Ian S. Dundas

Development of PCR markers for the selection of wheat stem rust resistance genes Sr24 and Sr26 in diverse wheat germplasm

The use of major resistance genes is the most cost-effective strategy for preventing stem rust epidemics in Australian wheat crops. The long-term success of this strategy is dependent on combining resistance genes that are effective against all predominant races of the pathogen, a task greatly assisted by the use of molecular markers linked to individual resistance genes. The wheat stem rust resistance genes Sr24 and Sr26 (derived from Agropyron elongatum) and SrR and Sr31 (derived from rye) are available in wheat as segments of alien chromosome translocated to wheat chromosomes. Each of these genes provides resistance to all races of wheat stem rust currently found in Australia .We have developed robust PCR markers for Sr24 and Sr26 (this study) and SrR and Sr31 (previously reported) that are applicable across a wide selection of Australian wheat germplasm. Wheat lines have recently become available in which the size of the alien segments containing Sr26, SrR and Sr31 has been reduced. Newly developed PCR-markers can be used to identify the presence of the shorter alien segment in all cases. Assuming that these genes have different gene-for-gene specificities and that the wheat industry will discourage the use of varieties carrying single genes only, the newly developed PCR markers will facilitate the incorporation of two or more of the genes Sr24, Sr26, SrR and Sr31 into wheat lines and have the potential to provide durable control to stem rust in Australia and elsewhere.

New sources of rust resistance from alien species : meliorating linked defects and discovery

This paper presents a review of projects undertaken over the past 2 decades aimed at improving the yield and/or quality attributes of translocation lines carrying rust resistance genes from species related to wheat, so as to make these lines more suitable for use in breeding programs. Homeologous recombination between the alien chromosome segments and normal wheat chromosomes was induced in a ph1bph1b background. Lines with shortened alien chromatin were selected using dissociation patterns of molecular-based markers. A new line of bread wheat was developed containing a shortened chromosome 1RS segment carrying rust resistance gene SrR (Secale cereale L.), in which a defect affecting dough-quality appears to have been deleted. In addition, several advanced lines were produced with modified 6Ae#1L chromosome segments with Sr26 (Thinopyrum ponticum), 2S#1 chromosome segments with Sr32, and a previously unnamed gene, a 2S#2 chromosome segment with Sr39 (Triticum speltoides), 4G#1 chromosome segments with Sr37, and 2G#2 chromosome segments with Sr40 (T. timopheevii).

Development of wheat lines carrying stem rust resistance gene Sr39 with reduced Aegilops speltoides chromatin and simple PCR markers for marker-assisted selection

The use of major resistance genes is a cost-effective strategy for preventing stem rust epidemics in wheat crops. The stem rust resistance gene Sr39 provides resistance to all currently known pathotypes of Puccinia graminis f. sp. tritici (Pgt) including Ug99 (TTKSK) and was introgressed together with leaf rust resistance gene Lr35 conferring adult plant resistance to P. triticina (Pt), into wheat from Aegilops speltoides. It has not been used extensively in wheat breeding because of the presumed but as yet undocumented negative agronomic effects associated with Ae. speltoides chromatin. This investigation reports the production of a set of recombinants with shortened Ae. speltoides segments through induction of homoeologous recombination between the wheat and the Ae. speltoides chromosome. Simple PCR-based DNA markers were developed for resistant and susceptible genotypes (Sr39#22r and Sr39#50s) and validated across a set of recombinant lines and wheat cultivars. These markers will facilitate the pyramiding of ameliorated sources of Sr39 with other stem rust resistance genes that are effective against the Pgt pathotype TTKSK and its variants.

Isolation of wheat–rye 1RS recombinants that break the linkage between the stem rust resistance gene SrR and secalin

Chromosome 1R of rye is a useful source of genes for disease resistance and enhanced agronomic performance in wheat. One of the most prevalent genes transferred to wheat from rye is the stem rust resistance gene Sr31. The recent emergence and spread of a stem rust pathotype virulent to this gene has refocused efforts to find and utilize alternative sources of resistance. There has been considerable effort to transfer a stem rust resistance gene, SrR, from Imperial rye, believed to be allelic to Sr31, into commercial wheat cultivars. However, the simultaneous transfer of genes at the Sec-1 locus encoding secalin seed storage proteins and their association with quality defects preclude the deployment of SrR in some commercial wheat breeding programs. Previous attempts to induce homoeologous recombination between wheat and rye chromosomes to break the linkage between SrR and Sec-1 whilst retaining the tightly linked major loci for wheat seed storage proteins, Gli-D1 and Glu-D3, and recover good dough quality characteristics, have been unsuccessful. We produced novel tertiary wheat-rye recombinant lines carrying different lengths of rye chromosome arm 1RS by inducing homoeologous recombination between the wheat 1D chromosome and a previously described secondary wheat-rye recombinant, DRA-1. Tertiary recombinant T6-1 (SrR+ Sec-1-) carries the target gene for stem rust resistance from rye and retains Gli-D1 but lacks the secalin locus. The tertiary recombinant T49-7 (SrR- Sec-1+) contains the secalin locus but lacks the stem rust resistance gene. T6-1 is expected to contribute to wheat breeding programs in Australia, whereas T49-7 provides opportunities to investigate whether the presence of secalins is responsible for the previously documented dough quality defects.

