In Vivo Persistence of Codominant Human CD8+ T Cell Clonotypes Is Not Limited by Replicative Senescence or Functional Alteration.

T cell responses to viral epitopes are often composed of a small number of codominant clonotypes. In this study, we show that tumor Ag-specific T cells can behave similarly. In a melanoma patient with a long lasting HLA-A2/NY-ESO-1-specific T cell response, reaching 10% of circulating CD8 T cells, we identified nine codominant clonotypes characterized by individual TCRs. These clonotypes made up almost the entire pool of highly differentiated effector cells, but only a fraction of the small pool of less differentiated "memory" cells, suggesting that the latter serve to maintain effector cells. The different clonotypes displayed full effector function and expressed TCRs with similar functional avidity. Nevertheless, some clonotypes increased, whereas others declined in numbers over the observation period of 6 years. One clonotype disappeared from circulating blood, but without preceding critical telomere shortening. In turn, clonotypes with increasing frequency had accelerated telomere shortening, correlating with strong in vivo proliferation. Interestingly, the final prevalence of the different T cell clonotypes in circulation was anticipated in a metastatic lymph node withdrawn 2 years earlier, suggesting in vivo clonotype selection driven by metastases. Together, these data provide novel insight in long term in vivo persistence of T cell clonotypes associated with continued cell turnover but not replicative senescence or functional alteration.

Ssr-based Genetic Diversity And Structure Of Garlic Accessions From Brazil.

Garlic is a spice and a medicinal plant; hence, there is an increasing interest in 'developing' new varieties with different culinary properties or with high content of nutraceutical compounds. Phenotypic traits and dominant molecular markers are predominantly used to evaluate the genetic diversity of garlic clones. However, 24 SSR markers (codominant) specific for garlic are available in the literature, fostering germplasm researches. In this study, we genotyped 130 garlic accessions from Brazil and abroad using 17 polymorphic SSR markers to assess the genetic diversity and structure. This is the first attempt to evaluate a large set of accessions maintained by Brazilian institutions. A high level of redundancy was detected in the collection (50 % of the accessions represented eight haplotypes). However, non-redundant accessions presented high genetic diversity. We detected on average five alleles per locus, Shannon index of 1.2, HO of 0.5, and HE of 0.6. A core collection was set with 17 accessions, covering 100 % of the alleles with minimum redundancy. Overall FST and D values indicate a strong genetic structure within accessions. Two major groups identified by both model-based (Bayesian approach) and hierarchical clustering (UPGMA dendrogram) techniques were coherent with the classification of accessions according to maturity time (growth cycle): early-late and midseason accessions. Assessing genetic diversity and structure of garlic collections is the first step towards an efficient management and conservation of accessions in genebanks, as well as to advance future genetic studies and improvement of garlic worldwide.

NEW MICROSATELLITE LOCI FOR WATER YAM (DIOSCOREA ALATA, DIOSCOREACEAE) AND CROSS-AMPLIFICATION FOR OTHER DIOSCOREA SPECIES

Premise of the study: Dioscorea alata L. is one of the most widely distributed species of the genus in the humid and semihumid tropics and is associated with traditional agriculture. Only a few microsatellite markers have been developed so far for this and other Dioscorea species. Methods and Results: We isolated 14 codominant polymorphic microsatellite markers using a microsatellite-enriched genomic library technique. Ten microsatellite loci were selected, and 80 D. alata accessions from different regions in Brazil were evaluated with nine polymorphic loci. The polymorphism information content (PIC) varied from 0.39 to 0.78 and the power discrimination (PD) ranged from 0.15 to 0.91. Six of the markers showed transferability for the species D. bulbifera, D. cayenensis-D. rotundata, and D. trifida. Conclusions: The SSR markers obtained are an important tool for further studies aiming to characterize the genetic diversity in D. alata and other Dioscorea spp. accessions.

