Cryptosporidium parvum mixed genotypes detected by PCR-restriction fragment length polymorphism analysis.

Combinations of 10 Cryptosporidium parvum oocysts, with various ratios of genotype I to genotype II, were isolated and subjected to PCR-restriction fragment length polymorphism analysis. Amplification of both genotypes in these samples ranged from 31 to 74% and yielded no information about the genotype proportions. In addition, since both genotypes were not always detected, amplification of a single genotype is not conclusive evidence that the sample contains only a single genotype.     

Imputation of ungenotyped parental genotypes in dairy and beef cattle from progeny genotypes

The objective of this study was to quantify the accuracy of imputing the genotype of parents using information on the genotype of their progeny and a family-based and population-based imputation algorithm. Two separate data sets were used, one containing both dairy and beef animals (n=3122) with high-density genotypes (735 151 single nucleotide polymorphisms (SNPs)) and the other containing just dairy animals (n=5489) with medium-density genotypes (51 602 SNPs). Imputation accuracy of three different genotype density panels were evaluated representing low (i.e. 6501 SNPs), medium and high density. The full genotypes of sires with genotyped half-sib progeny were masked and subsequently imputed. Genotyped half-sib progeny group sizes were altered from 4 up to 12 and the impact on imputation accuracy was quantified. Up to 157 and 258 sires were used to test the accuracy of imputation in the dairy plus beef data set and the dairy-only data set, respectively. The efficiency and accuracy of imputation was quantified as the proportion of genotypes that could not be imputed, and as both the genotype concordance rate and allele concordance rate. The median proportion of genotypes per animal that could not be imputed in the imputation process decreased as the number of genotyped half-sib progeny increased; values for the medium-density panel ranged from a median of 0.015 with a half-sib progeny group size of 4 to a median of 0.0014 to 0.0015 with a half-sib progeny group size of 8. The accuracy of imputation across different paternal half-sib progeny group sizes was similar in both data sets. Concordance rates increased considerably as the number of genotyped half-sib progeny increased from four (mean animal allele concordance rate of 0.94 in both data sets for the medium-density genotype panel) to five (mean animal allele concordance rate of 0.96 in both data sets for the medium-density genotype panel) after which it was relatively stable up to a half-sib progeny group size of eight. In the data set with dairy-only animals, sufficient sires with paternal half-sib progeny groups up to 12 were available and the within-animal mean genotype concordance rates continued to increase up to this group size. The accuracy of imputation was worst for the low-density genotypes, especially with smaller half-sib progeny group sizes but the difference in imputation accuracy between density panels diminished as progeny group size increased; the difference between high and medium-density genotype panels was relatively small across all half-sib progeny group sizes. Where biological material or genotypes are not available on individual animals, at least five progeny can be genotyped (on either a medium or high-density genotyping platform) and the parental alleles imputed with, on average, ⩾96% accuracy.     

Transfer of intracolonial genetic variability through gametes in Acropora hyacinthus corals

In recent years, the new phenomenon of intracolonial genetic variability within a single coral colony has been described. This connotes that coral colonies do not necessarily consist of only a single genotype, but may contain several distinct genotypes. Harboring more than one genotype could improve survival under stressful environmental conditions, e.g., climate change. However, so far it remained unclear whether the intracolonial genetic variability of the adult coral is also present in the gametes. We investigated the occurrence of intracolonial genetic variability in 14 mature colonies of the coral Acropora hyacinthus using eight microsatellite loci. A grid was placed over each colony before spawning, and the emerging egg/sperm bundles were collected separately in each grid. The underlying tissues as well as the egg/sperm bundles were genotyped to determine whether different genotypes were present. Within the 14 mature colonies, we detected 10 colonies with more than one genotype (intracolonial genetic variability). Four out of these 10 mature colonies showed a transfer of different genotypes via the eggs to the next generation. In two out of these four cases, we found additional alleles, and in the two other cases, we found only a subset of alleles in the unfertilized eggs. Our results suggest that during reproduction of A. hyacinthus, more than one genotype per colony is able to reproduce. We discuss the occurrence of different genotypes within a single coral colony and the ability for those to release eggs which are genetically distinct.     

