Clonal Genotypes Research Articles

In-depth genome analyses of viruses from vaccine-derived rabies cases and corresponding live-attenuated oral rabies vaccines

Live-attenuated rabies virus strains such as those derived from the field isolate Street Alabama Dufferin (SAD) have been used extensively and very effectively as oral rabies vaccines for the control of fox rabies in both Europe and Canada. Although these vaccines are safe, some cases of vaccine-derived rabies have been detected during rabies surveillance accompanying these campaigns. In recent analysis it was shown that some commercial SAD vaccines consist of diverse viral populations, rather than clonal genotypes. For cases of vaccine-derived rabies, only consensus sequence data have been available to date and information concerning their population diversity was thus lacking.In our study, we used high-throughput sequencing to analyze 11 cases of vaccine-derived rabies, and compared their viral population diversity to the related oral rabies vaccines using pairwise Manhattan distances. This extensive deep sequencing analysis of vaccine-derived rabies cases observed during oral vaccination programs provided deeper insights into the effect of accidental in vivo replication of genetically diverse vaccine strains in the central nervous system of target and non-target species under field conditions. The viral population in vaccine-derived cases appeared to be clonal in contrast to their parental vaccines. The change from a state of high population diversity present in the vaccine batches to a clonal genotype in the affected animal may indicate the presence of a strong bottleneck during infection. In conclusion, it is very likely that these few cases are the consequence of host factors and not the result of the selection of a more virulent genotype. Furthermore, this type of vaccine-derived rabies leads to the selection of clonal genotypes and the selected variants were genetically very similar to potent SAD vaccines that have undergone a history of in vitro selection.

Environmental uniformity, site quality and tree competition interact to determine stand productivity of clonal Eucalyptus

Abstract The major determinants of productivity in Eucalyptus plantations include the site productive capacity, local environmental uniformity, and the tree genetics, which impact their growth potential and competitiveness. While these factors have received a lot of attention individually, less attention has been given to the relative importance of these factors and the interactions between them. We examined these factors and their interactions using eight experiments located along a gradient in site productivity, consisting of 54 clonal genotypes (Eucalyptus urophylla × E. grandis hybrids) at two plant spacings (3 × 3.33 and 3 × 2 m). Stand productivity was quantified as the mean annual increment at age 6 years (m3 ha−1 year−1). Site productive capacity was represented using a site index based on mean dominant height at age 6 years. Environmental uniformity was calculated from within-plot variability in growth of very young stands using residual spatial autocorrelation. The competitive ability of a given phenotype was calculated as the percent loss in potential growth due to neighbor competition. Productivity increased with all variables but site quality had the greatest effect, followed by environmental uniformity and then competitive ability, with corresponding changes in productivity of about 30, 20 and 10 m3 ha−1 year−1, respectively, across the range of the given variable. With higher environmental heterogeneity, competition among genotypes increased, especially at dense spacing, showing that in such situations, highly competitive genotypes may display productivities similar to the less competitive genotypes in uniform environments. Experimental plots distributed across site quality and uniformity gradients provide opportunity for greater genetic differentiation than plots covering more homogeneous environments, reinforcing the recommendation of establishing genetic tests in locations with extreme site qualities. Our work suggests that the G × E interaction is linked to site quality and, to a lesser degree, to the local environmental uniformity. This work shows the value of adopting environmental gradient-based approaches in tree genetic testing and clone recommendation as a way to more accurately match genotypes to specific sites.

Rooting of cuttings of selected Diospyros virginiana clonal rootstocks and bud growth in rooted cuttings

