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PCR-based assays for validation of single nucleotide polymorphism markers in rice and mungbean
No full textAbstract
Background
Single nucleotide polymorphism (SNP) markers are the method of choice for genetic analyses including diversity and quantitative trait loci (QTL) studies. Marker validation is essential for QTL studies, but the cost and workload are considerable when large numbers of markers need to be verified. Marker systems with low development costs would be most suitable for this task.
Results
We have tested allele specific polymerase chain reaction (PCR), tetra markers and a genotyping tool based on the single strand specific nuclease CEL-I to verify randomly selected SNP markers identified previously either with a SNP array or by genotyping by sequencing in rice and mungbean, respectively. The genotyping capacity of allele-specific PCR and tetra markers was affected by the sequence context surrounding the SNP; SNPs located in repeated sequences and in GC-rich stretches could not be correctly identified. In contrast, CEL-I digestion of mixed fragments produced from test and reference DNA reliably pinpointed the correct genotypes, yet scoring of the genotypes became complicated when multiple SNPs were present in the PCR fragments. A cost analysis showed that as long the sample number remains small, CEL-I genotyping is more cost-effective than tetra markers.
Conclusions
CEL-I genotyping performed better in terms of genotyping accuracy and costs than tetra markers. The method is highly useful for validating SNPs in small to medium size germplasm panels
A new enzyme-linked immunosorbent assay (ELISA) for human free and bound kallikrein 9
No full textAbstract
Background
Kallikrein 9 (KLK9) is a member of the human kallikrein-related peptidases family, whose physiological role and implications in disease processes remain unclear. The active form of the enzyme is predicted to have chymotryptic activity. In the present study, we produced for the first time the active recombinant protein and monoclonal antibodies, and developed novel immunoassays for the quantification of free and bound KLK9 in biological samples.
Methods
The coding sequence of mature KLK9 isoform (mat-KLK9) was expressed in an Expi293F mammalian system and the synthesized polypeptide was purified through a two-step protocol. The purified protein was used as an immunogen for production of monoclonal antibodies in mice. Hybridomas were further expanded and antibodies were purified. Newly-produced monoclonal antibodies were screened for reaction with the KLK9 recombinant protein by a state-of-the-art immunocapture/parallel reaction monitoring mass spectrometry-based methodology.
Results
Anti-KLK9 antibodies were combined in pairs, resulting in the development of a highly sensitive (limit of detection: 15\ua0pg/mL) and specific (no cross-reactivity with other KLKs) sandwich-type ELISA. Highest KLK9 protein levels were found in tonsil and sweat and lower levels in the heart, kidney and liver. Hybrid immunoassays using an anti-KLK9 antibody for antigen capture and various anti-serine protease inhibitor polyclonal antibodies, revealed the presence of an a1-antichymotrypsin-bound KLK9 isoform in biological samples.
Conclusions
The ELISAs for free and bound forms of KLK9 may be highly useful for the detection of KLK9 in a broad range of biological samples, thus enabling the clarification of KLK9 function and use as a potential disease biomarker
Whole-genome sequence-based genomic prediction in laying chickens with different genomic relationship matrices to account for genetic architecture
No full textAbstract
Background
With the availability of next-generation sequencing technologies, genomic prediction based on whole-genome sequencing (WGS) data is now feasible in animal breeding schemes and was expected to lead to higher predictive ability, since such data may contain all genomic variants including causal mutations. Our objective was to compare prediction ability with high-density (HD) array data and WGS data in a commercial brown layer line with genomic best linear unbiased prediction (GBLUP) models using various approaches to weight single nucleotide polymorphisms (SNPs).
Methods
A total of 892 chickens from a commercial brown layer line were genotyped with 336\ua0K segregating SNPs (array data) that included 157\ua0K genic SNPs (i.e. SNPs in or around a gene). For these individuals, genome-wide sequence information was imputed based on data from re-sequencing runs of 25 individuals, leading to 5.2 million (M) imputed SNPs (WGS data), including 2.6\ua0M genic SNPs. De-regressed proofs (DRP) for eggshell strength, feed intake and laying rate were used as quasi-phenotypic data in genomic prediction analyses. Four weighting factors for building a trait-specific genomic relationship matrix were investigated: identical weights, \u2212(log
10
P ) from genome-wide association study results, squares of SNP effects from random regression BLUP, and variable selection based weights (known as BLUP|GA). Predictive ability was measured as the correlation between DRP and direct genomic breeding values in five replications of a fivefold cross-validation.