Host preference of plant genotypes is altered by intraspecific competition in a phytophagous insect

Variation among aphid genotypes leads them to preferentially colonize different host-plant genotypes. In a natural community, different genotypes within a species are expected to coexist on a single host plant, and these aphids can interact, potentially, altering host-plant preferences. Using a model aphid (Sitobion avenae) and barley (Hordeum vulgare) system, we compared aphid preference and performance in one- or two-genotype colonies in pots with genetically diverse host plants (6 genotypes) or genetically uniform host plants (1 genotype per pot). Aphid host preference was shown to differ when a second aphid genotype was present, with one aphid genotype exhibiting a preference change due to the genotypic identity of the second aphid. The population growth rate of the aphids was not influenced by the competitor, and thus, we conclude that these effects are due to aphid distribution (preference) rather than effects through performance. Our work demonstrates that within a complex ecological community, an individual’s behavior can be influenced by interactions with other genotypes within the same species, as well as interactions with genotypes of other species.     

Restriction of Francisella novicida Genetic Diversity during Infection of the Vector Midgut

The genetic diversity of pathogens, and interactions between genotypes, can strongly influence pathogen phenotypes such as transmissibility and virulence. For vector-borne pathogens, both mammalian hosts and arthropod vectors may limit pathogen genotypic diversity (number of unique genotypes circulating in an area) by preventing infection or transmission of particular genotypes. Mammalian hosts often act as “ecological filters” for pathogen diversity, where novel variants are frequently eliminated because of stochastic events or fitness costs. However, whether vectors can serve a similar role in limiting pathogen diversity is less clear. Here we show using Francisella novicida and a natural tick vector of Francisella spp. (Dermacentor andersoni), that the tick vector acted as a stronger ecological filter for pathogen diversity compared to the mammalian host. When both mice and ticks were exposed to mixtures of F. novicida genotypes, significantly fewer genotypes co-colonized ticks compared to mice. In both ticks and mice, increased genotypic diversity negatively affected the recovery of available genotypes. Competition among genotypes contributed to the reduction of diversity during infection of the tick midgut, as genotypes not recovered from tick midguts during mixed genotype infections were recovered from tick midguts during individual genotype infection. Mediated by stochastic and selective forces, pathogen genotype diversity was markedly reduced in the tick. We incorporated our experimental results into a model to demonstrate how vector population dynamics, especially vector-to-host ratio, strongly affected pathogen genotypic diversity in a population over time. Understanding pathogen genotypic population dynamics will aid in identification of the variables that most strongly affect pathogen transmission and disease ecology.     

Geographic and temporal distributions of four genotypes found in Erysiphe gracilis var. gracilis,a powdery mildew of evergreen oaks (Erysiphales)

A survey of 402 samples of Erysiphe gracilis var. gracilis on evergreen oaks collected from a wide area of western Japan showed that they were divided into four distinct genotypes each forming a separate clade with high bootstrap support, which were referred to as E. hiratae (genotype I), E. uncinuloides (genotype II), E. gracilis s. str. (genotype III), and E. pseudogracilis (genotype IV) in a separate taxonomic treatment. However, there are no clear differences in geographic distributions among these four genotypes. Quercus myrsinifolia was only infected by genotype II and Q. salicina only by genotype IV, whereas Q. glauca was infected by all four. These results strongly suggest an association between host species and speciation of these genotypes. A further 312 samples of Q. glauca with E. gracilis s. lat. colonies were collected from four locations in the Mie University campus to investigate frequency of genotypes I and II every month from May 2015 to January 2016. No temporal isolation was found in genotype frequencies. These genotypes frequently co-existed on a single leaf surface, especially at the locations disturbed by human activities. Two oak powdery mildews, E. gracilis s. lat. and Cystotheca wrightii, produced conidia only one month a year and their life cycle differed from most other powdery mildew species. This suggests that these oak mildews developed their unique life cycles to synchronize with the life cycle of evergreen oaks.     