Abstract Diospyros virginiana is a common rootstock in several countries growing commercial plantations of persimmon. However, trees grafted on this rootstock, which is exclusively propagated from seeds, are not uniform in both vegetative and reproductive growth. The aim of the present research was to study the effect of various factors on the rooting of D. virginiana cuttings, in order to develop an efficient rooting procedure for selected clonal rootstocks. The clone genotype had a significant effect on the rooting rate, which ranged from zero to close to 100% in the various clones examined. Cuttings collected from different positions on the mother plant shoots did not differ significantly in their rooting rate, suggesting that juvenility does not play an important role in rooting of D. virginiana cuttings. Collecting cuttings at the early stage of the growing season yielded the highest rooting rate, which gradually decreased as the growing season progressed. Using three to 4-node cuttings and treatment with 6000 mg L−1 indole-3-butyric acid (IBA) by quick-dip in solution led to a relatively high rooting rate. Anatomical analysis demonstrated that adventitious root primordia were initiated at the phloem side of the fascicular cambium, with no relationship to the callus tissue that was formed on the cut surface of the cutting base. Out of the examined clones, rooted cuttings of only one clone exhibited a high rate of bud growth, a phenomenon which was also found to be genotype-dependent. Increasing the applied IBA concentration to cuttings of a clone with a high bud growth rate inhibited bud growth, whereas increasing the length of cuttings of clones with a low bud growth rate promoted bud growth. These observations suggest that the IBA applied to promote rooting is transported acropetally and inhibits bud growth. No cutting mortality was observed during rooting and above 90% of the rotted cuttings which exhibited bud growth survived after transplanting into the growing pots.

Widespread generalist clones are associated with range and niche expansion in allopolyploids of Pacific Northwest Hawthorns (Crataegus L.).

Range and niche expansion are commonly associated with transitions to asexuality, polyploidy and hybridity (allopolyploidy) in plants. The ability of asexual polyploids to colonize novel habitats may be due to widespread generalist clones, multiple ecologically specialized clones, or may be a neutral by-product of multiple, independent origins of asexual polyploids throughout the range. We have quantified niche size and divergence for hawthorns of the Pacific Northwest using data from herbarium vouchers with known cytotypes. We find that all polyploid niches diverge from that of the diploid range, and allopolyploids have the broadest niches. Allotetraploids have the largest niche and the widest geographic distribution. We then assessed the genetic mechanism of range expansion by surveying the ecological and geographic distribution of genotypes within each cytotype from sites in which fine-scale habitat assessments were completed. We find no isolation by either geographic or ecological distance in allopolyploids, suggesting high dispersal and colonization ability. In contrast, autotriploids and diploids show patterns of isolation by geographic distance. We also compared the geographic and ecological distributions of clonal genotypes with those of randomly drawn sites of the most widespread cytotype. We found that most clones are geographically widespread and occur in a variety of habitats. We interpret these findings to suggest that patterns of range and niche expansion in Pacific Northwest Hawthorns may stem from these widespread, ecologically generalist clones of hybrid origin.

A modified multilocus sequence typing protocol to genotype Kingella kingae from oropharyngeal swabs without bacterial isolation

BackgroundOutbreaks of Kingella kingae infection are an emerging public health concern among daycare attendees carrying epidemic clones in the oropharynx. However, genotyping of such epidemic clones from affected cases is limited by the low performance of current methods to detect K. kingae from blood samples and lack of specimens available from infected sites. We aimed at developing a modified multilocus sequence typing (MLST) method to genotype K. kingae strains from oropharyngeal samples without prior culture. We designed in silico MLST primers specific for K. kingae by aligning whole nucleotide sequences of abcZ, adk, aroE, cpn60, recA, and gdh/zwf genes from closely related species belonging to the Kingella and Neisseria genera. We tested our modified MLST protocol on all Kingella species and N. meningitidis, as well as 11 oropharyngeal samples from young children with sporadic (n = 10) or epidemic (n = 1) K. kingae infection.ResultsWe detected K. kingae-specific amplicons in the 11 oropharyngeal samples, corresponding to sequence-type 6 (ST-6) in 6 children including the epidemic cases, ST-25 in 2 children, and 3 possible novel STs (ST-67, ST-68, and ST-69). No amplicon was obtained from other Kingella species and N. meningitidis.ConclusionsWe herein developed a specific MLST protocol that enables genotyping of K. kingae by MLST directly from oropharyngeal samples. This discriminatory tool, with which we identified the first K. kingae outbreak caused by ST-6 in Europe, may be used in further epidemiological investigations.

