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Simple Summary: Circadian clocks are molecular pacemakers that drive daily rhythms in physiology, metabolism, and behavior. They are present in a diverse range of organisms, from cyanobacteria to humans. In the last few decades, enormous progress has been made in understanding the molecular details of the circadian clock. The fruit-fly Drosophila has been instrumental in identifying 'clock' genes, which are well conserved in mammals, both in sequence and function. Many 'clock' genes have been identified using laboratory mutagenesis screens, which look for mutants that show aberrant circadian behavior. The recent accumulation of genomic sequences from wild populations of Drosophila provides an opportunity to identify natural genetic variations in 'clock' genes, which serve as molecular adaptations to local habitats. Here, we focused on nine polymorphic sites in genes associated with the clock and tested their impact on diurnal behavior. We found seven sites whose alleles confer different circadian behavior or seasonal response. This analysis of natural variation can provide information about fine-tuning of the clock, and it is essential for understanding the evolution of the circadian system. Our understanding of the gene regulatory network that constitutes the circadian clock has greatly increased in recent decades, notably due to the use of Drosophila as a model system. In contrast, the analysis of natural genetic variation that enables the robust function of the clock under a broad range of environments has developed more slowly. In the current study, we analyzed comprehensive genome sequencing data from wild European populations of Drosophila, which were densely sampled through time and space. We identified hundreds of single nucleotide polymorphisms (SNPs) in nine genes associated with the clock, 276 of which exhibited a latitudinal cline in their allele frequencies. While the effect sizes of these clinal patterns were small, indicating subtle adaptations driven by natural selection, they provided important insights into the genetic dynamics of circadian rhythms in natural populations. We selected nine SNPs in different genes and assessed their impact on circadian and seasonal phenotypes by reconstructing outbred populations fixed for either of the SNP alleles, from inbred DGRP strains. The circadian free-running period of the locomotor activity rhythm was affected by an SNP in doubletime (dbt) and eyes absent (Eya). The SNPs in Clock (Clk), Shaggy (Sgg), period (per), and timeless (tim) affected the acrophase. The alleles of the SNP in Eya conferred different levels of diapause and the chill coma recovery response. [ABSTRACT FROM AUTHOR]

Our understanding of the gene regulatory network that constitutes the circadian clock has greatly increased in recent decades, notably due to the use of Drosophila as a model system. In contrast, the analysis of natural genetic variation that enables the robust function of the clock under a broad range of environments has developed more slowly. In the current study, we analyzed comprehensive genome sequencing data from wild European populations of Drosophila, which were densely sampled through time and space. We identified hundreds of single nucleotide polymorphisms (SNPs) in nine genes associated with the clock, 276 of which exhibited a latitudinal cline in their allele frequencies. While the effect sizes of these clinal patterns were small, indicating subtle adaptations driven by natural selection, they provided important insights into the genetic dynamics of circadian rhythms in natural populations. We selected nine SNPs in different genes and assessed their impact on circadian and seasonal phenotypes by reconstructing outbred populations fixed for either of the SNP alleles, from inbred DGRP strains. The circadian free-running period of the locomotor activity rhythm was affected by an SNP in doubletime (dbt) and eyes absent (Eya). The SNPs in Clock (Clk), Shaggy (Sgg), period (per), and timeless (tim) affected the acrophase. The alleles of the SNP in Eya conferred different levels of diapause and the chill coma recovery response.

Cryptochrome (CRY) is a conserved protein associated with the circadian clock in a broad range of organisms, including plants, insects, and mammals. In Drosophila , cry is a pleiotropic gene that encodes a blue light-dedicated circadian photoreceptor, as well as an electromagnetic field sensor and a geotaxis behavior regulator. We have generated a panel of nearly-isogenic strains that originated from various wild populations and which carry different natural alleles of cry. Sequencing of these alleles revealed substantial polymorphism, the functional role of which was elusive. To link this natural molecular diversity to gene function, we relied on association mapping. Such analysis revealed two major haplogroups consisting of six linked nucleotides associated with circadian phase (haplotypes All1/All2). We also generated a maximum-likelihood gene-tree that uncovered an additional pair of haplogroups (B1/B2). Behavioral analysis of the different haplotypes indicated significant effect on circadian phase and period, as well on the amount of activity and sleep. The data also suggested substantial epistasis between the All and B haplogroups. Intriguingly, circadian photosensitivity, assessed by light-pulse experiments, did not differ between the genotypes. Using CRISPR-mediated transgenic flies, we verified the effect of B1/B2 polymorphism on circadian phase. The transgenic flies also exhibited substantially different levels of cry transcription. We, moreover, analyzed the geographical distribution of the B1/B2 haplotypes, focusing on a 12 bp insertion/deletion polymorphism that differentiates the two haplotypes. Analysis of cry sequences in wild populations across Europe revealed a geographical cline of B1/B2 indel frequency, which correlated with seasonal bioclimatic variables. This spatial distribution of cry polymorphism reinforces the functional importance of these haplotypes in the circadian system and local adaptation. [ABSTRACT FROM AUTHOR]

To understand the genetic diversity and differentiation of Vietnamese melon (Cucumis melo L.), we collected 64 landraces from the central and southern parts of the country and assessed molecular polymorphism using simple sequence repeat and random amplified polymorphic DNA markers. The Vietnamese melon was divided into seven cultivar groups, namely "Dua le", "Dua vang", "Dua bo", "Dua gang-andromonoecious", "Dua gang-monoecious", "Dua thom", "Montok", and the weedy-type melon "Dua dai". Among these, Dua le, Dua vang, Dua bo, and Dua gang-andromonoecious are cultivated on plains and they formed cluster II along with the reference accessions of Conomon and Makuwa. Based on genetic distance, Dua le and Dua vang were regarded as Makuwa and Dua bo and Dua gang-andromonoecious as Conomon. In contrast, Dua thom and Montok are cultivated in highlands, and they formed cluster III along with landraces from the southern and eastern foot of the Himalayas. Dua gang-monoecious which is commonly cultivated in the southern parts of Vietnam, exhibited the greatest genetic diversity, as explained by its possible origin through the hybridization between Dua gang-andromonoecious and Montok. Genetic differences in melon landraces between plains and highlands and hybridization between these two geographical groups have contributed to the enhancement of genetic diversity in Vietnamese melon. [ABSTRACT FROM AUTHOR]

