Detection and validation of genomic regions associated with resistance to rust diseases in a worldwide hexaploid wheat landrace collection using BayesR and mixed linear model approaches

Key message

BayesR and MLM association mapping approaches in common wheat landraces were used to identify genomic regions conferring resistance to Yr, Lr, and Sr diseases.

Abstract

Deployment of rust resistant cultivars is the most economically effective and environmentally friendly strategy to control rust diseases in wheat. However, the highly evolving nature of wheat rust pathogens demands continued identification, characterization, and transfer of new resistance alleles into new varieties to achieve durable rust control. In this study, we undertook genome-wide association studies (GWAS) using a mixed linear model (MLM) and the Bayesian multilocus method (BayesR) to identify QTL contributing to leaf rust (Lr), stem rust (Sr), and stripe rust (Yr) resistance. Our study included 676 pre-Green Revolution common wheat landrace accessions collected in the 1920–1930s by A.E. Watkins. We show that both methods produce similar results, although BayesR had reduced background signals, enabling clearer definition of QTL positions. For the three rust diseases, we found 5 (Lr), 14 (Yr), and 11 (Sr) SNPs significant in both methods above stringent false-discovery rate thresholds. Validation of marker–trait associations with known rust QTL from the literature and additional genotypic and phenotypic characterisation of biparental populations showed that the landraces harbour both previously mapped and potentially new genes for resistance to rust diseases. Our results demonstrate that pre-Green Revolution landraces provide a rich source of genes to increase genetic diversity for rust resistance to facilitate the development of wheat varieties with more durable rust resistance.

     

Target cell directed NK inactivation. Concomitant loss of NK and antibody-dependent cellular cytotoxicity activities

We investigated the inactivation of human NK cells, a population of large granular lymphocytes (LGL), with K562, an NK-sensitive target cell (TC) and KLCL, an NK-resistant TC, but which can be lysed by NK cells via antibody (Ab)-dependent cellular cytotoxicity. NK-enriched effector cells (ECc) were first treated with either K562 or Ab-coated KLCL (Ab-KLCL). After incubation, ECc were separated from their TC then examined for residual NK and ADCC activities, phenotypic changes, and changes in LGL morphology. K562-treated ECc and Ab-KLCL-treated ECc, when retested against the inactivating TC, respectively, lost greater than 90% of their lytic activities. However, K562-treated ECc lost 60 to 70% of their activity against Ab-KLCL, whereas Ab-KLCL-treated ECc lost less than 10% of their activity against K562. In contrast to what we observed with K562-treated ECc, we detected significant reductions in plasma membrane expression of Leu-11a and Leu-11b on Ab-KLCL-treated ECc. Although the proportion of OKM1+ cells remained unchanged after the inactivation process, the density of OKM1 on both K562-treated ECc and Ab-KLCL-treated ECc increased significantly. Morphologic analysis revealed no apparent differences in the percentages of LGL before and after treatment with K562 or Ab-KLCL. Finally, IL-2 restored lytic potential to both K562-treated ECc and Ab-KLCL-treated ECc and, in addition, IL-2-induced enhancement of Ab-KLCL-treated ECc was accompanied by a partial reexpression of Leu-11a. These data support the hypothesis that NK-cell-mediated cytotoxicity and antibody-dependent cellular cytotoxicity may result from a common lytic mechanism, although the initiation steps and regulation of the pathway are distinct.     

Pulsed 86Sr-labeling and NanoSIMS imaging to study coral biomineralization at ultra-structural length scales

A method to label marine biocarbonates is developed based on a concentration enrichment of a minor stable isotope of a trace element that is a natural component of seawater, resulting in the formation of biocarbonate with corresponding isotopic enrichments. This biocarbonate is subsequently imaged with a NanoSIMS ion microprobe to visualize the locations of the isotopic marker on sub-micrometric length scales, permitting resolution of all ultra-structural details. In this study, a scleractinian coral, Pocillopora damicornis, was labeled 3 times with ⁸⁶Sr-enhanced seawater for a period of 48?h with 5?days under normal seawater conditions separating each labeling event. Two non-specific cellular stress biomarkers, glutathione-S-transferase activity and porphyrin concentration plus carbonic anhydrase, an enzymatic marker involved in the physiology of carbonate biomineralization, as well as unchanged levels of zooxanthellae photosynthesis efficiency indicate that coral physiological processes are not affected by the ⁸⁶Sr-enhancement. NanoSIMS images of the ⁸⁶Sr/⁴⁴Ca ratio in skeleton formed during the experiment allow for a determination of the average extension rate of the two major ultra-structural components of the coral skeleton: Rapid Accretion Deposits are found to form on average about 4.5 times faster than Thickening Deposits. The method opens up new horizons in the study of biocarbonate formation because it holds the potential to observe growth of calcareous structures such as skeletons, shells, tests, spines formed by a wide range of organisms under essentially unperturbed physiological conditions.     

