Identification of sex-specific markers based on gene expression in adult Colorado potato beetles.

The Colorado potato beetle, Leptinotarsa decemlineata, (Coleoptera: Chrysomelidae) is an economically important pest insect of potatoes. Understanding how the mechanisms driving its invasiveness vary between sexes will be critical for developing modern control methods. However, the currently available methods for sexing adult Colorado potato beetles are either inefficient or unsuitable for projects that require RNA as an input, like those measuring gene expression. Therefore, the development of simple molecular tools that are tailored to these studies is important. In this study, we used publicly available RNA-seq data to select 5 candidate genes for sex-specific markers in adult Colorado potato beetles. We confirmed that our 5 marker candidates exhibit a sex-specific expression pattern and can be used as PCR markers for sex determination. This method of sex detection will allow researchers to distinguish the sex of the individual with a simple PCR reaction using cDNA as the template and assign sex to RNA-seq samples post hoc.

Permanent Spreading of 1RS.1AL and 1RS.1BL Translocations in Modern Wheat Breeding.

Wheat-rye translocations 1RS.1BL and 1RS.1AL are used in bread wheat breeding worldwide because a short arm of rye chromosome 1 (1RS) when introgressed into the wheat genome confers resistance to diseases, pests and better performance under drought-stress conditions. However, in durum wheat genotypes, these translocations occur only in experimental lines, although their advantages could enhance the potential of this crop. P.P. Lukyanenko National Grain Centre (NGC) has successfully developed commercially competitive cultivars of bread and durum wheat demanded by many agricultural producers in the South of Russia for decades. Here, 94 accessions of bread and 343 accessions of durum wheat, representing lines and cultivars from collection, competitive variety trials and breeding nursery developed at NGC were screened for 1RS using PCR markers and genomic in situ hybridization. The 1RS.1BL and 1RS.1AL translocations were detected in 38 and 6 bread wheat accessions, respectively. None of the durum wheat accessions showed translocation, despite the fact that some of them had 1RS.1BL donors in their pedigree. The absence of translocations in the studied durum wheat germplasm can be caused by the negative selection of 1RS carriers at different stages of the breeding process due to low quality and difficulties in transferring rye chromatin through wheat gametes.

RppM, Encoding a Typical CC-NBS-LRR Protein, Confers Resistance to Southern Corn Rust in Maize.

Southern corn rust (SCR) caused by Puccinia polysora Underw. poses a major threat to maize production worldwide. The utilization of host SCR-resistance genes and the cultivation of resistant cultivars are the most effective, economical strategies for controlling SCR. Here, we identified and cloned a new SCR resistance gene, RppM, from the elite maize inbred line Jing2416K. RppM was found to encode a typical CC-NBS-LRR protein localized in both the nucleus and cytoplasm. This gene was constitutively expressed at all developmental stages and in all tissues examined, with the strongest expression detected in leaves at the mature stage. A transcriptome analysis provided further evidence that multiple defense systems were initiated in Jing2416K, including pathogen-associated molecular pattern-triggered immunity and effector-triggered immunity, reinforcement of cell walls, accumulation of antimicrobial compounds, and activation of phytohormone signaling pathways. Finally, we developed functional Kompetitive allele-specific PCR markers for RppM using two conserved SNP sites and successfully applied these functional markers for the detection of RppM and the cultivation of resistant maize cultivars, demonstrating their great potential utility in maize breeding.

Photosystem I P700 chlorophyll a apoprotein A1 as PCR marker to identify diatoms and their associated lineage.

The morphological characteristics of diatoms are useful for studying their taxonomy. However, the distinction between closely related diatom taxa can be very difficult, especially when the morphological characters are modified by environmental constraints. In the present study, 13 fresh water diatoms were identified morphologically and cultured under axenic conditions. To check this, PCR primers specific for multilocus genes were designed to amplify and screen 13 fresh water diatom monocultures. Multilocus PCR primers (DRR3, scfcpA, Lhcf11, SIT1, SIT3, SIT4, LOC101218388, COI-5P, rbcL, rbcL-3P, LSU D2/D3, UPA, psaA, and 18S rRNA) were tested. It was found that psaA gene, a plant pigment chlorophyll-based PCR marker, amplified in all the diatoms. Out of 13 diatom amplicons, only two fresh water diatoms DNA were sequenced. This included Cyclotella meneghiniana and Sellaphora pupula. The Sanger sequencing results thus established that morphologically identified diatom, Sellaphora pupula, exhibited close phylogeny to Sellaphora whereas fresh water Cyclotella meneghiniana has close lineage to marine diatom Thallosiosira.

