Search

Background: Y chromosome microdeletions provide a pivotal role in the control of spermatogenesis. These are located on the q arm of it, specifically, the azoospermia factor (AZF) region, hence named as Yq microdeletions. The mutations in this region are related to testicular pathologies such as azoospermia, oligozoospermia, and other semen categories. Objectives: This study was undertaken to review these microdeletions, screening, and their correlation to semen categories in relation to the Indian and Global populations in context to male infertility, future strategies, and its implications. Methodology: These deletions are screened using sequence-tagged sites (STS) of the European Academy of Andrology (EAAs) and Non-EAAs with polymerase chain reaction technology. The data included were Indian and Global studies obtained from research articles, abstracts, and reviews (75) indexed in Google Scholar, PubMed, and Scopus. Results: A number of total 45,562 Y chromosomes were analyzed. In India, this frequency contributes to 8.33% (453/5435) against the average frequency of 6.96% (3170/ 45562) worldwide. Globally, this frequency was higher (9.1%) in the North American continent. Among all types of deletions, AZFc deletions are higher followed by b, a, b+c, and others as well as are related to azoospermia than oligospermia and other semen cases/categories globally including India. In India, the data on partial deletions are scanty. Further, cases with AZFc are better suitable for assisted reproductive technologies after counseling. Conclusion: Evaluation of correct deletion type to a specific testis pathologic phenotype is suggested for sperm retrieval to correct the male partner. This study is hence better suitable for azoospermia with AZFc deletion around the Global and Indian scenario in association with male infertility. Future strategies are thus called for successful treatment of a specific microdeletion clinically in the male.

Sequence-based markers have added a new dimension in the efficiency of identifying alien introgressions in wheat. Expressed sequence tag-sequence tagged sites (EST-STS) markers have proved useful in tracing alien chromatin. In this study, we report the development of Thinopyrum bessarabicum- and Secale anatolicum-specific EST-STS markers and their application in tracing respective alien chromatin introgressions in wheat. The parental lines, Chinese Spring (CS), ISR991.1 (CS/Th. bessarabicum amphidiploid), and ISR1049.2 (CS/Secale anatolicum amphidiploid), were used as core experimental materials. Using comparative analysis of RNA-Seq data, 10 903 and 10 660 candidate sequences specific to Th. bessarabicum and S. anatolicum, respectively, were assembled and identified. To validate the genome specificity of these candidate sequences, 68 and 64 EST-STS markers were developed from randomly selected candidate sequences of Th. bessarabicum and S. anatolicum, respectively, and tested on sets of alien addition lines. Fifty-five and 53 markers for Th. bessarabicum and S. anatolicum chromatin, respectively, were assigned to chromosomal location(s), covering all seven chromosomes. Approximately 83% of S. anatolicum-specific markers were transferable to S. cereale. The genome-specific candidate sequences identified and the EST-STS markers developed will be valuable resources for exploitation of Th. bessarabicum and Secale species diversity in wheat and triticale breeding.

A-3-year- old Bangladeshi pediatric patient named Tasin was presented with a diagnosed case of congenital hypothyroidism (CH). This type of hypothyroidism may occur due to the alteration in the nucleotide sequences of the Thyroid transcription factor 2 gene. Few studies are present on the genetic basis of this disease. CH is common in Bangladesh, may be due to geographical variation or other causes. Therefore, this study was conducted to identify whether there was any genetic alteration in the exon2 of Thyroid transcription factor 2 gene. With due procedure and permission from the guardian of the pediatric patient, socio-demographic data was collected. Isolation of DNA, quantitation and qualitation of DNA was ensured, polymerase chain reaction (PCR) was performed, the amplicons that was obtained from PCR; validated visually by gel electrophoresis methods; cycle sequencing was performed by Sanger sequencing. The chromatogram data that was obtained from Sanger sequencing was analyzed and compared with the National Center for Biotechnology Information database by Basic Local Alignment Search Tool search. Sanger sequencing revealed substitution (c.1051G>T) in the Sequence Tagged Site of the exon2 of Thyroid transcription factor 2 gene and this is new variants and not reported in National Center for Biotechnology Information database.

Background Etiology of male infertility is intriguing and Y chromosome microdeletion within azoospermia factor (AZF) sub‐regions is considered major cause. We conducted a screening for Y chromosome microdeletion in an infertile male cohort from West Bengal, India to characterize Y chromosome microdeletion among infertile men. Methods We recruited case subjects that were categorized on the basis of sperm count as azoospermia (N = 63), severe oligozoospermia (N = 38), and oligozoospermia (N = 17) and compared them with age, demography, and ethnicity matched healthy proven fertile control males (N = 84). Sequence Tagged Site makers and polymerase chain reaction based profiling of Y chromosome was done for AZF region and SRY for cases and controls. Results We scored 16.1% of cases (19 out of 118) that bear one or more microdeletions in the studied loci and none among the controls. The aberrations were more frequent among azoospermic males (17 of 19) than in severe oligozoospermic subjects (2 of 19). Conclusion Our study provides the results of screening of the largest Bengali infertile men sample genotyped with the maximum number of STS markers spanning the entire length of Y chromosome long arm. Y chromosome microdeletion is a significant genetic etiology of infertility among Bengali men., Y chromosome microdeletion (YCMD) is a major genetic cause of infertility in men but not yet explored in the Bengali population from India. We characterized YCMD among infertile Bengali men with microsatellite markers and found this may be causative factors of azoospermia and severe oligozoospermia.

Background MDR bacteria including carbapenem-resistant Pseudomonas aeruginosa are recognized as an important cause of hospital-acquired infections worldwide. This investigation seeks to determine the molecular characterization and antibiotic resistance genes associated with carbapenem-resistant P. aeruginosa. Methods We conducted WGS and phylogenetic analysis of 72 carbapenem-resistant P. aeruginosa isolated from hospital-acquired infection patients from August 2011 to March 2015 in three major hospitals in Hanoi, Vietnam. Results We identified three variants of IMP gene, among which blaIMP-15 was the most frequent (n = 34) in comparison to blaIMP-26 (n = 2) and blaIMP-51 (n = 12). We observed two isolates with imipenem MIC >128 mg/L that co-harboured blaIMP-15 and blaDIM-1 genes and seven isolates (imipenem MIC > 128 mg/L) with a blaKPC-1 gene from the same hospital. MLST data shows that these 72 isolates belong to 18 STs and phylogenetic tree analysis has divided these isolates into nine groups. Conclusions Our results provide evidence that not only blaIMP-26 but other IMP variants such as blaIMP-15 and blaIMP-51 genes and several STs (ST235, ST244, ST277, ST310, ST773 and ST3151) have been disseminating in healthcare settings in Vietnam. In addition, we report the emergence of two isolates belonging to ST1240 and ST3340 that harboured two important carbapenemase genes (blaIMP-15 and blaDIM-1) and seven isolates belonging to ST3151 of P. aeruginosa that carried the blaKPC-1 gene in Vietnam, which could potentially cause serious restricted availability of treatment options in healthcare settings.

