Newton O. Otecko

Identification of valid reference genes for mRNA and microRNA normalisation in prostate cancer cell lines

RT-qPCR offers high sensitivity, for accurate interpretations of qPCR results however, normalisation using suitable reference genes is fundamental. Androgens can regulate transcriptional expression including reference gene expression in prostate cancer. In this study, we evaluated ten mRNA and six non-protein coding RNA reference genes in five prostate cell lines under varied dihydrotestosterone (DHT) treatments. We validated the effects of DHT-treatments using media containing charcoal-stripped serum prior to DHT stimulation on the test samples by Western blot experiments. Reference gene expression stability was analysed using three programs (geNorm, NormFinder and BestKeeper), and the recommended comprehensive ranking is provided. Our results reveal that ACTB and GAPDH, and miR-16 and miR-1228-3p are the most suitable mRNA and miRNA reference genes across all cell lines, respectively. Considering prostate cancer cell types, ACTB/GAPDH and ACTB/HPRT1 are the most suitable reference gene combinations for mRNA analysis, and miR-16/miR-1228-3p and RNU6-2/RNU43 for miRNA analysis in AR+, and AR− and normal cell lines, respectively. Comparison of relative target gene (PCA3 and miR-141) expression reveals different patterns depending on reference genes used for normalisation. To our knowledge, this is the first report on validation of reference genes under different DHT treatments in prostate cancer cells. This study provides insights for discovery of reliable DHT-regulated genes in prostate cells.

Out of Southern East Asia of the Brown Rat Revealed by Large-Scale Genome Sequencing

The geographic origin and migration of the brown rat (Rattus norvegicus) remain subjects of considerable debate. In this study, we sequenced whole genomes of 110 wild brown rats with a diverse world-wide representation. We reveal that brown rats migrated out of southern East Asia, rather than northern Asia as formerly suggested, into the Middle East and then to Europe and Africa, thousands of years ago. Comparison of genomes from different geographical populations reveals that many genes involved in the immune system experienced positive selection in the wild brown rat.

Genome-wide discovery of long intergenic noncoding RNAs and their epigenetic signatures in the rat

Long intergenic noncoding RNAs (lincRNAs) play a crucial role in many biological processes. The rat is an important model organism in biomedical research. Recent studies have detected rat lincRNA genes from several samples. However, identification of rat lincRNAs using large-scale RNA-seq datasets remains unreported. Herein, using more than 100 billion RNA-seq reads from 59 publications together with RefSeq and UniGene annotated RNAs, we report 39,154 lincRNA transcripts encoded by 19,162 lincRNA genes in the rat. We reveal sequence and expression similarities in lincRNAs of rat, mouse and human. DNA methylation level of lincRNAs is higher than that of protein-coding genes across the transcription start sites (TSSs). And, three lincRNA genes overlap with differential methylation regions (DMRs) which associate with spontaneously hypertensive disease. In addition, there are similar binding trends for three transcription factors (HNF4A, CEBPA and FOXA1) between lincRNA genes and protein-coding genes, indicating that they harbour similar transcription regulatory mechanisms. To date, this is the most comprehensive assessment of lincRNAs in the rat genome. We provide valuable data that will advance lincRNA research using rat as a model.

