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  • 1. Integrating Molecular Techniques to Maximise the Genetic Potential of Forage Legumes Derek R. Woodfield and E. Charles Brummer AgResearch Grasslands, Private Bag 11008, Palmerston North, New Zealand, and Department of Agronomy, Iowa State University, Ames IA50011, USA Key words: breeding methods, genetic gains, heterosis, backcrossing, marker-assisted selection, interspecific hybridisation, genomics, transgenics Abstract: Genetic gains from forage legume breeding have exceede d 1% per year for white clover but are more variable for alfalfa and red clover depending on the trait. A change from synthetic to hybrid varieties would provide better control of economic traits in most out- crossing forage species. New breeding strategies that capture heterosis are essential in forage legumes with historically slow yield improvement and where producing hybrids is difficult. More emphasis is needed on characterisation and evaluation of genetic resources, particularly to identify heterotic groups, using both classical and molecular methods. Application of molecular marker- and genomics-based techniques will advance our understanding of the genetic control underpinning phenotypic traits. Markers will be used to understand and capture heterosis, to identify quantitative trait loci, to develop detailed genetic linkage maps, to introgress unique genetic variation from conventional and transgenic sources, to conduct marker-assisted selection, and to determine the factors involved in genotype by environment interactions. In conjunction with international genomics efforts in higher plants, economically important phenotypes will ultimately be linked through their underlying physiology and metabolism to the genes responsible for them. Combining molecular genetic innovations with strong population improvement programs will enable the maximum genetic potential of forage legumes to be realised. 1. INTRODUCTION In most temperate regions, and particularly in Australasia, grazed pastures are the preferred nutritive source for animal production because of their low cost and competitive trade advantage. Forage legumes provide nitrogen and improve nutritive value, quality and intake rates of grazed forage. Forage breeding has traditionally focused on increasing animal productivity by providing high yielding, persistent forages resistant to a range of pests and 1
  • 2. 2 Woodfield and Brummer diseases. While these remain important, breeding objectives have diversified to improve feeding value, and to manipulate compounds that affect animal and human health, animal welfare, reproductive fertility, animal product quality, flavour and texture. Phenotypic recurrent selection has been the predominant breeding method for forage legumes; however, in most forage species and particularly those with slower genetic improvement there is a clear need for new breeding methods that provide reliable improvement (Brummer 1999). We extend previous reviews of breeding methods used for herbaceous, out-crossing, perennial forage legumes (Taylor 1987; Bowley 1997) by providing information on methods to develop transgenic varieties, methods to capture heterosis, applications of molecular markers and the potential of genomics. 2. POPULATION IMPROVEMENT Population improvement strategies focus on identification of elite genotypes with good general combining ability. By crossing these genotypes, breeders aim to accumulate favourable genes in subsequent generations while maximising heterozygosity. Open-pollinated populations are a hetero- geneous mixture of highly heterozygous individuals, with high levels of genetic variation both within and between populations. This variation has made good selection responses possible with simple breeding methods. However, traits with lower heritability require other recurrent selection schemes involving progeny testing. Intra-population recurrent selection methods, including mass selection, phenotypic, half-sib, full-sib and inbred progeny (S 1 or S 2) recurrent selection schemes, are much more widely used than inter-population methods due to their low cost and simplicity. Mass selection has been the driving force behind the array of natural ecotype populations that have provided unique genetic variation in many forage legumes (Williams 1987; Taylor 1987). Bulked seed from the population of interest is grown in the target environment, and after natural selection has occurred, seed is harvested, bulked, and the process repeated. Phenotypic recurrent selection differs from mass selection in that breeders have pollen control over one and commonly both parents. A good example is its use to develop resistance to clover cyst nematode (Heterodera trifolii) and clover root-knot nematode ( eloidogyne trifoliophila) in white M clover. Five cycles of phenotypic selection for clover cyst nematode resistance and seven cycles for clover root-knot nematode were conducted with approximately 1000 plants screened in each cycle. For clover cyst nematode resistance, an average reduction of 122 cysts per gram of root