Mink astrovirus infection remains a poorly understood disease entity, and the aetiological agent itself causes disease with a heterogeneous course, including gastrointestinal and neurological symptoms. This paper presents cases of astrovirus infection in mink from continental Europe. RNA was isolated from the brains and intestines of animals showing symptoms typical of shaking mink syndrome (n = 6). RT-PCR was used to amplify astrovirus genetic material, and the reaction products were separated on a 1% agarose gel. The specificity of the reaction was confirmed by sequencing fragment coding RdRP protein (length of sequencing product 170 bp) from all samples. The presence of astrovirus RNA was detected in each of the samples tested. Sequencing and bioinformatic analysis indicated the presence of the same variant of the virus in all samples. Comparison of the variant with the sequences available in bioinformatics databases confirmed that the Polish isolates form a separate clade, closely related to Danish isolates. The dissimilarity of the Polish variant to those isolated in other countries ranged from 2.4% (in relation to Danish isolates) to 7.1% (in relation to Canadian isolates). Phylogenetic relationships between variants appear to be associated with the geographic distances between them. To our knowledge, this work describes the first results on the molecular epidemiology of MAstV in continental Europe. The detection of MAstV in Central Europe indicates the need for further research to broaden our understanding of the molecular epidemiology of MAstV in Europe. [ABSTRACT FROM AUTHOR]

The T-cell receptor (TCR) is a specific molecule on the surface of all T cells that mediates cellular adaptive immune responses to antigens. Hucho bleekeri is a critically endangered species and is regarded as a glacial relict that has the lowest-latitude distribution compared with any Eurasian salmonid. In the present study, two TCR genes, namely, TCR α and β , were identified and characterized in H. bleekeri. Both TCR α and TCR β have typical TCR structures, including the IgV domain, IgC domain, connecting peptide, transmembrane and cytoplasmic domains. The two TCR genes were constitutionally expressed in various tissues, with the highest expression found in the spleen for TCR α and in the trunk kidney for TCR β. Challenge of H. bleekeri with LPS or poly(I:C) resulted in significant upregulation of both TCR α and β expression in headkidney and spleen primary cells, indicating their potential roles in the immune response. Molecular polymorphism analysis of the whole ORF regions of TCR α and β in different individuals revealed high diversity of IgV domains of these two genes, especially in complementarity-determining region (CDR) 3. The ratio of nonsynonymous substitution occurred at a significantly higher frequency than synonymous substitution in the CDR of TCR α and β , demonstrating the existence of positive selection. The results obtained in the present study enhance our understanding of TCR roles in regulating immune mechanisms and provide new information for the study of TCR lineage diversity in fish. • T-cell receptors were isolated from critically endangered Hucho bleekeri for the first time. • TCR genes were constitutionally expressed with the highest expression found immune tissues. • LPS or Poly(I:C) challenge resulted in significant upregulation of TCR genes. • Different selection modes were found in the different regions TCR α and TCR β. [ABSTRACT FROM AUTHOR]

The BK polyomavirus (BKPyV), a representative of the family Polyomaviridae, is widespread in the human population. While the virus does not cause significant clinical symptoms in immunocompetent individuals, it is activated in cases of immune deficiency, both pharmacological and pathological. Infection with the BKPyV is of particular importance in recipients of kidney transplants or HSC transplantation, in which it can lead to the loss of the transplanted kidney or to haemorrhagic cystitis, respectively. Four main genotypes of the virus are distinguished on the basis of molecular differentiation. The most common genotype worldwide is genotype I, with a frequency of about 80%, followed by genotype IV (about 15%), while genotypes II and III are isolated only sporadically. The distribution of the molecular variants of the virus is associated with the region of origin. BKPyV subtype Ia is most common in Africa, Ib-1 in Southeast Asia, and Ib-2 in Europe, while Ic is the most common variant in Northeast Asia. The development of molecular methods has enabled significant improvement not only in BKPyV diagnostics, but in monitoring the effectiveness of treatment as well. Amplification of viral DNA from urine by PCR (Polymerase Chain Reaction) and qPCR Quantitative Polymerase Chain Reaction) is a non-invasive method that can be used to confirm the presence of the genetic material of the virus and to determine the viral load. Sequencing techniques together with bioinformatics tools and databases can be used to determine variants of the virus, analyse their circulation in populations, identify relationships between them, and investigate the directions of evolution of the virus.

Abstract Background Passionflowers Passiflora edulis and Passiflora alata are diploid, outcrossing and understudied fruit bearing species. In Brazil, passion fruit cultivation began relatively recently and has earned the country an outstanding position as the world’s top producer of passion fruit. The fruit’s main economic value lies in the production of juice, an essential exotic ingredient in juice blends. Currently, crop improvement strategies, including those for underexploited tropical species, tend to incorporate molecular genetic approaches. In this study, we examined a set of P. edulis transcripts expressed in response to infection by Xanthomonas axonopodis, (the passion fruit’s main bacterial pathogen that attacks the vines), aiming at the development of putative functional markers, i.e. SSRs (simple sequence repeats) and SNPs (single nucleotide polymorphisms). Results A total of 210 microsatellites were found in 998 sequences, and trinucleotide repeats were found to be the most frequent (31.4%). Of the sequences selected for designing primers, 80.9% could be used to develop SSR markers, and 60.6% SNP markers for P. alata. SNPs were all biallelic and found within 15 gene fragments of P. alata. Overall, gene fragments generated 10,003 bp. SNP frequency was estimated as one SNP every 294 bp. Polymorphism rates revealed by SSR and SNP loci were 29.4 and 53.6%, respectively. Conclusions Passiflora edulis transcripts were useful for the development of putative functional markers for P. alata, suggesting a certain level of sequence conservation between these cultivated species. The markers developed herein could be used for genetic mapping purposes and also in diversity studies.

