Several studies report about intra-specific trait variation of nitrogen-metabolism related traits, such as N(itrogen)-use efficiency, protein content, N-storage and remobilization in barley and related grass species. The goal of this study was to assess the intra-specific genetic diversity present in primary N-metabolism genes of barley and to investigate the associations of the detected haplotype diversity with malting and kernel quality related traits.
Partial sequences of five genes related to N-metabolism in barley (Hordeum vulgare L.) were obtained, i.e. nitrate reductase 1, glutamine synthetase 2, ferredoxin-dependent glutamate synthase, aspartate aminotransferase and asparaginase. Two to five haplotypes in each gene were discovered in a set of 190 various varieties. The development of 33 SNP markers allowed the genotyping of all these barley varieties consisting of spring and winter types. Furthermore, these markers could be mapped in several doubled haploid populations. Cluster analysis based on haplotypes revealed a more uniform pattern of the spring barleys as compared to the winter barleys. Based on linear model approaches associations to several malting and kernel quality traits including soluble N and protein were identified.
A study was conducted to investigate the presence of sequence variation of several genes related to the primary N-metabolism in barley. The detected diversity could be related to particular phenotypic traits. Specific differences between spring and winter barleys most likely reflect different breeding aims. The developed markers can be used as tool for further genetic studies and marker-assisted selection during breeding of barley.
Natural polyploidy has played an important role during the speciation and evolution of vertebrates, including anurans, with more than 55 described cases. The species of the Phyllomedusa burmeisteri group are mostly characterized by having 26 chromosomes, but a karyotype with 52 chromosomes was described in P. tetraploidea. This species was found in sintopy with P. distincta in two localities of São Paulo State (Brazil), where triploid animals also occur, as consequence of natural hybridisation. We analyse the chromosomes of P. distincta, P. tetraploidea, and their triploid hybrids, to enlighten the origin of polyploidy and to obtain some evidence on diploidisation of tetraploid karyotype.
Phyllomedusa distincta was 2n = 2x = 26, whereas P. tetraploidea was 2n = 4x = 52, and the hybrid individuals was 2n = 3x = 39. In meiotic phases, bivalents were observed in the diploid males, whereas both bivalents and tetravalents were observed in the tetraploid males. Univalents, bivalents or trivalents; metaphase II cells carrying variable number of chromosomes; and spermatids were detected in the testis preparations of the triploid males, indicating that the triploids were not completely sterile. In natural and experimental conditions, the triploids cross with the parental species, producing abnormal egg clutches and tadpoles with malformations. The embryos and tadpoles exhibited intraindividual karyotype variability and all of the metaphases contained abnormal constitutions. Multiple NORs, detected by Ag-impregnation and FISH with an rDNA probe, were observed on chromosome 1 in the three karyotypic forms; and, additionally, on chromosome 9 in the diploids, mostly on chromosome 8 in the tetraploids, and on both chromosome 8 and 9 in the triploids. Nevertheless, NOR-bearing chromosome 9 was detected in the tetraploids, and chromosome 9 carried active or inactive NORs in the triploids. C-banding, base-specific fluorochrome stainings with CMA3 and DAPI, FISH with a telomeric probe, and BrdU incorporation in DNA showed nearly equivalent patterns in the karyotypes of P. distincta, P. tetraploidea, and the triploid hybrids.
All the used cytogenetic techniques have provided strong evidence that the process of diploidisation, an essential step for stabilising the selective advantages produced by polyploidisation, is under way in distinct quartets of the tetraploid karyotype.
Polyploidy; Diploidisation; Chromosome banding; FISH; Molecular cytogenetics
Many native Atlantic salmon populations have been invaded by domesticated escapees for three decades or longer. However, thus far, the cumulative level of gene-flow that has occurred from farmed to wild salmon has not been reported for any native Atlantic salmon population. The aim of the present study was to investigate temporal genetic stability in native populations, and, quantify gene-flow from farmed salmon that caused genetic changes where they were observed. This was achieved by genotyping historical and contemporary samples from 20 populations covering all of Norway with recently identified single nucleotide polymorphism markers that are collectively diagnostic for farmed and wild salmon. These analyses were combined with analysis of farmed salmon and implementation of Approximate Bayesian computation based simulations.
Five of the populations displayed statistically significant temporal genetic changes. All five of these populations became more similar to a pool of farmed fish with time, strongly suggesting introgression of farmed fish as the primary cause. The remaining 15 populations displayed weak or non-significant temporal genetic changes. Estimated introgression of farmed fish ranged from 2-47% per population using approximate Bayesian computation. Thus, some populations exhibited high degrees of farmed salmon introgression while others were more or less unaffected. The observed frequency of escapees in each population was moderately correlated with estimated introgression per population R2 = 0.47 P < 0.001. Genetic isolation by distance existed within the historical and contemporary data sets, however, the among-population level of divergence decreased with time.
This is the first study to quantify cumulative introgression of farmed salmon in any native Atlantic salmon population. The estimations demonstrate that the level of introgression has been population-specific, and that the level of introgression is not solely predicted by the frequency of escapees observed in the population. However, some populations have been strongly admixed with farmed salmon, and these data provide policy makers with unique information to address this situation.
