Candidate Gene Analysis Research Articles

Quantitative trait locus mapping and candidate gene analysis for salt tolerance at bud stage in rice.

Soil salinization has a serious influence on rice yield and quality. How to enhance salt tolerance in rice is a topical issue. In this study, 120 recombinant inbred line populations were generated through nonstop multi-generation selfing using a male indica rice variety Huazhan (Oryza sativa L. subsp. indica cv. 'HZ') and a female variety of Nekken2 (Oryza sativa L. subsp. japonica cv. 'Nekken2') as the parents. Germination under 80 mM NaCl conditions was measured and analyzed, and quantitative trait locus (QTL) mapping was completed using a genetic map. A total of 16 salt-tolerance QTL ranges were detected at bud stage in rice, which were situated on chromosomes 3, 4, 6, 8, 9, 10, 11, and 12. The maximum limit of detection was 4.69. Moreover, the qST12.3 was narrowed to a 192 kb region on chromosome 12 using map-based cloning strategy. Statistical analysis of the expression levels of these candidate genes under different NaCl concentrations by qRT-PCR revealed that qST12.3 (LOC_Os12g25200) was significantly down-regulated with increasing NaCl concentration, and the expression level of the chlorine-transporter-encoding gene LOC_Os12g25200 in HZ was significantly higher than that of Nekken2 under 0 mM NaCl. Sequencing analysis of LOC_Os12g25200 promoter region indicated that the gene expression difference between parents may be due to eight base differences in the promoter region. Through QTL mining and analysis, a plurality of candidate genes related to salt tolerance in rice was obtained, and the results showed that LOC_Os12g25200 might negatively regulate salt tolerance in rice. The results provide the basis for further screening and cultivation of salt-tolerant rice varieties and have laid the foundation for elucidating further molecular regulation mechanisms of salt tolerance in rice.

Discovering Loci for Breeding Prospective and Phenology in Wheat Mediterranean Landraces by Environmental and eigenGWAS.

Knowledge of the genetic basis of traits controlling phenology, differentiation patterns, and environmental adaptation is essential to develop new cultivars under climate change conditions. Landrace collections are an appropriate platform to study the hidden variation caused by crop breeding. The use of genome-wide association analysis for phenology, climatic data and differentiation among Mediterranean landraces led to the identification of 651 marker-trait associations that could be grouped in 46 QTL hotspots. A candidate gene analysis using the annotation of the genome sequence of the wheat cultivar 'Chinese Spring' detected 1097 gene models within 33 selected QTL hotspots. From all the gene models, 42 were shown to be differentially expressed (upregulated) under abiotic stress conditions, and 9 were selected based on their levels of expression. Different gene families previously reported for their involvement in different stress responses were found (protein kinases, ras-like GTP binding proteins and ethylene-responsive transcription factors). Finally, the synteny analysis in the QTL hotspots regions among the genomes of wheat and other cereal species identified 23, 21 and 7 ortho-QTLs for Brachypodium, rice and maize, respectively, confirming the importance of these loci.

Development of a set of novel binary expression vectors for plant gene function analysis and genetic transformation.

With the advent of multiple omics and Genome-Wide Association Studies (GWAS) technology, genome-scale functional analysis of candidate genes is to be conducted in diverse plant species. Construction of plant binary expression vectors is the prerequisite for gene function analysis. Therefore, it is of significance to develop a set of plant binary expression vectors with highly efficient, inexpensive, and convenient cloning method, and easy-to-use in screening of positive recombinant in Escherichia coli. In this study, we developed a set of plant binary expression vectors, termed pBTR vectors, based on Golden Gate cloning using BsaI restriction site. Foreign DNA fragment of interest (FDI) can be cloned into the destination pBTR by one-step digestion-ligation reaction in a single tube, and even the FDI contains internal BsaI site(s). Markedly, in one digestion-ligation reaction, multiple FDIs (exemplified by cloning four soybean Glyma.02g025400, Glyma.05g201700, Glyma.06g165700, and Glyma.17g095000 genes) can be cloned into the pBTR vector to generate multiple corresponding expression constructs (each expression vector carrying an FDI). In addition, the pBTR vectors carry the visual marker, a brightness monomeric red fluorescent protein mScarlet-I, that can be observed with the unaided eye in screening of positive recombinants without the use of additional reagents/equipment. The reliability of the pBTR vectors was validated in plants by overexpression of AtMyb75/PAP1 in tomato and GUSPlus in soybean roots via Agrobacterium rhizogenes-mediated transformation, promoter activity analysis of AtGCSpro in Arabidopsis via A. tumefaciens-mediated transformation, and protein subcellular localization of the Vitis vinifera VvCEB1opt in tobacco, respectively. These results demonstrated that the pBTR vectors can be used in analysis of gene (over)expression, promoter activity, and protein subcellular localization. These vectors will contribute to speeding up gene function analysis and the process of plant molecular breeding.