Results
Averaged over the three traits, the highest predictive ability (0.366\ua0\ub1\ua00.075) was obtained when only genic SNPs from WGS data were used. Predictive abilities with genic SNPs and all SNPs from HD array data were 0.361\ua0\ub1\ua00.072 and 0.353\ua0\ub1\ua00.074, respectively. Prediction with \u2212(log
10
P ) or squares of SNP effects as weighting factors for building a genomic relationship matrix or BLUP|GA did not increase accuracy, compared to that with identical weights, regardless of the SNP set used.
Conclusions
Our results show that little or no benefit was gained when using all imputed WGS data to perform genomic prediction compared to using HD array data regardless of the weighting factors tested. However, using only genic SNPs from WGS data had a positive effect on prediction ability
Derivation of economic values for production traits in aquaculture species
No full textAbstract
Background
In breeding programs for aquaculture species, breeding goal traits are often weighted based on the desired gains but economic gain would be higher if economic values were used instead. The objectives of this study were: (1) to develop a bio-economic model to derive economic values for aquaculture species, (2) to apply the model to determine the economic importance and economic values of traits in a case-study on gilthead seabream, and (3) to validate the model by comparison with a profit equation for a simplified production system.
Methods
A bio-economic model was developed to simulate a grow-out farm for gilthead seabream, and then used to simulate gross margin at the current levels of the traits and after one genetic standard deviation change in each trait with the other traits remaining unchanged. Economic values were derived for the traits included in the breeding goal: thermal growth coefficient ( TGC ), thermal feed intake coefficient ( TFC ), mortality rate ( M ), and standard deviation of harvest weight (
\u3c3
H
W
). For a simplified production system, improvement in TGC was assumed to affect harvest weight instead of growing period. Using the bio-economic model and a profit equation, economic values were derived for harvest weight, cumulative feed intake at harvest, and overall survival.
Results
Changes in gross margin showed that the order of economic importance of the traits was: TGC , TFC , M , and
\u3c3
H
W
. Economic values in \u20ac (kg production)
\u22121
(trait unit)
\u22121
were: 0.40 for TGC , \u22120.45 for TFC , \u22127.7 for M , and \u22120.0011 to \u22120.0010 for
\u3c3
H
W
. For the simplified production system, similar economic values were obtained with the bio-economic model and the profit equation. The advantage of the profit equation is its simplicity, while that of the bio-economic model is that it can be applied to any aquaculture species, because it can include any limiting factor and/or environmental condition that affects production.
Conclusions
We confirmed the validity of the bio-economic model. TGC is the most important trait to improve, followed by TFC and M , ..
An SNP-based saturated genetic map and QTL analysis of fruit-related traits in Zucchini using Genotyping-by-sequencing
No full textAbstract
Background
Cucurbita pepo is a cucurbit with growing economic importance worldwide. Zucchini morphotype is the most important within this highly variable species. Recently, transcriptome and Simple Sequence Repeat (SSR)- and Single Nucleotide Polymorphism (SNP)-based medium density maps have been reported, however further genomic tools are needed for efficient molecular breeding in the species. Our objective is to combine currently available complete transcriptomes and the Zucchini genome sequence with high throughput genotyping methods, mapping population development and extensive phenotyping to facilitate the advance of genomic research in this species.
Results
We report the Genotyping-by-sequencing analysis of a RIL population developed from the inter subspecific cross Zucchini x Scallop (ssp. pepo x ssp. ovifera ). Several thousands of SNP markers were identified and genotyped, followed by the construction of a high-density linkage map based on 7,718 SNPs (average of 386 markers/linkage group) covering 2,817.6\ua0cM of the whole genome, which is a great improvement with respect to previous maps. A QTL analysis was performed using phenotypic data obtained from the RIL population from three environments. In total, 48 consistent QTLs for vine, flowering and fruit quality traits were detected on the basis of a multiple-environment analysis, distributed in 33 independent positions in 15 LGs, and each QTL explained 1.5\u201362.9% of the phenotypic variance. Eight major QTLs, which could explain greater than 20% of the phenotypic variation were detected and the underlying candidate genes identified.