Comparative analysis of genetic diversity in the mangrove species Avicennia marina (Forsk.) Vierh. (Avicenniaceae) detected by AFLPs and SSRs

Avicennia marina is an important mangrove species with a wide geographical and climatic distribution which suggests that large amounts of genetic diversity are available for conservation and breeding programs. In this study we compare the informativeness of AFLPs and SSRs for assessing genetic diversity within and among individuals, populations and subspecies of A. marina in Australia. Our comparison utilized three SSR loci and three AFLP primer sets that were known to be polymorphic, and could be run in a single analysis on a capillary electrophoresis system, using different-colored fluorescent dyes. A total of 120 individuals representing six populations and three subspecies were samplcd. At the locus level, SSRs were considerably more variable than AFLPs, with a total of 52 alleles and an average heterozygosity of 0.78. Average heterozygosity for AFLPs was 0.193, but all of the 918 bands scored were polymorphic. Thus, AFLPs were considerably more efficient at revealing polymorphic loci than SSRs despite lower average heterozygosities. SSRs detected more genetic differentiation between populations (19 vs 9%) and subspecies (35 vs 11%) than AFLPs. Principal co-ordinate analysis revealed congruent patterns of genetic relationships at the individual, population and subspecific levels for both data sets. Mantel testing confirmed congruence between AFLP and SSR genetic distances among, but not within, population comparisons, indicating that the markers were segregating inde- pendently but that evolutionary groups (populations and subspecies) were similar. Three genetic criteria of importance for defining priorities for ex situ collections or in situ conservation programs (number of alleles, number of locally common alleles and number of private alleles) were correlated between the AFLP and SSR data sets. The congruence between AFLP and SSR data sets suggest that either method, or a combination, is applicable to expanded genetic studies of mangroves. The codominant nature of SSRs makes them ideal for further population-based investigations, such as mating-system analyses, for which the dominant AFLP markers are less well suited. AFLPs may be particularly useful for monitoring propagation programs and identifying duplicates within collections, since a single PCR assay can reveal many loci at once.

A genetic map of macadamia based on randomly amplified DNA fingerprinting (RAF) markers

The first genetic linkage map of macadamia (Macadamia integrifolia and M. tetraphylla) is presented. The map is based on 56 F-1 progeny of cultivars 'Keauhou' and 'A16'. Eighty-four percent of the 382 markers analysed segregated as Mendelian loci. The two-way pseudo-testcross mapping strategy allowed construction of separate parental cultivar maps. Ninety bridging loci enabled merging of these maps to produce a detailed genetic map of macadamia, 1100 cM in length and spanning 70-80% of the genome. The combined map comprised 24 linkage groups with 265 framework markers: 259 markers from randomly amplified DNA fingerprinting (RAF), five random amplified polymorphic DNA (RAPD), and one sequence-tagged microsatellite site (STMS). The RAF marker system unexpectedly revealed 16 codominant markers, one of them a putative microsatellite locus and exhibiting four distinct alleles in the cross. This molecular study is the most comprehensive examination to date of genetic loci of macadamia, and is a major step towards developing marker-assisted selection for this crop.

Biology of amazonian mosquitoes. III. Esterase isozymes in Anopheles darlingi.

Six esterase isozymes were studied during the development of Anopheles darlingi by using polyacrylamide gel electrophoresis and two different substrates, a-naphthylcelate and a-naphthylpropionate. Esterases 5 and 6'were detected in all developmental stages esterases 1 and 2 were more intensively stained if larvae, while esterases 3 and 4 were better visualized in pupae and adults. Strong differences in intensity of some of the isozymes were observed during the pupal stage.Four out of the six isozymes showed variation in the electrophoretic mobility. Esterase-2 was choosed for genetic studies, because was the best stained isozyme in the gels. Two codominant alleles {Est2*S and Est2*F) code for this polymorphic system, with the Est*S frequency equal to 0.521. Phenotypic distribution is in agreement with hardy-Weinberg expectations.

ELECTROPHORETIC ANALYSIS OF 11 ENZYMES IN NATURAL POPULATIONS OFROOT, 1926 (DIPTERA: CULICIDAE) IN THE AMAZON REGION

Of eleven proteins analyzed in four Amazonian populations, the esterases showed the greatest variation, with five activity zones. EST1, EST2 and EST5 showed variation in each of the populations studied. EST1 and EST2 are each controlled by two, and EST5 by four, codomi-nant alleles. LAP presented six activity zones, with codominant variation in LAP5and LAP6.oc—GPDH was monomorphic with one activity band on starch gel and two on polyacrylamide gel. 1DH presented two activity zones, with variation in the IDHl region. PGM had a single activity zone, with variation in all populations. The Ariquemes populations showed five alleles and the other populations three, all of then codominant. Three activity zones with two codominant alleles were observed for ODH. Aldehyde Oxidase showed two activity zones, with variation in AOl only in the Ariquemes and Porto Velho/Samuel populations. 6-PGDH showed only one activity zone and variation only in the Ariquemes population. The remaing systems - XDH, G-6-PDH and GDH. was monomorphic.

Tests for sex-biased dispersal using bi-parentally inherited genetic markers.