Growth differentiation factor 15 is dispensable for acetaminophen-induced liver injury in mice.

Acetaminophen (APAP)-induced liver injury (AILI) has been recognized as a pivotal contributor to drug-induced liver failure in Western countries, but its molecular mechanism remains poorly understood. Growth differentiation factor 15 (GDF15) is a pleiotropic factor that alleviates non-alcoholic liver steatohepatitis, liver fibrosis and liver injury. The aim of the present study was to examine the possibility whether GDF15 confers protection against AILI. We found that the gene expression of Gdf15 was increased significantly after APAP overdose in mice. Next, the role of Gdf15 in AILI was evaluated by hepatic Gdf15 overexpression (using adeno-associated virus serotype 8), injection with recombinant human GDF15 (rhGDF15) and Gdf15 knockout mice after challenge with APAP. A marked elevation of Gdf15 was observed after AILI. However, there were no significant differences in AILI-related liver injury and JNK phosphorylation after Gdf15 overexpression, rhGDF15 injection or Gdf15 deficiency. Together, we conclude that, despite a noticeable elevation of Gdf15 level after AILI, Gdf15 is dispensable for APAP-induced AILI. Our study further suggests that genomic analysis of mRNA expression after APAP overdose is of limited relevance unless followed up by a functional analysis of candidate genes in vivo.

Molecular characterization of seed longevity in soybean [Glycine max (L.) Merrill] using SSR markers

Soybean is the most important oilseed in the tropical and subtropical parts of the world unfortunately has poor competence for seed longevity. It?s strongly affected by environment (Temperature and RH) during seed formation, harvest and storage. Tagging of seed longevity associated markers in the crosses (Birsa Soya1 ? DS-228) which are good and poor storer genotypes resp. by using 26 SSR primers in genotypes contrasting for these traits in soybean. The polymorphism was observed between genotypes differing for seed longevity trait for six primer pairs, viz; Sat_162, Satt632, Sat_202, Satt523, Satt184 and Satt460, among them seed coat hardiness QTL, SCH 1-2 linked Satt632 amplified distinct markers. Bulked segregant analysis was carried out by using SSR markers in segregating 93 F2 populations. The Satt632-237 bp marker was further validated in individual F2 plant population. The genotype, Birsa Soya1 and it segregants Satt632-237 bp marker was found to be closely associated with high seed longevity in soybean. Hence, the primer Satt632 is impending marker for marker assisted breeding for high seed longevity marker to be linked to seed coat hardiness loci qSCH1-2 like gene present on chromosome A2. Seed longevity trait is governed by one or few gene with observed segregation ratio of 3.65:1 as against expected segregated ratio 3:1. Candidate gene analysis at qSCH1-2 loci, indicated for role of either a brassinosteriod regulated protein or pentatricopeptide repeat gene to regulate seed longevity.

Ovarian expression analysis of fecundity candidate genes in Goat breeds differing in prolificacy

To understand the genetics of goat fertility, goat breeds differing in prolificacy were used for exploring candidate genes using quantitative polymerase chain reaction. Ovaries (both left and right) of Black Bengal, Osmanabadi and Sirohi goats (n=3 from each breed) were collected from the local abattoir located in their respective breeding tracts. The RNA isolated from antral follicles was converted into cDNA. The β-Actin (ACTB) was used as a reference gene for quantifying the expression of BMP15, GDF9, BMPR1B, KISS1 and GPR54 mRNAs in ovaries. The results showed that BMPR1B, GDF9 and BMP15 genes were highly expressed whereas KISS1 and GPR54 were slightly expressed in the ovaries. Significant differences (P<0.05) were recorded in the gene expression of GDF9 and BMP15. Expression of these two genes varied in prolificacy being highest in Black Bengal followed by Osmanabadi and least in Sirohi. BMPR1B expression did not vary significantly among the goat breeds. In conclusion, the study provides baseline data and contributes toward the understanding of the molecular basis of prolificacy in goat breeds.