Tunisia is one of the world’s largest producers of olive oil, and it preserves pools of olive genetic diversity that are still unexplored. A recent prospection and collection program of the National Gene Bank of Tunisia (NGBT) focused on the vast oasis of Degache, in the south west part of Tunisia, where 47 samples were collected and genetically characterized through simple sequence repeat (SSR) markers. Identification and authentication of genotypes were obtained through comparison with reference cultivars belonging to the Olive National Collection of Tunisia (IOC) and with cultivars from Algeria, Italia, Syria and Lebanon. Degache olive genotypes showed large genetic variability, a significant diversity from the reference germplasm, and a clear differentiation from modern varieties. The population structure analysis identified four gene pools characterizing genotypes from different area of origin. Two gene pools appear to be more represented in germplasm from southern Tunisia, where environmental conditions at critical plant development phases, are harsher. This suggests that this germplasm might present traits of adaptation useful for breeding to improve resilience to abiotic stresses. Our results will support ex situ and in situ conservation activities of Tunisian olive germplasm pursued by the National Gene Bank of Tunisia.

Olive is one of the oldest cultivated species in the Mediterranean Basin, including Tunisia, where it has a wide diversity, with more than 200 cultivars, of both wild and feral forms. Many minor cultivars are still present in marginal areas of Tunisia, where they are maintained by farmers in small local groves, but they are poorly characterized and evaluated. In order to recover this neglected germplasm, surveys were conducted in different areas, and 31 genotypes were collected, molecularly characterized with 12 nuclear microsatellite (simple sequence repeat (SSR)) markers, and compared with 26 reference cultivars present in the Tunisian National Olive collection. The analysis revealed an overall high genetic diversity of this olive’s germplasm, but also discovered the presence of synonymies and homonymies among the commercialized varieties. The structure analysis showed the presence of different gene pools in the analyzed germplasm. In particular, the marginal germplasm from Ras Jbal and Azmour is characterized by gene pools not present in commercial (Nurseries) varieties, pointing out the very narrow genetic base of the commercialized olive material in Tunisia, and the need to broaden it to avoid the risk of genetic erosion of this species in this country.

Eighteen Vetiveria zizanioides accessions were analyzed for genetic diversity by Random Amplified Polymorphic DNA (RAPD) including CIM-Vridhi, an elite released variety. Twenty MAP primers were used to detect the DNA polymorphism and a total of 152 bands were obtained, out of which 45 were monomorphic, 137 polymorphic, 15 unique bands and 73.1% polymorphism were estimated. The average polymorphic information content (PIC) value obtained with RAPD markers was 0.44; marker index (MI), effective multiplex ratio (EMR), and resolving power (RP) were 2.39, 5.09, and 22.3, respectively. Principal component analysis (PCA) derived for RAPD markers illustrated the first three principal coordinate components accounted for 76.75% of the genetic similarity variance. [ABSTRACT FROM AUTHOR]

Background: Passionflowers Passiflora edulis and Passiflora alata are diploid, outcrossing and understudied fruit bearing species. In Brazil, passion fruit cultivation began relatively recently and has earned the country an outstanding position as the world's top producer of passion fruit. The fruit's main economic value lies in the production of juice, an essential exotic ingredient in juice blends. Currently, crop improvement strategies, including those for underexploited tropical species, tend to incorporate molecular genetic approaches. In this study, we examined a set of P. edulis transcripts expressed in response to infection by Xanthomonas axonopodis, (the passion fruit's main bacterial pathogen that attacks the vines), aiming at the development of putative functional markers, i.e. SSRs (simple sequence repeats) and SNPs (single nucleotide polymorphisms). Results: A total of 210 microsatellites were found in 998 sequences, and trinucleotide repeats were found to be the most frequent (31.4%). Of the sequences selected for designing primers, 80.9% could be used to develop SSR markers, and 60.6% SNP markers for P. alata. SNPs were all biallelic and found within 15 gene fragments of P. alata. Overall, gene fragments generated 10,003 bp. SNP frequency was estimated as one SNP every 294 bp. Polymorphism rates revealed by SSR and SNP loci were 29.4 and 53.6%, respectively. Conclusions: Passiflora edulis transcripts were useful for the development of putative functional markers for P. alata, suggesting a certain level of sequence conservation between these cultivated species. The markers developed herein could be used for genetic mapping purposes and also in diversity studies. [ABSTRACT FROM AUTHOR]

Purpose:To develop elite genotypes inMucuna pruriens(L.) DC with high L-DOPA (L-3, 4 dihydroxyphenylalanine) yields, with non-itching characteristics and better adaptability by applying γ-irradiation. Molecular and chemical analysis was performed for screening based on specific characteristics desired for developing suitable genotypes. Materials and methods:Developed, mutant populations were analyzed for L-DOPA % in seeds through TLC (thin layer chromatography), and the results obtained were validated with the HPLC (High performance liquid chromatography). The DNA (Deoxyribonucleic acid) was isolated from the leaf at the initial stage and used for DNA polymorphism. RNA (Ribonucleic acid) was isolated from the leaf during maturity and used for expression analysis. Results:The selected mutant T-I-7 showed 5.7% L-DOPA content compared to 3.18% of parent CIM-Ajar. The total polymorphism obtained was 57% with the molecular marker analysis. The gene expression analysis showed higher fold change expression of the dopadecarboxylase gene (DDC) in control compared to selected mutants (T-I-7, T-II-23, T-IV-9, T-VI-1). Conclusion:DNA polymorphism was used for the screening of mutants for efficient screening at an early stage. TLC was found suitable for the large-scale comparative chemical analysis of L-DOPA. The expression profile of DDC clearly demonstrated the higher yields of L-DOPA in selected mutants developed by γ-irradiation in the seeds of the control. [ABSTRACT FROM AUTHOR]

