Heterozygosity Rate Research Articles

Genome-wide analysis indicates association between heterozygote advantage and healthy aging in humans

BackgroundGenetic diversity is known to confer survival advantage in many species across the tree of life. Here, we hypothesize that such pattern applies to humans as well and could be a result of higher fitness in individuals with higher genomic heterozygosity.ResultsWe use healthy aging as a proxy for better health and fitness, and observe greater heterozygosity in healthy-aged individuals. Specifically, we find that only common genetic variants show significantly higher excess of heterozygosity in the healthy-aged cohort. Lack of difference in heterozygosity for low-frequency variants or disease-associated variants excludes the possibility of compensation for deleterious recessive alleles as a mechanism. In addition, coding SNPs with the highest excess of heterozygosity in the healthy-aged cohort are enriched in genes involved in extracellular matrix and glycoproteins, a group of genes known to be under long-term balancing selection. We also find that individual heterozygosity rate is a significant predictor of electronic health record (EHR)-based estimates of 10-year survival probability in men but not in women, accounting for several factors including age and ethnicity.ConclusionsOur results demonstrate that the genomic heterozygosity is associated with human healthspan, and that the relationship between higher heterozygosity and healthy aging could be explained by heterozygote advantage. Further characterization of this relationship will have important implications in aging-associated disease risk prediction.

Development of canine X-chromosome inactivation pattern analysis for the detection of cell clonality by incorporating the examination of the SLIT and NTRK-like family member 4 (SLITRK4) gene

X-chromosome inactivation pattern (XCIP) analysis can be used to assess the clonality of cell populations of various origin by distinguishing the methylated X chromosome from the unmethylated X chromosome. In this study, the utility of XCIP analysis was improved by incorporating the examination of AC dinucleotide repeats in SLIT and NTRK-like family member 4 (SLITRK4) gene into the previously reported CAG repeat examination of androgen receptor (AR) gene in dogs. The rate of heterozygosity when both genes were analysed (125/150, 83.3%) was higher than AR gene examination alone (86/150, 57.3%). Blood samples from heterozygous dogs in either AC-1 or AC-2 of SLITRK4 gene were examined for the corrected inactivation allele ratio (CIAR), resulting in the determination of a reference range of CIAR <3.8 in non-neoplastic cell/tissue samples. Using this analytical method, 49% (21/43) of neoplastic tissue samples from dogs showed a CIAR >3.8, indicating the presence of a clonal population. Through the present study, the availability of canine XCIP analysis was improved by incorporating the examination of the SLITRK4 gene, providing a highly useful laboratory examination system for the detection of the clonality of various cell/tissue samples in dogs.

Genome survey sequencing and genetic background characterization of yellow horn based on next-generation sequencing.

Yellowhorn (Xanthoceras sorbifolium Bunge) is an important wood oil tree species, with high ornamental and medicinal value. Nevertheless, genomic information of yellowhorn is currently unavailable. Here, for the first time, we conducted a genome survey of two yellowhorn cultivars, Zhongshi 4 and Zhongshi 9, which had distinct differences on the phenotype and drought resistance, to obtain knowledge on the genomic information by next generation sequencing (NGS). Meanwhile, its genome size was estimated using flow cytometry. As a result, the whole genome survey of Zhongshi 4 and Zhongshi 9 generated 34.40 and 39.55GB sequence data. The genome size of Zhongshi 4 and Zhongshi 9 estimated were about 536.58Mb and 569.52Mb, which were closed to results of flow cytometry. The heterozygosity rates were calculated to be 0.75% and 0.89%, and the repeat rates were 60.08% and 62.00%. These reads were assembled into 1024,373 and 885,404 contigs with a N50 length of 1005bp and 1219bp, respectively, which were further assembled into 714,369 and 686,128 scaffolds with scaffold N50 length of ~ 1963bp and ~ 1938bp, total length of 386,915Kb and 391,904Kb. These results indicated that there was little difference in genome size and complexity among different cultivars. In addition, 63137 and 65271 high-quality genomic simple sequence repeat (SSR) markers in Zhongshi 4 and Zhongshi 9 were generated. We suggest that the technologies combining Illumina and PacBio, assisted by Hi-C and matching assemble software should be used to one of two yellowhorn cultivars genome sequencing. The result will help to design whole genome sequencing strategies for yellowhorn, and provided a large amount of gene resources for further excavation and utilization of yellowhorn.