Application of an Improved 2-Dimensional High-Throughput Soybean Root Phenotyping Platform to Identify Novel Genetic Variants Regulating Root Architecture Traits.

Nutrient-efficient root system architecture (RSA) is becoming an important breeding objective for generating crop varieties with improved nutrient and water acquisition efficiency. Genetic variants shaping soybean RSA is key in improving nutrient and water acquisition. Here, we report on the use of an improved 2-dimensional high-throughput root phenotyping platform that minimizes background noise by imaging pouch-grown root systems submerged in water. We also developed a background image cleaning Python pipeline that computationally removes images of small pieces of debris and filter paper fibers, which can be erroneously quantified as root tips. This platform was used to phenotype root traits in 286 soybean lines genotyped with 5.4 million single-nucleotide polymorphisms. There was a substantially higher correlation in manually counted number of root tips with computationally quantified root tips (95% correlation), when the background was cleaned of nonroot materials compared to root images without the background corrected (79%). Improvements in our RSA phenotyping pipeline significantly reduced overestimation of the root traits influenced by the number of root tips. Genome-wide association studies conducted on the root phenotypic data and quantitative gene expression analysis of candidate genes resulted in the identification of 3 putative positive regulators of root system depth, total root length and surface area, and root system volume and surface area of thicker roots (DOF1-like zinc finger transcription factor, protein of unknown function, and C2H2 zinc finger protein). We also identified a putative negative regulator (gibberellin 20 oxidase 3) of the total number of lateral roots.

Research Note: Polymorphisms of gonadotrophin-releasing hormone gene and their association with growth traits in quail (Coturnix Coturnix)

This study aimed to identify polymorphisms of gonadotropin-releasing hormone (GnRH) gene and their association with growth traits in quail by PCR and direct sequencing. Genomic DNA was extracted from quail blood samples of 36 from Savimalt (SV) and 49 from French Giant (FG). Growth traits were measured and used for candidate gene analysis, as body weight (BW), shank length (SL), chest width (CW), chest depth (CD), breastbone length (BBL), body length (BL), and shank circumference (SC). The results showed that a total of 20 SNPs were detected in GnRH gene, whereas 8 SNPs were significantly associated with growth traits (P < 0.05). The T215C, G279A, C458T, A520G, and C547G were significantly associated with SL at 3 wk of age in the FG strain, whereas A583T was significantly related to BBL and BL, and C591T was significantly related to SL, BBL, and BL, whereas A592G was significantly correlated with SL, CW, CD, BBL, and BL (P < 0.05). The 8 SNPs were significantly related to CW, CD, and BBL at 3 wk of age in the SV strain, whereas A583T, C591T, and A592G were significantly associated with BW (P < 0.05). The G279A showed significant correlations with SL at 5 wk of age in FG, whereas A583T showed significant associations with SC in FG, and C591T was significantly associated with BW and SC in FG, whereas A592T was significantly related to BW, SL, and CD in FG (P < 0.05). The T215C, G279A, C458T, A520G, and C547G were significantly correlated with BW, CW, BBL, and BL at 5 wk of age in SV, whereas A583T, C591T, and A592G were significantly related to BW, SL, CW, BBL, and BL (P < 0.05). Haplotypes based on 8 SNPs showed significant correlation with BW, SL, CW, CD, BBL, BL, and SC in FG (P < 0.05). In conclusion, the GnRH gene could be used as a molecular genetic marker to provide theoretical foundation to improve growth traits in quail.

Comparative Meta-analysis of Adipose Tissue Transcriptomics Data in PCOS Patients and Healthy Control Women.