Genetic diversity and antibiotic susceptibility of Staphylococcus aureus isolates from wild boars.

We here report the occurrence of S. aureus in wild boars and characterize isolates genotypically and phenotypically in order to get knowledge about the occurrence of clonal lineages and genotypes in free-living wild animals. Forty-one S. aureus isolates obtained from 111 wild boars hunted in Lower Saxony, Germany, were investigated and compared to human and livestock isolates. The S. aureus belonged to multilocus sequence types ST1, ST7, ST30, ST133, ST425, ST804, ST890 and to the new ST3237, ST3238, ST3255 and ST3369. The livestock associated CC398-MRSA lineage, however, was not found. In addition to well-known spa types, the new types t14999, t15000, t15001 and t15002 were detected. Macrorestriction analysis revealed a variety of different SmaI fragment patterns. Most isolates were susceptible to all antimicrobials tested, including methicillin, and resistance was detected only to ampicillin, penicillin and erythromycin. PCR analysis confirmed the presence of staphylococcal enterotoxin genes (seh) in all t127-ST1 isolates. A high degree of genetic diversity was detected with many spa types and clonal lineages previously reported in humans and livestock animals.

Segregation of Hydroxycinnamic Acid Esters Mediating Sweetpotato Weevil Resistance in Storage Roots of Sweetpotato.

Resistance to sweetpotato weevils (Cylas spp.) has been identified in several sweetpotato (Ipomoea batatas) landraces from East Africa and shown to be conferred by hydroxycinnamic acids that occur on the surface of storage roots. The segregation of resistance in this crop is unknown and could be monitored using these chemical traits as markers for resistance in F1 offspring from breeding programs. For the first time in a segregating population, we quantified the plant chemicals that confer resistance and evaluated levels of insect colonization of the same progeny in field and laboratory studies. We used a bi-parental mapping population of 287 progenies from a cross between I. batatas ‘New Kawogo,’ a weevil resistant Ugandan landrace and I. batatas ‘Beauregard’ a North American orange-fleshed and weevil susceptible cultivar. The progenies were evaluated for resistance to sweetpotato weevil, Cylas puncticollis at three field locations that varied climatically and across two seasons to determine how environment and location influenced resistance. To augment our field open-choice resistance screening, each clone was also evaluated in a no choice experiment with weevils reared in the laboratory. Chemical analysis was used to determine whether differences in resistance to weevils were associated with plant compounds previously identified as conferring resistance. We established linkage between field and laboratory resistance to Cylas spp. and sweetpotato root chemistry. The data also showed that resistance in sweetpotato was mediated by root chemicals in most but not all cases. Multi-location trials especially from Serere data provided evidence that the hydroxycinnamic acid esters are produced constitutively within the plants in different clonal genotypes and that the ecological interaction of these chemicals in sweetpotato with weevils confers resistance. Our data suggest that these chemical traits are controlled quantitatively and that ultimately a knowledge of the genetics of resistance will facilitate management of these traits, enhance our understanding of the mechanistic basis of resistance and speed the development of new sweetpotato varieties with resistance to sweetpotato weevil.

Virulence traits associated with Burkholderia cenocepacia ST856 epidemic strain isolated from cystic fibrosis patients