  • 3. Maximising the Genetic Potential of Forage Legumes 3 dryweight was achieved per cycle. This equates to a genetic response of 7.3±2.0% per cycle of selection. Over 7 cycles of selection for clover root- knot nematode resistance, the mean decrease per cycle was 164 galls per gram of root dry-weight, which equates to a genetic response of 2.3±0.2% per cycle of selection (Mercer et al. 2000). Recurrent selection methods involving half-sib, full-sib or inbred progeny testing provide better information on the relative importance of additive and non-additive genetic effects. Half-sib and full-sib progeny tests are being used extensively but with many modifications to suit the crop and trait. Bowley (1997) presented a combined among- and within -HS progeny test method that was more effective than half-sib selection for improving birdsfoot trefoil seed yield. Inter-population recurrent selection schemes such as reciprocal recurrent selection (RRS) can exploit all types of gene action responsible for heterosis (Brummer 1999). In RRS, each population serves as the tester for the other population, and improvement is carried out within each population separately but based upon test-cross performance. In alfalfa, Rowe and Hill (1981) results suggested that inter-population hybrid performance could be improved by RRS or top-cross progeny testing. No published information is available on the effectiveness of RRS relative to other breeding methods in forage legumes; however, RRS has been partially integrated into some commercial alfalfa breeding programmes (Moutray pers comm.). Backcrossing has been under-utilised in most out-crossing species due to perceptions that performance gains in backcross-derived populations may not justify the time and resource involved. Concerns about inbreeding depression occurring during backcrossing have also been expressed, but this can generally be avoided by changing the recurrent parents for each backcross generation (Bingham 1990). Backcrossing is frequently used for introgressing traits from wild relatives such as potato leaf hopper resistance transferred from Medicago sativa subsp. glandulosa to cultivated alfalfa (M. sativa subsp. sativa) (Shade and Kitch 1986; McCaslin 1998). Similarly, tetraploid M. sativa subsp. falcata germplasm, WISFAL, was developed by backcrossing between ploidy levels and sub-species (Bingham 1990). Backcrossing is particularly attractive for autotetraploid species such as alfalfa as it works with equal efficiency for diploid, allotetraploid (disomic) or autotetraploid (tetrasomic) inheritance (Micallef and Bingham 1995). The number of backcross generations required depends on the relative performance of the donor parent. The better the performance of the donor parent the fewer backcrosses that are required. Field Evaluation: Evaluating forages as spaced plants in the absence of companion species has decreas ed. Spaced plant evaluations provide useful morphological information and are essential for PVR descriptions but the
  • 4. 4 Woodfield and Brummer agronomic information is frequently a poor indicator of performance in on-farm situations. An example is the poor correlation between performance of white clover genotypes under cutting and their performance under intensive grazing (Evans et al. 1992). Cutting in white clover favours large-leaved genotypes, while small- and medium -leaved genotypes are more persistent and productive under intensive grazing. Similarly in alfalfa, evaluating material under grazing has enabled breeders to identify superior genotypes and populations. The first true dual-purpose alfalfa cultivar, Alfagraze, was developed by selecting surviving plants after two cycles of intensive grazing by beef cattle (Bouton et al. 1991). Alfagraze had superior grazing tolerance but yielded slightly less than the top entries under cutting and had low resistance to phytophthora root rot. Newer varieties developed by integrating the protocol developed with Alfagraze (Bouton and Smith 1998) into commercial breeding programs have yields and disease resistance profiles equal to or better than the best commercially available varieties (Brummer and Moore 2000). Genetic Gains: While there are few published reports, genetic gains have been achieved in the major temperate forage legumes. Genetic gain for white clover yield under various management systems, stolon density and nitrogen fixation has exceeded 1% but genetic gains have been more variable in red clover and alfalfa (Table 1). With the exception of McKersie (1997), genetic improvement in alfalfa has consistently been less than 0.35% yr -1 (Table 1). Part of the reason for lower rates of genetic improvement is tetrasomic inheritance, which increases the number of possible allelic combinations at a single heterozygous locus (Hill et al. 1988). However, other reasons may have been more important, such as an emphasis on disease and insect resistance breeding and a near total disregard for developing populations that provide heterotic yield increases (see below). Improvements in animal performance were lower than the increases in forage yield and quality (Table 1). Achieving animal production responses relies upon the ability to utilise additional forage produced, and this often requires higher stock rates and better grazing management. Poor management can erase all the gains obtained from breeding. Other on-farm benefits can also be observed when animal health is improved through better nutrition or elimination of an anti-quality factor. An example of this is the strong selection response for reducing formononetin content of red clover (Table 1), that subsequently provided higher ovulation rates in ewes grazing these low-formononetin red clovers (Rumball et al. 1997). Accumulation of favourable alleles, with both additive and dominant effects, in linkage blocks along with a decreased frequency of deleterious recessive alleles has provided population improvements in alfalfa and is consistent with explanations for improvements in various self - and cross- pollinated crops (Bingham 1998).