Polycystic ovary syndrome (PCOS) is a common condition in reproductive-aged women that induces reproductive and metabolic derangements. Women with PCOS seem to have disturbances in lipid metabolism in the adipose tissue. Nevertheless, gene expression in adipose tissue of PCOS women and its relation to other disturbances have been fragmentarily investigated. We utilized microarray data to identify the most important up- and down-regulated candidate genes in adipose tissue of PCOS women in contrast to healthy women using the meta-analysis technique. Microarray data produced from three independent experiments (n = 3) conducted on adipose tissue in women with PCOS were retrieved from ArrayExpress. Then, the datasets were merged using the metaSeq package in Rstudio and differentially expressed genes (DEGs) were selected in the studies. The integrative bioinformatics analyses of candidate genes were performed by gene ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, and protein-protein interaction (PPI) network construction. Of these, 12 up-regulated genes and 12 down-regulated genes were identified and assessed as the most highly up-regulated and down-regulated genes in adipose tissue of women with PCOS. These DEGs that were annotated by KEGG analysis were mainly involved in PI3K-Akt, MAPK, Rap1, and Ras signaling pathways, and pathways in cancer such as hepatocellular carcinoma and gastric cancer, as well as metabolic pathways, and brain disorder pathways such as Alzheimer's disease and Huntington disease pathways. In the PPI networks, PRDM10, FGFR2, IGF1R, and FLT1 were the key nodes in the up-regulated networks, while the NDUFAB1 and NME2 proteins were key in the down-regulated networks. Overall, these findings provide insight into the gene expression in adipose tissue of PCOS women and its relation to other disturbances.

Identification of genomic regions governing moisture and heat stress tolerance employing association mapping in rice (Oryza sativa L.).

Rice crop is damaged extremely by abiotic stress world-wide. The best approach to enhance drought tolerance in rice varieties is to identify and introgress yield QTLs with major effects. The Association mapping approach helps in the identification of genomic regions governing physiological, yield and yield attributes under moisture and heat stress conditions in diverse collections of crop germplasm, based on historic recombination events and linkage disequilibrium across the genome. The association mapping panel of 110 rice germplasm lines exhibited significant variation for all the traits in both irrigated and moisture stress conditions. The extent of yield reduction ranged to 83% during rabi, 2018-19, 53% in rabi, 2019-20 and 68% in pooled analysis. The genotypes Badami, Badshabhog, Pankaj, Varalu, Vasundhara, Vivekdhan, Krishna and Minghui63 exhibited drought tolerance with least yield penalty under moisture stress conditions. The genotypes Konark, MTU3626, NLR33671, PR118 and Triguna exhibited minimal reduction in heat stress tolerance traits. Association mapping of germplasm using 37808 SNP markers detected a total of 10 major MTA (Marker-trait association) clusters distributed on chromosomes 1, 3, 4 and 11 through mixed linear model (MLM) governing multiple traits from individual data analysis which are consistent across the years and situations. The pooled data generated a total of five MTA clusters located on chromosome 6. In addition, several novel unique MTAs were also identified. Heat stress analysis generated a total of 23 MTAs distributed on chromosomes 1, 5, 6 and 11. Candidate gene analysis detected a total of 53 and 38 genes under individual and pooled data analysis for various yield and yield attributes under control and moisture stress conditions, respectively and a total of 11 candidate genes in heat stress Conditions. The major and novel MTAs identified in the present investigation for various drought and heat tolerant traits can be utilized for breeding climate-resilient rice varieties. The candidate genes predicted for key MTAs are of great value to deploy into the rice breeding after functional characterization.

Comparative Analysis of the Liver Transcriptome of Beijing You Chickens and Guang Ming Broilers under Salmonella enterica Serovar Typhimurium Infection.

