Molecular Characterization of Cryptosporidium molnari Reveals a Distinct Piscine Clade

Multilocus phylogenetic analysis of small-subunit (SSU) rRNA and actin from Cryptosporidium molnari clustered this species with the C. molnari-like genotype of an isolate from the guppy, although the two fish isolates seem to be distinct species. The analysis of available piscine genotypes provides some support for cladistic congruence of the genus Piscicryptosporidium, but additional piscine genotypes are needed.Recent reviews accept more than 20 valid cryptosporidium species (7, 20), and characterization of additional isolates is expanding this list rapidly (http://www.vetsci.usyd.edu.au/staff/JanSlapeta/icrypto/index.htm). In addition, numerous morphotypes or genotypes have been proposed whose taxonomic affiliation is unsettled due to incomplete characterization according to minimum consensus standards (5, 7, 24). Five species have been proposed for fish isolates (15), but only Cryptosporidium molnari and Cryptosporidium scophthalmi (2, 4) stand as valid species (20), although not without discussion (7). Fish cryptosporidia present some unique features, which have even led to the genus Piscicryptosporidium being proposed (13). However, lack of genetic support keeps this genus and several fish morphotypes as incertae sedis (12, 15, 24). Detailed biological data on C. molnari and C. scophthalmi have been previously presented (3, 18, 19), but no molecular characterization has yet been conducted, thus hampering species identification of other fish isolates (7, 24) and evaluation of their relationships within the genus (15). Ribosomal and actin gene data on an isolate from guppy fish (Poecilia reticulata) have been obtained, and preliminary analyses of these sequences indicated a basal position in the cryptosporidial tree (17). Although it was regarded as C. molnari-like, biological characterization of this isolate was limited. The purpose of this work was to provide the necessary C. molnari comparative genetic data and to clarify the relationship of available fish isolates in a phylogenetic context.     

Epidemiology of Cryptosporidium molnari in Spanish Gilthead Sea Bream (Sparus aurata L.) and European Sea Bass (Dicentrarchus labrax L.) Cultures: from Hatchery to Market Size

A long-term epidemiological study of Cryptosporidium molnari in aquacultured European sea bass (ESB) and gilthead sea bream (GSB) was performed in different types of facilities on the Atlantic, Cantabric, and Mediterranean coasts. Four types of studies were carried out. In study A, fish raised from juveniles to marketable size (ongrowing stage) were periodically sampled in three different types of cultures. Studies B and C focused on hatchery and nursery facilities. In study D, occasional samplings were performed during mortality or morbidity outbreaks. As a general trend, C. molnari was more prevalent in GSB than in ESB. Data on the distribution pattern of C. molnari in total sampled GSB (studies A, B, and D) had a variance higher than the mean (overdispersion). In GSB (study A), the type of ongrowing system (sea cages, earth ponds, or indoor tanks) was found to have no significant effect. There was a significant relationship between the presence of the parasite and both fish weight and season. The highest infection values were recorded in spring. Prevalence and intensity had convex weight profiles, with a peak in 30- to 100-g fish. In study D, the prevalence of infection was higher in fish recently introduced in sea cages and in preongrowing systems. In studies B and C, fish were almost never infected before entering the postlarval and nursery facilities. The parasite seems to enter the host mainly through the water in production steps with less stringent water treatment. Recirculation systems and fish cannibalism could contribute to oocyst concentration and dispersion in aquaculture facilities.     

Genetic structure and diversity of Coscinium fenestratum: a critically endangered liana of Western Ghats,India

