SNP-based pool genotyping and haplotype analysis accelerate fine-mapping of the wheat genomic region containing stripe rust resistance gene <Emphasis Type="Italic">Yr26</Emphasis>

Key message

NGS-assisted super pooling emerging as powerful tool to accelerate gene mapping and haplotype association analysis within target region uncovering specific linkage SNPs or alleles for marker-assisted gene pyramiding.

Abstract

Conventional gene mapping methods to identify genes associated with important agronomic traits require significant amounts of financial support and time. Here, a single nucleotide polymorphism (SNP)-based mapping approach, RNA-Seq and SNP array assisted super pooling analysis, was used for rapid mining of a candidate genomic region for stripe rust resistance gene Yr26 that has been widely used in wheat breeding programs in China. Large DNA and RNA super-pools were genotyped by Wheat SNP Array and sequenced by Illumina HiSeq, respectively. Hundreds of thousands of SNPs were identified and then filtered by multiple filtering criteria. Among selected SNPs, over 900 were found within an overlapping interval of less than 30 Mb as the Yr26 candidate genomic region in the centromeric region of chromosome arm 1BL. The 235 chromosome-specific SNPs were converted into KASP assays to validate the Yr26 interval in different genetic populations. Using a high-resolution mapping population (>?30,000 gametes), we confined Yr26 to a 0.003-cM interval. The Yr26 target region was anchored to the common wheat IWGSC RefSeq v1.0 and wild emmer WEWSeq v.1.0 sequences, from which 488 and 454 kb fragments were obtained. Several candidate genes were identified in the target genomic region, but there was no typical resistance gene in either genome region. Haplotype analysis identified specific SNPs linked to Yr26 and developed robust and breeder-friendly KASP markers. This integration strategy can be applied to accelerate generating many markers closely linked to target genes/QTL for a trait of interest in wheat and other polyploid species.

     

Membrane-associated phosphoinositides-specific phospholipase C forms from <Emphasis Type="Italic">Catharanthus roseus</Emphasis> transformed roots

We have previously reported that Catharanthus roseus transformed roots contain at least two phosphatidylinositol 4,5-bisphosphate-phospholipase C (PLC) activities, one soluble and the other membrane associated. Detergent, divalent cations, and neomycin differentially regulate these activities and pure protein is required for a greater understanding of the function and regulation of this enzyme. In this article we report a partia purification of membrane-associated PLC. We found that there are at least two forms of membrane-associated PLC in transformed roots of C. roseus. These forms were separated on the basis of their affinity for heparin. One form shows an affinity for heparin and elutes at approx 600 mM KCl. This form has a molecular mass of 67 kDa by size exclusion chromatography and Western blot analysis, whereas the other form does not bind to heparin and has a molecular mass of 57 kDa. Possible differential regulation of these forms during transformed root growth is discussed.     

Suppression of Grb2 expression improved hepatic steatosis, oxidative stress, and apoptosis induced by palmitic acid in vitro partly through insulin signaling alteration

In this study, we aimed to study the role of growth factor receptor-bound protein 2 (Grb2) in palmitic acid-induced steatosis and other “fatty liver” symptoms in vitro. HepG2 cells, with or without stably suppressed Grb2 expression, were incubated with palmitic acid for 24 h to induce typical clinical “fatty liver” features, including steatosis, impaired glucose metabolism, oxidative stress, and apoptosis. MTT and Oil Red O assays were applied to test cell viability and fat deposition, respectively. Glucose uptake assay was used to evaluate the glucose utilization of cells. Quantitative polymerase chain reaction and Western blot were used to measure expressional changes of key markers of insulin signaling, lipid/glucose metabolism, oxidative stress, and apoptosis. After 24-h palmitic acid induction, increased fat accumulation, reduced glucose uptake, impaired insulin signaling, enhanced oxidative stress, and increased apoptosis were observed in HepG2 cells. Suppression of Grb2 in HepG2 significantly reduced fat accumulation, improved glucose metabolism, ameliorated oxidative stress, and restored the activity of insulin receptor substrate-1/Akt and MEK/ERK pathways. In addition, Grb2 deficiency attenuated hepatic apoptosis shown by reduced activation of caspase-3 and fluorescent staining. Modulation of Bcl-2 and Bak1 also contributed to reduced apoptosis. In conclusion, suppression of Grb2 expression in HepG2 cells improved hepatic steatosis, glucose metabolism, oxidative stress, and apoptosis induced by palmitic acid incubation partly though modulating the insulin signaling pathway.     

