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PubMed2000

Heritability of stigma position and the effect of stigma-anther separation on outcrossing in a predominantly self-fertilizing weed, Datura stramonium (Solanaceae).

A polymorphism for anthocyanin production was used as a genetic marker to document the relationship between anther-stigma separation and outcrossing rate in the predominantly self-fertilizing weed Datura stramonium. White-flowered plants that differed in anther-stigma separation were placed into populations consisting exclusively of purple-flowered plants. Self vs. outcross origin of progeny was evident in the hypocotyl color of the seedlings. Outcrossing rates measured for single flowers were significantly positively correlated with anther-stigma separation, albeit with some scatter around the regression line, especially for flowers with exserted stigmas. We also performed an 8 × 8 diallel cross to determine whether anther-stigma separation is genetically determined. Heritability in two field plots was ∼0.3 and in the greenhouse was ∼0.2. Maternal effects, epistasis, and dominance appeared to be relatively unimportant. Genotypes performed consistently across the three environments, although total plant size varied more than fivefold. It appears that the mixed-mating system of D. stramonium has a heritable basis and would be capable of responding to selection.

Journal Article↗

PubMed1999

[Distribution of the genotypes of hepatitis C virus in intravenous drug addicts in Argentina].

Intravenous drug addiction (IVD) is an unfrequent risk factor in Argentina, representing less than 10% of patients (pts) with chronic HCV infection seen in our Unit. In order to study the genotypes (Gt) in IVD and compare them with a non drug addicted control population, 68 pts with a history of IVD were enrolled in this study and compared with 68 non drug addict (NDA) pts with chronic HCV, with similar age and gender distribution. In all pts a liver biopsy was performed. Genotyping was done by INNO LiPA (Innogenetics, Belgium). Mean age in both groups was 35 +/- 7.8 years and 50 were males. No difference was observed between both groups in the prevalence of Gt1a, Gt2a/c and in those with mixed infections. The prevalence of Gt1b in IVD was 19.1% and in NDA 38.2% (p = 0.0228). A highly significant difference was also observed in the prevalence of Gt3a, of 42.6% in IVD and only 11.8% in NDA (p = 0.0001). Gt1a was the second most frequent genotype in IVD pts (26.5%). Simultaneous HIV infection was present in 8 IVD pts (11.8%) and in none of NDA group. Liver biopsies showed a higher prevalence of mild chronic hepatitis in NDA (57.3%) than in IVD (32.4%) (p = 0.0058). Severe chronic hepatitis with advanced fibrosis or cirrhosis was more frequent in the Gt3 of the group with IVD when compared with Gt3 of the NDA group. It can be concluded that in accordance with other geographical areas, Gt3a is far more prevalent in intravenous drugs addicts than in the general population in Argentina where Gt1b is more frequent. Mild forms of chronic hepatitis are less frequent in IVD. In spite of the relatively small group with HCV co-infection with HIV, it seems important to note that 2/8 (25%) showed severe hepatitis C or cirrhosis.

Adult↗

PubMed2006

Clinical performance of the new rRoche COBAS TaqMan HCV Test and High Pure System for extraction, detection and quantitation of HCV RNA in plasma and serum.

We evaluated the Roche COBAS TaqMan HCV Test For Use With The High Pure System (TaqMan HPS; Roche Diagnostics), for the extraction, detection and quantitation of hepatitis C virus (HCV) RNA in serum or plasma of HCV-infected individuals. The TaqMan HPS is a real-time PCR assay with a reported linear dynamic range of 3.0x10(1) to 2.0x10(8) HCV RNA IU/ml, and a reported lower limit of detection (LLD) of 10 IU/ml. Calculation of the HCV RNA titre is based upon an external standard curve in the presence of an internal control. Intra-assay and inter-assay variation were small in reference panel members with HCV RNA > or =100 IU/ml. Genotype performance and quantitative correlation between the TaqMan HPS and the bDNA (VERSANT HCV 3.0 assay; Bayer Diagnostics), assessed in 59 patient samples, were good for HCV genotype 1 but poor for genotypes 2, 3 and 4. For genotypes 2, 3 and 4, values obtained from the TaqMan HPS were in general 0.5 log lower than those from the bDNA. Sensitivity was poor in low viral titre samples of genotypes 1, 2, 3 and 4. The LLD (95%) was estimated at 41 HCV RNA IU/ml for genotype 4. The TaqMan HPS underestimates HCV RNA at all levels in plasma and serum from HCV-infected individuals, and the LLD should be reconsidered. This is clinically relevant because underestimation of HCV RNA levels during therapy may lead physicians into making incorrect treatment decisions.