BackgroundBurkholderia cenocepacia is considered one of the most problematic cystic fibrosis (CF) pathogens. Colonization prevalence in the Serbian CF population is high and virtually exclusively limited to a single highly transmissible clone of B. cenocepacia ST856 which is positive for both the B. cepacia epidemic strain marker (BCESM) and cable pilin, and is closely related to the epidemic strain CZ1 (ST32).MethodsBiofilm formation for 182 isolates, and adhesion to components of the host extracellular matrix, proteolytic activity, mucoidy and motility of selected ST856 representatives, as well as B. cenocepacia ST858 and ST859, and B. stabilis ST857, novel STs isolated from Serbian CF patients, were investigated in this study. The presence of the cepI, cepR, fliG, llpE, wbiI, and bcscV genes was analyzed.ResultsBiofilm-formation ability of analyzed strains was poor under standard laboratory conditions, but changed in stress conditions (cold stress) and conditions that mimic CF milieu (increased CO2). All strains expressed ability to bind to collagen and fibronectin albeit with different intensity. Representatives of ST856 exhibited gelatinase activity. ST858, ST859 and 9/11 of ST856 genotypes were positive for swimming and twitching motility whereas ST857 was non-motile. Mucoidy was demonstrated in all ST856 genotypes, ST857 was semi-mucoid, and ST858 and ST859 were non-mucoid. Molecular analysis for major virulence factors revealed that ST856 and ST857 carried the six analyzed genes, while ST858 and ST859 were negative for the llpE gene.ConclusionVariations in virulence phenotypes in different genotypes of epidemic B. cenocepacia ST856 clone, in vitro, could be a consequence of diversification driven by pathoadaptation. Diversity of epidemic clone genotypes virulence, could be challenging for accurate diagnosis and treatment, as well as for infection control.

Clonal Complexes and Antimicrobial Susceptibility Profiles of Staphylococcus pseudintermedius Isolates from Dogs in the United States.

Staphylococcus pseudintermedius is the primary cause of canine pyoderma and has been associated with diseases in other animals, including human beings. A high prevalence of methicillin and multidrug resistance has been reported in this bacterium in some geographic regions of the United States. Multilocus sequence type (MLST) 68 was implicated, initially, as the major clonal genotype based on a limited number of samples. The objectives of this study were to determine the population genetics of S. pseudintermedius isolated from a cross-section of the United States using a seven-locus multilocus sequence typing method, to identify clonal complexes (CCs), and to correlate sequence types with antimicrobial susceptibility profiles. A total of 190 S. pseudintermedius with 86 different MLSTs were detected and the constituents of three major CCs of methicillin-resistant S. pseudintermedius (MRSP), CC68, CC71, and CC84, were identified. Different patterns of resistance were associated with each CC. CC71 from the United States had notable differences with CC71 studied on other continents with chloramphenicol, tetracycline, and trimethoprim/sulfamethoxazole resistance. Some isolates with resistance to the broadest range of drugs tested, including that to chloramphenicol, had STs unrelated to the major CCs, suggesting the potential for the emergence of new clonal populations of MRSP that are resistant to most therapeutically useful antimicrobials.

Relevant units of analysis for applied and basic research dealing with neglected transmissible diseases: The predominant clonal evolution model of pathogenic microorganisms.

The predominant clonal evolution (PCE) model seeks to formulate a common population genetics framework for all micropathogens (namely, parasitic protozoa, fungi and yeasts, bacteria, and viruses). It relies on a definition of clonality that is only based on population structure features (namely, strongly restrained genetic recombination). Its clear-cut properties make it of strong interest for applied and basic research, since it permits the definition of stable, clearly delimited units of analysis below the species level: clonal genotypes and discrete genetic subdivisions (“near-clades”). These units of analysis can be used for clinical and epidemiological studies, vaccine and drug design, species description, and evolutionary studies on natural and experimental populations.In this review, the evolutionary and population genetics background of the model will be only briefly mentioned, while considerable emphasis will be given to its practical significance for the study and control of neglected tropical diseases. The goal of the paper is to make this practical usefulness accessible to a broad audience of readers, including scientists who are not evolution specialists, such as epidemiologists, field scientists, and clinicians. For extensive developments about the evolutionary background of the model, see our previous papers [1–9]. Citations of these former articles lead to the many references quoted in them, which cannot be listed again here.