Admixture; Aquaculture; Environmental impact; Escapees; Simulation; ABC; Fish farming; Migration
DNA profiling is essential for individual identification. In forensic medicine, the likelihood ratio (LR) is commonly used to identify individuals. The LR is calculated by comparing two hypotheses for the sample DNA: that the sample DNA is identical or related to a reference DNA, and that it is randomly sampled from a population. For multiple-fatality cases, however, identification should be considered as an assignment problem, and a particular sample and reference pair should therefore be compared with other possibilities conditional on the entire dataset.
We developed a new method to compute the probability via permanents of square matrices of nonnegative entries. As the exact permanent is known as a #P-complete problem, we applied the Huber–Law algorithm to approximate the permanents. We performed a computer simulation to evaluate the performance of our method via receiver operating characteristic curve analysis compared with LR under the assumption of a closed incident. Differences between the two methods were well demonstrated when references provided neither obligate alleles nor impossible alleles. The new method exhibited higher sensitivity (0.188 vs. 0.055) at a threshold value of 0.999, at which specificity was 1, and it exhibited higher area under a receiver operating characteristic curve (0.990 vs. 0.959, P = 9.6E-15).
Our method therefore offers a solution for a computationally intensive assignment problem and may be a viable alternative to LR-based identification for closed-incident multiple-fatality cases.
DNA polymorphism; DNA-based identification; Multiple-fatality cases; Permanent of square matrix; Assignment problem
Adaptations to different habitats across the globe and consequent genetic variation within rice have resulted in more than 120,000 diverse accessions including landraces, which are vital genetic resources for agronomic and quality traits. In India the rice landraces of the states West Bengal, Assam, Mizoram, Manipur and Nagaland are worthy candidates for genetic assessment. Keeping the above in view, the present study was conducted with the aim to (i) calculate the genetic distances among the accessions of 83 landraces collected from these states along with 8 check accessions (total 91 accessions) using 23 previously mapped SSR markers and (ii) examine the population structure among the accessions using model-based clustering approach.
Among the 91 accessions, 182 alleles were identified which included 51 rare and 27 null alleles. The average PIC value was 0.7467/marker. The non-aromatic landraces from West Bengal was most diverse with 154 alleles and an average PIC value of 0.8005/marker, followed by the aromatic landraces from West Bengal with 118 alleles and an average PIC value of 0.6524/marker, while the landraces from North East ranked third with 113 alleles and an average PIC value of 0.5745/marker. In the dendrogram distinct clusters consisting of predominantly aromatic landraces and predominantly North East Indian landraces were observed. The non-aromatic landraces from West Bengal were interspersed within these two clusters. The accessions were moderately structured, showing four sub-populations (A-D) with an Fst value of 0.398, 0.364, 0.206 and 0.281, respectively. The assigned clustering of accessions was well in agreement in both distance-based and model-based approaches.
Each of the accessions could be identified unequivocally by the SSR profiles. Genetically the non aromatic landraces from West Bengal were most diverse followed by the aromatic landraces from the same state. The North Eastern accessions ranked third. Further, grouping of accessions based on their agronomic traits may serve as a resource for future studies, leading to the improvement of rice. Moreover in-situ preservation of the landraces is also a means of protection of biodiversity and cultural heritage.
Oryza sativa; Landrace; Genetic diversity; SSR polymorphism; Population structure
Collection of high-quality DNA is essential for molecular epidemiology studies. Methods have been evaluated for optimal DNA collection in studies of adults; however, DNA collection in young children poses additional challenges. Here, we have evaluated predictors of DNA quantity in buccal cells collected for population-based studies of infant leukemia (N = 489 mothers and 392 children) and hepatoblastoma (HB; N = 446 mothers and 412 children) conducted through the Children’s Oncology Group. DNA samples were collected by mail using mouthwash (for mothers and some children) and buccal brush (for children) collection kits and quantified using quantitative real-time PCR. Multivariable linear regression models were used to identify predictors of DNA yield.
Median DNA yield was higher for mothers in both studies compared with their children (14 μg vs. <1 μg). Significant predictors of DNA yield in children included case–control status (β = −0.69, 50% reduction, P = 0.01 for case vs. control children), brush collection type, and season of sample collection. Demographic factors were not strong predictors of DNA yield in mothers or children in this analysis.
The association with seasonality suggests that conditions during transport may influence DNA yield. The low yields observed in most children in these studies highlight the importance of developing alternative methods for DNA collection in younger age groups.
DNA collection; Buccal cells; Pediatric epidemiology
The taxonomic and phylogenetic relationships of the genus Phyllomedusa have been amply discussed. The marked morphological similarities among some species hamper the reliable identification of specimens and may often lead to their incorrect taxonomic classification on the sole basis of morphological traits. Phenotypic variation was observed among populations assigned to either P. azurea or P. hypochondrialis. In order to evaluate whether the variation observed in populations assigned to P. hypochondrialis is related to that in genotypes, a cytogenetic analysis was combined with phylogenetic inferences based on mitochondrial and nuclear sequences.