Understanding why dispersal is sex-biased in many taxa is still a major concern in evolutionary ecology. Dispersal tends to be male-biased in mammals and female-biased in birds, but counter-examples exist and little is known about sex bias in other taxa. Obtaining accurate measures of dispersal in the field remains a problem. Here we describe and compare several methods for detecting sex-biased dispersal using bi-parentally inherited, codominant genetic markers. If gene flow is restricted among populations, then the genotype of an individual tells something about its origin. Provided that dispersal occurs at the juvenile stage and that sampling is carried out on adults, genotypes sampled from the dispersing sex should on average be less likely (compared to genotypes from the philopatric sex) in the population in which they were sampled. The dispersing sex should be less genetically structured and should present a larger heterozygote deficit. In this study we use computer simulations and a permutation test on four statistics to investigate the conditions under which sex-biased dispersal can be detected. Two tests emerge as fairly powerful. We present results concerning the optimal sampling strategy (varying number of samples, individuals, loci per individual and level of polymorphism) under different amounts of dispersal for each sex. These tests for biases in dispersal are also appropriate for any attribute (e.g. size, colour, status) suspected to influence the probability of dispersal. A windows program carrying out these tests can be freely downloaded from http://www.unil.ch/izea/softwares/fstat.html

Characterization of microsatellite loci and reliable genotyping in a polyploid plant, Mercurialis perennis (Euphorbiaceae).

For many applications in population genetics, codominant simple sequence repeats (SSRs) may have substantial advantages over dominant anonymous markers such as amplified fragment length polymorphisms (AFLPs). In high polyploids, however, allele dosage of SSRs cannot easily be determined and alleles are not easily attributable to potentially diploidized loci. Here, we argue that SSRs may nonetheless be better than AFLPs for polyploid taxa if they are analyzed as effectively dominant markers because they are more reliable and more precise. We describe the transfer of SSRs developed for diploid Mercurialis huetii to the clonal dioecious M. perennis. Primers were tested on a set of 54 male and female plants from natural decaploid populations. Eight of 65 tested loci produced polymorphic fragments. Binary profiles from 4 different scoring routines were used to define multilocus lineages (MLLs). Allowing for fragment differences within 1 MLL, all analyses revealed the same 14 MLLs without conflicting with merigenet, sex, or plot assignment. For semiautomatic scoring, a combination of as few as 2 of the 4 most polymorphic loci resulted in unambiguous discrimination of clones. Our study demonstrates that microsatellite fingerprinting of polyploid plants is a cost efficient and reliable alternative to AFLPs, not least because fewer loci are required than for diploids.

An 18-kDa translocator protein (TSPO) polymorphism explains differences in binding affinity of the PET radioligand PBR28

[(11)C]PBR28 binds the 18-kDa Translocator Protein (TSPO) and is used in positron emission tomography (PET) to detect microglial activation. However, quantitative interpretations of signal are confounded by large interindividual variability in binding affinity, which displays a trimodal distribution compatible with a codominant genetic trait. Here, we tested directly for an underlying genetic mechanism to explain this. Binding affinity of PBR28 was measured in platelets isolated from 41 human subjects and tested for association with polymorphisms in TSPO and genes encoding other proteins in the TSPO complex. Complete agreement was observed between the TSPO Ala147Thr genotype and PBR28 binding affinity phenotype (P value=3.1 x 10(-13)). The TSPO Ala147Thr polymorphism predicts PBR28 binding affinity in human platelets. As all second-generation TSPO PET radioligands tested hitherto display a trimodal distribution in binding affinity analogous to PBR28, testing for this polymorphism may allow quantitative interpretation of TSPO PET studies with these radioligands.

Reliable selfing rate estimates from imperfect population genetic data.

Genotypic frequencies at codominant marker loci in population samples convey information on mating systems. A classical way to extract this information is to measure heterozygote deficiencies (FIS) and obtain the selfing rate s from FIS = s/(2 - s), assuming inbreeding equilibrium. A major drawback is that heterozygote deficiencies are often present without selfing, owing largely to technical artefacts such as null alleles or partial dominance. We show here that, in the absence of gametic disequilibrium, the multilocus structure can be used to derive estimates of s independent of FIS and free of technical biases. Their statistical power and precision are comparable to those of FIS, although they are sensitive to certain types of gametic disequilibria, a bias shared with progeny-array methods but not FIS. We analyse four real data sets spanning a range of mating systems. In two examples, we obtain s = 0 despite positive FIS, strongly suggesting that the latter are artefactual. In the remaining examples, all estimates are consistent. All the computations have been implemented in a open-access and user-friendly software called rmes (robust multilocus estimate of selfing) available at http://ftp.cefe.cnrs.fr, and can be used on any multilocus data. Being able to extract the reliable information from imperfect data, our method opens the way to make use of the ever-growing number of published population genetic studies, in addition to the more demanding progeny-array approaches, to investigate selfing rates.