Salmonella enterica serovar Typhimurium (ST) is a food-borne pathogen that can infect animals and humans. It is currently the most common bacterial pathogen that negatively affects the poultry industry. Although different chicken breeds have been observed to exhibit diverse resistance to ST infection, the underlying genetic mechanisms remain unclear and the genes involved in this differential disease resistance need to be identified. To overcome this knowledge gap, we used a liver transcriptome analysis to screen differentially expressed genes (DEGs) in two different chicken breeds (local Beijing You (BY) and commercial Guang Ming No. 2 broiler line B (GM)) before and after ST infection. We also performed weighted gene co-expression network analysis (WGCNA) to detect hub genes, and employed selection signal analysis of candidate genes. Three promising genes (EGR1, JUN and FOS) were eventually identified, and were significantly and differentially expressed in the same breed under different conditions, and in the two breeds after ST infection. Hub genes, such as PPFIA4 and ZNF395, were identified using WGCNA, and were associated with the ratio of heterophils to lymphocytes (H/L), an indicator of disease resistance. the present study identified several genes and pathways associated with resistance to ST infection, and found that BY had greater resistance to ST infection than GM. The results obtained provide valuable resources for investigating the mechanisms of resistance to ST infection in different chicken breeds.

Quantitative trait locus mapping and identification of candidate genes for resistance to Verticillium wilt in four recombinant inbred line populations of Gossypium hirsutum

Improving resistance to Verticillium wilt is of great significance for achieving high and stable yields of Upland cotton (Gossypium hirsutum). To deeply understand the genetic basis of cotton resistance to Verticillium wilt, Verticillium wilt-resistant Upland Lumianyan 28 and four Verticillium wilt-susceptible Acala cotton cultivars were used to create four recombinant inbred line (RIL) populations of 469 families through nested hybridization. Phenotypic data collected in five stressful environments were used to select resistant and sensitive lines and create a mixed pool of extreme phenotypes for BSA-seq. A total of 8 QTLs associated with Verticillium wilt resistance were identified on 4 chromosomes, of which qVW-A12–5 was detected simultaneously in the RIL populations and in one of the RIL populations and was identified for the first time. According to the sequence comparison and transcriptome analysis of candidate genes in the QTL interval between parents and pools, 4 genes were identified in the qVW-A12–5 interval. qRT–PCR of parental and phenotypically extreme lines revealed that Gh_CPR30 was induced by and may be a candidate gene for resistance to Verticillium wilt in G. hirsutum. Furthermore, VIGS technology revealed that the disease severity index (DSI) of the Gh_CPR30-silenced plants was significantly higher than that of the control. These results indicate that the Gh_CPR30 gene plays an important role in the resistance of G. hirsutum to Verticillium wilt, and the study provides a molecular basis for analyzing the molecular mechanism underlying G. hirsutum resistance to Verticillium wilt.

Primary Mapping and Analysis of the CmARM14 Candidate Gene for Mature Fruit Abscission in Melon

Mature fruit abscission (MFA) is an important trait in terms of both harvest and yield. MFA can affect the production and economic value of melon fruit. An F3 population segregating for a single gene and derived from a cross between line M2-10, which shows no mature fruit abscission (None MFA), and the MFA line ZT00091 was used to map candidate genes. Specific length amplified fragment (SLAF) sequencing, in conjunction with bulked-segregant analysis (BSA), was used to map loci governing the natural fruit abscission of plants composing the F3-57 family. A candidate locus, mfa10.1, located on chromosome 10 between genomic positions 73,229 and 818,251, was obtained. An insertion-deletion (InDel) marker and 46 recombinant individuals were used to narrow the candidate region to within 35 kb at the genomic position of 650,203 to 685,250; this region included six candidate genes. qRT–PCR gene expression and gene sequence data showed that the CmARM14 gene, which encodes a RING-type E3 ubiquitin transferase (MELO3C012406), was a candidate for melon MFA. Subcellular localization observations revealed that the CmARM14 fusion protein was localized to the golgi apparatus. Taken together, these results provide a molecular basis for melon breeding.

