A Case-Only Genome-wide Association Study of Gender- and Age-specific Risk Markers for Childhood Leukemia

Males and age group 1 to 5 years show a much higher risk for childhood acute lymphoblastic leukemia (ALL). We performed a case-only genome-wide association study (GWAS), using the Illumina Infinium HumanCoreExome Chip, to unmask gender- and age-specific risk variants in 240 non-Hispanic white children with ALL recruited at Texas Children’s Cancer Center, Houston, Texas. Besides statistically most significant results, we also considered results that yielded the highest effect sizes. Existing experimental data and bioinformatic predictions were used to complement results, and to examine the biological significance of statistical results. Our study identified novel risk variants for childhood ALL. The SNP, rs4813720 (RASSF2), showed the statistically most significant gender-specific associations (P < 2 x 10-6). Likewise, rs10505918 (SOX5) yielded the lowest P value (P < 1 x 10-5) for age-specific associations, and also showed the statistically most significant association with age-at-onset (P < 1 x 10-4). Two SNPs, rs12722042 and 12722039, from the HLA-DQA1 region yielded the highest effect sizes (odds ratio (OR) = 15.7; P = 0.002) for gender-specific results, and the SNP, rs17109582 (OR = 12.5; P = 0.006), showed the highest effect size for age-specific results. Sex chromosome variants did not appear to be involved in gender-specific associations. The HLA-DQA1 SNPs belong to DQA1*01:07and confirmed previously reported male-specific association with DQA1*01:07. Twenty one of the SNPs identified as risk markers for gender- or age-specific associations were located in the transcription factor binding sites and 56 SNPs were non-synonymous variants, likely to alter protein function. Although bioinformatic analysis did not implicate a particular mechanism for gender- and age-specific associations, RASSF2 has an estrogen receptor-alpha binding site in its promoter. The unknown mechanisms may be due to lack of interest in gender- and age-specificity in associations. These results provide a foundation for further studies to examine the gender- and age-differential in childhood ALL risk. Following replication and mechanistic studies, risk factors for one gender or age group may have a potential to be used as biomarkers for targeted intervention for prevention and maybe also for treatment.

Analisi di 3 microdelezioni implicate nel disturbo autistico

Nella Tesi viene riportata l’analisi genetica di un campione di 128 famiglie con Disturbo dello Spettro Autistico, tramite il sistema di SNP array “PsychArray” (Illumina ), contenente oltre 500.000 sonde sull’intero genoma. Questi dati sono stati utilizzati per individuare Copy Number Variants (CNVs) rari e rilevanti da un punto di vista clinico. Sono stati quindi selezionati tre CNVs per un ulteriore approfondimento: due microdelezioni già descritte come patologiche (rispettivamente nella regione 1p36.32 e 22q13.33 comprendente il gene SHANK3) sono risultate essere “de novo”, mentre una terza microdelezione nel gene CTNNA3 è ereditata dalla madre. Tutti e tre i CNV sono stati validati tramite Real Time-PCR, definendone i confini. Per quanto riguarda la microdelezione in CTNNA3, poiché difetti di questo gene sono stati implicati nell’autismo con un meccanismo recessivo, è stata anche condotta un’analisi di sequenza di tutti gli esoni del gene negli individui della famiglia interessata, al fine di ricercare eventuali mutazioni puntiformi sull’allele non deleto. Questa analisi non ha individuato nessuna variante potenzialmente dannosa, pertanto il difetto in CTNNA3 non risulta essere la causa principale del fenotipo autistico in questa famiglia, anche se potrebbe avere un ruolo come fattore di suscettibilità.