The inter- and intra-population variation in the external morphology observed among the specimens analyzed in the present study do not reflect the phylogenetic relationships among populations. A monophyletic clade was recovered, grouping all the specimens identified as P. hypochondrialis and specimens assigned P. azurea from Minas Gerais state. This clade is characterized by conserved chromosomal morphology and a common C-banding pattern. Extensive variation in the nucleolar organizing region (NOR) was observed among populations, with four distinct NOR positions being recognized in the karyotypes. Intra-population polymorphism of the additional rDNA clusters observed in specimens from Barreiras, Bahia state, also highlights the marked genomic instability of the rDNA in the genome of this group. Based on the topology obtained in the phylogenetic analyses, the re-evaluation of the taxonomic status of the specimens from the southernmost population known in Brazil is recommended.
The results of this study support the need for a thorough revision of the phenotypic features used to discriminate P. azurea and P. hypochondrialis. The phylogenetic data presented here also contribute to an extension of the geographic range of P. hypochondrialis, which is known to occur in the Amazon basin and neighboring areas of the Cerrado savanna, where it may be sympatric with P. azurea, within contact zones. The misidentification of specimens may have led to inconsistencies in the original definition of the geographic range of P. azurea. The variability observed in the NOR of P. hypochondrialis reinforces the conclusion that these sites represent hotspots of rearrangement. Intraspecific variation in the location of these sites is the result of constant rearrangements that are not detected by classical cytogenetic methods or are traits of an ancestral, polymorphic karyotype, which would not be phylogenetically informative for this group.
Phyllomedusa; Morphological variation; Chromosome; Phylogenetic inference
Periodontal infection (Periodontitis) is a chronic inflammatory disease, which results in the breakdown of the supporting tissues of the teeth. Previous epidemiological studies have suggested that resistance to chronic periodontitis is controlled to some extent by genetic factors of the host. The aim of this study was to determine the phenotypic response of inbred and Collaborative Cross (CC) mouse populations to periodontal bacterial challenge, using an experimental periodontitis model. In this model, mice are co-infected with Porphyromonas gingivalis and Fusobacterium nucleatum, bacterial strains associated with human periodontal disease. Six weeks following the infection, the maxillary jaws were harvested and analyzed for alveolar bone loss relative to uninfected controls, using computerized microtomography (microCT). Initially, four commercial inbred mouse strains were examined to calibrate the procedure and test for gender effects. Subsequently, we applied the same protocol to 23 lines (at inbreeding generations 10–18) from the newly developed mouse genetic reference population, the Collaborative Cross (CC) to determine heritability and genetic variation of control bone volume prior to infection (CBV, naïve bone volume around the teeth of uninfected mice), and residual bone volume (RBV, bone volume after infection) and loss of bone volume (LBV, the difference between CBV and RBV) following infection.
BALB/CJ mice were highly susceptible (P<0.05) whereas DBA/2J, C57BL/6J and A/J mice were resistant. Six lines of the tested CC population were susceptible, whereas the remaining lines were resistant to alveolar bone loss. Gender effects on bone volume were tested across the four inbred and 23 CC lines, and found not to be significant. Based on ANOVA analyses, broad-sense heritabilities were statistically significant and equal to 0.4 for CBV and 0.2 for LBV.
The moderate heritability values indicate that the variation in host susceptibility to the disease is controlled to an appreciable extent by genetic factors. These results strongly support the possibility of using the Collaborative Cross, as well as developing dedicated F2 (resistant x susceptible inbred strains) resource populations, for future dissection of genetic factors in periodontitis.
Periodontal infection; Experimental periodontitis; microCT; Collaborative cross; Genes; Heritability
Penstemon’s unique phenotypic diversity, hardiness, and drought-tolerance give it great potential for the xeric landscaping industry. Molecular markers will accelerate the breeding and domestication of drought tolerant Penstemon cultivars by, creating genetic maps, and clarifying of phylogenetic relationships. Our objectives were to identify and validate interspecific molecular markers from four diverse Penstemon species in order to gain specific insights into the Penstemon genome.
We used a 454 pyrosequencing and GR-RSC (genome reduction using restriction site conservation) to identify homologous loci across four Penstemon species (P. cyananthus, P. davidsonii, P. dissectus, and P. fruticosus) representing three diverse subgenera with considerable genome size variation. From these genomic data, we identified 133 unique interspecific markers containing SSRs and INDELs of which 51 produced viable PCR-based markers. These markers produced simple banding patterns in 90% of the species × marker interactions (~84% were polymorphic). Twelve of the markers were tested across 93, mostly xeric, Penstemon taxa (72 species), of which ~98% produced reproducible marker data. Additionally, we identified an average of one SNP per 2,890 bp per species and one per 97 bp between any two apparent homologous sequences from the four source species. We selected 192 homologous sequences, meeting stringent parameters, to create SNP markers. Of these, 75 demonstrated repeatable polymorphic marker functionality across the four sequence source species. Finally, sequence analysis indicated that repetitive elements were approximately 70% more prevalent in the P. cyananthus genome, the largest genome in the study, than in the smallest genome surveyed (P. dissectus).
