Phylogeny and Patterns of Diversity of Goat mtDNA Haplogroup A Revealed by Resequencing Complete Mitogenomes

We sequenced to near completion the entire mtDNA of 28 Sardinian goats, selected to represent the widest possible diversity of the most widespread mitochondrial evolutionary lineage, haplogroup (Hg) A. These specimens were reporters of the diversity in the island but also elsewhere, as inferred from their affiliation to each of 11 clades defined by D-loop variation. Two reference sequences completed the dataset. Overall, 206 variations were found in the full set of 30 sequences, of which 23 were protein-coding non-synonymous single nucleotide substitutions. Many polymorphic sites within Hg A were informative for the reconstruction of its internal phylogeny. Bayesian and network clustering revealed a general similarity over the entire molecule of sequences previously assigned to the same D-loop clade, indicating evolutionarily meaningful lineages. Two major sister groupings emerged within Hg A, which parallel distinct geographical distributions of D-loop clades in extant stocks. The pattern of variation in protein-coding genes revealed an overwhelming role of purifying selection, with the quota of surviving variants approaching neutrality. However, a simple model of relaxation of selection for the bulk of variants here reported should be rejected. Non-synonymous diversity of Hg''s A, B and C denoted that a proportion of variants not greater than that allowed in the wild was given the opportunity to spread into domesticated stocks. Our results also confirmed that a remarkable proportion of pre-existing Hg A diversity became incorporated into domestic stocks. Our results confirm clade A11 as a well differentiated and ancient lineage peculiar of Sardinia.     

A Rare Y Chromosome Missense Mutation in Exon 25 of Human USP9Y Revealed by Pyrosequencing

Ubiquitin-specific protease 9, Y-linked (USP9Y), is a protein encoded by the Y chromosome. Its precise function in the cell is unknown, although a role in the regulation of protein turnover has been postulated. Nonetheless, mutations in this gene could result in the over- or under-abundance of proteins involved in the regulation of spermatogenesis. We have identified a novel mutation, SM1, located in exon 25 of USP9Y (c.3642G→A), which results in an amino acid substitution (p.V1214I). The mutation is in close linkage (four bases distant) from a silent mutation, referred to as M222 (p.E1212E, c.3636G→A). In our male population (n = 374), SM1 was found in one individual (0.3%) who belongs to the recently described haplogroup R1b3h, defined by the U152 SNP. This new mutation is expected to represent a new haplogroup, (R1b1c10a); therefore, within our population of individuals from haplogroup R1b3h (R1b110) (n = 16), it has a frequency of 6.3% (95% CI: 2.7–9.9%).     

SNPs Altering Ammonium Transport Activity of Human Rhesus Factors Characterized by a Yeast-Based Functional Assay

Proteins of the conserved Mep-Amt-Rh family, including mammalian Rhesus factors, mediate transmembrane ammonium transport. Ammonium is an important nitrogen source for the biosynthesis of amino acids but is also a metabolic waste product. Its disposal in urine plays a critical role in the regulation of the acid/base homeostasis, especially with an acid diet, a trait of Western countries. Ammonium accumulation above a certain concentration is however pathologic, the cytotoxicity causing fatal cerebral paralysis in acute cases. Alteration in ammonium transport via human Rh proteins could have clinical outcomes. We used a yeast-based expression assay to characterize human Rh variants resulting from non synonymous single nucleotide polymorphisms (nsSNPs) with known or unknown clinical phenotypes and assessed their ammonium transport efficiency, protein level, localization and potential trans-dominant impact. The HsRhAG variants (I61R, F65S) associated to overhydrated hereditary stomatocytosis (OHSt), a disease affecting erythrocytes, proved affected in intrinsic bidirectional ammonium transport. Moreover, this study reveals that the R202C variant of HsRhCG, the orthologue of mouse MmRhcg required for optimal urinary ammonium excretion and blood pH control, shows an impaired inherent ammonium transport activity. Urinary ammonium excretion was RHcg gene-dose dependent in mouse, highlighting MmRhcg as a limiting factor. HsRhCG^R202C may confer susceptibility to disorders leading to metabolic acidosis for instance. Finally, the analogous R211C mutation in the yeast ScMep2 homologue also impaired intrinsic activity consistent with a conserved functional role of the preserved arginine residue. The yeast expression assay used here constitutes an inexpensive, fast and easy tool to screen nsSNPs reported by high throughput sequencing or individual cases for functional alterations in Rh factors revealing potential causal variants.     

