M. J. Hayden

Genetic mapping of 66 new microsatellite (SSR) loci in bread wheat.

Abstract.In hexaploid bread wheat (Triticum aestivum L. em. Thell), ten members of the IWMMN (International Wheat Microsatellites Mapping Network) collaborated in extending the microsatellite (SSR = simple sequence repeat) genetic map. Among a much larger number of microsatellite primer pairs developed as a part of the WMC (Wheat Microsatellite Consortium), 58 out of 176 primer pairs tested were found to be polymorphic between the parents of the ITMI (International Triticeae Mapping Initiative) mapping population W7984 × Opata 85 (ITMIpop). This population was used earlier for the construction of RFLP (Restriction Fragment Length Polymorphism) maps in bread wheat (ITMImap). Using the ITMIpop and a framework map (having 266 anchor markers) prepared for this purpose, a total of 66 microsatellite loci were mapped, which were distributed on 20 of the 21 chromosomes (no marker on chromosome 6D). These 66 mapped microsatellite (SSR) loci add to the existing 384 microsatellite loci earlier mapped in bread wheat.

Mapping of durable adult plant and seedling resistances to stripe rust and stem rust diseases in wheat

Doubled haploid populations of CD87/Katepwa, Cranbrook/Halberd, and Sunco/Tasman were assessed for seedling response to stem rust and stripe rust. The CD87/Katepwa population was also screened as adult plants in the field against stripe rust. The respective parents differed in presence or absence of various stem rust and stripe rust resistance genes. At least 4 resistance loci controlled adult plant resistance to stripe rust in the CD87/Katepwa population, and based on quantitative trait loci mapping results, two of these were contributed by CD87. Pedigree information indicated that these regions correspond to durable adult plant stripe rust resistance genes Yr18 and Yr29. Yr29 was mapped to the distal region of chromosome 1BL. The third gene, contributed by Katepwa, YrKat, was located in chromosome arm 2DS. Sr30 mapped distal to markers abg3 and P36/M61-170 in chromosome arm 5DL. Genes Yr7 and Pbc (completely linked with durable stem rust resistance gene Sr2) showed close associations with markers in chromosome arms 2BL and 3BS, respectively. A distally located genomic region in chromosome 6AS also affected the expression of Pbc. The temperature-sensitive stripe rust resistance gene, YrCK, carried by Sunco showed monogenic inheritance and was located in chromosome arm 2DS. Several markers showed complete association with Triticum timopheevi derived stem rust resistance gene Sr36. Microsatellite markers stm773 and gwm271A were validated on a set of wheat genotypes and were found to be diagnostic for the detection of Sr36. TheSr36-linked Xstm773 allele showed better amplification than the Sr36-linked Xgwm271A allele. These markers could be used for marker assisted identification of Sr36 in breeding populations.

Genetic Diversity in Australian Populations of Puccinia graminis f. sp. avenae

ABSTRACT Sequence-tagged microsatellite profiling was used to develop 110 microsatellites for Puccinia graminis f. sp. tritici (causal agent of wheat stem rust). Low microsatellite polymorphism was exhibited among 10 pathogenically diverse P. graminis f. sp. tritici isolates collected from Australian cereal growing regions over a period of at least 70 years, with two polymorphic loci detected, each revealing two alleles. Limited cross-species amplification was observed for the wheat rust pathogens, P. triticina (leaf rust) and P. striiformis f. sp. tritici (stripe rust). However, very high transferability was revealed with P. graminis f. sp. avenae (causal agent of oat stem rust) isolates. A genetic diversity study of 47 P. graminis f. sp. avenae isolates collected from an Australia-wide survey in 1999, and a historical group of 16 isolates collected from Australian cereal growing regions from 1971 to 1996, revealed six polymorphic microsatellite loci with a total of 15 alleles. Genetic analysis revealed the presence of several clonal lineages and subpopulations in the pathogen population, and wide dispersal of identical races and genotypes throughout Australian cereal-growing regions. These findings demonstrated the dynamic population structure of this pathogen in Australia and concur with the patterns of diversity observed in pathogenicity studies.

Genetic mapping of commercially significant starch characteristics in wheat crosses