The genomic basis of copper tolerance in Drosophila is shaped by a complex interplay of regulatory and environmental factors

Escalation in industrialization and anthropogenic activity have resulted in an increase of pollutants released into the environment. Of these pollutants, heavy metals such as copper are particularly concerning due to their bio-accumulative nature. Due to its highly heterogeneous distribution and its dual nature as an essential micronutrient and toxic element, the genetic basis of copper tolerance is likely shaped by a complex interplay of genetic and environmental factors. In this study, we utilized the natural variation present in multiple populations of Drosophila melanogaster collected across Europe to screen for variation in copper tolerance. We found that latitude and the degree of urbanization at the collection sites, rather than any other combination of environmental factors, were linked to copper tolerance. While previously identified copper-related genes were not differentially expressed in tolerant vs. sensitive strains, genes involved in metabolism, reproduction, and protease induction contributed to the differential stress response. Additionally, the greatest transcriptomic and physiological responses to copper toxicity were seen in the midgut, where we found that preservation of gut acidity is strongly linked to greater tolerance. Finally, we identified transposable element insertions likely to play a role in copper stress response. Overall, by combining genome-wide approaches with environmental association analysis, and functional analysis of candidate genes, our study provides a unique perspective on the genetic and environmental factors that shape copper tolerance in natural D. melanogaster populations and identifies new genes, transposable elements, and physiological traits involved in this complex phenotype.

QTL mapping and candidate gene analysis of microspore embryogenesis in Capsicum

QTL mapping and candidate gene analysis of microspore embryogenesis in Capsicum

Probing prenatal bisphenol exposures and tissue-specific DNA methylation responses in cord blood, cord tissue, and placenta

The early-gestational fetal epigenome establishes the landscape for fetal development and is susceptible to disruption via environmental stressors including chemical exposures. Research has explored how cell- and tissue-type-specific epigenomic signatures contribute to human disease, but how the epigenome in each tissue comparatively responds to environmental exposures is largely unknown. This pilot study compared DNA methylation in four previously identified genes across matched cord blood (CB), cord tissue (CT), and placental (PL) samples from 28 mother-infant pairs in tthe Michigan Mother Infant Pairs study; evaluated association between prenatal exposure to bisphenols (BPA, BPF, and BPS) and DNA methylation (DNAm) by tissue type; compared epigenome-wide DNAm of CB and PL; and explored associations between prenatal bisphenol exposures and epigenome-wide DNAm in PL. Bisphenol concentrations were quantified in first-trimester maternal urine. DNAm was assessed at four genes via pyrosequencing in three tissues; epigenome-wide DNAm analysis via Infinium MethylationEPIC array was completed on CB and PL. Candidate gene analysis revealed tissue-specific differences across all genes. In adjusted linear regression, BPA and BPF were associated with DNAm across candidate genes in PL but not CB and CT. Epigenome-wide comparison of matched CB and PL DNAm revealed tissue-specific differences at most CpG sites and modest associations between maternal first-trimester bisphenol exposures and PL but not CB DNAm. These data endorse inclusion of a variety of tissues in prenatal exposure studies. Overlapping and divergent responses in CB, CT, and PL demonstrate their utility in combination to capture a fuller picture of the epigenetic effect of developmental exposures.

Cis-eQTL Analysis and Functional Validation of Candidate Genes for Carcass Yield Traits in Beef Cattle.

Carcass yield traits are of considerable economic importance for farm animals, which act as a major contributor to the world’s food supply. Genome-wide association studies (GWASs) have identified many genetic variants associated with carcass yield traits in beef cattle. However, their functions are not effectively illustrated. In this study, we performed an integrative analysis of gene-based GWAS with expression quantitative trait locus (eQTL) analysis to detect candidate genes for carcass yield traits and validate their effects on bovine skeletal muscle satellite cells (BSCs). The gene-based GWAS and cis-eQTL analysis revealed 1780 GWAS and 1538 cis-expression genes. Among them, we identified 153 shared genes that may play important roles in carcass yield traits. Notably, the identified cis-eQTLs of PON3 and PRIM2 were significantly (p < 0.001) enriched in previous GWAS loci for carcass traits. Furthermore, overexpression of PON3 and PRIM2 promoted the BSCs’ proliferation, increased the expression of MYOD and downregulated the expression of MYOG, which indicated that these genes may inhibit myogenic differentiation. In contrast, PON3 and PRIM2 were significantly downregulated during the differentiation of BSCs. These findings suggested that PON3 and PRIM2 may promote the proliferation of BSCs and inhibit them in the pre-differentiation stage. Our results further contribute to the understanding of the molecular mechanisms of carcass yield traits in beef cattle.

Targeted bisulfite sequencing analysis of candidate genes associated with Alzheimer’s disease.

