild and Rare Self-Incompatibility Allele S17 Found in 24 Sweet Cherry (Prunus avium L.) Cultivars

The pollination of self-incompatible diploid sweet cherry is determined by the S-locus alleles. We resolved the S-alleles of 50 sweet cherry cultivars grown in Estonia and determined their incompatibility groups, which were previously unknown for most of the tested cultivars. We used consensus primers SI-19/20, SI-31/32, PaConsI, and PaConsII followed by allele-specific primers and sequencing to identify sweet cherry S-genotypes. Surprisingly, 48% (24/50) of the tested cultivars, including 17 Estonian cultivars, carry the rare S-allele S17, which had initially been described in wild sweet cherries in Belgium and Germany. The S17-allele in Estonian cultivars could originate from ‘Leningradskaya tchernaya’ (S6|S17), which has been extensively used in Estonian sweet cherry breeding. Four studied cultivars carrying S17 are partly self-compatible, whereas the other 20 cultivars with S17 have not been reported to be self-compatible. The recommended pollinator of seven self-incompatible sweet cherries is of the same S-genotype, including four with S17-allele, suggesting heritable reduced effectiveness of self-infertility. We classified the newly genotyped sweet cherry cultivars into 15 known incompatibility groups, and we proposed four new incompatibility groups, 64–67, for S-locus genotypes S3|S17, S4|S17, S5|S17, and S6|S17, respectively, which makes them excellent pollinators all across Europe. Alternatively, the frequency of S17 might be underestimated in Eastern European populations and some currently unidentified sweet cherry S-alleles might potentially be S17.

Presence of the Hmq system and production of 4-hydroxy-3-methyl-2-alkylquinolines is heterogeneously distributed between Burkholderia cepacia complex species and more prevalent among environmental than clinical isolates

Some Burkholderia cepacia complex (Bcc) strains have been reported to produce 4-hydroxy-3-methyl-2-alkylquinolines (HMAQs), analogous to the 4-hydroxy-2-alkylquinolines of Pseudomonas aeruginosa. Using in silico analyses, we previously showed that the hmqABCDEFG operon, which encodes enzymes involved in the biosynthesis of HMAQs, is carried by about one-third of Bcc strains, with considerable inter- and intra-species variability. In the present study, we investigated by PCR, using consensus primers, the distribution of hmqABCDEFG in a collection of 313 Bcc strains (222 of clinical and 91 of environmental origins) - belonging to 18 Bcc species. We confirmed that the distribution is species-specific, although not all strains within a species carry the hmqABCDEFG operon. Among the 30% of strains bearing the hmqABCDEFG operon, we measured the total HMAQs production and showed that 90% of environmental isolates and 68% of clinically isolated Bcc produce detectable levels of HMAQs when cultured in TSB medium. For the strains having the hmqABCDEFG operon but not producing HMAQs, we studied the transcription and showed that none expressed the hmqA gene under the specified culture conditions. Interestingly, the hmqABCDEFG operon is more prevalent among plant root environment species (e.g. B. ambifaria, B. cepacia) and absent in species commonly found in chronically colonized individuals with cystic fibrosis (e.g. B. cenocepacia, B. multivorans), suggesting that the Hmq system could play a role in niche adaptation by influencing rhizosphere microbial community and could have been lost through evolution. Understanding the Hmq system and its regulation will provide clues concerning the production of HMAQs and their functions in Bcc.

Self-Incompatibility Alleles in Iranian Pear Cultivars

In the present study, the S-alleles of eighteen pear cultivars, (including fourteen cultivars planted commercially in Iran and four controls) are determined. 34 out of 36 S-alleles are detected using nine allele-specific primers, which are designed for amplification of S101/S102, S105, S106, S107, S108, S109, S111, S112 and S114, as well as consensus primers, PycomC1F and PycomC5R. S104, S101 and S105 were the most common S-alleles observed, respectively, in eight, seven and six cultivars. In 16 cultivars, (‘Bartlett’ (S101S102), ‘Beurre Giffard’ (S101S106), ‘Comice’ (S104S105), ‘Doshes’ (S104S107), ‘Koshia’ (S104S108), ‘Paskolmar’ (S101S105), ‘Felestini’ (S101S107), ‘Domkaj’ (S104S120), ‘Ghousi’ (S104S107), ‘Kaftar Bache’ (S104S120), ‘Konjoni’ (S104S108), ‘Laleh’ (S105S108), ‘Natanzi’ (S104S105), ‘Sebri’ (S101S104), ‘Se Fasleh’ (S101S105) and ‘Louise Bonne’ (S101S108)) both alleles are identified but in two cultivars, (‘Pighambari’ (S105) and ‘Shah Miveh Esfahan’ (S107)) only one allele is recognized. It is concluded that allele-specific PCR amplification can be considered as an efficient and rapid method to identify S-genotype of Iranian pear cultivars.