Thinopyrum intermedium (2n = 6x = 42, JJJSJSStSt) is one of the important resources for the wheat improvement. So far, a few Th. intermedium (Thi)-specific molecular markers have been reported, but the number is far from enough to meet the need of identifying alien fragments in wheat-Th. intermedium hybrids. In this study, 5,877,409 contigs were assembled using the Th. intermedium genotyping-by-sequencing (GBS) data. We obtained 5,452 non-redundant contigs containing mapped Thi-GBS markers with less than 20% similarity to the wheat genome and developed 2,019 sequence-tagged site (STS) molecular markers. Among the markers designed, 745 Thi-specific markers with amplification products in Th. intermedium but not in eight wheat landraces were further selected. The distribution of these markers in different homologous groups of Th. intermedium varied from 47 (7/12/28 on 6J/6St/6JS) to 183 (54/62/67 on 7J/7St/7JS). Furthermore, the effectiveness of these Thi-specific markers was verified using wheat-Th. intermedium partial amphidiploids, addition lines, substitution lines, and translocation lines. Markers developed in this study provide a convenient, rapid, reliable, and economical method for identifying Th. intermedium chromosomes in wheat. In addition, this set of Thi-specific markers can also be used to estimate genetic and physical locations of Th. intermedium chromatin in the introgression lines, thus providing valuable information for follow-up studies such as alien gene mining.

This study aims to analyze the length polymorphisms in sequence-tagged-site (STS) sY1291 of the Y chromosome in Vietnamese men of infertile couples. All 322 DNA samples were amplified with the sY1291 primer by the quantitative fluorescent polymerase chain reaction (QF-PCR) assay. DNA sequencing technique was employed to evaluate the accuracy of QF-PCR results. The study showed 273 out of 322 DNA samples had the presence of STS sY1291, accounted for 84.78%. The QF-PCR results showed that there were various lengths in STS sY1291: 507 bp, 512 bp, 523 bp and 527 bp. The most prevalent length in STS sY1291 was 507 bp (87.5%), the others were 512 bp (4.8%), 523 bp (4.8%) and 527 bp (2.9%). We found that the observed length polymorphisms derived from differences in the number of mononucleotide Thymine (T) repeats in its structure. It stretched from 22 T to 39 T. DNA sequencing results identified that the number of mononucleotide T repeats causes these polymorphisms. However, the pair-wise alignment between the obtained and reference sequence was 77%. It can be seen that the length polymorphisms in STS sY1291 observed in QF-PCR results was accurate but it is still difficult to sequence fragments with mononucleotide repeats.

Objectives To describe the prevalence of poxtA among clinical linezolid-resistant enterococci (LRE) collected in France from 2016 to 2020 and to extensively characterize its genetic supports and environments. Methods All LRE clinical isolates received at the National Reference Centre for Enterococci from French hospitals between 2016 and 2020 were included. LRE isolates were screened for linezolid resistance genes (cfr-like, optrA and poxtA) by real-time PCR and phenotypically characterized. A collection of 11 representative poxtA-positive isolates (10 Enterococcus faecium and 1 Enterococcus faecalis) underwent WGS by hybrid assembly combining short-read (Illumina MiSeq) and long-read (MinION) approaches. Transferability of poxtA was attempted by filter-mating experiments. Results Out of 466 LRE received at the National Reference Centre for Enterococci over the period, 47 (10.1%) were poxtA-positive, including 42 E. faecium. The 11 isolates characterized by WGS were confirmed to be epidemiologically unrelated by core genome analysis and eight different STs were assigned to E. faecium isolates. The poxtA gene was found to be plasmid carried and flanked by IS1216E transposase genes in all isolates and frequently linked with fexB, tet(M) and tet(L). A total of seven distinct poxtA-harbouring plasmids were obtained after hybrid assembly and plasmid transfer of poxtA was successful in three cases. For the two poxtA/optrA-positive isolates, those genes were carried by different plasmids. Conclusions The poxtA gene has been circulating among clinical enterococci in France since at least 2016, mostly in E. faecium and independently from optrA. The poxtA-carrying plasmids often co-carried resistance genes to phenicols and tetracyclines, and could have been co-selected through their veterinary use.

Objectives Carbapenemase-producing Enterobacterales (CPE) have spread widely into health care facilities (HCF) but clusters caused by carbapenemase-producing (CP) Citrobacter freundii have been uncommon until recent years. Here we describe CP C. freundii clusters detected in Finland during 2016–20. Methods As a part of the national CPE surveillance, clinical microbiology laboratories send potential CP C. freundii isolates to the reference laboratory for confirmation and further characterization. Whole genome sequencing (WGS) with Illumina MiSeq sequencer was used to detect clusters. Resistance genes and STs were analysed using SRST2 and typing with core genome (cg) MLST. A case was defined as a patient with a CP C. freundii isolate belonging to one of the detected clusters. Results We detected three CP C. freundii clusters: cluster 1 included 16 cases in five HCFs during 2016–20, cluster 2 had two cases in two HCFs during 2018–19 and cluster 3 had two cases in one HCF in 2020. The isolates (11 clinical and 5 screening) in cluster 1 had KPC-2 carbapenemase and were sequence type (ST)18. Cluster 2 (2 clinical isolates) had OXA-181/GES-5 carbapenemases and were ST604 and cluster 3 (two screening isolates) had KPC-3 carbapenemase and were ST116. None of the cases had a history of recent travel abroad. Conclusions CP C. freundii also causes outbreaks and can be a reservoir of carbapenemase genes. The long intervals between successive cases, mostly found in clinical specimens in two clusters, suggest that besides unknown carriers, environmental contamination may play a role in transmission.

Context and rationale: Congenital hypothyroidism is a prevalent endocrine disease that may occur due to the alteration in the sequence of nucleotides of the NKX2.5 gene. Though congenital hypothyroidism is quite common among the Bangladeshi pediatric population, there are few studies on the genetic basis of this disease. Objective: This study aimed to identify any mutation in the exon2 of the NKX2.5 gene in Bangladeshi pediatric patients with congenital hypothyroidism. Methods: Forty (40) Bangladeshi pediatric patients with congenital hypothyroidism were recruited, the sociodemographic data were collected and analyzed, DNA was isolated, quantity and quality of DNA were checked, polymerase chain reaction (PCR) was done, the amplicons were visually validated by gel electrophoresis and cycle sequencing was done by Sanger sequencing. The raw chromatogram data were analyzed and compared with the NCBI database by BLAST (Basic Local Alignment Search Tool) search. Results: Sanger sequencing revealed two types of alteration in the nucleotide sequence. Nine patients showed substitutions (c.1051G>T) and eight patients showed deletions (c.1143 delT-), and both substitution and deletion were present in four patients. This substitution and deletion occurred in the Sequence Tagged Site (STS) of the exon2 of the NKX2.5 gene and these are new variants and not reported in NCBI database. Conclusion: In the present study, two types of variants were identified. So, further study to find out mutational status among Bangladeshi children might be helpful in enriching the database of mutational spectra of pediatric patients with congenital hypothyroidism.