Branched DNA Signal Amplification Assay↗

PubMed1999

Inconsistency of association between type 1 von Willebrand disease phenotype and genotype in families identified in an epidemiological investigation.

In a previous epidemiological investigation among schoolchildren of Northern Italy, a conservative 1% prevalence of type 1 von Willebrand disease (VWD) was found. Diagnosis was based on a positive family history and low von Willebrand factor (VWF) ristocetin cofactor activity. To investigate whether the type 1 VWD phenotype as detected by our original methodology cosegregates with one or more specific alleles of the VWF gene, we have performed genotype analysis in affected subjects and their family members. Eleven of the 14 subjects previously identified as having VWD, all with mild personal bleeding symptoms, agreed to participate in the genetic study. Remarkably, the laboratory measurements of the previous investigation were completely confirmed in 10 of the 11 subjects. Clear cosegregation of the VWD type 1 and a specific VWF allele was observed in one family and was likely in the family of two other pro-bands. In three additional propositi and their families a possible association of the phenotype with a VWF allele was found. No association was observed in the remaining five subjects and their families. During 13-year follow-up few additional bleeding episodes were recorded among investigated subjects, most often occurring in the one family manifesting clear cosegregation. The results of this study illustrate that a personal and family bleeding history and persistently low VWF ristocetin cofactor activity, fitting the usual criteria for type 1 VWD, may not cosegregate with genetic markers at the VWF gene locus. Thus the prevalence of VWD defined as a disorder involving the VWF locus might be overestimated in population study. However, phenotypic diagnosis still remains fundamental to identify patients at risk of bleeding. Further research should clarify whether in families with more severe clinical and laboratory phenotype a clear association with markers of VWF is found.

Alleles↗

PubMed2000

Detection of structural gene mutations and promoter polymorphisms in the mannan-binding lectin (MBL) gene by polymerase chain reaction with sequence-specific primers.

This study describes a new approach to the determination of all known mannan-binding lectin (MBL) mutations. The distribution of known variants of the MBL gene in a population of healthy unrelated Danes was determined and the genotype was correlated with the plasma MBL concentrations. The following genetic polymorphisms were studied: three point mutations in the promoter region at position -550 (H/L variants), -221 (X/Y variants), -70 (nt C or T), one point mutation in the 5' untranslated (UT) region at position +4 (P/Q variants) and three point mutations located at codons 52, 54 and 57 in exon 1 of the MBL gene, at nucleotide positions 223, 230 and 239, respectively. To perform genotyping, we designed sequence specific primers for a polymerase chain reaction (PCR-SSP). PCR-SSP is a powerful technique for the discrimination of alleles resulting from single base substitutions and is a widely used technique. Another major advantage of the PCR-SSP method is its ability to determine whether sequence motifs are in cis or trans. The frequencies of variants in exon 1 obtained by PCR-SSP were completely comparable to results obtained by previously described PCR methods, restriction fragment length polymorphism (RFLP) and site-directed mutagenesis (SDM). This PCR-SSP method is performed with standard laboratory equipment and has the capacity to detect all genetic variants in 100 samples in 2 days at an estimated total cost of GBP 11 per sample. Analysing the correlation between MBL haplotype and plasma MBL levels, we confirmed that three different structural variants, B, C and D and the promoter haplotypes HY, LY and LX have a dominant effect on the concentration of MBL. The HY haplotype is associated with the highest plasma concentration, the LY haplotype with intermediate levels and the LX haplotype with the lowest levels. The LX haplotype was found to be associated with very low levels of MBL similar to those found in association with the structural B genotype. The gene frequencies of variants in the MBL gene in the Danish population studied correspond to previous reports on Caucasian populations.

Carrier Proteins↗

PubMed2005

SNP Cutter: a comprehensive tool for SNP PCR-RFLP assay design.

The Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) is a relatively simple and inexpensive method for genotyping single nucleotide polymorphisms (SNPs). It requires minimal investment in instrumentation. Here, we describe a web application, 'SNP Cutter,' which designs PCR-RFLP assays on a batch of SNPs from the human genome. NCBI dbSNP rs IDs or formatted SNPs are submitted into the SNP Cutter which then uses restriction enzymes from a pre-selected list to perform enzyme selection. The program is capable of designing primers for either natural PCR-RFLP or mismatch PCR-RFLP, depending on the SNP sequence data. SNP Cutter generates the information needed to evaluate and perform genotyping experiments, including a PCR primers list, sizes of original amplicons and different allelic fragment after enzyme digestion. Some output data is tab-delimited, therefore suitable for database archiving. The SNP Cut-ter is available at http://bioinfo.bsd.uchicago.edu/SNP_cutter.htm.

DNA Primers↗

PubMedDate not supplied

Comparison of the Velogene Rapid MRSA Identification Assay, Denka MRSA-Screen Assay, and BBL Crystal MRSA ID System for rapid identification of methicillin-resistant Staphylococcus aureus.

Early detection of methicillin-resistant S.aureus (MRSA) is critical for both the management of infected patients, and the timely institution of appropriate infection control measures. Although detection of the mecA gene by PCR remains the gold standard, this technology is inaccessible for many laboratories. Therefore, we sought to evaluate several new rapid identification systems and compare them to PCR. A total of 71 methicillin-susceptible S. aureus (MSSA), 25 borderline oxacillin-resistant S. aureus (BORSA), and 213 MRSA were selected for study. S.aureus was identified using standard methods. Initial screening was performed on a Mueller-Hinton agar plate with 6 mg/L of oxacillin. MRSA strains were identified using PCR with primers specific for the mecA gene. PCR was used as the reference method. All isolates were tested using the BBL Crystal MRSA ID System (Becton Dickinson Microbiology Systems, Maryland, USA), the MRSA-Screen Assay (Denka Seiken Co., Ltd., Tokyo, Japan), and the Velogene Rapid MRSA Identification Assay (ID Biomedical Corp, Vancouver, BC). With minor modifications, all assays were performed according to manufacturers' instructions. Overall, the 3 commercial assays performed well. The sensitivity and specificity of the BBL, Denka, and Velogene systems were 99%/100%, 99%/100%, and 96%/100% respectively. The advantages of the phenotypic tests-BBL Crystal Kit and Denka MRSA-Screen Assay include lower cost per test, shelf-life, ease of use, and rapid turn-around times. Advantages of the Velogene Rapid MRSA include ability to perform genotypic high-volume testing without the equipment requirements and technical complexity involved with PCR. Turn-around times ranged from 15 min for the Denka MRSA-Screen Assay, 2 h for the Velogene Rapid MRSA, and 4 h for the BBL Crystal. The BBL Crystal, Denka MRSA-Screen, and Velogene Rapid MRSA identification systems are rapid, easy to perform, and provide accurate identification of MRSA. These rapid kits offer an acceptable alternative for smaller, non-reference, laboratories and reduce the dependency on PCR in larger laboratories for routine confirmation.

Bacterial Typing Techniques↗

PubMed2000

D2 dopamine receptor gene polymorphisms among African-Americans and Mexican-Americans: a lung cancer case-control study.