We demonstrated the utility of GR-RSC to identify homologous loci across related Penstemon taxa. Though PCR primer regions were conserved across a broadly sampled survey of Penstemon species (93 taxa), DNA sequence within these amplicons (12 SSR/INDEL markers) was highly diverse. With the continued decline in next-generation sequencing costs, it will soon be feasible to use genomic reduction techniques to simultaneously sequence thousands of homologous loci across dozens of Penstemon species. Such efforts will greatly facilitate our understanding of the phylogenetic structure within this important drought tolerant genus. In the interim, this study identified thousands of SNPs and over 50 SSRs/INDELs which should provide a foundation for future Penstemon phylogenetic studies and breeding efforts.
Breeding domesticated Penstemon; Genome reduction; Homologous sequences; LTR retroelements; Plantaginaceae; Pyrosequencing; Repetitive elements
The primary objective of this study is to identify novel copy number variations (CNVs) associated with familial ankylosing spondylitis (AS). A customized genome-wide microarray was designed to detect CNVs and applied to a multiplex AS family with six (6) affected family members. CNVs were detected using the built-in DNA analytics aberration detection method-2 (ADM-2) algorithm. Gene enrichment analysis was performed to observe the segregation. Subsequent validation was performed using real time quantitative fluorescence polymerase reaction (QF-PCR). The frequency of copy number variation for the UGT2B17 gene was then performed on two well-defined AS cohorts. Fisher exact test was performed to quantify the association.
Our family-based analysis revealed ten gene-enriched CNVs that segregate with all six family members affected with AS. Based on the proposed function and the polymorphic nature of the UGT2B17 gene, the UGT2B17 gene CNV was selected for validation using real time QF-PCR with full concordance. The frequency of two copies of the UGT2B17 gene CNV was 0.41 in the Newfoundland AS cases and 0.35 in the Newfoundland controls (OR = 1.26(0.99-1.59); p < 0.05)), whereas the frequency of two (2) copies of the UGT2B17 gene CNV was 0.40 in the Alberta AS cases and 0.39 in the Alberta controls (OR = 1.05(95% CI: 0.83-1.33); p < 0.71)).
A genome-wide microarray interrogation of a large multiplex AS family revealed segregation of the UGT2B17 gene CNV among all affected family members. The association of the UGT2B17 CNV with AS is particularly interesting given the recent association of this CNV with osteoporosis and the proposed function as it encodes a key enzyme that inhibits androgens. However, two copies of the UGT2B17 gene CNV were only marginally significant in a uniplex AS cohort from Newfoundland but not in a uniplex AS cohort from Alberta.
Copy number variants (CNV); UGT2B17; Ankylosing spondylitis; Complex autoimmune disease
The Africanized honey bee is one of the most spectacular invasions in the Americas. African bees escaped from apiaries in Brazil in 1956, spread over Americas and by 1994 they were reported in Puerto Rico. In contrast to other places, the oceanic island conditions in Puerto Rico may mean a single introduction and different dynamics of the resident European and new-coming Africanized bees.
To examine the genetic variation of honey bee feral populations and colonies from different locations in Puerto Rico, we used eight known polymorphic microsatellite loci.
In Puerto Rico, gAHB population does not show any genetic structure (Fst = 0.0783), and is best described as one honey bee population, product of hybridization of AHB and EHB. The genetic variability in this Africanized population was similar to that reported in studies from Texas. We observed that European private allele frequencies are high in all but one locus. This contrasts with mainland Africanized populations, where European allele frequencies are diminished. Two loci with European private alleles, one on Linkage Group 7, known to carry two known defensiveness Quantitative Trait Loci (QTLs), and the other on Linkage Group 1, known to carry three functionally studied genes and 11 candidate genes associated with Varroa resistance mechanisms were respectively, significantly greater or lower in European allele frequency than the other loci with European private alleles.
Genetic structure of Puerto Rico gAHB differs from mainland AHB populations, probably representing evolutionary processes on the island.
Apis mellifera; Honeybee population; Hybridization; Africanized; European bees
Kisspeptins are the peptide products of KISS1 gene, which operate via the G - protein-coupled receptor GPR54. These peptides have emerged as essential upstream regulators of neurons secreting gonadotropin-releasing hormone (GnRH), the major hypothalamic node for the stimulatory control of the hypothalamic–pituitary– gonadal (HPG) axis. The present study detected the polymorphisms of caprine KISS1 gene in three goat breeds and investigated the associations between these genetic markers and litter size.
Three goat breeds (n = 680) were used to detect single nucleotide polymorphisms (SNPs) in the coding regions with their intron–exon boundaries and the proximal flanking regions of KISS1 gene by DNA sequencing and PCR–RFLP. Eleven novel SNPs (g.384G>A, g.1147T>C, g.1417G>A, g.1428_1429delG, g.2124C>T, g.2270C>T, g.2489T>C, g.2510G>A, g.2540C>T, g.3864_3865delCA and g.3885_3886insACCCC) were identified. It was shown that Xinong Saanen and Guanzhong goat breeds were in Hardy-Weinberg disequilibrium at g.384G>A locus (P < 0.05). Both g.2510G>A and g.2540C>T loci were closely linked in Xinong Saanen (SN), Guanzhong (GZ) and Boer (BG) goat breeds (r2 > 0.33). The g.384G>A, g.2489T>C, g.2510G>A and g.2540C>T SNPs were associated with litter size (P<0.05). Individuals with AATTAATT combinative genotype of SN breed (SC) and TTAATT combinative genotype of BG breed (BC) had higher litter size than those with other combinative genotypes in average parity. The results extend the spectrum of genetic variation of the caprine KISS1 gene, which might contribute to goat genetic resources and breeding.