No rest for the laurels: symbiotic invaders cause unprecedented damage to southern USA forests

Laurel wilt is an extraordinarily destructive exotic tree disease in the southeastern United States that involves new-encounter hosts in the Lauraceae, an introduced vector (Xyleborus glabratus) and pathogen symbiont (Raffaelea lauricola). USDA Forest Service Forest Inventory and Analysis data were used to estimate that over 300 million trees of redbay (Persea borbonia sensu lato) have succumbed to the disease since the early 2000s (ca 1/3 of the pre-invasion population). In addition, numerous native shrub and tree species in the family are susceptible and threatened in the Western Hemisphere. Genetic markers were used to test the hypothesis that the vector and pathogen entered North America as a single introduction. With a portion of the cytochrome oxidase I gene, a single X. glabratus haplotype was detected in the USA. Similarly, Amplified Fragment Length Polymorphisms indicated that 95% (54 of 57) of the isolates of R. lauricola that were examined were of a single clonal genotype; only minor variation was detected in three polymorphic isolates. Similar levels of disease developed after swamp bay (P. palustris) was inoculated with each of the four genotypes of R. lauricola. It is proposed that a single founding event is responsible for the laurel wilt epidemic in the United States.

Prevalence and genetic characterization of Toxoplasma gondii in free-range chickens from grocery stores and farms in Maryland, Ohio and Massachusetts, USA

Chickens are considered important in the epidemiology of Toxoplasma gondii. Chicken hearts (n=1185) obtained from grocery stores were tested for T. gondii infection. Antibodies to T. gondii were assayed in fluid removed from the heart cavity using the modified agglutination test (MAT) at 1:5, 1:25, and 1:100 dilutions. MAT antibodies were detected in 222 hearts at 1:5 dilution and 8 hearts at 1:25 dilution, but none were positive at 1:100 dilution. Seropositive (n=230, 19.4%) chicken hearts were bioassayed in mice and seronegative (n=157) chickens were bioassayed in cats. Viable T. gondii was not isolated from any hearts by bioassays in mice. The 2 cats fed 60 and 97 hearts did not excrete T. gondii oocysts. The results indicate a low prevalence of viable T. gondii in chickens from grocery stores. Molecular typing of 23 archived T. gondii strains isolated from free-range chickens from Ohio and Massachusetts using the 10 PCR-RFLP markers including SAG1, SAG2 (5'-3'SAG2 and altSAG2), SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico revealed that seven were ToxoDB PCR-RFLP genotype #1, 11 were genotype #2, one was genotype #3, three were genotype #170, and one was mixed genotype. These results indicate that the clonal genotypes #1 (type II), #2 (type III), and #3 (type II variant) are common in free-range chickens.

English

In the Western Amazon, Brazil, Coffea canephora Pierre is the main species cultivated because it has good adaptation to the climate and soils of the region. Among the factors that limit the yield of this crop, Meloidogyne or root-knot nematode, caused by the root knot nematode, is an aggressive disease present in the state of Rondonia. The aim of this study was to evaluate the reaction of fifteen clones of C. canephora, belonging to the cultivar BRS Ouro Preto, to Meloidogyne incognita (Est I2). Therefore, six seedlings with six months of growth in a nursery from each one of the clones of the cultivar were transferred to pots containing a sterilized substrate in a greenhouse. Following the same design, three clonal genotypes selected from the cultivar IAC Apoatã (C. canephora botanical variety Robusta) served as resistant controls, and seedlings of C. arabica of the Obatã line and tomato (Solanum lycopersicum) cv. Santa Clara (20 days after germination) as susceptible controls, for a total of 120 pots. A 10 ml suspension containing 5000 eggs of M. incognita was inoculated on each plant, and after 150 days, they were evaluated with regards to root fresh weight (FWR), number of galls (NG), number of eggs (NE), and the nematode reproduction factor (RF = final population/initial population). Except for the clone K98M-0061, which exhibited galls (29.17), all the clones of the variety BRS Ouro Preto showed resistance at levels equivalent to those of the controls of the var. Apoatã (RF <1) and good root development. Thus, it can be concluded that the total composition of clones of the variety BRS Ouro Preto is resistant to M. incognita in Rondonia. Key words: Coffee, Meloidogyne, plant selection.

