Development of co-dominant KASP markers co-segregating with Ug99 effective stem rust resistance gene <Emphasis Type="Italic">Sr26</Emphasis> in wheat

Stem rust of wheat, caused by Puccinia graminis f. sp. tritici (Pgt), is a threat to global food security due to its ability to cause total crop failures. The Pgt race TTKSK (Ug99) and its derivatives detected in East Africa carry virulence for many resistance genes present in modern cultivars. However, stem rust resistance gene Sr26 remains effective to all races of Pgt worldwide. Sr26 is carried on the Agropyron elongatum (syn. Thinopyrum ponticum) segment 6Ae#1L translocated to chromosome 6AL of wheat. In this study, a recombinant inbred line (RIL) population derived from a cross between the landrace Aus27969 and Avocet S, which carries Sr26, was used to develop co-dominant kompetitive allele-specific polymerase chain reaction (KASP) markers that co-segregate with Sr26. Four KASP markers (sunKASP_216, sunKASP_218, sunKASP_224 and sunKASP_225) were also shown to co-segregate with Sr26 in four additional RIL populations. When tested on Australian cultivars and breeding lines, these markers amplified alleles alternate to that linked with Sr26 in all cultivars known to lack this gene and Sr26-linked alleles in cultivars and genotypes known to carry Sr26. Genotypes WA-1 and WA-1/3*Yitpi carrying the shortest Sr26 translocation segment were positive only for markers sunKASP_224 and sunKASP_225. Our results suggest the four KASP markers are located on the original translocation and sunKASP_224 and sunKASP_225 are located on the shortened version. Therefore, sunKASP_224 and sunKASP_225 can be used for marker-assisted pyramiding of Sr26 with other stem rust resistance genes to achieve durable resistance in wheat.     

Molecular markers for adult plant leaf rust resistance gene <Emphasis Type="Italic">Lr48</Emphasis> in wheat

Leaf rust of wheat, caused by Puccinia triticina, is an important disease throughout the world. The adult plant leaf rust resistance gene Lr48 reported in CSP44 was previously mapped in chromosome 2B, but the marker–gene association was weak. In this study, we confirmed the location of Lr48 to be in the short arm of chromosome 2B and identified closely linked markers suitable for use in breeding. The CSP44/WL711 recombinant inbred line (RIL) population (90 lines) showed monogenic segregation for Lr48. Twelve resistant and 12 susceptible RILs were used for selective genotyping using an iSelect 90K Infinium SNP assay. Closely linked SNPs were converted into Kompetitive allele-specific primers (KASP) and tested on the parental lines. KASP markers giving clear clusters for alternate genotypes were assayed on the entire RIL population. SNP markers IWB31002, IWB39832, IWB34324, IWB72894 and IWB36920 co-segregated with Lr48 and the marker IWB70147 was mapped 0.3 cM proximal to this gene. Closely linked KASP markers were tested on a set of Australian and Nordic wheat genotypes. The amplification of SNP alleles alternate to those linked with Lr48 in the majority of the Australian and Nordic wheat genotypes demonstrated the usefulness of these markers for marker-assisted pyramiding of Lr48 with other rust resistance genes.     

Fine mapping of the chromosome 5B region carrying closely linked rust resistance genes <Emphasis Type="Italic">Yr47</Emphasis> and <Emphasis Type="Italic">Lr52</Emphasis> in wheat

Key message

Fine mapping of Yr47 and Lr52 in chromosome arm 5BS of wheat identified close linkage of the marker sun180 to both genes and its robustness for marker-assisted selection was demonstrated.