Ocimum (Lamiaceae) is an important source of essential oils and aroma chemicals especially eugenol, methyl eugenol, linalool, methyl chavicol etc. An elite evergreen hybrid has been developed from Ocimum kilimandscharicum and Ocimum basilicum, which demonstrated adaptive behavior towards cold stress. A comparative molecular analysis has been done through RAPD, AFLP, and ISSR among O. basilicum and O. kilimandscharicum and their evergreen cold-tolerant hybrid. The RAPD and AFLP analyses demonstrated similar results, i.e., the hybrid of O. basilicum and O. kilimandscharicum shares the same cluster with O. kilimandscharicum, while O. basilicum behaves as an outgroup, whereas in ISSR analysis, the hybrid genotype grouped in the same cluster with O. basilicum. Ocimum genotypes were analyzed and compared for their trichome density. There were distinct differences on morphology, distribution, and structure between the two kinds of trichomes, i.e., glandular and non-glandular. Glandular trichomes contain essential oils, polyphenols, flavonoids, and acid polysaccharides. Hair-like trichomes, i.e., non-glandular trichomes, help in keeping the frost away from the living surface cells. O. basilicum showed less number of non-glandular trichomes on leaves compared to O. kilimandscharicum and the evergreen cold-tolerant hybrid. Trichomes were analyzed in O. kilimandscharicum, O. basilicum, and their hybrid. An increased proline content at the biochemical level represents a higher potential to survive in a stress condition like cold stress. In our analysis, the proline content is quite higher in tolerant variety O. kilimandscharicum, low in susceptible variety O. basilicum, and intermediate in the hybrid. Gene expression analysis was done in O. basilicum, O. kilimandscharicum and their hybrid for TTG1, GTL1, and STICHEL gene locus which regulates trichome development and its formation and transcription factors WRKY and MPS involved in the regulation of plant responses to freezing and cold. The analysis showed that O. kilimandscharicum and the hybrid were very close to each other but O. basilicum was more distinct in all respects. The overexpression of the WRKY coding gene showed high expression in the hybrid as compared to O. kilimandscharicum and O. basilicum and the transcription factor microspore-specific (MPS) promoter has also shown overexpression in the hybrid for its response against cold stress. The developed evergreen interspecific hybrid may thus provide a base to various industries which are dependent upon the bioactive constituents of Ocimum species. [ABSTRACT FROM AUTHOR]

Information about the natural patterns of genetic variability and their evolutionary bases are of fundamental practical importance for sustainable forest management and conservation. In the present study, the genetic diversity of 164 individuals from fourteen natural populations of Crataegus pontica K.Koch was assessed for the first time using three genome-based molecular techniques; inter-retrotransposon amplified polymorphism (IRAP); inter-simple sequence repeats (ISSR) and start codon targeted (SCoT) polymorphism. IRAP, ISSR and SCoT analyses yielded 126, 254 and 199 scorable amplified bands, respectively, of which 90.48, 93.37 and 83.78% were polymorphic. ISSR revealed efficiency over IRAP and SCoT due to high effective multiplex ratio, marker index and resolving power. The dendrograms based on the markers used and combined data divided individuals into three major clusters. The correlation between the coefficient matrices for the IRAP, ISSR and SCoT data was significant. A higher level of genetic variation was observed within populations than among populations based on the markers used. The lower divergence levels depicted among the studied populations could be seen as evidence of gene flow. The promotion of gene exchange will be very beneficial to conserve and utilize the enormous genetic variability. [ABSTRACT FROM AUTHOR]

Start Codon Targeted markers were used to establish phylogenetic relationship among seven species from Citrus L. genus. Twelve SCoT primers were used for their ability to reveal polymorphism of the targeted codon of initiation. A total of 132 amplicons were generated and 93.9% of them were polymorphic. The polymorphism information content of 0.884 and the resolving power of 75.22 illustrate the efficiency of the tested SCoT primers in highlighting polymorphism. The average Nei's (1973) gene diversity (0.376), the Schannon's index (0.548) and the Gst parameter (0.346) describe an important polymorphism at the interspecies level in Citrus genus. Analysis of molecular variance suggested significant genetic differences within species. In fact, 84% of variance occurs within the species, whereas 16% of the variation was recorded among the species of Citrus . The limited gene flow (Nm = 0.941) was recognized as a major factor to explain the partition of the observed diversity. The principal coordinates analyses, Neighbor Joining and the Bayesian clustering approach based on the SCoT markers also confirm the discrimination of the species of Citrus . Our results confirm the relevance and suggest the effectiveness of the SCoT markers for assessing genetic diversity, characterization and identification of the species of Citrus . [ABSTRACT FROM AUTHOR]

The molecular polymorphisms of chlorpyrifos (CPF)-degrading strains in soil treated with microcapsule (MC) and emulsifiable concentrate (EC) formulations of CPF were studied by laboratory experiments. Results showed that CPF residue in MC-treated soil was maintained at a certain level during 60 days, and the residual period reached 120 days. CPF residue decreased via a first-order model with a residual period of 60 days in EC-treated soil. A total of 16 and 9 degrading strains capable of utilizing CPF as a sole source of carbon and energy were isolated from these two treatments, respectively. All degrading strains were grouped into 16 different types based on enterobacterial repetitive intergenic consensus (ERIC)-PCR pattern similarities. Strains isolated from MC- and EC-treated soils belonged to 14 and 8 ERIC-types, respectively. Six groups of strains that were isolated from MC- and EC-treated soils belonged to similar ERIC-types. All ERIC-type strains were similar to nine known species according to BLAST analysis at the NCBI website. The results indicated that the population and degradation abilities of CPF-degrading strains in soil were visibly strengthened by the controlled-release formulation of CPF compared with those by the non-controlled release formulation. [ABSTRACT FROM AUTHOR]

In species with separate sexes, social systems can differ in the relative variances of male versus female reproductive success. Papionin monkeys (macaques, mangabeys, mandrills, drills, baboons, and geladas) exhibit hallmarks of a high variance in male reproductive success, including a female-biased adult sex ratio and prominent sexual dimorphism. To explore the potential genomic consequences of such sex differences, we used a reduced representation genome sequencing approach to quantifying polymorphism at sites on autosomes and sex chromosomes of the tonkean macaque (Macaca tonkeana), a species endemic to the Indonesian island of Sulawesi. The ratio of nucleotide diversity of the X chromosome to that of the autosomes was less than the value (0.75) expected with a 1:1 sex ratio and no sex differences in the variance in reproductive success. However, the significance of this difference was dependent on which outgroup was used to standardize diversity levels. Using a new model that includes the effects of varying population size, sex differences in mutation rate between the autosomes and X chromosome, and GC-biased gene conversion (gBGC) or selection on GC content, we found that themaximum-likelihood estimate of the ratio of effective population size of the X chromosome to that of the autosomes was 0.68, which did not differ significantly from 0.75. We also found evidence for 1) a higher level of purifying selection on genic than nongenic regions, 2) gBGC or natural selection favoring increased GC content, 3) a dynamic demography characterized by population growth and contraction, 4) a higher mutation rate in males than females, and 5) a very low polymorphism level on the Y chromosome. These findings shed light on the population genomic consequences of sex differences in the variance in reproductive success, which appear to be modest in the tonkean macaque; they also suggest the occurrence of hitchhiking on the Y chromosome. [ABSTRACT FROM AUTHOR]