Starch properties were measured on the doubled haploid progeny of 2 crosses, one between Cranbrook and Halberd and the other between CD87 and Katepwa. Properties studied included starch peak and final viscosity measured by Rapid Visco Analyser, starch granule size distribution measured by laser light scattering, starch gelatinisation temperature by differential scanning calorimetry, and flour swelling volume. In the Cranbrook × Halberd cross (samples from 2 environments), a highly significant quantitative trait locus (QTL) was located on chromosome 4A for both starch peak viscosity and starch/flour swelling volume, centred around the Wx-B1 locus. In previous studies, this locus has been found to be linked to Japanese noodle quality. The increases in starch peak viscosity and flour swelling volume are derived from the Halberd parent, consistent with the fact that Halberd is null for the Wx-B1 locus on chromosome 4A and is missing the respective granule-bound starch synthase protein, whereas Cranbrook is a wheat line carrying the normal 3 Wx loci. The final starch viscosity also showed an association with the Wx-B1 locus. In the CD87 × Katepwa cross, the progeny showed an association between peak viscosity and a marker on chromosome 7A. This appeared to be near the Wx-A1 locus. In some experiments, flour viscosity showed a highly significant QTL on chromosome 7B, apparently at the same locus as the late maturity α- amylase derived from the Cranbrook parent. Starch gelatinisation onset temperature indicated a significant QTL on chromosomes 2B and 7A (LOD = 2.58 and 3.66, respectively, in interval analyses). Starch gelatinisation peak temperatures indicated a QTL on chromosome 7A, which, although not in the significant (P = 0.05) class based on regression analyses, indicated a LOD score of 3.06 in interval analyses. Heat of gelatinisation (∆H) indicated a suggestive QTL (LRS = 14.5 with a threshold of 14.7 for P < 0.05, LOD = 2.65 for interval analysis), on chromosome 4A, at the Wx-B1 locus with an increased effect coming from the Halberd parent. The A:B granule ratio analysis indicated a significant QTL on chromosome 4B, but this was not observed in all environments and may be due to the fact that the QTL corresponded to the position of a major QTL controlling plant growth. Additional keywords: viscosity, gelatinisation, granule size, genetics, quantitative traits. I M S. h C. K. . A Genetic mapping ofstarch characteristics

Microsatellites as markers for Australian Wheat improvement

Microsatellite markers have been shown to be highly polymorphic and simple to use in hexaploid wheat. This study aimed to establish microsatellites as informative markers for Australian wheat improvement. By screening microsatellites developed as part of the Wheat Microsatellite Consortium and other available microsatellite sources, 257 informative microsatellites for Australian wheat varieties were identified and reported in the Australian National Wheat Molecular Marker Program microsatellite database (http://www.scu.edu.au/research/cpcg/). Of these, 151 microsatellites identifying 172 loci were scored on at least 1 of 4 double haploid mapping populations and were then integrated, where possible, into existing genetic maps. Polymorphism information content values were calculated for most microsatellites to establish a reference for their value for future investigations. The mapping of available microsatellites enhances the quality of the genetic maps and may provide useful genetic markers for traits of interest to the Australian wheat breeding programs.

A new approach to extending the wheat marker pool by anchored PCR amplification of compound SSRS

A study was undertaken to determine the utility in bread wheat of anchored PCR for the development of single locus SSR markers targeted at compound repeat motifs. In anchored PCR, microsatellite amplification is achieved using a single primer complementary to the flanking sequence, and one which anchors to the repeat junction of the compound SSR. The recovery rate of useable markers was found to be similar (43%) to that reported for conventionally generated SSRs. Thus, anchored PCR can be used to reduce the costs of marker development, since it requires that only half the number of primers be synthesised. Where fluorescence-based platforms are used, marker deployment costs are lower, since only the anchoring primers need to be labelled. In addition, anchored PCR improves the recovery of useful markers, as it allows assays to be generated from microsatellite clones with repeat sequences located close to their ends, a situation where conventional PCR amplification fails as two flanking primers cannot be designed. Strategies to permit the large-scale development of compound SSR markers amplified by anchored PCR are discussed.

Inheritance and molecular mapping of a gene conferring seedling resistance against Puccinia hordei in the barley cultivar Ricardo

Genetic studies were undertaken to determine the inheritance and genomic location of uncharacterised seedling resistance to leaf rust, caused by Puccinia hordei, in the barley cultivar Ricardo. The resistance was shown to be conferred by a single dominant gene, which was tentatively designated RphRic. Bulk segregant analysis (BSA) and genetic mapping of an F3 mapping population using multiplex-ready SSR genotyping and Illumina GoldenGate SNP assay located RphRic in chromosome 4H. Given that this is the first gene for leaf rust resistance mapped on chromosome 4H, it was designated Rph21. The presence of an additional gene, Rph2, in Ricardo, was confirmed by the test of allelism. The seedling gene Rph21 has shown effectiveness against all Australian pathotypes of P. hordei tested since at least 1992 and hence represents a new and useful source of resistance to this pathogen.