Olfactory genes in Tibetan wild boar

were assembled in Li et al.1 (total of 216 Gb) to the Duroc pig reference genome (Ssc10.2) and evaluated read depth for the 1,301 reported olfactory receptor genes7. We found that all these genes were supported by Tibetan wild boar sequencing reads, with an average depth of 100.4×, and more than 59% of these genes had sequencing depth greater than the average depth for the genome (~65.8×) (Fig. 1a,b). All these analyses imply that most olfactory receptor genes in Tibetan wild boar. We identified only 581 of these genes (longer than 900 bp), 30 of which were pseudogenes (Table 1). Olfactory receptor genes comprise the largest gene family, and members in each subfamily exhibit high levels of similarity, mainly emanating from segmental duplications7 whose complete assembly via WGSA is problematic. In our analysis, we unsurprisingly found a remarkably smaller number of segmental duplications in Tibetan wild boar than in Duroc pig (Table 1 and Supplementary Table 1). The Duroc pig genome was assembled by combining both BAC and WGSA approaches8, hence representing a more comprehensive strategy. This analysis therefore demonstrates the possibility that the contraction in olfactory receptor genes observed in Tibetan wild boar may be a property of methodological difference. To verify this, we further studied olfactory receptor genes in the genome of Wuzhishan pig, a non-high-altitude pig9. Its genome was assembled by WGSA, similarly to the Tibetan wild boar genome. We interestingly observed a contraction of both olfactory receptor genes and segmental duplications in Wuzhishan pig, as was also observed in Tibetan wild boar (Table 1 and Supplementary Table 1). In addition, we mapped the paired-end sequencing reads from Tibetan wild boar that To the Editor: In a previous study, Li et al.1 performed a genome sequencing and comparative analysis for Tibetan wild boars, which provided new insight into the genetic landscape for the high-altitude adaptation of this species. In their study, they performed de novo assembly with 131× coverage of the genome from a female Tibetan wild boar employing a whole-genome shotgun sequencing and assembly (WGSA) strategy with the SOAPdenovo program2. They reported an interesting finding of contraction of olfactory receptor genes in Tibetan wild boar and inferred that it was an adaptation attributable to the high-altitude environment with limited food variety. They also cited lower barometric pressure and lower humidity, which tend to decrease the capacity of air to carry odorants bound to vapor molecules, as well as lower temperature, which slows the diffusion of odorous molecules, as contributing factors. Their contextualization and explanation of this finding gives their conclusion quite some plausibility. However, in stark contrast, Qiu et al.3 demonstrated an expansion of olfactory receptor genes in yak (native to and the symbol of Tibet) in comparison to cattle. Two additional studies on the ground tit genome assembly via WGSA4,5 reported contradictory findings about the evolution of olfactory receptor genes in high altitude. Thus far, this discrepancy remains unresolved and unexplained. It is known that de novo genome assembly approaches based on WGSA perform poorly in assembling homogenous regions, such as segmental duplications and repeats6. We therefore postulated that the apparent contraction of olfactory receptor genes in Tibetan wild boar may be an illusion caused by limitations of WGSA and not an actual adaptation to high altitude. To test our speculation, we used 1,301 olfactory receptor genes (longer than 900 bp) reported by Nguyen et al.7 in Duroc pig8 (the reference genome of Sus scrofa, Ssc10.2) as queries to search olfactory receptor genes against the genome assembly of Tibetan wild boar (Supplementary Note). Consistent with the result of Li et al.1, we observed a contraction in Olfactory genes in Tibetan wild boar

Whole-Genome Sequencing of African Dogs Provides Insights into Adaptations against Tropical Parasites

&NA; Natural selection in domestic dogs is of great interest in evolutionary biology since dogs have migrated to every inhabited continent of the world alongside humans, and adapted to diverse environments. Here, we explored their demographic history and genetic basis of adaptation to the tropical African environment using whole genome analyses of 19 African indigenous dogs from Nigeria. Demographic analysis suggests that the ancestors of these dogs migrated into Africa from Eurasia 14,000 years ago and underwent a severe founder effect before population expansion. Admixture analysis further reveals that African dog genomes contain about 1.88–3.50% introgression from African golden wolves (Canis anthus). Population genetic analysis identifies 50 positively selected genes linked with immunity, angiogenesis, ultraviolet protection, as well as insulin secretion and sensitivity that may contribute to adaptation to tropical conditions. One of the positively selected genes, adhesion G protein‐coupled receptor E1 (ADGRE1), has also been found to be association with severe malaria resistance in African human populations. Functional assessments showed that ADGRE1 provides protective host defense against Plasmodium infections. This result, together with the fact that the inflammatory response to canine babesiosis is similar to complicated falciparum malaria in humans, support the dogs as a model for the study of malaria control and treatment.

Rapid Evolution of Genes Involved in Learning and Energy Metabolism for Domestication of the Laboratory Rat

The laboratory rat, widely used in biomedical research, is domesticated from wild brown rat. The origin and genetic mechanism underlying domestication of the laboratory rat remain largely elusive. In the present study, large scale genomes supported a single origin for the laboratory rat, possibly from a sister group to wild rats from Europe/Africa/Middle East. Genomic and transcriptomic analyses uncovered many artificially selected genes (e.g., FOXP2, B3GAT1, and CLOCK) involved in the nervous system. These genes associate with learning ability and regulation of circadian rhythm, which likely enabled the successful domestication of the laboratory rat. Particularly, many genes, including mitochondrial genes responsible for energy metabolism, displayed a substantially increased expression in the brain of laboratory rats compared with wild rats. Our findings demystify the origin and evolution of this model animal, and provide insight into the process of its domestication.

