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Marker Implementation in the Department of Agriculture, Western Australia Wheat Breeding Program

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Marker Implementation in the Department of Agriculture, Western Australia Wheat Breeding Program
Iain Barclay(a), Robyn McLean(a), Robin Wilson(a), Rudi Appels(b), Mehmet Cakir(b), Gabby Devlin(b), Dora Li(b),

(a) Department of Agriculture, South Perth, WA 6151, Australia

(b) WA State Agricultural Biotechnology Center, Murdoch University, Murdoch, WA 6150, Australia.
Department of Agriculture Wheat Breeding Program
The Department of Agriculture, Western Australia (DAWA) wheat breeding program has three wheat breeders with complementary targets in quality grades and different environments. There is a high degree of co-operation, collaboration and exchange of germplasm between the plant breeders, but each breeder controls a separate germplasm pool. The program aims to produce commercially competitive cultivars in five major quality grades for a target environment in Western Australia currently producing wheat on 4.5m. ha./year. Currently cultivars from the program are sown on 85% of that area. The wheat breeders have very limited emphasis on basic research, but are involved in research via linkages with other groups.
The breeding program primarily uses the F2 bulk progeny method, but uses backcrossing, doubled haploids, modified single seed descent and bulk methods as appropriate. We are currently making approximately 3,000 crosses each year and manage a field program of 180,000 plots across 14 sites. In the breeding program Marker Assisted Selection (MAS) is seen as complementing the existing screening for yield, quality and disease resistance.
Molecular marker technologies offer a range of novel approaches to improve the efficiency of a breeding program. The DAWA breeding program uses molecular markers to accelerate wheat breeding and hence to increase genetic gain per year by way of optimising parental selection in crossing program and enriching the populations for the desired traits (Cakir et. al., 2003). The success of these strategies is often dependent on the availability of polymorphic and also closely linked markers to the trait of interest. During the past ten years marker discovery and linkage studies have identified a large number of QTL regions that could be used for the breeding of wheat. However, these QTL studies are not easily applicable in breeding programs and therefore further validation experiments for the markers within those QTL regions need to be carried out using breeding populations.
There are three steps in developing MAS; discovery, validation and refinement, and implementation.
Marker Discovery
The majority of wheat marker development in Australia occurs through the Australian Winter Cereal Molecular Marker Program (AWCMMP). The AWCMMP is a national research and development effort funded by the Grains Research and Development Corporation (GRDC) that has projects across 14 research organisations. The aim of the AWCMMP is to provide overall coordination of wheat and barley molecular marker development and implementation in Australia, specifically to:

  • Identify markers linked of traits of importance to wheat/barley improvement

  • Apply markers in all Australian wheat/barley breeding programs

  • Provide molecular marker service laboratories associated with each of the major publicly funded wheat/barley improvement programs

  • Provide the most effective research effort and most efficient utilisation of resources through a nationally coordinated effort (

Within the AWCMMP breeders are involved in the prioritization of target traits of most significance to breeding programs and in the development and phenotyping of populations for marker development and validation. Access to information is available to all participants via regular meetings and an electronic site. Current target traits include disease resistances, agronomic and physiologic traits, and end-use quality.

Marker/Trait Validation
Marker validation is a key step to marker implementation which has been largely neglected when funding and resource allocations are made. It has fallen in a gap between marker discovery and implementation projects. To take a newly discovered marker from another laboratory to routine implementation within a breeding program involves considerable effort from both molecular biologists and breeders. Experience has shown that there are considerable phenotyping and laboratory costs in this process and sufficient time also needs to be allocated to the process. Because of these costs it is important to have close collaboration between breeders and molecular biologists to prioritise marker validation for the program. Close involvement of breeders in validation has also proved valuable in explaining some of the early results with a new marker because of their experience and understanding of the germplasm and populations being used.
The first step in validation is to understand the genetic distance between a linked marker and the gene of interest to determine likely recombination rate and to evaluate the usefulness of the marker to a breeding program. Some reported marker/trait linkages are so far apart that their value in a breeding program is low as high recombination rates make the marker an unreliable screening tool. If the marker is close enough to the gene of interest the next step is to transfer the specific conditions for the assay developed in one laboratory to another. When this has been achieved the marker has to be validated in relevant germplasm for that breeding program. In Australia the major breeding programs have quite different germplasm bases, and for linked markers a marker that may work in the germplasm of one program may not be applicable in another breeding program due to lack of polymorphism at the marker site. Another consideration at this stage is that it may be necessary to convert a marker to a more appropriate marker type for the large scale implementation used in that laboratory, or to facilitate the opportunities for multiplexing.
Often there is an unnecessary delay between marker discovery and the validation process. Researchers often wish to continue work on marker development until a publishable stage, and not make information more broadly available until then. This needlessly delays the validation process in breeding programs, and delays implementation. In Australia this is an area where the process of implementing markers could be made more efficient, and could also help develop more robust markers.
The validation step can identify some unexpected results. For example the Lr24 and Sr24-linked marker, developed by Schachermayr et al (1995) on red-grained wheat, was not diagnostic in white grained Australian cultivars carrying these genes. In Australia breeders have utilized a shortened segment version of the original Thinopyrum ponticum (Podp.) Z.-W. Liu & R.-C. Wang (Agropyron elongatum (Host.) Beauv.) derived translocation where the genes for red grain are not present, and the linked marker is not able to be used.