This study explored the genetic polymorphism of KISS1 gene, and indicated that four SNPs may play an important role in litter size. Their genetic mechanism of reproduction in goat breeds should be further investigated. The female goats with SC1 (AATTAATT) and BC7 (TTAATT) had higher litter size than those with other combinative genotypes in average parity and could be used for the development of new breeds of prolific goats. Further research on a large number of animals is required to confirm the link with increased prolificacy in goats.
Combinative genotype; SNP; PCR-RFLP; Candidate gene
Calluna vulgaris is one of the most important landscaping plants produced in Germany. Its enormous economic success is due to the prolonged flower attractiveness of mutants in flower morphology, the so-called bud-bloomers. In this study, we present the first genetic linkage map of C. vulgaris in which we mapped a locus of the economically highly desired trait “flower type”.
The map was constructed in JoinMap 4.1. using 535 AFLP markers from a single mapping population. A large fraction (40%) of markers showed distorted segregation. To test the effect of segregation distortion on linkage estimation, these markers were sorted regarding their segregation ratio and added in groups to the data set. The plausibility of group formation was evaluated by comparison of the “two-way pseudo-testcross” and the “integrated” mapping approach. Furthermore, regression mapping was compared to the multipoint-likelihood algorithm. The majority of maps constructed by different combinations of these methods consisted of eight linkage groups corresponding to the chromosome number of C. vulgaris.
All maps confirmed the independent inheritance of the most important horticultural traits “flower type”, “flower colour”, and “leaf colour”. An AFLP marker for the most important breeding target “flower type” was identified. The presented genetic map of C. vulgaris can now serve as a basis for further molecular marker selection and map-based cloning of the candidate gene encoding the unique flower architecture of C. vulgaris bud-bloomers.
Bud-bloomer; Flower architecture; Linkage map; ML mapping; Molecular marker
Fatty acid composition of oil extracted from peanut (Arachis hypogaea L.) seed is an important quality trait because it may affect the flavor and shelf life of resulting food products. In particular, a high ratio of oleic (C18:1) relative to linoleic (C18:2) fatty acid (O/L ≥ 10) results in a longer shelf life. Previous reports suggest that the high oleic (~80%) trait was controlled by recessive alleles of ahFAD2A and ahFAD2B, the former of which is thought to have a high frequency in US runner- and virginia-type cultivars. Functional mutations, G448A in ahFAD2A and 442insA in ahFAD2B eliminate or knock down desaturase activity and have been demonstrated to produce peanut oil with high O/L ratios. In order to employ marker assisted selection (MAS) to select a high oleic disease resistant peanut and to evaluate genotypic and phenotypic variation, crosses were made between high oleic (~80%) and normal oleic (~50%) peanuts to produce segregating populations.
A total of 539 F2 progenies were randomly selected to empirically determine each ahFAD2 genotype and the resulting fatty acid composition. Five of the six crosses segregated for the high oleic trait in a digenic fashion. The remaining cross was consistent with monogenic segregation because both parental genotypes were fixed for the ahFAD2A mutation. Segregation distortion was significant in ahFAD2A in one cross; however, the remaining crosses showed no distortion. Quantitative analyses revealed that dominance was incomplete for the wild type allele of ahFAD2, and both loci showed significant additive effects. Oleic and linoleic acid displayed five unique phenotypes, based on the number of ahFAD2 mutant alleles. Further, the ahFAD2 loci did exhibit pleiotropic interactions with palmitic (C16:0), oleic (C18:1), linoleic (C18:2) acids and the O/L ratio. Fatty acid levels in these progeny were affected by the parental genotype suggesting that other genes also influence fatty acid composition in peanut. As far as the authors are aware, this is the first study in which all of the nine possible ahFAD2 genotypes were quantitatively measured.
The inheritance of the high oleic trait initially was suggested to be controlled by dominant gene action from two homoeologous genes (ahFAD2A and ahFAD2B) exhibiting complete recessivity. Analyzing the ahFAD2 genotypes and fatty acid compositions of these segregating peanut populations clearly demonstrated that the fatty acid contents are quantitative in nature although much of the variability in the predominant fatty acids (oleic, linoleic, and palmitic) is controlled by only two loci.
Real-time PCR; Gas chromatography; ahFAD2; High oleic peanuts; Fatty acids; Segregation ratios
The Butyrophilin-like (BTNL) proteins are likely to play an important role in inflammation and immune response. Like the B7 protein family, many human and murine BTNL members have been shown to control T lymphocytes response, and polymorphisms in human BTNL2 have been linked to several inflammatory diseases, such as pulmonary sarcoidosis, inflammatory bowel disease and neonatal lupus.
In this study we provide a comprehensive population, genomic and transcriptomic analysis of a 56-kb deletion copy number variant (CNV), located within two segmental duplications of two genes belonging to the BTNL family, namely BTNL8 and BTNL3. We confirm the presence of a novel BTNL8*3 fusion-protein product, and show an influence of the deletion variant on the expression level of several genes involved in immune function, including BTNL9, another member of the same family. Moreover, by genotyping HapMap and human diversity panel (HGDP) samples, we demonstrate a clear difference in the stratification of the BTNL8_BTNL3-del allele frequency between major continental human populations.