Biomass production and potential water stress increase with planting density in four highly productive clonal Eucalyptus genotypes

The choice of planting density and tree genotype are basic decisions when establishing a forest stand. Understanding the interaction between planting density and genotype, and their relationship with biomass production and potential water stress, is crucial as forest managers are faced with a changing climate. However, few studies have investigated this relationship, especially in areas with highly productive forests. This study aimed to determine the interaction between biomass production and leaf water potential, as a surrogate of potential water stress, in different clonal Eucalyptus genotypes across a range of planting densities. Four clones (two clones of E. urophylla × E. grandis, one clone of E. urophylla, and one clone of E. grandis × E. camaldulensis) and four planting densities (ranging from 591 to 2 949 trees ha−1) were evaluated in an experimental stand in south-eastern Brazil. Biomass production was estimated 2.5 years after planting and predawn (ψpd) and midday (ψmd) leaf water potential were measured 2 and 2.5 years after planting, in February (wet season) and August (dry season) in 2014. For all clones, total stand stemwood biomass production increased and leaf water potential decreased with planting density, and their interaction was significant. Thus, wood biomass at tighter spacings was higher but exhibited lower leaf water potentials, resulting in a trade-off between productivity and potential water stress. These are preliminary findings and still need to be supported by more experimental evidence and repetitions. However, in light of the increased frequency of extreme climate events, silvicultural practices that are tailored to the potential productivity of each region and that result in low potential water stress should be considered.

Influence of low light intensity and soil flooding on cacao physiology

Growth and development of plants frequently are limited by multiple abiotic stresses that occur simultaneously in the environment. ‘Cabruca’ an agroforestry system is a main cropping system invariably adapted for cultivation of cacao in southern Bahia, Brazil. In this system of management, cacao is grown under the shade of native species of the Atlantic Forest. However, growing cacao under the shade of trees is still questionable due to higher incidence of pests and diseases and difficulty of evaporation of excess soil water in high rainfall periods that often causes flooding. The objective of the current study is to evaluate the performance of two clonal genotypes of cacao, contrasting for tolerance to soil flooding (TSA 792 – tolerant and TSH 774 – intolerant), subjected to four levels of light intensity and two water regimes, control and flooded, in order to elucidate the mechanisms of tolerance to these stresses under ‘Cabruca’ environment. All the plants survived the period of flooding, however, with flooding stress visual symptoms like leaf chlorosis, lenticels hypertrophied on the stem base, and decomposition of roots were observed. Obtained results demonstrated morpho-physiological and molecular changes in response to low light intensity and low light intensity×soil flooding interaction. Significant effects (p<0.05) of isolated factors, double interaction and inter- and intra-genotypic differences (p<0.05) were observed for leaf gas exchange, chlorophyll fluorescence, and growth parameters. Differences among light intensities, water regime, plant organ (p<0.001), and for the interactions low light intensity×water regime and water regime×plant organ (p<0.01) were observed with respect to total soluble sugar content. Isolated factors and double and triple interactions (p<0.01) had significant effects on the starch accumulation. Triple interaction was also observed in the expression of genes coding for ADH, LDH and PDC enzymes and for aquaporin PIP1;2. Low light intensity interacts with soil flooding, limiting the damages caused by flooding in cacao plants under lowest light intensity. Flooding was the dominant factor for the most physiological responses observed during the interaction between light intensity and soil flooding whose physiological adjustments were different amoung the clonal cocoa genotypes assessed.

Abstract PR07: Complex sub-clonal populations in colorectal cancer lymph node metastasis