Joint Estimates of Heterozygosity and Runs of Homozygosity for Modern and Ancient Samples.

Both the total amount and the distribution of heterozygous sites within individual genomes are informative about the genetic diversity of the population they belong to. Detecting true heterozygous sites in ancient genomes is complicated by the generally limited coverage achieved and the presence of post-mortem damage inflating sequencing errors. Additionally, large runs of homozygosity found in the genomes of particularly inbred individuals and of domestic animals can skew estimates of genome-wide heterozygosity rates. Current computational tools aimed at estimating runs of homozygosity and genome-wide heterozygosity levels are generally sensitive to such limitations. Here, we introduce ROHan, a probabilistic method which substantially improves the estimate of heterozygosity rates both genome-wide and for genomic local windows. It combines a local Bayesian model and a Hidden Markov Model at the genome-wide level and can work both on modern and ancient samples. We show that our algorithm outperforms currently available methods for predicting heterozygosity rates for ancient samples. Specifically, ROHan can delineate large runs of homozygosity (at megabase scales) and produce a reliable confidence interval for the genome-wide rate of heterozygosity outside of such regions from modern genomes with a depth of coverage as low as 5–6× and down to 7–8× for ancient samples showing moderate DNA damage. We apply ROHan to a series of modern and ancient genomes previously published and revise available estimates of heterozygosity for humans, chimpanzees and horses.

Genetic Diversity of Benin Cattle Populations Using Microsatellite Markers

The genetic characteristics of cattle breeds of Benin are little known. This study aimed to assess the genetic diversity of major cattle breeds of Benin. For this purpose, blood samples from 179 cattle including 31 Girolando, 30 White Fulani cattle, 31 Borgou, 31 Lagunaire, 43 Somba and 13 crossbreed Azawak x Lagunaire were characterized using 12 microsatellites markers. All used microsatellites were polymorphic within each breed of cattle. The TGLA53 marker presented the highest number of alleles (15) in the Borgou cattle while the lowest number of alleles (2) was found at the INRA 63 locus for crossbreed Azawak x Lagunaire. The observed heterozygosity rates ranged from 0.79 to 0.57 in Girolando and Somba populations respectively. The highest expected heterozygosity rates were observed in the Girolando cattle population (0.79) while the the lowest (0.63) was encountered in Lagunaire and Somba populations. The lowest value of the differentiation indice between population pairs (0.084) was observed between Borgou and White Fulani cattle. Genetic structure analysis clustered Somba and Lagunaire cattle breeds while White Fulani and Borgou cattle breeds were very close. The crossbreed populations (Borgou and Azawak x Lagunaire) were between taurine and zebu. However, taurine were introgressed (20% on average) by the zebu blood. This study is a basic information in undertaking a conservation program and/or for support in the elaboration of a genetic improvement program for local cattle breeds of Benin.

Genetic Analysis of Turkey Common Bean (Phaseolus vulgaris L.) Genotypes by Simple Sequence Repeats Markers