Despite tremendous progress in the field of structural variation, rather few CNVs have been functionally characterized so far. Here, we show clear functional consequences of a new deletion CNV (BTNL8_BTNL3-del) with potentially important implication in the human immune system and in inflammatory and proliferative disorders. In addition, the marked population differences found of BTNL8_BTNL3-del frequencies suggest that this deletion CNV might have evolved under positive selection due to environmental conditions in some populations, with potential phenotypic consequences.
The Leporinus genus, belonging to the Anostomidae family, is an interesting model for studies of sex chromosome evolution in fish, particularly because of the presence of heteromorphic sex chromosomes only in some species of the genus. In this study we used W chromosome-derived probes in a series of cross species chromosome painting experiments to try to understand events of sex chromosome evolution in this family.
W chromosome painting probes from Leporinus elongatus, L. macrocephalus and L. obtusidens were hybridized to each others chromosomes. The results showed signals along their W chromosomes and the use of L. elongatus W probe against L. macrocephalus and L. obtusidens also showed signals over the Z chromosome. No signals were observed when the later aforementioned probe was used in hybridization procedures against other four Anostomidae species without sex chromosomes.
Our results demonstrate a common origin of sex chromosomes in L. elongatus, L. macrocephalus and L. obtusidens but suggest that the L. elongatus chromosome system is at a different evolutionary stage. The absence of signals in the species without differentiated sex chromosomes does not exclude the possibility of cryptic sex chromosomes, but they must contain other Leporinus W sequences than those described here.
FISH; Zoo-FISH; Microdissection; Sex chromosome evolution
Dendropsophus is a monophyletic anuran genus with a diploid number of 30 chromosomes as an important synapomorphy. However, the internal phylogenetic relationships of this genus are poorly understood. Interestingly, an intriguing interspecific variation in the telocentric chromosome number has been useful in species identification. To address certain uncertainties related to one of the species groups of Dendropsophus, the D. microcephalus group, we carried out a cytogenetic analysis combined with phylogenetic inferences based on mitochondrial sequences, which aimed to aid in the analysis of chromosomal characters. Populations of Dendropsophus nanus, Dendropsophus walfordi, Dendropsophus sanborni, Dendropsophus jimi and Dendropsophus elianeae, ranging from the extreme south to the north of Brazil, were cytogenetically compared. A mitochondrial region of the ribosomal 12S gene from these populations, as well as from 30 other species of Dendropsophus, was used for the phylogenetic inferences. Phylogenetic relationships were inferred using maximum parsimony and Bayesian analyses.
The species D. nanus and D. walfordi exhibited identical karyotypes (2n = 30; FN = 52), with four pairs of telocentric chromosomes and a NOR located on metacentric chromosome pair 13. In all of the phylogenetic hypotheses, the paraphyly of D. nanus and D. walfordi was inferred. D. sanborni from Botucatu-SP and Torres-RS showed the same karyotype as D. jimi, with 5 pairs of telocentric chromosomes (2n = 30; FN = 50) and a terminal NOR in the long arm of the telocentric chromosome pair 12. Despite their karyotypic similarity, these species were not found to compose a monophyletic group. Finally, the phylogenetic and cytogenetic analyses did not cluster the specimens of D. elianeae according to their geographical occurrence or recognized morphotypes.
We suggest that a taxonomic revision of the taxa D. nanus and D. walfordi is quite necessary. We also observe that the number of telocentric chromosomes is useful to distinguish among valid species in some cases, although it is unchanged in species that are not necessarily closely related phylogenetically. Therefore, inferences based on this chromosomal character must be made with caution; a proper evolutionary analysis of the karyotypic variation in Dendropsophus depends on further characterization of the telocentric chromosomes found in this group.
Chromosome; Phylogeny; Dendropsophus; Anura
Vasa is a member of the DEAD-box protein family that plays an indispensable role in mammalian spermatogenesis, particularly during meiosis. Bovine vasa homology (Bvh) of Bos taurus has been reported, however, its function in bovine testicular tissue remains obscure. This study aimed to reveal the functions of Bvh and to determine whether Bvh is a candidate gene in the regulation of spermatogenesis in bovine, and to illustrate whether its transcription is regulated by alternative splicing and DNA methylation.
Here we report the molecular characterization, alternative splicing pattern, expression and promoter methylation status of Bvh. The full-length coding region of Bvh was 2190 bp, which encodes a 729 amino acid (aa) protein containing nine consensus regions of the DEAD box protein family. Bvh is expressed only in the ovary and testis of adult cattle. Two splice variants were identified and termed Bvh-V4 (2112 bp and 703 aa) and Bvh-V45 (2040 bp and 679 aa). In male cattle, full-length Bvh (Bvh-FL), Bvh-V4 and Bvh-V45 are exclusively expressed in the testes in the ratio of 2.2:1.6:1, respectively. Real-time PCR revealed significantly reduced mRNA expression of Bvh-FL, Bvh-V4 and Bvh-V45 in testes of cattle-yak hybrids, with meiotic arrest compared with cattle and yaks with normal spermatogenesis (P < 0.01). The promoter methylation level of Bvh in the testes of cattle-yak hybrids was significantly greater than in cattle and yaks (P < 0.01).