Biodegradation of Chlorpyrifos by a Newly Isolated Bacillus subtilis Strain,Y242

A bacterial strain Y242 isolated from agricultural wastewater was found to be highly effective in degrading chlorpyrifos. On the basis of morphology, physiological characteristics, biochemical tests, and phylogenetic analysis of 16S rRNA sequence, the isolate was identified as Bacillus subtilis. The efficiency of the B. subtilis Y242 isolate as a chlorpyrifos degrader was examined under different culture conditions formulated according to the Plackett-Burman experimental design. It was observed that B. subtilis Y242 was able to utilize chlorpyrifos as a sole carbon and energy source and grows on a medium containing concentration up to 150 mg/L. A growth medium formulated based on the results of the Plackett-Burman experiment and supplied with 150 mg/L chlorpyrifos recorded 95.12% pesticide decomposition within 48 h. Degradation study of chlorpyrifos by B. subtilis Y242 was examined by gas chromatography–mass spectrometer (GC-MS) and high-performance liquid chromatography (HPLC). These results suggest that B. subtilis Y242 will be potentially useful in the cleanup of contaminated pesticide waste in the environment.     

Homologous Series by Chromosome Number and the Genome Rearrangements in the Phylogeny of Salmonoidei

Published data on chromosome numbers of Salmonoidei are summarized. The existence of homologous variation of chromosome number in different phyletic lines of this suborder is substantiated. It is suggested that the origin of homologous series is related to major genome rearrangements (simultaneous fusion of several chromosomes).     

Design of a High Density SNP Genotyping Assay in the Pig Using SNPs Identified and Characterized by Next Generation Sequencing Technology

Background

The dissection of complex traits of economic importance to the pig industry requires the availability of a significant number of genetic markers, such as single nucleotide polymorphisms (SNPs). This study was conducted to discover several hundreds of thousands of porcine SNPs using next generation sequencing technologies and use these SNPs, as well as others from different public sources, to design a high-density SNP genotyping assay.

Methodology/Principal Findings

A total of 19 reduced representation libraries derived from four swine breeds (Duroc, Landrace, Large White, Pietrain) and a Wild Boar population and three restriction enzymes (AluI, HaeIII and MspI) were sequenced using Illumina''s Genome Analyzer (GA). The SNP discovery effort resulted in the de novo identification of over 372K SNPs. More than 549K SNPs were used to design the Illumina Porcine 60K+SNP iSelect Beadchip, now commercially available as the PorcineSNP60. A total of 64,232 SNPs were included on the Beadchip. Results from genotyping the 158 individuals used for sequencing showed a high overall SNP call rate (97.5%). Of the 62,621 loci that could be reliably scored, 58,994 were polymorphic yielding a SNP conversion success rate of 94%. The average minor allele frequency (MAF) for all scorable SNPs was 0.274.

Conclusions/Significance

Overall, the results of this study indicate the utility of using next generation sequencing technologies to identify large numbers of reliable SNPs. In addition, the validation of the PorcineSNP60 Beadchip demonstrated that the assay is an excellent tool that will likely be used in a variety of future studies in pigs.     

A New 4.8-kDa Polypeptide Intrinsic to the PS II Reaction Center, as Revealed by Modified SDS-PAGE with Improved Resolution of Low-Molecular-Weight Proteins