  • 5. Table 1. Estimates of rates of genetic gains in temperate forage legume species . Species/Trait Benchmark Variety Trials/Varieties Genetic gain Reference -1 Alfalfa (%. yr ) Forage Yield Vernal 14 trials (150 var) 0.26 Hill et al. 1988 Forage Yield Cossack, Ladak 2 trials (12 var) 0.18 Holland & Bingham 1994 Forage Yield Saranac Multiple trials (80 var) 0.18 -1.07 McKersie 1997 Forage Yield Wairau 3 trials (5 NZ var) 0.35 Woodfield 1999 Red Clover Forage Yield G. Turoa G.Hamua, Pawera 0.43 Anderson 1973 Forage Yield G. Hamua (2x) G. Colenso (2x) 0.21 Claydon et al. 1993 Forage Yield G. Pawera (4x) G27 (4x) 1.39 Rumball et al. 1997 Formonenetin G. Pawera (4x) G27 (4x) 2.83 Rumball et al. 1997 White Clover Forage Yield -Sheep grazing G. Huia 8 trials (10 var) 1.49 Woodfield 1999 Forage yield -Cattle grazing G. Huia 5 trials (9 var) 1.21 Woodfield 1999 Stolon density G. Huia 5 trials (9 var) 1.09 Woodfield 1999 Clover content G. Huia 110 var 0.60 Woodfield & Caradus 1994 Nitrogen fixation G. Huia 3 trials (7 var) 1.19 Woodfield 1999 Lamb growth G. Huia G. Demand 0.33 Ryan & Widdup 1997 Lamb growth G. Huia G. Tahora 0.48 Chapman et al. 1993 5
  • 6. 3. CAPTURING HETEROSIS IN FORAGES Severe inbreeding depression and heterosis have been reported for most cross-pollinated forage legumes (Jones and Bingham 1995; Brummer 1999). In diploids and allotetraploids heterosis is maximised in the single-cross generation, while autotetraploids exhibit progressive heterosis with double- cross performance exceeding that of single-crosses from inbred parents (Bingham et al. 1994). Heterosis results from alleles or linkage blocks with partial to complete dominance and with different frequencies between populations (Bingham 1998; Brummer 1999). The presence of heterosis in forage legumes has led to considerable discussion on developing hybrid varieties. Currently all commercially available varieties of white clover, red clover, birdsfoot trefoil and alfalfa are synthetics, developed by intermating selected parents for 4 to 6 generations. Hybrid varieties have potential advantages for reducing the number of generations of seed multiplication, capitalising on both additive and non-additive gene action, and providing higher yield and better environmental stability. Hybrids would also be simpler for deploying and pyramiding transgenes. Pollination control mechanisms are required to develop hybrid varieties. Male-sterility systems have been identified in alfalfa, rose clover and birdsfoot trefoil (Viands et al. 1988; Molina-Freaner and Jain 1992; Negri and Rosselini 1996), while he gametophytic self-incompatibility system of white and red t clover also provides excellent pollination control. Breeding schemes for producing hybrids using self-incompatibility have been described by Williams (1987) and Taylor (1987). Despite suitable pollination control mechanisms, development of hybrid alfalfa varieties has not been viable due to reduced pollination of male-sterile rows by bees (Viands et al. 1988). Such difficulties suggest new breeding strategies to capture hybrid vigour are essential in forage legumes such as alfalfa. Concurrently, more emphasis is required on characterisation and evaluation of genetic resources, particularly identifying heterotic groups, using both classical and molecular methods. Brummer (1999) proposed a semihybrid breeding scheme that uses population improvement within separate heterotic populations. This scheme relies on the identification of heterotic groups, which unfortunately has received almost no attention in forage legumes. In alfalfa, Kidwell et al. (1994a) demonstrated that M. sativa subsp. falcata and, to a lesser degree, Peruvian germplasm were genetically distinct from other subsp. sativa germplasm pools. Riday and Brummer (1999) have confirmed that sativa x falcata interpopulation hybrids performed better than both intra-population 6
  • 7. Maximising the Genetic Potential of Forage Legumes 7 crosses, while Ray et al. (1999) reported good heterosis between non- dormant and dormant alfalfa germplasm pools. Identifying and using similar heterotic patterns in other forage legumes is urgently required. The semihybrid scheme also relies on independent population improvement continuing within the respective populations (Brummer 1999). This is consistent with reciprocal recurrent selection approaches used for maize. Developing more than two heterotic groups may be required for crops or breeding programs that cover a wide geographical or environmental range. Current breeding strategies that essentially bulk all germplasm into one large gene pool need urgent re-evaluation, or identification and development of heterotic groups will become almost impossible. In order to identify heterotic groups, crosses among germplasm sources need to be made. Numerous diallel analyses in other forage legumes have partitioned additive and dominance genetic variance into general (GCA) and specific (SCA) combining ability. However, this information has not been used to identify heterotic groups. Two populations whose hybrids have higher average SCA than crosses within either population are assigned to different heterotic groups. Thus, assessing germplasm in order to develop heterotic groups is a non-trivial task. The development of conventional F hybrids is still of interest to forage 1 breeders. Inbreeding programs have been initiated in most forage legumes (Jones and Bingham 1995; Taylor 1987) but whether these will result in successful hybrid varieties remain s uncertain. Regardless of its utility for hybrid production, inbreeding remains an important tool for reducing the genetic load within cross-pollinated species. 