Published in Crop Sci. 43:1736-1740 (2003).
© 2003 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
CROP BREEDING, GENETICS & CYTOLOGY
Shifts in Pest Resistance, Fall Dormancy, and Yield in 12-, 24-, and 120-Parent Grazing Tolerant Synthetics Derived from CUF 101 Alfalfa
M. K. Sledge*,a,
J. H. Boutonb and
G. Kochertc
a The Samuel Roberts Noble Foundation, Ardmore, OK 73401
b Dep. of Crop and Soil Sciences, Univ. of Georgia, Athens, GA 30602-7272
c Dep. of Botany, Univ. of Georgia, Athens, GA 30602-7271
* Corresponding author (mksledge{at}noble.org).
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ABSTRACT
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Selection for grazing tolerance has been reported to be accompanied by changes in resistance to blue alfalfa aphid (Acyrthosiphon kondoi Shinji) and fall dormancy in alfalfa (Medicago sativa L.). This study was conducted to determine changes in pest resistance, fall dormancy, grazing tolerance, and yield between ‘CUF 101’ alfalfa and three experimental, grazing tolerant synthetics. A 120 parent experimental synthetic was derived from CUF 101 after two cycles of selection for grazing tolerance. Two synthetics composed of 12 and 24 parents were selected from the 120 grazing tolerant parents on the basis of the genetic dissimilarity of amplified fragment length polymorphism (AFLP) markers. There were no significant differences between the three experimental synthetics for grazing tolerance; however, all experimental synthetics were more grazing tolerant than CUF 101. All experimental synthetics had lower levels of resistance to blue alfalfa aphid, but generally higher levels of resistance to five other diseases in comparison to CUF 101. All experimental synthetics were more fall dormant than CUF 101. No consistent differences in yield were documented among the synthetics at testing locations in Georgia or California. In conclusion, this study underscores the need for further evaluation, after selection for grazing tolerance, to document fall dormancy and blue alfalfa aphid resistance ratings and to practice reselection for these traits if necessary.
Abbreviations: AFLP, amplified fragment length polymorphism • AN, anthracnose • APH, Aphanomyces root rot • BAA, blue alfalfa aphid • BW, bacterial wilt • FW, Fusarium wilt • PRR, Phytophthora root rot • RFLP, restriction fragment length polymorphism • VW, Verticillium wilt
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INTRODUCTION
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ALFALFA is the most important and widely grown forage legume in the world. It produces more protein per hectare than grain or oilseed crops, making it highly desirable for hay production and pasture for livestock. In addition, alfalfa's ability to fix atmospheric nitrogen makes it valuable for use in crop rotations, increasing the productivity of subsequent crops (Barnes, 1993). In North America, alfalfa is grown primarily as a hay crop (Smith and Bouton, 1993), and has generally not been used for grazing because of rapid stand loss (Bouton et al., 1998). The development of grazing tolerant cultivars of alfalfa has been a major achievement in the improvement of grassland utilization in the USA (Hoveland, 2000).
Grazing tolerance, disease resistance, and winter survival are the major factors affecting alfalfa stand persistence under grazing (Smith et al., 2000). Cultivars with high levels of disease resistance, however, are not necessarily grazing tolerant. Smith et al. (2000), therefore, concluded that grazing tolerance was the primary trait affecting stand persistence under grazing, and disease resistance was secondary. Furthermore, previous research showed that selection for grazing tolerance did not result in consistent changes in insect or disease resistance (Smith and Bouton, 1993; Bouton et al., 1998), with the exception of blue alfalfa aphid resistance, which decreased (Bouton et al., 1998). Moutray (2000) reported that grazing tolerant cultivars of alfalfa showed increased resistance to Phoma crown rot (caused by Phoma medicaginis Malbr. & Roum. in Roum.), reduced resistance to blue alfalfa aphid, and little changes in other pest and disease resistance levels. Fall dormancy may also be affected by selection for grazing tolerance. Smith and Bouton (1993) reported no shift in fall dormancy after one cycle of selection for grazing tolerance, but with the second cycle of selection, fall dormancy increased by one class (Bouton et al., 1998).