Low-molecular-weight polypeptides in various PS II preparationsfrom spinach and wheat were analyzed by modified SDS-PAGE, whichgave good resolution of low-molecular-weight proteins with minimizedinterference by lipids. PS II membrane fragments contained atleast nine low-molecular-weight polypeptides of between 3.9kDa and 11 kDa, and all of them were identified in thylakoidmembranes. Of these nine polypeptides, the 10-kDa phosphoprotein,the 5-kDa, 4.8-kDa, and 4.1-kDa polypeptides, and the two subunitsof cytochrome b₅₅₉ were commonly found in O₂-evolving core complexesof wheat and spinach. In contrast, PS II reaction center complexesthat consisted of D1, D2 and two cytochrome b₅₅₉ polypeptidesretained only the 4.8- kDa polypeptide. Analysis by Westernblotting revealed that the 4.8-kDa polypeptide is an intrinsiccomponent of the PS II reaction center. (Received May 30, 1988; Accepted August 19, 1988)     

Spatial Genetic Heterogeneity in Populations of a Newly Invasive Whitefly in China Revealed by a Nation-Wide Field Survey

Background

Even though introductions of exotic species provide ready-made experiments of rapid evolution, few studies have examined the genetic structure of an exotic species shortly after its initial introduction and subsequent spread. To determine the genetic structure of its populations during the initial introduction, we investigated the invasive sweet potato whitefly (Bemisia tabaci Q, commonly known as B. tabaci biotype Q) in China, which was introduced in approximately 2003. A total of 619 B. tabaci Q individuals in 20 provinces throughout China were collected and analyzed using five microsatellite loci.

Results

The introduced populations of B. tabaci Q in China represent eight genetic clusters with different geographic distributions. The populations in Yunnan Province, where B. tabaci Q was first detected, are genetically different from the other populations in China.

Conclusion

The introduced populations of B. tabaci Q in China have high spatial genetic heterogeneity. Additional research is required to determine whether the heterogeneity results from multiple introductions, rapid evolution following one or few introductions, or some combination of multiple introductions and rapid evolution. The heterogeneity, however, is inconsistent with a single introduction at Yunnan Province, where B. tabaci Q was first detected, followed by spread.     

High-Throughput Screening for Spermatogenesis Candidate Genes in the AZFc Region of the Y Chromosome by Multiplex Real Time PCR Followed by High Resolution Melting Analysis

Microdeletions in the AZF region of the Y chromosome are among the most frequent genetic causes of male infertility, although the specific role of the genes located in this region is not fully understood. AZFa and AZFb deletions impair spermatogenesis since no spermatozoa are found in the testis. Deletions of the AZFc region, despite being the most frequent in azoospermic patients, do not correlate with spermatogenic failure. Therefore, the aim of this work was to develop a screening method to ascertain the presence of the main spermatogenesis candidate genes located in the AZFc region in the light of the identification of those responsible for spermatogenic failure. DAZ, CDY, BPY2, PRY, GOLGA2LY and CSGP4LY genes were selected on the basis of their location in the AZFc region, testis-only expression, and confirmed or predicted protein codification. AMEL and SRY were used as amplification controls. The identification of Real Time PCR products was performed by High Resolution Melting analysis with SYTO 9 as intercalating dye. The herein described method allows a rapid, simple, low-cost, high-throughput screening for deletions of the main AZFc genes in patients with spermatogenic failure. This provides a strategy that would accelerate the identification of spermatogenesis candidate genes in larger populations of patients with non-obstructive idiopathic azoospermia.     

A Biochip for Genotyping Polymorphisms Associated with Eye,Hair, Skin Color,AB0 Blood Group,Sex, Y Chromosome Core Haplogroup,and Its Application to Study the Slavic Population

Molecular Biology - This paper presents a method for genotyping a panel of 60 single nucleotide polymorphisms (SNPs) using single-stage PCR followed by hybridization on a hydrogel biochip. The pool...     

High Differentiation among Eight Villages in a Secluded Area of Sardinia Revealed by Genome-Wide High Density SNPs Analysis

To better design association studies for complex traits in isolated populations it''s important to understand how history and isolation moulded the genetic features of different communities. Population isolates should not “a priori” be considered homogeneous, even if the communities are not distant and part of a small region. We studied a particular area of Sardinia called Ogliastra, characterized by the presence of several distinct villages that display different history, immigration events and population size. Cultural and geographic isolation characterized the history of these communities. We determined LD parameters in 8 villages and defined population structure through high density SNPs (about 360 K) on 360 unrelated people (45 selected samples from each village). These isolates showed differences in LD values and LD map length. Five of these villages show high LD values probably due to their reduced population size and extreme isolation. High genetic differentiation among villages was detected. Moreover population structure analysis revealed a high correlation between genetic and geographic distances. Our study indicates that history, geography and biodemography have influenced the genetic features of Ogliastra communities producing differences in LD and population structure. All these data demonstrate that we can consider each village an isolate with specific characteristics. We suggest that, in order to optimize the study design of complex traits, a thorough characterization of genetic features is useful to identify the presence of sub-populations and stratification within genetic isolates.     