Effective cytochrome P450 (CYP) inhibitor isolated from thyme (Thymus saturoides) purchased from a Japanese market

A highly polymethylated flavone that effectively inhibited cytochrome P450s (CYPs) 1A2 and 3A4 (IC(50) = 2.41 and 1.71 μM) in vitro was isolated from thyme leaves (Thymus saturoides) purchased from a Japanese market. Its structure was spectroscopically identified as 4',5-dihydroxy-3',6,7,8-tetramethoxy flavone (8-methoxycirsilineol, 1). This is the first report describing a strong inhibitor of CYP1A2 and 3A4 isolated from Thymus saturoides.     

Development of wheat–Aegilops speltoides recombinants and simple PCR-based markers for Sr32 and a new stem rust resistance gene on the 2S#1 chromosome

Key message

Wheat– Aegilops speltoides recombinants carrying stem rust resistance genes Sr32 and SrAes1t effective against Ug99 and PCR markers for marker-assisted selection.

Abstract

Wild relatives of wheat are important resources for new rust resistance genes but underutilized because the valuable resistances are often linked to negative traits that prevent deployment of these genes in commercial wheats. Here, we report ph1b-induced recombinants with reduced alien chromatin derived from E.R. Sears’ wheat–Aegilops speltoides 2D-2S#1 translocation line C82.2, which carries the widely effective stem rust resistance gene Sr32. Infection type assessments of the recombinants showed that the original translocation in fact carries two stem rust resistance genes, Sr32 on the short arm and a previously undescribed gene SrAes1t on the long arm of chromosome 2S#1. Recombinants with substantially shortened alien chromatin were produced for both genes, which confer resistance to stem rust races in the TTKSK (Ug99) lineage and representative races of all Australian stem rust lineages. Selected recombinants were back crossed into adapted Australian cultivars and PCR markers were developed to facilitate the incorporation of these genes into future wheat varieties. Our recombinants and those from several other labs now show that Sr32, Sr39, and SrAes7t on the short arm and Sr47 and SrAes1t on the long arm of 2S#1 form two linkage groups and at present no rust races are described that can distinguish these resistance specificities.     

A new leaf rust resistance gene <Emphasis Type="Italic">Lr79</Emphasis> mapped in chromosome 3BL from the durum wheat landrace Aus26582

Key message

A new leaf rust resistance gene Lr79 has been mapped in the long arm of chromosome 3B and a linked marker was identified for marker-assisted selection.

Abstract

Aus26582, a durum wheat landrace from the A. E. Watkins Collection, showed seedling resistance against durum-specific and common wheat-specific Puccinia triticina (Pt) pathotypes. Genetic analysis using a recombinant inbred line (RIL) population developed from a cross between Aus26582 and the susceptible parent Bansi with Australian Pt pathotype showed digenic inheritance and the underlying loci were temporarily named LrAW2 and LrAW3. LrAW2 was located in chromosome 6BS and this study focused on characterisation of LrAW3 using RILs lacking LrAW2. LrAW3 was incorporated into the DArTseq map of Aus26582/Bansi and was located in chromosome 3BL. Markers linked with LrAW3 were developed from the chromosome survey sequence contig 3B_10474240 in which closely-linked DArTseq markers 1128708 and 3948563 were located. Although bulk segregant analysis (BSA) with the 90 K Infinium array identified 51 SNPs associated with LrAW3, only one SNP-derived KASP marker mapped close to the locus. Deletion bin mapping of LrAW3-linked markers located LrAW3 between bins 3BL11-0.85-0.90 and 3BL7-0.63. Since no other all stage leaf rust resistance gene is located in chromosome 3BL, LrAW3 represented a new locus and was designated Lr79. Marker sun786 mapped 1.8 cM distal to Lr79 and Aus26582 was null for this locus. However, the marker can be reliably scored as it also amplifies a monomorphic fragment that serves as an internal control to differentiate the null status of Aus26582 from reaction failure. This marker was validated among a set of durum and common wheat cultivars and was shown to be useful for marker-assisted selection of Lr79 at both ploidy levels.