An Evolutionary Genomic Perspective on the Breeding of Dwarf Chickens

The evolutionary history for dwarfism in chickens remains an enigma. Herein, we explore the evolution of the Serama, the smallest breed of chicken. Leveraging comparative population genomics, analyses identify several genes that are potentially associated with the growth and development of bones and muscles. These genes, and in particular both POU1F1 and IGF1, are under strong positive selection. Three allopatric dwarf bantams (Serama, Yuanbao, and Daweishan) with different breeding-histories, form distinct clusters and exhibit unique population structures. Parallel genetic mechanisms underlay their variation in body size. These findings provide insights into the multiple and complex pathways, depending on genomic variation, that chicken can take in response to aviculture selection for dwarfism.

PigVar: a database of pig variations and positive selection signatures

Abstract Pigs are excellent large-animal models for medical research and a promising organ donor source for transplant patients. Next-generation sequencing technology has yielded a dramatic increase in the volume of genomic data for pigs. However, the limited amount of variation data provided by dbSNP, and non-congruent criteria used for calling variation, present considerable hindrances to the utility of this data. We used a uniform pipeline, based on GATK, to identify non-redundant, high-quality, whole-genome SNPs from 280 pigs and 6 outgroup species. A total of 64.6 million SNPs were identified in 280 pigs and 36.8 million in the outgroups. We then used LUMPY to identify a total of 7 236 813 structural variations (SVs) in 211 pigs. Positively selected loci were identified through five statistical tests of different evolutionary attributes of the SNPs. Combining the non-redundant variations and the evolutionary selective scores, we built the first pig-specific variation database, PigVar (http://www.ibiomedical.net/pigvar/), which is a web-based open-access resource. PigVar collects parameters of the variations including summary lists of the locations of the variations within protein-coding and long intergenic non-coding RNA (lincRNA) genes, whether the SNPs are synonymous or non-synonymous, their ancestral and derived states, geographic sampling locations, as well as breed information. The PigVar database will be kept operational and updated to facilitate medical research using the pig as model and agricultural research including pig breeding. Database URL: http://www.ibiomedical.net/pigvar/

Annotating long intergenic non-coding RNAs under artificial selection during chicken domestication

BackgroundNumerous biological functions of long intergenic non-coding RNAs (lincRNAs) have been identified. However, the contribution of lincRNAs to the domestication process has remained elusive. Following domestication from their wild ancestors, animals display substantial changes in many phenotypic traits. Therefore, it is possible that diverse molecular drivers play important roles in this process.ResultsWe analyzed 821 transcriptomes in this study and annotated 4754 lincRNA genes in the chicken genome. Our population genomic analysis indicates that 419 lincRNAs potentially evolved during artificial selection related to the domestication of chicken, while a comparative transcriptomic analysis identified 68 lincRNAs that were differentially expressed under different conditions. We also found 47 lincRNAs linked to special phenotypes.ConclusionsOur study provides a comprehensive view of the genome-wide landscape of lincRNAs in chicken. This will promote a better understanding of the roles of lincRNAs in domestication, and the genetic mechanisms associated with the artificial selection of domestic animals.

Identifying molecular signatures of hypoxia adaptation from sex chromosomes: A case for Tibetan Mastiff based on analyses of X chromosome.

Genome-wide studies on high-altitude adaptation have received increased attention as a classical case of organismal evolution under extreme environment. However, the current genetic understanding of high-altitude adaptation emanated mainly from autosomal analyses. Only a few earlier genomic studies paid attention to the allosome. In this study, we performed an intensive scan of the X chromosome of public genomic data generated from Tibetan Mastiff (TM) and five other dog populations for indications of high-altitude adaptation. We identified five genes showing signatures of selection on the X chromosome. Notable among these genes was angiomotin (AMOT), which is related to the process of angiogenesis. We sampled additional 11 dog populations (175 individuals in total) at continuous altitudes in China from 300 to 4,000 meters to validate and test the association between the haplotype frequency of AMOT gene and altitude adaptation. The results suggest that AMOT gene may be a notable candidate gene for the adaptation of TM to high-altitude hypoxic conditions. Our study shows that X chromosome deserves consideration in future studies of adaptive evolution.