Marker Implementation – Target Traits
The experience in Australia has been that marker implementation has been evolutionary rather than revolutionary. Breeding programs have been using markers in situations where they are not necessarily the most efficient selection tool, but are considered more as an exercise in ‘building for the future’ to develop marker technology and strategies. It is also important to remember that markers are just one of many screening tools available to plant breeders, and that different tools may be appropriate for different traits, or stages of the breeding program.
Breeders see the most important role for marker assisted selection (MAS) in targeting traits that are difficult or expensive to phenotype. The reality is that these are also the most difficult markers to develop. Consequently most of the markers available are for simply inherited and phenotyped traits, though progress in developing markers for pre-harvest sprouting, cereal cyst nematode (Heterodera avenae Woll.) and Barley Yellow Dwarf Virus (BYDV) are examples of more difficult/expensive to measure traits. There are still efficiencies for a breeding program in using markers for simpler traits where markers can be multiplexed, or where phenotyping can cause plant setback (e.g. Al tolerance screening), or phenotyping can not be done until late in the plants development (e.g. polyphenol oxidase, while simple to score, can only be scored on the grain produced so could not be phenotyped before crossing).
Other target traits for markers are those controlled by recessive genes, where a co-dominant marker can enable tracking of genes without the need for progeny testing. Pre-emptive breeding for diseases that may be expected to arrive in the future (“Quarantine traits”), which in Australia include diseases and insects such as Karnal bunt (Tilletia indica Mitra), Russian Wheat Aphid (Diuraphis noxia Mordvilko) and Hessian Fly (Mayetiola destructor Say), can be handled efficiently with the use of markers. With these traits international collaboration for phenotyping is essential.
Markers currently implemented in the DAWA wheat breeding program mainly for disease resistance (rusts, BYDV, cereal cyst nematode, and yellow spot (tan spot) (Pyrenophora tritici-repentis (Died.) Drechs.)), grain quality (grain hardness, flour colour on 7AL, GBSS Null 4A, and pre-harvest sprouting on 4A) and phenology (Rht1 and Rht2). The program currently has a list of 20 markers at various stages in the validation process.
Marker Implementation – Place in the Breeding Program
Marker assisted selection (MAS) will be increasingly applied to three major areas within the DAWA breeding program.
Choice of parents. First is the use of MAS in choice of parents in crossing programs, particularly screening of F1s in backcross and topcross situations. The efficiency over conventional crossing strategies is greatest where crossing targets multiple traits, and particularly in tracking recessively inherited traits where the need for progeny testing is removed. Breeders have considered the use of MAS in accelerated backcrossing by selection for the recurrent parent background genotype. At this stage the high cost of whole genome screening and the need to handle larger numbers of plants within a substantial backcrossing program has resulted in the decision to not use this strategy within the breeding program. However, in situations such as the introduction of a new disease where new cultivars were urgently required this would be a desirable strategy. At the end of a complex crossing process MAS is also used to select F1 plants which carry targeted genes for production of F2 seed or doubled haploids. Experience in the breeding program has also shown the advantage of pre-screening of parents prior to crossing. In several instances new parents have unexpectedly been found to be mixed for the gene(s) of interest.