     

Role of lipoxygenation in human natural killer cell activation

Nordihydroguaiaretic acid (NDGA), quercetin, eicosatetraynoic acid (ETYA), phenidone, and esculetin, agents known to inhibit cellular lipoxygenase (LO) activity, also inhibit human natural killer cell-mediated cytotoxicity (NK-CMC) of K562 tumor target cells (TC) in a dose-dependent fashion. Kinetic analysis demonstrated that LO inhibitors blocked an early event in the activation of the lytic mechanism but did not impair conjugate formation. LO inhibitors also did not affect subsequent chromium release, indicating that their site of inhibition was the NK cell and not the TC. The lipoxygenase products 5-hydroperoxyeicosatetraenoic acid (5-HPETE) and leukotriene-B4 significantly enhanced NK activity, with 5-HPETE being the more effective. Other LO products tested included 15-HPETE and the hydroxy derivatives 15-hydroxyeicosatetraenoic acid (15-HETE) and 5-HETE. These LO metabolites were either without effect on NK-CMC or inhibitory, depending upon the concentration. Additionally, we examined the ability of 5-HPETE to circumvent the effects of LO inhibitors and found that, in the presence of NDGA, ETYA or quercetin, 5-HPETE significantly (p less than 0.001) restored lytic activity. Inhibitors of LTB4 and LTC4 synthesis, diethylcarbamazine and U-60,257 respectively, produced no inhibition of NK activity. In fact, U-60,257 significantly (p less than 0.05) enhanced NK-CMC. Previous studies in our laboratory, with a new technique which allows for the separation of NK cells from K562 cells, have shown that K562-treated effector cells are greater than 90% inactivated when retested against fresh K562 in the standard chromium release assay. Lipids were extracted from K562-treated, Percoll-purified LGL and evaluated by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). No significant increases were seen in the arachidonic acid-derived LO products evaluated. Thus, our studies indicate that lipoxygenation may be required in the activation of NK-CMC, possibly as a means to generate oxygen radicals which have been previously implicated in NK-CMC.     

A comparison of lung metastases and natural killer cell activity in daily fractions and weekly fractions of radiation therapy on murine B16a melanoma

C57BL/6J male mice were inoculated with 5 X 10(5) B16a melanoma cells. Seven days post-inoculation, when the tumor had grown to 8.0-10.0 mm in diameter, 120 tumor-bearing mice were randomly divided into three groups: (1) sham-irradiated controls, (2) mice receiving 200 cGy five times a week for 6 weeks, and (3) mice receiving 800 cGy once a week for 4 weeks. Thirty mice in each group were sacrificed 47 days postinoculation. Ten mice in each group were observed for the survival time data. The primary tumor was significantly smaller and the number of lung metastases were significantly fewer in mice treated with 800 cGy once a week compared to mice treated with 200 cGy five times a week. When natural killer (NK) cell activity was assessed against YAC-1 tumor targets, it was found to be significantly higher in mice treated with a single large weekly dose of irradiation. These results show that B16a melanoma responds more favorably to a single large dose of irradiation administered once a week compared to the smaller conventional fraction administered five times a week. This beneficial effect correlates with an increase in NK activity, indicating that there may be a causal relationship.     

Characterization of <Emphasis Type="Italic">Lr75</Emphasis>: a partial,broad-spectrum leaf rust resistance gene in wheat

Key message

Here, we describe a strategy to improve broad-spectrum leaf rust resistance by marker-assisted combination of two partial resistance genes. One of them represents a novel partial adult plant resistance gene, named Lr75.

Abstract

Leaf rust caused by the fungal pathogen Puccinia triticina is a damaging disease of wheat (Triticum aestivum L.). The combination of several, additively-acting partial disease resistance genes has been proposed as a suitable strategy to breed wheat cultivars with high levels of durable field resistance. The Swiss winter wheat cultivar ‘Forno’ continues to show near-immunity to leaf rust since its release in the 1980s. This resistance is conferred by the presence of at least six quantitative trait loci (QTL), one of which is associated with the morphological trait leaf tip necrosis. Here, we used a marker-informed strategy to introgress two ‘Forno’ QTLs into the leaf rust-susceptible Swiss winter wheat cultivar ‘Arina’. The resulting backcross line ‘ArinaLrFor’ showed markedly increased leaf rust resistance in multiple locations over several years. One of the introgressed QTLs, QLr.sfr-1BS, is located on chromosome 1BS. We developed chromosome 1B-specific microsatellite markers by exploiting the Illumina survey sequences of wheat cv. ‘Chinese Spring’ and mapped QLr.sfr-1BS to a 4.3 cM interval flanked by the SSR markers gwm604 and swm271. QLr.sfr-1BS does not share a genetic location with any of the described leaf rust resistance genes present on chromosome 1B. Therefore, QLr.sfr-1BS is novel and was designated as Lr75. We conclude that marker-assisted combination of partial resistance genes is a feasible strategy to increase broad-spectrum leaf rust resistance. The identification of Lr75 adds a novel and highly useful gene to the small set of known partial, adult plant leaf rust resistance genes.