In the present study, Bvh was isolated and characterized. These data suggest that Bvh functions in bovine spermatogenesis, and that transcription of the gene in testes were regulated by alternative splice and promoter methylation.
Epidemiological studies indicate a substantial excess familial recurrence of non-syndromic Tetralogy of Fallot (TOF), implicating genetic factors that remain largely unknown. The Rho induced kinase 1 gene (ROCK1) is a key component of the planar cell polarity signalling pathway, which plays an important role in normal cardiac development. The aim of this study was to investigate the role of genetic variation in ROCK1 on the risk of TOF.
ROCK1 was sequenced in a discovery cohort of 93 non-syndromic TOF probands to identify rare variants. TagSNPs were selected to capture commoner variation in ROCK1. Novel variants and TagSNPs were genotyped in a discovery cohort of 458 TOF cases and 1331 healthy controls, and positive findings were replicated in a further 209 TOF cases and 1290 healthy controls. Association between genotypes and TOF was assessed using LAMP.
A rare SNP (c.807C > T; rs56085230) discovered by sequencing was associated with TOF risk (p = 0.006) in the discovery cohort. The variant was also significantly associated with the risk of TOF in the replication cohort (p = 0.018). In the combined cohorts the odds ratio for TOF was 2.61 (95% CI 1.58-4.30); p < 0.0001. The minor allele frequency of rs56085230 in the cases was 0.02, and in the controls it was 0.007. The variant accounted for 1% of the population attributable risk (PAR) of TOF. We also found significant association with TOF for an uncommon TagSNP in ROCK1, rs288979 (OR 1.64 [95% CI 1.15-2.30]; p = 1.5x10-5). The minor allele frequency of rs288979 in the controls was 0.043, and the variant accounted for 11% of the PAR of TOF. These association signals were independent of each other, providing additional internal validation of our result.
Low frequency intermediate penetrance (LFIP) variants in the ROCK1 gene predispose to the risk of TOF.
Congenital heart disease; Tetralogy of fallot; Genetics; Planar cell polarity pathway
Gene identification and sequence determination are critical requirements for many biological, genomic, and bioinformatic studies. With the advent of next generation sequencing (NGS) technologies, such determinations are predominantly accomplished in silico for organisms for which the genome is known or for which there exists substantial gene sequence information. Without detailed genomic/gene information, in silico sequence determination is not straightforward, and full coding sequence determination typically involves time- and labor-intensive PCR-based amplification and cloning methods.
An improved method was developed with which to determine full length gene coding sequences in silico using de novo assembly of RNA-Seq data. The scheme improves upon initial contigs through contig-to-gene identification by BLAST nearest–neighbor comparison, and through single-contig refinement by iterative-binning and -assembly of reads. Application of the iterative method produced the gene identification and full coding sequence for 9 of 12 genes and improved the sequence of 3 of the 12 genes targeted by benzimidazole, macrocyclic lactone, and nicotinic agonist classes of anthelminthic drugs in the swine nodular parasite Oesophagostomum dentatum. The approach improved upon the initial optimized assembly with Velvet that only identified full coding sequences for 2 genes.
Our reiterative methodology represents a simplified pipeline with which to determine longer gene sequences in silico from next generation sequence data for any nematode for which detailed genetic/gene information is lacking. The method significantly improved upon an initial Velvet assembly of RNA-Seq data that yielded only 2 full length sequences. The identified coding sequences for the 11 target genes enables further future examinations including: (i) the use of recombinant target protein in functional assays seeking a better understanding of the mechanism of drug resistance, and (ii) seeking comparative genomic and transcriptomic assessments between parasite isolates that exhibit varied drug sensitivities.
Transcriptome; In silico sequence; Nematode; Anthelminthic; Drug resistance
As a well-known epigenomic modification, DNA methylation is found to be common in plants and plays an important role in many biological processes. Relying on the unique feature of methylation-dependent digestion, the family of methylation-requiring restriction-like endonuclease, such as MspJI and its homologs, was suggested for a potential usage in methylation detection.
In this study, we combine MspJI digestion and electrophoretic band selection with next generation high-throughput sequencing technology to detect 5-methylcytosines in Arabidopsis genome. By developing a bioinformatics workflow to attribute the CNNR sites recognized by MspJI to the reference genome, we fulfilled the systematic assessment of this method.
According to the assessment, here we provide the method for generating a detailed map of plant methylome that could be feasible, reliable and economical in methylation investigation.
MspJI; DNA methylation; Arabidopsis
Certain mutations in the Deadend1 (Dnd1) gene are the most potent modifiers of testicular germ cell tumor (TGCT) susceptibility in mice and rats. In the 129 family of mice, the Dnd1Ter mutation significantly increases occurrence of TGCT-affected males. To test the hypothesis that he Dnd1Ter allele is a loss-of-function mutation; we characterized the consequences of a genetically-engineered loss-of-function mutation in mice, and compared these results with those for Dnd1Ter.