Common bean (Phaseolus vulgaris L.), besides being an agricultural product that can be consumed as fresh vegetable, is a significant legume widely being planted in both Turkey and world. Because of having different usage areas, it is being considered as a valuable plant for human nutrition, trade and in many respects. In this study, we aimed at genetically characterization of the local and registered common bean genotypes and population structure of genotype groups belong to these common bean genotypes in Turkey. For this purpose, total 102 common bean genotypes including 93 local genotypes from 8 provinces, 7 cultivars and 2 reference cultivars were analyzed by 13 fluorescent SSR markers. As the result of the study, it was determined that the total SSR allele number was 192 and the average allele number was 14.8. While it was found that there were no synonymous genotypes, the highest heterozygosity rate was determined in three loci. Factorial correspondence analysis partially demonstrated substructure among common bean genotype groups. Structure analysis showed the same results as the Nm values and the Fst values. In the study, it was observed that SSR markers could be easily used in the molecular studies of common bean germplasm. The obtained results will be able to be used at the conservation, utilization of local common bean genetic resources and at the marker assisted selection studyings.

Genome survey on invasive veined rapa whelk (Rapana venosa) and development of microsatellite loci on large scale

The veined rapa whelk (Rapana venosa) is an economically important gastropod in China, but is considered as an invasive species globally. Only a few studies have examined the R. venosa genome, a genomewide survey is necessary for improving our understanding of the genome structure and size of this organism. Microsatellite markers are powerful tools for characterizing germplasms, genetic diversity and kinship among individuals. The resultant data are applicable to breeding efforts in commercial aquaculture or for understanding invasion mechanisms. Here, we investigated the genome structure of R. venosa on an Illumina Hi-seq platform with ∼92× sequencing depth. We performed a K-mer analysis to estimate genome size, repeat sequences and heterozygosity. Clean reads were de novo assembled for the identification of simple-sequence repeat (SSR) loci that are suitable as markers. The estimated genome size of R. venosa was 2200.07 Mb, with a 1.41% heterozygosity rate and 67.04% repeats. We detected 5,477,450 simple-sequence repeats (SSRs), with 3,400,602 loci present as pure tandem repeats and 2,076,848 as compound motifs. We further selected and characterized 28 polymorphic markers in 78 individuals from Dandong, Laizhou, Weihai and Zhoushan in China. The range of alleles was 2-28 and the observed heterozygosity was 0.2857-0.8676. The data obtained from our genomic survey will aid the design of R. venosa whole-genome sequencing projects and advance the identification of SSR markers. Both these developments are valuable for further studies on ecological, evolutionary and genetic breeding in R. venosa.

Identification of biparental and diploid blastocysts from monopronuclear zygotes with the use of a single-nucleotide polymorphism array

To select normal fertilized diploid blastocysts in patients who had only monopronucleated (1PN) embryos for transfer. Experimental study. University-affiliated center. Couples who were undergoing intracytoplasmic sperm injection treatment and had 1PN blastocysts. In a preliminary test, limited cells of parthenogenetic human embryonic stem cells (phESCs) and normal fertilized blastocysts were analyzed with the use of a low-density single-nucleotide polymorphism (SNP) array to identify the distribution pattern and rate of heterozygosity. In the clinical application, 1PN blastocysts were analyzed with the use of the SNP array. Only diagnosed normal blastocysts were transferred. The diagnosed uniparental blastocysts were validated by imprinted gene expression. Distribution pattern and rate of heterozygosity between parthenogenesis and normal fertilization. In the pretest, phESCs exhibited distinct distribution pattern and lower rate of heterozygosity, compared with normal fertilized blastocysts after SNP analysis. In particular, homozygous hESCs showed a panhomozygosity distribution pattern, hybrid phESCs showed a partial homozygosity distribution pattern, and normal fertilized blastocysts exhibited a panheterozygosity distribution pattern with an average of 20.21% heterozygosity rate; 13.6% was found to be the minimum cutoff to predict normal fertilized samples. In the clinical application, 24 1PN blastocysts were analyzed; 10/24 showed chromosomal abnormalities, 3/24 showed panhomozygosity with 0.45%-0.8% heterozygosity, and 1/24 showed partial homozygosity with 6.54% heterozygosity. The remaining 10 blastocysts, with a panheterozygosity distribution pattern and higher genomic heterozygosity rate, were diagnosed as normal-fertilization diploid embryos; three were transferred and resulted in two healthy newborns. The low-density SNP array might serve as a cost-effective method to identify biparental origin and diploid 1PN blastocysts for transfer.