ObjectivesSurveillance of antimicrobial resistance (AMR) in Neisseria gonorrhoeae, supported by molecular typing, ideally through genome sequencing, is imperative. We defined N. gonorrhoeae Sequence Typing for Antimicrobial Resistance (NG-STAR) clonal complexes (CCs) and validated their usefulness in gonococcal AMR surveillance.MethodsAll NG-STAR alleles and STs available in the public database (https://ngstar.canada.ca/) were analysed using PHYLOViZ 2.0 to define CCs according to the closest founder ST with ≥5 identical alleles and founding ST with the highest number of links. The published 2013 European gonococcal dataset (n = 1054), the 2016 WHO reference strain panel (n = 14) and N. gonorrhoeae isolates with ceftriaxone resistance determinant penA-60.001 (n = 7) from several countries were used for validation.ResultsThe majority of the isolates (n = 1063) were designated to 71 CCs. The most common CC was CC90 (n = 194), followed by CC63 (n = 166), CC139 (n = 73), CC158 (n = 73) and CC127 (n = 62). CC90 included isolates belonging to the internationally spread MDR clone N. gonorrhoeae Multi-Antigen Sequence Typing (NG-MAST) G1407 (predominantly MLST ST1901). The ceftriaxone-resistant isolates with penA-60.001 (n = 7) belonged to CC73 or STs linking between CC90 and CC73 (ST233 and ST1133). Phylogenomic analysis revealed that NG-STAR CCs more appropriately correlated to phylogenomic AMR clusters compared with MLST STs, NG-MAST STs, NG-MAST genogroups and NG-STAR STs.ConclusionsNG-STAR CCs: are consistent with the gonococcal genome phylogeny; allow rapid visualizations with limited computational requirements; provide a simple, reproducible and portable nomenclature (for WGS and conventional Sanger sequencing data); and predict AMR lineages. Phenotypic AMR surveillance, supplemented with WGS, is imperative and NG-STAR CCs can effectively support this.

Blastocystis is an enteric microorganism commonly found in humans and animals worldwide. Its pathogenic role in humans and transmission patterns has not been fully explained. However, nine subtypes (ST1&ndash, 8, ST12) are considered as potentially zoonotic. Studies from various regions of the world show that pigs are mainly infected with ST5. Although pigs are important farmed animals in Poland, the question of Blastocystis infection in these animals has not yet been investigated. Herein, 149 pig stool samples from 10 Polish pig farms were analyzed using sequence-tagged-site PCR and barcode region sequencing. The percentage of samples in which Blastocystis was identified using each method separately was similar: 38.25% and 37.58%, respectively. However, the percentage of positive results obtained by combining both methods was 46.97%, which means that, depending on the method used, the number of undetected samples varied between 8.72% and 9.39%. This shows the methodological limitations of up-to-date molecular approaches commonly used in Blastocystis research. A moderate infection rate (44.4&ndash, 50%) observed in different pig age groups with a vital predominance of ST5 (94.28%) in every age group shows that pigs are a likely natural host of ST5. A small percentage of mixed infections, namely ST5/ST1 (5.26%), ST5/ST3 (1.75%), and ST3/ST1 (1.75%), was observed only in animals of older age, suggesting that ST3 and ST1 can be acquired by pigs during contact with humans. This study provides the first data on the prevalence and Blastocystis subtypes (STs) distribution in pigs in Poland. The results also highlight the need for the development of new methods capable of detecting highly genetically diverse Blastocystis isolates and mixed infections.

In human gamete development, the important period is spermatogenesis, which is organized by specific genes on Y chromosome. In some cases, the infertile men have shown microdeletions on Y chromosome, which seemed as if the structural chromosome variance is linked to the reduction of sperm count. This study aimed to determine the frequency and patterns of Y chromosome microdeletions in azoospermia factor (AZF) of Iraqi infertile males. Here, 90 azoospermic infertile males as a study group and 95 normal fertile males as control group were investigated for the microdeletion of AZF loci using numerous sequence-tagged sites. Of these 90 infertile male patients, 43 (47.8%) demonstrated Y chromosome microdeletions, in which AZFb region was the most deleted section inazoospermia patients (33.3%) followed by deletions in the AZFc region (23%), while there were no microdeletion in the AZFa region. The largest microdeletion involved in both AZFb and AZFc was detected in six azoospermic patients (6.7%). The present study demonstrated a high frequency of Y chromosome microdeletions in the infertile Iraqi patients which is not reported previously. The high frequency of deletions may be due to the association of ethnic and genetic factors. PCR-based Y chromosome screening for microdeletions has a potential to be used in infertility clinics for genetic counselling and assisted reproduction.

In this study, 52 Streptococcus suis isolates from pigs in southern China were divided into four known sequence types (STs) and six new STs, using multilocus sequence typing. Ten representative isolates were selected from 10 STs for the analysis of whole genome sequences. Virulence was assessed in 10 isolates, which were classified into three pathogenic groups. The prevalence of virulence-associated factors in the moderately pathogenic group was higher than that in the highly pathogenic group. The isolates from ST1 complex and serotype 2 were allocated to the moderately pathogenic group, while the isolates from the highly pathogenic group belonged to the non-ST1 complex and non-serotype 2. Three clusters were obtained based on multilocus sequence typing sequences: cluster III isolates from the nasal cavity of healthy pigs were classified into the highly pathogenic group and showed many peculiarities compared with cluster I and II isolates in virulence genotypes, genetic typing and pathogenesis, indicating a potential independent evolutionary line. Our results suggest that S. suis infections in China are becoming more complicated with constantly mutating isolates, which makes it difficult to distinguish their virulence by recognized typing methods. Thus, increased investigation and monitoring of these infections should be a priority for the swine industry in China.

International audience; The genetic control of self-incompatibility (SI) has been recently disclosed in olive. Intervarietal crossing confirmed the presence of only two incompatibility groups (G1 and G2), suggesting a simple Mendelian inheritance of the trait. A double digest restriction associated DNA (ddRAD) sequencing of a biparental population segregating for incompatibility groups has been performed and high-density linkage maps were constructed in order to map the SI locus and identify gene candidates and linked markers. The progeny consisted of a full-sib family of 229 individuals derived from the cross ‘Leccino’ (G1) × ‘Dolce Agogia’ (G2) varieties, segregating 1:1 (G1:G2), in accordance with a diallelic self-incompatibility (DSI) model. A total of 16,743 single nucleotide polymorphisms was identified, 7,006 in the female parent ‘Leccino’ and 9,737 in the male parent ‘Dolce Agogia.’ Each parental map consisted of 23 linkage groups and showed an unusual large size (5,680 cM in ‘Leccino’ and 3,538 cM in ‘Dolce Agogia’). Recombination was decreased across all linkage groups in pollen mother cells of ‘Dolce Agogia,’ the parent with higher heterozygosity, compared to megaspore mother cells of ‘Leccino,’ in a context of a species that showed exceptionally high recombination rates. A subset of 109 adult plants was assigned to either incompatibility group by a stigma test and the diallelic self-incompatibility (DSI) locus was mapped to an interval of 5.4 cM on linkage group 18. This region spanned a size of approximately 300 Kb in the olive genome assembly. We developed a sequence-tagged site marker in the DSI locus and identified five haplotypes in 57 cultivars with known incompatibility group assignment. A combination of two single-nucleotide polymorphisms (SNPs) was sufficient to predict G1 or G2 phenotypes in olive cultivars, enabling early marker-assisted selection of compatible genotypes and allowing for a rapid screening of inter-compatibility among cultivars in order to guarantee effective fertilization and increase olive production. The construction of high-density linkage maps has led to the development of the first functional marker in olive and provided positional candidate genes in the SI locus.