Recent research suggests that variant alleles (A1 and B1) of the DRD2 gene play a role in determining smoking status. However, no studies have evaluated these variant alleles in African-Americans and Mexican-Americans. The primary objective of this study, therefore, was to test the hypothesis that ever smokers in these ethnic groups are more likely than never smokers to have the DRD2 alleles associated with tobacco use (A1 and B1). Furthermore, because of a predicted higher prevalence of smokers in a family because of the patterns of inheritance of the genotypes associated with tobacco use, we also anticipated that individuals with these at-risk DRD2 alleles would be more likely to have a family history of smoking-related cancers. Because other inherited genetic variants may interact with smoking on cancer risk, we also hypothesized that this association might differ between cancer patients and control subjects. PCR was used to perform genotyping on peripheral WBC DNA from 140 lung cancer patients (43 Mexican-Americans and 97 African-Americans) and 222 age-, sex-, and ethnicity-matched controls (111 Mexican-Americans and 111 African-Americans). A personal family history was obtained from each participant. There were no statistically significant differences in the distribution of the DRD2 genotypes between cases and controls, although the frequency of the B1 genotype significantly differed by ethnicity (P = 0.002 for controls and P = 0.001 for cases). The DRD2 genotypes and smoking status showed a correlation among Mexican-American controls, although not among African-American controls. The cigarette pack-years in control subjects for the two ethnic groups combined were 30.8, 21.9, and 18.6 for the A1A1, A1A2, and A2A2 genotypes and 36.5, 20.8, and 18.5 for the B1B1, B1B2, and B2B2 genotypes, respectively. Similar trends were found for the number of cigarettes smoked per day among control subjects. From the standpoint of polymorphisms, however, there was a borderline significantly increased (3.6 times greater) frequency of smoking-related cancers among the first-degree relatives of case subjects with an A1 allele than among those without an A1 allele. There was also an elevated (1.8 times greater) frequency of smoking-related cancer among first-degree relatives of case subjects with a B1 allele compared with patients without a B1 allele, but this finding was not statistically significant. This phenomenon was not observed among control subjects. We noted a trend toward interaction of DRD2 A1 genotypes and case status for increased risk of smoking-related cancer among first-degree relatives. These findings suggest that the variant DRD2 genotypes are associated with a greater likelihood to smoke and a greater smoking intensity, as well as with a familial aggregation of smoking-related cancers. However, a large study is needed to confirm this finding.

Aged↗

PubMedDate not supplied

The effect of proinflammatory cytokine gene and angiotensin-converting enzyme polymorphisms on erythropoietin requirements in patients on continuous ambulatory peritoneal dialysis.

BACKGROUND: The correction of anemia by recombinant human erythropoietin (rHuEPO) improves quality of life and prolongs life in end-stage renal failure. rHuEPO requirements for an individual are determined by a range of factors, including iron deficiency and inflammation. Single nucleotide polymorphisms in the promoter sequence of several proinflammatory cytokines have been shown, in different fields of medicine, to influence the cytokine response to different stimuli, with effects on clinical outcome. METHODS: The angiotensin-converting enzyme (ACE) insertion/deletion polymorphism and polymorphisms in the promoter regions of the genes for tumor necrosis factor alpha (-308 A/G), interleukin-6 (-174 G/C), and interferon gamma were examined for their association with rHuEPO requirements in 112 patients on continuous ambulatory peritoneal dialysis (CAPD). Genomic DNA was extracted from peripheral blood leukocytes and genotyping performed with ARMS-PCR methodology, with sequence-specific primers. We examined rHuEPO requirements and C-reactive protein at baseline and during a 6-month study period. RESULTS: We found no significant effect of proinflammatory cytokine polymorphisms on rHuEPO responsiveness. However, throughout the study, we observed that there was a significantly higher rHuEPO requirement in the II and ID ACE genotypes compared with the DD group, which remained an independent association following multivariate analysis. CONCLUSIONS: ACE insertion/deletion polymorphism may determine rHuEPO responsiveness in CAPD patients and should be considered in relative rHuEPO resistance.

Adolescent↗

PubMed2005

Molecular study of FMF patients in Armenia.

Familial Mediterranean Fever (FMF, MIM 249100), or Periodic disease, is a recessively transmitted and ethnically restricted condition prevalent in population from the Mediterranean decent. FMF notoriously has been hard to diagnose until mutations in the MEFV gene have been identified and as a tremendous help are used for the diagnosis of difficult cases. Since FMF can be controlled by medication, it is extremely desirable to have a firm diagnosis. The aim of this study was to establish the frequency of the most common mutations and genotypes in Armenian population. Molecular analysis of MEFV gene mutations in 3000 Armenian patients has demonstrated direct correlation between the clinical severity and the molecular diagnostic criteria of the disease, including the development of renal amyloidosis with MEFV genotypes. MEFV genotyping performed in the framework of a genetic counseling may reveal and identify affected individuals in presymptomatic phase, providing the possibility of a precocious start of the therapy.

Adult↗

PubMed1996

Polymorphism of the apolipoprotein E and angiotensin-converting enzyme genes in Japanese subjects with silent myocardial ischemia.