A 200K SNP chip reveals a novel Pacific salmon louse genotype linked to differential efficacy of emamectin benzoate

Antiparasitic drugs such as emamectin benzoate (EMB) are relied upon to reduce the parasite load, particularly of the sea louse Lepeophtheirus salmonis, on farmed salmon. The decline in EMB treatment efficacy for this purpose is an important issue for salmon producers around the world, and particularly for those in the Atlantic Ocean where widespread EMB tolerance in sea lice is recognized as a significant problem. Salmon farms in the Northeast Pacific Ocean have not historically experienced the same issues with treatment efficacy, possibly due to the relatively large population of endemic salmonid hosts that serve to both redistribute surviving lice and dilute populations potentially under selection by introducing naïve lice to farms. Frequent migration of lice among farmed and wild hosts should limit the effect of farm-specific selection pressures on changes to the overall allele frequencies of sea lice in the Pacific Ocean. A previous study using microsatellites examined L. salmonis oncorhynchi from 10 Pacific locations from wild and farmed hosts and found no population structure. Recently however, a farm population of sea lice was detected where EMB bioassay exposure tolerance was abnormally elevated. In response, we have developed a Pacific louse draft genome that complements the previously-released Atlantic louse sequence. These genomes were combined with whole-genome re-sequencing data to design a highly sensitive 201,279 marker SNP array applicable for both subspecies (90,827 validated Pacific loci; 153,569 validated Atlantic loci). Notably, kmer spectrum analysis of the re-sequenced samples indicated that Pacific lice exhibit a large within-individual heterozygosity rate (average of 1 in every 72 bases) that is markedly higher than that of Atlantic individuals (1 in every 173 bases). The SNP chip was used to produce a high-density map for Atlantic sea louse linkage group 5 that was previously shown to be associated with EMB tolerance in Atlantic lice. Additionally, 478 Pacific louse samples from farmed and wild hosts obtained between 2005 and 2014 were also genotyped on the array. Clustering analysis allowed us to detect the apparent emergence of an otherwise rare genotype at a high frequency among the lice collected from two farms in 2013 that had reported elevated EMB tolerance. This genotype was not observed in louse samples collected from the same farm in 2010, nor in any lice sampled from other locations prior to 2013. However, this genotype was detected at low frequencies in louse samples from farms in two locations reporting elevated EMB tolerance in 2014. These results suggest that a rare genotype present in Pacific lice may be locally expanded in farms after EMB treatment. Supporting this hypothesis, 437 SNPs associated with this genotype were found to be in a region of linkage group 5 that overlaps the region associated with EMB resistance in Atlantic lice. Finally, five of the top diagnostic SNPs within this region were used to screen lice that had been subjected to an EMB survival assay, revealing a significant association between these SNPs and EMB treatment outcome. To our knowledge this work is the first report to identify a genetic link to altered EMB efficacy in L. salmonis in the Pacific Ocean.

Haplotypes inside the beta-globin gene: use as new biomarkers for beta-thalassemia prenatal diagnosis in north of Iran