Targeted sequencing for high-resolution evolutionary analyses following genome duplication in salmonid fish : Proof of concept for key components of the insulin-like growth factor axis

Acknowledgements This study was funded by a Natural Environment Research Council grant (NERC, project code: NBAF704). FML is funded by a NERC Doctoral Training Grant (Project Reference: NE/L50175X/1). RLS was an undergraduate student at the University of Aberdeen and benefitted from financial support from the School of Biological Sciences. DJM is indebted to Dr. Steven Weiss (University of Graz, Austria), Dr. Takashi Yada (National Research Institute of Fisheries Science, Japan), Dr. Robert Devlin (Fisheries and Oceans Canada, Canada), Prof. Samuel Martin (University of Aberdeen, UK), Mr. Neil Lincoln (Environment Agency, UK) and Prof. Colin Adams/Mr. Stuart Wilson (University of Glasgow, UK) for providing salmonid material or assisting with its sampling. We are grateful to staff at the Centre for Genomics Research (University of Liverpool, UK) (i.e. NERC Biomolecular Analysis Facility – Liverpool; NBAF-Liverpool) for performing sequence capture/Illumina sequencing and providing us with details on associated methods that were incorporated into the manuscript. Finally, we are grateful to the organizers of the Society of Experimental Biology Satellite meeting 'Genome-powered perspectives in integrative physiology and evolutionary biology' (held in Prague, July 2015) for inviting us to contribute to this special edition of Marine Genomics and hosting a really stimulating meeting.

Genome-wide association studies in oesophageal adenocarcinoma and Barrett's oesophagus: a large-scale meta-analysis

Background: Esophageal adenocarcinoma (EA) is one of the fastest rising cancers in western countries. Barrett’s Esophagus (BE) is the premalignant precursor of EA. However, only a subset of BE patients develop EA, which complicates the clinical management in the absence of valid predictors. Genetic risk factors for BE and EA are incompletely understood. This study aimed to identify novel genetic risk factors for BE and EA.Methods: Within an international consortium of groups involved in the genetics of BE/EA, we performed the first meta-analysis of all genome-wide association studies (GWAS) available, involving 6,167 BE patients, 4,112 EA patients, and 17,159 representative controls, all of European ancestry, genotyped on Illumina high-density SNP-arrays, collected from four separate studies within North America, Europe, and Australia. Meta-analysis was conducted using the fixed-effects inverse variance-weighting approach. We used the standard genome-wide significant threshold of 5×10-8 for this study. We also conducted an association analysis following reweighting of loci using an approach that investigates annotation enrichment among the genome-wide significant loci. The entire GWAS-data set was also analyzed using bioinformatics approaches including functional annotation databases as well as gene-based and pathway-based methods in order to identify pathophysiologically relevant cellular pathways.Findings: We identified eight new associated risk loci for BE and EA, within or near the CFTR (rs17451754, P=4·8×10-10), MSRA (rs17749155, P=5·2×10-10), BLK (rs10108511, P=2·1×10-9), KHDRBS2 (rs62423175, P=3·0×10-9), TPPP/CEP72 (rs9918259, P=3·2×10-9), TMOD1 (rs7852462, P=1·5×10-8), SATB2 (rs139606545, P=2·0×10-8), and HTR3C/ABCC5 genes (rs9823696, P=1·6×10-8). A further novel risk locus at LPA (rs12207195, posteriori probability=0·925) was identified after re-weighting using significantly enriched annotations. This study thereby doubled the number of known risk loci. The strongest disease pathways identified (P<10-6) belong to muscle cell differentiation and to mesenchyme development/differentiation, which fit with current pathophysiological BE/EA concepts. To our knowledge, this study identified for the first time an EA-specific association (rs9823696, P=1·6×10-8) near HTR3C/ABCC5 which is independent of BE development (P=0·45).Interpretation: The identified disease loci and pathways reveal new insights into the etiology of BE and EA. Furthermore, the EA-specific association at HTR3C/ABCC5 may constitute a novel genetic marker for the prediction of transition from BE to EA. Mutations in CFTR, one of the new risk loci identified in this study, cause cystic fibrosis (CF), the most common recessive disorder in Europeans. Gastroesophageal reflux (GER) belongs to the phenotypic CF-spectrum and represents the main risk factor for BE/EA. Thus, the CFTR locus may trigger a common GER-mediated pathophysiology.