Alfalfa suffers from inbreeding depression; therefore, a large number of selected parents are intercrossed to create synthetic cultivars. The level of inbreeding in a synthetic cultivar decreases as the number of parents increases. In a theoretical study, Busbice (1969) showed that there is little increase in inbreeding among synthetic cultivars of autotetraploids when the number of parents is increased from 16 to 64 parents, when the parents are unrelated, and noninbred in the Syn 0. Hill and Elgin (1981) concluded that the optimum number of parents for a synthetic cultivar was greater than four but less than 16. In practice, the majority of synthetic cultivars of alfalfa produced today have more than 100 parents. New cultivars submitted by commercial breeders for certification with the Association of Official Seed Certifying Agencies in 1999 and 2000 averaged 139 parents per synthetic cultivar, with as few as 10 parents and as many as 450 parents (Association of Official Seed Certifying Agencies 1999, 2000). Parental numbers of grazing tolerant synthetic cultivars have ranged from 32 with Alfagraze (Bouton et al., 1991) to over 100 with ABT 805 (Bouton et al., 1997).
A method to reduce the number of parents while maintaining selected characteristics, and avoiding yield depression, would be desirable in alfalfa cultivar development. Molecular markers may provide such a tool. Molecular marker diversity and yield are positively correlated in alfalfa. Kidwell et al. (1994) measured genetic diversity in alfalfa by comparing levels of polymorphism among parental genotypes for restriction fragment length polymorphism (RFLP) markers. Genetic dissimilarities among the parents were estimated on the basis of the presence or absence of restriction fragments, and in tetraploid alfalfa, were highly correlated with yield of their single-cross progenies.
The objective of this study was to determine differences in grazing tolerance, pest resistance, fall dormancy, and yield between the nondormant cultivar CUF 101, and three experimental, grazing tolerant synthetics. A secondary objective of this study was to document the effect of narrowing the parental population size on forage yield, grazing tolerance, fall dormancy, and pest and disease resistances. This was accomplished by comparing the performance of a 120 parent synthetic with synthetics composed of 12 and 24 parents that were selected from the 120 parents based on genetic dissimilarity of AFLP markers.
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MATERIALS AND METHODS
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Plant Material
Using standard protocols (Bouton and Smith, 1998; Smith and Bouton, 1993), we isolated 120 genotypes after two cycles of selection for grazing tolerance at Tifton, GA, from the cultivar CUF 101 (Nielson and Lehman, 1977). Three synthetics were then produced. Twelve parents were chosen for the most narrow-based synthetic since this number is within the optimum range of four to 16 identified by Hill and Elgin (1981). Twenty-four parents were chosen as an intermediate number of parents, outside of the four to 16 range, but still a small number by current standards. One hundred-twenty parents were selected to form the broad-based synthetic, since this number represents a parent number typically used in the development of current alfalfa cultivars (Association of Official Seed Certifying Agencies 1999, 2000). GA-CUF-120 was a broad-based synthetic composited from all 120 genotypes. GA-CUF-24 and GA-CUF-12 were narrow-based synthetics composited from 24 and 12 genotypes that were selected on the basis of AFLP diversity and clustering of genetic dissimilarity scores from among the original GA-CUF-120 genotypes. GA-CUF-24 was composed of the 12 GA-CUF-12 genotypes, plus an additional 12 genotypes. Three and six cuttings were made from each genotype chosen for GA-CUF-24 and GA-CUF-12, respectively. Syn1 seed was produced from these cuttings in cage isolation in Idaho with leafcutter bees (Megachile spp.), and by hand intermating in the greenhouse. Seed from these two sources were pooled. Syn1 seed was limited; therefore, 5 g of Syn1 seed were used to produce Syn2 seed in cage isolation in Idaho with leafcutter bees.
Grazing Tolerance Trials
Two grazing tolerance tests were conducted at the UGA Coastal Plain Experiment Station, Tifton, GA, on a Tifton sandy loam soil (fine-loamy, siliceous, thermic, Plinthic Kandiudult), using standard protocols for field evaluation (Bouton and Smith, 1998). The experimental design was a randomized complete block with six replicates (blocks) and alfalfa entries as treatments. Syn1 seed of each synthetic was planted in the fall of 1997, and Syn2 seed was planted in the fall of 1998. Plots were allowed to reach an early flowering stage before grazing began. After an initial graze down, stand counts were taken in each plot by counting the number of plants in six 0.09-m2 quadrats within each plot. For the rest of the season, the plots were continuously grazed. Nondormant grazing tolerant and intolerant checks (‘ABT805’ and CUF101, respectively) were included and monitored by plant counts throughout the season. When statistically significant (P < 0.05) differences in plant counts between the checks occurred, and these were within acceptable ranges for these checks (Bouton and Smith, 1998), grazing was stopped. At this point, plant counts were taken on all plots. The Syn1 grazing trial was grazed for two seasons, from May 1998 until September 1998, and from May 1999 until September 1999. The Syn2 grazing trial was grazed for two seasons, from May 1999 until September 1999, and from May 2000 until September 2000. Grazing tolerance for each entry was determined by comparison of its final plant counts and survival with those of the check cultivars.