Abstract

The widely effective and genetically linked rust resistance genes Yr47 and Lr52 have previously been mapped in the short arm of chromosome 5B in two F₃ populations (Aus28183/Aus27229 and Aus28187/Aus27229). The Aus28183/Aus27229 F₃ population was advanced to generate an F₆ recombinant inbred line (RIL) population to identify markers closely linked with Yr47 and Lr52. Diverse genomic resources including flow-sorted chromosome survey sequence contigs representing the orthologous region in Brachypodium distachyon, the physical map of chromosome arm 5BS, expressed sequence tags (ESTs) located in the 5BS6-0.81-1.00 deletion bin and resistance gene analog contigs of chromosome arm 5BS were used to develop markers to saturate the target region. Selective genotyping was also performed using the iSelect 90 K Infinium wheat SNP assay. A set of SSR, STS, gene-based and SNP markers were developed and genotyped on the Aus28183/Aus27229 RIL population. Yr47 and Lr52 are genetically distinct genes that mapped 0.4 cM apart in the RIL population. The SSR marker sun180 co-segregated with Lr52 and mapped 0.4 cM distal to Yr47. In a high resolution mapping population of 600 F₂ genotypes Yr47 and Lr52 mapped 0.2 cM apart and marker sun180 was placed 0.4 cM distal to Lr52. The amplification of a different sun180 amplicon (195 bp) than that linked with Yr47 and Lr52 (200 bp) in 204 diverse wheat genotypes demonstrated its robustness for marker-assisted selection of these genes.

     

Identification of a robust molecular marker for the detection of the stem rust resistance gene Sr45 in common wheat

Key message

Fine mapping of the Ug99 effective stem rust resistance gene Sr45 introgressed into common wheat from the D -genome goatgrass Aegilops tauschii.

Abstract

Stem rust resistance gene Sr45, discovered in Aegilops tauschii, the progenitor of the D -genome of wheat, is effective against commercially important Puccinia graminis f. sp. tritici races prevalent in Australia, South Africa and the Ug99 race group. A synthetic hexaploid wheat (RL5406) generated by crossing Ae. tauschii accession RL5289 (carrying Sr45 and the leaf rust resistance gene Lr21) with a tetraploid experimental line ‘TetraCanthatch’ was previously used as the source in the transfer of these rust resistance genes to other hexaploid cultivars. Previous genetic studies on hexaploid wheats mapped Sr45 on the short arm of chromosome 1D with the following gene order: centromere–Sr45–Sr33–Lr21–telomere. To identify closely linked markers, we fine mapped the Sr45 region in a large mapping population generated by crossing CS1D5406 (disomic substitution line with chromosome 1D of RL5406 substituted for Chinese Spring 1D) with Chinese Spring. Closely linked markers based on 1DS-specific microsatellites, expressed sequence tags and AFLP were useful in the delineation of the Sr45 region. Sequences from an AFLP marker amplified a fragment that was linked with Sr45 at a distance of 0.39 cM. The fragment was located in a bacterial artificial chromosome clone of contig (ctg)2981 of the Ae. tauschii accession AL8/78 physical map. A PCR marker derived from clone MI221O11 of ctg2981 amplified 1DS-specific sequence that harboured an 18-bp indel polymorphism that specifically tagged the Sr45 carrying haplotype. This new Sr45 marker can be combined with a previously reported marker for Lr21, which will facilitate selecting Sr45 and Lr21 in breeding populations.     

Inhibition of human natural killer cell activity by the protein kinase C inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine is an early but post-binding event

Recent evidence has demonstrated a protein kinase C (PKC)-dependent step in cytotoxic T lymphocyte activation. Here, we examined the influence of PKC in the lytic response of human NK cells to K562, an NK-sensitive tumor target cell. We used the known protein kinase inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) and HA1004. H-7 caused a dose-related inhibition of NK cell-mediated cytolysis (CMC) when the inhibitor was present throughout the course of the 3-h chromium release assay. The 50% inhibitory concentration for H-7 was 7 microM. In contrast, HA1004, which exerts a greater inhibitory effect on cyclic nucleotide-dependent protein kinases than PKC, had no effect on NK-CMC. The suppression of NK-CMC by H-7 was not due to inhibition of binding of the effector cells to target cells and could be reversed by the addition of PMA. H-7 was most effective in abrogating NK-CMC when added to the assay within the first 30 min and treatment of the effector and target cells with H-7 resulted in no loss of NK-CMC. Because nearly 50% of the normal NK lytic activity had taken place by 30 min, this suggested that H-7 inhibited an early event. H-7 exerted a dose-related suppression of antibody-dependent cell-mediated cytotoxicity (ADCC) suggesting that NK-CMC and ADCC share the utilization of PKC, however, HA1004 did not inhibit ADCC. Treating NK cells with IL-2 or IFN-beta did not overcome the inhibition of NK-CMC by H-7. In this study, we have thus demonstrated the presence of a PKC-dependent step in NK-CMC and ADCC.     