Host ecology determines the relative fitness of virus genotypes in mixed-genotype nucleopolyhedrovirus infections

Mixed‐genotype infections are common in many natural host–parasite interactions. Classical kin‐selection models predict that single‐genotype infections can exploit host resources prudently to maximize fitness, but that selection favours rapid exploitation when co‐infecting genotypes share limited host resources. However, theory has outpaced evidence: we require empirical studies of pathogen genotypes that naturally co‐infect hosts. Do genotypes actually compete within hosts? Can host ecology affect the outcome of co‐infection? We posed both questions by comparing traits of infections in which two baculovirus genotypes were fed to hosts alongside inocula of the same or a different genotype. The host, Panolis flammea, is a herbivore of Pinus sylvestris and Pi. contorta. The pathogen, PfNPV (a nucleopolyhedrovirus), occurs naturally as mixtures of genotypes that differ, when isolated, in pathogenicity, speed of kill and yield. Single‐genotype infection traits failed to predict the ‘winning’ genotypes in co‐infections. Co‐infections infected and caused lethal disease in more hosts, and produced high yields, relative to single‐genotype infections. The need to share with nonkin did not cause fitness costs to either genotype. In fact, in hosts feeding on Pi. sylvestris, one genotype gained increased yields in mixed‐genotype infections. These results are discussed in relation to theory surrounding adaptive responses to competition with nonkin for limited resources.     

Locked nucleic acid (LNA) single nucleotide polymorphism (SNP) genotype analysis and validation using real-time PCR

With an increased emphasis on genotyping of single nucleotide polymorphisms (SNPs) in disease association studies, the genotyping platform of choice is constantly evolving. In addition, the development of more specific SNP assays and appropriate genotype validation applications is becoming increasingly critical to elucidate ambiguous genotypes. In this study, we have used SNP specific Locked Nucleic Acid (LNA) hybridization probes on a real-time PCR platform to genotype an association cohort and propose three criteria to address ambiguous genotypes. Based on the kinetic properties of PCR amplification, the three criteria address PCR amplification efficiency, the net fluorescent difference between maximal and minimal fluorescent signals and the beginning of the exponential growth phase of the reaction. Initially observed SNP allelic discrimination curves were confirmed by DNA sequencing (n = 50) and application of our three genotype criteria corroborated both sequencing and observed real-time PCR results. In addition, the tested Caucasian association cohort was in Hardy-Weinberg equilibrium and observed allele frequencies were very similar to two independently tested Caucasian association cohorts for the same tested SNP. We present here a novel approach to effectively determine ambiguous genotypes generated from a real-time PCR platform. Application of our three novel criteria provides an easy to use semi-automated genotype confirmation protocol.     

Population Structure of Mixed Mycobacterium tuberculosis Infection Is Strain Genotype and Culture Medium Dependent

Background

Molecular genotyping methods have shown infection with more than one Mycobacterium tuberculosis strain genotype in a single sputum culture, indicating mixed infection.

Aim

This study aimed to develop a PCR-based genotyping tool to determine the population structure of M. tuberculosis strain genotypes in primary Mycobacterial Growth Indicator Tubes (MGIT) and Löwenstein–Jensen (LJ) cultures to identify mixed infections and to establish whether the growth media influenced the recovery of certain strain genotypes.

Method

A convenience sample of 206 paired MGIT and LJ M. tuberculosis cultures from pulmonary tuberculosis patients resident in Khayelitsha, South Africa were genotyped using an in-house PCR-based method to detect defined M. tuberculosis strain genotypes.

Results

The sensitivity and specificity of the PCR-based method for detecting Beijing, Haarlem, S-family, and LAM genotypes was 100%, and 75% and 50% for detecting the Low Copy Clade, respectively. Thirty-one (15%) of the 206 cases showed the presence of more than one M. tuberculosis strain genotype. Strains of the Beijing and Haarlem genotypes were significantly more associated with a mixed infection (on both media) when compared to infections with a single strain (Beijing MGIT p = 0.02; LJ, p<0.01) and (Haarlem: MGIT p<0.01; LJ, p = 0.01). Strains with the Beijing genotype were less likely to be with “other genotype” strains (p<0.01) while LAM, Haarlem, S-family and LCC occurred independently with the Beijing genotype.

Conclusion

The PCR-based method was able to identify mixed infection in at least 15% of the cases. LJ media was more sensitive in detecting mixed infections than MGIT media, implying that the growth characteristics of M. tuberculosis on different media may influence our ability to detect mixed infections. The Beijing and Haarlem genotypes were more likely to occur in a mixed infection than any of the other genotypes tested suggesting pathogen-pathogen compatibility.     