Generation and molecular marker and cytological characterization of wheat - Secale strictum subsp. anatolicum derivatives.

Cereal rye and its wild forms are important sources of genetic diversity for wheat breeding due to their resistances to biotic and abiotic stresses. Secale strictum subsp. anatolicum (Boiss.) K. Hammer (SSA) is a weedy relative of cultivated rye, S. cereale. Meiotic chromosome pairing in F1 hybrids of SSA and S. cereale reveals strong genomic affinity between the two genomes. A study of the transferability of S. cereale sequence-based markers to SSA and hexaploid triticale demonstrated their applicability for tracing SSA chromatin in wheat. The transferability of the markers was over 80% from homoeologous groups 1, 2, and 3, and greater than 70% from groups 4 to 7. This study focused on the generation and molecular and cytogenetic characterization of wheat-SSA alien derivatives. Twelve were identified using combinations of non-denaturing fluorescence in situ hybridization (ND-FISH), genomic in situ hybridization (GISH), and molecular marker analysis. All SSA chromosomes, except 3Ra and 6Ra, were transferred to wheat either in the form of monosomic additions (MA), mono-telosomic additions (MtA), double-mono-telosomic additions (dMtA), or double-monosomic additions (dMA). The germplasm developed in this study will help to enhance the genetic base of wheat and facilitate molecular breeding of wheat and triticale.

Molecular epidemiology of Pseudomonas aeruginosa isolated from lower respiratory tract of ICU patients / Epidemiologia molecular de Pseudomonas aeruginosa isolada em pacientes com infecções do trato respiratório inferior admitidos em UTI

Lower respiratory tract infections (LRTIs) caused by Pseudomonas aeruginosa are the most common infection among hospitalized patients, associated with increased levels of morbidity, mortality and attributable health care costs. Increased resistant Pseudomonas worldwide has been quite meaningful to patients, especially in intensive care unit (ICUs). Different species of Pseudomonas exhibit different genetic profile and varied drug resistance. The present study determines the molecular epidemiology through DNA fingerprinting method and drug resistance of P. aeruginosa isolated from patients with LTRIs admitted in ICU. A total of 79 P. aeruginosa isolated from patients with LRTIs admitted in ICU were characterized by Restriction Fragment Length Polymorphism (RFLP), Random Amplified Polymorphic DNA (RAPD) and Repetitive Extrapalindromic PCR (REP-PCR). Antibiotic resistance was determined by minimum inhibitory concentration (MIC) assay while MDR genes, viz, blaTEM, blaOXA, blaVIM, blaCTX-M-15 were detected by polymerase chain reaction (PCR). Of the 137 Pseudomonas sp isolated from ICU patients, 57.7% of the isolates were reported to be P. aeruginosa. The overall prevalence of P. aeruginosa among the all included patients was 34.5%. The RAPD analysis yielded 45 different patterns with 72 clusters with 57% to 100% similarity level. The RFLP analysis yielded 8 different patterns with 14 clusters with 76% to 100% similarity level. The REP PCR analysis yielded 37 different patterns with 65 clusters with 56% to 100% similarity level. There was no correlation among the different DNA patterns observed between the three different methods. Predominant of the isolates (46.8%) were resistant to amikacin. Of the 79 isolates, 60.8% were positive for blaTEM gene and 39.2% were positive for blaOXA gene. P. aeruginosa was predominantly isolated from patients with LRTIs admitted in ICU. The difference in the similarity level observed between the three DNA fingerprinting methods indicates that there is high inter-strain variability. The high genetic variability and resistance patterns indicates that we should continuously monitor the trend in the prevalence and antibiotic resistance of P. aeruginosa especially in patients with LRTIs admitted in ICU.