Abstract: The Montepulciano d'Abruzzo “Colline Teramane” premium wine DOCG is produced in the Teramo province (Abruzzo, Italy). This region has a great tradition in winemaking and the wine is produced by a spontaneous fermentation so it could represent a reservoir of wine natural yeasts with important oenological features. The aim of this study was to characterize the yeast community of this wine grape growing region in order to create a Saccharomyces cerevisiae bank, providing data on oenological properties for potential industrial applications. A total of 430 yeasts were isolated at the end of spontaneous fermentation. PCR-RFLP was applied for the identification at the species level and underlined that 14 strains exhibited unusual and characteristic restriction patterns different from those typical of the species S. cerevisiae. This difference was due to the insertion of base C at a position 138 in the ITS1 region that determined an additional cleavage site for the enzyme HaeIII. This insertion could be associated to the fermentative performance and associated to the relationship existing between yeasts and a viticulture region or ‘terroir’. [Copyright &y& Elsevier]

Current knowledge of wild Lactuca L. species, their taxonomy, biogeography, gene-pools, germplasm collection quality and quantity, and accession availability is reviewed in this paper. Genetic diversity of Lactuca spp. is characterized at the level of phenotypic and phenological variation, variation in karyology and DNA content, biochemical traits, and protein and molecular polymorphism. The reported variation in reaction to pathogens and pests of wild Lactuca spp. is summarized, including the viral pathogens (Lettuce mosaic virus- LMV, Mirafiori lettuce virus/Lettuce big vein virus- LBV, Beet western yellows virus- BWYV, Tomato spotted wilt virus- TSWV, Cucumber mosaic virus- CMV, Lettuce necrotic stunt virus- LNSV), bacterial pathogens (corky root- Rhizomonas suberifaciens, bacterial leaf spot- Xanthomonas campestris pv. vitians), fungal pathogens (downy mildew- Bremia lactucae, powdery mildew- Golovinomyces cichoracearum, anthracnose- Microdochium panattoniana, stemphylium leaf spot- Stemphylium spp., sclerotinia drop- Sclerotinia spp., verticillium wilt- Verticillium dahliae, fusarium wilt- Fusarium spp., pythium wilt- Pythium tracheiphylum, P. uncinulatum), nematodes (potato cyst nematode- Globodera rostochiensis, root-knot nematode- Meloidogyne spp., incognita, hapla, javanica, enterolobii), insects and mites (the green lettuce aphid- Nasonovia ribisnigri, the green peach aphid- Myzus persicae, the potato aphid- Macrosiphum euphorbiae, leafminer- Liriomyza spp., L. langei). The approaches used to exploit wild Lactuca spp. in lettuce breeding (interspecific hybridization, cell and tissue culture, transformation) are dicussed, and known examples of lettuce cultivars with traits derived from wild Lactuca spp. are described. [ABSTRACT FROM AUTHOR]

Background: Grain protein content (GPC) is an important quality determinant for barley used as malt, feed as well as food. It is controlled by a complex genetic system. GPC differs greatly among barley genotypes and is also variable across different environments. It is imperative to understand the genetic control of barley GPC and identify the genotypes with less variation under the different environments. Results: In this study, 59 cultivated and 99 Tibetan wild barley genotypes were used for a genome-wide association study (GWAS) and a multi-platform candidate gene-based association analysis, in order to identify the molecular markers associated with GPC. Tibetan wild barley had higher GPC than cultivated barley. The significant correlation between GPC and diastatic power (DP), and malt extract confirmed the importance of GPC in determining malt quality. Diversity arrays technology (DArT) markers associated with barley GPC were detected by GWAS. In addition, GWAS revealed two HvNAM genes as the candidate genes controlling GPC. No association was detected between HvNAM1 polymorphism and GPC, while a single nucleotide polymorphism (SNP) (798, P < 0.01), located within the second intron of HvNAM2, was associated with GPC. There was a significant correlation between haplotypes of HvNAM1, HvNAM2 and GPC in barley. Conclusions: The GWAS and candidate gene based-association study may be effectively used to determine the genetic variation of GPC in barley. The DArT markers and the polymorphism of HvNAM genes identified in this study are useful in developing high quality barley cultivars in the future. HvNAM genes could play a role in controlling barley GPC. [ABSTRACT FROM AUTHOR]

Abstract: Major histocompatibility complex (MHC) is a large genomic region characterized by extremely high polymorphism, and it plays an important role in the immune response of vertebrates. In the present study, we isolated MHC class II genes from Nile tilapia in order to investigate the immune mechanism in tilapia and develop better strategies for disease prevention. Moreover, we cloned the full-length cDNA sequences of MHC IIA and IIB from Nile tilapia by the RACE approach. In addition, the genomic structure, molecular polymorphism and expression patterns of MHC II genes in Nile tilapia were also examined. Compared with that of other teleosts, Nile tilapia MHC class IIA contained four exons and three introns. The deduced amino acid sequence of the MHC IIA molecule shared 25.4–64.5% similarity with those of other teleosts and mammals. Six exons and five introns were identified from Nile tilapia MHC IIB, and the deduced amino acid sequence shared 26.9–74.7% similarity with those of other teleosts and mammals. All the characteristic features of MHC class II chain structure could be identified in the deduced sequences of MHC IIA and IIB molecules, including the leader peptide, α1/β1 and α2/β2 domains, connecting peptide and transmembrane and cytoplasmic regions, as well as conserved cysteines and N-glycosylation site. A total of 12 MHC IIA alleles were identified from six individuals. Four alleles originating from a single individual suggested that at least four MHC IIA loci existed. Moreover, 10 MHC IIB alleles were identified, among which four were detected in a single individual, suggesting that at least four MHC IIB loci existed. The expression of MHC IIA and IIB at the mRNA level in 10 types of normal tissues was determined using quantitative real-time PCR analysis. The highest expression level was detected in stomach and gill, whereas the lowest expression was detected in muscle and brain. Furthermore, MHC IIA and IIB were probably two candidate immune molecules involved in the resistance against streptococcosis, because their expression was significantly up-regulated in gill, kidney, intestine and spleen after the intraperitoneal injection of Streptococcus agalactiae. [Copyright &y& Elsevier]