Coscinium fenestratum is a critically endangered medicinal plant, well-known for its bioactive isoquinoline alkaloid berberine. The species has been over harvested from its natural habitats to meet the huge requirement of raw drug market and industrial consumption. This has lead to a rapid decline in the population size and has also led to local population extinction at few locations in the Western Ghats, India. In this study, inter-simple sequence repeat markers were used to investigate the genetic variation and population structure of seven extant populations of C. fenestratum from the central Western Ghats, India. Eight primer combination produced a total of 57 unambiguous bands, of which (47.1 %) were polymorphic. The species exhibited a moderate to low level of intra population genetic diversity (H _s = 0.347 ± 0.008; H _t = 0.378 ± 0.006 (POPGENE) and H _s = 0.262 ± 0.0028; H _t = 0.204 ± 0.020 (HICKORY)). The populations were low to moderately differentiated from one another (G _ST = 0.221) and geographical distance was not significantly correlated with genetic distance, suggesting that these long-lived, geographically distant remnant populations were once connected through gene flow. There was a significant amount of genetic variation among populations (19.85 %). The Bayesian software STRUCTURE and HICKORY were used to further reveal the genetic structure of C. fenestratum. The results revealed weak population structure (K = 2) with one single widespread gene pool, and indicated that gene flow and inbreeding are likely to be the major driving force in shaping current population genetic structure of C. fenestratum. Thus, an understanding of the genetic diversity and population structure of C. fenestratum can provide insight into the conservation and management of this species.     

Genetic mapping of a new race specific resistance allele effective to Puccinia hordei at the Rph9/Rph12locus on chromosome 5HL in barley

Background

Tea is one of the most popular beverages in the world. Many species in the Thea section of the Camellia genus can be processed for drinking and have been domesticated. However, few investigations have focused on the genetic consequence of domestication and geographic origin of landraces on tea plants using credible wild and planted populations of a single species. Here, C. taliensis provides us with a unique opportunity to explore these issues.

Results

Fourteen nuclear microsatellite loci were employed to determine the genetic diversity and domestication origin of C. taliensis, which were represented by 587 individuals from 25 wild, planted and recently domesticated populations. C. taliensis showed a moderate high level of overall genetic diversity. The greater reduction of genetic diversity and stronger genetic drift were detected in the wild group than in the recently domesticated group, indicating the loss of genetic diversity of wild populations due to overexploitation and habitat fragmentation. Instead of the endangered wild trees, recently domesticated individuals were used to compare with the planted trees for detecting the genetic consequence of domestication. A little and non-significant reduction in genetic diversity was found during domestication. The long life cycle, selection for leaf traits and gene flow between populations will delay the emergence of bottleneck in planted trees. Both phylogenetic and assignment analyses suggested that planted trees may have been domesticated from the adjacent central forest of western Yunnan and dispersed artificially to distant places.

Conclusions

This study contributes to the knowledge about levels and distribution of genetic diversity of C. taliensis and provides new insights into genetic consequence of domestication and geographic origin of planted trees of this species. As an endemic tea source plant, wild, planted and recently domesticated C. taliensis trees should all be protected for their unique genetic characteristics, which are valuable for tea breeding.     

Host-Size-Matching in a Sperm-Dependent Asexual Fish

Among vertebrates, there are approximately fifty sperm-dependent asexual species, all of which are of hybrid origin. Sperm-dependent asexual vertebrates use sperm from one, or both, of their parental species for reproduction. To address the hypothesis that selection has resulted in asexual phenotypes that resemble their host’s phenotype, I studied size, shape, and genetic variation (using six microsatellite loci) in nine populations of a sperm-dependent asexual fish, Chrosomus eos-neogaeus, in Alberta, Canada. These nine populations differed in the presence or absence of each of the two parental species: three populations coexist with C. eos, two populations coexist with C. neogaeus, and four populations coexist with both parental species. Consistent with my hypothesis, I found that C. eos-neogaeus tended to match the body size of the parental species with which they coexist, and that C. eos-neogaeus that coexist with only C. eos were genetically divergent from C. eos-neogaeus that are syntopic with only C. neogaeus. The genetic divergence among C. eos-neogaeus populations was independent of geographic distance among populations, and estimates of quantitative trait divergence among C. eos-neogaeus populations exceeded neutral expectations. These observations suggest that processes other than migration, mutation, and drift are likely shaping the diversity of C. eos-neogaeus in the lakes sampled for this study. For example, sexual selection leading to host mimicry, or natural selection leading to environmental adaptation, may explain the observed pattern of host-size matching.     