Infecções do trato respiratório inferior (ITRIs) são as infecções mais comuns entre pacientes internados em unidade de terapia intensiva (UTI). Pseudomonas aeruginosa é a causa mais comum de ITRIs e está associada ao aumento da mortalidade. Diferentes espécies de Pseudomonas exibem diferentes perfis genéticos e resistência variada as drogas. O presente estudo determina a epidemiologia molecular através do método de fingerprinting de DNA e resistência as drogas de P. aeruginosa isoladas de pacientes com LTRIs internados em UTI. Um total de 79 P. aeruginosa isoladas de pacientes com ITRIs internados em UTI foram caracterizados por Polimorfismo de Comprimento de Fragmentos de Restrição (RFLP), DNA Polimórfico Amplificado ao Acaso (RAPD) e PCR Extrapalindrômico Repetitivo (REP-PCR). A resistência aos antibióticos foram determinadas pelos ensaios de concentrações inibitória mínima (MIC), enquanto os genes MDR, blaTEM, blaOXA, blaVIM, blaCTX-M-15 foram detectados pela reação em cadeia da polimerase (PCR). Das 137 Pseudomonas sp isoladas de pacientes de UTI, 57,7% dos isolados foram relatados como P. aeruginosa. A prevalência geral de P. aeruginosa entre os pacientes incluídos foram de 34,5%. A análise RAPD renderam 45 padrões diferentes com 72 clusters com nível de similaridade de 57% a 100%. A análise RFLP renderam 8 padrões diferentes com 14 clusters com 76% a 100% de similaridade. A análise de PCR do REP produziram 37 padrões diferentes com 65 clusters com nível de similaridade de 56% a 100%. Não houveram correlações entre os diferentes padrões de DNA observados entre os três diferentes métodos. Predominantes dos isolados (46,8%) eram resistentes à amicacina. Dos 79 isolados, 60,8% foram positivos para o gene blaTEM e 39,2% foram positivos para o gene blaOXA. P. aeruginosa foi predominantemente isolado de pacientes com ITRIs internados em UTI. A diferença no nível de similaridade observado entre os três métodos de fingerprinting do DNA indica que há alta variabilidade inter-strain. A alta variabilidade genética e os padrões de resistência indicam que devemos monitorar continuamente a tendência na prevalência e resistência a antibióticos de P. aeruginosa, especialmente em pacientes com ITRIs internados em UTI.

The Use of Pentaploid Crosses for the Introgression of Amblyopyrum muticum and D-Genome Chromosome Segments Into Durum Wheat.

The wild relatives of wheat provide an important source of genetic variation for wheat improvement. Much of the work in the past aimed at transferring genetic variation from wild relatives into wheat has relied on the exploitation of the ph1b mutant, located on the long arm of chromosome 5B. This mutation allows homologous recombination to occur between chromosomes from related but different genomes, e.g. between the chromosomes of wheat and related chromosomes from a wild relative resulting in the generation of interspecific recombinant chromosomes. However, the ph1b mutant also enables recombination to occur between the homologous genomes of wheat, e.g. A/B, A/D, B/D, resulting in the generation of wheat intergenomic recombinant chromosomes. In this work we report on the presence of wheat intergenomic recombinants in the genomic background of hexaploid wheat/Amblyopyrum muticum introgression lines. The transfer of genomic rearrangements involving the D-genome through pentaploid crosses provides a strategy by which the D-genome of wheat can be introgressed into durum wheat. Hence, a pentaploid crossing strategy was used to transfer D-genome segments, introgressed with either the A- and/or the B-genome, into the tetraploid background of two durum wheat genotypes Karim and Om Rabi 5 in either the presence or absence of different Am. muticum (2n = 2x = 14, TT) introgressions. Introgressions were monitored in backcross generations to the durum wheat parents via multi-color genomic in situ hybridization (mc-GISH). Tetraploid lines carrying homozygous D-genome introgressions, as well as simultaneous homozygous D- and T-genome introgressions, were developed. Introgression lines were characterized via Kompetitive Allele-Specific PCR (KASP) markers and multi-color fluorescence in situ hybridization (FISH). Results showed that new wheat sub-genomic translocations were generated at each generation in progeny that carried any Am. muticum chromosome introgression irrespective of the linkage group that the segment was derived from. The highest frequencies of homologous recombination were observed between the A- and the D-genomes. Results indicated that the genotype Karim had a higher tolerance to genomic rearrangements and T-genome introgressions compared to Om Rabi 5. This indicates the importance of the selection of the parental genotype when attempting to transfer/develop introgressions into durum wheat from pentaploid crosses.