The apolipoprotein epsilon4 allele and homozygous deletion allele (DD) of the angiotensin-converting enzyme gene are reported to be associated with an increase in the incidence of ischemic heart disease. In this study, we examined whether the apolipoprotein epsilon4 genotype and angiotensin-converting enzyme/DD allele are associated with silent myocardial ischemia. We screened 3920 subjects undergoing general checkups who no symptoms of ischemic heart disease. Seventy subjects (2 percent) showed ischemic ST-segment depression during the double two-step exercise test. One hundred and twenty control subjects without ischemic ST-segment depression were recruited from the same population and matched for sex, age, and blood pressure. We performed genotyping of the apolipoprotein E gene (epsilon2, epsilon3, and epsilon4) and angiotensin-converting enzyme gene (I and D) using polymerase chain reaction-restriction fragment length polymorphism and polymerase chain reaction, respectively. Allele frequently of epsilon4 of the apolipoprotein E gene was higher in the ischemic group (11 percent) than the nonischemic group (5 percent) (chi2 = 5.35, P < .05), but there was no significant association between the allele or the genotype frequency of the angiotensin-converting enzyme gene and the incidence of ischemic ST-segment depression. Furthermore, stepwise multiple regression analysis also revealed that total cholesterol level and epsilon4 genotype were predictors of ischemic change in the exercise tolerance test (chi2 = 12.8, P < .005, R(2) = .051). These results suggest that the apolipoprotein epsilon4 allele is an independent genetic risk factor for silent myocardial ischemia in Japanese subjects.

Adult↗

PubMed2005

Performance characteristics of the ARCHITECT anti-HCV assay.

INTRODUCTION: The ARCHITECT Anti-HCV assay is a fully automated high throughput chemiluminescent microparticle immunoassay (CMIA) for the detection of antibodies to structural and nonstructural proteins of the hepatitis C virus (HCV). To further enhance the performance of this test, the assay was modified to improve the specificity for blood donor specimens. METHODS: The specificity of the enhanced ARCHITECT Anti-HCV assay was evaluated by screening blood donor samples randomly collected from various German blood banks, as well as hospitalized patient samples derived from Germany and the US. Additionally, antibody sensitivity was determined on commercially available anti-HCV seroconversion panels and on a commercially available worldwide anti-HCV genotype performance panel. RESULTS: Apparent specificity of the modified ARCHITECT Anti-HCV assay in a blood donor population consisting of 3811 specimens was 99.92%, compared to 99.76% for the current on-market assay. Additionally, antibody sensitivity was determined on commercially available anti-HCV seroconversion panels. Seroconversion sensitivity equivalent to or better than the current on-market product was observed by testing 33 seroconversion panels. CONCLUSION: This study demonstrates that the modified version of the ARCHITECT Anti-HCV assay shows improved specificity for blood donor specimens compared to the current assay on market without compromising sensitivity. With the availability of the improved ARCHITECT Anti-HCV assay and the recent launch of the ARCHITECT HIV Ag/Ab Combo assay, the ARCHITECT system now offers a full hepatitis/retrovirus menu with excellent performance on a high throughput, random access, automated analyzer, ideally suited for blood screening and diagnostic applications.

Automation↗

PubMedDate not supplied

Serotonin transporter polymorphism and harm avoidance personality in chronic tension-type headache.

OBJECTIVE: To investigate the 5-HTT-gene-linked polymorphic region (5-HTTLPR) genotypes and harm avoidance (HA) dimension in chronic tension-type headache (CTH). BACKGROUND: Serotonin transporter protein (5-HTT) is a key modulating protein in synaptic serotonergic neurotransmission. Among serotonin gene-linked polymorphism, promoter located in the regulatory region of the 5-HTTLPR has two alleles (short and long) with different transcriptional efficiencies. The HA personality trait may be heritable and associated with serotonergic neurotransmitter activity. DESIGN: We amplified the 5-HTTLPR by means of polymerase chain reaction and performed genotype polymorphism analyses and we investigated the serotonin-related personality trait by evaluating the HA dimension in tridimensional personality questionnaire (TPQ) in 107 patients with CTH and in 100 healthy controls. RESULTS: We found an excess frequency of the short allele and a different genotype distribution in patients with CTH. S/S genotype frequency was significantly higher in patients with CTH (76%) than in those with controls (59%; P=.02). Patients with CTH had significantly higher HA scores (21.4+/6.3) than controls (16.3+/-6.1). CONCLUSIONS: This suggests a serotonergic activity might be involved in the development of CTH and 5-HTTLPR might be one of the genetically contributing factors.

