Spatial isolation and genetic differentiation in naturally fragmented plant populations of the Swiss Alps

Aims The effect Of anthropogenic landscape fragmentation on the genetic diversity and adaptive potential of plant populations is a major issue in conservation biology. However, little is known about the partitioning of genetic diversity in alpine species, which occur in naturally fragmented habitats. Here, we, investigate molecular patterns of three alpine plants (Epilobium fleischeri, Geum reptans and Campanula thyrsoides) across Switzerland and ask whether Spatial isolation has led to high levels of populations differentiation, increasing over distance, and a decrease of within-population variability. We further hypothesize that file contrasting potential for long-distance dispersal (LDD) of Seed in these Species will considerably influence and explain diversity partitioning. Methods For each study species, we Sampled 20-23 individuals from each of 20-32 populations across entire Switzerland. We applied Random Amplified Polymorphic Dimorphism markers to assess genetic diversity within (Nei's expected heterozygosity, H-e; percentage of polymorphic hands, P-P) and among (analysis of molecular variance, Phi(st)) populations and correlated population size and altitude with within-populalion diversity. Spatial patterns of genetic relatedness were investigated using Mantel tests and standardized major axis regression as well as unweighted pair group method with arithmetic mean cluster analyses and Monmonier's algorithm. To avoid known biases, We standardized the numbers of populations, individuals and markers using multiple random reductions. We modelled LDD with a high alpine wind data set using the terminal velocity and height of seed release as key parameters. Additionally, we assessed a number of important life-history traits and factors that potentially influence genetic diversity partitioning (e.g. breeding system, longevity and population size). Important findings For all three species, We found a significant isolation-by-distance relationship but only a moderately high differentiation among populations (Phi(st): 22.7, 48 and 16.8%, for E. fleischeri, G. reptans and C. thyrsoides, respectively). Within-population diversity (H-c: 0.19-0.21, P-p: 62-75%) was not reduced in comparison to known results from lowland species and even small populations with < 50 reproductive individuals contained high levels of genetic diversity. We further found no indication that a high long-distance seed dispersal potential enhances genetic connectivity among populations. Gene flow seems to have a strong stochastic component causing large dissimilarity between population pairs irrespective of the spatial distance. Our results suggest that other life-history traits, especially the breeding System, may play an important role in genetic diversity partitioning. We conclude that spatial isolation in the alpine environment has a strong influence on population relatedness but that a number of factors can considerably influence the strength of this relationship.

Chromosomal organization and evolutionary conservation of the human chromosome 19q linkage group containing three DNA repair genes

A series of human-rodent somatic cell hybrids were investigated by Southern blot analysis for the presence or absence of twenty-six molecular markers and three isozyme loci from human chromosome 19. Based on the co-retention of these markers in the various independent hybrid clones containing portions of human chromosome 19 and on pulsed field mapping, chromosome 19 is divided into twenty ordered regions. The most likely marker order for the chromosome is: (LDLR, C3)-(cen-MANNB)-D19S7-PEPD-D19S9-GPI-TGF$ \beta$-(CYP2A, NCA, CGM2, BCKAD)-PSG1a-(D19S8, XRCC1)-(D19S19, ATP1A3)-(D19S37, APOC2)-CKMM-ERCC2-ERCC1-(D19S62, D19S51)-D19S6-D19S50-D19S22-(CGB, FTL)-qter.^ The region of 19q between the proximal marker D19S7 and the distal gene coding for the beta subunit of chorionic gonadotropin (CGB) is about 37 Mb in size and covers about 37 cM genetic distance. The ration of genetic to physical distance on 19q is therefore very close to the genomic average OF 1 cM/Mb. Estimates of physical distances for intervals between chromosome 19 markers were calculated using a mapping function which estimates distances based on the number of breaks in hybrid clone panels. The consensus genetic distances between individual markers (established at HBM10) were compared to these estimates of physical distances. The close agreement between the two estimates suggested that spontaneously broken hybrids are as appropriate for this type of study as radiation hybrids.^ All three DNA repair genes located on chromosome 19 were found to have homologues on Chinese hamster chromosome 9, which is hemizygous in CHO cells, providing an explanation for the apparent ease with which mutations at these loci were identified in CHO cells. Homologues of CKMM and TGF$\beta$ (from human chromosome 19q) and a mini-satellite DNA specific to the distal region of human chromosome 19q were also mapped to Chinese hamster 9. Markers from 19p did not map to this hamster chromosome. Thus the q-arm of chromosome 19, at least between the genes PEPD and ERCC1, appears to be a linkage group which is conserved intact between humans and Chinese hamsters. ^