Abstract A number of studies have highlighted the role of sub-clones in tumor progression across multiple cancer types including colorectal cancer (CRC). However, the genetic origin of lymph node (LN) metastasis is not well understood. Given that the prognosis for CRC patients worsens once metastasis occurs, we aimed to better understand the relationship between detected sub-clones in the primary tumor and the LN metastasis. Two or three spatially disparate regions from each of 5 colon cancers were dissected and subjected to next-generation whole exome DNA sequencing (average read depth of 50x) followed by targeted confirmatory deep sequencing (average read depth of 420x). The number of mutations per tumor ranged from 95 to 334 in the 4 non-hypermutated tumors and was 1208 in the single hypermutated tumor. DNA was then isolated from the metastatic areas of two to four matched metastatic LN's from each tumor. Deep-targeted sequencing was also performed on the LN samples targeting the same somatic variant locations identified in the primary tumors. Affymetrix OncoScan SNP arrays were performed on all primary tumor and LN samples to assess copy number (CN) variation and to permit more accurate inference of the cellular frequency of each somatic variant using the computational method PyClone. The PyClone software implements a probabilistic model to estimate the cellular frequency of each mutation in a population of cells using observed alternate allele frequencies as well as copy number and loss of heterozygosity (LOH) information, resulting in a clustering of variants based on the cellular frequency patterns of variants in each sample. Sub-clones are inferred corresponding to the distinct groupings of variants across all tumor and LN samples from a single patient. Additionally, the Clomial software was utilized to complement and validate PyClone results. Clomial implements an expectation-maximization algorithm to infer, given a count of distinct clones expected in a sample, profiles of the variant genotypes of each sample and sample clonal compositions. Generating Clomial profiles at several possible expected clone counts provides an orthogonal measure of clonal genotypes, as well as generating tumor purity estimates and facilitating phylogenetic profiling of variants and clones. CN and LOH differences were identified between different spatial locations in some individual tumors as well as between different LN's from the same tumor. Some LN's contained the same CN changes as a particular spatial region of the matched primary tumor and others contained CN variants that were distinct from the tested regions of the primary tumor. As defined by PyClone, each region in the primary tumor contained multiple (2–13, average 6.1) distinct sub-clones and each LN contained 3 - 13 sub-clones (average 5.9). We demonstrate evidence that individual LN metastasis are comprised of multiple sub-clones from the primary tumor. When there are multiple independent LN metastases in an individual patient, they do not all have the same composition of sub-clones. Some metastatic sub-clones were shared among all of the LN's from the same tumor but there were also sub-clones that were private to a portion of the tumor and to an individual LN. This may be evidence of individual sub-clones metastasizing at different times to different LN's. Further study of the origins of metastasis may help elucidate how to better target it therapeutically. This abstract is also being presented as Poster A10. Citation Format: Karin M. Hardiman, Peter J. Ulintz, Joel K. Greenson, Rong Wu, Eric R. Fearon. Complex sub-clonal populations in colorectal cancer lymph node metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr PR07.

Morphological descriptors and ISSR molecular markers in the evaluation of genetic variability of Tectona grandis genotypes.

This study aimed to evaluate the genetic variability of the teak germplasm bank, using morphological traits and inter-simple sequence repeat molecular markers. Thirty clones were evaluated in a randomized complete block design with three replicates, and each plot was composed of three plants. A joint analysis of quantitative and qualitative variables was performed using the Gower algorithm. Quantitative, qualitative, and molecular variables were analyzed simultaneously using the Ward-MLM procedure. There is genetic variability among the 30 teak genotypes studied, considering the quantitative, qualitative, and molecular variables by the Ward-MLM statistical procedure. Morphological traits used proved to be efficient for the study of genetic variability; however, it was not possible to compose a descriptor table for clonal teak genotypes based on the traits evaluated. The Gower method was efficient in discriminating the groups, demonstrating that the simultaneous analysis of qualitative and quantitative data is feasible and can allow greater efficiency in the knowledge of the variability among teak genotypes. The genotype 22 showed to be the most divergent compared to the other genotypes, except for the cluster of genotypes by the UPGMA method based on the Gower distance obtained by the Ward-MLM procedure, which formed a group with genotypes 9 and 30, in the morphological and molecular analyses and was grouped alone.

Molecular characterization of Toxoplasma gondii in pork meat from different production systems in the Czech Republic

Toxoplasmosis is a major public health issue, due to the presence of Toxoplasma gondii, mainly in pork. The aim of this study was to determine the occurrence of T. gondii in pigs and wild boars bred in different production systems in the Czech Republic using ELISA and qPCR methods. Our results show that T. gondii infection is widespread in pigs and wild boars bred and slaughtered in the Czech Republic and that there is a higher exposure to T. gondii in backyard slaughter operations and organic pig farming, indicating a potential risk for meat consumption. Additionally, genotyping of amplified loci for Type II suggests the presence of one clonal genotype circulating in these animals.