The content and composition of seed storage proteins is largely responsible for wheat end-use quality. They mainly consist of polymeric glutenins and monomeric gliadins. According to their electrophoretic mobility, gliadins and glutenins are subdivided into several fractions. Glutenins are classified as high molecular weight or low molecular weight glutenin subunits (HMW-GSs and LMW-GSs, respectively). LMW-GSs are encoded by multigene families located at the orthologous Glu-3 loci. We designed a set of 16 single-nucleotide polymorphism (SNP) markers that are able to detect SDS-PAGE alleles at the Glu-A3 and Glu-B3 loci. The SNP markers captured the diversity of alleles in 88 international reference lines and 27 Mexican cultivars, when compared to SDS-PAGE and STS markers, however, showed a slightly larger percent of multiple alleles, mainly for Glu-B3. SNP markers were then used to determine the Glu-1 and Glu-3 allele composition in 54 CIMMYT historical lines and demonstrated to be useful tool for breeding programs to improve wheat end product properties.

Background Emerging antimicrobial-resistant Escherichia coli represent mainly the nested (fluoroquinolone-resistant [FQR]) H30R and H30Rx subclones within sequence type 131 (ST131). Intestinal colonization and within-household transmission may underlie H30R’s emergence. Methods We screened fecal samples from 741 volunteers (383 veterans, 358 household members, including pets) for ST131 and FQR E. coli (FQREC) and used molecular profiling to resolve unique strains. Selected strains underwent PCR-based detection of phylogroups, sequence types (STs), H30, H30Rx, and 53 virulence genes (VGs). Within-household strain sharing was compared with household, host, and bacterial characteristics. Fecal isolates were compared with clinical isolates. Results Colonization prevalence was 5.1% for H30R, 8% for ST131 (67% FQREC), and 10% for FQREC (52% ST131). ST131 isolates exhibited more VGs than non-ST131 isolates. Strain sharing (27% of multisubject households, 18% of corresponding subjects) was associated with the elderly, FQREC, H30R, H30Rx, ST73, and specific VGs. Fecal ST131 and FQREC isolates resembled contemporaneous and historical clinical isolates according to all studied traits. Conclusions Veterans and their human household members commonly carry and extensively share FQREC, predominantly H30R, thereby likely facilitating the ST131 pandemic. Strain sharing corresponds with multiple bacterial characteristics, including FQ resistance and specific VGs, which may promote intestinal colonization and/or host-to-host transmission.

Kernel texture (grain hardness) is a key trait that influences the milling and baking quality of wheat ( Triticum aestivum L.). Herein, hard red spring wheat varieties Butte86 and ND2603, possessing the Pina-D1b and Pinb-D1b alleles, respectively, were used as parents in the development of 132 recombinant inbred lines (RIL). The RIL population and its parents were phenotyped for single kernel characterization system (SKCS) and NIR kernel texture, grain protein content, test weight, SKCS weight, and SKCS diameter, and genotyped using next-generation genotyping-by-sequencing (GBS) technology, SSR markers and allele-specific sequence tagged site markers. Linkage maps for the entire RIL population and for two subsets of RILs, grouped on the basis of the Puroindoline-D1 alleles, were developed using a total of 695 markers. Multiple QTL mapping identified 10 QTLs for SKCS and NIR kernel texture with significant loci on 1AS, 1BS, 1BL, 5AL, 5BL, 6BL and 7BS. Of these, the 1BS QTL was associated with the Glu-B3 amplicon 691, and one of the 1BL QTL with Glu-B1 . All QTL conferred an additive effect of ∼3–8 hardness units, a difference in kernel texture similar to or greater than the difference between the Pina-D1b and Pinb-D1b hardness mutations.

Background Healthcare-associated infections (HAIs) caused by Acinetobacter baumannii, an opportunistic gram-negative pathogen, often contribute to morbidity and mortality among hospitalized patients. To determine molecular epidemiology of circulating hospital Acinetobacter strains in Detroit area in a non-outbreak situation, we performed whole genome sequencing (WGS) and whole genome multilocus sequence typing (wgMLST) analysis to characterize genomic diversity. Methods We performed WGS on patient isolates from two disparate, geographically distinct tertiary care Detroit hospitals admitted to 16 intensive care units (ICU) and non-ICU wards between 2017-2019. The samples were obtained 48 hours following admission and WGS was performed using the Illumina NextSeq instrument. The contigs were de novo assembled using SPAdes and WgMLST analysis was performed using BioNumerics software v7.6. Minimum spanning tree (MST) was constructed to demonstrate the clusters in each hospital and their wards. Results WgMLST analysis was performed on a total of 62 Acinetobacter baumannii patient isolates, 33 of which were from hospital # 1 (H1) and 29 were from hospital # 2 (H2). ST2 (52%) was the predominant sequence type for both hospitals. In addition to ST2, ST93 (8%) and ST406 (8%) clusters were also found in H1. Most of the isolates were predominantly clustered in the intensive care unit (ICU) setting. Several identical sequence types of ST93 (2 identical in H1), ST36 (2 identical in H1) and ST2 (5 identical for H1 and 2 identical for H2) were also observed from different patients in both hospitals. Unlike H1, H2 only has ST2 as a predominant cluster. Other sporadic sequence types such as ST119, ST36, ST49, ST212, ST667 for H1 and ST395, ST214, ST427 and ST268 for H2 were also observed. Figure 1. MInimum Spanning Tree of Acinetobacter strains in hospitals (H1 and H2) and different wards (U1-U12). For identical strains circles are marked with dividing lines. Different sequence types (STs) are marked om the side bar. Conclusion Our data suggests that ST2 is the predominant sequence type circulating in both the geographically distinct hospitals and is endemic to the ICU. There was in hospital spread of this distinct Acinetobacter sequence type from one patient to the other, raising concerns about infection control practices and the role of environment in the spread of these infections. Early detection of these endemic strains within the hospital may be critical in preventing healthcare-associated infections in the future. Disclosures Chetan Jinadatha, MD, MPH, AHRQ (Research Grant or Support)Department of Veterans Affairs (Other Financial or Material Support, Owner: Department of Veterans Affairs. Licensed to: Xenex Disinfection System, San Antonio, TX)Inventor (Other Financial or Material Support, Methods for organizing the disinfection of one or more items contaminated with biological agents)NiH/NINR (Research Grant or Support)NSF (Research Grant or Support)Xenex Healthcare Services (Research Grant or Support) Mark Stibich, PhD MHS, Xenex Disinfection Services, Inc (Board Member, Employee)