Fall Dormancy, Pest, and Disease Resistance
Fall dormancy trials were conducted at Kingsburg, CA, and at Northfield, MN, according to standard tests (Teuber et al., 1998) with Syn2 seed. Evaluations were conducted according to standard tests for the following insects and diseases: blue alfalfa aphid (BAA), (Berberet et al., 1991); anthracnose (AN) (caused by Colletotrichum trifolii Bain & Essary) (O'Neill, 1991); Aphanomyces root rot (APH) (caused by Aphanomyces euteiches Drechs.) (Fitzpatrick et al., 1998); bacterial wilt (BW) [caused by Clavibacter michiganensis subsp. insidiosus (McCulloch, 1925) Davis et al., 1984 = Corynebacterium insidiosum (McCulloch, 1925) Jensen, 1934] (Fox and Thies, 1991); Fusarium wilt (FW) [caused by Fusarium oxysporum Schlechtend.: Fr. f. sp. medicaginis (Weimer) W.C. Snyder & H.N. Hans.] (Nygaard and Barnes, 1991); Phytophthora root rot (PRR) (caused by P. megasperma Drechs. f. sp. medicaginis T. Kuan & D.C. Erwin) (Thies and Barnes, 1991); and Verticillium wilt (VW) (caused by Verticillium albo-atrum Reinke & Berthier) (Grau, 1991).
Yield Trials
Yield testing was conducted in replicated small plots at three locations. The experimental design was a randomized complete block with six replicates (blocks) and alfalfa entries as treatments. Entries were GA-CUF-120, GA-CUF-24, GA-CUF-12, the parental cultivar CUF 101, and ABT 805, a high yielding, grazing tolerant check cultivar developed for use in the Southeast (Bouton et al., 1997). Plots of each entry (1.5 x 3.5 m) were established with Syn1 seed at the Central Georgia Branch Experiment Station near Eatonton, GA, on a Cecil sandy loam soil (clayey, kaolinitic, thermic, Typic Hapludult) in the fall of 1997. Plots were harvested five times in 1998. At the UGA Plant Sciences Farm near Athens, GA, plots were established with Syn1 seed on a Cecil sandy loam soil (clayey, kaolinitic, thermic, Typic Hapludult) in the spring of 1998. Plots were harvested twice in 1998, and three times in 1999. Syn2 seed was planted in Kingsburg, CA, in the spring of 1999, and plots were harvested five times that year. Plots were cut with a flail harvester, weighed, and subsampled for dry matter percentage. Dry matter yield data were analyzed by ANOVA. Although two additional forage yield trials were also planted with Syn2 seed, these plots were lost either to disease or flooding, so Syn2 forage yield data is only available for 1999 in California.
DNA Isolation and AFLP Markers
Leaf material was collected from each of the 120 genotypes growing in pots in the greenhouse. This leaf material was frozen in liquid nitrogen, and lyophilized for 24 to 48 h. Dried leaves were ground in a mortar and pestle with liquid nitrogen and a small amount of glass beads. DNA was isolated according to the CTAB (cetyltrimethylammonium bromide) procedure of Saghai-Maroof et al. (1984). AFLP assays (Vos et al., 1997), were performed with the Gibco-BRL AFLP Analysis System I (Invitrogen Corporation, Carlsbad, CA) using the reagents and instructions provided by the manufacturer.
AFLP Data Analysis
Each AFLP band was assigned a marker number, and the 120 parent plants were manually assigned a score of 1 for presence and 0 for absence of each band. Genetic dissimilarity was estimated for pairs of genotypes by subtracting from 1 a similarity value calculated by comparing banding patterns over markers by means of the Dice coefficient (Dice, 1945):
in which GDij is the genetic dissimilarity between a pair of genotypes (i and j), a is the number of bands common to both i and j, b is the number of bands present in i and absent in j, and c is the number of bands absent in i and present in j.
Genotypes were clustered, on the basis of genetic dissimilarity scores, into 12 and 24 clusters, by PROC CLUSTER of the Statistical Analysis System (SAS, 1989). Individuals within the clusters were genetically similar, whereas individuals from different clusters were genetically dissimilar. Within each cluster, the genotype with the highest total number of AFLP bands was chosen to represent that cluster in the GA-CUF-12 and GA-CUF-24 experimental synthetics.
