Construction and application for QTL analysis of a Restriction Site Associated DNA (RAD) linkage map in barley

<p>Abstract</p> <p>Background</p> <p>Linkage maps are an integral resource for dissection of complex genetic traits in plant and animal species. Canonical map construction follows a well-established workflow: an initial discovery phase where genetic markers are mined from a small pool of individuals, followed by genotyping of selected mapping populations using sets of marker panels. A newly developed sequence-based marker technology, Restriction site Associated DNA (RAD), enables synchronous single nucleotide polymorphism (SNP) marker discovery and genotyping using massively parallel sequencing. The objective of this research was to assess the utility of RAD markers for linkage map construction, employing barley as a model system. Using the published high density EST-based SNP map in the Oregon Wolfe Barley (OWB) mapping population as a reference, we created a RAD map using a limited set of prior markers to establish linakge group identity, integrated the RAD and prior data, and used both maps for detection of quantitative trait loci (QTL).</p> <p>Results</p> <p>Using the RAD protocol in tandem with the Illumina sequence by synthesis platform, a total of 530 SNP markers were identified from initial scans of the OWB parental inbred lines - the "dominant" and "recessive" marker stocks - and scored in a 93 member doubled haploid (DH) mapping population. RAD sequence data from the structured population was converted into allele genotypes from which a genetic map was constructed. The assembled RAD-only map consists of 445 markers with an average interval length of 5 cM, while an integrated map includes 463 RAD loci and 2383 prior markers. Sequenced RAD markers are distributed across all seven chromosomes, with polymorphic loci emanating from both coding and noncoding regions in the <it>Hordeum </it>genome. Total map lengths are comparable and the order of common markers is identical in both maps. The same large-effect QTL for reproductive fitness traits were detected with both maps and the majority of these QTL were coincident with a dwarfing gene (<it>ZEO) </it>and the <it>VRS1 </it>gene, which determines the two-row and six-row germplasm groups of barley.</p> <p>Conclusions</p> <p>We demonstrate how sequenced RAD markers can be leveraged to produce high quality linkage maps for detection of single gene loci and QTLs. By combining SNP discovery and genotyping into parallel sequencing events, RAD markers should be a useful molecular breeding tool for a range of crop species. Expected improvements in cost and throughput of second and third-generation sequencing technologies will enable more powerful applications of the sequenced RAD marker system, including improvements in <it>de novo </it>genome assembly, development of ultra-high density genetic maps and association mapping.</p

Inscuteable Regulates the Pins-Mud Spindle Orientation Pathway

During asymmetric cell division, alignment of the mitotic spindle with the cell polarity axis ensures that the cleavage furrow separates fate determinants into distinct daughter cells. The protein Inscuteable (Insc) is thought to link cell polarity and spindle positioning in diverse systems by binding the polarity protein Bazooka (Baz; aka Par-3) and the spindle orienting protein Partner of Inscuteable (Pins; mPins or LGN in mammals). Here we investigate the mechanism of spindle orientation by the Insc-Pins complex. Previously, we defined two Pins spindle orientation pathways: a complex with Mushroom body defect (Mud; NuMA in mammals) is required for full activity, whereas binding to Discs large (Dlg) is sufficient for partial activity. In the current study, we have examined the role of Inscuteable in mediating downstream Pins-mediated spindle orientation pathways. We find that the Insc-Pins complex requires Gαi for partial activity and that the complex specifically recruits Dlg but not Mud. In vitro competition experiments revealed that Insc and Mud compete for binding to the Pins TPR motifs, while Dlg can form a ternary complex with Insc-Pins. Our results suggest that Insc does not passively couple polarity and spindle orientation but preferentially inhibits the Mud pathway, while allowing the Dlg pathway to remain active. Insc-regulated complex assembly may ensure that the spindle is attached to the cortex (via Dlg) before activation of spindle pulling forces by Dynein/Dynactin (via Mud)

Van Gogh and Frizzled Act Redundantly in the Drosophila Sensory Organ Precursor Cell to Orient Its Asymmetric Division

Drosophila sensory organ precursor cells (SOPs) divide asymmetrically along the anterior-posterior (a-p) body axis to generate two different daughter cells. Planar Cell Polarity (PCP) regulates the a-p orientation of the SOP division. The localization of the PCP proteins Van Gogh (Vang) and Frizzled (Fz) define anterior and posterior apical membrane domains prior to SOP division. Here, we investigate the relative contributions of Vang, Fz and Dishevelled (Dsh), a membrane-associated protein acting downstream of Fz, in orienting SOP polarity. Genetic and live imaging analyses suggest that Dsh restricts the localization of a centrosome-attracting activity to the anterior cortex and that Vang is a target of Dsh in this process. Using a clone border assay, we provide evidence that the Vang and fz genes act redundantly in SOPs to orient its polarity axis in response to extrinsic local PCP cues. Additionally, we find that the activity of Vang is dispensable for the non-autonomous polarizing activity of fz. These observations indicate that both Vang and Fz act as cues for downstream effectors orienting the planar polarity axis of dividing SOPs