A Validation Study of Vascular Cognitive Impairment Genetics Meta-Analysis Findings in an Independent Collaborative Cohort

Vascular cognitive impairment (VCI), including its severe form, vascular dementia (VaD), is the second most common form of dementia. The genetic etiology of sporadic VCI remains largely unknown. We previously conducted a systematic review and meta-analysis of all published genetic association studies of sporadic VCI prior to 6 July 2012, which demonstrated that APOE (ɛ4, ɛ2) and MTHFR (rs1801133) variants were associated with susceptibility for VCI. De novo genotyping was conducted in a new independent relatively large collaborative European cohort of VaD (nmax = 549) and elderly non-demented samples (nmax = 552). Where available, genotype data derived from Illumina's 610-quad array for 1210 GERAD1 control samples were also included in analyses of genes examined. Associations were tested using the Cochran-Armitage trend test: MTHFR rs1801133 (OR = 1.36, 95% CI 1.16-1.58, p = <0.0001), APOE rs7412 (OR = 0.62, 95% CI 0.42-0.90, p = 0.01), and APOE rs429358 (OR = 1.59, 95% CI 1.17-2.16, p = 0.003). Association was also observed with APOE epsilon alleles; ɛ4 (OR = 1.85, 95% CI 1.35-2.52, p = <0.0001) and ɛ2 (OR = 0.67, 95% CI 0.46-0.98, p = 0.03). Logistic Regression and Bonferroni correction in a subgroup of the cohort adjusted for gender, age, and population maintained the association of APOE rs429358 and ɛ4 allele.

Novel Genetic Determinants of Diabetic Kidney Disease

Diabetic kidney disease (DKD) is a devastating diabetes complication, with known heritability not fully revealed by previous genetics studies. We performed the largest genome-wide association study of type 1 DKD to date, in a 13-cohort consortium of 15,590 individuals of European ancestry genotyped on the Illumina HumanCoreExome Beadchip, which allows exploration of coding variation in addition to genomic markers.

As prior work has shown that different characterizations of the DKD phenotype highlight distinct genetic associations, we investigated a spectrum of DKD definitions based on proteinuria and renal function criteria. Controls were DKD-free after a minimum of 15 years diabetes duration; cases had diabetes for at least 10 years prior to DKD diagnosis. We also performed a quantitative trait analysis of estimated glomerular filtration rate in all participants.

Our top finding was a missense mutation in COL4A3, rs55703767 (Asp326Tyr); the minor allele is common in Europeans (20%) and East Asians (13%) but not Africans (2%). This SNP had a genome-wide significant association with traditionally defined DKD (macroalbuminuria or end-stage renal disease [ESRD], (OR= 0.79, P=1.9×10-9), and a suggestive association with macroalbuminuria (OR= 0.79, P=1.6×10-6) and ESRD (OR= 0.79, P=4.5×10-5) individually. Though its PolyPhen score is 0.3 (benign), this SNP has been implicated as a splice site disruptor.

The COL4A3 gene encodes the alpha 3 subunit of Type IV collagen, the major structural component of basement membranes. Pathogenic mutations in COL4A3 have been identified in thin basement membrane nephropathy, familial focal segmental glomerulosclerosis, and Alport syndrome. A proxy (r2=0.6) for rs55703767 had no significant associations in the CKDGen consortium, suggesting its pathogenicity occurs solely in the setting of hyperglycemia.

By significantly increasing sample size we have discovered a novel locus underlying DKD risk, paving the way for better understanding of pathology, prevention, and treatment.

Genometa - A Fast and Accurate Classifier for Short Metagenomic Shotgun Reads

Metagenomic studies use high-throughput sequence data to investigate microbial communities in situ. However, considerable challenges remain in the analysis of these data, particularly with regard to speed and reliable analysis of microbial species as opposed to higher level taxa such as phyla. We here present Genometa, a computationally undemanding graphical user interface program that enables identification of bacterial species and gene content from datasets generated by inexpensive high-throughput short read sequencing technologies. Our approach was first verified on two simulated metagenomic short read datasets, detecting 100% and 94% of the bacterial species included with few false positives or false negatives. Subsequent comparative benchmarking analysis against three popular metagenomic algorithms on an Illumina human gut dataset revealed Genometa to attribute the most reads to bacteria at species level (i.e. including all strains of that species) and demonstrate similar or better accuracy than the other programs. Lastly, speed was demonstrated to be many times that of BLAST due to the use of modern short read aligners. Our method is highly accurate if bacteria in the sample are represented by genomes in the reference sequence but cannot find species absent from the reference. This method is one of the most user-friendly and resource efficient approaches and is thus feasible for rapidly analysing millions of short reads on a personal computer.