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RESULTS
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All three experimental synthetics were significantly more grazing tolerant than the grazing sensitive check cultivar CUF 101, but only GA-CUF-120 showed plant survival equal to the grazing tolerant check ABT 805 (Table 1). The GA-CUF synthetics did not differ significantly (P < 0.05) from each other for grazing tolerance in either the Syn1 or Syn2 generation (Table 1), although GA-CUF-12 and GA-CUF-24 were numerically lower than GA-CUF-120 in both experiments.
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Table 1. Grazing survival of Syn1 and Syn2 generations of 12-, 24-, and 120-parent experimental synthetics selected for grazing tolerance from CUF 101 alfalfa.
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GA-CUF-120, GA-CUF-24, and GA-CUF-12 all had a fall dormancy rating of 8 in Kingsburg, CA and 7 at Northfield, MN (data not shown). Differences in fall dormancy ratings between locations cannot be explained, but it is noted that different dormancy class check cultivars were used at the two locations. CUF 101 is the fall dormancy class 9 check cultivar. This change in fall dormancy class score from a 9 to a 7 or 8 represents an increase in fall dormancy.
Concerning changes in disease resistance that occurred during selection for grazing tolerance, the GA-CUF experimental synthetics had consistently higher resistance to AN, APH, BW, and VW than CUF 101 (Table 2). Resistance to PRR was similar between CUF 101 and GA-CUF-120, but was higher for GA-CUF-24 and GA-CUF-12. Resistance to FW was similar between CUF 101 and the GA-CUF experimental synthetics. Significant differences (P < 0.05) between synthetics with differing numbers of parents were seen in resistance to AN, BW, and PRR, however, no one synthetic was consistently more resistant than another. There was an increase in resistance to all diseases between the Syn 1 and Syn 2 generation for all synthetics tested with the exception of GA-CUF-24 for resistance to anthracnose.
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Table 2. Disease and insect resistance levels in Syn1 and Syn2 generations of 12-, 24-, and 120-parent experimental synthetics selected for grazing tolerance from CUF 101 alfalfa.
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CUF 101 (Nielson and Lehman, 1977) is considered highly resistant to BAA with a resistance level of 55% (Table 2). All experimental synthetics had lower levels of resistance to blue alfalfa aphid than did CUF 101. There was no trend for increased resistance in the Syn2 generation.
There were no significant differences (P < 0.05) in yield between the broad-based and narrow-based synthetics (Table 3) at Athens, GA, or Kingsburg, CA. At Eatonton, GA-CUF-24 yielded less than the other experimental synthetics. Problems at Eatonton, including poor stand establishment, drought, and deer grazing, may have contributed to these variable results. This testing environment was dropped from the study after 1998 for these reasons. There was no depression in yield associated with decreasing the number of parents for the synthetic from 120 to 24 or 12 parents, suggesting a lack of detrimental inbreeding in the low parent number populations. There were also no differences in yield between the adapted, high yielding check cultivar ABT 805 and the three GA-CUF synthetics. ABT 805 and all three synthetics yielded significantly higher than CUF 101, the cultivar from which the synthetics were selected, at Athens and Eatonton, but were not significantly different from CUF 101 at Kingsburg, CA.
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Table 3. Dry matter yield for 12-, 24-, and 120-parent experimental synthetics selected for grazing tolerance from CUF 101 alfalfa at three locations.
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A total of 177 AFLP markers, from four primer pairs, were scored for presence or absence among the 120 GA-CUF-120 genotypes. Genetic dissimilarity scores ranged from a low of 0.09 to 0.52. The most common scores were between 0.2 and 0.3 (Fig. 1).
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Fig. 1. Distribution of genetic dissimilarity of pair wise comparisons of all GA-CUF-120 genotypes determined on the basis of dissimilarity of AFLP markers.
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DISCUSSION
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CUF 101 is a nondormant synthetic cultivar with 91 parents, adapted to southern California and southwestern Arizona. It was selected from genotypes growing in an over-irrigated hay production field that had been infested with BAA for more than 6 wk. The parentage of CUF 101 can be traced to the nine basic germplasm sources of alfalfa in the following percentages: M. falcata L. 0, Ladak 1, M. varia (T. Martyn) Arcang. 2, Turkistan 11, Flemish 1, Chilean 7, Peruvian 2, Indian 23, and African 53. CUF 101 has moderate resistance to PRR, high resistance to FW, high resistance to spotted alfalfa aphid, and is susceptible to BW. CUF 101 is currently used as the nondormant grazing sensitive check in the standard test for grazing tolerance (Bouton and Smith, 1998).