A Phylogenetic Analysis of Human Syntenies Revealed by Chromosome Painting in Euarchontoglires Orders

To search for cytogenetic signatures that can help to clarify evolutionary affinities among the five orders within the Euarchontoglires clade, we focused on associations of conserved syntenic blocks that have been accumulated in the karyotypes of Primates (Strepsirhini and Haplorhini), five families of Rodentia, Scandentia (Tupaia belangeri), Dermoptera (Galeopterus variegatus) and Lagomorpha (Oryctolagus cuniculus). We examined available chromosome painting data to identify conserved chromosomes and chromosomal segments, and syntenic associations likely to have characterized the ancestral eutherian karyotype. The data set includes 161 characters that have been subjected to a concatenated analysis using maximum parsimony (MP) and Bayesian inference (BI). The phylogenetic pattern recovered is generally consistent with reconstructions based on molecular and morphological data (particularly with respect to higher systematic groupings), but there are several anomalies (e.g., in the position of the lagomorphs). Both MP and BI topologies have weak statistical support, as a consequence of the high number of autapomorphic and homoplastic character states that have evolved during the history of the clade. The vast majority of derived associations are located on the terminal portions of the branches, and very few can be identified to support deeper divergences in the tree, indicating that chromosomal structures are far more fluid that was previously recognized. The high levels of homoplasy reflected in our data suggest that the number of possible syntenic character states is limited by chromosomal structures, and the same associations occur repeatedly.     

Recent Spread of a Retrotransposon in the Silene latifolia Genome, Apart From the Y Chromosome

Transposable elements often accumulate in nonrecombining regions, such as Y chromosomes. Contrary to this trend, a new Silene retrotransposon described here, has spread recently all over the genome of plant Silene latifolia, except its Y chromosome. This coincided with the latest steps of sex chromosome evolution in this species.     

Catabolism of N-Acetylneuraminic Acid,a Fitness Function of the Food-Borne Lactic Acid Bacterium Lactobacillus sakei,Involves Two Newly Characterized Proteins

In silico analysis of the genome sequence of the meat-borne lactic acid bacterium (LAB) Lactobacillus sakei 23K has revealed a repertoire of potential functions related to the adaptation of this bacterium to the meat environment. Among these functions, the ability to use N-acetyl-neuraminic acid (NANA) as a carbon source could provide a competitive advantage for growth on meat in which this amino sugar is present. In this work, we proposed to analyze the functionality of a gene cluster encompassing nanTEAR and nanK (nanTEAR-nanK). We established that this cluster encoded a pathway allowing transport and early steps of the catabolism of NANA in this genome. We also demonstrated that this cluster was absent from the genome of other L. sakei strains that were shown to be unable to grow on NANA. Moreover, L. sakei 23K nanA, nanT, nanK, and nanE genes were able to complement Escherichia coli mutants. Construction of different mutants in L. sakei 23K ΔnanR, ΔnanT, and ΔnanK and the double mutant L. sakei 23K Δ(nanA-nanE) made it possible to show that all were impaired for growth on NANA. In addition, two genes located downstream from nanK, lsa1644 and lsa1645, are involved in the catabolism of sialic acid in L. sakei 23K, as a L. sakei 23K Δlsa1645 mutant was no longer able to grow on NANA. All these results demonstrate that the gene cluster nanTEAR-nanK-lsa1644-lsa1645 is indeed involved in the use of NANA as an energy source by L. sakei.     