Alleles↗

PubMed2000

Mapping a quantitative trait locus via the EM algorithm and Bayesian classification.

Mapping a locus controlling a quantitative genetic trait (e.g., blood pressure) to a specific genomic region is of considerable interest. Data on the quantitative trait under consideration and several codominant genetic markers with known genomic locations are collected from members of families and statistically analyzed to draw inferences on the genomic position of the trait locus. The vector of parameters of interest comprises the pairwise recombination fractions, theta, between the putative quantitative trait locus and the marker loci. One of the major complications in estimating theta for a quantitative trait in humans is the lack of haplotype information on members of families. The purpose of this study was to devise a computationally simple and efficient method of estimation of theta in the absence of haplotype information. We have proposed a two-stage estimation procedure using the expectation-maximization (EM) algorithm. In the first stage, parameters of the QTL are estimated based on data of a sample of unrelated individuals. From estimates thus obtained, we have used a Bayes' rule to infer QTL genotypes of parents in families. Finally, in the second stage of the procedure, we have proposed an EM algorithm for obtaining the maximum likelihood estimate of theta based on data of informative families (which are identified upon inferring parental QTL genotypes performed in the first stage). We have shown, using simulated data, that the proposed procedure is cost-effective, computationally simple, and statistically efficient. As expected, analysis of data on multiple markers jointly is more efficient than the analysis based on single markers.

Algorithms↗

PubMed2005

Response to long-term lamivudine treatment (up to 5 years) in patients with severe chronic hepatitis B, role of genotype and drug resistance.

Lamivudine is effective in suppressing viral replication, normalizing alanine aminotransferase (ALT), and improving histological appearance in HBe positive and negative hepatitis. It is unclear whether hepatitis B virus (HBV) genotype influences the response to lamivudine. We report the long-term response of patients with chronic hepatitis B with and without cirrhosis at baseline treated with lamivudine according to HBV genotype. Retrospective review of charts of all patients treated with lamivudine monotherapy between 1993 and 2002. Response to therapy defined as ALT in the normal range, undetectable HBV DNA, and in the HBeAg positive group loss of HBeAg and/or the development of anti-HBe. HBV DNA measured by the Digene Hybrid capture assay (sensitivity 1.4 x 10(6) copies/mL). YMDD mutation at rtL180M and rtM204V/I measured by restriction digest of amplified products. Genotyping performed by sequencing and phylogenetic tree analysis of the preS region of the virus genome. Seventy-one patients treated with lamivudine for 6 months or more, 53 (75%) were male, average age 47 years, 38 (54%) were HBeAg+ and 33 (46%) HBeAg-. Mean baseline HBV DNA viral titre was 1280.2 copies/mL and 518 copies/mL respectively. Cirrhosis was present in 30 (42%). Sera were examined for YMDD mutations at last patient visit in 61 (86%), and were detected in 45 (74%), there being no association with a particular genotype. Data from up to 5 years on lamivudine indicated no difference in biochemical or virological response between genotypes. Cirrhosis was more prevalent with specific genotypes. We found no influence of HBV genotype on the development of resistance to lamivudine, however liver disease severity was influenced by genotype.

Alanine Transaminase↗

PubMed2001

Natural selection for grazer resistance to toxic cyanobacteria: evolution of phenotypic plasticity?