Discovery and trait association of single nucleotide polymorphisms from gene regions of influence on meat tenderness and long-chain omega-3 fatty acid content in Australian lamb

Whole genome association studies in humans have shown a strong relationship between omega-3 levels in plasma and single nucleotide polymorphisms (SNP) located close to genes whose protein products are involved in the biosynthesis of long-chain omega-3 fatty acids. In sheep and other livestock species, the calpain/calpastatin system is the principal influence on natural variation in meat tenderness between animals. Using targeted next generation sequencing, we sequenced the fatty acid desaturase locus (FADS1/2/3), ELOVL2 and SLC26A10 and the calpain/calpastatin (CAPN1/2/3 and CAST) gene loci of 35 industry sires from the Australian flock. A total of 753 SNP were identified and 182 of these SNP were selected for incorporation onto a research SNP panel that represented the genetic variation across the nine genes. A total of 1252 animals were genotyped from the Australian Sheep CRC Information Nucleus Flock for these SNP and the genomic association was calculated for omega-3 fatty acid content and objective meat tenderness in lamb. Six SNP within CAST and CAPN2 showed association with shear force at Day 5 post-mortem at a significance level of P ≤ 0.01. When these were fitted simultaneously in a mixed-model analysis with fixed effects and covariates, three SNP remained significant. These SNP each had an unfavourable effect on shear force of between 1.1 and 1.8 N, with a combined effect of 4.1 N. The frequency of the favourable alleles in the progeny measured indicates that these SNP hold good potential for improving the management of meat tenderness across Merino, Border Leicester and Terminal sire types. No SNP within the FADS1/2/3, ELOVL2 and SLC26A10 gene regions were associated with lamb muscle omega-3 levels. This indicates that genetic variation in the long-chain omega-3 biosynthesis pathway genes analysed here may not be important for omega-3 content in lamb within the Information Nucleus Flock population.

Accelerating wheat breeding for end-use quality with multi-trait genomic predictions incorporating near infrared and nuclear magnetic resonance-derived phenotypes

Key messageUsing NIR and NMR predictions of quality traits overcomes a major barrier for the application of genomic selection to accelerate improvement in grain end-use quality traits of wheat.AbstractGrain end-use quality traits are among the most important in wheat breeding. These traits are difficult to breed for, as their assays require flour quantities only obtainable late in the breeding cycle, and are expensive. These traits are therefore an ideal target for genomic selection. However, large reference populations are required for accurate genomic predictions, which are challenging to assemble for these traits for the same reasons they are challenging to breed for. Here, we use predictions of end-use quality derived from near infrared (NIR) or nuclear magnetic resonance (NMR), that require very small amounts of flour, as well as end-use quality measured by industry standard assay in a subset of accessions, in a multi-trait approach for genomic prediction. The NIR and NMR predictions were derived for 19 end-use quality traits in 398 accessions, and were then assayed in 2420 diverse wheat accessions. The accessions were grown out in multiple locations and multiple years, and were genotyped for 51208 SNP. Incorporating NIR and NMR phenotypes in the multi-trait approach increased the accuracy of genomic prediction for most quality traits. The accuracy ranged from 0 to 0.47 before the addition of the NIR/NMR data, while after these data were added, it ranged from 0 to 0.69. Genomic predictions were reasonably robust across locations and years for most traits. Using NIR and NMR predictions of quality traits overcomes a major barrier for the application of genomic selection for grain end-use quality traits in wheat breeding.

Genetic Relationship of Stripe Rust Resistance Genes Yr34 and Yr48 in Wheat and Identification of Linked KASP Markers

The Australian continent was free from wheat stripe rust caused by Puccinia striiformis f. sp. tritici until exotic incursions occurred in 1979 and 2002. The 2002 incursion enabled the identification of a new stripe rust resistance gene (Yr34) in the advanced breeding line WAWHT2046. In this study, we developed and validated markers closely linked with Yr34, which is located in the distal region in the long arm of chromosome 5A. Four kompetitive allele-specific polymerase chain reaction (KASP) and three sequence-tagged site (STS) markers derived from the International Wheat Genome Sequencing Consortium RefSeq v1.0 scaffold-77836 cosegregated with Yr34. Markers sun711, sun712, sun725, sunKASP_109, and sunKASP_112 were shown to be suitable for marker-assisted selection in a validation panel of 71 Australian spring wheat genotypes, with the exception of cultivar Orion that carried the Yr34-linked alleles for sunKASP_109 and sunKASP_112. Markers previously reported to be linked with adult plant stripe rust resistance gene Yr48 also cosegregated with Yr34. Wheat genotypes carrying Yr34 and Yr48 produced identical haplotypes for the Yr34-linked markers identified in this study and those previously reported to be linked with Yr48. Phenotypic testing of genotypes carrying Yr34 and Yr48 showed that both genes conferred similar seedling responses to pre-2002 and post-2002 P. striiformis f. sp. tritici pathotypes. Further testing of 600 F2 plants from a cross between WAWHT2046 and RIL143 (Yr48) with P. striiformis f. sp. tritici pathotype 134 E16A+Yr17+Yr27+ failed to reveal any susceptible segregants. Our results strongly suggest that Yr34 and Yr48 are the same gene, and that Yr48 should be considered a synonym of Yr34.