Effect of Leafy Spurge (Euphorbia esula) Genotype on Feeding Damage and Reproduction of Aphthona spp.: Implications for Biological Weed Control

North American leafy spurge (Euphorbia esula L.) is genetically diverse and composed of multiple genotypes introduced from several areas of Europe and Asia. Five species of leafy spurge flea beetle (Aphthona spp.) have been introduced as biological control agents for leafy spurge, but were collected in a relatively small region of Europe. Greenhouse and field experiments were conducted to determine if observed variation in feeding preference and reproduction of Aphthona spp. on North American leafy spurge may be due in part to leafy spurge genotype. Leafy spurge genotypes were collected from Austria; Manitoba in Canada; and the states of Montana, Nebraska, North Dakota, South Dakota, and Wyoming in the United States. Leafy spurge genotype affected feeding but not egg laying by Aphthona spp. adults. Aphthona czwalinae/lacertosa fed slightly less in a free-choice test on a genotype from Manitoba (7%) compared to genotypes from Nebraska and North Dakota (14%). Aphthona flava tended to feed less on the Nebraska genotype than any other genotype evaluated. Reproduction of Aphthona spp. was greatly affected by leafy spurge genotype. For instance, A. czwalinae/lacertosa produced 72 adults per plant from a Nebraska genotype compared to 11 to 32 adults per plant from all other genotypes evaluated. Thus, some observed variation in establishment and reproduction of Aphthona spp. can be attributed to leafy spurge genotype.     

中国美利奴羊H-FABP基因多态性及其与部分肉质性状的相关性

利用聚合酶链式反应-单链构象多态（PCR-SSCP）技术检测中国美利奴羊（Ovis aries var. Merino）心型脂肪酸结合蛋白基因（H-FABP）外显子2的单核苷酸多态性（SNPs）和遗传多态性,分析其与肌内脂肪（IMF）含量、肌纤维直径和肌纤维密度的相互关系,为该品种绵羊的分子标记辅助选择提供理论依据。结果显示,H-FABP基因外显子2有AA、AB和BB 3种基因型,AA型和BB型在778位均发生了C缺失,939位均发生了A→G转换,BB型还在789位发生了T→C转换,该突变导致所编码氨基酸发生了缬氨酸→丙氨酸的替换;BB型为IMF的优势基因型,与AB型相比差异显著（P<0.05）,与AA型相比差异极显著（P<0.01）;BB型对肌纤维直径存在负相关。结果提示,中国美利奴羊H-FABP基因外显子2具有多态性,该基因可能是中国美利奴羊肉质性状的主效基因,或者与控制肉质性状的主效基因相连锁。     

Maternal Obesity and Tobacco Use Modify the Impact of Genetic Variants on the Occurrence of Conotruncal Heart Defects

Conotruncal heart defects (CTDs) are among the most severe birth defects worldwide. Studies of CTDs indicate both lifestyle behaviors and genetic variation contribute to the risk of CTDs. Based on a hybrid design using data from 616 case-parental and 1645 control-parental triads recruited for the National Birth Defects Prevention Study between 1997 and 2008, we investigated whether the occurrence of CTDs is associated with interactions between 921 maternal and/or fetal single nucleotide polymorphisms (SNPs) and maternal obesity and tobacco use. The maternal genotypes of the variants in the glutamate-cysteine ligase, catalytic subunit (GCLC) gene and the fetal genotypes of the variants in the glutathione S-transferase alpha 3 (GSTA3) gene were associated with an elevated risk of CTDs among obese mothers. The risk of delivering infants with CTDs among obese mothers carrying AC genotype for a variant in the GCLC gene (rs6458939) was 2.00 times the risk among those carrying CC genotype (95% confidence interval: 1.41, 2.38). The maternal genotypes of several variants in the glutathione-S-transferase (GST) family of genes and the fetal genotypes of the variants in the GCLC gene interacted with tobacco exposures to increase the risk of CTDs. Our study suggests that the genetic basis underlying susceptibility of the developing heart to the adverse effects of maternal obesity and tobacco use involve both maternal and embryonic genetic variants. These results may provide insights into the underlying pathophysiology of CTDs, and ultimately lead to novel prevention strategies.     