We found that intercrossing Dnd1+/KO heterozygotes to generate a complete loss-of-function led to absence of Dnd1KO/KO homozygotes and significantly reduced numbers of Dnd1+/KO heterozygotes. Further crosses showed that Dnd1Ter partially rescues loss of Dnd1KO mice. We also found that loss of a single copy of Dnd1 in Dnd1KO/+ heterozygotes did not affect baseline occurrence of TGCT-affected males and that Dnd1Ter increased TGCT risk regardless whether the alternative allele was loss-of-function (Dnd1KO) or wild-type (Dnd1+). Finally, we found that the action of Dnd1Ter was not limited to testicular cancer, but also significantly increased polyp number and burden in the Apc+/Min model of intestinal polyposis.
These results show that Dnd1 is essential for normal allelic inheritance and that Dnd1Ter has a novel combination of functions that significantly increase risk for both testicular and intestinal cancer.
Testicular cancer; Allelic segregation; Intestinal neoplasia; DND1
Skeletal muscle is one of the most important economic traits in agricultural animals, especially in pigs. In the modern pig industry, lean type pigs have undergone strong artificial selection for muscle growth, which has led to remarkable phenotypic variations compared with fatty type pigs, making these different breeds an ideal model for comparative studies.
Here, we present comprehensive gene expression profiling for the white (longissimus dorsi muscle) and the red (psoas major muscle) skeletal muscles among male and female fatty Rongchang, feral Tibetan and lean Landrace pigs, using a microarray approach. We identified differentially expressed genes that may be associated the phenotypic differences of porcine muscles among the breeds, between the sexes and the anatomical locations. We also used a clustering method to identify sets of functionally coexpressed genes that are linked to different muscle phenotypes. We showed that, compared with the white muscles, which primarily modulate metabolic processes, the red muscles show a tendency to be a risk factor for inflammation and immune-related disorders.
This analysis presents breed-, sex- and anatomical location-specific gene expression profiles and further identified genes that may be associated with the phenotypic differences in porcine muscles among breeds, between the sexes and the anatomical locations.
Gene expression; Microarray; Muscle; Pig
Birth weight (BW) is an economically important trait in beef cattle, and is associated with growth- and stature-related traits and calving difficulty. One region of the cattle genome, located on Bos primigenius taurus chromosome 14 (BTA14), has been previously shown to be associated with stature by multiple independent studies, and contains orthologous genes affecting human height. A genome-wide association study (GWAS) for BW in Brazilian Nellore cattle (Bos primigenius indicus) was performed using estimated breeding values (EBVs) of 654 progeny-tested bulls genotyped for over 777,000 single nucleotide polymorphisms (SNPs).
The most significant SNP (rs133012258, PGC = 1.34 × 10-9), located at BTA14:25376827, explained 4.62% of the variance in BW EBVs. The surrounding 1 Mb region presented high identity with human, pig and mouse autosomes 8, 4 and 4, respectively, and contains the orthologous height genes PLAG1, CHCHD7, MOS, RPS20, LYN, RDHE2 (SDR16C5) and PENK. The region also overlapped 28 quantitative trait loci (QTLs) previously reported in literature by linkage mapping studies in cattle, including QTLs for birth weight, mature height, carcass weight, stature, pre-weaning average daily gain, calving ease, and gestation length.
This study presents the first GWAS applying a high-density SNP panel to identify putative chromosome regions affecting birth weight in Nellore cattle. These results suggest that the QTLs on BTA14 associated with body size in taurine cattle (Bos primigenius taurus) also affect birth weight and size in zebu cattle (Bos primigenius indicus).
GWAS; Birth weight; Bos primigenius indicus; Nellore cattle; Stature
Paspalum (Poaceae) is an important genus of the tribe Paniceae, which includes several species of economic importance for foraging, turf and ornamental purposes, and has a complex taxonomical classification. Because of the widespread interest in several species of this genus, many accessions have been conserved in germplasm banks and distributed throughout various countries around the world, mainly for the purposes of cultivar development and cytogenetic studies. Correct identification of germplasms and quantification of their variability are necessary for the proper development of conservation and breeding programs. Evaluation of microsatellite markers in different species of Paspalum conserved in a germplasm bank allowed assessment of the genetic differences among them and assisted in their proper botanical classification.
Seventeen new polymorphic microsatellites were developed for Paspalum atratum Swallen and Paspalum notatum Flüggé, twelve of which were transferred to 35 Paspalum species and used to evaluate their variability. Variable degrees of polymorphism were observed within the species. Based on distance-based methods and a Bayesian clustering approach, the accessions were divided into three main species groups, two of which corresponded to the previously described Plicatula and Notata Paspalum groups. In more accurate analyses of P. notatum accessions, the genetic variation that was evaluated used thirty simple sequence repeat (SSR) loci and revealed seven distinct genetic groups and a correspondence of these groups to the three botanical varieties of the species (P. notatum var. notatum, P. notatum var. saurae and P. notatum var. latiflorum).
The molecular genetic approach employed in this study was able to distinguish many of the different taxa examined, except for species that belong to the Plicatula group, which has historically been recognized as a highly complex group. Our molecular genetic approach represents a valuable tool for species identification in the initial assessment of germplasm as well as for characterization, conservation and successful species hybridization.
Cross-species amplification; Genetic diversity; Germplasm evaluation; Microsatellite markers; Paspalum botanical varieties