Onkolyyttiset pikornavirukset ja echovirus 7:n käyttö viroterapiassa

Echovirus 7 (E-7) on enterovirus, joka kuuluu pikornaviruksiin, jotka ovat pieniä ja vaipattomia viruksia ja joilla on ikosahedraalinen rakenne. E-7-viruksen positiivissäikeisen ja yksijuosteisen RNA-genomin koko on noin 7500 emäsparia. Se käyttää solupinnan reseptorina DAF-molekyyliä, jota ilmentyy useissa solutyypeissä ja erityisesti syöpäso¬luissa. Onkolyyttiset virukset tuhoavat syöpäsoluja viruksen lyyttisen kierron ansiosta ja kohdistuvat spesifisesti soluihin, jotka ilmentävät viruksen käyttämää reseptoria. Rigvir on onkolyyttinen, elävä, geneettisesti muokkaamaton, soluadaptoitu E-7, joka on Latviassa hyväksytty viruslääkkeeksi melanoomaa vastaan. Työssä tutkittiin, miten Rigvir eroaa muista E-7-isolaateista genomiltaan ja infektiokyvyltään. E-7-viruskannat valittiin kokeisiin virusneutralisaatiotestin perusteella. Työn aikana virukset testattiin myös geneettisellä tyypitystestillä. E-7-virukset (Rigvir, E-7 Wallace ja 7 kliinistä isolaattia) infektoitiin kymmeneen eri solulinjaan ja virusinfektiota ja virusten aiheuttamaa solutuhoa seurattiin mikroskooppisesti ja RT-qPCR:n avulla. Pitkä-PCR-menetelmä kehitettiin virusgenomin kloonaamiseksi geenivektoriin. Illumina MiSeq -NGS-genomisekvensointimenetelmällä sekvensoitiin virus-RNA:ta ja pitkä-PCR-tuotteita, ja tällä pyrittiin näkemään isolaattien välisiä eroja genomitasolla. Rigvir ei eronnut infektiokyvyltään muista E-7-viruksista lukuun ottamatta SW480-paksusuolisyöpäsoluja. Yksikään virusisolaateista ei infektoinut rintasyöpäsoluja (MDA-MB-231 ja MCF-7). Kohdunkaulasyöpäsoluja (HeLa Ohio) infektoivat huonosti kaikki paitsi yksi kliinisistä E-7-isolaateista. Tyypityksessä selvisi, että kolme solukokeissa käytetyistä isolaateista oli E-19-tyyppiä, jotka infektoivat hyvin glioomasoluja. Tutkimuksessa todettiin, että E-7:llä on syöpäsoluja tuhoavia ominaisuuksia, mutta tulokset eivät osoittaneet Rigvirin tuhoavan syöpäsoluja paremmin kuin muut E-7-isolaatit. NGS-sekvensointi ajettiin kerran E-7-kannoista eristetyistä vRNA-paloista, joista kaksi saatiin sopimaan verrokkina käytettyyn Wallace-sekvenssiin. Eroja näkyi varsinkin 5’-pään alueella ja noin 6000 bp vastaavalla alueella. Pitkä-PCR-tuotteista ajettaessa saatiin kaikki näytteet sopimaan verrokkiin, ja eroja näkyi odotetusti 5’-pään alueella. PCR-tuotteet toimivat NGS:ssä paremmin kuin RNA-näytteet, mutta RNA-näytteiden käsittely on huomattavasti helpompaa kuin PCR-näytteiden tuottaminen virus-RNA:sta.