Background Suvratoxumab is a human monoclonal antibody that neutralizes S. aureus (SA) alpha toxin (AT). SAATELLITE, a phase 2 study of the safety and efficacy of suvratoxumab for reducing the incidence of SA pneumonia (NCT02296320), was conducted within the consortium for Combatting Bacterial Resistance in Europe. Methods A total of 304 SA isolates (baseline, onset and last available isolates from suspected serious bacterial infections, SSBIs) collected from the lower respiratory tract samples from 165 subjects during SAATELLITE were subjected to whole genome sequencing. AT gene (hla) sequences were translated and amino acid variation was identified in comparison to the reference SA USA300 FPR3757. Phylogenetic analysis, genomic annotation and ST analysis were performed. AT expression in SA culture supernatants was performed by ELISA. Representative isolates with novel AT subtypes that had not been identified in previous studies were tested for hemolytic activity and suvratoxumab neutralizing activity. Wilcoxon rank sum test and Fisher’s exact test were performed, respectively: a) to compare difference in baseline AT expression in relation to SA pneumonia incidence; b) to evaluate the association between occurrence of AT stop codons and incidence of SA pneumonia at baseline, as well as the association between occurrence of AT stop codons and treatment arms at post baseline. Results We identified a total of 44 sequence types (STs) and 21 unique AT subtypes, 7 of which have not been described previously. No substitutions were located in the suvratoxumab binding region and all novel AT subtypes displaying lytic activity were neutralized by suvratoxumab. We detected stop codons Q113B and W205B in AT sequences in 53 and 2 SA isolates, respectively. We uncovered no significant associations of: 1) baseline AT expression with SA pneumonia incidence [p=0.967]; 2) occurrence of AT gene stop codon with either SA pneumonia incidence [p >0.999] or suvratoxumab treatment [p=0.103; lower frequency of stop codons in suvratoxumab arm versus placebo]. Conclusion Our data indicated that: 1) suvratoxumab target region in (AT) remains conserved; 2) suvratoxumab is active against all AT variants identified to date; 3) suvratoxumab did not exert pressure on SA clinical isolates for selection of escape mutants. Disclosures David E. Tabor, PhD, AstraZeneca (Employee, Shareholder) Andrey Tovchigrechko, PhD, AstraZeneca (Employee, Shareholder)KitePharma, a Gilead company (Employee, Shareholder) Bret R. Sellman, PhD, AstraZeneca (Employee, Shareholder) Michael McCarthy, n/a, AstraZeneca (Employee) Kathryn Shoemaker, MS, AstraZeneca (Employee) Hasan S. Jafri, MD, FAAP, AstraZeneca (Employee) Mark T. Esser, PhD, AstraZeneca (Employee) Alexey Ruzin, PhD, AstraZeneca (Employee, Shareholder)

BRRI dhan52 is a uniquire submergence tolerant rice variety containing prominant genetic background of BR11, a mega rainfed lowland rice (RLR) variety of Bangladesh, but is susceptible to bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae ( Xoo ). The variety is considerably popular in the southern part of Bangladesh due to its high yield in flash flood condition and comparatvely medium slender grain. Molecular markers linked to BB resistance genes ( Xa genes) and submergence QTL ( SUB1 ) were utilized in a marker-aided selection program to develop elite breeding lines with broad-spectrum resistance to bacterial blight. Sequence tagged site (STS) and simple sequence repeat (SSR) markers were essentially used to detect the genes for BB and submergence as well . In backcross generation, markers closely linked to Xa21 and SUB1 QTL were used to select desirable plants possessing these resistance genes (foreground selection) and microsatellite markers polymorphic between donor and recurrent parent were also used to select plants that have maximum contribution from the recurrent parent genome (background selection). In BC1F1 generation, three best plants consiquently were selected from previously selected ten double heterozygous ( Xa21 and SUB1 QTL) plants. The percentage of recipient genome recovery in the best plant 1, 2 and 3 were 78.7%, 75.83% and 75.4%, respectively. Eventually this work illustrates the successful application of marker-assisted breeding for introgression of bacterial blight resistant gene into a rice variety of Bangladesh. Bangladesh J. Agril. Res. 42(3): 403-411, September 2017

Genotyping-by-sequencing (GBS) utilizes Next-Generation sequencing to genetically and physically map traits of interest. Here we use GBS to identify QTLs and SNP markers associated with milling and end-use quality traits in an hard red spring wheat recombinant inbred line (RIL) population. The RIL population and parents were phenotyped for eleven milling and end-use quality traits, and genotyped using GBS technology, simple sequence repeat (SSR) and allele specific sequence tagged site markers. A genetic map comprising 696 markers was used to map the end-use quality traits. Multiple QTL mapping identified 79 QTLs, 19 of which were declared ‘major’ as they explained greater than 15% of the phenotypic variance each. Transgressive segregants were observed for all phenotypes and co-locating QTLs controlling multiple quality traits were confirmed on chromosomes 1BL, 5AS, 7AS, 7AL and 7BS. To date, no GBS analysis to locate end-use quality QTLs in wheat has been conducted, thus the reporting and validation of these GBS sequence tags and their associated QTLs will shed light on the genetic architecture underlying these quantitative traits and assist wheat breeders in developing cultivars with favorable alleles through the use of marker assisted selection.

Background K. pneumoniae (KPN) has emerged as a major hospital associated pathogen in the recent years. Clonal complex (CC) 258 constitutes an international epidemic clone responsible for spread of extended spectrum beta-lactamases (ESBL) and carbapenemases. We evaluated the core genome (cg) MLST, resistance (R) gene and plasmid profiles of a worldwide collection of KPN using the 1928 bioinformatic cloud platform. Methods A total of 155 KPN clinical isolates (CC258, n=129; non-CC258, n=26) collected from 19 countries during 2018 of SENTRY Program were analyzed. Isolates included carbapenem resistant (CR; n=120) and non-CR (n=35). Whole genome sequencing FASTQ files were uploaded to the 1928 pipeline for analysis. Results Most CC258 isolates belonged to ST258, ST11 and ST512 (Table), and separated from unrelated ST by allelic distance (ad) > 2000. cgMLST grouped together isolates from the same STs, and those from similar geographies had greater homology, except isolates from US showed greater heterogenicity (ad 620). Applying an ad cutoff of < 100, ST258 isolates grouped in 4 clades with a predominance of either KPC-2 or -3. An ad cutoff of < 200 identified 7 clades within ST11 that were related by geography and R genes. Among CC258 isolates KPC was the major carbapenemase (58.1%) and associated with Tn4401 (a or b in 84% KPC); NDM-1 was detected (8.5%) only in ST11 and ST395. KPC-2 was more prevalent in Latin America and the isolates were closely related (ad 104) among ST258 compared to ST11 (ad 355). KPC-3 CC258 isolates showed ad of 164 and were from Italy, Russia, Greece, and US. CTX-M-14 was prevalent in ST258 while CTX-M-15 was common in other STs, except ST512 which carried no CTX-M despite clustering within ST258. Conclusion 1928 generated cgMLST showed good correlation with MLST in classifying all KPN isolates. ST258 isolates showed tight clustering, no NDM genes and distribution in the Americas while ST11 showed global dissemination and diversity of carbapenemases. Table 1 Disclosures Fredrik Dyrkell, n/a, 1928 Diagnostics (Employee) Dimitrios Arnellos, n/a, 1928 Diagnostics (Employee) Mariana Castanheira, PhD, 1928 Diagnostics (Research Grant or Support)A. Menarini Industrie Farmaceutiche Riunite S.R.L. (Research Grant or Support)Allergan (Research Grant or Support)Allergan (Research Grant or Support)Amplyx Pharmaceuticals (Research Grant or Support)Cidara Therapeutics (Research Grant or Support)Cidara Therapeutics (Research Grant or Support)Cipla Ltd. (Research Grant or Support)Cipla Ltd. (Research Grant or Support)Fox Chase Chemical Diversity Center (Research Grant or Support)GlaxoSmithKline (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Merck (Research Grant or Support)Merck (Research Grant or Support)Merck & Co, Inc. (Research Grant or Support)Merck & Co, Inc. (Research Grant or Support)Paratek Pharma, LLC (Research Grant or Support)Pfizer (Research Grant or Support)Qpex Biopharma (Research Grant or Support)