4. INTRODUCING NEW GENETIC VARIATION Recurrent selection and backcross methods rely primarily o additive genetic n variation present in the primary gene pool. When the required genetic variation is absent, or has very low heritability, interspecific hybridisation and transformation have been used to generate new genetic variation. Mutagenesis has been largely unsuccessful in forages (Bowley 1997). Interspecific hybridisation : Gene transfer from related species in the secondary gene pool has proven difficult in most forage legumes due to strong hybridisation barriers. Embryo rescue has frequently been required to obtain hybrids following failure of the endosperm to develop, and many of the resulting hybrids have either been sterile or have had meiotic instability. Interspecific hybridisation has been more successful in white clover than in red clover. Red clover has been crossed with four other species but none has resulted in transfer of useful variation (Taylor 1987). White clover has
  • 8. 8 Woodfield and Brummer been successfully hybridised with Trifolium nigrescens, to transfer clover cyst nematode resistance and increased seed production (Hussain et al. 1997; Marshall et al. 1998). Similarly, several research groups have obtained fertile hybrids between Caucasian (kura) clover (T. ambiguum M. Bieb) and white clover, with virus resistance and the ability to form both rhizomes and stolons present in the hybrids. Hussain and Willams (1997) developed a ploidy series of hybrids from 4x to 8x between these two species by developing a fertile bridge cross. The initial hybrids required embryo rescue and chromosome doubling to provide a fertile octoploid (8x H-435), however, an array of congruity backcross progenies were subsequently generated from this genotype. Recent crosses have produced the desired recombinants with both rhizomes and stolons (Hussain pers comm.). Similarly, Abberton et al. (1998) used conventional backcrossing of the initial interspecific hybrid to transfer rhizomes into white clover. Interestingly, Abberton et al. (1998) predominantly used tetraploid hybrids, while Hussain and Williams (1997) have achieved their successful recombinants at the hexaploid level. Interspecific hybridisation has successfully reduced the coumarin levels in yellow sweetclover (Melilotus officinalis L.) and white sweetclover (Melilotus alba Desr.) by transferring a recessive alle le (cu) from a wild relative (M. dentata Walst et Kit.)(reviewed by Bowley 1997). Similarly, interspecific hybridisation of serredella (Ornithopus ssp.) resulted in a commercial variety (cv. G. Spectre) that combined the prostrate growth habit and hard seed of yellow serredella (O. compressus Mill.) with the higher forage yield of pink serredella (O. sativus Brot.) (Williams et al. 1987). Transgenics: The insertion of foreign genes to provide unique genetic variation has received considerable interest for forage legume improvement (this volume). Transformation and regeneration protocols have been developed for all the major forage legumes and a range of cloned genes have been introduced. Si gle-gene transformants in alfalfa (Austin and Bingham n 1997) and white clover (White et al. 2000) have been field tested; however, no transgenic forage legumes have been commercially released yet. Developing synthetic transgenic varieties in cross-pollinating species is more difficult than in species where clonal, inbred or hybrid varieties are used (Conner and Christey 1994). The development of transgenic varieties relies on adequate expression and stable inheritance of the introduced gene(s), and the ability to cost-effectively conduct field and laboratory testing prior to commercial release. Screening primary transformants for transgene expression levels is standard practice, however, few attempts have been made to modify expression levels through conventional selection. Instead most effort has gone into modifying gene constructs to increase expression. Identifying genetic backgrounds that enhance the function of the
