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.

     

Association of myostatin deficiency with collagen related disease-umbilical hernia and tippy toe standing in pigs

Transgenic Research - Herein, we investigate the high incidence of umbilical hernia and tippy-toe standing and their underlying changes in gene expression and proliferation in myostatin knockout...     

Autocrine tumor necrosis factor alpha links endoplasmic reticulum stress to the membrane death receptor pathway through IRE1alpha-mediated NF-kappaB activation and down-regulation of TRAF2 expression

NF-kappaB is critical for determining cellular sensitivity to apoptotic stimuli by regulating both mitochondrial and death receptor apoptotic pathways. The endoplasmic reticulum (ER) emerges as a new apoptotic signaling initiator. However, the mechanism by which ER stress activates NF-kappaB and its role in regulation of ER stress-induced cell death are largely unclear. Here, we report that, in response to ER stress, IKK forms a complex with IRE1alpha through the adapter protein TRAF2. ER stress-induced NF-kappaB activation is impaired in IRE1alpha knockdown cells and IRE1alpha(-/-) MEFs. We found, however, that inhibiting NF-kappaB significantly decreased ER stress-induced cell death in a caspase-8-dependent manner. Gene expression analysis revealed that ER stress-induced expression of tumor necrosis factor alpha (TNF-alpha) was IRE1alpha and NF-kappaB dependent. Blocking TNF receptor 1 signaling significantly inhibited ER stress-induced cell death. Further studies suggest that ER stress induces down-regulation of TRAF2 expression, which impairs TNF-alpha-induced activation of NF-kappaB and c-Jun N-terminal kinase and turns TNF-alpha from a weak to a powerful apoptosis inducer. Thus, ER stress induces two signals, namely TNF-alpha induction and TRAF2 down-regulation. They work in concert to amplify ER-initiated apoptotic signaling through the membrane death receptor.     

Genetic co-transfer of CCR7 ligands enhances immunity and prolongs survival against virulent challenge of pseudorabies virus

The CC chemokine receptor 7 (CCR7) and cognate CCR7 ligands, CCL19 and CCL21, help establish microenvironments in lymphoid tissue that can facilitate encounters between naive T cells and mature dendritic cells (DCs). This study was conducted to determine if CCR7 ligands can augment the immunogenicity of a DNA vaccine that expresses glycoprotein B (gB) of the pseudorabies virus (PrV). The genetic co-transfer of CCR7 ligands along with a PrV DNA vaccine increased the levels of serum PrV-specific immunoglobulin (Ig) G by 2- to 2.5-fold. In addition, the level of PrV-specific IgG2a isotype was significantly enhanced by co-injection of CCR7 ligand DNA, which indicates that CCR7 ligand biases the humoral immunity toward the Th1-type pattern. The co-injection of CCR7 ligand DNA consistently enhanced the level of Th1-type cytokines (IL-2 and IFN-gamma) produced by stimulated immune cells when compared with a group that was vaccinated with the PrV DNA vaccine. Also, the genetic co-transfer of CCR7 ligand DNAs with PrV DNA vaccine provided prolonged survival against a virulent challenge by PrV. Moreover, the co-administration of CCR7 ligand DNA increased the number of mature DCs into the secondary lymphoid tissues, which appeared to enhance the proliferation of PrV-immune CD4(+) T cells. Taken together, these findings indicate that CCR7 ligands are an attractive adjuvant for a PrV DNA vaccine that can offer protective immunity against the PrV.     

A triple mutation in the second transmembrane domain of mouse dopamine transporter markedly decreases sensitivity to cocaine and methylphenidate

Previously, we reported that Phe105 in transmembrane domain 2 of the mouse dopamine transporter (DAT) is crucial for high-affinity cocaine binding. In the current study, we investigated whether other residues surrounding Phe105 also affect the potency of cocaine inhibition. After three rounds of sequential random mutagenesis at these residues, we found a triple mutant (L104V, F105C and A109V) of mouse DAT that retained over 50% uptake activity and was 69-fold less sensitive to cocaine inhibition when compared with the wild-type mouse DAT. The triple mutation also resulted in a 47-fold decrease in sensitivity to methylphenidate inhibition, suggesting that the binding sites for cocaine and methylphenidate may overlap. In contrast, the inhibition of dopamine uptake by amphetamine or methamphetamine was not significantly changed by the mutations, suggesting that the binding sites for the amphetamines differ from those for cocaine and methylphenidate. Such functional but cocaine-insensitive DAT mutants can be used to generate a knock-in mouse line to study the role of DAT in cocaine addiction.     

Temporary effect of postharvest UV-C irradiation on gene expression profile in tomato fruit

To obtain an overall view on gene expression during the early stage (24 h) of tomato fruit in response to postharvest UV-C irradiation (4 kJ/m(2)), we performed a microarray analysis by using Affymetrix Tomato Genechip. The results showed that 274 and 403 genes were up- or down-regulated, respectively, more than two folds in postharvest tomato fruit irradiated with UV-C as compared with that in control fruit. The up-regulated genes mainly involve in signal transduction, defense response and metabolism. Conversely, genes related to cell wall disassembly, photosynthesis and lipid metabolism were generally down-regulated. These results opened ways to probe into the molecular mechanisms of the effects of postharvest UV-C irradiation on increased disease resistance, delayed softening, better quality maintenance and prolonged postharvest life in tomato fruit.     

<Emphasis Type="Italic">Sphingomonas humi</Emphasis> sp. nov., isolated from soil

A Gram-negative, non-motile, non-spore-forming, small, orange, rod-shaped bacterium was isolated from soil in South Korea and characterized to determine its taxonomic position. Phylogenetic analysis based on 16S rRNA gene sequence examination revealed that strain PB323^T belongs to the family Sphingomonadaceae. The highest degree of sequence similarity was found with Sphingomonas kaistensis PB56^T (98.9%), followed by Sphingomonas astaxanthinifaciens TDMA-17^T (98.3%). Chemotaxonomic characteristics (the G+C content of the genomic DNA 69.0 mol%, Q-10 quinone system, C_18:1 ω7c/ω9t/ω12t, C_16:1 ω7c/C_15:0 iso 2OH, C_17:1 ω6c, and C16:0 as the major fatty acids) corroborated assignment of strain PB323^T to the genus Sphingomonas. Results of physiological and biochemical tests clearly demonstrate that strain PB323^T represents a distinct species and support its affiliation with the genus Sphingomonas. Based on these data, PB323^T (=KCTC 12341^T =JCM 16603T =KEMB 9004-003^T) should be classified as a type strain of a novel species, for which the name Sphingomonas humi sp. nov. is proposed.