Fine-scale spatial genetic structure of a fungal parasite of coffee scale insects

The entomopathogenic fungus Lecanicillium lecanii persists in a highly dynamic network of habitat patches (i.e., a metapopulation) formed by its primary host, the green coffee scale Coccus viridis. Lecanicillium lecanii is an important biological control of both C. viridis and the coffee rust, Hemileia vastatrix. Successfully managing this biocontrol agent will depend on an increased understanding of the characteristics of its dispersal, as migration between occupied and unoccupied patches is essential for the persistence of this metapopulation. In the present study, we employ a population genetics approach, and show that in our study system, a coffee farm in the Soconusco region of southern Mexico, L. lecanii is characterized by clear spatial genetic structure among plots within the farm but a lack of apparent structure at smaller scales. This is consistent with dispersal dominated by highly localized transport, such as by insects or rain splash, and less dependence on longer distance dispersal such as wind transport. The study site was dominated by a few multi-locus microsatellite genotypes, and their identities and large-scale locations persist across both study years, suggesting that local epizootics (outbreaks) are initiated each wet season by residual propagules from the previous wet season, and not by long-distance transport of propagules from other sites. The index of association, a measure of linkage disequilibrium, indicates that epizootics are primarily driven by asexual, clonal reproduction, which is consistent with the apparent lack of a teleomorph in the study site and the presence of only a single mating type across the site (MAT-1-2-1). Although the same predominant clonal genotypes were found across years, a drastic difference in genotypic diversity was witnessed across two sites between the two years, suggesting that interclonal selection was occurring. In light of the dispersal limitation of L. lecanii, spatial structure may be an essential axis of management to ensure the persistence of L. lecanii and preserve the ecosystem services provided by this versatile biocontrol agent in this and similar coffee farms.

Abstract 2373: Assessing intra-tumor heterogeneity and tracking longitudinal and spatial clonal evolution by next-generation sequencing

Abstract Cancer is a disease driven by genetic and epigenetic alterations that follows Darwinian evolution. Recently, there have been increasing efforts to sequence the tumor from the same patient at multiple time points and/or from multiple spatially separated resections. Different snapshots of the same tumor have proved invaluable for identifying subclonal populations and inferring the tumor's history. We propose a method, Canopy, for estimating the clonal history and for longitudinal and spatial comparison of mutation profiles from one or more samples derived from a single patient. Canopy accounts for normal cell contamination and reconstructs subclonal phylogeny utilizing both somatic copy number alterations (CNAs) and somatic single nucleotide alterations (SNAs). Canopy provides a general mathematical framework that enumerates all possible CNA-SNA phases and temporal orderings. Taking as input the mutant and reference allele frequencies for SNAs, and depth of coverage for CNAs, Canopy gives confidence assessments of all possible configurations of the cancer's clonal evolution. Canopy is applied to three cancer sequencing datasets of varying study design, as well as to an extensive simulation study. On a whole-exome study of a transplantable metastasis model derived from human breast cancer cell line MDA-MB-231, Canopy successfully deconvolutes the mixed cell sublines, using the single cell sublines as ground truth, and identifies DNA signatures that can be prognostic of distant metastasis. On a whole-genome sequencing dataset of the primary tumor and relapse genome of a leukemia patient, Canopy predicts phylogenetic histories in concordance with existing knowledge. On a whole-genome sequencing dataset of the breast cancer tumor and its subsequent metastatic xenograft, Canopy's inferred clonal phylogeny is concordant with genomic markers of major clonal genotype and is confirmed by single-cell sequencing. Finally, through simulations, we explore the effects of various parameters on deconvolution accuracy, and evaluate performance with comparison against existing methods. Collectively, Canopy provides a rigorous foundation for statistical inference on repeated sequencing experiments from evolving populations delineated temporally and spatially. Citation Format: Yuchao Jiang, Yu Qiu, Andy J. Minn, Nancy R. Zhang. Assessing intra-tumor heterogeneity and tracking longitudinal and spatial clonal evolution by next-generation sequencing. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2373.