The molecular and genetic characterization of the MADS box transcription factor {\it Drosophila\/} MEF2

The myocyte enhancer factor (MEF)-2 family of transcription factors has been implicated in the regulation of muscle transcription in vertebrates, but the precise position of these regulators within the genetic hierarchy leading to myogenesis is unclear. The MEF2 proteins bind to a conserved A/T-rich DNA sequence present in numerous muscle-specific genes, and they are expressed in the cells of the developing somites and in the embryonic heart at the onset of muscle formation in mammals. The MEF2 genes belong to the MADS box family of transcription factors, which control specific programs of gene expression in species ranging from yeast to humans. Each MEF2 family member contains two highly conserved protein motifs, the MADS domain and the MEF2-specific domain, which together provide the MEF2 factors with their unique DNA binding and dimerization properties. In an effort to further define the function of the MEF2 proteins, and to evaluate the degree of conservation shared among these factors and the phylogenetic pathways that they regulate, we sought to identify MEF2 family members in other species. In Drosophila, a homolog of the vertebrate MEF2 genes was identified and termed D-mef2. The D-MEF2 protein binds to the consensus MEF2 element and can activate transcription through tandem copies of that site. During Drosophila embryogenesis, D-MEF2 is specific to the mesoderm germ layer of the developing embryo and becomes expressed in all muscle cell types within the embryo. The role of D-mef2 in Drosophila embryogenesis was examined by generating a loss-of-function mutation in the D-mef2 gene. In embryos homozygous for this mutant allele, somatic, cardiac, and visceral muscles fail to differentiate, but precursors of these myogenic lineages are normally specified and positioned. These results demonstrate that different muscle cell types share a common myogenic differentiation program controlled by MEF2 and suggest that this program has been conserved from Drosophila to mammals. ^

Role of {\it ERCC1\/} in DNA repair and genetic recombination

The ERCC1 (Excision Repair Cross-Complementing-1) gene is the presumptive mammalian homolog of the Saccharomyces cerevisiae RAD10 gene. In mammalian NER, the Ercc1/XpF complex functions as an endonuclease that specifically recognizes 5$\sp\prime$ double-strand-3$\sp\prime$ single-strand structures. In yeast, the analogous function is performed by the Rad1/Rad10 complex. These observations and the conservation of amino acid homology between the Rad1 and XpF and the Rad10 and Ercc1 proteins has led to a general assumption of functional homology between these genes.^ In addition to NER, the Rad1/Rad10 endonuclease complex is also required in certain specialized mitotic recombination pathways in yeast. However, a similiar requirement for the endonuclease function of the Ercc1/XpF complex during genetic recombination in mammalian cells has not been directly demonstrated. The experiments performed in these studies were designed to determine if ERCC1 deficiency would produce recombination-deficient phenotypes in CHO cells similar to those observed in RAD10 deletion mutants, including: (1) decreased single-reciprocal exchange recombination, and (2) inability to process 5$\sp\prime$ sequence heterology in recombination intermediates.^ Specifically, these studies describe: (1) The isolation and characterization of the ERCC1 locus of Chinese hamster ovary cells; (2) The production of an ERCC1 null mutant cell line by targeted knock-out of the endogenous ERCC1 gene in a Chinese hamster ovary cell line, CHO-ATS49tg, which contains an endogenous locus, APRT, suitable as a chromosomal target for homologous recombination; (3) The characterization of mutant ERCC1 alleles from a panel of Chinese hamster ovary cell ERCC1 mutants derived by conventional mutagenesis; (4) An investigation of the effects of ERCC1 mutation on mitotic recombination through targeting of the APRT locus in an ERCC1 null background.^ The results of these studies strongly suggest that the role of ERCC1 in homologous recombination in mammalian cells is analogous to that of the yeast RAD10 gene. ^

Novel genetic markers associate with atrial fibrillation risk in Europeans and Japanese