Molecular Characterization of 87 Functional Genes in Wheat Diversity Panel and Their Association With Phenotypes Under Well-Watered and Water-Limited Conditions.

Modern breeding imposed selection for improved productivity that largely influenced the frequency of superior alleles underpinning traits of breeding interest. Therefore, molecular diagnosis for the allelic variations of such genes is important to manipulate beneficial alleles in wheat molecular breeding. We analyzed a diversity panel largely consisted of advanced lines derived from synthetic hexaploid wheats for allelic variation at 87 functional genes or loci of breeding importance using 124 high-throughput KASP markers. We also developed two KASP markers for water-soluble carbohydrate genes (TaSST-D1 and TaSST-A1) associated with plant height and thousand grain weight (TGW) in the diversity panel. KASP genotyping results indicated that beneficial alleles for genes underpinning flowering time (Ppd-D1 and Vrn-D3), thousand grain weight (TaCKX-D1, TaTGW6-A1, TaSus1-7B, and TaCwi-D1), water-soluble carbohydrates (TaSST-A1), yellow-pigment content (Psy-B1 and Zds-D1), and root lesion nematodes (Rlnn1) were fixed in diversity panel with frequency ranged from 96.4 to 100%. The association analysis of functional genes with agronomic and biochemical traits under well-watered (WW) and water-limited (WL) conditions revealed that 21 marker-trait associations (MTAs) were consistently detected in both moisture conditions. The major developmental genes such as Vrn-A1, Rht-D1, and Ppd-B1 had the confounding effect on several agronomic traits including plant height, grain size and weight, and grain yield in both WW and WL conditions. The accumulation of favorable alleles for grain size and weight genes additively enhanced grain weight in the diversity panel. Graphical genotyping approach was used to identify accessions with maximum number of favorable alleles, thus likely to have high breeding value. These results improved our knowledge on the selection of favorable and unfavorable alleles through unconscious selection breeding and identified the opportunities to deploy alleles with effects in wheat breeding.

Allelic variation at high-molecular weight and low-molecular weight glutenin subunit genes in Moroccan bread wheat and durum wheat cultivars.

Glutenin is a major protein fraction contributing to the functional properties of gluten and dough. The glutenin constitutes 30-40% of the protein in wheat flour and about half of that in gluten. It is essential to identify correct glutenin alleles and to improve wheat quality by selecting alleles that exert favorable effects. Moroccan wheat cultivars are unique in West Asia and North Africa region, since many of them possess resistance to Hessian fly, a pest, which is becoming important in other countries in the region. Hence, these cultivars are being used as donor for the resistance in the breeding program. Here, we determine the allelic variation in high-molecular weight glutenin subunits (HMW-GS) and low-molecular weight glutenin subunits (LMW-GS) in Moroccan cultivars of bread and durum wheat using the gene-specific PCR markers. In 20 cultivars of bread wheat, 9 different allele variants were detected at HMW-GS and 13 different allele variants were detected at LMW-GS, in which the alleles Glu-A1b (2*), Glu-B1i (17 + 18), Glu-B1c (7*/7 + 9), Glu-D1d (5 + 10), Glu-A3c, Glu-B3 h, and Glu-D3b were the most frequents. In 26 cultivars of durum wheat, less allelic variation was found: seven different allele variants at HMW-GS and six different allele variants at LMW-GS were identified, in which the major alleles were Glu-A1c (null), Glu-B1b (7 + 8), Glu-B1e (20), Glu-A3c, and Glu-B3d. The mean value of the genetic diversity for the glutenin loci was 0.502 in bread wheat and 0.449 in durum wheat. Most of the glutenin alleles carried by Moroccan bread wheat cultivars impart good bread-making quality. Most of the durum wheat glutenin alleles were related to low strength dough or poor quality and need to be improved. To improve quality of Moroccan durum wheat, essentially, Glu-A1c and Glu-B3d alleles of the genes should be replaced with the better alleles through breeding.