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 messageWheat–Aegilops speltoidesrecombinants carrying stem rust resistance genesSr32andSrAes1teffective against Ug99 and PCR markers for marker-assisted selection.AbstractWild 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.

First report of meiotic chromosome number and karyotype analysis of an accession of Trigonella balansae (Leguminosae)

Abstract This study represents the first report of the number of meiotic chromosomes and morphology of the mitotic chromosomes of Trigonella balansae. The haploid complement of chromosomes of T. balansae consists of eight chromosomes (2n = 16). Of these, only the satellited chromosome and possibly a subtelocentric chromosome are identifiable. The remaining six chromosomes of each haploid chromosome complement cannot be reliably identified using arm ratio or relative length measurements.

Morphological characterisation and genetic analysis of a bi-pistil mutant (bip) in Medicago truncatula Gaertn.

A floral organ mutant was observed in transgenic Medicago truncatula Gaertn. plants that had two separate stigmas borne on two separate styles that emerged from a single superior carpel primordium. We propose the name bi-pistil, bip for the mutation. We believe this is the first report of such a mutation in this species. Genetic and molecular analyses of the mutant were conducted. The mutant plant was crossed to a mtapetala plant with a wild-type pistil. Expression of the mutant trait in the F1 and F2 generations indicates that the bi-pistil trait is under the control of a single recessive gene. Other modifying genes may influence its expression. The mutation was associated with the presence of a T-DNA insert consisting of the Alfalfa mosaic virus (AMV) coat protein gene in antisense orientation and the nptII selectable marker gene. It is suggested that the mutation is due to gene disruption because multiple copies of the T-DNA were observed in the mutant. The bi-pistil gene was found to be independent of the male-sterile gene, tap. This novel mutant may assist in understanding pistil development in legumes.

Three group 16SrII phytoplasma variants detected in co-located pigeonpea, lucerne and tree medic in South Australia

Legume-infecting phytoplasmas identified in symptomatic tree medic (Medicago arborea), lucerne (M. sativa) and pigeonpea (Cajanus cajan) growing at a single trial site at Urrbrae, South Australia, were all shown to belong to the 16SrII phytoplasma taxonomic group, but each host species was found to be infected with a different genotype. Within one species (M. sativa) the genotypes were more uniform as demonstrated by comparing five isolates from lucerne plots. These lucerne genotypes differed from the Australian lucerne yellows phytoplasma genotype previously described in New South Wales, and the pigeonpea phytoplasma isolate was more similar to the non-legume infecting Bonamia little leaf (BoLL) than to pigeonpea little leaf phytoplasma. This diversity supports the view that these legumes were infected from different reservoir phytoplasma hosts rather than from a single perennial host such as tree medic.

Single nucleotide polymorphic marker enabling rapid and early screening for the homoeolocus of β-amylase-R1: a gene linked to copper efficiency on 5RL

This study describes the development of a PCR marker to detect the β-amylase-R1 gene of rye. It provides an easy and rapid means for the identification of plants containing the β-amylase-R1. Because rye chromosome segments do not normally recombine with wheat chromosomes, this marker provides a means for tracking all linked genes on that alien 5RL chromosome segment. Reaction conditions were optimised for an annealing temperature of 60°C for a high stringency. The reaction was also optimised for low reaction volumes reducing the cost of the reagents required for the reaction. This PCR test can be used in breeding or mapping programs for the rapid screening of progeny containing translocations of 5RL and hence select for the copper efficiency trait of rye.

Chromosome Engineering Techniques for Targeted Introgression of Rust Resistance from Wild Wheat Relatives

Hexaploid wheat has relatively narrow genetic diversity due to its evolution and domestication history compared to its wild relatives that often carry agronomically important traits including resistance to biotic and abiotic stresses. Many genes have been introgressed into wheat from wild relatives using various strategies and protocols. One of the important issues with these introgressions is linkage drag, i.e., in addition to beneficial genes, undesirable or deleterious genes that negatively influence end-use quality and grain yield are also introgressed. Linkage drag is responsible for limiting the use of alien genes in breeding programs. Therefore, a lot of effort has been devoted to reduce linkage drag. If a gene of interest is in the primary gene pool or on a homologous chromosome from species in the secondary gene pool, it can be introgressed into common wheat by direct crosses and homologous recombination. However, if a gene of interest is on a homoeologous chromosome of a species belonging to the secondary or tertiary gene pools, chromosome engineering is required to make the transfer and to break any linkage drag. Four general approaches are used to transfer genes from homoeologous chromosomes of wild relatives to wheat chromosomes, namely, spontaneous translocations, radiation, tissue culture, and induced homoeologous recombination. The last is the method of choice provided the target gene(s) is not located near the centromere where recombination is lacking or is suppressed, and synteny between the alien chromosome carrying the gene and the recipient wheat chromosome is conserved. In this chapter, we focus on the homoeologous recombination-based chromosome engineering approach and use rust resistance genes in wild relatives of wheat as examples. The methodology will be applicable to other alien genes and other crops.