Human Papillomavirus Genome based Detection and Typing: A Holistic Molecular Approach

Human Papillomavirus (HPV) is a species specific double-stranded DNA virus infecting human cutaneous or mucosal tissues. The genome structure of HPV is extremely polymorphic hence making it difficult to discriminate between them. HPV exhibits numerous dissimilar types that can be subdivided into high-risk (HR), probably high-risk and low-risk (LR), causing numerous types of cancers and warts around the genital organs in humans. Several screening methods are performed in order to detect cytological abnormalities and presence or absence of HPV genome. Currently available commercial kits and methods are designed to detect only a few HR/LR-HPV types, which are expensive adding to the economic burden of the affected individual and are not freely available. These gaps could be minimized through Polymerase Chain reaction (PCR) method, which is a gold standard and a cost-effective technique for the detection of most HPV (both known and unknown) types by using specific consensus primers in minimal lab setup. In this context, numerous studies have validated the effectiveness of different sets of consensus primers in the screening of HPVs. Numerous consensus primers, such as E6, E6/E7, GP-E6/E7, MY09/11, GP5+/GP6+, SPF10, and PGMY09/11 have been developed to detect the presence of HPV DNA. In addition, HPV detection sensitivity could be achieved through consensus primer sets targeting specific ORF regions like L1 and E6, which may finally assist in better diagnosis of several unknown HR-HPVs. The present review, provides a summary of the available methods, kits and consensus primer sets for HPV genome based detection, their advantages and limitations along with future goals to be set for HPV detection.

Self-incompatibility allele identification in Ukrainian sweet cherry (Prunus avium L.) cultivars

Aim. Ukrainian breeders have created a large number of sweet cherry cultivars, which still remain almost unexplored at the molecular level. The aim of our study was to identify the self-incompatibility alleles (S-alleles) in Ukrainian sweet cherry cultivars and landraces, and to elucidate, to which cross-incompatibility group the cultivars belong. Methods. The PCR was conducted using consensus primers to the first and second introns of S-RNAse gene and to the single intron of SFB gene. The electrophoretic analysis of the PCR products of the second intron of S-RNAse was carried out in agarose gel, whereas detection of fluorescently labeled DNA fragments of the first S-RNAse intron and the SFB intron was performed using a genetic analyzer. Results. The S-alleles of 25 Ukrainian sweet cherry cultivars and 10 landraces were identified. The S-alleles frequencies and affiliation of cultivars and landraces to the groups of cross-incompatibility were determined. The obtained data can be used in breeding programs and by planning of industrial plantings. Conclusions. In the study, 12 different S-alleles and 79 S-haplotypes were identified. The S1, S3, S4, S5, S6 and S9 alleles are the most widespread among Ukrainian sweet cherry cultivars and landraces. The high frequencies of S5 and especially of S9 alleles are characteristic for the Ukrainian cultivars and distinguish them from other European ones. For the Ukrainian sweet cherry cultivars, the XXXVII (S5S9) cross-incompatibility group appeared to be the most numerous.Keywords: Ukrainian sweet cherry cultivars, S-locus, Sgenotypes, self- and cross-incompatibility, Prunus avium.