High genetic diversity and significant population structure in Cedrus brevifolia Henry, a narrow endemic Mediterranean tree from Cyprus

Endemic island plant species with a narrow distribution are often, but not always, linked to low genetic variation within populations and a lack of differentiation among populations. Cedrus brevifolia is a narrow endemic island tree species of Cyprus. Its range is restricted to a single forest, divided into five neighbouring sites. This study, using biparentally inherited nuclear microsatellites and paternally inherited plastid (chloroplast) microsatellites, assessed the genetic variation of C. brevifolia within its sole population and the level of genetic differentiation among formed sites. The results from both markers showed high diversity (nuclear H _T?=?0.70; plastid H _T?=?0.93), strongly suggesting that the species did not experience severe bottleneck events or extensive genetic drift. Besides, the maintenance of a high genetic diversity in C. brevifolia may suggest that it originates from a widespread congener species. Significant genetic differentiation at nuclear (G _ST?=?0.052) and plastid (G _ST?=?0.119) markers was found among the formed sites. Remarkably, the relatively high genetic differentiation found at plastid markers was comparable to values observed in two widespread congener cedar species. The genetic differentiation probably occurred due to fragmentation of a previously uniform population. This would lead to the shaping of different genetic groups (Bayesian analysis) and to significant population substructure. Furthermore, significant values observed for both isolation by distance and large-scale spatial genetic structure could indicate ineffective gene flow among sites and the early geographical isolation of the more isolated sites from the core population.     

A Genetic System for Clostridium ljungdahlii: a Chassis for Autotrophic Production of Biocommodities and a Model Homoacetogen

Methods for genetic manipulation of Clostridium ljungdahlii are of interest because of the potential for production of fuels and other biocommodities from carbon dioxide via microbial electrosynthesis or more traditional modes of autotrophy with hydrogen or carbon monoxide as the electron donor. Furthermore, acetogenesis plays an important role in the global carbon cycle. Gene deletion strategies required for physiological studies of C. ljungdahlii have not previously been demonstrated. An electroporation procedure for introducing plasmids was optimized, and four different replicative origins for plasmid propagation in C. ljungdahlii were identified. Chromosomal gene deletion via double-crossover homologous recombination with a suicide vector was demonstrated initially with deletion of the gene for FliA, a putative sigma factor involved in flagellar biogenesis and motility in C. ljungdahlii. Deletion of fliA yielded a strain that lacked flagella and was not motile. To evaluate the potential utility of gene deletions for functional genomic studies and to redirect carbon and electron flow, the genes for the putative bifunctional aldehyde/alcohol dehydrogenases, adhE1 and adhE2, were deleted individually or together. Deletion of adhE1, but not adhE2, diminished ethanol production with a corresponding carbon recovery in acetate. The double deletion mutant had a phenotype similar to that of the adhE1-deficient strain. Expression of adhE1 in trans partially restored the capacity for ethanol production. These results demonstrate the feasibility of genetic investigations of acetogen physiology and the potential for genetic manipulation of C. ljungdahlii to optimize autotrophic biocommodity production.     

Phylogenetic analysis of nuclear and mitochondrial DNA reveals a complex of cryptic species in Crassicutis cichlasomae (Digenea: Apocreadiidae), a parasite of Middle-American cichlids

We obtained nuclear ITS-1 and mitochondrial cox1 sequences from 225 Crassicutis cichlasomae adults collected in 12 species of cichlids from 32 localities to prospect for the presence of cryptic species. This trematode is commonly found in species of cichlids over a wide geographic range in Middle-America. Population-level phylogenetic analyses of ITS-1 and cox1, assessments of genetic and haplotype diversity, and morphological observations revealed that C. cichlasomae represents a complex of seven cryptic species for which no morphological diagnostic characters have been discovered thus far. Bayesian and Maximum Likelihood analyses of concatenated datasets (906 bp) recovered eight lineages of C. cichlasomae, all with high posterior probabilities and bootstrap branch support. Values of genetic divergence between clades ranged from 1.0% to 5.2% for ITS-1, and from 7.2% to 30.0% for cox1. Morphological study of more than 300 individuals did not reveal structural diagnostic traits for the species defined using molecular evidence. These observations indicate that some traditional morphological characters (e.g., testes position) have substantial intra-specific variation, and should be used with caution when classifying C. cichlasomae and their sister taxa. Additionally, phylogenetic analyses did not reveal a strict correlation between these cryptic species and their host species or geographic distribution, however it appears that genetic distinctiveness of these cryptic species was influenced by the diversification and biogeographical history of Middle-American cichlids.     