Effects of nitrogen forms on dry matter partitioning and nitrogen metabolism in two contrasting genotypes of Catasetum fimbriatum (Orchidaceae)

The effects of either organic (urea and glutamine) or inorganic nitrogen forms (nitrate and ammonium) on dry matter accumulation in shoots and roots and on nitrogen assimilatory enzyme activities were studied in two Catasetum fimbriatum genotypes. Both genotypes, which had inverse patterns of dry matter partitioning between shoots and roots, were aseptically incubated in gelled culture media containing 6 mol m⁻³ of nitrogen and incubated in growth chamber for 30 and 60 days. In vivo nitrate reductase, glutamine synthetase, glutamate dehydrogenase activities as well as free ammonium contents were determined in shoots and roots of plants grown in four different nitrogen sources. Nitrogen assimilatory enzyme activities showed the highest values in the genotype that accumulated dry matter predominantly in the shoots. The nitrogen sources supplied affected dry matter accumulation in shoots and roots of both C. fimbriatum genotypes; however, they were not enough to change the characteristic pattern of dry matter partitioning of each genotype. On the other hand, the differences in the root/shoot ratio found among nitrogen treatments were relatively higher in the genotype that directed dry matter mainly to roots than in the genotype that allocates biomass to shoots. Our results suggest that NADH-dependent glutamate dehydrogenase plays an important role in ammonium assimilation in C. fimbriatum plants, particularly in the root system. Nitrogen metabolism and the dry matter partitioning of the two genotypes are discussed.     

Molecular phylogeny of the psittacid herpesviruses causing Pacheco's disease: correlation of genotype with phenotypic expression

Fragments of 419 bp of the UL16 open reading frame from 73 psittacid herpesviruses (PsHVs) from the United States and Europe were sequenced. All viruses caused Pacheco's disease, and serotypes of the European isolates were known. A phylogenetic tree derived from these sequences demonstrated that the PsHVs that cause Pacheco's disease comprised four major genotypes, with each genotype including between two and four variants. With the exception of two viruses, the serotypes of the virus isolates could be predicted by the genotypes. Genotypes 1 and 4 corresponded to serotype 1 isolates, genotype 2 corresponded to serotype 2 isolates, and genotype 3 corresponded to serotype 3 isolates. The single serotype 4 virus mapped to genotype 4. DNA from a virus with a unique serotype could not be amplified with primers that amplified DNA from all other PsHVs, and its classification remains unknown. Viruses representing all four genotypes were found in both the United States and Europe, and it was therefore predicted that serotypes 1, 2, and 3 were present in the United States. Serotype 4 was represented by a single European isolate that could not be genetically distinguished from serotype 1 viruses; therefore, the presence of serotype 4 in the United States could not be predicted. Viruses of genotype 4 were found to be the most commonly associated with Pacheco's disease in macaws and conures and were least likely to be isolated in chicken embryo fibroblasts in the United States. All four genotypes caused deaths in Amazon parrots, but genotype 4 was associated with Pacheco's disease only in Amazons in Europe. Genotypes 2, 3, and 4, but not 1, were found in African grey parrots. Although parrots from the Pacific distribution represent a relatively small percentage of the total number of birds with Pacheco's disease, all four genotypes were found to cause disease in these species.     

Patterns of genetic diversity in natural populations of Paspalum agamic complexes

Paspalum has many multiploid species displaying a wide range of ploidy levels and reproductive systems including apomixis. However, not much is known about the genetic structure of natural populations of the apomictic species of Paspalum. The aim of this work was to evaluate the genetic diversity of several natural populations belonging to five species of Paspalum. A total of 13 populations were analyzed using amplified fragment length polymorphism (AFLP). The AFLP data revealed maximal genotypic diversity and significant levels of genetic diversity in diploid and mixed diploid–tetraploid populations of P. denticulatum and P. rufum, where all individuals represent different genotypes. This may be mainly due to the reproductive system of diploid members and the gene flow from diploids to polyploids. The pure populations of tetraploids consist of either multiple genotypes (P. nicorae) or of one dominant genotype with a few deviated genotypes (P. denticulatum and P. lividum). Here, the main source of variability may be the residual sexuality, which continues generating new genotypic combinations. The hexaploid populations of P. buckleyanum consist of a single AFLP genotype and each population represents a particular genotype suggesting that populations arose from independent polyploidization events. This study represents one of the first reports of genetic diversity in natural populations of several Paspalum agamic complexes. Apomixis in these five species may be acting as a successful method for the dispersion of better adapted genotypes.     