Differences in the Detection of BrdU/EdU Incorporation Assays Alter the Calculation for G1, S,and G2 Phases of the Cell Cycle in Trypanosomatids

Trypanosomatids are the etiologic agents of various infectious diseases in humans. They diverged early during eukaryotic evolution and have attracted attention as peculiar models for evolutionary and comparative studies. Here, we show a meticulous study comparing the incorporation and detection of the thymidine analogs BrdU and EdU in Leishmania amazonensis, Trypanosoma brucei, and Trypanosoma cruzi to monitor their DNA replication. We used BrdU‐ and EdU‐incorporated parasites with the respective standard detection approaches: indirect immunofluorescence to detect BrdU after standard denaturation (2 M HCl) and “click” chemistry to detect EdU. We found a discrepancy between these two thymidine analogs due to the poor detection of BrdU, which is reflected on the estimative of the duration of the cell cycle phases G1, S, and G2. To solve this discrepancy, we increase the exposure of incorporated BrdU using different concentrations of HCl. Using a new value for HCl concentration, we re‐estimated the phases G1, S, G2 + M, and cytokinesis durations, confirming the values found by this approach using EdU. In conclusion, we suggest that the studies using BrdU with standard detection approach, not only in trypanosomatids but also in others cell types, should be reviewed to ensure an accurate estimation of DNA replication monitoring.     

Synchronous and early activation of planarian Hox genes and the re-specification of body axes during regeneration in Dugesia (G.) tigrina (Turbellaria; Tricladida)

Seven Hox cluster-related genes (Dthox-A to -G) have been isolated from the freshwater triclad Dugesia (G.) tigrina, their sequence compared to other Hox genes and their expression in intact and regenerating organisms analyzed by whole mount in situ hybridization. Sequence comparison analyses show high similarities of D. tigrina Hox genes to anterior and medial groups of coelomate Hox genes. Expression analyses show very early, synchronous, and overlapping expression of Dthox -A, -E, -G and -F in anterior, posterior and lateral regenerative tissues. At one hour of regeneration all Dthox genes studied showed a neat, clear expression at the wound boundary. Later, as the blastema grows, the expression area expands to more proximal regions covering the blastema and the distal postblastema regions. Blastemas formed by intercalary regeneration also show a synchronous expression of the same Hox genes though the onset of activation is much delayed. The finding that the same set of Hox genes is synchronously activated in anterior, posterior, intercalary and lateral regeneration is in sharp contrast to its well established role in specifying antero-posterior pattern during embryonic development. The implications of these results as regards ancestral versus co-opted roles of Hox genes in development and regeneration are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Ursolic acid prevents doxorubicin‐induced cardiac toxicity in mice through eNOS activation and inhibition of eNOS uncoupling

In addition to the known antitumour effects of ursolic acid (UA), increasing evidence indicates that this molecule plays a role in cardiac protection. In this study, the effects of ursolic acid on the heart in mice treated with doxorubicin (DOX) were assessed. The results showed that ursolic acid improved left ventrical fractional shortening (LVFS) and left ventrical ejection fraction (LVEF) of the heart, increased nitrogen oxide (NO) levels, inhibited reactive oxygen species (ROS) production and decreased cardiac apoptosis in mice treated with doxorubicin. Mechanistically, ursolic acid increased AKT and endothelial nitric‐oxide synthase (eNOS) phosphorylation levels, and enhanced eNOS expression, while inhibiting doxorubicin induced eNOS uncoupling through NADPH oxidase 4 (NOX4) down‐regulation. These effects of ursolic acid resulted in heart protection from doxorubicin‐induced injury. Therefore, ursolic acid may be considered a potential therapeutic agent for doxorubicin‐associated cardiac toxicity in clinical practice.