Genetic diversity of Crocus antalyensis B. Mathew (Iridaceae) and a new subspecies from southern Anatolia

Crocus antalyensis B. Mathew is a bulbous plant endemic to Turkey. It is morphologically variable within the western part of Anatolia. Amplified fragment length polymorphism (AFLP) marker system was used to detect genetic variation among the Crocus taxa. Twenty-two primer combinations were used to screen for polymorphism among the samples. Genetic variation ranged from 0.44 to 0.69. We demonstrated the efficiency of the AFLP marker system for discriminating between individual C. antalyensis specimens. A high level of genetic variation was present among C. antalyensis specimens collected from different locations in Turkey. We also observed that C. antalyensis subspp. are genetically distinct from their relative Crocus flavus Haw. subsp. dissectus Baytop & B. Mathew. A new subspecies of C. antalyensis B. Mathew from southern Turkey is described. It is characterized by striped outer perianth segments, waist-shaped flowers, and glabrous throat of the perianth. A composite image of the new subspecies is presented.     

Chloroplast diversity and population differentiation of Castanopsis fargesii (Fagaceae): a dominant tree species in evergreen broad-leaved forest of subtropical China

Subtropical forests in China constitute the major expanse of evergreen broad-leaved forest in East Asia. The significant genetic divergence of the keystone tree species should be expected due to the huge geomorphological and environmental changes from west to east in subtropical China. In this study, a total of 652 individuals from 27 populations of Castanopsis fargesii throughout its natural range in mainland China were genotyped with eight chloroplast microsatellite markers to investigate genetic diversity, population differentiation, and demographic history of C. fargesii. Phylogeographic structure among populations of C. fargesii was evidenced by the permutation test, revealing that N_ST was significantly higher than G_ST . The strong genetic differentiation found among populations was well in accordance with isolation-by-distance model. In addition, significant isolation by elevation was detected among populations. Significant genetic differentiations were revealed among the west, center, and east regions by approximate Bayesian computations (ABC). The genetic divergence might reflect the regional responses to the fast and dramatic uplift of Yunnan-Guizhou Plateau and Wuyi mountain range in the Pleistocene. In the present study, contraction-expansion process was detected in the west, center, and east regions, indicating that geomorphological remodeling together with climatic changes in the Pleistocene had strong impact on genetic structure of C. fargesii.     

Effect of cliff connectivity on the genetic population structure of a rock-dwelling land snail species with frequent self-fertilization

To examine the effect of habitat connectivity on the genetic differentiation in the frequently selfing gastropod Chondrina avenacea we assessed RAPD profiles in 12 subpopulations from continuous cliffs and in 12 subpopulations from small, naturally fragmented cliffs in the Swiss Jura mountains. Subpopulations of C. avenacea on continuous cliffs showed a smaller number of polymorphic RAPD loci and a lower gene diversity (Nei's h = 0.049) than subpopulations on fragmented cliffs (h = 0.078). Genetic distance was not correlated with geographical distance between subpopulations. G_ST analysis indicated strong isolation effects. Neither the number of polymorphic loci nor Nei's h was correlated with local population density. Our analysis indicates that the local population structure of C. avenacea is a combined result of frequent self-fertilization, genetic drift and low dispersal. Similar findings have been reported in studies on spatial population structure of plants. We suggest that future management actions (selective tree cutting, and controlled access of sport climbers) should also consider small, isolated limestone cliffs to preserve the genetic diversity of C. avenacea.     

