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Raymond F. Baker Center for Plant Breeding, Dep. of Agronomy, Iowa State Univ., Ames, IA 50011
* Corresponding author (brummer{at}iastate.edu)
ALFALFA, Medicago sativa L., is a herbaceous, perennial forage crop grown extensively throughout temperate and dry tropical regions of the world for hay, pasture, and silage. More than any other forage crop adapted to these regions, alfalfa combines high biomass productivity, optimal nutritional profiles, and adequate survival, making its cultivation ideal for dairy and livestock enterprises. Within the context of a cropping system, alfalfa controls soil erosion, improves water quality, mitigates pest outbreaks, and contributes significant amounts of nitrogen to succeeding crops. Although commercial breeding programs often target marketable traits for improvement, major agronomic traits of importance are biomass yield and winter hardiness. To be profitably grown, alfalfa needs to sustain the production of high yields over a several year period. Corn silage represents the primary alternative forage to alfalfa for dairy cattle rations, and though it can produce more dry matter yield than alfalfa it requires protein supplementation. Further, its cultivation leaves cropland devoid of cover during the winter months and susceptible to significant wind and water soil erosion. Thus, improved alfalfa cultivars can have positive economic and environmental impacts on the agricultural sector.
A negative relationship exists between biomass yield and winter hardiness, complicating improvement efforts on both traits simultaneously. In response to temperature and photoperiod cues in autumn, alfalfa plants acclimate for winter, making physiological alterations that affect obvious phenotypes such as plant height and biomass production. Nondormant plants have little or no acclimation response and are typically more productive than dormant plants during autumn. Plant height in October is typically used in the upper midwestern USA as an indirect measure of dormancy response, with shorter plants being more dormant. Height is associated with winter injury when considering the entire spectrum of dormancy responses, but we have found no genetic correlation between the traits in a population derived from dormant parents but which segregated for both traits (Brummer et al., 2000). Thus, to some extent, these traits can be selected independently, although new selection methods may be needed to do it efficiently.
We are attempting to address the biomass yield and winter injury trade-off by augmenting traditional selection methods with genomics. Our efforts are focused in three areas: (i) identification of quantitative trait loci (QTL) and candidate loci associated with the traits, (ii) profiling germplasm sources to identify novel alleles and to structure breeding programs to capture heterosis, and (iii) isolation of genes associated with dormancy control to eventually hasten adaptation of genetic resources to diverse environments.
Effective selection for yield and winter hardiness requires multiple year field evaluations to ensure long-term persistence and sustained yield; consequently, genetic improvements accrue slowly. Selection based on molecular markers offers one means of decreasing the cycle time, and we are attempting to identify loci associated with these traits using standard genetic mapping and QTL detection procedures (Robins et al., 2003). In addition to mapping these ultimate phenotypes, we are dissecting both traits by concurrently mapping morphological and physiological components, such as sugar, starch, and protein content in roots during autumn (Alarcón-Zúñiga et al., 2004). Our hope is that these component traits, which may have a simpler genetic basis, will allow us to manipulate the overall complex trait in a targeted, modular manner. Finally, we are mapping candidate genes known or suspected to be associated with winter injury in these same populations to assign putative functions to QTL. These mapping studies, being conducted in both tetraploid and diploid populations, will provide a picture of the genomic landscape of these important traits and identify options for improving them.
Besides a marker-assisted selection approach, a possible means to increase yield is to capture heterosis, which currently is not being done in commercially available synthetic cultivars (Brummer, 1999). To capture heterosis, genetically distinct populations, or heterotic groups, need to be identified and improved independently. Three possible heterotic groups include M. sativa subsp. falcata, dormant subsp. sativa, and nondormant subsp. sativa. We have recently shown that crosses between falcata and dormant sativa results in significant heterosis for biomass yield (Riday and Brummer, 2002). Molecular markers have not been successful at predicting heterosis, but were more effective at differentiating the subspecies (Riday et al., 2003; Riday and Brummer, 2004). Although clear morphological differences can easily separate falcata and sativa, our results suggest that markers may not be useful to place germplasm into possible heterotic groups within each of the subspecies.
Both falcata and nondormant sativa have agronomic weaknesses for the upper Midwest: the subsp. falcata has slow regrowth, particularly in the late summer and autumn, and nondormant sativa is not winter hardy. Even though hybrids between falcata and elite cultivars produce high yields and exhibit heterosis for seasonal total yield, their superior performance dissipates as the growing season progresses and disappears under a harvest regime with short regrowth periods. Hybrids of dormant and nondormant sativa cannot be evaluated effectively, as winter injury obfuscates the results. These problems are associated with the dormancy response—too strong in falcata, and not strong enough in nondormant sativa. Traditional breeding, possibly aided by markers, may be useful to improve autumn growth in falcata germplasm and is clearly effective in improving winter hardiness of nondormant sativa (Weishaar et al., 2005).
Modifying the dormancy response of falcata or nondormant sativa through transgenic means might offer a way to engineer nonadapted germplasm for use directly in breeding programs outside its area of adaption. We are attempting to differentiate between genes involved in temperature and photoperiod sensing using M. truncatula Gaertner microarrays probed with RNA from dormant and nondormant alfalfa genotypes pre- and postacclimation in the field and in the growth chamber where one of the variables (temperature or photoperiod) was held constant. Genes identified from this screen could also be useful for marker-assisted selection aimed at altering the dormancy response of various populations.