BackgroundBeta-thalassemia is common in the Mediterranean area as well as the Middle East and India. Official report in Iran revealed the average prevalence rate of carriers about 4%. More than 20 restriction fragment length polymorphisms (RFLPs) are known in the beta-globin gene cluster and used in the prenatal diagnosis (PND) services. Some of these locations may have low allele frequency and are not informative in the prenatal diagnosis. The current study aims to find new haplotypes and polymorphisms with high allele frequency in the local population.MethodsTwo thousand three hundred fifty samples (1,321 male and 1,029 female) from the northern Iran, whom suspected to be the carriers either for alpha or beta thalassemia and referred to the local diagnostic laboratory as a routine services were investigated during five years, (2010–2015). The beta-globin gene was sequenced for all samples.ResultsHeterozygosity for five SNPs in the beta-globin gene was calculated separately. 383 individuals (16.29%) showed no sign of nucleotide change in the beta-globin gene sequence. In total, codon2 (C/T) 31.72%, IVSII-16 (C/G) 31.72%, IVSII-74 (G/T) 54.71%, IVSII-81 (C/T) 19.47%, and IVSII-666 (T/C) 31.72% were seen respectively. Although all five polymorphisms showed reasonably high heterozygosity, IVSII-74 (G/T) [GG wild type (36.5%), G/T (54.71%) and TT (8.8%)] revealed the highest heterozygosity rate. Four combinations of these five SNPs were defined as new haplotypes named M1 to M4. ARMS-PCR also were designed and applied to detect IVSII-74 (G/T) nucleotide position.ConclusionsThis study represents an intragenic polymorphism, IVSII-74, a reliable position with high heterozygosity rates in Iranian population for PND analysis.Trial registrationRetrospectively registered.

Gain of function in the mouse model of a recurrent mutation p53N236S promotes the formation of double minute chromosomes and the oncogenic potential of p19ARF.

The mutation p53N236S (p53S) has been identified as one of the recurrent mutations in human cancers by TCGA database. Our in vitro data revealed the oncogenic gain of function of p53S. To understand the function of p53S in vivo, we generated the p53S knock-in mouse. The p53S/S mice manifested highly invasive lymphomas and metastatic sarcomas with dramatically increased double minute chromosomes. The survival curve, the incidence of tumors and the tumor spectrum of p53S/S mice is very similar to the p53R172H mouse model. The p53S/+ mice showed delayed onset of tumorigenesis and a high metastasis rate (40%) and low loss of heterozygosity rate (2/16). The activation of CDKN2A pathway in p53S/S MEF and tumors, and the accumulation of p19ARF protein in tumor tissues suggested p19ARF might contribute to the accumulation of mutant p53S protein in the tumor and promote tumorigenesis. The high expression of p19ARF correlated with mutant p53 accumulation and tumor progression, suggesting a dual role of p19ARF in tumor promotion or suppression that might depend on the p53 mutation status in tumor cells. The oncogenic gain of function of this recurrent mutation p53S prompts the reconsideration of p53 mutations function that occurs at a low frequency.

Molluscan Genomics: Implications for Biology and Aquaculture

As a result of advances in DNA sequencing technology, molluscan genome research, which initially lagged behind that of many other animal groups, has recently seen a rapid succession of decoded genomes. Since molluscs are highly divergent, the subjects of genome projects have been highly variable, including evolution, neuroscience, and ecology. In this review, recent findings of molluscan genome projects are summarized, and their applications to aquaculture are discussed. Recently, 14 molluscan genomes have been published. All bivalve genomes show high heterozygosity rates, making genome assembly difficult. Unique gene expansions were evident in each species, corresponding to their specialized features, including shell formation, adaptation to the environment, and complex neural systems. To construct genetic maps and to explore quantitative trait loci (QTL) and genes of economic importance, genome-wide genotyping using massively parallel, targeted sequencing of cultured molluscs was employed. Molluscan genomics provides information fundamental to both biology and industry. Modern genomic studies facilitate molluscan biology, genetics, and aquaculture.

CRISPR/Cas9: A Practical Approach in Date Palm Genome Editing.

