A robust KASP marker for selection of four pairs of linked leaf rust and stripe rust resistance genes introgressed on chromosome arm 5DS from different wheat genomes

Molecular Biology Reports - Stripe rust and leaf rust are among the most devastating diseases of wheat, limiting its production globally. Wheat wild relatives harbour genetic diversity for new...     

Mapping of flag smut resistance in common wheat

Flag smut, caused by Urocystis agropyri, has been a problem in wheat production, but its incidence has declined with the use of resistant varieties and seed dressing. Diamondbird, an Australian wheat cultivar that carries high levels of resistance to flag smut, was crossed with susceptible Chinese landrace TH3929 and a doubled haploid (DH) population was developed. A linkage map comprising 386 markers was used for detection of genomic regions controlling flag smut resistance. Composite interval mapping identified five quantitative trait loci (QTL) with significant effects for flag smut resistance. QTL QFs.sun-3AL, QFs.sun-6AS, QFs.sun-1BL and QFs.sun-5BS were contributed by Diamondbird. Although TH3929 was susceptible, it contributed a minor QTL QFs.sun-3AS. QTL QFs.sun-3AL and QFs.sun-6AS were detected in both seasons and each explained more than 17 % of the variation in flag smut response. Other QTL QFs.sun-3AS, QFs.sun-1BL and QFs.sun-5BS explained 5–10 % of the phenotypic variation. DH lines that showed low flag smut levels carried combinations of three or more QTL. This is the first report on chromosomal location of flag smut resistance in a modern common wheat cultivar.     

Generation and characterization of a lipopolysaccharide-specific murine monoclonal antibody to <Emphasis Type="Italic">Proteus vulgaris</Emphasis> OX19

The three strains of non-pathogenic Proteus species namely, Proteus vulgaris OX2, P. vulgaris OX19 and Proteus mirabilis OXK used in the Weil–Felix test are the group-specific cross-reactive antigens for Rickettsia and Orientia species. Earlier studies have revealed that the group specific and cross-reactive antigens responsible for the Weil–Felix test lie mostly in the lipopolysaccharide (LPS) moiety of the bacterial cell wall [Amano et al. (1993a) Infect Immun 61:4350–4355, (1993b) Microbiol Immunol 37:927–933, (1998) Infect Immun 66:923–926]. The three Proteus strains (OX2, OX19 and OXK) were used to raise murine monoclonal antibodies (MAbs) by hybridoma technology. Several MAb-producing hybridomas could be stabilized following limiting dilution. Affinity and specificity of these MAbs were checked by indirect ELISA using a battery of homologous and heterologous antigens including LPS. Amongst these, one MAb was found to be specific for P. vulgaris OX19 LPS. Since the Weil–Felix reaction is based on the cross-reactivity between the LPS based epitopes, this MAb could be of potential use in mapping of epitopes on the cross-reactive LPS and may also be useful as a potential diagnostic reagent.     

Thidiazuron-induced morphogenesis in tamarind seedlings

Summary Germination of tamarind seeds in medium containing thidiazuron (TDZ) resulted in induction of nodular protrusions in and around the cotyledonary node meristem. The structures developed radially in well-defined circles and subsequently spread towards the cotyledonary bridge and also in the proximal part of the hypocotyl. The structures developed into shoots on transfer to medium devoid of growth regulators. Histological studies revealed that the protrusions initiated from the nodal meristem and extended to the non-meristematic region between the two meristems and also in the proximal part of the hypocotyl in seedlings germinated in 9.08 μM TDZ. Newly formed cell layers and less-differentiated meristematic protrusions were also seen. With the increase in the distance from the meristem, the buds were less differentiated; in the proximal part of the hypocotyl only the multiple layers of meristematic cells were noted. With extension of the period of incubation, the TDZ-induced meristematic activity extended laterally in circles towards the neighboring region. The radial spread of the meristematic activity from the center of the nodal meristem was also evident at 18.16 μM TDZ. From the pattern of the morphogenic development and the histological studies it may be hypothesized that in tamarind, TDZ influences the existing meristems specifically. Subsequently de novo organogenesis is triggered in the neighboring cells.     

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.