Structure of an Enzyme-Derived Phosphoprotein Recognition Domain

Membrane Associated Guanylate Kinases (MAGUKs) contain a protein interaction domain (GKdom) derived from the enzyme Guanylate Kinase (GKenz). Here we show that GKdom from the MAGUK Discs large (Dlg) is a phosphoprotein recognition domain, specifically recognizing the phosphorylated form of the mitotic spindle orientation protein Partner of Inscuteable (Pins). We determined the structure of the Dlg-Pins complex to understand the dramatic transition from nucleotide kinase to phosphoprotein recognition domain. The structure reveals that the region of the GKdom that once served as the GMP binding domain (GBD) has been co-opted for protein interaction. Pins makes significantly more contact with the GBD than does GMP, but primarily with residues that are conserved between enzyme and domain revealing the versatility of the GBD as a platform for nucleotide and protein interactions. Mutational analysis reveals that the GBD is also used to bind the GK ligand MAP1a, suggesting that this is a common mode of MAGUK complex assembly. The GKenz undergoes a dramatic closing reaction upon GMP binding but the protein-bound GKdom remains in the ‘open’ conformation indicating that the dramatic conformational change has been lost in the conversion from nucleotide kinase to phosphoprotein recognition domain

Waumananyi: The Song on the Wind

Uti Kulintjaku [UK] is an innovative, Aboriginal-led mental health literacy project that takes its name from a Pitjantjatjara phrase that means ‘to listen, think and understand clearly’. Formed from the Ngangkari traditional healers and artists of the NPY Women’s Council, the UK addresses community issues of mental health from both Aboriginal and Western perspectives. “What makes (the UK project) especially compelling”, writes Kim Mahood in The Monthly, “is that it provides a framework for a conversation about the underlying psychological forces that drive human behaviour.”Working with the Big Anxiety and fEEL, the Uti Kulintjaku team have created two new virtual reality works, sharing their healing practices through creative visualisation, including Waumananyi: The Song on the Wind, an Anangu-led response to the experiences of constraint, entrapment, and depression through the traditional story (or ‘tjukurpa’) of ‘The Man in the Log’.Thes VR work was presented at:Winda Film Festival, University of Technology, Sydney. 21/11/2019 - 24/11/2019.Siggraph Asia, Brisbane Convention & Exhibition Centre, Sydney. 17/11/2019 - 20/11/2019.VRST 25th ACM Symposium on Virtual Reality Software and Technology, Western Sydney University, Parramatta. 12/11/2019 - 15/11/2019.Pop up exhibition, UNSW Library, Sydney. 28/10/2019 - 31/10/2019.Art after Hours. The Big Anxiety virtual reality station., Art Gallery NSW, Sydney. 16/10/2019.The DAX Centre, University of Melbourne, Melbourne. 10/10/2019 - 08/11/2019.Big Anxiety Festival, UNSW Art and Design, Sydney. 27/09/2019 - 09/11/2019

Uti Kulintjaku meditation VR

The Uti Kulintjaku [UK] is an innovative, Aboriginal-led mental health literacy project that takes its name from a Pitjantjatjara phrase that means ‘to listen, think and understand clearly’. Formed from the Ngangkari traditional healers and artists of the NPY Women’s Council, the UK addresses community issues of mental health from both Aboriginal and Western perspectives. “What makes (the UK project) especially compelling”, writes Kim Mahood in The Monthly, “is that it provides a framework for a conversation about the underlying psychological forces that drive human behaviour.”Working with the Big Anxiety and fEEL, the Uti Kulintjaku team have created two new virtual reality works, sharing their healing practices through creative visualisation, including Waumananyi: The Song on the Wind, an Anangu-led response to the experiences of constraint, entrapment, and depression through the traditional story (or ‘tjukurpa’) of ‘The Man in the Log’.This VR work was presented at:Pop up exhibition, UNSW Library, Sydney. 28/10/2019 - 31/10/2019.Art after Hours. The Big Anxiety virtual reality station., Art Gallery NSW, Sydney. 16/10/2019.Big Anxiety Festival, UNSW Art and Design, Sydney. 27/09/2019 - 09/11/2019

Asymmetric cortical extension shifts cleavage furrow position in Drosophila neuroblasts

A spindle-independent, myosin II–containing domain causes asymmetric cortical extension during anaphase of asymmetrically dividing Drosophila neuroblasts. Heterotrimeric G-proteins regulate the activity of the myosin II domain, and their loss leads to symmetric expansion and daughter cell size