The approaches we are using will identify loci involved in biomass yield and winter hardiness. These markers, QTL, and genes can be used as selection tools or as transgenes to aid the improvement of these genetically complex traits. Applying this information to a breeding program will not be as straightforward in alfalfa as in many other crops. Commercial alfalfa cultivars are synthetic populations, consisting of a heterogeneous mix of heterozygous genotypes (inbred lines are not available). Further, cultivated alfalfa has an autotetraploid (2n = 4x = 32) genome. Both of these characteristics complicate the application of genomics solutions to practical breeding problems.
Identifying most of the loci within a breeding population involved in biomass yield, winter hardiness, and other complex traits will require more complicated pedigree structures than simple biparental crosses. Methods to construct and analyze pedigree based populations have been developed for diploid organisms (Yi and Xu, 2001), but extension to autotetraploids remains to be done. Regardless of the theoretical possibilities, building complex populations and accumulating phenotypic and genotypic data to successfully identify important loci will require an effort beyond the capacities of most public and private alfalfa laboratories. Selection based on markers alone will have to consider linkage disequilibrium (about which little is known) unless the markers are the genes of interest. Given the difficulties of identifying haplotypes in autotetraploid plants, all efforts to use candidate genes should be pursued. Association mapping may be a more sensible approach than linkage analysis, although it too will be complicated by tetraploidy and by relatively low marker density. Perhaps marker-assisted selection will be more useful for the identification and introgression of novel alleles from exotic germplasm into elite breeding populations. Manipulating the frequency of introduced alleles and minimizing linkage drag could be done rather expeditiously even in tetraploid populations.
The recent expansion of basic research on the model legume M. truncatula bodes well for the alfalfa community if the genomics tools developed in the model can be usefully applied to crop improvement. The major traits of winter survival, multiyear persistence, regrowth, biomass yield, and seed yield cannot be thoroughly assessed in the autogamous, annual, diploid model species. Thus, in order for genomics tools to be used to develop better alfalfa cultivars for farmers, breeders will need to apply the technology directly in alfalfa improvement programs. However, the infrastructure developed in M. truncatula, including the development of genetic markers and the analysis of certain biochemical pathways, will be directly applicable to alfalfa.
Manipulation of single gene traits by biotechnological approaches has been clearly successful, even in alfalfa. Glyphosate-tolerant (Roundup Ready, Monsanto Corp., St. Louis, MO) alfalfa will be commercialized in the near future and other value-added traits lie on the horizon (M. McCaslin, Forage Genetics, Intl., pers. comm.). Commercial emphasis on the use of genomics will lie in those traits which provide a marketing advantage, but serious efforts to improve fundamentally important but inherently complex traits such as winter hardiness or biomass yield likely will not be conducted in the private sector. For long-term improvement of alfalfa, research should be focused in four main areas: (i) constructing a comprehensive picture of both cultivated and wild genetic resources, (ii) streamlining selection procedures to shorten cycle time and increase heritabilities, (iii) developing alternative cultivar types that harness the genetic potential within and among germplasm groups, and (iv) facilitating the creation and maintenance of genetic variation for major quantitative traits in diverse breeding populations. The use of genomics methods to characterize exotic germplasm, dissect quantitative traits, and identify candidate loci could address each of these problems. The incorporation of genomics techniques into the breeding process will be challenging, but many of these problems can be overcome with enough effort.
As we consider using genomics to improve crop breeding, we need to ask whether we are using genomics tools to solve serious breeding problems, or inventing breeding problems that are solvable by genomics methods. Many essential breeding goals can continue to be met with traditional methods if effort is devoted to them. The paradox of the genomics age is that funding for plant breeding programs is decreasing at the same time that the potential of genomics is being realized. Thus, genomics initiatives have replaced, rather than augmented, breeding programs, with the result that many technological advances in genomics may not be applied to cultivar development at all! Many breeding programs in both the public and private sector have limited funding that constrains their ability to conduct extensive multienvironment selection and evaluation programs and to produce varieties. Given financial constraints, emphasis is often diverted to genomics projects for which money can be procured. This results in the somewhat ridiculous situation of high technology solutions being developed for a crop that lacks the most basic breeding capabilities. Further, minor crops, including many forage and noncommodity species, have little focused breeding effort devoted to them in the public sector and virtually none from industry. Without active and effective breeding programs, genomics will not contribute to genetic gains in any traits.
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ACKNOWLEDGMENTS |
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Received for publication July 23, 2003.
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  B. Thapa, R. Arora, A. D. Knapp, and E. C. Brummer Applying Freezing Test to Quantify Cold Acclimation in Medicago truncatula J. Amer. Soc. Hort. Sci., September 1, 2008; 133(5): 684 - 691. [Abstract] [Full Text] [PDF]
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 B. C.Y Collard and D. J Mackill Marker-assisted selection: an approach for precision plant breeding in the twenty-first century Phil Trans R Soc B, February 12, 2008; 363(1491): 557 - 572. [Abstract] [Full Text] [PDF]
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