The genetic modifications through breeding of crop plants have long been used to improve the yield and quality. However, precise genome editing (GE) could be a very useful supplementary tool for improvement of crop plants by targeted genome modifications. Various GE techniques including ZFNs (zinc finger nucleases), TALENs (transcription activator-like effector nucleases), and most recently clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 (CRISPR-associated protein 9)-based approaches have been successfully employed for various crop plants including fruit trees. CRISPR/Cas9-based approaches hold great potential in GE due to their simplicity, competency, and versatility over other GE techniques. However, to the best of our knowledge no such genetic improvement has ever been developed in date palm—an important fruit crop in Oasis agriculture. The applications of CRISPR/Cas9 can be a challenging task in date palm GE due to its large and complex genome, high rate of heterozygosity and outcrossing, in vitro regeneration and screening of mutants, high frequency of single-nucleotide polymorphism in the genome and ultimately genetic instability. In this review, we addressed the potential application of CRISPR/Cas9-based approaches in date palm GE to improve the sustainable date palm production. The availability of the date palm whole genome sequence has made it feasible to use CRISPR/Cas9 GE approach for genetic improvement in this species. Moreover, the future prospects of GE application in date palm are also addressed in this review.

Low-depth genotyping-by-sequencing (GBS) in a bovine population: strategies to maximize the selection of high quality genotypes and the accuracy of imputation

BackgroundGenotyping-by-sequencing (GBS) has emerged as a powerful and cost-effective approach for discovering and genotyping single-nucleotide polymorphisms. The GBS technique was largely used in crop species where its low sequence coverage is not a drawback for calling genotypes because inbred lines are almost homozygous. In contrast, only a few studies used the GBS technique in animal populations (with sizeable heterozygosity rates) and many of those that have been published did not consider the quality of the genotypes produced by the bioinformatic pipelines. To improve the sequence coverage of the fragments, an alternative GBS preparation protocol that includes selective primers during the PCR amplification step has been recently proposed. In this study, we compared this modified protocol with the conventional two-enzyme GBS protocol. We also described various procedures to maximize the selection of high quality genotypes and to increase the accuracy of imputation.ResultsThe in silico digestions of the bovine genome showed that the combination of PstI and MspI is more suitable for sequencing bovine GBS libraries than the use of single digestions with PstI or ApeKI. The sequencing output of the GBS libraries generated a total of 123,666 variants with the selective-primer approach and 272,103 variants with the conventional approach. Validating our data with genotypes obtained from mass spectrometry and Illumina’s bovine SNP50 array, we found that the genotypes produced by the conventional GBS method were concordant with those produced by these alternative genotyping methods, whereas the selective-primer method failed to call heterozygotes with confidence. Our results indicate that high accuracy in genotype calling (>97%) can be obtained using low read-depth thresholds (3 to 5 reads) provided that markers are simultaneously filtered for genotype quality scores. We also show that factors such as the minimum call rate and the minor allele frequency positively influence the accuracy of imputation of missing GBS data. The highest accuracies (around 85%) of imputed GBS markers were obtained with the FIMPUTE program when GBS and SNP50 array genotypes were combined (80,190 to 100,297 markers) before imputation.ConclusionsWe discovered that the conventional two-enzyme GBS protocol could produce a large number of high-quality genotypes provided that appropriate filtration criteria were used. In contrast, the selective-primer approach resulted in a substantial proportion of miscalled genotypes and should be avoided for livestock genotyping studies. Overall, our study demonstrates that carefully adjusting the different filtering parameters applied to the GBS data is critical to maximize the selection of high quality genotypes and to increase the accuracy of imputation of missing data. The strategies and results presented here provide a framework to maximize the output of the GBS technique in animal populations and qualified the PstI/MspI GBS assay as a low-cost high-density genotyping platform. The conclusions reported here regarding read-depth and genotype quality filtering could benefit many GBS applications, notably genome-wide association studies, where there is a need to increase the density of markers genotyped across the target population while preserving the quality of genotypes.

GenomeScope: fast reference-free genome profiling from short reads.

GenomeScope is an open-source web tool to rapidly estimate the overall characteristics of a genome, including genome size, heterozygosity rate and repeat content from unprocessed short reads. These features are essential for studying genome evolution, and help to choose parameters for downstream analysis. We demonstrate its accuracy on 324 simulated and 16 real datasets with a wide range in genome sizes, heterozygosity levels and error rates. http://genomescope.org , https://github.com/schatzlab/genomescope.git . mschatz@jhu.edu. Supplementary data are available at Bioinformatics online.