Genetic variability of marine shrimp in the Brazilian industry

The objective of this work was to estimate the genetic variability level and distribution in Brazilian broodstocks of marine shrimp (Litopenaeus vannamei). Nine of the country's largest hatcheries were evaluated using codominant and highly polymorphic microsatellite markers. The results obtained from genotyping of ten microsatellite loci are indicative of genetic variability that is compatible with that found in wild populations of L. vannamei in Mexico and Central America. A possible explanation is the highly diversified and relatively recent origin of the available broodstocks. Bayesian analysis detected a signal for five founding populations. The distribution of genetic distances partially reflects geographical location, and this information will be useful for the creation of new broodstocks. Therefore, L. vannamei genetic variability among nine of the largest national hatcheries can be considered high.

Orosomucoid (alpha-1 acid glycoprotein) phenotyping by use of immobilized pH gradients with 8 M urea and immunoblotting. A new variant encountered in a population study.

Orosomucoid (ORM) phenotyping has been performed on 329 unrelated Swiss subjects, using immobilized pH gradients with 8 M urea and 2% v/v 2-mercaptoethanol followed by immunoblotting. After desialylation the band patterns of ORM confirmed that the polymorphism of the structural locus ORM1 is controlled by three codominant autosomal alleles (ORM1*F1, ORM1*S and ORM1*F2). One rare and one new allele were detected. The rare variant, tentatively assigned to the second structural locus ORM2, is observed in a cathodal position and named ORM2 B1. The new variant, tentatively assigned to the first structural locus ORM1, is observed in a region located between ORM1 S and ORM1 F2, and named ORM1 F3. Moreover, the pI values of the ORM variants have been measured accurately with Immobiline Dry Plates (LKB): they were found to be within the pH range 4.93-5.14.

A new allele of peptidase-B in cattle

Electrophoretic analyses of peptidase-B were carried out on red cell hemolysates from Holstein, Mantiqueira and Gyr cattle, using cornstarch, known in Brazil as Penetrose-30. We describe a new peptidase-B allele, denoted Pep-B1, in Mantiqueira cattle, belonging to the Bos taurus group, which are the result of a cross of native cattle of Portuguese origin introduced in Brazil during colonial times (16th century) with Holstein and Caracu cattle. The genetic control of peptidase-B was determined by typing parents and progeny segregating for all three alleles, confirming that peptidase B is controlled by a single autosomal locus with three codominant alleles, denoted Pep-B1, Pep-B2 and Pep-B3 The use of the citrate-phosphate buffer system, at pH 5.9, on 14% gel, under the electrophoretic conditions standardized in this study permitted good visualization of all peptidase-B variants.

Absence of linkage between MHC and a gene involved in susceptibility to human schistosomiasis

Six hundred million people are at risk of infection by Schistosoma mansoni. MHC haplotypes have been reported to segregate with susceptibility to schistosomiasis in murine models. In humans, a major gene related to susceptibility/resistance to infection by S. mansoni (SM1) and displaying the mean fecal egg count as phenotype was detected by segregation analysis. This gene displayed a codominant mode of inheritance with an estimated frequency of 0.20-0.25 for the deleterious allele and accounted for more than 50% of the variance of infection levels. To determine if the SM1 gene segregates with the human MHC chromosomal region, we performed a linkage study by the lod score method. We typed for HLA-A, B, C, DR and DQ antigens in 11 informative families from an endemic area for schistosomiasis in Bahia, Brazil, by the microlymphocytotoxicity technique. HLA-DR typing by the polymerase chain reaction with sequence-specific primers (PCR-SSP) and HLA-DQ were confirmed by PCR-sequence-specific oligonucleotide probes (PCR-SSOP). The lod scores for the different q values obtained clearly indicate that there is no physical linkage between HLA and SM1 genes. Thus, susceptibility or resistance to schistosomiasis, as defined by mean fecal egg count, is not primarily dependent on the host's HLA profile. However, if the HLA molecule plays an important role in specific immune responses to S. mansoni, this may involve the development of the different clinical aspects of the disease such as granuloma formation and development of hepatosplenomegaly.