Family-specific vs. universal PCR primers for the study of mitochondrial DNA in plants

Mitochondrial genomes (mtDNAs or mitogenomes) of seed plants are characterized by a notoriously unstable organization on account of which available so-called universal or consensus primers may fail to fulfil their foreseen function - amplification of various mtDNA regions in a broad range of plant taxa. Thus, the primers developed for groups assumed to have similar organization of their mitogenomes, such as families, may facilitate a broader usage of more variable non-coding portions of these genomes in group members. Using in silico PCR method and six available complete mitogenomes of Fabaceae, it has been demonstrated that only three out of 36 published universal primer and three Medicago sativa-specific primer pairs that amplify various mtDNA regions are suitable for six representatives of the Fabaceae family upon minor modifications, and develop 21 Fabaceae-specific primer pairs for amplification of all 14 cis-splicing introns in genes of NADH subunits (nad genes) which represent the most commonly used non-coding mtDNA regions in various studies in plants. Using the same method and six available complete mitogenomes of representatives of related families Cucurbitaceae, Euphorbiaceae and Rosaceae and a model plant, Arabidopsis thaliana, it has further been demonstrated that applicability of newly developed primer pairs for amplification of nad introns in more or less related taxa was dependent not only on species evolutionary distances but also on their genome sizes. A reported set of 24 primer pairs is a valuable resource which may facilitate a broader usage of mtDNA variability in future studies at both intra- and inter-specific levels in Fabaceae, which is the third largest family of flowering plants rarely studied at the mtDNA level, and in other more or less related taxa.

Sequencing-Based Detection of Circulating Tumor DNA in the Autologous Stem Cell Grafts of Patients with Diffuse Large B-Cell Lymphoma Undergoing Hematopoietic Stem Cell Transplantation

Introduction: High dose chemotherapy followed by autologous stem cell transplantation (ASCT) cures a subset of patients with chemosensitive relapsed or refractory (rel/ref) diffuse large B-cell lymphoma (DLBCL). Several factors associated with post-ASCT outcome have been identified, including pre-ASCT PET status, but better biomarkers are needed in order to optimally select candidates for the procedure. In other lymphoma subtypes with defining chromosomal translocations, PCR detection of pre- and post-ASCT minimal residual disease (MRD) in peripheral blood and of tumor contamination in the stem cell product is associated with inferior outcome. Until recently, MRD detection in DLBCL was limited by the rarity of detectable circulating disease using conventional techniques. The immunosequencing platform (Adaptive Biotechnologies, Corp.) is a next-generation-sequencing (NGS)-based MRD assay that detects small amounts of circulating tumor DNA (CTD) in patients with lymphoid malignancies. The assay detects CTD at diagnosis in most DLBCL patients and CTD levels track with response to induction therapy (Armand, 2013). Persistence of CTD or recurrence of CTD after completion of therapy is highly associated with DLBCL relapse (Kurtz, 2015; Roschewski, 2015). We evaluated whether CTD in autologous stem cell grafts was predictive of outcome in patients with rel/ref DLBCL undergoing ASCT. Methods: We retrospectively studied patients with rel/ref DLBCL, including transformed indolent lymphoma (TIL), who had paired archival tumor and autologous stem cell specimens and underwent ASCT at Brigham and Women's Hospital/Dana-Farber Cancer Institute from 2003-2013. Genomic tumor DNA was extracted from archival formalin-fixed paraffin-embedded (FFPE) tissue and analyzed using the NGS-based MRD assay. PCR amplification of IGH-VDJ, IGH-DJ,and IGK regions using universal consensus primers was performed followed by NGS to determine the tumor clonotype(s), defined as having a frequency > 5% in the tumor specimen. DNA from all available autologous peripheral blood or bone marrow stem cell specimens from each patient was amplified using universal consensus primers and sequenced to determine the level of CTD, defined as the number of lymphoma molecules per diploid genome. Results: We identified 98 eligible patients with rel/ref DLBCL/TIL. The median age was 60 (range 22-77) years; 63% were male; 65% had DLBCL, 29% had TIL, and 5% had primary mediastinal DLBCL; the median number of prior lines of therapy was 2 (range 2-5); all had received prior rituximab; 38% had primary refractory disease; 60% were in complete remission at ASCT; 96% received CBV conditioning. Median follow-up was 56 (range 19-123) months. The 4y progression-free survival (PFS) and overall survival (OS) in the entire cohort were 46% and 64%, respectively. Among 83 patients (85%) with sufficient DNA for clonotype determination, a clonotype was identified in 59 (71%). CTD data was complete in 53 patients (52 received peripheral blood stem cells (PBSC) and 1 received bone marrow). Eight patients (15%) had detectable CTD (CTD+) in the stem cell autografts (all PBSC) and 6/8 relapsed after ASCT. One CTD+ patient had early non-relapse mortality less than 1 month after ASCT and was never restaged. Seven of 8 CTD+ patients had TIL histology, 5 of whom relapsed (4 with aggressive lymphoma). The 4y PFS and OS in CTD+ v CTD- patients were 13% v 48% (p=0.01), and 38% v 67% (p=0.013), respectively [Figure 1]. In multivariable models including CTD status and pre-ASCT characteristics, CTD+ was the only factor associated with OS (HR 3.1, p=0.018), but was not significantly associated with PFS. Discussion: In patients with rel/ref DLBCL undergoing ASCT, the presence of CTD in the autologous stem cell graft is associated with inferior survival. CTD detection in the autograft may be more common in patients with TIL. In studies evaluating CTD detection in DLBCL, the plasma compartment has been more sensitive for detecting CTD than mononuclear cells. The use of concentrated cell specimens in this study may have decreased the sensitivity of the assay. Nevertheless, if the present findings are confirmed in a larger population, CTD detection may permit the identification of a subgroup of patients with a particularly poor outcome after ASCT, for whom alternative approaches could be considered. Figure 1. Overall (A) and Progression-Free (B) Survival in CTD+ vs CTD- Patients Figure 1. Overall (A) and Progression-Free (B) Survival in CTD+ vs CTD- Patients Figure 2. Figure 2. Disclosures Herrera: Sequenta, Inc.: Research Funding; Pharmacyclics: Research Funding; Genentech: Research Funding. Kong:Adaptive Biotechnologies, Corp.: Employment, Other: Stockholder. Davids:Genentech: Other: ad board; Pharmacyclics: Consultancy; Janssen: Consultancy. Rodig:Perkin Elmer: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Research Funding. Faham:Adaptive Biotechnologies Corp.: Employment, Other: Stockholder. Armand:BMS: Research Funding; Infinity: Consultancy, Research Funding; Merck: Consultancy, Research Funding; Sequenta, Inc.: Research Funding.