Background A few extraintestinal pathogenic Escherichia coli (ExPEC) multilocus sequence types (STs) cause the majority of community-acquired urinary tract infections (UTIs). We examine the genomic epidemiology of major ExPEC lineages, specifically factors associated with intestinal acquisition. Methods A total of 385 women with UTI caused by E. coli across Canada were asked about their diet, travel, and other exposures. Genome sequencing was used to determine both ST and genomic similarity. Logistic regression was used to identify factors associated with the acquisition of and infection with major ExPEC STs relative to minor ExPEC STs. Results ST131, ST69, ST73, ST127, and ST95 were responsible for 54% of all UTIs. Seven UTI clusters were identified, but genomes from the ST95, ST127, and ST420 clusters exhibited as few as 3 single nucleotide variations across the entire genome, suggesting recent acquisition. Furthermore, we identified a cluster of UTIs caused by 6 genetically-related ST1193 isolates carrying mutations in gyrA and parC. The acquisition of and infection with ST69, ST95, ST127, and ST131 were all associated with increased travel. The consumption of high-risk foods such as raw meat or vegetables, undercooked eggs, and seafood was associated with acquisition of and infection with ST69, ST127, and ST131, respectively. Conclusions Reservoirs may aid in the dissemination of pandemic ExPEC lineages in the community. Identifying ExPEC reservoirs may help prevent future emergence and dissemination of high-risk lineages within the community setting.

Sequence tagged microsatellite site (STMS) are useful PCR based DNA markers. Wide genome coverage, high polymorphic index and co-dominant nature make STMS a preferred choice for marker assisted selection (MAS), genetic diversity analysis, linkage mapping, seed genetic purity analysis etc. Routine STMS analysis involving low-throughput, laborious and time-consuming polyacrylamide/agarose gels often limit their full utility in crop breeding experiments that involve large populations. Therefore, convenient, gel-less marker detection methods are highly desirable for STMS markers. The present study demonstrated the utility of SYBR Green dye based melt-profiling as a simple and convenient gel-less approach for detection of STMS markers (referred to as GLADS) in bread wheat and rice. The method involves use of SYBR Green dye during PCR amplification (or post-PCR) of STMS markers followed by generation of a melt-profile using controlled temperature ramp rate. The STMS amplicons yielded characteristic melt-profiles with differences in melting temperature (Tm) and profile shape. These characteristic features enabled melt-profile based detection and differentiation of STMS markers/alleles in a gel-less manner. The melt-profile approach allowed assessment of the specificity of the PCR assay unlike the end-point signal detection assays. The method also allowed multiplexing of two STMS markers with non-overlapping melt-profiles. In principle, the approach can be effectively used in any crop for STMS marker analysis. This SYBR Green melt-profiling based GLADS approach offers a convenient, low-cost (20–51%) and time-saving alternative for STMS marker detection that can reduce dependence on gel-based detection, and exposure to toxic chemicals.

Aim To assess the association between azoospermia factor c microrearrangements and semen quality, and between Y-chromosome background with distinct azoospermia factor c microrearrangements and semen quality impairment. Methods This retrospective study, carried out in the Research Center for Genetic Engineering and Biotechnology “Georgi D. Efremov,” involved 486 men from different ethnic backgrounds referred for couple infertility from 2002- 2017: 338 were azoospermic/oligozoospermic and 148 were normozoospermic. The azoospermia factor c microrearrangements were analyzed with sequence tagged site and sequence family variant markers, quantitative fluorescent polymerase chain reaction, and multiplex ligation probe amplification analysis. The Y-haplogroups of all participants were determined with direct single nucleotide polymorphism typing and indirect prediction with short tandem repeat markers.Results Our participants had two types of microdeletions: gr/gr and b2/b3; three microduplications: b2/b4, gr/gr, and b2/b3; and one complex rearrangement gr/gr deletion + b2/b4 duplication. Impaired semen quality was not associated with microrearrangements, but b2/b4 and gr/ gr duplications were significantly associated with haplogroup R1a (P < 0.001 and P = 0.003, respectively) and b2/b3 deletions with haplogroup E (P = 0.005). There were significantly more b2/b4 duplication carriers in Albanians than in Macedonians with haplogroup R1a (P = 0.031). Conclusion Even though azoospermia factor c partial deletions/duplications and Y-haplogroups were not associated with impaired semen quality, specific deletions/ duplications were significantly associated with distinct haplogroups, implying that the Y chromosome background may confer susceptibility to azoospermia factor c microrearrangements.

Deletions in the AZoospermia Factor (AZF) regions (spermatogenesis loci) on the human Y chromosome are reported as one of the most common causes of severe testiculopathy and spermatogenic defects leading to male infertility, yet not much data is available for Indian infertile men. Therefore, we screened for AZF region deletions in 973 infertile men consisting of 771 azoospermia, 105 oligozoospermia and 97 oligoteratozoospermia cases, along with 587 fertile normozoospermic men. The deletion screening was carried out using AZF-specific markers: STSs (Sequence Tagged Sites), SNVs (Single Nucleotide Variations), PCR-RFLP (Polymerase Chain Reaction - Restriction Fragment Length Polymorphism) analysis of STS amplicons, DNA sequencing and Southern hybridization techniques. Our study revealed deletion events in a total of 29.4% of infertile Indian men. Of these, non-allelic homologous recombination (NAHR) events accounted for 25.8%, which included 3.5% AZFb deletions, 2.3% AZFbc deletions, 6.9% complete AZFc deletions, and 13.1% partial AZFc deletions. We observed 3.2% AZFa deletions and a rare long AZFabc region deletion in 0.5% azoospermic men. This study illustrates how the ethnicity, endogamy and long-time geographical isolation of Indian populations might have played a major role in the high frequencies of deletion events.

Anthracnose, caused by Colletotrichum spp. is the most devastating disease of chili (Capsicum annuum) in the tropical and subtropical regions of the world. The present study aimed at molecular mapping and development of markers linked to a new gene for anthracnose resistance in the chili cultivar ‘Punjab Lal’. Phenotypic evaluation of F1, F2, and BC1F1 populations derived from a cross between ‘Punjab Lal’ and susceptible cultivar ‘Arka Lohit’ against a virulent isolate of C. truncatum revealed that anthracnose resistance in Punjab Lal is governed by a monogenic-dominant gene designated as RCt1. Forty-four (28 ISSRs and 16 AFLPs) out of 201 markers exhibited parental polymorphism and were used in bulk segregant analysis. Three ISSRs (ISSR411493, ISSR581485, and ISSR1121857) and one AFLP marker (E-ACA/M-CTG516) showed precise polymorphism between resistant and susceptible bulks, and were used for genotyping F2 and BC1 populations. The four putative fragments were converted into sequence-tagged site (STS) markers and southern blotting confirmed their association with the resistance locus. Molecular mapping revealed that the STS markers CtR-431 and CtR-594 were closely linked to the RCt1 locus in coupling at distances of 1.8 and 2.3 cM, respectively. Furthermore, both of these markers showed the presence of resistance-linked allele in seven genotypes including the highly resistant C. chinnese ‘PBC932’ and C. baccatum ‘PBC80’ while negatively validated in 32 susceptible genotypes. Therefore, CtR431 and CtR-594 could be recommended as efficient diagnostic markers to facilitate the introgression of RCt1 locus into susceptible chili variants towards the development of high-yielding anthracnose resistance genotypes in C. annuum background.