Natural variation in CIRCADIAN CLOCK ASSOCIATED 1 is associated with flowering time in Brassica rapa.

Flowering time is a very important agronomic trait and the development of molecular markers associated with this trait can facilitate crop breeding. CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), a core oscillator component of circadian rhythms that affect metabolic pathways in plants, has been implicated in flowering time control in species of Brassica. CCA1 gene sequences from three Brassica rapa inbred lines, showing either early flowering or late flowering phenotypes, were analyzed and a high level of sequence variation was identified, especially within the fourth intron. Using this information, three PCR primer sets were designed and tested using various inbred lines of B. rapa. The usage of InDel markers was further validated by evaluation of flowering time and high resolution melting (HRM) analysis. Both methods, PCR and HRM, validated the use of newly developed markers. Additional sequence analyses of Brassica plants with diploid (AA, BB, or CC) and allotetraploid genomes further confirmed a large number of sequence polymorphisms in the CCA1 gene, including insertions/deletions in the fourth intron. Our results demonstrated that sequence variations in CCA1 can be used to develop valuable trait-related molecular markers for Brassica crop breeding.

Molecular epidemiology of Pseudomonas aeruginosa isolated from lower respiratory tract of ICU patients

Abstract Lower respiratory tract infections (LRTIs) caused by Pseudomonas aeruginosa are the most common infection among hospitalized patients, associated with increased levels of morbidity, mortality and attributable health care costs. Increased resistant Pseudomonas worldwide has been quite meaningful to patients, especially in intensive care unit (ICUs). Different species of Pseudomonas exhibit different genetic profile and varied drug resistance. The present study determines the molecular epidemiology through DNA fingerprinting method and drug resistance of P. aeruginosa isolated from patients with LTRIs admitted in ICU. A total of 79 P. aeruginosa isolated from patients with LRTIs admitted in ICU were characterized by Restriction Fragment Length Polymorphism (RFLP), Random Amplified Polymorphic DNA (RAPD) and Repetitive Extrapalindromic PCR (REP-PCR). Antibiotic resistance was determined by minimum inhibitory concentration (MIC) assay while MDR genes, viz, blaTEM, blaOXA, blaVIM, blaCTX-M-15 were detected by polymerase chain reaction (PCR). Of the 137 Pseudomonas sp isolated from ICU patients, 57.7% of the isolates were reported to be P. aeruginosa. The overall prevalence of P. aeruginosa among the all included patients was 34.5%. The RAPD analysis yielded 45 different patterns with 72 clusters with 57% to 100% similarity level. The RFLP analysis yielded 8 different patterns with 14 clusters with 76% to 100% similarity level. The REP PCR analysis yielded 37 different patterns with 65 clusters with 56% to 100% similarity level. There was no correlation among the different DNA patterns observed between the three different methods. Predominant of the isolates (46.8%) were resistant to amikacin. Of the 79 isolates, 60.8% were positive for blaTEM gene and 39.2% were positive for blaOXA gene. P. aeruginosa was predominantly isolated from patients with LRTIs admitted in ICU. The difference in the similarity level observed between the three DNA fingerprinting methods indicates that there is high inter-strain variability. The high genetic variability and resistance patterns indicates that we should continuously monitor the trend in the prevalence and antibiotic resistance of P. aeruginosa especially in patients with LRTIs admitted in ICU.