Abstract: Specific microsatellites (SSRs) markers were used to characterize a set of 32 Tunisian pomegranate (Punica granatum L.) cultivars. Using 13 SSR primers, a total of 40 alleles and 46 genotypes have been identified. As a result, data proved that a high level of polymorphism characterizes the Tunisian pomegranate germplasm at the DNA level. The derived Neighbour-joining (NJ) dendrogram constructed using DAS genetic distances exhibited a genetic diversity structured independently from the geographical origin of cultivars and their denomination. This result suggested that a common genetic basis may characterize Tunisian pomegranate cultivars despite their phenotypic divergences. Furthermore, based on the multilocus genotypes a cultivar''s identification key has been established and permitted to unambiguously differentiate between varieties. The obtained results are discussed in term of establishment and management of a national collection of pomegranate varieties, conformity checks, identification of homonyms and synonyms, and screening of the local resources. Furthermore, this microsatellite-based key is a first step towards a marker-assisted identification pomegranate database. [Copyright &y& Elsevier]

Little is known of intron sequences' variation in cases where eukaryotic gene coding regions undergo strong balancing selection. Phosphoglucose isomerase, PGI, of Colias butterflies offers such a case. Its 11 introns include many point mutations, insertions, and deletions. This variation changes with intron position and length, and may leave little evidence of homology within introns except for their first and last few basepairs. Intron position is conserved between PGIs of Colias and the silkmoth, but no intron sequence homology remains. % GC content and length are functional properties of introns which can affect whole-gene transcription; we find a relationship between these properties which may indicate selection on transcription speed. Intragenic recombination is active in these introns, as in coding sequences. The small extent of linkage disequilibrium (LD) in the introns decays over a few hundred basepairs. Subsequences of Colias introns match subsequences of other introns, untranslated regions of cDNAs, and insect-related transposons and pathogens, showing that a diverse pool of sequence fragments is the source of intron contents via turnover due to deletion, recombination, and transposition. Like Colias PGI's coding sequences, the introns evolve reticulately with little phylogenetic signal. Exceptions are coding-region allele clades defined by multiple amino acid variants in strong LD, whose introns are closely related but less so than their exons. Similarity of GC content between introns and flanking exons, lack of small introns despite mutational bias toward deletion, and findings already mentioned suggest constraining selection on introns, possibly balancing transcription performance against advantages of higher recombination rate conferred by intron length. [ABSTRACT FROM AUTHOR]

The review considers various aspects of the biology of the yeast Saccharomyces bayanus, which is distantly related to the cultured yeast S. cerevisiae. The cryotolerant S. bayanus strains found in wine-making became the second most important yeast for basic and applied studies. Introduction of natural and experimental hybrids of S. cerevisiae × S. bayanus in a range of fermentation processes indicates the high breeding importance of S. bayanus. The biological species S. bayanus acts as a new gene pool for the scientific and breeding projects. [ABSTRACT FROM AUTHOR]

With 1 table Abstract Microsatellite markers are needed for distinguishing cultivars and mapping disease and pest resistance genes in caladiums, one of the most important ornamental aroids. Through sequencing of a GA-enriched partial genomic library, 173 unique sequences suitable for designing simple sequence repeat primers were identified. A majority of the sequences (82.7%) contained perfect repeats of GA/TC motif, and 86.8% of the primers amplified discrete PCR products of expected sizes, resulting in development of the first 99 microsatellite markers in caladiums. These markers detected differences at 41.8% and 68.8% of the amplified alleles between two pairs of caladium cultivars that are most difficult to distinguish morphologically. The markers revealed high percentages (70.0% to 78.2%) of polymorphic alleles among three parents of segregating populations to be used for mapping genes for resistance to three major diseases/pests. High rates of transferability (51.7-100%) were observed across seven Caladium species. The availability of these highly polymorphic, robust markers will provide a powerful tool for differentiating cultivars, anchoring genetic maps and understanding the genetic relationships in caladiums. [ABSTRACT FROM AUTHOR]

Abstract: Improvement of malt quality is the most important objective in malt barley breeding. The current experiments investigated the variation of malt quality characters among barley genotypes and the difference in genetic variants of HvGlb1, encoding β-glucanase isoenzyme I, between Tibetan annual wild barley and cultivated barley. The correlation between the gene variants and malt quality showed that there was a large difference in the four malt quality parameters, i.e. Kolbach index, diastatic power (DP), viscosity and malt extract, among the analyzed barley cultivars. Kolbach index was negatively and positively correlated with viscosity and malt extract, respectively, while malt extract was negatively correlated with viscosity. Malt β-glucan content was a major determinant of malt quality, and was significantly correlated with Kolbach index (−0.633), malt extract (−0.333) and viscosity (0.672). On the other hand, malt β-glucan content was mainly controlled by malt β-glucanase activity. The correlation analysis showed that the HvGlb1 gene was correlated with malt β-glucan content and three of four main malt quality parameters, except DP. In addition, we also found that the HvGlb1 of Tibetan barley had wider diversity in haplotype than that of the cultivated barley, supporting the hypothesis that Tibet is one of the original centers of cultivated barley. [Copyright &y& Elsevier]