Natural genetic variation as a tool for discovery in Caenorhabditis nematodes

Over the last 20 years, studies of Caenorhabditis elegans natural diversity have demonstrated the power of quantitative genetic approaches to reveal the evolutionary, ecological, and genetic factors that shape traits. These studies complement the use of the laboratory-adapted strain N2 and enable additional discoveries not possible using only one genetic background. In this chapter, we describe how to perform quantitative genetic studies in Caenorhabditis, with an emphasis on C. elegans. These approaches use correlations between genotype and phenotype across populations of genetically diverse individuals to discover the genetic causes of phenotypic variation. We present methods that use linkage, near-isogenic lines, association, and bulk-segregant mapping, and we describe the advantages and disadvantages of each approach. The power of C. elegans quantitative genetic mapping is best shown in the ability to connect phenotypic differences to specific genes and variants. We will present methods to narrow genomic regions to candidate genes and then tests to identify the gene or variant involved in a quantitative trait. The same features that make C. elegans a preeminent experimental model animal contribute to its exceptional value as a tool to understand natural phenotypic variation.     

Testing for hybridization and assessing genetic diversity in Morelet’s crocodile (Crocodylus moreletii) populations from central Veracruz

Among the loss of genetic diversity due to population declines, population fragmentation and habitat loss, hybridization also stands as a threat to Morelet’s crocodile (Crocodylus moreletii) populations. Genetic surveys in Belize and the Yucatan Peninsula have detected evidence of hybridization with the American crocodile (C. acutus). Admixture between these two species is most likely driven by human-mediated translocations. Along the central gulf coast of Mexico, C. moreletii populations are presumed to be purebred. To test this, we use nine microsatellite loci and sequence data from the mitochondrial control region to detect if C. acutus alleles have introgressed into populations of C. moreletii from central Veracruz. In 2010, C. moreletii was transferred from Appendix I to II of CITES based on a whole species demographic analysis, which indicated that populations had recovered across its range. Our study shows that populations in central Veracruz are purebred, although they exhibit low levels of genetic diversity most likely caused by inbreeding. Our data also suggest there is fragmentation among populations of C. moreletii, which may lead to further loss of genetic variation. Due the purity and low genetic diversity of C. moreletii populations from central Veracruz, we recommend increased protection and active management practices that take genetic data into account.     

Molecular mapping of a stripe rust resistance gene in wheat line C51

Stripe rust, a major disease in areas where cool temperatures prevail, can strongly influence grain yield. To control this disease, breeders have incorporated seedling resistance genes from a variety of sources outside the primary wheat gene pool. The wheat line C51, introduced from the International Center for Agricultural Research in the Dry Areas (ICARDA), Syria, confers resistance to all races of Puccinia striiformis f. sp. tritici (PST) in China. To map the resistant gene(s) against stripe rust in wheat line C51, 212 F ₈ recombinant inbred lines (RILs) derived from the cross X440 × C51 were inoculated with Chinese PST race CYR33 (Chinese yellow rust, CYR) in the greenhouse. The result showed that C51 carried a single dominant gene for resistance (designated YrC51) to CYR33. Simple sequence repeat (SSR) and resistance gene-analogue polymorphism (RGAP) markers that were polymorphic between the parents were used for genotyping the 212 F ₈ RILs. YrC51was closely linked to two SSR loci on chromosome 2BS with genetic distances of 5.1 cM (Xgwm429) and 7.2 cM (Xwmc770), and to three RGAP markers C51R1 (XLRR For / NLRR For), C51R2 (CLRR Rev / Cre3LR-F) and C51R3 (Pto kin4/ NLRR-INV2) with genetic distances of 5.6, 1.6 and 9.2 cM, respectively. These RGAP-linked markers were then converted into STS markers. Among them, one STS marker, C51STS-4, was located at a genetic distance of 1.4 cM to YrC51 and was closely associated with resistance when validated in several populations derived from crosses between C51 and Sichuan cultivars. The results indicated that C51STS-4 can be used for marker assisted selection (MAS) and would facilitate the pyramiding of YrC51 with other genes for stripe rust resistance.     