Pyramiding Resistance genes. Second is the use of MAS for the pyramiding of disease resistance genes. In particular the breeding program is using MAS to pyramid genes for rust resistance. The wheat breeding program currently has a major effort in upgrading resistance to the three rusts, and in the future aims to produce cultivars with multiple effective genes to each of the three rusts to reduce the risk of development and multiplication of new rust races via simple step-wise mutations resulting in loss of effectiveness of resistance genes.
Population enrichment. Third is the enrichment of populations for target traits earlier in the breeding process. Greatest efficiency is achieved by selection as early as possible in the breeding process, but this is also where number of individual lines is greatest. As the affordability, throughput and range of assays improves the amount of screening the breeding program can do in F2 and F3 generations, and in the F5 and F6 generations after re-selection will increase significantly. Effective selection at early generations increases the overall efficiency of the breeding process as lines entering expensive replicated, multi-site yield trials are more targeted and have a greater probably of success. The DAWA breeding program has integrated MAS in both the F2 bulk progeny and single seed descent breeding systems.
MAS and more traditional screening methods are often complementary, and both utilised depending on the generation. As an example MAS may be utilised to select for rust resistance genes at an early generation and truncate a population. At later stages in the breeding program, where the number of lines is significantly reduced, screening nurseries may be used to evaluate the impact of background genotype on gene expression and to evaluate the resistance against multiple rust races. Similarly for a more complex trait controlled by multiple QTL one or a few components may be selected for at early generations through MAS. At later generations more expensive testing may be utilised to evaluate the trait overall. This strategy is particularly relevant for complex traits such as yield and end product quality, however a better understanding of epistatic interactions will be needed to best use markers for these traits.
Marker Implementation – In Practice
Taking markers from use in experimental populations to implementation in a large breeding program is proving to be a considerable challenge to both breeders and molecular biologists. This can be attributed to the significantly greater number of assays required by a breeding program, and the more complex crossing structure used in a breeding program compared with the simple crosses between two fixed line parents of most experimental populations. Timeliness of results is also a significant issue to the breeding program. Late arrival of marker information in a large backcrossing program creates immense problems and additional work for the breeding program.
Most experimental crosses are made between relatively genetically divergent parents, whilst in a breeding program crosses may be made between more closely related individuals which may result in difficulties in finding polymorphic markers. There is therefore an advantage in testing parents as soon as possible to find appropriate polymorphic markers to use before time critical results are required from segregating populations. From this year breeders will be attempting to sample all crossing parents for storage of DNA samples. As early as possible the breeders will document critical crosses where MAS is planned to enable the marker laboratory to determine appropriate markers to use prior to testing the cross populations. Earlier work on parents will also help to identify problems where some parents are unexpectedly mixed for a target trait.
The most obvious need for efficiency is in the laboratory to improve assay throughput, but producing and sampling leaf tissue on a large scale has proved challenging. The program has used both glasshouse and field grown leaf material and cutting leaf material to insert into 96 well plates for large numbers of individuals has proved very demanding of resources. Some progress has been

made towards developing a sampling tool that automatically punches a leaf disc directly into a cell of a 96 well plate to improve sampling efficiency and accuracy. More recently preliminary studies have shown the potential of extracting DNA directly from seed germinated in the 96 well plates in cases where it is not necessary to keep the tested plants. Successful development of this method will remove the need to sample leaf material, but will also have advantages of evening work flows across the year. Currently marker samples are being taken at a few major times during the year that fit in with plant breeding workloads. This results in significant workload peaks and troughs for the marker laboratory. With DNA extraction from germinated seed plant breeding staff can load seed into 96 well plates at the same time as they are handling seed for trial preparation. Plates can then be stored with dry seed for processing throughout the year, with plates being brought out of storage and germinated in plates as required.

Good data handling and laboratory management systems become essential to handle the larger numbers of lines and assays required by a breeding program. A new plant breeding database is being developed to help handle the additional data being generated, and to track samples and results between plant breeders and the marker laboratory. It has been necessary to develop more standardised procedures and processes with better documentation and labelling of samples and data between plant breeding and laboratory. In particular systems of standardised plate numbering and bar coded labelling and electronic transfer files accompanying each plate have been developed. The transfer file contains all information required by the laboratory, such as plate layout of lines, controls, pedigrees, generation, target traits, date results are required by and other relevant information. Results are then returned to breeders via the transfer file. With three wheat breeders all submitting samples for assay it has been important to develop a common prioritisation classification system so laboratory staff can schedule work according to the urgency with which results are required. For example samples relating to a crossing program are given a high rating to ensure results are available in time for crossing.
There is a need for good communications and a close working relationship between plant breeders and molecular biologists, but this relationship also extends to the technicians in the respective groups. Laboratory staff should understand the need for timely and accurate results for plant breeding operations, and plant breeding staff should appreciate the requirement for timely submission of correctly sampled and documented samples with realistic timelines for results. To develop this relationship the technical groups have arranged tours of both workplaces to gain some insight of work carried out. Close location of plant breeding facilities and the molecular marker laboratory has been an advantage in developing communications. Particularly at this early stage of integrating molecular markers into breeding programs having molecular marker service laboratories associated with each of the major publicly funded breeding programs has been a much better model than a single large national marker laboratory.
Marker Implementation – A Wish List
A plant breeder’s wish list would include:

  • The ability to target more traits through development of more marker/trait associations

  • Better markers, ideally this would be perfect markers, but also covers more co-dominant markers to distinguish homozygotes from heterozygotes and more tightly linked markers

  • Better distinguishing of alleles at a locus

  • Greater laboratory throughput and cheaper costs

  • Improved and more efficient sampling methods

  • Cheaper whole genome systems

  • Better data handling.

Cakir, M., Appels, R., Carter, M., Loughman, R., Francki, M., Li, C., Johnson, J., Bhave, M., Wilson, R., McLean, R., Barclay, I. 2003 Accelerated wheat breeding using molecular markers. In 'Proceedings of 10th International Wheat Genetics Symposium'. Vol.1, pp. 117-120. Istituto Sperimentale per la Cerealicoltura, Roma Italy.
Schachermayr, G.M., Messmer, M.M., Feuillet, C., Winzeler, H., Winzeler,

M. and Keller, B. 1995 Identification of molecular markers linked to the Agropyron elongatum-derived leaf rust resistance gene Lr24 in wheat. Theoretical and Applied Genetics 90: 982-990.

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