Independent introductions and admixtures have contributed to adaptation of European maize and its American counterparts.

Through the local selection of landraces, humans have guided the adaptation of crops to a vast range of climatic and ecological conditions. This is particularly true of maize, which was domesticated in a restricted area of Mexico but now displays one of the broadest cultivated ranges worldwide. Here, we sequenced 67 genomes with an average sequencing depth of 18x to document routes of introduction, admixture and selective history of European maize and its American counterparts. To avoid the confounding effects of recent breeding, we targeted germplasm (lines) directly derived from landraces. Among our lines, we discovered 22,294,769 SNPs and between 0.9% to 4.1% residual heterozygosity. Using a segmentation method, we identified 6,978 segments of unexpectedly high rate of heterozygosity. These segments point to genes potentially involved in inbreeding depression, and to a lesser extent to the presence of structural variants. Genetic structuring and inferences of historical splits revealed 5 genetic groups and two independent European introductions, with modest bottleneck signatures. Our results further revealed admixtures between distinct sources that have contributed to the establishment of 3 groups at intermediate latitudes in North America and Europe. We combined differentiation- and diversity-based statistics to identify both genes and gene networks displaying strong signals of selection. These include genes/gene networks involved in flowering time, drought and cold tolerance, plant defense and starch properties. Overall, our results provide novel insights into the evolutionary history of European maize and highlight a major role of admixture in environmental adaptation, paralleling recent findings in humans.

Comparative Genomics Integrated with Association Analysis Identifies Candidate Effector Genes Corresponding to Lr20 in Phenotype-Paired Puccinia triticina Isolates from Australia.

Leaf rust is one of the most common and damaging diseases of wheat, and is caused by an obligate biotrophic basidiomycete, Puccinia triticina (Pt). In the present study, 20 Pt isolates from Australia, comprising 10 phenotype-matched pairs with contrasting pathogenicity for Lr20, were analyzed using whole genome sequencing. Compared to the reference genome of the American Pt isolate 1-1 BBBD Race 1, an average of 404,690 single nucleotide polymorphisms (SNPs) per isolate was found and the proportion of heterozygous SNPs was above 87% in the majority of the isolates, demonstrating a high level of polymorphism and a high rate of heterozygosity. From the genome-wide SNPs, a phylogenetic tree was inferred, which consisted of a large clade of 15 isolates representing diverse presumed clonal lineages including 14 closely related isolates and the more diverged isolate 670028, and a small clade of five isolates characterized by lower heterozygosity level. Principle component analysis detected three distinct clusters, corresponding exactly to the two major subsets of the small clade and the large clade comprising all 15 isolates without further separation of isolate 670028. While genome-wide association analysis identified 302 genes harboring at least one SNP associated with Lr20 virulence (p < 0.05), a Wilcoxon rank sum test revealed that 36 and 68 genes had significant (p < 0.05) and marginally significant (p < 0.1) differences in the counts of non-synonymous mutations between Lr20 avirulent and virulent groups, respectively. Twenty of these genes were predicted to have a signal peptide without a transmembrane segment, and hence identified as candidate effector genes corresponding to Lr20. SNP analysis also implicated the potential involvement of epigenetics and small RNA in Pt pathogenicity. Future studies are thus warranted to investigate the biological functions of the candidate effectors as well as the gene regulation mechanisms at epigenetic and post-transcription levels. Our study is the first to integrate phenotype-genotype association with effector prediction in Pt genomes, an approach that may circumvent some of the technical difficulties in working with obligate rust fungi and accelerate avirulence gene identification.