Genetic Diversity and Conservation Implications of Four Cupressus Species in China as Revealed by Microsatellite Markers

Understanding the extent and distribution of genetic diversity is crucial for the conservation and management of endangered species. Cupressus chengiana, C. duclouxiana, C. gigantea, and C. funebris are four ecologically and economically important species in China. We investigated their genetic diversity, population structure, and extant effective population size (35 populations, 484 individuals) employing six pairs of nuclear microsatellite markers (selected from 53). Their genetic diversity is moderate among conifers, and genetic differentiation among populations is much lower in C. gigantea than in the other three species; the estimated effective population size was largest for C. chengiana, at 1.70, 2.91, and 3.91 times the estimates for C. duclouxiana, C. funebris, and C. gigantea, respectively. According to Bayesian clustering analysis, the most plausible population subdivision scheme within species is two groups in C. chengiana, three groups in C. duclouxiana, and a single group for both C. funebris and C. gigantea. We propose a conservation strategy for these cypress species.     

Host-Specific Phenotypic Plasticity of the Turtle Barnacle Chelonibia testudinaria: A Widespread Generalist Rather than a Specialist

Turtle barnacles are common epibionts on marine organisms. Chelonibia testudinaria is specific on marine turtles whereas C. patula is a host generalist, but rarely found on turtles. It has been questioned why C. patula, being abundant on a variety of live substrata, is almost absent from turtles. We evaluated the genetic (mitochondrial COI, 16S and 12S rRNA, and amplified fragment length polymorphism (AFLP)) and morphological differentiation of C. testudinaia and C. patula from different hosts, to determine the mode of adaptation exhibited by Chelonibia species on different hosts. The two taxa demonstrate clear differences in shell morphology and length of 4–6^th cirri, but very similar in arthropodal characters. Moreover, we detected no genetic differentiation in mitochondrial DNA and AFLP analyses. Outlier detection infers insignificant selection across loci investigated. Based on combined morphological and molecular evidence, we proposed that C. testudinaria and C. patula are conspecific, and the two morphs with contrasting shell morphologies and cirral length found on different host are predominantly shaped by developmental plasticity in response to environmental setting on different hosts. Chelonibia testudinaria is, thus, a successful general epibiotic fouler and the phenotypic responses postulated can increase the fitness of the animals when they attach on hosts with contrasting life-styles.     

Development of simple sequence repeat markers and construction of a high-density linkage map of Capsicum annuum

To facilitate marker-assisted breeding and genetic analyses of pepper (Capsicum annuum), we developed non-redundant 2- or 3-base simple sequence repeat (SSR) markers from enriched C. annuum genomic libraries and from C. annuum cDNA sequences in public databases. The SSR-enriched libraries were constructed using combinations of three restriction enzymes (AluI, HaeIII, and RsaI) and two biotinylated oligonucleotides [b(GA)₁₅ and b(CA)₁₅]. Ultimately, we obtained 1,736 genomic SSR markers and 1,344 cDNA-derived SSR markers from 6,528 clones and 13,003 sequences, respectively. We mapped 597 markers, including 265 of the newly developed SSR markers, onto a linkage map by using doubled-haploid (DH) lines derived from an intraspecific cross of two pure lines of C. annuum (K9-11 × MZC-180). The map, designated as the KL-DH map, consisted of 12 linkage groups. The map covered a genetic distance of 2,028 cM, and the average distance between markers was less than 4 cM. The frame structure of the KL-DH map was compared with the published standard conserved ortholog set II (COSII) map, which was derived from an interspecific F₂ population (C. frutescens × C. annuum), by using tomato (Solanum lycopersicum) chromosomal sequences to bridge the two maps. The intraspecific KL-DH map constructed in this study and the interspecific COSII map were similar in map length and marker distribution, suggesting that the KL-DH map covers nearly the whole genome of C. annuum.