Analyse transcriptomique de l'interaction tripartite "Pseudozyma flocculosa-Blumeria graminis f.sp. hordei-Hordeum vulgare"

Afin d’améliorer nos pratiques agricoles dans le contexte d’une agriculture durable, plusieurs agents de lutte biologique (ALB) ont été développés, testés et sont maintenant utilisés dans le monde pour combattre les pertes de rendements causées par les maladies. Blumeria graminis f. sp. hordei ( Bgh) est l’agent pathogène responsable du blanc de l’orge et peut réduire les rendements de cette culture jusqu’à 40%. Un champignon épiphyte, Pseudozyma flocculosa, a été découvert et identifié en 1987 en association étroite avec le blanc du trèfle. Les chercheurs ont alors remarqué que ce champignon exhibait une forte activité antagoniste contre le blanc en détruisant les structures de l’agent pathogène. Suite à d’autres travaux, il est apparu que ce comportement antagoniste était dirigé contre tous les membres des Erysiphales et semblait lié à la synthèse d’un glycolipide antifongique soit la flocculosine. Toutefois, on n’est toujours pas parvenus à associer l’efficacité de l’ALB avec la production de ce glycolipide. Ces observations suggèrent que d’autres facteurs seraient impliqués lorsque les deux protagonistes, l’ALB et le blanc, sont en contact. L’objectif principal de ce projet était donc de chercher d’autres mécanismes moléculaires pouvant expliquer l’interaction P. flocculosa-blanc et orge, en faisant une analyse transcriptomique complète des trois protagonistes en même temps. L’interaction tripartite a été échantillonnée à différents temps suivant l’inoculation de P. flocculosa sur des feuilles d’orge présentant déjà une intensité de blanc d’environ 50%. Les échantillons de feuilles prélevés ont ensuite été utilisés pour l’extraction de l’ARN qui ont été ensuite transformés en ADNc pour la préparation des librairies. Cinq répliquats ont été effectués pour chaque temps et le tout a été séquencé à l’aide de séquençage par synthèse Illumina HiSeq. Les séquences obtenues (reads) ont ensuite été analysées à l’aide du logiciel CLC Genomics Workbench. Brièvement, les séquences obtenues ont été cartographiées sur les trois génomes de référence. Suite à la cartographie, les analyses d’expression ont été conduites et les gènes exprimés de façon différentielle ont été recherchés. Cette étape a été conduite en portant une attention particulière aux gènes codant pour un groupe de protéines appelées CSEP pour “candidate secreted effector proteins” qui seraient possiblement impliquées dans l’interaction tripartite. Parmi les protéines exprimées de façon différentielle en présence du blanc ou en absence de ce dernier, nous avons pu constater que certaines CSEP étaient fortement exprimées en présence du blanc. Ces résultats sont prometteurs et nous offrent une piste certaine pour l’élucidation des mécanismes impliqués dans cette interaction tripartite.

Annotation of Gyrodactylus salaris transcriptome

Relationship between organisms within an ecosystem is one of the main focuses in the study of ecology and evolution. For instance, host-parasite interactions have long been under close interest of ecology, evolutionary biology and conservation science, due to great variety of strategies and interaction outcomes. The monogenean ecto-parasites consist of a significant portion of flatworms. Gyrodactylus salaris is a monogenean freshwater ecto-parasite of Atlantic salmon (Salmo salar) whose damage can make fish to be prone to further bacterial and fungal infections. G. salaris is the only one parasite whose genome has been studied so far. The RNA-seq data analyzed in this thesis has already been annotated by using LAST. The RNA-seq data was obtained from Illumina sequencing i.e. yielded reads were assembled into 15777 transcripts. Last resulted in annotation of 46% transcripts and remaining were left unknown. This thesis work was started with whole data and annotation process was continued by the use of PANNZER, CDD and InterProScan. This annotation resulted in 56% successfully annotated sequences having parasite specific proteins identified. This thesis represents the first of Monogenean transcriptomic information which gives an important source for further research on this specie. Additionally, comparison of annotation methods interestingly revealed that description and domain based methods perform better than simple similarity search methods. Therefore it is more likely to suggest the use of these tools and databases for functional annotation. These results also emphasize the need for use of multiple methods and databases. It also highlights the need of more genomic information related to G. salaris.

