Bilateral BBSRC-Embrapa: Uncovering the genetic and functional basis of the unique leaf rust resistance in the Brazilian wheat variety Toropi

The global demand for wheat is increasing, but so is the distribution and aggressiveness of the pathogens that seriously impact on wheat productivity. The Food Agricultural Organisation (FAO) estimates that wheat production must increase by 60% by 2050 to meet the demand from the growing human population. The fungal pathogens responsible for rust of wheat are a major constraint to achieving this increase in production and in recent years we have seen numerous epidemics due to the breakdown of rust race-specific resistance (R-) genes.

There are three rust pathogens of wheat, stripe (yellow-Puccinia striiformis), leaf (brown-P. triticina) and stem (black-P. graminis) rust. In the UK stripe rust is the most prevalent, occurring every year. Leaf rust has been confined to warmer regions, being prominent in the south-west of England. However, in recent years the UK Cereal Pathogen Virulence Survey has detected leaf rust on wheat as far north as the border with Scotland. This is considered to be as a result of milder winters increasing the ability of the pathogen to overwinter, and warmer springs allowing leaf rust to take hold in regions of the UK where previously it has not been considered a problem.

In Brazil leaf rust is the major problem. Many sources of leaf rust resistance used in wheat varieties have proven to be race-specific, with virulence rapidly arising in the P. triticina population, overcoming the resistance after only a few years of deployment in new wheat varieties. However, a small number of slow-rusting, adult plant resistance (APR) genes effective against leaf rust have been reported. These include Lr34, Lr46, Lr67 and Lr68. These leaf rust APR genes have proven effective over long periods of time, large acreages and under high disease pressure. Some of these durable, leaf rust APR genes have now been cloned, confirming, as suspected, that they do not function in the same way at race-specific, leaf rust R-genes.

The old Brazilian cv. Toropi contains a unique, durable source of leaf rust APR. Preliminary studies have identified two genes in Toropi which account for 71% of the resistance. However these two genes are poorly defined and we know very little about their mode of action, e.g. at what plant growth stages the leaf rust APR becomes effective and whether environmental factors such as temperature can effect the action of these resistance genes

In this study we will improve the genetic resolution of each gene by generating a high-density genetic map of Toropi using state-of-the-art DNA marker systems, and wheat genomic resources. We will develop wheat genetic materials that will enable us to unravel the mode of action of each gene, and marker tools that will enable each leaf rust APR gene to be identified and followed during the process of breeding new improved wheat varieties. We also aim to incorporate these Toropi resistance genes into elite Brazilian wheat varieties, using the marker tools we develop, thereby providing the Brazilian wheat breeder with materials they can enter directly into their wheat breeding programs.

To help understand the mode of action and biology underlying these leaf rust APR genes we will study the development of the leaf rust fungal pathogen in wheat at the microscopic level, infecting wheat plants at different stages of it growth and when grown at different temperatures.

This project also focusses on the training of young Brazilian researchers, and on broadening the international collaborative research network of the Brazilian team.

To identify and develop diagnostic markers linked to trp-1 and trp-2 we will fine mapping each genetic region in a large F2 population of the cross Toropi x IAC 13.

DArT-Seq Genotype by Sequencing (http://www.diversityarrays.com) of a smaller Toropi x IAC 13 RIL population is currently underway. The SNPs and indels identified will be converted to a PCR-based marker system using KASAPar technology. KASPar markers linked to trp-1 and trp-2 will be mapped in the F2 population to identify recombinants in each genetic region.

Additional DNA markers will be identified within each genetic region using 3 methodologies:
(I) A genome comparative analysis approach. The markers defining each gene will be used to identify the corresponding genomic regions in the related grass species rice, barley and Brachypodium for which full genome sequence data is available. We will also use the Chinese Spring wheat chromosome sequence scaffolds/contigs to identify predicted gene sequences lying within each genetic interval.

(II) RNA-Seq data sets are available for Toropi. The RNA-Seq libraries of Toropi have generated nearly 500k gene contigs. These contigs will be Blast aligned against the Chinese Spring genome sequence (http://www.wheatgenome.org) to identify which gene contigs lie within the chromosomal regions containing each leaf rust resistance gene.

(III) We will isolate and sequence chromosomes 1A (trp-1) and 4D (trp-2) directly from Toropi and IAC 13 to identify polymorphisms between the parental varieties of the mapping population in which the leaf rust resistance genes were identified.

The KASPar markers will be used to develop near-isogenic lines carrying each Toropi leaf rust resistance gene singularly and in combinations. Near-isogenic lines will be developed in rust susceptible wheat genetic backgrounds and in elite Brazilian wheat varieties.

The global demand for wheat is increasing, with the Food Agricultural Organisation estimating that wheat production must increase by 60% by 2050 to meet this demand. Both the UK and Brazil are net importers of wheat, so stabilisation of global wheat production is essential to ensure cheap imports of wheat to meet internal demand. The rust pathogens of wheat are major biotic factors responsible for significant losses every year, despite the availability of effective fungicides. The impact of rusts for wheat production is exemplified by the establishment of the Borlaug Global Rust Initiative (BGRI) in 2008, which focused the international wheat-rust community on the need to work together to find solutions to fight these devastating diseases.

The ToropiAPR UK-Brazilian partnership will generate knowledge, tools and resources that will enable wheat breeders in both countries to produce superior disease resistant wheat varieties, requiring less chemical inputs, being both environmentally friendly and financially sustainable. The program of work will contribute significantly to our understanding of rust resistance in wheat, providing wheat breeders with a detailed characterisation of the adult plant leaf rust resistance in the Brazilian wheat variety Toropi and how best to deploy this resistance alongside other rust resistance genes. The identification of genetic marker haplotypes for each leaf rust Adult Plant Resistance (APR) will allow the development of diagnostic DNA markers. This will be done using the KASPar technology. KASPar markers will provide the wheat breeders with the tools required to stack the Toropi rust resistance genes into new, improved wheat varieties as part of a Marker Assisted Breeding program. During ToropiAPR the KASPar markers will be used to develop near-isogenic lines carrying the Toropi rust APR genes individually and in combinations, providing genetic materials for further, detailed functional studies. The Toropi rust resistance genes will also be backcrossed into five elite Brazilian wheat varieties to produce wheat lines that can be immediately incorporated into Embrapa-Wheat's wheat breeding programs.

The specific deliverables that will result from this project are:

(1) Highly versatile KASPar markers for the Toropi leaf rust APR genes trp-1 and trp-2, and any other rust APR genes identified, that can be used in a Marker Assisted Selection wheat breeding program.

(2) Wheat near-isogenic lines carrying trp-1 and trp-2 that will provide materials for biological functional studies, both within this project and by the wider research community.

(3) Brazilian elite wheat varieties into which the Toropi leaf rust APR genes have been backcrossed, providing ready-to-use breeding materials for the wheat breeders.

(4) Extensive knowledge of the functional characteristics of trp-1 and trp-2 that will inform how to best deploy the Toropi rust resistance alongside other rust resistance genes.

(5) Advanced training of a new generation of Brazilian and UK researchers in advanced wheat functional genomics.