Molecular Markers in Alfalfa Improvement

Joe H. Bouton and Mary K. Sledge

Department of Crop and Soil Sciences

University of Georgia, Athens, GA 30602 USA

The ultimate result of any alfalfa improvement program is the successful development and farmer use of a new cultivar. By definition, a cultivar is a group of plants distinct for specific traits which remain uniform for these traits through generations of multiplication. In its purest form, a cultivar is the package or vessel containing a collection of favorable genes and a minimum number of unfavorable genes. Therefore, in cultivar development, alfalfa breeders simply try and add or increase the frequency of those genes which add value of this package or vessel.

Molecular markers and maps based on them now have great utility in crop improvement programs to aid in increasing the efficiency of selection for value-added genes. These genetic maps may allow one to identify and select chromosome fragments associated with the gene of interest and can even be applied to traits governed by multiple genes or quantitative trait loci (QTL) (Paterson et al., 1991). Molecular markers have therefore become a very powerful tool for plant breeders to solve very specific problems. Although still in its infancy, molecular markers are now being assessed for their practical use in alfalfa cultivar development.

Recent Trends in Alfalfa Cultivar Development

The trend in breeding the more recent alfalfa cultivars has been for improved multiple pest resistance and proper fall dormancy. This has been especially true in North America. Since insects and diseases are numerous in alfalfa, development of a cultivar with the proper fall dormancy and a broad genetic base for resistance to many pests is felt to give more persistence and higher yield. Therefore, in the USA, the current Alfalfa Council's List records the 276 cultivars into 9 dormancy groups for up to 12 pest resistance ratings. To measure the success of this approach, one has only to consult performance trials in their respective state or region to see that most of the new, multiple-pest resistant cultivars currently on the market are outperforming the old public checks (sometimes as much a 30 to 40% in yield).

Nearly all alfalfa cultivars are synthetic cultivars, so the approach taken is to identify, using standard screening procedures, the individual plants (e.g. genotypes) which contain the gene(s) of interest. These genotypes are viewed as parents and are then composited via replicated random mating (allowing all possible of crosses with all possible parents) in isolation into the first generation synthetic seed (syn 1). After its final release, each further generation (Syn2, Syn 3, Syn 4, etc.) of seed increase of the synthetic is then done in isolation and random mating is assumed. It is up to the breeder to then decide which Syn generation will be breeders, foundation, or certified seed.

The approach used by most alfalfa breeders has been to use commercially successful or elite material as much as possible. This then allows one to capitalize on all the good agronomic traits already in this material. If not possible in elite material, then most breeders progress to plant introductions and related subspecies, but are aware that one will probably introgress several unacceptable traits when this material is used. A good example of this has been the recent introduction of potato leaf hopper resistance via glandular hairs into cultivated alfalfa from the related subspecies (spp. glutinosa and spp. glandulosa). The original crosses were weak and not acceptable from an agronomic point of view, but did possess the glandular hairs from the subspecies which were felt to be responsible for insect deterrence. However, several years of breeding and re-selection were necessary to develop this material into the acceptable cultivars on the market today. More of this type of complex introgression is envisioned for future cultivar development which should increase the value and use of molecular markers to assist during selection.

There has also been a movement lately to introduce more complex (from a genetic point of view) traits into these multiple pest, dormancy specific cultivars. Cultivars with traits such as grazing tolerance, high nutritive quality, and salt tolerance are now marketed. The next steps will be use of transgenics for the addition of genes such as herbicide resistance (M. McCaslin, 1998, personal communication) or the use of molecular markers to assist during selection for intractable traits such as aluminum tolerance, winter-hardiness, and yield (Brummer et al. 1999).

Use of Molecular Markers in Alfalfa Cultivar Development

Cultivated alfalfa is a tetraploid (2n=4X=32) with polysomic inheritance which complicates the genetic analysis required for mapping. There are diploid subspecies available and diploid genotypes have been developed from cultivated alfalfa. These are all cross fertile with cultivated alfalfa and have greatly simplified the analyses required for developing diploid maps.

Five genetic maps of alfalfa have been published, four of which utilize diploid germplasm (Brummer et al., 1993, 1999; Kiss et al., 1993; Echt et al., 1994; and Tavoletti et al., 1996) and one which utilizes tetraploid germplasm (Brouwer and Osborn, 1999). The recent reports of using single dose restriction fragments (SDRF) (Brouwer and Osborn, 1999) or simple sequence repeats (SSR) with single dose allele (SDA) analysis (Diwan et al. 1996) also have potential to greatly simplify mapping in tetraploids. However, one problem with the alfalfa maps constructed with diploid F2 inbred populations is the high degree of segregation distortion favoring heterozygotes. In some cases, upwards of 50% of the markers show a skewed distortion. One explanation for this is inbreeding has uncovered deleterious recessive alleles (Osborn et al. 1998). The heterozygous loci tended to cluster together in linkage groups, and it is possible that not all linkages are valid. The non-inbred map constructed by Tavoletti et al. (1996) in an F1 population showed only 8.8% segregation distortion and indicated the use of non-inbred populations may overcome this problem.