In this review, we present a critical analysis of the current status of wild Lactuca L. germplasm in relation to its utility for lettuce breeding. We discuss wild Lactuca germplasm in ex situ collections from the perspectives of taxonomy, biogeography, biology and ecology, gene pools, field exploration and acquisition, descriptor development, characterization and evaluation, and enhancement. Future research and other activities related to wild Lactuca germplasm and their continued exploitation in lettuce breeding are considered. [ABSTRACT FROM AUTHOR]

A molecular evolutionary explanation of natural genetic variation requires analysis of specific variants’ evolutionary dynamics. To pursue this for phosphoglucose isomerase (PGI) of Colias butterflies, whose polymorphism is maintained by strong natural selection, we assembled a large data set of wild haplotypes, highly variable at the amino acid and DNA levels. The most common electrophoretic, i.e., charge macrostate, allele class, 3, is conserved in its pattern of charged amino acid residues. The next most common macrostate, 4, has multiple patterns of charge, i.e., microstates, while less common (1, 2, 5, 6) macrostates are very diverse. Macrostate 4 shows significant linkage disequilibrium (LD) among its variants, especially for two groups of five haplotypes each. We find extensive intragenic recombination among all haplotypes except the two high-LD groups of macrostate 4, which display none. Phyletic relations among haplotypes are largely reticulate, again except for the high-LD groups of macrostate 4, which form clades with strong bootstrap support. Charge-changing and linked charge-neutral amino acid variants occur in diverse parts of PGI’s sequence. Homology-based modeling of PGI’s structure shows that these regions are related spatially in ways suggesting functional interaction. The high-LD groups of macrostate 4 display parallel amino acid variation in these regions. This pattern of haplotype clades with high LD among multiple varying sites, emerging from chaotically recombining variation, may be a “signature” of refinement of complex adaptive sequences by recombination and selection. It should be tested further in this study system and others as a possibly general feature of the evolution of living complexity. [ABSTRACT FROM AUTHOR]

Aims: To study the yeast diversity of Nigerian palm wines by comparison with other African strains. Methods and Results: Twenty-three Saccharomyces cerevisiae strains were obtained from palm wine samples collected at four locations in eastern Nigeria, and characterized using different molecular techniques: internal transcribed spacer restriction fragment length polymorphism and sequence analysis, pulsed field gel electrophoresis, inter delta typing and microsatellite multilocus analysis. These techniques revealed that palm wine yeasts represent a group of closely related strains that includes other West African isolates (CBS400, NCYC110, DVPG6044). Population analysis revealed an excess of homozygote strains and an allelic richness similar to wine suggestive of local domestication. Several other African yeast strains were not connected to this group. Ghana sorghum beer strains and other African strains (DBVPG1853 and MUCL28071) displayed strikingly high relatedness with European bread, beer or wine strains, and the genome of strain MUCL30909 contained African and wine-type alleles, indicating its hybrid origin. Conclusions: Nigerian palm wine yeast represents a local specific yeast flora, whereas a European origin or hybrid was suspected for several other Africa isolates. Significance and Impact of the Study: This study presents the first genetic characterization of an autochthonous African palm wine yeast population and confirms the idea that human intervention has favoured yeast migration. [ABSTRACT FROM AUTHOR]

Abstract: The QuasiChemical Nonideal Associated Solution (QCNAS) model was used to analyze the dielectric permittivity and thermodynamic mixing functions of binary mixtures hydrocarbon (cyclohexane, hexane) — n-alkanol (pentan-1-ol, hexan-1-ol and heptan-1-ol) in the entire range of compositions at 298 K. The new model of supramolecular organization was substantiated, taking into account both chain and cyclic aggregation of alcohol with the formation of chain aggregates of arbitrary size with two possible states of H-bonds in the alcohol aggregates. This model describes a set of physicochemical properties of these mixtures with complicated behavior of both dielectrics and thermodynamics. Equilibrium constants, thermodynamic parameters of H-bonding with cooperative character, integral and differential characteristics of aggregation, and the structural parameters of aggregates were determined. It was established that the role of non-polar cyclic structures becomes more important with decreasing alcohol concentration. The proposed model describes the permittivity of the solutions in the entire range of compositions and reproduces the minimum of the dipolar correlation factor in the range of dilute alcohol solutions. The new data obtained on the supramolecular ordering characterized by the long-range molecular correlations due to H-bonding are discussed in detail. [Copyright &y& Elsevier]

In our researches we tried to convert the P. infestans RAPD markers into SCAR markers for a more operative diagnosis of potato late blight in the infected material. For this goal, several monomorphic and polymorphic RAPD sequences were recuperated from the agarose gel, and cloned into Ecoli bacteria. The cloned monomorphic sequences were sequenced, establishing the nucleotide sequence. Relying on this several pair of primers was designed. One pair from them, with the following nucleotide sequences AGC CCA CAC TGA AAT TAC GC/GGC TAC CGG ATC AGT ACC AA), gave amplification product only with P.infestans DNA. Being specific, this pair pf primer could be used for a straightforward potato late blight diagnosis from the infected material, and our objective concerning the conversion of P. infestans RAPD markers into SCAR markers could be considered accomplished. [ABSTRACT FROM AUTHOR]

Aims:  To guarantee the endemic genetic background of the isolates obtained in yeast isolation programs, it is necessary to differentiate between endemic and commercial strains because the progressive use of commercial yeast in wine areas around the world would affect the autochthonous yeast populations. Methods and Results:  Mitochondrial DNA restriction analysis, electrophoretic karyotyping and random amplification of polymorphic DNA (RAPD) were evaluated as experimental approaches to correlate genomic polymorphism and geographic origin of native wine yeast strains. The three molecular methods were capable of detecting a European commercial strain among native Chilean strains; however, RAPD proved to have the best performance. Conclusions:  The molecular polymorphism analysis is useful to evaluate the geographical origin of native yeast isolates and confirms or refutes the genetic background of currently marketed strains. Significance and Impact of the Study:  This study permits a genetic characterization of native yeast populations and confirms its utility as a tool for evaluating if a native isolate derives from the region where it was collected, permitting, furthermore, to develop studies on the evolution of native yeast populations and to evaluate the effect of introduced yeasts on these populations. [ABSTRACT FROM AUTHOR]