First complete genome sequence of a capsicum chlorosis tospovirus isolate from Australia with an unusually large S RNA intergenic region

The first complete genome sequence of capsicum chlorosis virus (CaCV) from Australia was determined using a combination of Illumina HiSeq RNA and Sanger sequencing technologies. Australian CaCV had a tripartite genome structure like other CaCV isolates. The large (L) RNA was 8913 nucleotides (nt) in length and contained a single open reading frame (ORF) of 8634 nt encoding a predicted RNA-dependent RNA polymerase (RdRp) in the viral-complementary (vc) sense. The medium (M) and small (S) RNA segments were 4846 and 3944 nt in length, respectively, each containing two non-overlapping ORFs in ambisense orientation, separated by intergenic regions (IGR). The M segment contained ORFs encoding the predicted non-structural movement protein (NSm; 927 nt) and precursor of glycoproteins (GP; 3366 nt) in the viral sense (v) and vc strand, respectively, separated by a 449-nt IGR. The S segment coded for the predicted nucleocapsid (N) protein (828 nt) and non-structural suppressor of silencing protein (NSs; 1320 nt) in the vc and v strand, respectively. The S RNA contained an IGR of 1663 nt, being the largest IGR of all CaCV isolates sequenced so far. Comparison of the Australian CaCV genome with complete CaCV genome sequences from other geographic regions showed highest sequence identity with a Taiwanese isolate. Genome sequence comparisons and phylogeny of all available CaCV isolates provided evidence for at least two highly diverged groups of CaCV isolates that may warrant re-classification of AIT-Thailand and CP-China isolates as unique tospoviruses, separate from CaCV.

Quasispecies dynamics and treatment outcome during early hepatitis C infection in a cohort of HIV-infected men

Hepatitis C virus (HCV) is emerging as one of the leading causes of morbidity and mortality in individuals infected with HIV and has overtaken AIDS-defining illnesses as a cause of death in HIV patient populations who have access to highly active antiretroviral therapy. For many years, the clonal analysis was the reference method for investigating viral diversity. In this thesis, a next generation sequencing (NGS) approach was developed using 454 pyrosequencing and Illumina-based technology. A sequencing pipeline was developed using two different NGS approaches, nested PCR, and metagenomics. The pipeline was used to study the viral populations in the sera of HCV-infected patients from a unique cohort of 160 HIV-positive patients with early HCV infection. These pipelines resulted in an improved understanding of HCV quasispecies dynamics, especially regarding studying response to treatment. Low viral diversity at baseline correlated with sustained virological response (SVR) while high viral diversity at baseline was associated with treatment failure. The emergence of new viral strains following treatment failure was most commonly associated with emerging dominance of pre-existing minority variants rather than re-infection. In the new era of direct-acting antivirals, next generation sequencing technologies are the most promising tool for identifying minority variants present in the HCV quasispecies populations at baseline. In this cohort, several mutations conferring resistance were detected in genotype 1a treatment-naïve patients. Further research into the impact of baseline HCV variants on SVR rates should be carried out in this population. A clearer understanding of the properties of viral quasispecies would enable clinicians to make improved treatment choices for their patients.

A transcriptome approach towards understanding the development of ripening capacity in 'Bartlett' pears (Pyrus communis L.)