  • 9. Maximising the Genetic Potential of Forage Legumes 9 transgene(s) and selecting for enhanced transgene expression is feasible. Scott et al. (1998) found that both genetic background and selection influenced expression of a GUS (CaMV35S promoter-uidA) gene. Range of genotypes Single genotype homozygous from elite population for inserted transgene F 1 families (h e t e r o z y g o u s f o r t r a n s g e n e i n a h e t e r o g e n e o u s g e n e t i c b a c k g r o u n d ) Intercross within or among F 1 families Progeny test F 2 families N o n -t r a n s g e n i c ( 0 to 2 copies of transgene) X tester Identify homozygous F2 genotypes Intercross confirmed homozygous genotypes Further generations of seed multiplication Synthetic cultivar (h o m o z y g o u s f o r transgene in a heterogeneous background) Figure 1. A modified synthetic breeding scheme for transgenic varieties in outcrossing diploid and allotetraploid species. While it is probably unnecessary to have the transgene expressed in all progeny of a synthetic variety, breeding schemes must have the flexibility to produce varieties with predictable transgene(s) frequencies. For transgenic breeding strategies to succeed they must also avoid causing inbreeding depression. Two breeding strategies, a modified synthetic scheme (Fig. 1) and a backcross scheme, that provide predictable transgene inheritance in outcrossing disomic and tetrasomic populations have been successfully tested (Scott et al. 1998; Micallef and Bingham 1995). Scott et al. (1998) reported stable inheritance of a GUS gene through three generations of crossing to non-transgenic plants, while Micallef and Bingham (1995) reported stable inheritance of GUS and a recovery of agronomic performance through 3 generations of backcrossing. These results indicate transgenic plants containing a single transgene insert could be used as the basis of a breeding program. Backcrossing maintains the transgene in a heterozygous form in diploids and allotetraploids, and in a simplex condition in autotetraploids (Micallef and Bingham 1995). Approximately 50% of the
  • 10. 10 Woodfield and Brummer backcross progeny have the transgene and transgenic progeny must be identified in each generation for subsequent backcrossing. The main advantage of backcrossing is that a single transgene can be rapidly transferred into multiple genetic backgrounds. Strategies that minimize the generations of intermating required for variety development would also be beneficial and therefore direct transformation of inbred lines is an attractive longer-term option. Direct transformation of inbred lines (S2 or S3) would allow stabilization of the transgene during inbreeding to S5 or beyond. Inbreds with optimal transgene expression and from backgrounds with know n heterotic response could be combined during hybrid production to pyramid multiple transgenic traits in the final variety. I nbreeding approaches, like backcrossing, can give rise to inbreeding depression, but also offer the potential to capitalise on hybrid vigour. Once the transgene is fixed in a homozygous condition, transgenic varieties could be produced using a single- or double-cross hybrid system (Williams 1987). Transgene dosage can be manipulated using these methods to provide varieties with predictable genetic composition and reliable expression of the target trait. However, further erosion of potential heterotic groups is a concern with transgenics as a single genotype or chromosome block could be introgressed into a wide range of germplasm. More consideration of this potential problem is needed. 5. INTEGRATING MOLECULAR TECHNIQUES The application of molecular marker- and genomics-based techniques will advance our understanding of the genetic control of phenotypic traits. The development of various marker systems and detailed genetic linkage maps will enable molecular markers to be used to understand and capture heterosis, to identify quantitative trait loci, to introgress unique genetic variation from conventional and transgenic sources, to conduct marker- assisted selection, and to determine the factors involved in genotype by environment interactions. Genetic Diversity and Parental Selection: Molecular markers have proven useful in detecting genetic diversity between germplasm sources (Brummer et al. 1991; Kidwell et al. 1994a; Ghérardi et al. 1998) and their use could be extended to identifying heterotic groups and parents (Brummer 1999). The relationship between molecular marker heterozygosity and heterosis has been studied in alfalfa and white clover (Kidwell et al. 1994b; Woodfield et al. 1999). In alfalfa, genetic dissimilarities based on 244 restriction
  • 11. Maximising the Genetic Potential of Forage Legumes 11 fragments were poorly correlated with diploid progeny performance but highly correlated with the performance of tetraploid progeny (Kidwell et al. 1994b). Genetic dissimilarity estimates based on 861 restriction fragments were significantly correlated with single-cross performance for two white clover synthetics in year 1 but the correlations decreased to nearly zero by year 3 (Woodfield et al. 1999). Kidwell et al. (1994b) suggested that genotypes selected for maximum genetic dissimilarity could be used to improve forage yield in cross-pollinated species. However, the yield of selections based on the genetic similarity and dissimilarity of synthetic parents was inconsistent (Kidwell et al. 1999). Correlations between genetic