We studied the selection response of the freshwater grazing zooplankter, Daphnia galeata, to increased abundance of cyanobacteria in its environment. Cyanobacteria are a poor-quality and often toxic food. Distinct genotypes of D. galeata were hatched from diapausing eggs extracted from three time horizons in the sediments of Lake Constance, Europe, covering the period 1962 to 1997, a time of change in both the prevalence of planktonic cyanobacteria and levels of phosphorus pollution. We assessed whether the grazers evolved to become more resistant to dietary cyanobacteria by exposing genetically distinct clones to two diets, one composed only of the nutritious green alga, Scenedesmus obliquus (good food), and the other a mixture of S. obliquus and the toxic cyanobacterium Microcvstis aeruginosa (poor food). Genotype performance was measured as the specific rate of weight gain from neonate to maturity (gj). We evaluated evolutionary change in the Daphnia population using an analysis of reaction norms based on relative (log-transformed) changes in gj. Log(gj) is a measure of the proportional effect of dietary cyanobacteria on other fitness components of the Daphnia phenotype. For comparison, we also analyze absolute (i.e., nontransformed) changes in gj and discuss the interpretations of the two approaches. Statistical results using a general linear model demonstrate a significant effect of genotype (showing differences in gj among genotypes), a significant genotype x food-type interaction (showing differences in phenotypic plasticity among genotypes), and, in the case of log-transformed data, a significant sediment-genotype-age x food-type interaction. The latter shows that phenotypic plasticity evolved over the period studied. Two constraints act on response to selection in the D. galeata-Lake Constance system. First, gj on a diet containing poor food is highly correlated with gj on a diet of good food, thus evolving resistance also meant evolving an increase in gj on both diets. Second, because genotypes with a high gj also grow to a large adult body size, which in turn increases Daphnia vulnerability to fish predation, we suggest that selection only acted to favor genotypes possessing a high potential gj after cyanobacteria became prevalent. The presence of cyanobacteria depressed realized gj and led to animals of small adult body size even if their genotypes had the potential for high gj and large size. With realized gj reduced, genotypes with an inherently high value could be selected even in the presence of predatory fish. The joint action of selection by dietary cyanobacteria and vulnerability to fish predation provides an explanation for the observed evolution of resistance to poor food through reduced phenotypic plasticity.

Animals↗

PubMed2006

The usefulness of the gfp reporter gene for monitoring Agrobacterium-mediated transformation of potato dihaploid and tetraploid genotypes.

Potato is one of the main targets for genetic improvement by gene transfer. The aim of the present study was to establish a robust protocol for the genetic transformation of three dihaploid and four economically important cultivars of potato using Agrobacterium tumefaciens carrying the in vivo screenable reporter gene for green fluorescent protein (gfp) and the marker gene for neomycin phosphotransferase (nptII). Stem and leaf explants were used for transformation by Agrobacterium tumefaciens strain LBA4404 carrying the binary vector pHB2892. Kanamycin selection, visual screening of GFP by epifluorescent microscopy, PCR amplification of nptII and gfp genes, as well as RT-PCR and Southern blotting of gfp and Northern blotting of nptII, were used for transgenic plant selection, identification and analysis. Genetic transformation was optimized for the best performing genotypes with a mean number of shoots expressing gfp per explant of 13 and 2 (dihaploid line 178/10 and cv. 'Baltica', respectively). The nptII marker and gfp reporter genes permitted selection and excellent visual screening of transgenic tissues and plants. They also revealed the effects of antibiotic selection on organogenesis and transformation frequency, and the identification of escapes and chimeras in all potato genotypes. Silencing of the gfp transgene that may represent site-specific inactivation during cell differentiation, occurred in some transgenic shoots of tetraploid cultivars and in specific chimeric clones of the dihaploid line 178/10. The regeneration of escapes could be attributed to either the protection of non-transformed cells by neighbouring transgenic cells, or the persistence of Agrobacterium cells in plant tissues after co-cultivation.

Agrobacterium tumefaciens↗

PubMed2002

[Study of microsatellite polymorphisms between the Han nationality and French population by fluorescence-based genotype].

OBJECTIVE: To facilitate the application of the large scale genome scan and individual identification by investigating the differences of microsatellite polymorphisms of the genethon human genetic linkage map between the Han nationality and French population. METHODS: Six to seventeen pairs of primers were added in one single tube with 5 microliters reaction volume and several microsatellite markers were amplified simultaneously. The products were electrophoresited on the 377XL DNA sequencer. RESULTS: During the entire experiment 400 microsatellite loci were amplified in 32 tubes and 306 microsatellite loci were satisfactory. CONCLUSION: The size range of genetic polymorphisms are different in race, and the difference exists at 124 microsatellite loci between the Han nationality and French population, so 10 bp should be added to both sides of the genethor map size range when we perform genotype in Han nationality.

Adult↗