Background: The capacity of European pear fruit (Pyrus communis L.) to ripen after harvest develops during the final stages of growth on the tree. The objective of this study was to characterize changes in 'Bartlett' pear fruit physico-chemical properties and transcription profiles during fruit maturation leading to attainment of ripening capacity. Results: The softening response of pear fruit held for 14days at 20°C after harvest depended on their maturity. We identified four maturity stages: S1-failed to soften and S2- displayed partial softening (with or without ET-ethylene treatment); S3 - able to soften following ET; and S4 - able to soften without ET. Illumina sequencing and Trinity assembly generated 68,010 unigenes (mean length of 911bp), of which 32.8% were annotated to the RefSeq plant database. Higher numbers of differentially expressed transcripts were recorded in the S3-S4 and S1-S2 transitions (2805 and 2505 unigenes, respectively) than in the S2-S3 transition (2037 unigenes). High expression of genes putatively encoding pectin degradation enzymes in the S1-S2 transition suggests pectic oligomers may be involved as early signals triggering the transition to responsiveness to ethylene in pear fruit. Moreover, the co-expression of these genes with Exps (Expansins) suggests their collaboration in modifying cell wall polysaccharide networks that are required for fruit growth. K-means cluster analysis revealed that auxin signaling associated transcripts were enriched in cluster K6 that showed the highest gene expression at S3. AP2/EREBP (APETALA 2/ethylene response element binding protein) and bHLH (basic helix-loop-helix) transcripts were enriched in all three transition S1-S2, S2-S3, and S3-S4. Several members of Aux/IAA (Auxin/indole-3-acetic acid), ARF (Auxin response factors), and WRKY appeared to play an important role in orchestrating the S2-S3 transition. Conclusions: We identified maturity stages associated with the development of ripening capacity in 'Bartlett' pear, and described the transcription profile of fruit at these stages. Our findings suggest that auxin is essential in regulating the transition of pear fruit from being ethylene-unresponsive (S2) to ethylene-responsive (S3), resulting in fruit softening. The transcriptome will be helpful for future studies about specific developmental pathways regulating the transition to ripening. © 2015 Nham et al.

Characterization, development and multiplexing of microsatellite markers in three commercially exploited reef fish and their application for stock identification

Thirty-four microsatellite loci were isolated from three reef fish species; golden snapper Lutjanus johnii, blackspotted croaker Protonibea diacanthus and grass emperor Lethrinus laticaudis using a next generation sequencing approach. Both IonTorrent single reads and Illumina MiSeq paired-end reads were used, with the latter demonstrating a higher quality of reads than the IonTorrent. From the 1–1.5 million raw reads per species, we successfully obtained 10–13 polymorphic loci for each species, which satisfied stringent design criteria. We developed multiplex panels for the amplification of the golden snapper and the blackspotted croaker loci, as well as post-amplification pooling panels for the grass emperor loci. The microsatellites characterized in this work were tested across three locations of northern Australia. The microsatellites we developed can detect population differentiation across northern Australia and may be used for genetic structure studies and stock identification.

Transcriptome Analysis of Capsicum Chlorosis Virus-Induced Hypersensitive Resistance Response in Bell Capsicum

Background Capsicum chlorosis virus (CaCV) is an emerging pathogen of capsicum, tomato and peanut crops in Australia and South-East Asia. Commercial capsicum cultivars with CaCV resistance are not yet available, but CaCV resistance identified in Capsicum chinense is being introgressed into commercial Bell capsicum. However, our knowledge of the molecular mechanisms leading to the resistance response to CaCV infection is limited. Therefore, transcriptome and expression profiling data provide an important resource to better understand CaCV resistance mechanisms. Methodology/Principal Findings We assembled capsicum transcriptomes and analysed gene expression using Illumina HiSeq platform combined with a tag-based digital gene expression system. Total RNA extracted from CaCV/mock inoculated CaCV resistant (R) and susceptible (S) capsicum at the time point when R line showed a strong hypersensitive response to CaCV infection was used in transcriptome assembly. Gene expression profiles of R and S capsicum in CaCV- and buffer-inoculated conditions were compared. None of the genes were differentially expressed (DE) between R and S cultivars when mock-inoculated, while 2484 genes were DE when inoculated with CaCV. Functional classification revealed that the most highly up-regulated DE genes in R capsicum included pathogenesis-related genes, cell death-associated genes, genes associated with hormone-mediated signalling pathways and genes encoding enzymes involved in synthesis of defense-related secondary metabolites. We selected 15 genes to confirm DE expression levels by real-time quantitative PCR. Conclusion/Significance DE transcript profiling data provided comprehensive gene expression information to gain an understanding of the underlying CaCV resistance mechanisms. Further, we identified candidate CaCV resistance genes in the CaCV-resistant C. annuum x C. chinense breeding line. This knowledge will be useful in future for fine mapping of the CaCV resistance locus and potential genetic engineering of resistance into CaCV-susceptible crops.