Background As the pandemic SARS-CoV-2 virus has spread globally its genome has diversified and distinct clones can now be recognized, tracked, and traced. Identifying clonal groups allows for assessment of geographic spread, transmission events, and identification of more virulent or transmissible emerging strains. Methods All SARS-CoV-2 genomes (n=17,504) that are complete and high coverage were downloaded from GISAID on May 17th 2020. We developed a GNU-based Virus IDentification (GNUVID) tool that implements a whole genome multilocus sequence typing (wgMLST) scheme composed of all ten ORFs in the SARS-CoV-2 genome. The 10,422 genomes that passed our quality check were fed to the GNUVID tool, which assigned a ST profile to each genome. Global optimum eBURST was then used to cluster the STs in clonal complexes (CCs). Results Our ST/CC analysis uncovered strong associations of ST/CCs with certain geographical regions but also dynamic local changes in ST/CC prevalence. We also identified several unexpected putative global transmission events (e.g., from the US to the Middle East and reintroduction to China later in the pandemic). We have made our tool (GNUVID) available so that new WG sequences can be rapidly assigned to an ST/CC (https://github.com/ahmedmagds/GNUVID). Conclusion Our sequence typing system uncovered previously unappreciated transmission events and waves of expansion and replacement of SARS-CoV-2 STs and CCs in different geographical locations, suggesting complex dynamics in viral populations that previously seemed monomorphic. Because, our tool can be rapidly updated with new sequencing data it can track emerging clones and identifying new hotspots. Disclosures All Authors: No reported disclosures

The azoospermia factor c (AZFc) region harbors multi-copy genes that are expressed in the testis. Deletions of this region lead to reduced copy numbers of these genes. In this present study we aimed to determine the frequency of AZFc subdeletion in infertile and fertile men from Tunisia and to identify whether deletions of DAZ and CDY1 gene copies are deleterious on spermatogenesis and on semen quality. We studied a group of 241 infertile men and 115 fertile healthy males using a sequence tagged site (STS)±method. To gain insight into the molecular basis of the heterogeneous phenotype observed in men with the deletion we defined the type of DAZ and CDY1 genes deleted. We reported in the present study and for the first time a new type of AZFc deletion (gr/gr-DAZ2-DAZ4-CDY1b) and hypothesis that this new deletion is the result of two successive events. We also demonstrated that this deletion constitutes a relative high-risk factor for male infertility in Tunisian population.

Identification of alleles responsible for low molecular weight protein subunits (LMW-GS) is of immediate concern due to their large effect on wheat processing qualities, especially gluten elasticity. Allele-specific PCR (AS-PCR) molecular markers have been developed to screen wheat genotypes for appropriate LMW-GS alleles. In this study sequence tagged site AS-PCR markers were used for identification of alleles at Glu-A3 and Glu-B3 loci for LMW protein subunits in twenty-six wheat cultivars developed in Pakistan to assess their potential contribution to enhance bread-making quality. Three alleles at Glu-A3 and four alleles at Glu-B3 loci were explored in the wheat genotypes under study. Allelic richness (4) and genetic diversity (H) (0.7) was comparatively higher for Glu-B3 locus as compared to Glu-A3 locus. Glu-A3b and Glu-A3d alleles were observed in 11 (64%) cultivars, which were known to encode high sedimentation and protein contents, which made them the important

Buckwheat (Fagopyrum spp.) has been an integral part of cold arid agro-ecology of the Indian Himalayas by providing food, nutritional and medicinal utilities. Development of trait-specific DNA markers can accelerate characterization of buckwheat accessions conserved in genebank and their subsequent utilization in varietal development programmes as well as in genomic studies. Here, we report the development of eighteen gene-specific sequence tagged site (STS) markers and their possible association with phenotypic traits in buckwheat. Four markers—BW10, BW12, BW22 and BW27— showed considerable polymorphism (PIC > 0.5). Overall, the eighteen gene-specific STS markers tested against 91 buckwheat germplasm accessions produced 2.76 alleles per locus and showed moderate estimates of polymorphic information content (0.268). The buckwheat accessions were phenotyped for 14 quantitative and 10 qualitative descriptors. The metric traits showed moderate variation (mean CV = 32.4). Among qualitative descriptors, flower colour and seed traits (shape, colour and shattering) showed substantial diversity (99 and 64 % of Hmax respectively) computed as Shannon–Weaver diversity indices. Multivariate analysis of variance suggested eight of the STS loci—BW10 (8KD allergen protein), BW18 (Aspartic proteinase 9), BW13 (Legumin-like protein), BW17 (Chalcone synthase), BW22 (Declined protein during seed development), BW09 (13S globulin), BW25 (Cys peroxiredoxin) and BW24 (Fagopyritol synthase 1)—to have significant association with six of the phenotypic traits (test weight, days to maturity, leaf length, number of primary branches, plant height and seed shape).

DNA based molecular markers have enormous potential to improve the efficiency and precision of conventional plant breeding via marker-assisted selection. Most of the molecular markers are individually amplified by oligonucleotide primers using PCR. The efficiency of PCR depends on primer annealing temperature ( Ta) which optimize amplification and minimize non-specific binding of primers to the template. In contrast to the majority of the software programs available to calculate melting temperature (Tm) of primers/oligonucleotides which enables us to get Tm for single sequence/ input at a time. Hence, we developed an MS-Excel based programme to calculate Tm for a large set of primers with one click based on two formulae viz., basic and salt adjusted depending on the size of the primers. A wide range of oligonucleotides with size range between 10 to 30mer covering RAPD, AFLP, ISSR SSR and SNP markers were tested which are widely used for marker-assisted selection. Keywords: PCR, DNA based markers, Marker Assisted Selection (MAS,) Melting Temperature (Tm), Annealing Temperature (Ta), MS-Excel I Introduction One of the major concerns of modern agriculture is the identification and utilization of valuable genes of agronomic importance in crop plants. The development and use of DNA based molecular markers to track loci that are tightly linked to important genes have irrevocably changed the fields of genetics and plant breeding. Molecular markers exhibit a high degree of polymorphism and simple inheritance patterns with minimal influence of environment and epistasis. Therefore, in combination with conventional breeding approaches, DNA based molecular markers can be used to monitor the presence or lack of these genes in breeding populations and monitoring the level of gene expression under biotic and abiotic stress situation through Marker Assisted Selection (MAS). Several techniques have been developed for the past few years for distinguishing the individuals at DNA sequence level. Most of the markers are PCR (Polymerase Chain Reaction) based and are individually amplified by using oligonucleotide primers. If there is no prior sequence information available in the database for a particular species, random markers like Random Amplified polymorphic DNA (RAPD), Amplified Fragment Length Polymorphism (AFLP) and Inter Simple Sequence Repeats (ISSR) can be employed. These are multilocus, mostly dominant genetic markers usually produce multiple DNA fragments (each of which is considered as a locus) allowing the generation of a large number of loci across the genome without prior genome sequence information. On the other hand, if sequence information is available, then species specific markers like microsatellites or Simple Sequence Repeats (SSRs) and Single nucleotide polymorphisms (SNPs) can be used for genotyping. In all the PCR based markers, PCR will be efficient under optimal conditions like template DNA concentration, salt concentrations and primer annealing temperature (Ta). Keeping other parameters in PCR constant , the successful amplification of a particular locus depends on optimum Ta which in turn depends on primer sequence and GC content, complementarities of forward and reverse primers, reaction volume etc. The primers of different markers vary in length and sequence composition (GC content) which in turn leads to the variation in primer annealing temperature (Ta). Primer annealing is a physical process where the primers will jiggle around and hydrogen bonds will be constantly formed and broken between specific bases in the single stranded primers and the single stranded template DNA. More stable bonds last a little bit longer (primers that fit exactly) and on that little double-stranded piece the DNA polymerase can attach and start copying the template and thereby results in doubling target sequence.