OBJECTIVES This study sought to identify nonredundant atrial fibrillation (AF) genetic susceptibility signals and examine their cumulative relations with AF risk. BACKGROUND AF-associated loci span broad genomic regions that may contain multiple susceptibility signals. Whether multiple signals exist at AF loci has not been systematically explored. METHODS We performed association testing conditioned on the most significant, independently associated genetic markers at 9 established AF loci using 2 complementary techniques in 64,683 individuals of European ancestry (3,869 incident and 3,302 prevalent AF cases). Genetic risk scores were created and tested for association with AF in Europeans and an independent sample of 11,309 individuals of Japanese ancestry (7,916 prevalent AF cases). RESULTS We observed at least 4 distinct AF susceptibility signals on chromosome 4q25 upstream of PITX2, but not at the remaining 8 AF loci. A multilocus score comprised 12 genetic markers demonstrated an estimated 5-fold gradient in AF risk. We observed a similar spectrum of risk associated with these markers in Japanese. Regions containing AF signals on chromosome 4q25 displayed a greater degree of evolutionary conservation than the remainder of the locus, suggesting that they may tag regulatory elements. CONCLUSIONS The chromosome 4q25 AF locus is architecturally complex and harbors at least 4 AF susceptibility signals in individuals of European ancestry. Similar polygenic AF susceptibility exists between Europeans and Japanese. Future work is necessary to identify causal variants, determine mechanisms by which associated loci predispose to AF, and explore whether AF susceptibility signals classify individuals at risk for AF and related morbidity.

Evolutionary conservation of the U7 small nuclear ribonucleoprotein in Drosophila melanogaster.

The U7 snRNP involved in histone RNA 3' end processing is related to but biochemically distinct from spliceosomal snRNPs. In vertebrates, the Sm core structure assembling around the noncanonical Sm-binding sequence of U7 snRNA contains only five of the seven standard Sm proteins. The missing Sm D1 and D2 subunits are replaced by U7-specific Sm-like proteins Lsm10 and Lsm11, at least the latter of which is important for histone RNA processing. So far, it was unknown if this special U7 snRNP composition is conserved in invertebrates. Here we describe several putative invertebrate Lsm10 and Lsm11 orthologs that display low but clear sequence similarity to their vertebrate counterparts. Immunoprecipitation studies in Drosophila S2 cells indicate that the Drosophila Lsm10 and Lsm11 orthologs (dLsm10 and dLsm11) associate with each other and with Sm B, but not with Sm D1 and D2. Moreover, dLsm11 associates with the recently characterized Drosophila U7 snRNA and, indirectly, with histone H3 pre-mRNA. Furthermore, dLsm10 and dLsm11 can assemble into U7 snRNPs in mammalian cells. These experiments demonstrate a strong evolutionary conservation of the unique U7 snRNP composition, despite a high degree of primary sequence divergence of its constituents. Therefore, Drosophila appears to be a suitable system for further genetic studies of the cell biology of U7 snRNPs.

The scaling of genetic diversity in a changing and fragmented world

Most species do not live in a constant environment over space or time. Their environment is often heterogeneous with a huge variability in resource availability and exposure to pathogens or predators, which may affect the local densities of the species. Moreover, the habitat might be fragmented, preventing free and isotropic migrations between local sub-populations (demes) of a species, making some demes more isolated than others. For example, during the last ice age populations of many species migrated towards refuge areas from which re-colonization originated when conditions improved. However, populations that could not move fast enough or could not adapt to the new environmental conditions faced extinctions. Populations living in these types of dynamic environments are often referred to as metapopulations and modeled as an array of subdivisions (or demes) that exchange migrants with their neighbors. Several studies have focused on the description of their demography, probability of extinction and expected patterns of diversity at different scales. Importantly, all these evolutionary processes may affect genetic diversity, which can affect the chance of populations to persist. In this chapter we provide an overview on the consequences of fragmentation, long-distance dispersal, range contractions and range shifts on genetic diversity. In addition, we describe new methods to detect and quantify underlying evolutionary processes from sampled genetic data.

Evolutionary Genomics and Conservation of the Endangered Przewalski's Horse.