Comparative clustering and genotyping of <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> strains isolated from broiler and turkey feces by using RAPD-PCR and ERIC-PCR analysis

To compare the genetic profiles of Campylobacter jejuni (C. jejuni) isolates of broiler and turkey reservoirs sampled in Semnan City, Iran, 60 C. jejuni isolates (30 from broilers and 30 from turkeys) were genotyped by RAPD-PCR- and ERIC-PCR-based methods. RAPD-PCR identified 6 genotypes and ERIC-PCR identified 21 genotypes among the 60 C. jejuni isolates. Both techniques were able to discriminate the C. jejuni isolates. Results demonstrated that one single genotype was identical to broiler and one single genotype was identical to turkey isolates at 83% similarity level in RAPD UPGMA clustering. Also, one single profile was identical to turkey isolates at 73% similarity level in ERIC-PCR clustering. The existence of high genetic similarity in some C. jejuni isolates from both hosts suggests the presence of some overlap between isolates from different sources and boosts the power of RAPD-PCR- and ERIC-PCR-based methods in discriminating C. jejuni isolates from various sources.     

Protective Association of Tumor Necrosis Factor Superfamily 15 (TNFSF15) Polymorphic Haplotype with Ulcerative Colitis and Crohn's Disease in an Indian Population

Background

Tumor necrosis factor superfamily (TNFSF) proteins are involved in the genesis of inflammatory bowel disease (IBD). We examined the association of seven single nucleotide polymorphisms (SNP) in the TNFSF15 gene with Crohn''s disease (CD) and ulcerative colitis (UC) in the Indian population.

Methods

Seven SNPs in the TNFSF15 gene (rs10114470, rs3810936, rs6478108, rs4263839, rs6478109, rs7848647 and rs7869487) were genotyped in 309 CD patients, 330 UC patients and 437 healthy controls using the Sequenom iPLEX MassArray platform. Disease associations were evaluated for allelotypes and for genotypes.

Results

The minor T alleles and the TT genotypes of rs10114470 and rs3810936 were significantly protectively associated with both CD and UC. The CC genotype of rs6478108, AA genotype of rs4263839, the AA genotype of rs6478109, the TT genotype of rs7848647 and the CC genotype of rs7869487 were all protectively associated with CD but not with UC. Two haplotype blocks could be discerned, one where SNPs rs10114470 and rs3810936 were in tight LD (D′ = 0.8) and the other where rs6478108, rs4263839, rs6478109, rs7848647 and rs7869487 were in tight LD (D′ 0.92–1.00). The second block of haplotypes were not associated with CD or with UC. The first block of haplotypes was very significantly associated with both CD and UC.

Conclusions

Strong associations exist between TNFSF15 gene polymorphisms and IBD (both CD and UC) in the Indian population.     

Genomic Relatedness within Five Common Finnish Campylobacter jejuni Pulsed-Field Gel Electrophoresis Genotypes Studied by Amplified Fragment Length Polymorphism Analysis, Ribotyping, and Serotyping

Thirty-five Finnish Campylobacter jejuni strains with five SmaI/SacII pulsed-field gel electrophoresis (PFGE) genotypes selected among human and chicken isolates from 1997 and 1998 were used for comparison of their PFGE patterns, amplified fragment length polymorphism (AFLP) patterns, HaeIII ribotypes, and heat-stable (HS) serotypes. The discriminatory power of PFGE, AFLP, and ribotyping with HaeIII were shown to be at the same level for this selected set of strains, and these methods assigned the strains into the same groups. The PFGE and AFLP patterns within a genotype were highly similar, indicating genetic relatedness. The same HS serotypes were distributed among different genotypes, and different serotypes were identified within one genotype. HS serotype 12 was only associated with the combined genotype G1 (PFGE-AFLP-ribotype). These studies using polyphasic genotyping methods suggested that common Finnish C. jejuni genotypes form genetic lineages which colonize both humans and chickens.