The genetic maps developed in both diploid and tetraploid alfalfa have potential for utilization in alfalfa improvement programs for the following practical approaches (Brummer et al. 1999): 1) interspecific hybridization analyses, 2) estimating genetic variation among and within the different germplasm sources, 3) marker assisted selection, and 4) estimating genetic dissimilarity among parental genotypes for production of high yielding populations or synthetics. For practical cultivar development, especially development practiced by private seed companies, marker assisted selection and selection of parents for synthetics have the most value at this stage.

Marker Assisted Selection

In marker assisted selection, genes of agronomic importance may be "tagged" to molecular markers closely linked to them. Selection is then based on the marker(s). This is useful during backcrossing and to facilitate introgression of desired genes from wild or less agronomically acceptable species, including species at the diploid level. Molecular markers could be used to facilitate ploidy level gene transfers via 2n gametes, a process that has been important in transferring genes from diploid Medicago falcata into cultivated alfalfa.

Molecular markers can even be used for traits controlled by QTL. Good examples are initial reports of QTL identification for aluminum tolerance (Sledge et al. 1996), winter-hardiness (Brouwer and Osborn, 1999), and forage yield (Shah et al. 1999) for some of the existing maps. The next step needs to be the successful documentation of using markers to introgress these traits into elite germplasm.

Finally, use of marker assisted selection can be applied to agronomically important diploid Medicago species, the annual medics. Commonly grown in Mediterranean regions of the world, annual medics have potential for use in North America as smother crops for weed control, as green manure, and for direct use as forage. Currently, genotypes well suited for growth in the USA are just now being identified.

Genetic Dissimilarity and Selection of Parents for Synthetics

High levels of heterozygosity appear important for maximizing forage yield in alfalfa. The finding that using molecular markers to maximize genetic dissimilarity among tetraploid plants correlated with higher yield among their single crosses increases the potential use of molecular markers for selecting the best parents for synthetics (Kidwell et al. 1994). However, Osborn et al. (1998) reported inconsistent results among most synthetic combinations based on their genetic dissimilarity in a set of field trials, but some positive trends were seen for some combinations especially for populations from low parent numbers.

Working within non-dormant germplasm, Sledge et al. (1998) produced synthetics from the 12 and 24 most dissimilar genotypes based on AFLP markers from a population of 120 genotypes previously selected for grazing tolerance from CUF101. These 12 and 24 parent synthetics were then compared to a synthetic produced from the entire 120 genotypes. Preliminary results thus far have shown an increase in grazing survival, pest resistance, and yield of all the selected populations over the parental cultivar, but no difference in yield among the populations based on genetic dissimilarity.

Conclusions

For the future, two things are probably needed to increase the practical use of molecular markers for alfalfa cultivar development. First, development of a portable public framework map as is now available in soybean needs to be accomplished as soon possible. This should greatly increase the availability and use of a good genetic map for everyone. Availability of a public map in turn should lead to more research on application of molecular markers as a tool to solve practical breeding problems. Secondly, more funding from industry for applied uses of the existing maps is likewise needed.

References

1. Brouwer , D.J., and T.C. Osborn. 1999. A molecular marker linkage map of tetraploid alfalfa (Medicago sativa L.). Theor. Appl. Genet. In Press.

2. Brummer, E.C., J.H. Bouton, and G. Kochert. 1993. Development of an RFLP map in diploid alfalfa. Theoretical and Applied Genetics. 86: 329-32.

3. Brummer, E. C., M. Sledge, J. H. Bouton, and G. Kochert. 1999. Molecular marker analyses in alfalfa and related species. In R. L. Phillips and I.K. Vasil (eds.) DNA-Based Markers in Plants. 2nd Ed. Kluwer, Dordrecht, The Netherlands (In press).

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6. Kidwell, K.K., E.T. Bingham, D.R. Woodfield, and T.C. Osborn. 1994. Relationships among genetic distance, forage yield and heterozygosity in isogenic diploid and tetraploid alfalfa populations. Theoretical and Applied Genetics. 89:323-328.

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9. Paterson, A.H., Damon S., Hewitt, J.D., Zamir, D, Rabinowitch, H.D., Lincoln, S.E., Lander, E.S. and Tanksley, S.D. 1991. Mendelian factors underlying quantitative traits in tomato: Comparison across species, generations, and environments. Genetics 127:181-197.

10. Shah, M.M., D. Luth, E.C. Brummer, C.L. Council, and R.C. Kunz. 1999. Molecular mapping of QTLs for yield heterosis in tetraploid alfalfa. Proc. VII Plant and Animal Genome Conference. 17-21 January 1999. Sherago International, NY.

12. Sledge, M.K., Bouton, J.H., Tamulonis, J., Kochert, G., and Parrott, W.A. 1996. Aluminum tolerance QTL in diploid alfalfa. Report 35th North American Alfalfa Improvement Conference. (In press).

13. Tavoletti, S., F. Veronesi, and T.C. Osborn. 1996. RFLP linkage map of an alfalfa meiotic mutant based on an F₁ population. J. Hered. 87:167-170.

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