distance and hybrid or synthetic performance may be improved by only using markers linked to genes or QTL for yield in the computation (Bernardo 1992), but another factor that could be influencing the relationship is linkage equilibrium in the populations under study (Kidwell et al. 1999). Genome Mapping and Marker-Assisted Selection: Among the forage legumes, alfalfa genome mapping is most advanced. Several independent maps were developed from a variety of diploid crosses (Brummer et al. 2000a). The most complete linkage map is based on an F2 population derived from a cross between M. sativa subsp. falcata and subsp. coerulea that includes more than 900 RFLP, RAPD, isozyme, seed protein, and morphological markers (Kaló et al. 2000). Several genes or quantitative trait loci have been mapped, including unifoliolate leaves, aluminium tolerance, somatic embryogenesis, flower colour, dwarfness, yield, and winterhardiness (summarised in Brummer et al. 2000a). Genetic mapping of tetraploids has been more difficult, but two maps have been or are being developed (Brouwer and Osborn 1999; Brummer et al. 2000b). Mapping efforts are underway in other forage legumes but are less advanced (White et al. 2000). The advent of DNA markers such as RFLPs, AFLPs, RAPDs and micro- satellites has increased the scope and precision with which marker assisted selection (MAS) can be pursued. MAS relies on identifying markers at or closely linked t genes of interest that can be used to indirectly select for o traits that are difficult or expensive to evaluate. Combinations of markers could theoretically be used to select for multiple traits simultaneously, without time-consuming evaluations. Although MAS is being practised in many crops (Young 1999), the true benefits of the technology may be most successfully realised in perennial forage crops, where each cycle of selection takes several years. For example, selection for winter hardiness in alfalfa takes three to five years, because winter severity is variable. A MAS approach could decrease cycle time significantly if markers linked to winter survival could be identified. Perhaps the most important requirement for using MAS is the development of transportable maps based on microsatellite
  • 12. 12 Woodfield and Brummer and single dose restriction fragments that will facilitate moving mapping information across populations. Impact of genomics: International genomics efforts are underway in several forage legumes, and two of these, Medicago truncatula and Lotus japonicus, are being extensively studied as model species (Cook et al. 1997). Sequence information from these model species can be compared with those from other higher plants such as Arabidopsis and rice, for which nucleotide sequences are nearly complete, to assign a putative function for many genes. Complete sequencing of many forage legumes is impractical in the short- term due to prohibitively large genome size or limited perceived economic value, so alternative approaches such as random sequencing of expressed sequence tag libraries from various plant tissues are being pursued. These approaches may be less effective for identifying low copy number genes. Genomics has the potential to ultimately link economically important phenotypes through their underlying physiology and metabolism to the genes responsible for them (DeRisi et al. 1997; Somerville and Somerville 1999). This offers real opportunities for forage breeders to systematically accumulate the genes responsible for control of quantitative traits. Since complete sequences will be possible for these genes, markers can be developed to accurately transfer them into appropriate genetic backgrounds. The precision that genome sequencing provides should also enable the underlying genetic control of quantitative traits to be determined. In particular the role of epistasis in plants and the importance of linked genes on chromosome segments can be examined (Bingham 1998). We caution that the power of these molecular genetic innovations cannot be effectively used without strong population improvement programmes. However, used in concert, they will enable the maximum genetic potential of forage legumes to be achieved. 6. REFERENCES: Abberton MT, Michaelson-Yeates TPT, Marshall AH, Holdbrook-Smith K, Rhodes I (1998) Morphological characteristics of hybrids between white clover, Trifolium repens L., and caucasian clover, Trifolium ambiguum M. Bieb. Plant Breed 117: 494-496. Anderson LB (1973) Relative performance of the late-flowering tetraploid red clover ‘Grasslands 4706’, five diploid red clovers, and white clover. NZ J Exper Agric 1: 233- 237 Austin S, Bingham ET (1997) The potential use of transgenic alfalfa as a bioreactor for the production of industrial enzymes. I : McKersie BD, Brown DCW (eds). Biotechnology n and the improvement of forage legumes. CAB International, UK, pp. 409-424 Bernardo R (1992) Relationship between single-cross performance and molecular marker heterozygosity. Theor Appl Genet 83: 628-634
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