Resumo Infecções do trato respiratório inferior (ITRIs) são as infecções mais comuns entre pacientes internados em unidade de terapia intensiva (UTI). Pseudomonas aeruginosa é a causa mais comum de ITRIs e está associada ao aumento da mortalidade. Diferentes espécies de Pseudomonas exibem diferentes perfis genéticos e resistência variada as drogas. O presente estudo determina a epidemiologia molecular através do método de fingerprinting de DNA e resistência as drogas de P. aeruginosa isoladas de pacientes com LTRIs internados em UTI. Um total de 79 P. aeruginosa isoladas de pacientes com ITRIs internados em UTI foram caracterizados por Polimorfismo de Comprimento de Fragmentos de Restrição (RFLP), DNA Polimórfico Amplificado ao Acaso (RAPD) e PCR Extrapalindrômico Repetitivo (REP-PCR). A resistência aos antibióticos foram determinadas pelos ensaios de concentrações inibitória mínima (MIC), enquanto os genes MDR, blaTEM, blaOXA, blaVIM, blaCTX-M-15 foram detectados pela reação em cadeia da polimerase (PCR). Das 137 Pseudomonas sp isoladas de pacientes de UTI, 57,7% dos isolados foram relatados como P. aeruginosa. A prevalência geral de P. aeruginosa entre os pacientes incluídos foram de 34,5%. A análise RAPD renderam 45 padrões diferentes com 72 clusters com nível de similaridade de 57% a 100%. A análise RFLP renderam 8 padrões diferentes com 14 clusters com 76% a 100% de similaridade. A análise de PCR do REP produziram 37 padrões diferentes com 65 clusters com nível de similaridade de 56% a 100%. Não houveram correlações entre os diferentes padrões de DNA observados entre os três diferentes métodos. Predominantes dos isolados (46,8%) eram resistentes à amicacina. Dos 79 isolados, 60,8% foram positivos para o gene blaTEM e 39,2% foram positivos para o gene blaOXA. P. aeruginosa foi predominantemente isolado de pacientes com ITRIs internados em UTI. A diferença no nível de similaridade observado entre os três métodos de fingerprinting do DNA indica que há alta variabilidade inter-strain. A alta variabilidade genética e os padrões de resistência indicam que devemos monitorar continuamente a tendência na prevalência e resistência a antibióticos de P. aeruginosa, especialmente em pacientes com ITRIs internados em UTI.

Molecular diagnosis of cerebral toxoplasmosis: comparing markers that determine Toxoplasma gondii by PCR in peripheral blood from HIV-infected patients

As cerebral toxoplasmosis is the most common cerebral focal lesion in AIDS patients, this study evaluated three PCR markers for diagnosis, since some limitations remain present, such as low parasite levels in some clinical samples. The molecular markers were B22-B23 and Tg1-Tg2 (based on the B1 gene) and Tox4-Tox5 (non-coding fragment, repeated 200-300-fold). DNA samples from 102 AIDS patients with previously known diagnosis were analyzed. The cerebral toxoplasmosis group was constituted of DNA extracted from the blood of 66 AIDS patients, which was collected before or until the third day of the therapy for toxoplasmosis. DNA from the blood of 36 AIDS patients with other neurologic opportunistic infections was used as control group. Sensitivities of B22-B23, Tg1-Tg2, and Tox4-Tox5 markers were of 95.5 percent, 93.9 percent, and 89.3 percent, respectively. In the control group, the specificities were of 97.2 percent (B22-B23), 88.9 percent (Tg1-Tg2), and 91.7 percent (Tox4-Tox5). The association of at least two markers increased the PCR sensitivity and specificity. The concordance index between two markers varied from 83.3 percent to 93.1 percent. These data demonstrated that all markers evaluated here were highly sensitive for T. gondii determination, although B22-B23 has been shown to be the best. The association of two markers increases PCR sensitivity, but the procedure was more expensive and time-consuming.

Genetic structure of Aedes aegypti in the city of Córdoba (Argentina), a recently reinfested area

To understand the transmission of a vector-borne disease, knowledge of the magnitude of dispersal among vector populations is essential because of its influence on pathogen transfer. The principal vector of dengue, the most common arboviral disease in the world, is the mosquito Aedes aegypti (L.). This tropical and subtropical species is native to Africa but has dispersed worldwide since the XV century. In Argentina, the species was declared eradicated in 1963, but has reinfested the country in recent years. In the present work, we used RAPD-PCR markers to assess the levels of genetic variability and differentiation among populations of Ae. aegypti (the vector of dengue and yellow fever) in Córdoba, the second largest city in Argentina. We detected similar levels of genetic variability (He between 0.351-0.404) across samples and significant genetic differentiation between most population pairs within the city (F ST between 0.0013-0.0253). Genetic distances indicate that there are three distinct groups, formed predominantly by populations that are connected by, or near, main roads. This suggests that, in addition to other factors such as availability of oviposition sites or step-by-step migration, passive transport plays an important role in gene flow within the city.