Abstract: In the fungus Neurospora crassa we have used RIP to obtain a presumptive null mutation of the phospholipase C-1 gene, thought to be important in intracellular calcium signaling, notably maintenance of the tip-high calcium gradient. The mutant is viable but has slow, aberrant growth and branching. Hence plc-1 is not required for polar growth at the tip, but is necessary to modulate growth to give normal form. The mutant has residual PLC activity suggesting that this enzyme function can be provided from other parts of the genome. Sequencing natural isolates has shown that the plc-1 gene is highly variable in nature. A large proportion of the variable sites are in a region that is unique to Neurospora. A phylogeny for this gene shows that New and Old World strains have diverged the most. Within the Americas, morphs are found throughout the continent suggesting extensive strain dispersal. [Copyright &y& Elsevier]

Summary: A set of 30 Anabaena strains, isolated from diverse geographical regions of India, were characterized using morphological and physiochemical attributes as well as molecular marker profiles. Significant differences were observed among the Anabaena strains with regard to the shape and size of trichomes and individual cells within a filament, besides qualitative and quantitative aspects of phycobiliprotein accumulation and activities of enzymes involved in nitrogen metabolism. Analyses of molecular polymorphisms in a selected set of 13 Anabaena strains, using primers based on repetitive sequences in the genome, led to unambiguous differentiation of the strains as well as understanding of their genetic relationships. Informative morphological, physio-chemical and molecular characters have been identified that could aid in differentiation and utilization of Anabaena strains as bioinoculants or as sources of pigments. [Copyright &y& Elsevier]

The mode and tempo of host-parasite evolution depend on population structure and history and the strength of selection that the species exert on each other. Here we genetically and epidemiologically characterize populations of the mycophagous fly Drosophila innubila and its male-killing Wolbachia endosymbiont, with the aim of integrating the local through global nature of this association. Drosophila innubila inhabit the forested "sky island" regions of the of the southwestern United States and northern Mexico, where its distribution is highly fragmented. We examine geographically isolated sky island populations of D. innubila, surveying the frequency and expression of Wolbachia infection as well as the distribution of genetic variation within and among populations of the host and parasite. In all populations, Wolbachia infection is associated with virtually complete male-killing, thus providing no evidence for the evolution of population-specific interaction phenotypes or local resistance. Although Wolbachia infection occurs in each of the main populations, there is variation among populations in the prevalence of infection and the resulting population-level sex ratio of D. innubila. Among these populations, the nuclear genes of D. innubila show moderate, though significant, differentiation. In contrast, the host mitochondrial DNA (mtDNA), which shares transmission with Wolbachia, exhibits substantially greater geographic differentiation, even after accounting for differences in transmission between nuclear and mitochondrial genes. We suggest that this pattern is caused by local Wolbachia--but not D. innubila--fluctuations in prevalence that increase the severity of drift experienced only by the mtDNA. Overall, our data suggest that the association between D. innubila and male-killing Wolbachia is ecologically dynamic within local populations, but evolutionarily coherent across the species as a whole. [ABSTRACT FROM AUTHOR]

The present study looks at possible changes in the activity of acetylcholinesterase (AChE) in tissues (brain and white muscle) of the Mediterranean bony fish Sparus auratus after a 20 days exposure to sublethal concentrations (0.1 or 0.5 ppm) of copper in the marine water and on control untreated animals. The trials also included measurements of Cu concentration in the tissues to evaluate possible metal accumulation. Moreover, sedimentation analysis as well as Vmax and Km determination were carried out in tissue extracts of Cu-exposed or control animals. Vmax and Km were also determined with or without addition of Cu2+ in the assay. No Cu accumulation occurred in brain and muscle after Cu exposure. AChE showed in both tissues a molecular polymorphism with putative globular (G) and asymmetric (A) forms. Cu exposition led to an increased specific activity and improved catalytic efficiency of AChE in brain and muscle, seemingly regarding G forms. The increase in catalytic efficiency also resulted from the in vitro assay with tissue extracts and Cu2+ addition. The higher AChE activity and catalytic efficiency in both tissues after Cu exposition and without metal accumulation, suggests an increase of free Cu aliquot into the cells, likely due to mechanisms of metal homeostasis. [Copyright &y& Elsevier]

ABSTRACTTo characterize the molecular variation in the glucose-6-phosphate dehydrogenase gene (G6PD), 196 asymptomatic and unrelated male G6PD-deficient blood donors from Bele´m, an Amazonian metropolis (Brazil), were analyzed. This deficiency was detected by horizontal agarose gel electrophoresis and quantitative spectrophotometric assay for enzyme activity. The mutations were searched by PCR/RFLP, SSCP, and direct DNA sequencing. The most frequent G6PD variant was the widespread and common G6PD A− (202G → A, 376A → G) observed in 161 subjects (82.1%). Besides this, we found another form of G6PD A− (968T → C, 376A → G) in 14 (7.1%) individuals, G6PD Seattle (844G → C) in 4.6%, G6PD Santamaria (542A → T, 376A → G) in 2.5%, and G6PD Tokyo (1246G → A) in one blood donor. Four novel variants were also identified: G6PD Bele´m (409C → T; Pro137His), G6PD Ananindeua (376A → G, 871G → A; Asn126Asp, Val291Met), G6PD Crispim with four point mutations (375G → T, 379G → T, 383T → C, and 384C → T) leading to three amino acid substitutions (Met125Ile, Ala127Ser, and Leu128Pro), and G6PD Amazonia (185C → A; Pro62His). The reported frequencies do not reflect the real values for blood donors from Bele´m, since an excess of individuals with “non A−” phenotype was included in this study to enhance the probability to find rare variants. Haplotype analyses were carried out for the less common G6PD variants identified in our study using PCR/RFLP for five polymorphic sites (FokI, PvuII, PstI, BclI, NlaIII). G6PD Crispim and G6PD Amazonia variants presented the most common haplotype found in G6PD B (− − + − −). G6PD Bele´m presented two haplotypes (− − + + +, − + + + +) and G6PD Ananindeua was found with the + − + − + haplotype. The reported heterogeneity probably is due to the great miscegenation, characteristic of the population of the Amazonian region, besides the apparently common occurrence of recurrent mutations in the G6PD gene. [Copyright &y& Elsevier]