Conclusions
Here we report the first SNP saturated map in the species, anchored to the physical map. Additionally, several consistent QTLs related to early flowering, fruit shape and length, and rind and flesh color are reported as well as candidate genes for them. This information will enhance molecular breeding in C. pepo and will assist the gene cloning underlying the studied QTLs, helping to reveal the genetic basis of the studied processes in squash
Genetic structure in the Sherpa and neighboring Nepalese populations
No full textAbstract
Background
We set out to describe the fine-scale population structure across the Eastern region of Nepal. To date there is relatively little known about the genetic structure of the Sherpa residing in Nepal and their genetic relationship with the Nepalese. We assembled dense genotype data from a total of 1245 individuals representing Nepal and a variety of different populations resident across the greater Himalayan region including Tibet, China, India, Pakistan, Kazakhstan, Uzbekistan, Tajikistan and Kirghizstan. We performed analysis of principal components, admixture and homozygosity.
Results
We identified clear substructure across populations resident in the Himalayan arc, with genetic structure broadly mirroring geographical features of the region. Ethnic subgroups within Nepal show distinct genetic structure, on both admixture and principal component analysis. We detected differential proportions of ancestry from northern Himalayan populations across Nepalese subgroups, with the Nepalese Rai, Magar and Tamang carrying the greatest proportions of Tibetan ancestry.
Conclusions
We show that populations dwelling on the Himalayan plateau have had a clear impact on the Northern Indian gene pool. We illustrate how the Sherpa are a remarkably isolated population, with little gene flow from surrounding Nepalese populations
Diet, gonadal sex, and sex chromosome complement influence white adipose tissue miRNA expression
No full textAbstract
Background
MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate gene expression by targeting specific mRNA species for degradation or interfering with translation. Specific miRNAs are key regulators of adipogenesis, and are expressed at different levels in adipose tissue from lean and obese mice. The degree of lipid accumulation and distribution of white adipose tissue differs between males and females, and it is unknown whether sex differences in adipose tissue-specific miRNA expression may contribute to this dimorphism. Typically, sex differences are attributed to hormones secreted from ovaries or testes. However, the sex chromosome complement (XX versus XY) is also a determinant of sex differences and may regulate miRNA expression in adipocytes.
Results
To identify sex differences in adipose tissue miRNA expression and to understand the underlying mechanisms, we performed high-throughput miRNA sequencing in gonadal fat depots of the Four Core Genotypes mouse model. This model, which consists of XX female, XX male, XY female, and XY male mice, allowed us to assess independent effects of gonadal type (male vs . female) and sex chromosome complement (XX vs . XY) on miRNA expression profiles. We have also assessed the effects of a high fat diet on sex differences in adipose tissue miRNA profiles. We identified a male\u2013female effect on the overall miRNA expression profile in mice fed a chow diet, with a bias toward higher expression in male compared to female gonadal adipose tissue. This sex bias disappeared after gonadectomy, suggesting that circulating levels of gonadal secretions modulate the miRNA expression profile. After 16\ua0weeks of high fat diet, the miRNA expression distribution was shifted toward higher expression in XY vs . XX adipose tissue. Principal component analysis revealed that high fat diet has a substantial effect on miRNA profile variance, while gonadal secretions and sex chromosome complement each have milder effects.
Conclusions
Our results demonstrate that the overall miRNA expression profile in adipose tissue is influenced by gonadal hormones and the sex chromosome complement, and that expression profiles change in response to gonadectomy and high fat diet. Differential miRNA expression profiles may contribute to sex differences in adipose tissue gene expression, adipose tissue development, and diet-induced obesity
Conserved gene expression in sperm reservoirs between birds and mammals in response to mating
No full textAbstract
Background
Spermatozoa are stored in the oviductal functional sperm reservoir in animals with internal fertilization, including zoologically distant classes such as pigs or poultry. They are held fertile in the reservoir for times ranging from a couple of days (in pigs), to several weeks (in chickens), before they are gradually released to fertilize the newly ovulated eggs. It is currently unknown whether females from these species share conserved mechanisms to tolerate such a lengthy presence of immunologically-foreign spermatozoa. Therefore, global gene expression was assessed using cDNA microarrays on tissue collected from the avian utero-vaginal junction (UVJ), and the porcine utero-tubal junction (UTJ) to determine expression changes after mating (entire semen deposition) or in vivo cloacal/cervical infusion of sperm-free seminal fluid (SF)/seminal plasma (SP).
Results
In chickens, mating changed the expression of 303 genes and SF-infusion changed the expression of 931 genes, as compared to controls, with 68 genes being common to both treatments. In pigs, mating or SP-infusion changed the expressions of 1,722 and 1,148 genes, respectively, as compared to controls, while 592 genes were common to both treatments. The differentially expressed genes were significantly enriched for GO categories related to immune system functions (35.72-fold enrichment). The top 200 differentially expressed genes of each treatment in each animal class were analysed for gene ontology. In both pig and chicken, an excess of genes affecting local immune defence were activated, though frequently these were down-regulated. Similar genes were found in both the chicken and pig, either involved in pH-regulation ( SLC16A2, SLC4A9, SLC13A1, SLC35F1, ATP8B3, ATP13A3 ) or immune-modulation ( IFIT5, IFI16, MMP27, ADAMTS3, MMP3, MMP12 ).