Przewalski's horses (PHs, Equus ferus ssp. przewalskii) were discovered in the Asian steppes in the 1870s and represent the last remaining true wild horses. PHs became extinct in the wild in the 1960s but survived in captivity, thanks to major conservation efforts. The current population is still endangered, with just 2,109 individuals, one-quarter of which are in Chinese and Mongolian reintroduction reserves [1]. These horses descend from a founding population of 12 wild-caught PHs and possibly up to four domesticated individuals [2-4]. With a stocky build, an erect mane, and stripped and short legs, they are phenotypically and behaviorally distinct from domesticated horses (DHs, Equus caballus). Here, we sequenced the complete genomes of 11 PHs, representing all founding lineages, and five historical specimens dated to 1878-1929 CE, including the Holotype. These were compared to the hitherto-most-extensive genome dataset characterized for horses, comprising 21 new genomes. We found that loci showing the most genetic differentiation with DHs were enriched in genes involved in metabolism, cardiac disorders, muscle contraction, reproduction, behavior, and signaling pathways. We also show that DH and PH populations split ∼45,000 years ago and have remained connected by gene-flow thereafter. Finally, we monitor the genomic impact of ∼110 years of captivity, revealing reduced heterozygosity, increased inbreeding, and variable introgression of domestic alleles, ranging from non-detectable to as much as 31.1%. This, together with the identification of ancestry informative markers and corrections to the International Studbook, establishes a framework for evaluating the persistence of genetic variation in future reintroduced populations.

ARRANGEMENT OF GENE LOCI IN EUPLOID CHINESE HAMSTER CELLS AND GENETIC CONSEQUENCES OF REARRANGEMENT IN CHO CELLS

In both euploid Chinese hamster (Cricetulus griseus) cells and pseudodiploid Chinese hamster ovary (CHO) cells, gene assignments were accomplished by G band chromosome and isozyme analysis (32 isozymes) of interspecific somatic cell hybrids obtained after HAT selection of mouse CL 1D (TK('-)) cells which were PEG-fused with either euploid Chinese hamster cells or HPRT('-) CHO cells. Hybrids slowly segregated hamster chromosomes. Clone panels consisting of independent hybrid clones and subclones containing different combinations of Chinese hamster chromosomes and isozymes were established from each type of fusion.^ These clone panels enabled us to provisionally assign the loci for: nucleoside phosphorylase (NP), glyoxalase (GLO), glutathione reductase (GSR), adenosine kinase (ADK), esterase D (ESD), peptidases B and S (PEPB and -S) and phosphoglucomutase 2 (PGM2, human nomenclature) to chromosome 1; adenylate kinase 1 (AK1), adenosine deaminase (ADA) and inosine triosephosphatase (ITP) to chromosome 6; triosephosphate isomerase (TPI) to chromosome 8; and glucose phosphate isomerse (GPI) and peptidase D (PEPD) to chromosome 9.^ We also confirm the assignments of 6-phosphogluconate dehydrogenase (PGD), PGM1, enolase 1 (ENO1) and diptheria toxin sensitivity (DTS) to chromosome 2 as well as provisionally assign galactose-1-phosphate uridyl transferase (GALT) and AK2 to chromosome 2. Selection in either HAT or BrdU for hybrids that had retained or lost the chromosome carrying the locus for TK enabled us to assign the loci for TK, galactokinase (GALK) and acid phosphatase 1 (ACP1) to Chinese hamster chromosome 7.^ These results are discussed in relation to current theories on the basis for high frequency of drug resistant autosomal recessive mutants in CHO cells and conservation of mammalian autosomal linkage groups. ^

Contemporary genetic, historical genetic and morphological data sets for the Yellow-tufted Honeyeater Lichenostomus melanops

Understanding the evolutionary history of threatened populations can improve their conservation management. Re-establishment of past but recent gene flow could re-invigorate threatened populations and replenish genetic diversity, necessary for population persistence. One of the four nominal subspecies of the common yellow-tufted honeyeater, Lichenostomus melanops cassidix, is critically endangered despite substantial conservation efforts over 55 years. Using a combination of morphometric, genetic and modelling approaches we tested for its evolutionary distinctiveness and conservation merit. We confirmed that cassidix has at least one morphometric distinction. It also differs genetically from the other subspecies in allele frequencies but not phylogenetically, implying that its evolution was recent. Modelling historical distribution supported the lack of vicariance and suggested a possibility of gene flow among subspecies at least since the late Pleistocene. Multi-locus coalescent analyses indicated that cassidix diverged from its common ancestor with neighbouring subspecies gippslandicus sometime from the mid-Pleistocene to the Holocene, and that it has the smallest historical effective population size of all subspecies. It appears that cassidix diverged from its ancestor with gippslandicus through a combination of drift and local selection. From patterns of genetic subdivision on two spatial scales and morphological variation we concluded that cassidix, gippslandicus and (melanops + meltoni) are diagnosable as subspecies. Low genetic diversity and effective population size of cassidix may translate to low genetic fitness and evolutionary potential, thus managed gene flow from gippslandicus is recommended for its recovery.