Prediction of Genetic Variation in Stocks of Offspring and Its Correlation with Viability and Growth Rate

AbstractThis study tested the accuracy of predictions of genetic variation with Genassemblage 1.0 software and investigated whether offspring with greater genetic variation have higher survivability in aquaculture conditions than offspring with less genetic variation. To achieve these aims, microsatellite polymorphism was used to determine genetic variation in rainbow trout, Oncorhynchus mykiss, spawners and progeny. Spawning pairs were assembled based on the estimated genetic variation of their offspring. The offspring were divided into two groups: (H) group had higher expected values of heterozygosity (0.93) and allelic richness (41), whereas (L) group had lower expected values (0.63 and 31). The heterozygosity, survivability, and growth rate of these groups were recorded. The observed heterozygosity was 0.93 in (H) group and 0.62 in (L) group. (H) group had slightly higher survivability (89.8%) than (L) group (87.6%), but this difference was not significant (p ≥ 0.05), possibly due to residual tetrasomy in the rainbow trout genome. In the offspring, predicted and observed variations were very similar, indicating that Genassemblage 1.0 accurately predicts genetic variation in progeny.

Diagnosis and phenotypic assessment of Pakistani Haemophilia B carriers.

Objectives:1: To assess the diagnostic utility of three polymorphisms (DdeI, XmnI and TaqI) and direct sequencing in haemophilia B (HB) carrier detection in Pakistani families. 2: To compare phenotypes of HB carriers with those of healthy females.Methods:The study was conducted from March 2014 till February 2016 at Khyber Medical University Peshawar and National Institute of Blood Diseases, Karachi. Individuals from HB families of Khyber Pakhtunkhwa (KP) and Federally Administered Tribal Areas (FATA) with known F9 mutation in the proband were enrolled into the study. FIX activity (FIX: C) levels were determined in all the participants. Bleeding scores (BS) and complete blood counts were performed in the female participants. Linkage analysis followed by targeted Sanger sequencing was carried out in all the study participants. Heterozygosity rate was determined for each polymorphism. Healthy females and the carrier groups were compared for bleeding phenotypes.Results:A total of 30 males and 48 females from 13 HB families were studied. The polymorphisms had a low heterozygosity rate. Direct sequencing determined the carrier status in all cases. The mean FIX: C was reduced whereas BS was raised in the carriers when compared with healthy females. A significant raise in white blood cells (WBCs) count was observed in the carriers.Conclusion:The three polymorphisms have a low heterozygosity rate in HB families from KP and FATA. Sanger sequencing is conclusive in determining carrier status in all the cases. FIX: C is low and BS is raised in the HB carriers in comparison to that of normal females. The mean WBCs count is significantly higher in the HB carriers than the normal females.

Genetic susceptibility to Gilbert's syndrome in a Valencian population; efficacy of the fasting test

ObjectiveTo describe the population distribution of the UGT1A1*28 variant (genetic variant code rs8175347) located in the promotor of the UGT gene and correlate its genotypes with the results of the fasting test, as well as its relationship with the biochemical disorder of Gilbert's syndrome (GS) in a Valencian population. Patients and methodsWe studied the prevalence of the genotypes (TA)6/6 (TA)6/7 and (TA)7/7 of the deleterious variant rs8175347 in 144 patients with hyperbilirubinemia, 38 of whom had previously undergone the fasting test to diagnose GS, and in 150 control patients. By analysing the genomic region of the TATA box of the UGT1A1 gene promotor using Sanger sequencing, we established the correlation between the rs8175347 genotypes and the fasting test results and with the patients’ biochemical disorders. ResultsThe rate of heterozygosity of allele (TA)7 in the control population was 32% and increased to 87.59% among the patients with suspected GS. The rate of genotype TA7/7 was 81.94% among the patients with hyperbilirubinemia, compared with 11.33% in the control patients. The fasting test showed a 15.79% rate of false negatives and a 5.26% rate of false positives. ConclusionsThe high frequency of allele (TA)7 among the Valencian control population, almost double the 5% reported for European control patients, confirms the high rate of GS reported in the Spanish population, without observing significant differences between the geographical ends of the country. The efficacy and reliability of the fasting test for the diagnosis of GS is questionable.