Characterisation and mapping of adult plant stripe rust resistance in wheat accession Aus27284

Key message

A new adult plant stripe rust resistance gene, Yr80, was identified in a common wheat landrace Aus27284. Linked markers were developed and validated for their utility in marker-assisted selection.

Abstract

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is among the most important constraints to global wheat production. The identification and characterisation of new sources of host plant resistance enrich the gene pool and underpin deployment of resistance gene pyramids in new cultivars. Aus27284 exhibited resistance at the adult plant stage against predominant Pst pathotypes and was crossed with a susceptible genotype Avocet S. A recombinant inbred line (RIL) population comprising 121 lines was developed and tested in the field at three locations in 2016 and two in 2017 crop seasons. Monogenic segregation for adult plant stripe rust response was observed among the Aus27284/Avocet S RIL population and the underlying locus was temporarily designated YrAW11. Bulked-segregant analysis using the Infinium iSelect 90K SNP wheat array placed YrAW11 in chromosome 3B. Kompetitive allele specific PCR (KASP) primers were designed for the linked SNPs and YrAW11 was flanked by KASP_65624 and KASP_53292 (3 cM) proximally and KASP_53113 (4.9 cM) distally. A partial linkage map of the genomic region carrying YrAW11 comprised nine KASP and two SSR markers. The physical position of KASP markers in the pseudomolecule of chromosome 3B placed YrAW11 in the long arm and the location of markers gwm108 and gwm376 in the deletion bin 3BL2-0.22 supported this conclusion. As no other stripe rust resistance locus has been reported in chromosome 3BL, YrAW11 was formally designated Yr80. Marker KASP_ 53113 was polymorphic among 94% of 81 Australian wheat cultivars used for validation.

     

Target cell-directed inactivation and IL-2-dependent reactivation of LAK cells

In a previous study, we demonstrated that human natural killer cells (NK) lost their lytic activity after interaction with a sensitive target. The loss of NK activity also led to the loss of antibody-dependent cellular cytotoxicity (ADCC), prompting us to postulate that NK and ADCC activities may result from a common lytic mechanism. In this study, we examined whether nonadherent lymphocytes cultured 7 days in the presence of IL-2 (lymphokine-activated killer (LAK) cells) could also be inactivated and, subsequently, be reactivated in the presence of IL-2. We tested three populations of effector cells (EC): cells isolated from freshly drawn blood and tested immediately, cells cultured with IL-2 for 18 hr, and LAK cells. Once they have interacted with K562, all three cell populations lost greater than 90% of their NK-like lytic activity (NK-CMC) but only 80% of ADCC. However, when we treated the three cell types with antibody-coated K562, they lost 90-99% of NK-CMC and 90-97% of ADCC. In these inactivated effector cells we also observed: (i) a reduction in membrane expression of C-reactive protein; and (ii) a decrease in the expression of Leu-11a when EC were inactivated with antibody-coated K562. The loss of lytic activity against K562 was accompanied by a concomitant loss of activity against other LAK-sensitive targets as well as against antibody-coated targets (ADCC). In competitive inhibition experiments the inactivated effector cells failed to inhibit normal NK-CMC and ADCC activities mediated by fresh NK cells. As we have shown previously, this target-directed inactivation was not due to cell death or to lack of conjugate formation. Inactivated LAK cells regained their lytic potential when cultured with IL-2 and this effect was time dependent. By 72 hr, LAK cells inactivated with K562 regained 99% NK-CMC and 82% ADCC, whereas LAK cells inactivated with antibody-coated K562 regained only 80% NK-CMC and 70% ADCC. When we treated the effector cells with emetine, a potent inhibitor of protein synthesis, we could still inactivate the effector cells with K562 and with antibody-coated K562 but could not reactivate them with IL-2.