An image segmentation based on a genetic algorithm for determining soil coverage by crop residues

Determination of the soil coverage by crop residues after ploughing is a fundamental element of Conservation Agriculture. This paper presents the application of genetic algorithms employed during the fine tuning of the segmentation process of a digital image with the aim of automatically quantifying the residue coverage. In other words, the objective is to achieve a segmentation that would permit the discrimination of the texture of the residue so that the output of the segmentation process is a binary image in which residue zones are isolated from the rest. The RGB images used come from a sample of images in which sections of terrain were photographed with a conventional camera positioned in zenith orientation atop a tripod. The images were taken outdoors under uncontrolled lighting conditions. Up to 92% similarity was achieved between the images obtained by the segmentation process proposed in this paper and the templates made by an elaborate manual tracing process. In addition to the proposed segmentation procedure and the fine tuning procedure that was developed, a global quantification of the soil coverage by residues for the sampled area was achieved that differed by only 0.85% from the quantification obtained using template images. Moreover, the proposed method does not depend on the type of residue present in the image. The study was conducted at the experimental farm “El Encín” in Alcalá de Henares (Madrid, Spain).

Genetic diversity and relationships in accessions from different cultivar groups and origins in the tree tomato (Solanum betaceum Cav.)

Abstract Tree tomato (Solanum betaceum) is an Andean small tree cultivated for its juicy fruits. Little information is available on the characterization of genetic resources and breeding of this neglected crop. We have studied the molecular diversity with AFLP markers using 11 combinations of primers of a collection of 25 S. betaceum accessions belonging to four cultivar groups, most of which had been previously morphologically characterized, as well as one accession of the wild relative S. cajanumense.Atotal of 197 AFLP fragments were scored, of which 84 (43 %) were polymorphic. When excluding S. cajanumense from the analysis, the number of polymorphic AFLP fragments was 78 (40 %). Unique AFLP fingerprints were obtained for every accession, but no AFLP fragments specific and universal to any of the four cultivar groups were found. The total genetic diversity (HT) of cultivated accessions was HT = 0.2904, while for cultivar groups it ranged from HT = 0.1846 in the orange group to HT = 0.2498 in the orange pointed group. Genetic differentiation among cultivar groups (GST) was low (GST = 0.2248), which was matched by low values of genetic distance among cultivar groups. The diversity of collections from Ecuador, which we hypothesize is a center of diversity for tree tomato, was similar to that from other origins (HT = 0.2884 and HT = 0.2645, respectively). Cluster and PCoA analyses clearly separated wild S. cajanumense from the cultivated species. However, materials of different cultivar groups and origins were intermingled in both analyses. The Mantel test correlation coefficient of the matrices of morphological and AFLP distances was low (-0.024) and non-significant. Overall, the results show that a wide diversity is present in each of the cultivar groups, indicate that Ecuador may be regarded as a center of accumulation of diversity for this crop, and confirm that AFLP and morphological characterization data are complementary. The results obtained are of value for the conservation of genetic resources and breeding of tree tomato, as an assessment of the genetic diversity and relationships among differen

Genetic characterization of chestnut (Castanea sativa Mill.) orchards and traditional nut varieties in El Bierzo, a glacial refuge and major cultivation site in Northwestern Spain

This paper presents a detailed genetic study of Castanea sativa in El Bierzo, a major nut production region with interesting features. It is located within a glacial refuge at one extreme of the distribution area (northwest Spain); it has a centenary tradition of chestnut management; and more importantly, it shows an unusual degree of genetic isolation. Seven nuclear microsatellite markers were selected to analyze the genetic variability and structure of 169 local trees grafted for nut production. We analyzed in the same manner 62 local nuts. The selected loci were highly discriminant for the genotypes studied, giving a combined probability of identity of 6.1 × 10−6. An unprecedented density of trees was sampled for this project over the entire region, and nuts were collected representing 18 cultivars marketed by local producers. Several instances of misclassification by local growers were detected. Fixation index estimates and analysis of molecular variance (AMOVA) data are supportive of an unexpectedly high level of genetic differentiation in El Bierzo, larger than that estimated in a previous study with broader geographical scope but based on limited local sampling (Pereira-Lorenzo et al., Tree Genet Genomes 6: 701–715, 2010a). Likewise, we have determined that clonality due to grafting had been previously overestimated. In line with these observations, no significant spatial structure was found using both a model-based Bayesian procedure and Mantel’s tests. Taken together, our results evidence the need for more fine-scale genetic studies if conservation strategies are to be efficiently improved.