AbstractBackgroundRecent epigenome‐wide association studies (EWAS) have identified a number of loci in specific genes that show robust and reproducible alterations in DNA methylation in Alzheimer’s disease brain samples. The standard method to assess methylation in EWAS is via microarrays, however, these only target a limited number of methylation sites in each gene. Therefore, further analysis of methylation changes across the entire gene are required to determine the exact extent and pattern of methylation changes in disease. In this study we have performed targeted bisulfite sequencing for candidate genes in the Brains for Dementia Research (BDR) tissue sample resource, which is a highly characterized cohort containing tissue with a high degree of standardized pathological, clinical and administrative data available to allow comparative studies.MethodPrefrontal cortex brain samples from 60 individuals were selected from the BDR cohort and grouped by Braak stage (Control 0‐II; mild cognitive impairment III‐IV; AD V‐VI). The DNA was extracted, before 30 genomic regions of interest, identified from previous AD EWAS, were captured using Agilent Sure Select target baits. The DNA was next‐generation bisulfite sequenced, before the sequence reads were aligned and the methylation status of cytosine residues were called using the Bismark Bisulfite Mapper program. Differentially methylated positions (DMPs) were then analyzed across the three groups.ResultThe degree and extent of methylation within the targeted genomic regions were identified and quantified for each group before being analyzed using linear regression models.ConclusionThis study builds on previous work that identified differential methylation in several genomic regions that were associated with Braak stage. By identifying the exact positions that are subjected to differential methylation this work provides further evidence that dysregulation of methylation is associated with pathological changes in AD prefrontal cortex.

De novo transcriptome sequencing of Impatiens uliginosa and the analysis of candidate genes related to spur development

BackgroundSpur, a structure capable of producing and storing nectar, not only plays a vital role in the pollination process but also promotes the rapid diversification of some plant lineages, which is considered a key innovation in plants. Spur is the focus of many studies, such as evolution and ecological hypothesis, but the current understanding of spur development is limited. High-throughput sequencing of Impatiens uliginosa was carried out to study the molecular mechanism of its spur development, which is believed to provide some insights into the spur development of Impatiens.ResultsTranscriptomic sequencing and analysis were performed on spurs and limbs of I. uliginosa at three developmental stages. A total of 47.83 Gb of clean data were obtained, and 49,716 unigene genes were assembled. After comparison with NR, Swiss-Prot, Pfam, COG, GO and KEGG databases, a total of 27,686 genes were annotated successfully. Through comparative analysis, 19,356 differentially expressed genes were found and enriched into 208 GO terms and 146 KEGG pathways, among which plant hormone signal transduction was the most significantly enriched pathway. One thousand thirty-two transcription factors were identified, which belonged to 33 TF families such as MYB, bHLH and TCP. Twenty candidate genes that may be involved in spur development were screened and verified by qPCR, such as SBP, IAA and ABP.ConclusionsTranscriptome data of different developmental stages of spurs were obtained, and a series of candidate genes related to spur development were identified. The importance of genes related to cell cycle, cell division, cell elongation and hormones in spur development was clarified. This study provided valuable information and resources for understanding the molecular mechanism of spur development in Impatiens.

Bridging clinical care and research in Ontario, Canada: Maximizing diagnoses from reanalysis of clinical exome sequencing data.

We examined the utility of clinical and research processes in the reanalysis of publicly-funded clinical exome sequencing data in Ontario, Canada. In partnership with eight sites, we recruited 287 families with suspected rare genetic diseases tested between 2014 and 2020. Data from seven laboratories was reanalyzed with the referring clinicians. Reanalysis of clinically relevant genes identified diagnoses in 4% (13/287); four were missed by clinical testing. Translational research methods, including analysis of novel candidate genes, identified candidates in 21% (61/287). Of these, 24 families have additional evidence through data sharing to support likely diagnoses (8% of cohort). This study indicates few diagnoses are missed by clinical laboratories, the incremental gain from reanalysis of clinically-relevant genes is modest, and the highest yield comes from validation of novel disease-gene associations. Future implementation of translational research methods, including continued reporting of compelling genes of uncertain significance by clinical laboratories, should be considered to maximize diagnoses.