Iron (Fe) toxicity is one of the limiting factors for rice growth and production in paddy fields. The use of iron tolerant varieties is one of the most efficient way to solve this problems. Identification of molecular markers linked to the trait is very important to develop marker-assisted selection (MAS) to obtain tolerant lines. The objective of this study was to identify sequence tagged sites (STS) markers associated with iron tolerance trait in double haploid rice population. Forty-five double haploid lines derived from reciprocal double crossing, i.e. IR54/Parekaligolara//Bio110/ Markuti, were phenotypically screened in high Fe wetland rice field at Taman Bogo Experimental Station, Lampung. Molecular analysis performed using STS markers. The results of the association between the genetic and phenotypic analysis showed that there were three markers, i.e. OsIRT1 , OsIRT2 , and OsFRO2 , associated with iron tolerance trait in rice. The markers have potential as selection markers for iron toxicity tolerant lines.

Momordica dioica (family—Cucurbitaceae), a perennial, dioecious climber with homomorphic sex chromosomes has been recently identified as a strategic plant for studying the early evolution of sex in angiosperms. Further, there is a greater demand for early sex identification in M. dioica because of its sex-specific medicinal and economic values. In this study, we used inter simple sequence repeat (ISSR) primers for developing a reliable molecular marker linked to sporophytic expression in M. dioica . Screening of bulked DNA pooled from male and female genotypes using 27 ISSR primers resulted in the detection of a male specific marker MdMSM . A sex-linked sequence tagged site (STS) marker MdY1 designed from MdMSM could efficiently differentiate the male and female M. dioica genotypes across different growth stages and geographical regions. Genome walking across the length of the sex-linked marker showed that MdY1 is highly homologous to the reverse transcriptase domain of a polyprotein and nested within a Copia -like retrotransposable element, MdRE1. Southern blot analysis revealed multiple copy locus and exclusive presence of MdY1 and MdRE1 only in the male genome suggesting that it might be exhibiting prominent Y-chromosome specific characteristics with strong expressions in the evolution of male sex organs. Thus, the STS marker MdY1 together with the Y-specific retroelement MdRE1 could be used as an efficient tool for early sex diagnosis in M. dioica , as well as a starting material towards characterization of traits involved in sex determination and evolutionary pathways of sexual dimorphism.

Bacterial Leaf Blight (BLB) is the most important yield limiting factor in Nepalese rice. BLB resistance rice varieties are highly demanding in the country. Breeding efforts for developing disease resistant depends on availability and use of resistant gene donors. Nepalese rice landraces could be the source of resistant gene. Therefore, ninety six Nepalese rice accessions were screened using eight Simple Sequence Repeats (SSR) markers and one Sequence Tagged Sites (STS) marker for presence and absence of BLB resistance gene. We have detected BLB resistance gene Xa-10 on five accessions, Xa-13 on six accessions, Xa-7 on 23 accessions, Xa-3 and Xa-4 on 52 accessions, Xa-5 on 25 accessions, Xa-8 on 30 rice accessions. No any rice accessions tested have Xa-21. Similarly, 17 rice accessions showed three and more than three BLB resistance genes. Presence of Xa-13 on susceptible check variety CNTRL-85033 confirmed that this resistant gene is not working in Nepalese rice field. Therefore, Nepal need to pyramide the BLB resistant genes for durable resistance.Nepal Journal of Biotechnology. Dec. 2015 Vol. 3, No. 1: 35-39

YrSM139-1B maybe a new gene for effective resistance to stripe rust and useful flanking markers for marker-assisted selection were developed. Stripe rust, caused by Puccinia striiformis f. sp. tritici, is an important foliar disease of wheat. Two dominant stripe rust resistant genes YrSM139-1B and YrSM139-2D were pyramided in bread wheat cultivar Shaanmai 139; one from wild emmer and the other from Thinopyrum intermedium. Three near-isogenic F7:8 line pairs (contrasting RILs), N122-1013R/S, N122-185R/S, and N122-1812R/S, independently derived from different F2 plants and differing at the YrSM139-1B locus were generated from the cross Shaanmai 139 × Hu 901-19 through marker-assisted selection. A large F2:3 population from cross N122-1013R × N122-1013S tested for stripe rust response and subjected to analysis with markers in the 1BS10-0.5 bin region using SSR expressed sequence tags (EST) and site-specific sequence markers developed from the 90 K Illumina iSelect SNP array. Five EST–STS markers and four allele-specific PCR markers were mapped to the YrSM139-1B region. The 30.5 cM genetic map for YrSM139-1B consisted of nine markers, two of which were closer to YrSM139-1B than Xgwm273, which was used in producing the contrasting RIL pairs. Race response data and allelism tests showed that YrSM139-1B is different from Yr10, Yr15, and Yr24/26/CH42.

Rationale: This study aimed to report 1 family case with novel Y chromosome structural variations by an established next-generation sequencing (NGS) method using unique STSs. Patient concerns: The case studied was from a family with a father and son (the proband). G-band staining was used for karyotype analysis. Y chromosome microdeletions were detected by sequence-tagged site (STS)-PCR analysis and a new NGS screening strategy. Diagnoses: Semen analysis showed that the proband was azoospermic. The patient had an abnormal karyotype (45,X[48%]/46,XY[52%]). His father exhibited a normal karyotype. STS-PCR analysis showed that the proband had a deletion of the AZFb+c region, and his father had no deletion of STS markers examined. The sequencing method revealed that the patient had DNA sequence deletions from nt 20099846 to nt 28365090 (8.3 Mb), including the region from yel4 to the Yq terminal, and his father exhibited a deletion of b1/b3 and duplication of gr/gr. Interventions: The proband was advised to undergo genetic counseling, and consider the use of sperm from a sperm bank or adoption to become a father. Outcomes: The proband was azoospermic. AZFc partial deletions may produce a potential risk for large AZFb+c deletions or abnormal karyotypes causing spermatogenic failure in men. Lessons: The NGS method can be considered a clinical diagnostic tool to detect Y chromosome microdeletions. The partial AZFc deletions and/or duplications can be a risk of extensive deletions in offspring.

Candida glabrata is a genetically diverse human pathogenic yeast, whose subpopulations have been documented to vary geographically. Here, we report MLST genotypes and antifungal drug susceptibility of C. glabrata isolates from Africa. Among 47 mostly urogenital isolates, we found 13 sequence types, amounting to a 27% genetic population difference. More than half of the isolates were of novel sequence types. ST18 was most predominant and had reduced susceptibility to fluconazole. There was clear segregation of STs between urine and vaginal specimen. In Tanzania, the C. glabrata population is genetically diverse, and divergent from those seen in other countries.