Previously reported population changes associated with selection for grazing tolerance include an increase in fall dormancy (Bouton et al., 1998) and a decrease in resistance to BAA (Moutray, 2000). These changes were also observed in this study. Moutray (2000) reported that one cycle of selection for resistance to BAA could restore the population to levels of resistance observed before selection for grazing tolerance. Bouton et al. (1998) suggested that when selecting for grazing tolerance, one should start with a higher dormancy class than is desired for the final cultivar, to compensate for the change in dormancy. This study underscores the need for further evaluation, after selection for grazing tolerance, to document fall dormancy and BAA resistance ratings and to practice reselection for these traits if necessary.
With the exception of resistance to BAA and FW, all GA-CUF synthetics showed higher levels of disease resistance than did CUF 101 (Table 2). This is in contrast to previous findings in which no consistent changes in pest and disease resistance were observed as a result of selection for grazing tolerance (Smith and Bouton, 1993; Bouton et al., 1998; Moutray 2000). It is possible, however, that the changes in disease resistance levels observed in the current study are not a result of selection for grazing tolerance, but are due to field selection that may have occurred in the grazing trials from which CUF-120 was selected, or in the soil of the seed increase. Selection pressure due to the presence of a particular pest or pathogen could have resulted in increased levels of resistance, whereas lack of selection pressure due to absence of a pest or pathogen could have allowed resistance levels to decrease. Smith and Bouton (1993) suggested that close grazing imposes morphological and physiological stresses that can make plants more susceptible to pathogen pressure. Plants with stress avoidance characteristics, such as the deep set crowns and leaf retention associated with grazing tolerance, may be less likely to enter a weakened state that would encourage pathogen infection. Selecting smaller numbers of parents on the basis of genetic dissimilarity of AFLP markers had no consistent effect on levels of pest and disease resistance.
No difference was observed between Syn1 and Syn2 generations for grazing tolerance; however, there were shifts in disease resistance, with a trend toward higher levels of disease resistance in the Syn2 generation. This could have been due to presence of the diseases in the soil of the seed increase, resulting in a greater contribution of seed from resistant plants to the next generation.
The AFLPs used in this study to determine genetic diversity are unmapped; therefore, the genomic coverage of these markers is unknown. It has been suggested that using unmapped markers with unknown genome coverage may result in unreliable genetic diversity measurements (Karp et al., 1997; Laurie et al., 1997). Laurie et al. (1997) stated that a map-based approach to genetic diversity studies should be used, in which it is known that the entire genome, or a large part of it, is sampled to provide an overall measure of genetic diversity. Virk et al. (2000), however, found that there was no advantage in using mapped versus unmapped markers to assess genetic diversity, and that unmapped AFLP bands revealed patterns of variation that were consistent with those obtained with mapped markers.
Kidwell et al. (1999) used 61 RFLP markers to measure pair wise dissimilarity values for 93 tetraploid alfalfa genotypes derived from nine cultivars. They observed genetic dissimilarity scores between 0.33 and 0.58, with 0.46 being the most common score. In the current study, the genetic dissimilarity scores ranged from 0.09 to 0.52, with scores between 0.21 and 0.31 being the most common. The difference in the range of genetic dissimilarity scores may reflect a greater genetic diversity in the germplasm utilized by Kidwell et al. (1999) which consisted of a mixture of M. sativa subsp. sativa and M. sativa subsp. falcata, drawn from seven of the original nine germplasm sources (Barnes, 1993). In contrast, CUF 101 was derived largely from two of the nine germplasm sources, 53% African and 23% Indian, with no M. falcata germplasm. The lower genetic dissimilarity scores could be a reflection of the smaller number of germplasm sources used to derive CUF 101.
We found that selecting small numbers of parents on the basis of genetic dissimilarity had a negative impact on grazing tolerance. While there were no differences in grazing tolerance among the GA-CUF experimental synthetics, only GA-CUF-120 had grazing tolerance similar to the check cultivar ABT 805. Selecting small numbers of parents on the basis of genetic dissimilarity had no significant effect on forage yield. These results are consistent with those obtained by Kidwell et al. (1999) in which selection based on genetic dissimilarity was observed to have no consistent effect on forage yield.
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ACKNOWLEDGMENTS
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Evaluations for fall dormancy and for insect and disease resistances were conducted by ABI-Alfalfa and Crop Characteristics, Inc.
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NOTES
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Part of a Ph.D. thesis submitted by M.K. Sledge.
Received for publication August 13, 2002.
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REFERENCES
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ABSTRACT
INTRODUCTION