Allele-specific PCR detection of sweet cherry self-incompatibility alleles S3, S4 and S9 using consensus and allele-specific primers in the Czech Republic

  Prunus avium species of the Rosaceae family exhibit gametophytic self-incompatibility. Determination of the self-incompatibility genotype of individuals is essential for genetic studies and the development of informed management strategies. The PCR-based detection of S-allele helps to promote and speed up traditional breeding activity and hence molecular analysis of the perspective genotypes has become more intensive in all cherry growing countries. The alleles S₃, S₄ and S₉ from 34 accessions of Czech collections were determined using the polymerase chain reaction (PCR) method. Initially, DNA extracts were amplified with consensus primers that amplify across the first, second, or both introns of the S-ribonuclease gene which shows a considerable length polymorphism. The new allele specific primers were designed with the goal to overcome some occurring difficulties in the detection of expected alleles by previously published allele specific primers. S-alleles fragments of standard cultivars used in this study were PCR amplified, sequenced to validate the designed primers. The study demonstrates the advantage of newly designed primers application in testing of sweet cherry genotypes.    

Novel parvovirus from the worm lizard Trogonophis wiegmanni — First virus ever detected in amphisbaenian hosts

To explore the diversity of some DNA viruses in reptiles, a continuous screening is going on, in our laboratory, by PCR using different consensus primers designed for the detection of the most conserved genome regions of adeno-, herpes- and parvoviruses. The test material consists essentially of dead specimens collected randomly from private pet owners, local pet shops, or at occasional exotic pet fairs. Here we report the partial sequence of a putative novel parvovirus obtained from a dead checkerboard worm lizard(Trogonophis wiegmanni)that had been wild-caught in its native habitat. An in-house-developed PCR with consensus primers targeting the gene of the parvoviral capsid protein was used. Other PCRs, intended to detect certain large DNA viruses, remained negative. The sequence of the PCR product indicated the presence of a hitherto unknown parvovirus in the internal organs of the checkerboard worm lizard. In phylogeny reconstruction, the novel sequence clustered with the members of theDependovirusgenus of theParvoririnaesubfamily, closest to the branch of snake adeno-associated virus. Since we could not demonstrate the presence of a potential helper virus, the putative amphisbaenian parvovirus supposedly can replicate autonomously. This is the first virus infection ever detected in any members of the suborder Amphisbaenia, and only the third parvoviral sequence obtained from any reptilian host.