Conclusion
Despite being phylogenetically distant, chicken and pig appear to share some gene functions for the preservation of viable spermatozoa in the female reservoirs
Thromboelastometry analysis of thrombocytopenic dengue patients: a cross-sectional study
No full textAbstract
Background
Dengue virus infection (DVI) is a prevalent and potentially fatal viral disease associated with coagulopathy. So far, the coagulation profile of DVI patients with thrombocytopenia has not been assessed through a viscoelastic test such as rotational thromboelastometry. We aimed to describe the prevalence and characteristics of coagulation abnormalities in dengue fever outpatients with thrombocytopenia, addressed by both rotational thromboelastometry and conventional coagulation tests.
Methods
This was a cross-sectional study conducted between April 6
th
and May 5
th
2015 in S\ue3o Paulo, Brazil during a dengue outbreak. Thromboelastometry (ROTEM\uae) and the conventional coagulation tests prothrombin time (PT), international normalized ratio (INR), activated partial thromboplastin time (aPTT), thrombin time (TT), platelet count and fibrinogen levels were performed in 53 patients with DVI and thrombocytopenia.
Results
Despite a median interquartile range (IQR) platelet count of 77 (63\u201388) x 10
9
/L in DVI patients, conventional coagulation tests and plasma fibrinogen levels were within the normal range. Subjects demonstrated hypocoagulability in 71.7% (38/53) in INTEM and 54.7% (29/53) in EXTEM DVI patients. FIBTEM analyses detected only 5.7% (3/53) with hypocoagulability among this population. The median (IQR) clotting time (CT), clot formation time (CFT) and maximum clot firmness (MCF) on INTEM were, respectively, 177 (160\u2013207) sec, 144 (108\u2013178) sec and 48 (42\u201352) mm. On EXTEM, median (IQR) CT, CFT and MCF were, respectively, 69 (65\u201378) sec, 148 (126\u2013198) sec and 49 (44\u201355) mm. Median (IQR) MCF on FIBTEM was 15 (13\u201318) mm.
Conclusion
Thromboelastometry impairment is highly prevalent in DVI patients with thrombocytopenia, particularly in INTEM and EXTEM analyses, while standard coagulation tests are normal in this setting. Clinical implications remain to be established
When parents won\u2019t vaccinate their children: a qualitative investigation of australian primary care providers\u2019 experiences
No full textAbstract
Background
Increasingly, the experiences and perceptions of parents who decline vaccination are the subject of investigation. However, the experiences of clinicians who encounter these parents in the course of their work has received little academic attention to date. This study aimed to understand the challenges faced and strategies used when general practitioners and immunising nurses encounter parents who choose not to vaccinate their children.
Methods
Primary care providers were recruited from regions identified through the Australian Childhood Immunisation Register (ACIR) as having higher than national average rates of registered objection to childhood vaccination. Interviews began with an exploration of provider experiences with parents who accept, are hesitant towards, and who decline vaccination. Participants were asked specifically about how they addressed any difficulties they encountered in their interactions. Thematic analysis focused on encounters with parents \u2013 challenges and strategies.
Results
Twenty-six general practitioners (GPs), community and practice nurses (PNs) were interviewed across two regions in NSW, Australia. Providers\u2019 sense of professional identity as health advocates and experts became conflicted in their encounters with vaccine objecting parents. Providers were dissatisfied when such consultations resulted in a \u2018therapeutic roadblock\u2019 whereby provider-parent communication came to a standstill. There were mixed views about being asked to sign forms exempting parents from vaccinating their children. These ranged from a belief that completing the forms rewarded parents for non-conformity to seeing it as a positive opportunity for engagement. Three common strategies were employed by providers to navigate through these challenges; 1) to explore and inform, 2) to mobilise clinical rapport and 3) to adopt a general principle to first do no harm to the therapeutic relationship.
Conclusions
Many healthcare providers find consultations with vaccine objecting parents challenging and some, particularly more experienced providers, employ successful strategies to address this. Primary care providers, especially those more junior, could benefit from additional communication guidance to better the outcome and increase the efficiency of their interactions with such parents