Gene Conversion between the X Chromosome and the Male-Specific Region of the Y Chromosome at a Translocation Hotspot

Outside the pseudoautosomal regions, the mammalian sex chromosomes are thought to have been genetically isolated for up to 350 million years. However, in humans pathogenic XY translocations occur in XY-homologous (gametologous) regions, causing sex-reversal and infertility. Gene conversion might accompany recombination intermediates that resolve without translocation and persist in the population. We resequenced X and Y copies of a translocation hotspot adjacent to the PRKX and PRKY genes and found evidence of historical exchange between the male-specific region of the human Y and the X in patchy flanking gene-conversion tracts on both chromosomes. The rate of X-to-Y conversion (per base per generation) is four to five orders of magnitude more rapid than the rate of Y-chromosomal base-substitution mutation, and given assumptions about the recombination history of the X locus, tract lengths have an overall average length of ∼100 bp. Sequence exchange outside the pseudoautosomal regions could play a role in protecting the Y-linked copies of gametologous genes from degeneration.     

Cytogenetic Analysis of a Segment of the Y Chromosome of DROSOPHILA MELANOGASTER

Males carrying a large deficiency in the long arm of the Y chromosome known to delete the fertility gene kl-2 are sterile and exhibit a complex phenotype: (1) First metaphase chromosomes are irregular in outline and appear sticky; (2) spermatids contain micronuclei; (3) the nebenkerns of the spermatids are nonuniform in size; (4) a high molecular weight protein ordinarily present in sperm is absent; and (5) crystals appear in the nucleus and cytoplasm of spermatocytes and spermatids. In such males that carry Ste⁺ on their X chromosome the crystals appear long and needle shaped; in Ste males the needles are much shorter and assemble into star-shaped aggregates. The large deficiency may be subdivided into two shorter component deficiencies. The more distal is male sterile and lacks the high molecular weight polypeptide; the more proximal is responsible for the remainder of the phenotype. Ste males carrying the more proximal component deficiency are sterile, but Ste ⁺ males are fertile. Genetic studies of chromosome segregation in such males reveal that (1) both the sex chromosomes and the large autosomes undergo nondisjunction, (2) the fourth chromosomes disjoin regularly, (3) sex chromosome nondisjunction is more frequent in cells in which the second or third chromosomes nondisjoin than in cells in which autosomal disjunction is regular, (4) in doubly exceptional cells, the sex chromosomes tend to segregate to the opposite pole from the autosomes and (5) there is meiotic drive; i.e., reciprocal meiotic products are not recovered with equal frequencies, complements with fewer chromosomes being recovered more frequently than those with more chromosomes. The proximal component deficiency can itself be further subdivided into two smaller component deficiencies, both of which have nearly normal spermatogenic phenotypes as observed in the light microscope. Meiosis in Ste ⁺ males carrying either of these small Y deficiencies is normal; Ste males, however, exhibit low levels of sex chromosome nondisjunction with either deficient Y. The meiotic phenotype is apparently sensitive to the amount of Y chromosome missing and to the Ste constitution of the X chromosome.     

Normal Testis Determination in the Mouse Depends on Genetic Interaction of a Locus on Chromosome 17 and the Y Chromosome

We previously described a locus on chromosome (Chr) 17 of the mouse that is critical for normal testis development. This locus was designated "T-associated sex reversal" (Tas) because it segregated with the dominant brachyury allele hairpin tail (Thp) and caused gonads of C57BL/6J XY, Thp/+ individuals to develop as ovaries or ovotestes rather than as testes. To clarify the inheritance of Tas, we investigated the effects of T-Orleans (TOrl), another brachyury mutation, on gonad development. We found that gonads of C57BL/6J XY, Thp/+ and TOrl/+ mice develop ovarian tissue if the Y chromosome is derived from the AKR/J inbred strain, whereas normal testicular development occurs in the presence of a Y chromosome derived from the C57BL/6J inbred strain. From these observations we conclude that: (1) Tas is located in a region on Chr 17 common to the deletions associated with Thp, and TOrl, and (2) the Y-linked testis determining gene, Tdy, carried by the AKR/J inbred strain differs from that of the C57BL/6J inbred strain. We suggest that in mammals Tdy is not the sole testis determinant because autosomal loci must be genetically compatible with Tdy for normal testicular development.